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METHODS IN ENZYMOLOGY EDITORS-IN-CHIEF

John N. Abelson

Melvin I. Simon

DIVISION OF BIOLOGY CALIFORNIA INSTITUTE OF TECHNOLOGY PASADENA. CALIFORNIA

FOUNDING EDITORS

Sidney P. Colowick and Nathan 0. Kaplan

Methods in Enzymology Volume 182

Guide to Protein Purijication EDITED BY

Murray P. Deutscher DEPARTMENT OF BIOCHEMISTRY UNIVERSITY OF CONNECTICUT HEALTH CENTER FARMINGTON. CONNECTICUT

w

Academic Press

San Diego New York Boston London Sydney Tokyo Toronto

COPYRIGHT O 1990 BY ACADEMIC PRESS, INC. All Rights Reserved. No pan of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

Academic Press, Inc. A Division of Harcourt Brace & Company 525 B Street, Suite 1900 San Diego, California 92101-4495 United Kingdom Edition published by ACADEMIC PRESS LIMITED 24-28 Oval Road, London NW 1 7DX

LIBRARY OF CONGRESS CATALOG CARD NUMBER:

ISBN 0- 12-182083-1 (Hardcover)(alk. paper) ISBN 0-12-213585-7 (comb bound)(alk. paper)

PRINTED [N THE UNITED STATES OF AMERICA 94 95 96 97 QW 9 8 7 6 5

54-91 10

Table of Contents

1. Why Purify Enzymes?

ARTHUR KORNBERG

Section I. D e v e l o p i n g Purification P r o c e d u r e s 2. Strategies and Considerations for Protein Purifica- STUART LINN

tions

Section 11. G e n e r a l Methods for Handling Proteins and E n z y m e s 3. Setting Up a Laboratory

MURRAY P. DEUTSCHER

19

4. Buffers: Principles and Practice

VINCENT S. STOLLAND JOHNS. BLANCHARD

24

5. Measurement of Enzyme Activity

EDWARDF. ROSSOMANDO

38

6. Quantitation of Protein

CHRISTAM. STOSCHECK

50

7. Concentration of Proteins and Removal of Solutes THOMAS POHL

68

8. Maintaining Protein Stability

83

MURRAY P. DEUTSCHER

S e c t i o n 111. Optimization of Starting M a t e r i a l s 9. Overproduction of Proteins in Escherichia coli: ASISDAS Vectors, Hosts, and Strategies 10. Overexpression of Proteins in Eukaryotes

MARGARET K. BRADLEY

11. Secretion of Heterologous Proteins in Escherichia I. BARRYHOLLAND, coli BRENDAN KENNY, BORISSTEIPE,AND ANDREAS PL~~CKTHUN

112

132

vi

TABLE OF CONTENTS

Section IV. Preparation of Extracts and Subcellular Fractionation 12. Preparation of Extracts from Prokaryotes

MILLARD CULLAND CHARLES S. MCHENRY

147

13. Preparation of Extracts from Yeast

S. MICHALJAZWINSKI

154

14. Preparation of Extracts from Plants

PETERGEGENHEIMER

174

15. Preparation of Extracts from Higher Eukaryotes

JOHNDAVIDDIGNAM

194

16. Isolation of Subcellular Organelles

BRIANSTORRIE AND EDWARD A. MADDEN

203

JURISOZOLS

225

17. Preparation of Membrane Fractions

Section V. Solubilization P r o c e d u r e s 18. Detergents: An Overview

JUDITHM. NEUGEBAUER 239

19. Solubilization of Native Membrane Proteins

LEONARD M. HJELMELAND 253

20. Solubilization of Protein Aggregates

M. HJELMELAND 277 21. Removal of Detergents from Membrane Proteins LEONARD

Section VI. Purification Procedures: B u l k M e t h o d s 22. Precipitation Techniques 23. Precipitation of Proteins with Polyethylene Glycol KENNETH C. INGHAM

Section VII. Purification Procedures: Chromatographic Methods 24. Ion-Exchange Chromatography 25. Gel Filtration 26. Protein

Chromatography Columns

on

Hydroxyapatite MARINA J. GORBUNOFF

27. Hydrophobic Chromatography

ROBERT M. KENNEDY

28. Chromatography on Immobilized Reactive Dyes

EARLESTELLWAGEN

29. Affinity Chromatography: General Methods

STEVEN OSTROVE

30. Affinity Chromatography: Specialized Techniques STEVEN OSTROVE AND SHELLY WEISS

3 1. Chromatofocusing

LALI-AN GIN

301

TABLE OF CONTENTS

vii -

32. High-Performance Liquid Chromatography: Ef- ROMANM. CHICZAND fective Protein Purification by Various Chro- FREDE. REGNIER matographic Modes

Section VIII. Purification Procedures: Electrophoretic Methods 33. One-Dimensional Gel Electrophoresis

DAVIDE. GARFIN

34. Protein Analysis Using High-Resolution TwoDimensional Polyacrylamide Gel Electrophoresis

BONNIE S. DUNBAR. HITOMIKIMURA, AND THERESEM. TIMMONS

35. lsoelectric Focusing

DAVIDE. GARFIN

36. Gel Staining Techniques

CARLR. MERRIL

37. Elution of Protein from Gels

MICHAELG. HARRINGTON

Section IX. Specialized Purification Procedures 38. Purification of Membrane Proteins

THOMASC. THOMASAND MARKG. MCNAMEE

39. Purification of DNA-Binding Proteins by SiteSpecific DNA Affinity Chromatography

SHLOMOEISENBERG, STEPHENC. FRANCESCONI. CHRISCIVALIER, AND SCOTTS. WALKER

40. Purification of Glycoproteins

CRAIGGERARD

41. Purification of Multienzyme Complexes

PAULA. SREREAND CHRISTOPHER K. MATHEWS

Section X. Characterization of Purified Proteins 42. Determination of Purity 43. Determination of Size, Molecular Weight, and THOMASM. LAUEAND DAVIDG. RHODES Presence of Subunits 44. Amino Acid Analysis

JURISOZOLS

45. Limited N-Terminal Sequence Analysis

PAULMATSUDAIRA

46. Peptide Mapping

RALPHC. JUDD

47. Analysis for Protein Modifications and Nonpro- SAMSEIFTERAND tein Cofactors SASHAENGLARD 48. Protein Crystallization

viii

TABLEOFCONTENTS

Section XI. Immunological Procedures 49. Preparation of Polyclonal Antibodies

AND BONNIES. DUNBAR ERICD. SCHWOEBEL

50. Preparation of Monoclonal Antibodies

AND BONNIES. DUNBAR SHERIM. SKINNER

51. Protein Blotting and Immunodetection

THERESE M. TIMMONS AND BONNIES. DUNBAR

52. Immunoprecipitation of Proteins

GARYL. FIRESTONE AND SANDRA D. WINGUTH

53. Immunoassays

CHARLES W. PARKER

Section XII. Additional Techniques 54. Radiolabeling of Proteins

W. PARKER CHARLES

55. Using Purified Protein to Clone Its Gene

JOHNM. WOZNEY

56. Computer Analysis of Protein Structure

PATRICK ARGOS

Section XIII. C o n c l u d i n g Remarks 57. Rethinking Your Purification Procedure

MURRAY P. DEUTSCHER

Appendix: P e r s o n a l Perspectives on Enzyme Purification 58. The Private Life of DNA Polymerase I

ARTHUR KORNBERG

59. Purification and Crystallization of Transaldolase B. L. HORECKER from Candida utilis A N D 0 . TSOLAS 60. Discovery of Glutamine Synthetase Cascade

EARLR. STADTMAN

61. Discovery, Resolution, Purification, and Function KIVIEMOLDAVE

of Elongation Factors

Contributors to Volume 182 Article numbers are in parentheses following the names of contributors. AtEliations listed are current.

PATRICKARGOS(56), European Molecular Biology Laboratory, 6900 Heidelberg, Federal Republic of Germany JOHN S. BLANCHARD (4), Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461 MARGARETK. BRADLEY(lo), Department of Pathology, Dana-Farber Cancer Institute and the Harvard Medical School, Boston, Massachusetts 02115 ROMANM . CHICZ(32), Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138 CHRISCIVALIER (39), Department of Microbiology, University of Connecticut Health Center, Farmington, Connecticut 06032 MILLARDCULL (12). Department of Biochemistry, Biophysics and Genetics, University of Colorado Health Sciences Center, Denver, Colorado 80262 ASISDRS(9), Department of Microbiology, University of Connecticut Health Center, Farmington, Connecticut 06032 MURRAYP. DEUTSCHER (3, 8, 57), Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 06032 JOHN DAVIDDIGNAM (IS), Department of Biochemistry, Medical College of Ohio, Toledo, Ohio 43699 BONNIES. DUNBAR(34, 49-51), Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030 SHLOMOEISENBERG (39), Department of Microbiology, University of Connecticut Health Center, Farmington, Connecticut 06032 SASHAENGLARD (22, 47), Department of

Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461 GARY L. FIRESTONE (52), Department of Molecular and Cell Biology and Cancer Research Laboratory, University of California at Berkeley, Berkeley, California 94720 STEPHENC . FRANCESCONI (39), Department of Microbiology, University of Connecticut Health Center, Farmington, Connecticut 06032 DAVIDE . GARFIN(33, 3 9 , Chemical Division, Research Products Group, Bio-Rad Laboratories, Incorporated, Richmond, California 94804 PETER GEGENHEIMER(14), Departments ofBotany and Biochemistry, University of Kansas, Lawrence, Kansas 66045 CRAIGGERARD(40), Department of Pediatrics, Harvard Medical School, Children's Hospital Medical Center, Boston, Massachusetts 02115 LALLANGIRI(31), Quality Control Department, Connaught Laboratories, Inc., Swiftwater, P A 18370 MARINAJ. GORBUNOFF (26), Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254 MICHAELG . HARRINGTON (37), Biology Department, California Institute of Technology, Pasadena, California 91125 DONNAL. HARTLEY(ZO), Centre International de Recherche Daniel Carasso, 92350 Le Plessis-Robinson, Paris, France

LEONARDM . HJELMELAND (19, 21), Departments of Ophthalmology and Biological Chemistry, School of Medicine, University of California, Davis, Davis, California 95616

x

CONTRIBUTORS TO VOLUME

182

I. BARRY HOLLAND ( l l ) ,Department of Gelute, Massachusetts Institute of Technolnetics, University of Leicesrer, Leicester ogy, Cambridge, Massachusetts 02142 LEI 7RH, England CHARLES S. MCHENRY (12), Department of B . L. HORECKER (59), Department of BioBiochemistry, Biophysics and Genetics, chemistry, Cornell University Medical University of Colorado Health Sciences College, New York, New York 10021 Center, Denver, Colorcdo 80262 KENNETHC. INGHAM(231, Biochemistry MARKG. MCNAMEE(38), Department of Laboratory, American Red Cross HalBiochemistry and Biophysics, University land Laboratories, Rockville, Maryland of California, Davis, California 95616 20855 CARLR. MERRIL(36), Laboratory of BioS. MICHAL JAZWINSKI (131, Department of chemical Genetics, National Institute of Biochemistry and Molecular Biology, Mental Health, Bethesda, Maryland Louisiana State University Medical Cen20892 ter, New Orleans, Louisiana 70112 KIVIEMOLDAVE (61), Department of BiolRALPHC. JUDD (461, Division of Biological ogy, University of California, Santa Cruz, Sciences, University of Montana, Missunfacruz,california 95064 soula. Montana 59812 JUDITH M. NEUGEBAUER (la), Department ROBERT M. KENNEDY (271, Membrex Incorof chemistry and Institute of Colloid and porated, Garfield, New Jersey 07026 Surface Science, Clarkson University, BRENDANKENNY(111, Department of GePotsdam, New York I3676 netics, University of Leicesrer, Leicesrer DAVID OLLIS (48), Department of BiochemLEI 7RH, England istry, Molecular Biology and Cell Biology, HITOMIKIMURA(34), Department of BioNorthwestern University, Evanston, Illichemistry, State University of New' York nois 60208 at Stony Brook, Stony Brook, New York STEVENOSTROVE(29, 30), Davy McKee 11794 Corporation, Berkeley Heights, New JerARTHURKORNBERG (1, 58), Department of sey 07922 Riochemistry, Stanford University, StanJURIS OZOLS(17, 44), Department of Bioford, California 94305 chemistry, University of Connecticut THOMAS M. LAUE(42, 43), Department of Health Center, Farmington, Connecticut Biochemistry, University of New Hamp06032 shire, Durham, New Hampshire 03824 W. PARKER(53, 5417 STUARTLINN(2), Division of Biochemistry CHARLES of Medicine and ~ ~~ i ~university l l ~ ~o f~~ a ~l - ~, ~ and Microbiology, l ~ Washing~ ton University School of Medicine, St. ifornia, Berkeley, ~ ~ ~ california k ~ l ~ ~ , Louis, Missouri 63110 94720 EDWARDA. MADDEN(16), Department of ANDREASP L ~ ~ C K T H(11). U N Gen-Zentrum Biology, University of Indianapolis, Indider Universitat Miinchen, Max-Planck-lnanapolis, Indiana 46227 stitut fur Biochemie, 0-8033 Martinsried, Munich, Federal Republic of Germany FIONAA. 0. MARSTON(20), Celltech Limired, Slough. Berkshire SLI 4EN, En- THOMAS POHL(7,Abteilung fur Molekulare gland Neuroendokrinologie, Max-Planck-lnstirut fur Experimentelle Medizin, 3400 GotCHRISTOPHER K. MATHEWS(41), Departringen, Federal Republic of Germany ment of Biochemistry and Biophysics, Oregon State University, Coruallis, Oregon FREDE. REGNIER (32), Department o f ~ i o 97331 chemistry, Purdue University, West LaPAULMATSUDAIRA ( 4 9 , Whitehead Instifayette, Indiana 47907

DAVIDG . RHODES(42, 43), Biomolecular Structure Analysis Center, Department of Radiology, University of Connecticut Health Center, Farmington, Connecticut 06032 EDWARDF. ROSSOMANDO ( 5 , 24), Department of BioStructure and Function, Universify of Connecticut Health Center, Farmington, Connecticut 06032 EPJC D. SCHWOEBEL (49), Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030 SAMSEIFTER (22, 47), Department of Biochemistty, Albert Einstein College of Medicine, Bronx, New York I0461 SHERIM . SKINNER (SO),Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030 PAULA. SRERE(41), Research Service, Department of Veteran Affairs, University of Texas Southwestern Medical Center, Dallas, Texas 75216 EARL R. STADTMAN (60), National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892 BORISSTEIPE( I I ) , Gen-Zentrum der Universitat Munclzen, Max-Planck-lnstitut fur Biochemie, 0-8033 Martinsried, Munich, Federal Republic of Germany EARLESTELLWAGEN (25, 28), Department of Biochemistry, University of Iowa. Iowa City, Iowa 52242 VINCENT S . STOLL( 4 ) , Department of Biochemistry, Albert Einstein College of

Medicine, Bronx, New York 10461 BRIANSTORRIE (16), Biochemistry Department, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24060 CHRISTAM. STOSCHECK (6), Department of Medicine, Division of Dermatology, Veterans Administration, Nashville, Tennessee 37212 THOMASC. THOMAS(38), Department of Biochemistry and Biophysics, University of California, Davis, D u u i ~ , Culifornia 95616 THERESE M. TIMMONS ( 3 4 , 5 1 ) ,Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030 0 . TSOLAS(59), Laborarory of Biological Chemistry, University of loannina Medical School, loannina, GR 453 32 Greece SCOTTS . WALKER(39), Department of Microbiology, University of Connecticut Health Center, Farmington, Connecticut 06032 SHELLYWEISS(30), New Brunswick Scientific, Edison, New Jersey 08818 STEPHENWHITE(48), Department of Biology, Brookhuven National Laboratory, Upton, New York 11973 SANDRAD. WINGUTH(52), Department of Ophthalmology, Ocular Oncology Unit, University of California at San Francisco, San Francisco, California 94143 JOHNM. WOZNEY( 5 3 , Genetics Institute, Incorporated, Cambridge, Massachusetts 02140

Preface The explosion of work and interest in molecular biology in recent years has made protein purification something of a lost art, especially among younger biochemists and molecular biologists. At the same time, many of the more interesting biological problems have now reached a stage that requires work with purified proteins and enzymes. This has led to a situation in which many important studies stop at the demonstration of a physiological effect, and are not carried through to an understanding of the proteins responsible for the phenomenon. For these reasons a methods manual dealing with all aspects of protein purification should be a valuable addition to the Methods in Enzymology series and should be extremely useful to the scientific community. Although techniques for protein purification have been included in a few volumes in this series in the past, this Guide brings together in one source up-to-date procedures for purifying, characterizing, and working with proteins and enzymes. The volume begins with introductory chapters describing the rationale for studying proteins and enzymes with strategies for their purification, is followed by contributions that familiarize the reader with procedures for working with proteins and enzymes, and proceeds to describe in detail methods for their purification and characterization. Useful immunological procedures and other techniques that aid in the study of proteins are also included. In addition to the methods articles that make up the bulk of the Guide, a few retrospective chapters by eminent biochemists, which describe one of their famous studies in order to give a feeling for the "art" of enzyme purification that goes beyond techniques and mechanical procedures, have been included. The Guide is a self-contained volume covering all the important procedures for purifying proteins, as well as other more specialized techniques. However, to stay within the confines of a single volume, some details are dealt with by reference to other works, but these have been kept to a minimum. It is hoped that this volume will satisfy the needs of both the novice in protein purification and the more experienced researcher.

...

Xlll

[I]

WHY PURIFY ENZYMES?

1

[I] Why Purify Enzymes?

"Don't waste clean thinking on dirty enzymes" is an admonition of Efraim Racker's which is at the core of enzymology and good chemical practice. It says simply that detailed studies of how an enzyme catalyzes the conversion of one substance to another is generally a waste of time until the enzyme has been purified away from the other enzymes and substances that make up a crude cell extract. The mixture of thousands of different enzymes released from a disrupted liver, yeast, or bacterial cell likely contains several that direct other rearrangements of the starting material and the product of the particular enzyme's action. Only when we have purified the enzyme to the point that no other enzymes can be detected can we feel assured that a single type of enzyme molecule directs the conversion of substance A to substance B, and does nothing more. Only then can we learn how the enzyme does its work. The rewards for the labor of purifying an enzyme were laid out in a series of inspirational papers by Otto Warburg in the 1930s. From his laboratory in Berlin-Dahlem came the discipline and many of the methods of purifying enzymes and with those the clarification of key reactions and vitamin functions in respiration and the fermentation of glucose. Warburg's contributions strengthened the classic approach to enzymology inaugurated with Eduard Biichner's accidental discovery, at the turn of this century, of cell-free conversion of sucrose to ethanol. One tracks the molecular basis of cellular function-alcoholic fermentation in yeast, glycolysis in muscle, luminescence in a fly, or the replication of DNA-by first observing the phenomenon in a cell-free system. Then one isolates the responsible enzyme (or enzymes) by fractionation of the cell extract and purifies it to homogeneity. Then one hopes to learn enough about the structure of the enzyme to explain how it performs its catalytic functions, responds to regulatory signals, and is associated with other enzymes and structures in the cell. By a reverse approach, call it neoclassical, especially popular in recent decades, one first obtains a structure and then looks for its function. The protein is preferably small and stable, and has been amplified by cloning or is commercially available. By intensive study of the protein and homologous proteins, one hopes to get some clues to how it functions. As the popularity of the neoclassical approach has increased, so has there METHODS IN ENZYMOLOGY. VOL. 182

Copyright O 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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METHODS IN ENZYMOLOGY

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been a corresponding decrease in interest in the classical route: pursuit of a function to isolate the responsible structure. Implicit in the devotion to purifying enzymes is the faith of a dedicated biochemist of being able to reconstitute in a test tube anything a cell can do. In fact, the biochemist with the advantage of manipulating the medium: pH, ionic strength, etc., by creating high concentrations of reactants, by trapping products and so on, should have an easier time of it. Another article of faith is that everything that goes on in a cell is catalyzed by an enzyme. Chemists sometimes find this conviction difficult to swallow. On a recent occasion I was told by a mature and well-known physical chemist that what fascinated him most in my work was that DNA replication was catalyzed by enzymes! This reminded me of a seminar I gave to the Washington University chemistry department when I arrived in St. Louis in 1953. I was describing the enzymes that make and degrade orotic acid, and began to realize that my audience was rapidly slipping away. Perhaps they had been expecting to hear about an organic synthesis of erotic acid. In a last-ditch attempt to retrieve their attention, I said loudly that every chemical event in the cell depends on the action of an enzyme. At that point, the late Joseph Kennedy, the brilliant young chairman, awoke: "Do you mean to tell us that something as simple as the hydration of carbon dioxide (to form bicarbonate) needs an enzyme?" The Lord had delivered him into my hands. "Yes, Joe, cells have an enzyme, called carbonic anhydrase. It enhances the rate of that reaction more than a million fold. " Enzymes are awesome machines with a suitable level of complexity. One may feel ill at ease grappling with the operations of a cell, let alone those of a multicellular creature, or feel inadequate in probing the fine chemistry of small molecules. Becoming familiar with the personality of an enzyme performing in a major synthetic pathway can be just right. To gain this intimacy, the enzyme must first be purified to near homogeneity. For the separation of a protein species present as one-tenth or one-hundredth of 1% of the many thousands of other kinds in the cellular community, we need to devise and be guided by a quick and reliable assay of its catalytic activity. No enzyme is purified to the point of absolute homogeneity. Even when other proteins constitute less than 1% of the purified protein and escape detection by our best methods, there are likely to be many millions of foreign molecules in a reaction mixture. Generally, such contaminants do not matter unless they are preponderantly of one kind and are highly active on one of the components being studied.

[ll

WHY PURIFY ENZYMES?

3

Only after the properties of the pure enzyme are known is it profitable to examine its behavior in a crude state. "Don't waste clean thinking on dirty enzymes" is sound dogma. I cannot recall a single instance in which I begrudged the time spent on the purification of an enzyme, whether it led to the clarification of a reaction pathway, to discovering new enzymes, to acquiring a unique analytical reagent, or led merely to greater expertise with purification procedures. So, purify, purify, purify. Purifying an enzyme is rewarding all the way, from first starting to free it from the mob of proteins in a broken cell to having it finally in splendid isolation. It matters that, upon removing the enzyme from its snug cellular niche, one cares about many inclemencies: high dilution in unfriendly solvents, contact with glass surfaces and harsh temperatures, and exposure to metals, oxygen, and untold other perils. Failures are often attributed to the fragility of the enzyme and its ready denaturability, whereas the blame should rest on the scientist for being more easily denatured. Like a parent concerned for a child's whereabouts and safety, one cannot leave the laboratory at night without knowing how much of the enzyme has been recovered in that day's procedure and how much of the contaminating proteins still remain. To attain the goal of a pure protein, the cardinal rule is that the ratio of enzyme activity to the total protein is increased to the limit. Units of activity and amounts of protein must be strictly accounted for in each manipulation and at every stage. In this vein, the notebook record of an enzyme purification should withstand the scrutiny of an auditor or bank examiner. Not that one should ever regard the enterprise as a business or banking operation. Rather, it often may seem like the ascent of an uncharted mountain: the logistics like those of supplying successively higher base camps. Protein fatalities and confusing contaminants may resemble the adventure of unexpected storms and hardships. Gratifying views along the way feed the anticipation of what will be seen from the top. The ultimate reward of a pure enzyme is tantamount to the unobstructed and commanding view from the summit. Beyond the grand vista and thrill of being there first, there is no need for descent, but rather the prospect of even more inviting mountains, each with the promise of even grander views. With the purified enzyme, we learn about its catalytic activities and its responsiveness to regulatory molecules that raise or lower activity. Beyond the catalytic and regulatory aspects, enzymes have a social face that dictates crucial interactions with other enzymes, nucleic acids, and membrane surfaces. To gain a perspective on the enzyme's contributions to the cellular economy, we must also identify the factors that induce or

4

METHODS IN ENZYMOLOGY

[I]

repress the genes responsible for producing the enzyme. Tracking a metabolic or biosynthetic enzyme uncovers marvelous intricacies by which a bacterial cell gears enzyme production precisely to its fluctuating needs. Popular interest now centers on understanding the growth and development of flies and worms, their cells and tissues. Many laboratories focus on the aberrations of cancer and hope that their studies will furnish insights into the normal patterns. Enormous efforts are also devoted to AIDS, both to the virus and its destructive action on the immune system. In these various studies, the effects of manipulating the cell's genome and the actions of viruses and agents are almost always monitored with intact cells and organisms. Rarely are attempts made to examine a stage in an overall process in a cell-free system. This reliance in current biological research on intact cells and organisms to fathom their chemistry is a modern version of the vitalism that befell Pasteur and that has permeated the attitudes of generations of biologists before and since. It baffles me that the utterly simple and proven enzymologic approach to solving basic problems in metabolism is so commonly ignored. The precept that discrete substances and their interactions must be understood before more complex phenomena can be explained is rooted in the history of biochemistry and should by now be utterly commensensical. Robert Koch, in identifying the causative agent of an infectious disease, taught us a century ago that we must first isolate the responsible microbe from all others. Organic chemists have known even longer that we must purify and crystallize a substance to prove its identity. More recently in history, the vitamin hunters found it futile to try to discover the metabolic and nutritional roles of vitamins without having isolated each in pure form. And so with enzymes it is only by purifying enzymes that we can clearly identify each of the molecular machines responsible for a discrete

FIG. I . Personalized license plate expressing a commitment to enzymology.

metabolic operation. Convinced of this, one of my graduate students expressed it in a personalized license plate (Fig. 1). Acknowledgment This article borrows extensively from "For the Love of Enzymes: The Odyssey of a Biochemist," Harvard University Press, 1989.

Section I Developing Purification Procedures

[21

GENERAL STRATEGIES AND CONSIDERATIONS

9

[21 Strategies and Considerations for Protein Purifications By STUART LINN

The budding enzymologist is generally surprised by the time necessary to develop a protein purification procedure relative to the time required to accumulate information once the purified protein is available. While there is no magic formula for designing a protein purification, some forethought can help to expedite the tedious job of developing the purification scheme. This chapter is designed to point out some considerations to be undertaken prior to stepping up to the bench. Once at the bench, the subsequent chapters of this book as well as two other recent publications concerning enzyme purification1-2should serve as a guide. Preliminary Considerations

What Is the Protein To Be Used For In these days of the biotechnology revolution, the required amount of purified protein may vary from a few micrograms needed for a cloning endeavor to several kilograms required for an industrial or pharmaceutical application. Therefore, a very major consideration is the amount of material required. One should be aware of the scale-up ultimately expected, and the final scheme should be appropriate for expansion to those levels. There are very real limitations to how far a procedure can be scaled up. These limitations are brought about not only by considerations of cost and availability of facilities, but also by physical constraints of such factors as chromatographic resin support capabilities and electrophoresis heating factors. As outlined below, individual steps of the procedure should flow from high-capacityllow-cost techniques toward lowcapacitylhigh-cost ones. Nonetheless, in some cases two procedures may be required: for example, one to obtain microgram quantities for cloning and a second to produce kilogram amounts of the cloned material. The protein chemist should remain flexible for adopting new procedures when such changes are warranted. Another consideration is whether the protein must be active (an enzyme, a regulatory protein, or an antibody, for example), whether it must R. K. Scopes, "Protein Purification, Principles and Practice," 2nd Ed. Springer-Verlag, New York, 1987. R. Burges, ed., "Protein Purification, Micro to Macro." Alan R. Liss, New York, 1987. Copyright 0 1990 by Academic Press, Inc.

METHODS IN ENZYMOLOGY, VOL. 182

All rights of reproduction in any form reserved.

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DEVELOPING PURIFICATION PROCEDURES

[21

be in a native configuration, but not associated with an activity, or whether it need not be in any specific configuration (a small peptide or a peptide to be utilized only for obtaining sequence information, for example). The techniques employed should be as gentle as is necessary, but, whenever possible, some of the harsher but often spectacularly successful procedures such as those which involve extremes of pH, organic solvents, detergents, or hydrophobic or strong affinity chromatographic media should also be used. Assays

Possibly the most important preliminary step is to develop appropriate assays. The success of the purification is often most dependent on this. Five considerations come to mind: sensitivity, accuracy, precision, substrate availability, and cost. Sensitivity is often the limiting factor as the protein becomes diluted into column effluents, etc. Before beginning a step, the likely dilution and losses ought to be estimated and the ability to detect the protein after a reasonably successful procedure ought to be possible. Accuracy and precision are often compromised in these days of fast technology, but clearly these items must be controlled to the extent that the assay is reliable for assuring recovery of material and reproducibility. Specificity is usually a problem early in the purification. Often, however, substrates can be simplified or controls omitted as the purification progresses. Substrate availability and cost refer to the practicality of the assay: Can enough substrate be prepared to perform the entire purification without interruption? Stopping to prepare more substrate or skimping on material usually results in disaster. On the other hand, assays at certain steps in the purification might be modifiable, e.g., leaving out specificity controls at later stages or assaying alternate chromatography fractions. There is a recent trend not to use assays for protein activity, but to purify a gel band or an antigen instead. Although this tactic might be appropriate in instances where activity is not being sought, it is to be strongly discouraged when activity is in fact what is desired. It cannot be emphasized strongly enough that an activity assay is necessary to obtain optimal yields of activity, be it one associated with an enzyme, a DNAbinding protein, an antibody, or a hormone. A final comment pertains to the protein assay. Again, the goals are simplicity, reproducibility, specificity, and reliability. Accuracy is generally compromised, as no commonly used assay is absolute with regard to all proteins. With crude fractions, color reactions are probably best.

rrCl

[21

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11

GENERAL STRATEGIES AND CONSIDERATIONS

TABLE I ADDITIONS TO PROTEIN SOLVENTS Class Buffer Salt Detergents Surfactants Glycerol, sucrose

Sodium azide Metal chelators

Sulfhydryl agents Ligands

Protease inhibitors

Examples

KCI, NaCI, (NH4)2S04 Deoxycholate Triton X-100

EDTA (ethylenediaminetetraacetic acid), EGTA [ethylene glycol bis(&aminoethylether) N,N1-tetraacetic acid] 2-Mercaptoethanol, dithiothreitol Mg2+,ATP, phosphate PMSF (phenylmethylsulfonyl fluoride), TPCK (N-tosyl-L-phenylalanine chloromethyl ketone), TLCK (Na-p-tosyl-Llysine chloromethyl ketone)

Purpose Stability Stability Stability, solubility Stability Stability; allows storage below 0" in liquid state Bacteriostatic Stability

Stability Stability Stability

While the Bradford method3 is by far the simplest of these, in our laboratory we find it to be unreliable with crude fractions from animal cells or when detergents are present. For column effluents, ultraviolet absorption is optimal: it is simple, sensitive, and does not consume the material. For extremes of sensitivity, wavelengths between 210 and 230 nm can be ~ t i l i z e d . ~Again, ,~ protein assay procedures can and often must be changed as the purification progresses. What Should Be Added to the Buffers Once a purification scheme is developed, there is great resistance to modifying it, as modification requires laborious trial runs. The usual response to "why is the protein suspended in x?" is "if 1 leave it out, I don't know what will happen." The obvious lesson is to add something only with good reason in the first place. Solutes are added usually to improve stability, prevent the growth of microorganisms, reduce the freezing point, or keep the protein in solution. Table I lists several classes and examples of such additions. It is well M. M. Bradford, Anal. Biochem. 72, 248 (1976). W. J . Waddell, J . Lab. Clin. Med. 48, 311 (1956). M. P. Tombs, F. Souter, and N. F. MacLagan, Biochem. J. 73, 167 (1959).

12

DEVELOPING PURIFICATION PROCEDURES

[21

worth the effort to carry out stability studies (e.g., heat inactivation or storage trials) in order to learn how to maintain a stable protein. Two notes of caution: (1) optimal storage conditions'change with purification; (2) optimal storage conditions need not relate to optimal conditions for activity. Indeed, additions which stabilize a protein often inhibit it when added to activity mixes. Of course, the latter situation must be considered when utilizing the protein-interfering substances will have to be removed or "diluted out" during utilization of the protein. In our experience, reducing agents are particularly effective with bacterial enzymes which derive from a reducing environment, whereas mammalian cell enzymes take kindly to surfactants and protease inhibitors. Fungal proteins also respond to protease inhibitors. Optimal pH and salt concentrations vary. Most enzymes prefer the lowest temperature allowable: 0" (on ice, not in a refrigerator) or -20" with glycerol or sucrose present. If frozen, storage above liquid nitrogen or at -70" is often best. Special precautions which must be taken for purification and stabilization of large protein complexes are noted in Section IX of this volume. A final note concerns the containers used for purified proteins or purification fractions. Glass should not be used with very dilute solutions, plastic tubes being better. In our experience, polypropylene-based plastics are superior to polyethylene ones, and polystyrene or other clear plastics are less satisfactory. Be sure to have tight-fitting caps if storage is in "frost-free'' freezers. Contaminating Activities

Often proteins need not or cannot be obtained in a pure state, but particular interfering activities (e.g., nucleases, protease, phosphatases) must not be present. In our experience, attempting to purify one activity against one or more others by doing multiple types of activity assays as a criterion of purity is an extremely frustrating endeavor. Instead, purifying so as to optimize yield and specific activity (unitslmg protein) with selective choice of fractions only at the last or at most penultimate step is more likely to be satisfactory. Source of Protein

When the source of a protein is not absolutely dictated, careful consideration of the source is worth the time and effort, and trial extracts from a number of sources should be done. The cost and availability of the source, particularly if a largely scaled up preparation might be desirable in the future, should also be considered

121

GENERAL STRATEGIES AND CONSIDERATIONS

13

as well as the genetic knowledge and technology available for the organism should regulatory andlor gene sequence manipulations be envisioned. If the protein is to be overexpressed, is a bacterial or fungal cell better? Which one? What special precautions are necessary for each organism? Will the protein be appropriately processed? Once at the bench, several sources should be tested for total yield of activity (per gram of starting material or per unit cost), the starting specific activity (unitslmg protein), and the stability of the protein. In the extreme case, the classical microbiological approach of isolating microorganisms with unique growth requirements might lead to unexpected success. Preparing Extracts

Preparing extracts is discussed in Section IV of this volume so only general considerations will be noted here. In our experience, the manner in which cells are disrupted has a profound and unpredictable effect on the yield and quality of the protein preparation. Trials are clearly necessary. Thought should always be given toward scaling up the preparation, and how the disruption procedure will or will not adapt to being scaled up. Will the volumes or time required become unreasonable? Can a subsequent clarification step also be conveniently scaled up? In general, volumes should be kept as small as possible, i.e., extracts as concentrated as possible. Tissue, cell type, or organelle fractionation is almost always worthwhile prior to disruption. Finally, consideration should also be given to the substance in which the starting material is suspended so that the protein desired is soluble and/or stable. Of course, the contents of the suspension buffer should not interfere with the subsequent step(s) in the purification procedure. Bulk or Batch Procedures

These procedures are almost always utilized early in the purification as they are often most effective in removing nonprotein material and are most amenable to the large volume and amounts of material that exist in earlier stages of the preparation. A great deal of effort went into designing these steps in the early days of protein chemistry, and much frustration can probably be avoided by reinstituting some of these old-fashioned procedures. Section VI of this volume outlines some of these approaches. Drastic methods such as heat, extremes of pH, or phase partition with organic

14

DEVELOPING PURIFICATION PROCEDURES

121

solvents might be particularly effective with stable proteins, though subtle forms of damage may be difficult to foresee or to detect. Gentler procedures include phase partition with organic polymers, "salting out," or addition of ion-exchange resin as a slurry. Batch elution from large, highcapacity ion-exchange columns might also be effective. The time expended in developing and optimizing these early steps is always worthwhile-even a factor of two increase in specific activity may decide the feasibility of a subsequent step from both cost and technical considerations. Refined Procedures Once the bulk methods have yielded a protein preparation which is reasonably free of nucleic acids, polysaccharides, and lipids, the preparation becomes amenable to the more interesting and spectacular procedures which have been developed in recent years. The general strategy is to proceed from high- to low-capacity procedures and to attempt to exploit specific affinity materials whenever possible. Applications and technical details for these procedures are noted in Sections VII, VIII, IX, and XI of this volume, and will not be described here beyond citing examples. As a general consideration, in proceeding from one procedure to the next, one ought to reduce as much as possible the necessity for dialysis and concentration. Hence, procedures that separate by size can also be exploited to remove salt. Procedures utilizing high-capacity resins can concentrate proteins as well as purify them, or resins from which proteins elute at low-salt concentrations can be directly followed with resins to which the protein binds at higher salt concentrations. Also, some steps (e.g., sedimentation through gradients of sucrose or glycerol) may leave the protein in a medium which might be ideal for long-term storage, but difficult (or appropriate) for utilization in a subsequent step. Finally, interchanging the order of the steps of a procedure can, and often does, have a profound effect on the success of a purification scheme. Some procedures which cannot be effectively scaled up [e.g., sedimentation, or high-performance liquid chromatography (HPLC)] can be carried out with small aliquots of the preparation, but only if left to the final stages. (In some instances the utilization of aliquots is desirable, the less purified fractions may be more stable to long-term storage.) High-Capacity Steps

Generally, these include ion-exchange resins or very general affinity agents such as dyes or glass. When used for large amounts of material,

[21 ~~~~~

GENERAL STRATEGIES AND CONSIDERATIONS -

15

~

ion-exchange resins can often be successfully reutilized at a later stage for additional purification (especially if the pH is changed) or for concentration. Intermediate-Capacity Steps

These might include the hydrophobic resins for which long chromatographic times reduce activity yields. Many affinity agents (bulk DNA or simple DNA sequences, immunoaffinity, or ligands of a protein) fall into this class. In these instances, thought and effort must be given to finding materials that can successfully elute the protein without destabilizing or inactivating it. Gel filtration should also be considered as a step with intermediate capacity. Low-Capacity Steps

Affinity steps utilizing valuable ligands such as substrate analogs, complex DNA sequences, and lectins might be included here. Also included are isoelectric focusing (precipitation is often a problem with moderate amounts of protein), electrophoresis, HPLC (which in our hands is difficult to scale up without loss of resolution), and ultracentrifugation. Very small hydrophobic columns might also be successful where larger ones have failed. Conclusions

Though protein purification is often a difficult and frustrating process, its rewards are great. Moreover, with the continual development of new technology, the commercial availability of materials utilized for purification procedures, and the availability of genetically altered sources of material, the future bodes well for simpler procedures accompanied by greater rewards and indeed for protein chemistry as well.

Section I1 General Methods for Handling Proteins and Enzymes

TIC-

CCC

.r"

F

[31

SETTING UP A LABORATORY

19

[31 Setting Up a Laboratory

The aim of this chapter is to provide some general information on the basic equipment, chemicals, and supplies that should be present in any laboratory undertaking protein purification. Details relevant to individual pieces of equipment, information on apparatus and chemicals for specialized applications, useful vendors, etc., can be found in chapters throughout this volume. Although any laboratory engaged in protein purification may have many types of equipment, chemicals, and supplies, all these materials basically fall into three categories, those used for fractionation, those needed for detection and assay, and those that I call supporting materials. The supporting materials (e.g., tubes, pipets, baths, stirrers, timers, salts, buffers, and much more) are common to every biochemical laboratory. They are generally the least costly, used most frequently, required in largest numbers, and are the most essential. It is natural in setting up a laboratory to focus on the large, expensive apparatus, but in practice, available funds should first go to ensuring an adequate supply of supporting materials. (It obviously makes no sense to buy a sophisticated fraction collector, and not to have enough tubes.) Obtain the necessary amount of glassware, chemicals, disposables, etc., for the number of people who will be working in the laboratory. A representative (but not complete) list follows.

Supporting Materials

Glassn~areand Plasticware Tubes, beakers, flasks, bottles, cylinders, funnels, and pipets, in a wide range of sizes (disposable materials are often useful) Transfer (Pasteur) pipets Micropipets (especially for small volumes where automatic pipets are inaccurate) Baking dishes Plastic containers Large carboys and jars Ice buckets METHODS IN ENZYMOLOGY. VOL. 182

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

20

GENERAL METHODS FOR HANDLING PROTEINS AND ENZYMES

Chemicals

High-grade distilled H 2 0 Salts (generally chlorides) Sodium and potassium phosphates Enzyme-grade ammonium sulfate Tris and other organic buffers EDTA Acids and bases Reducing agents (2-mercaptoethanol, dithiothreitol, glutathione) Protease inhibitors Detergents Glycerol Disposables

Dialysis tubing Plastic gloves Weighing paper and boats Filter paper pH Paper Aluminum foil Glass wool Syringes and needles Marking tape and pens Small Equipment and Accessories Burners and flints Timers (including a stopwatch) Vortex mixers Magnetic stirrers and stirring bars of various sizes Forceps and scissors Ice buckets Equipment and Apparatus

Refrigerator Freezer (-20") and -70", if funds available Water baths (shaking and standing) Balances (top loading and analytical) Trip balance pH meter and electrode Accessibility to a cold room, autoclave, ice machine, lyophilizer

[31

[31

SETTING UP A LABORATORY

21

If funds still remain after filling the above list, obtain other items directly relevant to protein purification (although some of these could also be considered supporting materials), i.e., those necessary for detection and assay and for fractionation. In these areas, some sf the equipment could be quite costly and sophisticated. A great deal of thought should be given to the planned usage of such equipment to determine your actual needs. In some cases it might be essential to have the item in your immediate laboratory. However, in others, if only occasional use is contemplated, you might get by with nearby access to the piece of equipment. With limited funds, and the current cost of some equipment, a priority list is very helpful. In some instances, duplicating a frequently used item may be more advantageous than purchasing a new piece of equipment that will only be used infrequently. Thus, in my experience, a lab actively engaged in protein purification never has enough fraction collectors, columns, and gel electrophoresis apparatus.

Detection and Assay Requirements

Probably the most important detection device in the laboratory is the spectrophotometer. It can be used for determining protein concentrations, measuring the growth of bacterial cultures, as well as for a variety of enzymatic and colorimetric assays. The spectrophotometer should be equipped with both UV and visible optics and cover the range from about 200 to 800 nm to be of most use. Both glass and quartz cuvettes are necessary to cover the visible and UV range, respectively. It is often useful to have one set of microcuvettes for analysis of small volumes (- 0.2 ml). Disposable cuvettes are available, and are best for measuring cell growth. Most enzymatic assays rely on either spectrophotometry or the use of radioisotopes. In the latter case, a scintillation counter is a necessity. The use of a scintillation counter means that the supplies, chemicals, and other accessories needed for preparing radioactive samples will also be required. Scintillation counters are quite costly, and often are shared among several laboratories. If the use of radioactive material is contemplated, radiation monitors, shielding, and other precautions will be needed as well. Two other detection devices that often come in handy are a conductivity meter and a refractometer. These are used to measure salt gradients on chromatographic columns, and sucrose, glycerol, or CsCl centrifugal gradients.

22

GENERAL METHODS FOR HANDLING PROTEINS AND ENZYMES

[31

Fractionation Requirements

Protein purification means protein fractionation. What distinguishes a protein purification laboratory from the usual biochemistry or molecular biology laboratory is largely the number and types of fractionation apparatus and materials available. Subsequent chapters will discuss these items in detail; they will be mentioned here only briefly. Probably the most frequently used piece of equipment in the laboratory is the centrifuge. The workhorse of the protein purification laboratory is the refrigerated high-speed centrifuge which attains speeds up to 20,000 rpm. The usefulness of such a centrifuge is directly related to the presence in the laboratory of a wide variety of rotors and centrifuge tubes and bottles. Rotors are available that hold as small a volume as a few milliliters per tube to ones that hold six 500-ml bottles. The large rotors are invaluable for handling the large volumes of extracts often encountered in early steps of a protein purification. In instances in which one wants to remove or prepare subcellular organelles, access to an ultracentrifuge is desirable. Instruments are now available that can process reasonably large volumes at speeds as high as 80,000 rpm, and smaller machines on the market can go even faster. The availability of this instrumentation has greatly reduced the time required to prepare microsomal or high-speed supernatant fractions. In view of the cost of these machines, and their relatively infrequent use in most cases, they are often shared among laboratories. In recent years, the advent of many microanalytical techniques has made the minifuge or microcentrifuge a popular item. Though not really a fractionation apparatus in a protein purification laboratory, it is often a useful addition. In this regard, the larger centrifuges are also frequently used for assays of various types, rather than only for fractionation purposes. In order to isolate proteins, a means of rupturing cells is required. Various apparatuses are available for this purpose, including hand-held and motor-driven homogenizers, blenders, sonicators, pressure cells, etc. These will be discussed in detail in [12]-[17]. In general, it is desirable to have a variety of the less costly items in individual laboratories, with the remainder available as shared equipment. Column chromatography is the primary protein purification method in use in most laboratories. Every laboratory involved in protein fractionation should have available a large supply of columns of various lengths and diameters in anticipation of every conceivable need, since they will arise during the course of developing purification schemes. Columns are available in various degrees of sophistication (and cost). In our labora-

[31

SETTING UP A LABORATORY

23

tory, we have found that simple, open-top columns fitted with stoppers and syringe needles, or tubing, for fluid inlet and control, are satisfactory for most chromatographic procedures. A dependable fraction collector is one of the most important pieces of equipment in the laboratory. Failure of a fraction collector may result in the loss of several month's work. In this instance, extra money spent on a good, versatile machine is a wise investment. Instruments able to handle a large number of tubes, of various sizes, in different collection modes, are the most useful. Many different types of fraction collectors are available. Careful analysis of the various models, and matching to anticipated requirements, is good practice prior to purchase. A suitable strategy for many laboratories would be the purchase of one of the more sophisticated instruments for special needs, and one or more of the less costly, simple machines for routine use. A number of other accessories to column chromatography are useful, if not essential. These include aperistaltic pump, various sizes of gradient makers, and a UV monitor. Gradient makers can be homemade from flasks or bottles, if necessary. Following the protein elution profile during a chromatographic run provides important information. This can be done by determining the absorbance of individual fractions with a spectrophotometer, or automatically with an in-stream UV monitor. Dual-wavelength models with different size flow cells are the most versatile (and also most costly). Finally, every laboratory should also have on hand a basic supply of chromatographic gels and resins. These should include an anion and cation exchanger (e.g., DEAE and CM), various porosity gel filtration media, hydroxyapatite, a hydrophobic gel, and probably an immobilized dye resin. Nowadays, no protein purification laboratory is complete without the presence of gel electrophoresis equipment. These items are used to monitor a purification procedure or for fractionation itself. Generally, a vertical slab-gel apparatus with various-sized spacers and combs is satisfactory for most applications. An electrophoresis power supply unit is also required. If only one is to be purchased, a regulated constant-current (050 mA) constant-voltage (0-200 V) model is useful. In addition to the chemicals necessary for preparing gels, several protein standards should also be obtained. The instrumentation used for gel electrophoresis can also be used for isoelectric focusing. The equipment, chemicals, and supplies mentioned in this chapter should allow you to enter the field of protein purification. As you read through this volume, and actually begin to purify proteins, many other useful items will become apparent.

24

GENERAL METHODS FOR HANDLING PROTEINS AND ENZYMES

[41

[41 Buffers: Principles and Practice By VINCENTS. STOLLand JOHNS. BLANCHARD

The necessity for maintaining a stable pH when studying enzymes is well established.' Biochemical processes can be severely affected by minute changes, in hydrogen ion concentrations. At the same time many protons may be consumed or released during an enzymatic reaction. It has become increasingly important to find buffers to stabilize hydrogen ion concentrations while not interfering with the function of the enzyme being studied. The development of a series of N-substituted taurine and glycine buffers by Good et al. has provided buffers in the physiologically relevant range (6.1-10.4) of most enzymes, which have limited side effects with most enzyme^.^ It has been found that these buffers are nontoxic to cells at 50 mM concentrations and in some cases much higher.3 Theory

The observation that partially neutralized solutions of weak acids or weak bases are resistant to pH changes on the addition of small amounts of strong acid or strong base leads to the concept of "b~ffering".~ Buffers consist of an acid and its conjugate base, such as carbonate and bicarbonate, or acetate and acetic acid. The quality of a buffer is dependent on its buffering capacity (resistance to change in pH by addition of strong acid or base), and its ability to maintain a stable pH upon dilution or addition of neutral salts. Because of the following equilibria, additions of small amounts of strong acid or strong base result in the removal of only small amounts of the weakly acidic or basic species; therefore, there is little change in the pH: HA (acid) B (base) + H+

H+ + A- (conjugate base)

* BH+ (conjugate acid)

(1) (2)

The pH of a solution of a weak acid or base may be calculated from the Henderson-Hasselbalch equation:

' R . J . Johnson and D. E. Metzler. this series, Vol. 22, p. 3; N . E. Good and S. Izawa, Vol. 24, p. 53. N. E. Good. G. D. Winget, W. Winter, T. N. Connolly, S. Izawa, and R. M. M. Singh, Biochernisrry 5, 467 (1966). W. J. Ferguson er a / . , Anal. Biochem. 104, 300 (1980). D. D. Penin and B. Dempsey, "Buffers for pH and Metal Ion Control." Chapman & Hall, London, 1974.

METHODS IN ENZYMOLOGY. VOL. 182

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form resewed.

[41

BUFFERS: PRINCIPLES A N D PRACTICE

pH = pKA

+ logibasic species]/[acidic species]

25 (3)

The pK, of a buffer is that pH where the concentrations of basic and acidic species are equal, and in this basic form the equation is accurate between the pH range of 3 to 11. Below pH 3 and above pH 11 the concentrations of the ionic species of water must be included in the e q ~ a t i o nSince . ~ the pH range of interest here is generally in the pH 3-1 1 range, this will be ignored. From the Henderson-Hasselbalch equation an expression for buffer capacity may be deduced. If at some concentration of buffer, c, the sum [A-1 + [HA] is constant, then the amount of strong acid or base needed to cause a small change in pH is given by the relationship

In this equation K, refers to the ionic product of water, and the second and third terms are only significant below pH 3 or above pH 11. In the pH range of interest (pH 3-1 1) this equation yields the following expression:

which represents a maximum value for d [Blld pH when pH = pKa. The buffer capacity of any buffer is dependent on the concentration, c, and may be calculated over a buffer range of 1 pH unit around the pK to determine the buffer capacity, as shown in Fig. 1 for one of the Good buffers, HEPES. It can be seen that the buffer capacity is greatest at its

*

FIG.1. Buffer capacity ( P ) versus ApH over the range t 1 pH unit of the pK, for HEPES (0.05 M). Points calculated using Eq. (5), and data from D. D. Perrin and Dempsey, "Buffers for pH and Metal Ion Control" (Chapman and Hall, London, 1974).

26 -

GENERAL METHODS FOR HANDLING PROTEINS AND ENZYMES -

[41

-

pK, and drops off quickly 1 pH unit on either side of the pK. In practice, buffers should not be used beyond these values. Buffer Selection

1

There are many factors that must be considered when choosing a buffer. When studying an enzyme one must consider the pH optimum of the enzyme, nonspecific buffer effects on the enzyme, and interactions with substrates or metals. When purifying a protein, cost becomes an important consideration, as does the compatibility of the buffer with different purification techniques. Table I lists a wide variety of buffers covering a broad pH range. Determining the pH optimum of a protein is a first step in determining the best buffer to emp10y.~Since the buffering capacity is maximal at the pK, buffers should be used close to this value. When determining the pH optimum for an enzyme, it is useful to use a series of related buffers that span a wide pH range. Once an optimal pH has been approximated, different buffers within this pH range can be examined for specific buffer effects. The Good buffers have been shown to be relatively free of side effects. However, inorganic buffers do have a high potential for specific buffer effects. Many enzymes are inhibited by phosphate buffer, including carboxypeptidase, urease, as well as many kinases and dehydrogena~es.~ Borate buffers can form covalent complexes with mono- and oligosaccharides, the ribose moieties of nucleic acids, pyridine nucleotides, and other gem-diols. Tris and other primary amine buffers may form Schiff base adducts with aldehydes and ketones. Buffer complexation with metals may present additional problems. In this respect inorganic buffers can prove problematic in that they may remove, by chelation, metals essential to enzymatic activity (e.g., Mg2+ for kinases, Cu2+ or Fe2+ for hydroxylases). Release of protons upon chelation or precipitation of metal-buffer complexes may also be a potential problem. Where metal chelation presents a problem, the Good buffers are useful since they have been shown to have low metal-binding capabilitie~.~ Once a suitable buffer has been found (noninteracting, with an appropriate pK), a concentration should be chosen. Since high ionic strength may decrease enzyme activity, the buffer concentration should be as low as p ~ s s i b l eA. ~reasonable way to determine how low a concentration may be used is to examine the properties (reaction rate, or protein stability) at J. S. Blanchard, this series, Vol. 104, p. 404.

(41

27

BUFFERS: PRINCIPLES AND PRACTICE

TABLE I SELECTEDBUFFERSAND THEIRpK VALUESAT 25' -

-

Trivial name Phosphate (pK,) Malate (pKI) Formate Succinate (pK,) Citrate (pK2) Acetate Malate Pyridine Succinate (pKJ MES Cacodylate Dimethylglutarate Carbonate (pKI) Citrate (pK,) Bis-Tris ADA Pyrophosphate EDPs (PKI) Bis-Tris propane PIPES ACES

Imidazole BES MOPS Phosphate (pK2) EMTA TES HEPES

TEA POPS0 EPPS, HEPPS

Buffer name

pKa

d pKaldt

2-(N-Morpholino)ethanesulfonic acid Dimethylarsinic acid 3,3-Dimethylglutarate (pKZ) -

[Bis(2-hydroxyethyl)imino]tris(hydroxymethy1)methane N-2-Acetamidoiminodiacetic acid

propane Piperazine-N, N'-bis(2-ethanesulfonic acid) N-2-Acetamido-2-hydroxyethanesulfonic acid 3-(N-Morpho1ino)-2-hydroxypropanesulfonic acid -

N,N-Bis-(2-hydroxyethy1)-2-aminoethanesulfonic acid 3-(N-Morpholino)propanesulfonic acid

3,6-Endomethylene-l,2,3,6-tetrahydrophthalic acid 2-[Tris(hydroxymethyl)methylamino]ethanesulfonic acid N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid 3-[N-Bis(hydroxyethyI)amino]-2-hydroxypropanesulfonic acid Triethanolamine Piperazine-N, N1-bis(2-hydroxypropanesulfonic acid) N-2-Hydroxyethylpiperazine-N'-3-propanesulfonic acid --

(continued)

28

GENERAL METHODS FOR HANDLING PROTEINS AND ENZYMES

[41

TABLE I (continued) Trivial name Tris Tricine Glycinamide PIPPS Glycylglycine Bicine TAPS Morpholine PIBS AES Borate Ammonia Ethanolamine CHES Glycine (pKz) EDPS APS Carbonate (pK2) CAPS Pipendine Phosphate ( p 4 )

Buffer name

Tris(hydroxymethyl)aminomethane N-[Tris(hydroxymethyI)methyl]glycine

-

1,4-Bis(3-sulfopropy1)piperazine N,N-Bis(2-hydroxyethy1)glycine 3-{[Tris(hydroxymethyl)methyl]amino)propanesulfonic acid 1,4-Bis(4-sulfobutyl)piperazine 2-Aminoethylsulfonic acid, taurine Cyclohexylaminoethanesulfonic acid

-

N,N1-Bis(3-su1fopropyl)ethylenediamine 3-Aminopropanesulfonic acid

-

3-(Cyclohexylarnino)propanesulfonic acid

-

pK,

d pK,/dr

8.06 8.05 8.06 8.10 8.25 8.26 8.40

-0.028 -0.021 -0.029 -0.025 -0.018 0.018

8.49 8.60 9.06 9.23 9.25 9.50 9.55 9.78 9.80 9.89 10.33 10.40 11.12 12.33

-0.022 -0.008 -0.031 -0.029 0.029 -0.025 -0.009 0.032 -0.026

a low (10-20 mM) concentration of buffer. The pH prior to, and an adequate time after, addition of protein should not vary more than -t 0.05 pH. If the pH changes too drastically (greater than t 0.1 pH unit), then the buffer concentration should be raised to 50 mM. In cases where protons are consumed or released stoichiometrically with substrate utilization, pH stability becomes increasingly important. Buffers may be made up in stock solutions, then diluted for use. When stock solutions are made, it should be done close to the working temperature, and in glass bottles (plastic bottles can leach UV-absorbing material).4 Buffers have temperature-sensitive pK values, particularly amine buffers. The carboxylic acid buffers are generally the least sensitive to temperature, and the Good buffers have only a small inverse temperature dependence on pK. The effects of dilution of stock solutions, or addition of salts, on pH should be checked by measurement of the pH after addition of all components. Choosing a buffer for protein purification requires some special considerations. Large amounts of buffer will be needed for centrifugation,

BUFFERS: PRINCIPLES AND PRACTICE chromatographic separations, and dialysis, which makes cost a concern. Tris and many inorganic buffers are widely used since they are relatively inexpensive. Although buffers like Tris are inexpensive, and have been widely used in protein purification, they do have disadvantages. Tris is a poor buffer below pH 7.5 and its pK is temperature dependent (a solution made up to pH 8.06 at 2.5" will have a pH of 8.85 at 0"). Many primary amine buffers such as Tris and glycine6 will interfere with the Bradford dye-binding protein assay. Some of the Good buffers, HEPES, EPPS, and Bicine, give false-positive colors with Lowry assay. Spectroscopic measurement of enzyme rates is a commonly applied method. It may be important to use a buffer that does not absorb appreciably in the spectral region of interest. The Good buffers, and most buffers listed in Table I, can be used above 240 nm. Buffer Preparation

Once a suitable buffer has been chosen it must be dissolved and titrated to the desired pH. Before titrating a buffer solution the pH meter must be calibrated. Calibration should be done using commercially available pH standards, bracketing the desired pH. If monovalent cations interfere, or are being investigated, then titration with tetramethylammonium hydroxide can be done to avoid mineral cations. Similarly, the substitution of the most commonly used counteranion, chloride, with other anions such as acetate, sulfate, or glutamate, may have significant effects on enzyme activity or protein-DNA interactions.' Stock solutions should be made with quality water (deionized and double-distilled, preferably) and filtered through a sterile ultrafiltration system (0.22 pm) to prevent bacterial or fungal growth, especially with solutions in the pH 6-8 range. To prevent heavy metals from interfering, EDTA (10-100 pM) may be added to chelate any contaminating metals. Volatile Buffers

In certain cases it is necessary to remove a buffer quickly and completely. Volatile buffers make it possible to remove components that may interfere in subsequent procedures. Volatile buffers are useful in electrophoresis, ion-exchange chromatography, and digestion of proteins followed by separation of peptides or amino acids. Most of the volatile M. M. Bradford, Anal. Biochem. 22, 248 (1976).

' S. Leirmo, C. Hamson, D. S. Cayley, R. R. Burgess, and M. T. Record, Biochemistry 26, 2095 (1987).

30

GENERAL METHODS FOR HANDLING PROTEINS A N D ENZYMES

[41

TABLE I1 TYPESOF SYSTEMS FOR USE AS VOLATILE BUFFERS~ System 87 ml Glacial acetic acid + 25 ml 88% HCOOH in 11 liters 25 ml 88% HCOOH in I liter Pyridine-formic acid Trimethylamine-formic acid Triethylamine-formic (or acetic) acid 5 ml Pyridine + 100 ml glacial acetic acid in 1 liter 5 ml Pyridine + 50 ml glacial acetic acid in 1 liter Trimethylamine-acetic acid 25 ml Pyridine + 25 ml glacial acetic acid in 1 liter Collidine-acetic acid 100 ml Pyridine + 4 ml glacial acetic acid in 1 liter Triethanolamine-HC1 Ammonia-formic (or acetic) acid Trimethylamine-C02 Triethylamine-C02 24 g NH4HC03in 1 liter Ammonium carbonate-ammonia Ethanolamine-HC1 20 g (NHJ2C0, in 1 liter

pH range 1.9 2.1 2.3-3.5 3.0-5.0 3-6 3.1 3.5 4.0-6.0 4.7 5.5-7.0 6.5 6.8-8.8 7.0-10.0 7-12 7-12 7.9 8.0-10.5 8.5-10.5 8.9

" From D. D. Perrin and Boyd Dempsey, "Buffers for pH and Metal Ion Control." Chapman and Hall, London, 1974.

buffers (Table 11) are transparent in the lower UV range except for the buffers containing ~ y r i d i n eAn . ~ important consideration is interference in amino acid analysis (i.e., reactions with ninhydrin). Most volatile buffers will not interfere with ninhydrin if the concentrations are not too high (e.g., triethanolamine less than 0.1 M does not interfere). Broad-Range Buffers

There may be occasions where a single buffer system is desired that can span a wide pH range of perhaps 5 or more pH units. One method would be a mixture of buffers that sufficiently covers the pH range of interest. This may lead to nonspecific buffer interactions for which corrections must be made. Another common approach is to use a series of structurally related buffers that have evenly spaced pK values such that each pK is separated by approximately + 1 pH unit (the limit of buffering capacity). The Good buffers are ideal for this approach since they are structurally related and have relatively evenly spaced pK values. As the

BUFFERS: PRINCIPLES

[41

A N D PRACTICE

31

P

.

~

r"

pH passes the pK of one buffer it becomes nonparticipatory and therefore has no further function. These nonparticipating buffer components may show nonspecific buffer effects as well as raising the ionic strength with potential deleterious effects. A detailed description of buffer mixtures which provide a wide range of buffering capacity with constant ionic strength is a ~ a i l a b l e . ~

F

Recipes for Buffer Stock Solutions

Stock Solutions A: 6.2 M solution of glycine (15.01 g in 1000 ml) B: 0.2 M HCl 50 ml of A

+ x ml of B, diluted to a total of 200 ml:

2 . Citrate Bufferlo Stock Solutions A: 0.1 M solution of citric acid (21.O1 g in 1000 ml) B: 0.1 M solution of sodium citrate (29.41 g C6H507Na3-2H20 in 1000 ml) x ml of A

lo

+ y ml of B, diluted to a total of 100 ml:

K. J. Ellis and J. F. Morrison, this series, Vol. 87, p. 405. S. P. L. Sorensen, Biochem. Z. 21, 131 (1909); 22, 352 (1909). R. D. Lillie, "Histopathologic Technique." Blakiston, Philadelphia, Pennsylvania, 1948.

32

GENERAL METHODS FOR HANDLING PROTEINS AND ENZYMES

[41

3. Acetate Buffer" Stock Solutions A: 0.2 M solution of acetic acid (11.55 ml in 1000 ml) B: 0.2 M solution of sodium acetate (1 6.4 g of C2H302Naor 27.2 g of C2H302Na.3H20 in 1000 ml) x ml of A

+ y ml of B, diluted to a total of 100 ml:

4 . Citrate-Phosphate Buffer12

Stock Solutions A: 0.1 M solution of citric acid (19.21 g in 1000 ml) B: 0.2 M solution of dibasic sodium phosphate (53.65 g of Na2HP04.7H20 or 7 1.7 g of Na2HP04 12H20 in 1000 ml) Ii I2

G. S. Walpole, J. Chem. Soc. 105, 2501 (1914). T. C. McIlvaine, J. Biol. Chem. 49, 183 (1921).

[41

BUFFERS: PRINCIPLES AND PRACTICE

x ml of A

+ y ml of B, diluted to a total of 100 ml: X

Y

pH

5 . Succinate Buffer13 Stock Solutions

A: 0.2 M solution of succinic acid (23.6 g in 1000 ml) B: 0.2 M NaOH 25 ml of A

"

+ x ml of B, diluted to a total of 100 ml:

G . Gornori, unpublished observations.

33

34

GENERAL METHODS FOR HANDLING PROTEINS AND ENZYMES

141

6 . Cacodylate Buffer14

Stock Solutions

A: 0.2 M solution of sodium cacodylate (42.8 g of Na(CH3)2As02 3H20 in 1000 ml) B: 0.2 M NaOH

50 ml of A

+ x ml of B. diluted to a total of 200 ml:

7. Phosphate Buffer9 Stock Solutions

A: 0.2 M solution of monobasic sodium phosphate (27.8 g in 1000 ml) B: 0.2 M solution of dibasic sodium phosphate (53.65 g of Na2HP04.7H20 or 71.7 g of Na2HP04 12H20 in 1000 ml) x ml of A

l4

+ y ml of B, diluted to a total of 200 ml:

M. Plumel, Bull. Soc. Chim. Biol. 30, 129 (1949).

[41

BUFFERS: PRINCIPLES AND PRACTICE

35

8. Barbital Buffer" Stock Solutions A: 0.2 M solution of sodium barbital (veronal) (41.2 g in 1000 ml) B: 0.2 M HC1

50 ml of A

+ x ml of B. diluted to a total of 200 ml:

Solutions more concentrated than 0.05 M may crystallize on standing, especially in the cold. 9. Tris(hydroxymethy1)aminomethane (Tris) BufferI6

Stock Solutions A: 0.2 M solution of tris(hydroxymethyl)aminomethane (24.2 g in 1000 ml) B: 0.2 M HCl

50 ml of A

l5 Ih

+ x ml of B , diluted to a total of 200 ml:

L. Michaelis, J . Biol. Chem. 87, 33 (1930) 0.Hayaishi. this series, Vol. 1, p. 144.

36

GENERAL METHODS FOR HANDLING PROTEINS AND ENZYMES

[41

10. Boric Acid-Borax Buffer1' Stock Solutions

A: 0.2 M solution of boric acid (12.4g in 1000 ml) B: 0.05 M solution of borax (19.05 g in 1000 ml; 0.2 M in terms of sodium borate)

50 ml of A

+ x ml of B, diluted to a total of 200 ml:

1 1 . 2-Amino-2-methyl-1,3-propanediol (Ammediol) Buffer18 Stock Solutions

A: 0.2 M solution of 2-amino-2-methyl-l,3-propanediol(21.03 g in 1000 ml)

B: 0.2 M HCI 50 ml of A

"

+ x ml of B, diluted to a total of 200 ml:

W. Holmes, Anat. Rec. 86, 163 (1943). Gomori, Proc. Soc. Exp. Biol. Med. 62, 33 (1946).

l8 G.

[41

BUFFERS: PRINCIPLES AND PRACTICE

37

Stock Solutions A: 0.2 M solution of glycine (15.01 g in 1000 ml) B: 0.2 M NaOH 50 ml of A

+ x ml of B. diluted to a total of 200 ml:

Stock Solutions A: 0.05 M solution of borax (19.05 g in 1000 ml; 0.02 M in terms of sodium borate) B: 0.2 M NaOH 50 ml of A

+ x ml of B, diluted to a total of 200 ml:

14. Carbonate-Bicarbonate BufferZo

Stock Solutions A: 0.2 M solution of anhydrous sodium carbonate (21.2 g in 1000 ml) B: 0.2 M solution of sodium bicarbonate (16.8 g in 1000 ml) l9 Z0

W. M. Clark and H. A. Lubs, J. Bacteriol. 2, 1 (1917). G . E. Delory and E. J. King, Biochem. J. 39, 245 (1945).

38

GENERAL METHODS FOR HANDLING PROTEINS AND ENZYMES

x ml of A

[51

+ y ml of B , diluted to a total of 200 ml: X

Y

pH

[SI Measurement of Enzyme Activity

This chapter deals with the development of methods for the assay of enzyme activity in a cell lysate or in a partially purified enzyme preparation. They are also applicable during purification and for purified enzymes as well. Preparations that contain more than one protein will be referred to as multizymes. Concepts in the Measurement of Enzyme Activity

Anatomy of Enzyme Assay'

Dissection of a representative assay reveals several distinct parts (Fig. 1). However, some assays may not require all the components, and the absence of one or another of these can provide the basis for a classification scheme (see below). E. F. Rossomando, "High Performance Liquid Chromatography in Enzymatic Analysis." Wiley, New York, 1987.

METHODS IN ENZYMOLOGY, VOL. 182

Copyright D 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

[51

39

MEASUREMENT OF ENZYME ACTIVITY

ENZYME

MIX

Mix

Preparation

+

Initiation Incubation

Y! Termination

Separation

Detection

1

Data Reduction FIG. 1. Schematic of a representative enzymatic assay to illustrate its component parts. The reaction mixture is prepared (Mix Preparation) and the reaction can be started (Initiation) by the addition of the enzyme. During the reaction (Incubation), samples are removed at intervals labeled t , , t 2 , and t 3 , and the reaction is stopped (Termination) by inactivating the enzyme. The incubation mixture is fractionated (in the illustration a traditional chromatographic column is being used) and the product is isolated from the substrate (Separation). In the assay illustrated, a radiolabeled substrate has been used and therefore the amount of product that formed is determined by collection of fractions, the addition of scintillation fluid to each fraction, and the measurement of radioactivity in each fraction by scintillation counting (Detection). The progress of the reaction is given by the amount of radioactive product recovered (Data Reduction). (From Rossomando.' Reprinted with permission.)

The first component of an assay is the reaction mixture. The reaction mixture usually contains the buffer used to establish the correct pH, the substrate, and any cofactors that may be required for catalysis. Preparation of reaction mixtures involves mixing these ingredients in a reaction vessel such as a test tube or, for some assay methods, a cuvette. Often,

40

GENERAL METHODS FOR HANDLING PROTEINS AND ENZYMES

[51

the reaction mixture is brought to a specified temperature prior to initiation of the reaction. The enzyme preparation must also be obtained. This topic is discussed in detail in other chapters of this volume. The second part of an assay is initiationlincubation. A reaction is often started by the addition of the enzyme preparation to the substrate already present in the reaction mixture. This combination initiates the incubation phase, and all subsequent time points are referenced to this time as zero. Many reactions require termination, a step to stop the reaction. Termination usually involves inactivation of the enzyme. Termination can be followed by separation. Most often separation involves isolating the substrate from the reaction product. The next step, detection, refers to that process by which the product is located, identified, and the amount formed during a specific incubation interval quantitated. Finally, the last step in an assay involves reduction of the data. This step includes all procedures in which the data are analyzed and graphed to determine initial rates as well as kinetic constants. These various parts of an assay are illustrated in Fig. 1.

ClassiJication of Enzymatic Assay Methods Methods of assay have traditionally been divided into three classes: continuous, coupled, and discontinuous assays. The three methods differ with regard to the matter of separation. Thus, whereas the first two do not require the product to be separated from the substrate, the last does. In addition, the first two do not require a termination step, whereas the last usually does. Continuous methods do not require a separation step prior to detection. For assays using this method, the substrate and product must differ in some property such that either one may be measured directly in the incubation solution. For example, many phosphomonoesterases catalyze the conversion of 4-nitrophenyl phosphate (4NP), a colorless compound, to Cnitrophenol, which is yellow and has an absorption maximum at 410 nm. Since the substrate does not absorb in this region of the spectrum, the reaction can be carried out directly in a cuvette, and the formation of product followed continuously by measuring the change in optical density during the course of the reaction. In the coupled assay method,* activity is measured indirectly, and two reactions are involved: The first is the reaction of interest, such as A + B. H. N. Bergmeyer, K. Gawehn, and D. W. Moss, "Methods of Enzymatic Analysis," Vol. 1. Verlag Chemie, Deerfield Beach, Florida, 1974.

[51

MEASUREMENT OF ENZYME ACTIVITY

41

The second converts B to C, and is referred to as an indicator reaction, not only because it uses the product of the first reaction as a substrate, but also because the formation of C may be monitored by a continuous assay. In this way, the two reactions are coupled. For example, pyruvate kinase may be assayed by such a method. This enzyme catalyzes the reaction Phosphoenolpyruvate (PEP)

+ ADP + pyruvate + ATP

(1)

This is the reaction of interest. When a second enzyme, such as lactate dehydrogenase, is added together with pyruvate and NADH to the reaction mixture, a second reaction occurs and NAD forms in the cuvette as follows: Pyruvate

+ NADH + lactate + NAD

(2)

The formation of NAD may be followed continuously by the decrease in absorbance at 340 nm, and the progress of the kinase reaction may be followed through this coupling of the formation of pyruvate to the formation of NAD. In the discontinuous method, the product must be separated from the substrate in order to measure activity. Since separation often does not include detection, assays in this group usually require two steps. Assays that use radiochemical substrates are included in this group as radiochemical detectors are unable to differentiate between the radiolabel of the substrate and that of the product. As an illustration, consider the assay to measure the activity of the aminoacyl-tRNA synthetases. These enzymes catalyze the covalent attachment of an amino acid, usually radioactive (as indicated by the asterisk in the reaction), to the tRNA as follows: ATP + *AA + Enz -+ Enz(AMP-*AA) + PPi Enz(AMP-*AA) + tRNA + tRNA-*AA + AMP + Enz

(3) (4)

Activity is usually followed by measuring the amount of tRNA-"AA, the product of reaction (4), formed during the incubation. Since the radiochemical detector cannot differentiate the free radioactive amino acid used as the substrate from that bound covalently to the tRNA, the free and the bound amino acids must be separated prior to the detection or quantitation step. This separation step requires the addition of an acid such as trichloroacetic acid (TCA) to the sample, which also serves to terminate the enzyme reaction. As TCA also precipitates the tRNA and any radioactive amino acid covalently linked to it, the radioactive product can be separated from the free amino acid by a simple filtration step. The reaction

42

GENERAL METHODS FOR HANDLING PROTEINS AND ENZYMES

[51

product, which is trapped on the filter as a precipitate, can be detected by transferring the filter to a scintillation counter for quantitation of the amount of product formed. It should be noted that since assays of this design usually focus on one component at a time, no information is obtained about the amount of ATP, AMP, PPi, or free amino acid during the course of the reaction. Rate of Reaction

The concept of rate of reaction is important for obtaining accurate and reproducible data. The rate of a reaction is a measure of the conversion of substrate to product and describes how fast the reaction goes in a unit of time under specified conditions. While the maximum rate of an enzymatic reaction is a property of the enzyme molecule itself, the observed rate can be influenced by the reaction conditions. For example, the substrate concentration is particularly important when working with multizyme preparations, since activities catalyzing secondary reactions could reduce substrate levels and slow the rate of the primary reaction. Definitions of Units and Specijic Activity3

Standardized units and terms have been developed for the reporting of enzymatic activity. In describing activity three items should be included: the amount of substrate converted or product formed in some standard unit, usually the micromole (pmol); the time of the reaction, usually minutes; and the amount of enzyme, in some unit such as milligrams. The values used in the unit may be the actual ones measured or they can be normalized to some standard units. Thus, enzyme activity is often quoted as pmol/min and, for convenience, this is defined as a unit. The term unit should be read as the amount of enzyme which catalyzes the transformation of 1 mass unit of substrate (usually the micromole) per minute under defined conditions of pH and temperature. The amount of activity per unit of enzyme would be given as unitslmg and is called the specijic activity. During the course of study of enzyme activity, an assay will have been developed in which some variable will provide a quantitative measure of the progress of the reaction during the incubation. Usually, the units of the variable are arbitrary, that is they are machine units or detector units of some kind. The first task is to convert these to units of mass. (If a concentration term is involved this should be eliminated by calculating the total amount of product formed during the course of the incubation.) The conversion of machine units to some standard mass unit such as the M. Dixon, E. C. Webb, K. F. Tipton, and C. J. R. Thorne, "Enzymes," 3rd Ed., Chap. 2. Academic Press, New York, 1980.

[sl

MEASUREMENT OF ENZYME ACTIVITY

43

CL

TABLE I STEPSIN DESIGN OF AN ASSAY^ L.l

CI

c

1. 2. 3. 4.

Analyze the primary reaction Analyze all secondary reactions Select the method of analysis that will allow for measurement of products If a discontinuous assay method is chosen, select the method for termination and separation 5. Select appropriate detection system. Will it be necessary to collect fractions? From Rossomando.' Reprinted with permission.

mole is usually performed by construction of a calibration curve. It is also necessary for the unit of activity to specify the time of the reaction. If the rate is constant during the time interval, it is acceptable to divide the amount of product formed by the total time to obtain the amount per unit time. However, when it is not known if the rate is constant, as would be the case with a discontinuous assay in which only one or two time points have been taken, then it is best to specify the exact time. Next it is necessary to specify the amount of enzyme in the unit. In multizyme preparations this is usually expressed as milligrams protein since the enzyme is not pure. Quantitative Analysis of the Reaction As a result of the procedures described above, a graphical representation of the rate of product formation can be obtained. Such data can be analyzed visually or be subjected to statistical analysis. Initial rate data at different substrate concentrations can be used to produce derivative plots, such as [l/(initial velocity) versus l/(substrate concentration)], which can be used to calculate the K , and V,,, of the reaction. More detailed analyses can be used to differentiate among different reaction mechanisms. See Refs. 4 and 5 for more detailed information on enzyme kinetics. Strategy for Design of an Assay

In this section, a strategy will be presented for the design of an assay system. By focusing on one enzymatic reaction, the steps leading to the development of the assay for this activity can be described (Table I). W. W. Cleland, in "The Enzymes" (P. D. Boyer, ed.), 3rd Ed., Vol. 2. Academic Press, New York, 1970. F. B. Rudolph and H. J . Fromm, this series, Vol. 63, p. 138.

44

GENERAL METHODS FOR HANDLING PROTEINS AND ENZYMES

[51

Analysis of Primary Reaction The design of an assay system begins with a complete analysis of the primary reaction; i.e., the reaction catalyzed by the enzyme under study. To begin this analysis, indicate all substrates, products, and cofactors of the reaction. If metals are required for catalysis, include them. In the case of metals, however, it is useful to note whether they are an integral part of the substrate, for example, when the complex MgATP is the substrate, or whether they are required for some other function, such as activation of the enzyme. It is also useful to indicate the pH of the reaction as well as the type and concentration of buffer to be used. The goal of this analysis is to list all the components present in the reaction mixture before the start of the reaction. To illustrate this approach, consider the assay of a pyrophosphohydrolase, an enzyme that catalyzes the reaction MgATP --,MgAMP

+ PP,

(5)

MgATP is the substrate, and MgAMP and pyrophosphate (PPi) are the products. Since this activity is usually assayed at a pH of 7.5 using a TrisHCl buffer system, the reaction tube will contain ATP, Mg, and TrisHCl, pH 7.5.

Analysis of Secondary Reactions For those dealing with multizyme preparations, a useful distinction is that between the primary and secondary reactions. The primary reaction is the reaction catalyzed by the enzyme under study, while secondary reactions are catalyzed by other activities in the multizyme preparation. Secondary reactions may use the same substrate as the primary reaction or they may use the product of the primary reaction. In either case their presence can affect the analysis of the enzyme under study. For example, AMP, the product of the primary reaction, may undergo secondary reactions to form adenosine and phosphate or IMP and ammonia. In addition, other secondary reactions could involve ATP; an example is the degradation of ATP to ADP. While these secondary reactions can be eliminated or their significance minimized, they should not be overlooked in the analysis and design of the assay system.

Selection of Method of Analysis With the list of reactants, cofactors, and reaction conditions compiled, a method for the assay can be chosen. As mentioned above this can be either continuous, coupled, or discontinuous. In the present example a

[51

MEASUREMENT OF ENZYME ACTIVITY

45

continuous method is not possible since products and substrates are too similar. A coupled assay is possible using AMP deaminase as the indicator enzyme. This activity would convert AMP to IMP, which, because of its spectral differences, could be differentiated from AMP. Alternatively, a discontinuous method could be chosen. This would require the introduction of a termination and separation step. The choice of a discontinuous method of analysis would bring with it a requirement for two other decisions, a method for sampling and a method for terminati~nof the reaction. In the discontinuous technique, obtaining kinetic data requires multiple samples, each one representing a single time point. Reactions requiring multiple sampling can be arranged in one of two ways. In one arrangement, separate reaction mixtures are set up, each one representing a single time point. In this case, the total volume required for a single reaction mixture would be the volume required for a single analysis. The number of incubation tubes would be determined by the number of time points required by the experiment. In the second arrangement, a single incubation mixture is prepared, and samples are removed from it at suitable intervals for analysis. In this arrangement, the volume required for the reaction mixture would be determined as the product of the volume needed for each analysis multiplied by the total number of analyses. Since with both arrangements the volume of a single analysis is the important variable, it would appear that once this value is determined the overall reaction volume can be established. In designing a discontinuous assay for an enzyme reaction, it is often necessary to introduce a termination step into the protocol. There are a variety of ways to accomplish this, such as the addition of acids or bases, which must be removed, or chelating agent^.^ The latter technique is suitable only for reactions in which the enzymatic activities have an absolute requirement for a metal. An alternative method we have found useful for terminating reactions is to heat the incubation mixture to a temperature that results in rapid inactivation of the enzyme, but does not affect the product of the reaction. One of the techniques often used is to immerse the reaction tube in a bath of boiling water, although it is important that incubation mixtures be brought to 100" quickly enough to effect instantaneous termination. Commercially available heating blocks may also be used. In our laboratory a simple device, a sand bath, was found effective in terminating reactions instantly. We filled a stainless steel rectangular pan (about 8 x 10 in.) with about 2 in. of sand and placed it on a hot plate. The J. X. Khym, J. W. Bynum, and E. Volkin, Anal. Biochem. 77, 446 (1977).

46

GENERAL METHODS FOR HANDLING PROTEINS AND ENZYMES

[51

temperature of the sand bath is easily brought to 155", and this temperature can be maintained throughout the working day without fear of evaporation. There is never a problem of fitting the tubes-one simply thrusts any size capped glass tube directly into the sand. The insertion of an incubation tube containing as much as 500 p1 of incubation mixture resulted in the temperature inside the solution reaching 100" "instantly," thus terminating the reaction. Termination of most enzymatic reactions with heat results in precipitation of proteins present in the reaction mixture. Because this precipitation is generally irreversible, and with crude extracts the amount of protein may be considerable, it is often necessary to remove the precipitate prior to sampling. The precipitate can be removed either by filtration or by centrifugation. The small volumes usually present in reaction mixtures necessitate the use of microcentrifuges or filters with small "hold-up" volumes. Following the removal of the precipitate, a sample may be removed from the filtrate and analyzed. Assay Conditions

To obtain initial rate data, optimal assay conditions must be established. Optimization involves the determination of several variables, such as substrate concentration, pH, temperature, and enzyme concentration. Some idea of the optimal substrate concentration can be obtained from the value of the Michaelis constant (K,), that concentration of the substrate at which the rate will be one-half of its maximum rate. If the K , value can be obtained from the literature, then it is possible to begin with the substrate at a concentration two to three times the K , value (assuming the absence of "substrate inhibition"). Such a concentration would be adequate for use in early experiments. The literature can often provide information on starting values for other parameters, such as pH, any requirement for activators, and the optimal temperature for the incubation. Armed with this information, a trial reaction mixture can be prepared. What remain to be determined prior to the initiation of the reaction are the amount of enzyme to be added to the reaction mixture, the time course of the reaction, and, for discontinuous assays, the time between samplings and the volume of these samples. These questions can be answered by a process of trial and error using the following scheme. First, an arbitrary amount of enzyme is selected. While any concentration can be used, excess protein can be a problem for many types of assay. Therefore, choose the lowest amount possible to start the reaction. This reaction is then examined for product. If a product is detected, and

[5]

MEASUREMENT OF ENZYME ACTIVITY

47

its amount is small compared to the total amount of substrate, then a second sample can be withdrawn from the incubation mixture and analyzed. Again the amount of product formed should be determined. If this is more than 50% of that of the substrate, the reaction has progressed too far, and it is necessary to start again by preparing a new reaction mixture. In order to obtain more time points, the reaction rate should be slowed by using less enzyme. Alternatively, in the absence of any product, incubation could be continued for longer times or more enzyme added. If this does not result in the formation of detectable product, the possibility should be considered that the fraction being assayed contains no activity. Obtaining Initial Rate Data As a result of the preliminary trials described above, values will have been obtained for two parameters: the amount of the enzyme required to form sufficient detectable product and the incubation time required to form this amount of product. Sometimes it may be necessary to alter the reaction rate as follows. If the rate of product formation is too rapid, i.e., the reaction rate becomes nonlinear too soon, then the rate should be slowed by decreasing the amount of enzyme. Alternatively, if the rate of the reaction is too slow, the enzyme concentration should be increased so that a linear rate can be observed. Next, a second and third series of reaction mixtures should be prepared, with enzyme added at one-half and twice the amount used in the first. These reactions are started and the data presented as a function of reaction time. It should be noted that at this early stage in the optimization of the assay it is advisable to continue the incubations until the rate of product formation becomes nonlinear or the amount of substrate present is exhausted. This prolonged incubation provides information about the extent of the primary reaction and also allows time for the formation of products of secondary reactions. A determination of the rate of product formation becomes difficult at the lower limits of substrate concentration. However, certain changes can be made in the assay system that can solve this problem. One is to increase the sensitivity of the detector. Most detectors contain range switches that make this a simple matter. When range switching is carried out, it is useful to determine if the calibration curves constructed at one range setting are still valid at another. In the case of discontinuous assays, the amount of product being detected may be increased by increasing the volume of the reaction mixture. In some cases it may be necessary to concentrate the reaction mixture prior to analysis. It is also possible to increase sensitivity by using a more sensitive assay, such as one involving

48

GENERAL METHODS FOR HANDLING PROTEINS AND ENZYMES

[51

radiochemicals as substrates and to determine the amount of radioactive product that has formed. Understanding and Dealing with Secondary Reactions

When working with multizyme preparations, the importance of understanding secondary reactions cannot be overemphasized. This knowledge is invaluable to the interpretation of the results of enzymatic assays. "Beware of secondary reactions" is a rule that should always be kept in the forefront. How can secondary reactions be handled? These include purifying the activity of the primary reaction to homogeneity. However, this may not always be possible or desirable. Therefore, some other solution must be found. The use of analogs is one such s ~ l u t i o nFor . ~ example, if an analog of the substrate is used, then an analog of the product will be formed. If the latter is not a suitable substrate for the secondary enzyme, then no secondary reactions will occur. Alternatively, one can try to adjust the reaction conditions in such a way that the enzymes catalyzing the secondary reactions will not be active. For example, if the primary reaction does not require metals but the secondary reaction does, adding a chelator will inhibit the latter. Analogs can be used in another way. Consider the case of developing an assay procedure for adenosine kinase, the enzyme that catalyzes the primary reaction Ado + ATP + AMP + ADP. Problems will arise during the assay of this activity in crude extracts since other enzymes may be present that can form AMP directly from ATP. Radiochemical analogs such as radiolabeled adenosine are ideal for solving this problem because if the formation of radiolabeled AMP is monitored, it is possible to distinguish the AMP formed from adenosine from that formed from ATP, which, of course, would not be labeled. Alternatively, this same reaction can be assayed if adenosine is replaced by an analog whose fate can be followed by a separate detector. With such an analog as a substrate, its product would be different from the others. Quantitation

Internal standards, compounds added at any stage of the analytical procedure, can be useful in calibrating andlor calculating the effect of that procedure on the recovery of the substrate or product of the reaction. The

' E. F. Rossomando, G. A. Cordis, and G. D. Markham, Arch. Biochem. Biophys. 220,71 (1983).

[51

MEASUREMENT OF ENZYME ACTIVITY

49

compounds chosen as internal standards should have similar detection characteristics. One more potential problem concerns the question of selecting the range of substrate concentrations to be used throughout the study. Considering the sensitivity of most detectors and the apparent K , values of most enzyme activities, the selection of the upper limit of concentration is usually not a problem. A problem will develop, however, when rate determinations are made at low substrate concentrations, since at these concentrations the amount of product formed during the course of the reaction will be small and may be below the monitor's level of detection. Therefore, prior to executing any experimental protocols dealing with low substrate concentrations, it is prudent to ascertain the lower limits of the detector being used in order to determine what product concentrations can be detected. Developing an Assay for a Previously Undescribed Activity

Perhaps one of the most rewarding aspects to working with enzymes comes from the discovery of an activity not previously described. Yes, there remain many activities to be discovered! The sequence begins during the study of a physiologic process or a biochemical pathway with the realization that a change has occurred in some component. For example, the unwinding of DNA, the contraction of muscle, the oriented migration of cells. The next step is the key, and it involves guessing at what the reaction might be and either isolating the natural substrate or making a synthetic "substrate" to search for the postulated activity. Once a substrate is available, the activity can be measured and a strategy for the isolation and purification of the new enzyme developed. Finally, it is necessary to show that the new enzyme is not, in fact, an old activity in disguise. The use of more traditional substrates, as well as an analysis of the physical and chemical properties of the protein, including the production of antibodies, are all helpful. Also, the isolation of mutant cell lines that have altered functions, and correlation of the enzyme level in the mutant with the presence of the mutation, can be most convincing in such studies. Acknowledgments I wish to thank Ms. Jane Hadjimichael for her input and editorial assistance and Ms. Pamela Vachon for typing the manuscript.

50

GENERAL METHODS FOR HANDLING PROTEINS AND ENZYMES

161

[61 Quantitation of Protein

Introduction

Many methods for estimating protein concentration are available and the appropriate choice of method depends on five major criteria: the amount of protein available to assay, the concentration of the protein, the specificity of the assay, the presence of chemicals which may interfere with the assay, and the ease and reliability of performing the assay. An approximate range of sensitivity is given for each assay. It should be emphasized that this range is approximate since the sensitivity of each assay is highly dependent on the type of protein being measured and the assay volume. On the average, microtiter plate assays are 10 times more sensitive than the indicated range. Only those assays that are easy to perform, require simple instrumentation, and are highly sensitive will be discussed although there are many other excellent m e t h ~ d s . l -Methods ~ to concentrate samples or to eliminate interfering reagents are available and will also be discussed. Protein assay kits are available from Bio-Rad (Richmond, CA), Pierce (Rockford, IL), and Sigma (St. Louis, MO). Ultraviolet Absorption Methods

Ultraviolet light absorption methods have several advantages: ( I ) they can be performed directly on the sample without the addition of any reagents, (2) they can be performed very rapidly since no incubations are required, and (3) the relationship between protein concentration and absorbance is linear. Despite its technical simplicity there can be many

' E. Layne, this series, Vol. 3, p. 447.

'

G. L. Peterson, this series, Vol. 91, p. 95. D. H. Campbell, J. S. Garvey, N. E. Cremer, and D. H. Sussdorf, in "Methods in Immunology," 2nd Ed., p. 61. Benjamin, Reading, Massachusetts, 1970. G. Kresze, in "Methods of Enzymatic Analysis" (H. U. Bergmeyer, ed.), 3rd Ed., Vol. 2, p. 84. Verlag Chemie, Deerfield Beach, Florida, 1983. S. J. Jackson and E. L. McCandless, Anal. Biochem. 90, 802 (1978). S. B. Sheffield, D. Graff, and H. P. Li, Anal. Biochem. 166, 49 (1987). G. Krystal, Anal. Biochem. 167, 86 (1987). V. Neuhoff, K . Philipp, H. Zirnmer, and S . Mesecke, Hoppe-Seyler's Z. Physiol. Chem. 360, 1657 (1979). E. C. Butcher and 0. H. Lowry, Anal. Biochem. 76, 502 (1976).

METHODS IN ENZYMOLOGY, VOL. 182

Copyright O 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

rn

[61

QUANTITATION OF PROTEIN

51

Wavelength (rim) FIG.1. Ultraviolet spectrum of proteins and nucleic acids. Bovine immunoglobulin G (I), bovine serum albumin (B), and gelatin (G) were weighed and solubilized at 1 mglml in the following buffer: 0.01% Brij 35, 0.1 M K2SO4,and 5 mM KH,P04, pH 7. The scans of a 1 mglml solution are shown in the inset of (A) and the scans of a 15 pglml solution are shown in (A). RNA and DNA were weighed and solubilized at 10 pglml and the wavelength scans are shown in (B).

pitfalls if the principles of this technique are not understood. Several excellent reviews are available on this t ~ p i c . ' ~ , l ' Proteins actively absorb light in the ultraviolet region with two maxima, 280 and 200 nm (see Fig. 1). Absorption spectroscopy involves the absorption of a photon by an electron. Only those photons with a certain energy level can be absorbed as defined by the difference in energy between the orbital of the unexcited electron and a higher energy orbital. This is why there are absorption maxima. Photons with higher energy lo

l1

J. R. Little and H. Donahue, Methods Immunol. Zmmunochem. 2, 163 (1968). J. W. Donovan, in "Physical Principals and Techniques of Protein Chemistry," Part A (S. J. Leach, ed.), Academic Press, New York, 1969.

52

[61

GENERAL METHODS FOR HANDLING PROTEINS AND ENZYMES

have shorter wavelengths. Thus, electrons that are excited at 280 nm have absorbed less energy than those at 200 nm. Less energy is required for the electrons which absorb at 280 nm because these electrons lie within aromatic rings which stabilize the excited state due to resonance. Amino acids which have aromatic rings are phenylalanine, tryptophan, histidine, and tyrosine. It should be noted that those proteins with few of these amino acid residues would be expected to have little absorbance at 280 nm. This is shown to be the case for gelatin (Fig. 1). In addition to secondary structure, the tertiary structure of a protein can also play a role in its absorbance spectrum because interactions between different amino acids can further stabilize electron excited states. Consequently, conditions such as buffer pH, polarity, and ionic strength that alter tertiary structure can alter the absorbance spectrum of a protein. Buffers and buffer components can also interact directly with certain amino acids resulting in the stabilization or destabilization of electron orbitals. Although there is a high level of variability of absorbance at 280 nm from protein to protein, this wavelength has been found to be convenient for protein estimation because fewer chemicals absorb at this wavelength than at shorter wavelengths. The peptide bond absorbs photons below 210 nm. Because of the large number of peptide bonds in a protein, this is a highly sensitive area of the protein spectrum. Although protein conformation and some absorption by tryptophan and tyrosine residues occurs in this region, less variability between proteins is observed than at 280 nm. The disadvantage of this region is that many chemicals also absorb, especially those which contain double bonds between carbons or carbon and oxygen. However, buffers can be chosen carefully so that this highly sensitive region can be used (Table I).

TABLE 1 CONCENTRATION LIMITS OF CHEMICALS I N PROTEIN ASSAYS" Concentration limits UVI!

Substanceb Acids and bases HCI NaOH PCA TCA

Enhanced copper

> 1.25% >1.25%

BCAd

0.1 M 0.1 M < 1%
Dye'

0.1 M 0.1 M

Colloidal gold/

0.1 M 0.1 M

280 nm

>I M >I M 10% 10%

205 nm

0.5 M 25 mM 1M < 1%

TABLE I (continued) Concentration limits

uvp Substanceb Buffers Acetate Ammonium sulfate Borate Citrate Glycine HEPES Phosphate Tris Detergents Brij 35 CHAPS Deoxycholate Digitonin Lubrol PX Octylglucoside SDS Triton X-100 Triton X-100(R) Tween 20 Reductants Dithiothreitol 2-Mercaptoethanol Miscellaneous DNAIRN A DMSO EDTA Glycerol KCI NaCl Sucrose Urea

Enhanced copper'

>28 m M 2.5 m M 2.5 m M 2.5 p M 250 m M 250 m M

BCAd

0.2 M 20% 10 mM <1 m M 1M 100pM 250mM 0.1 M

Dye'

0.6 M I M 50 m M 0.1 M 100mM 2M 2M

Colloidal go1df

0.1 M 20mM 100 m M

1%

1.25% 0.25%

1%

0.10% 0.10%

5% 1M 1M 0.5 M

10 mM 9% >I00 mM <10 m M 5 mM <20 m M 50 mM 40 m M

1%

0.10% (0.01% 2% 0.10%

0.25% 1% 1% 1%

0.1 M >50%

205 nm

I% 10% 0.30% 10% 10% 10% 0.10% 0.02% >lo% 0.30%

I%

625 pgiml

280 nm

0.10% 1%

1% <0.1% 0.1%

0.10%

1%

50 p M 1.8 p M

< I mM < 1%

1M 1M

I p-M 10 p M

3 mM 10 mM

0.1 mM <10 m M

0.2 mg >6.2% 125 p M 25% 30 mM 1.75 M 50 m M >200mM

0.1 mg 5% 10 m M 10% t10mM IM 40% 3M

0.25 mg

10 ng 20% 10 mM 10%

1 Pg 20% 30 mM 40% 100 m M >1 M 2M >l M

0.1 M 100% I M 5M 1M 6M

<1 M 1M

" This table is a general guide. Test buffer mixtures as described in the text. More complete listings of chemicals may be obtained from Refs. 2,24,25, and 30. Figure preceded by (<) or (>) symbols indicate that the tolerable limit for the chemical is unknown but is, respectively, less than or greater than the amount shown. Blank spaces indicate that data were unavailable. PCA, Perchloric acid; TCA, trichloroacetic acid; HEPES, N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid; CHAPS. 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid: SDS, sodium dodecyl sulfate; (R), reduced; DMSO, dimethyl sulfoxide; EDTA, ethylenediaminetetraacetic acid. ' Figures indicate the final concentration of the chemical in the assay. Figures indicate the concentration of the chemical in a 50-p1 sample. Figures indicate the concentration of the chemical in a 25-pl sample. f Figures indicate the concentration of the chemical in a 10-p1 sample. 8 Figures indicate the final concentration of the chemical which does not produce an absorbance of 0.5 over water.

54

GENERAL METHODS FOR HANDLING PROTEINS AND ENZYMES -

-

-

-

[61

-

Avoid storing buffers in plastic containers since some plastics leach plasticizers which absorb at UV wavelengths. Detergents can also be troublesome since many absorb UV light. In some cases hydrogenated forms of the detergent are available where the carbon double bonds have been converted to single bonds, thus reducing their absorption in the UV range. For example, Calbiochem (La Jolla, CA) and Aldrich (Milwaukee, WI) now offer hydrogenated Triton X-100. Method 1: Absorbance at 280 nm Range: 20 to 3000 p g Procedure 1. Turn on the UV lamp of the spectrophotometer and warm up the machine (usually 15 min). Adjust the wavelength to 280 nm. 2. Zero the spectrophotometer using the buffer in which the protein is dissolved as a blank. 3. Measure the absorbance of the protein solution. 4. For unknown or protein mixtures, use the following formula for a rough estimate (when using a cuvette with a path length other than 1 cm, divide the absorbance reading at 280 nm by the path length in centimeters): Concentration (mglml) = absorbance of protein at 280 nm

(1)

For a protein whose absorbance coefficient is known, its value may be found in the reference cited be lo^.'^ The absorbance coefficient is usually given as A::$*', or EM(molar extinction coefficient). The absorbance coefficient is dependent on pH and ionic strength, so be sure to match these parameters with those in the tables. Protein concentration is then determined by the following formula when using a cuvette with a 1-cm path length: Concentration (mglml) = absorbancel~$"' Concentration (%) = absorbancel~ Concentration (M) = absorbance/EM

!zm

(2)

Percentage protein and molarity can easily be converted to milligrams per milliliter protein by the following formula: Concentration (mglml) = percentage protein110 = molaritylprotein molecular weight l2

(3)

D. M. Kirschenbaum, in "Handbook of Biochemistry and Molecular Biology" (G. D. Fasman, ed.), 3rd Ed., Vol. 2, p. 383. CRC Press, Cleveland, Ohio, 1976.

[61

QUANTITATION OF PROTEIN

55

Comments. If the buffer or protein solution is cold, the outside of the cuvette may need to be wiped between each reading with a lint-free wiper and the readings made quickly after placing the cold solution into the cuvette, because atmospheric moisture may condense on the outside of the cuvette producing an erroneously high reading. Warming of cold solutions also can release gas in the form of bubbles which will also produce erroneously high readings. Alternatively, use warmed solutions. If the absorbance of the protein solution is greater than 2, dilute the protein sample in the same buffer as the original solution. Particulates or lipid droplets cannot be present in the protein solution since they have a strong tendency to scatter light at short wavelengths. The range of sensitivity given above is extremely rough since it is dependent on the type of protein being measured. For a more accurate estimate of protein concentration use Method 2 or 3 if the buffer conditions permit. The absorbance coefficient (A),:; for some commonly used proteins are as follow: bovine serum albumin, 6.3; bovine, human, or rabbit immunoglobulin G, 13.8; and chicken ovalbumin, 7. l2 Nucleic acids absorb strongly at 280 nm. Thus, crude cell extracts containing RNA and/or DNA will produce erroneously high protein estimates. To correct for nucleic acid content perform the following steps in addition to those outlined above: Zero the cuvette containing the buffer at 260 nm. Next, place the solution containing the protein in the cuvette and read the absorbance. To determine the protein concentration of the solution, use the following formula (A is absorbance which is directly followed by the wavelength, in nanometers, at which it was obtained)13: Protein concentration (mglml) = 1.55A280- 0.76A260

(4)

Method 2: Absorbance a t 205 nmI4 Range: 1 to 100 pg Procedure. The procedure is the same as for Method 1 except for adjusting the spectrophotometer to 205 nm and including 0.01% Brij 35 in the buffer to prevent protein losses onto vessel surfaces. Proteins stick to both plastic and glass surfaces in both concentrated and dilute solutions, but losses are proportionately higher in dilute solutions. Proteins absorb much more strongly at 205 nm and there is less variability from protein to protein7 (see Fig. 1). Use Eq. (5) to estimate protein. l3 l4

W. E. Groves, F. C. Davis, Jr., and B . H. Sells, Anal. Biochem. 22, 195 (1968). R. K. Scopes, Anal. Biochem. 59, 277 (1974).

56

GENERAL METHODS FOR HANDLING PROTEINS AND ENZYMES

[61

Concentration (mglml) = 3 1 (absorbance at 205 nm)

(5)

Comments. See Method 1 for general comments. At normal cellular concentrations of proteins and nucleic acids, nucleic acids contribute relatively little toward the absorbance at 205 nm. This method is highly dependent on the accuracy of the wavelength setting of the spectrophotometer because 205 nm is on the shoulder of the protein peak (see Fig. 1). It is also dependent on the absence of stray light (this parameter is part of the mechanics of the spectrophotometer itself). These shortcomings can be overcome by using a 10 pglml solution of BSA as a standard and determining protein concentration from the straight line between the buffer blank and the absorbance of the BSA. Only one standard point is needed since the relationship between protein concentration and absorbance is linear. Protein concentration can also be determined at 210 nm. However, this wavelength is less sensitive and tends to vary more with buffer conditions than at 205 nm. The extinction coefficients for 1 mglml concentrations of proteins range between 20 and 24 at 210 nm.I4 Method 3: Determination of Extinction Coeficient for Protein of Unknown Concentration l 4 Range: 20 to 3000 pg Procedure

1. Determine the absorbance of the protein at 205 and 280 nm as outlined above. The protein will have to be diluted about 30-fold for the 205-nm reading. 2. Use the absorbances in the following formula. Do not forget to multiply the reading at 205 nm by the dilution factor before using the formula. 3. The concentration of the protein is then determined using Eq. (2) and the value derived in Eq. (7). 4. Now that the concentration of the protein is known, the extinction coefficient at 280 nm can be determined. Comments. These determinations are dependent on the protein having an "average" amino acid composition. Abnormal phenylalanine content will especially skew the result. Scopes claims an accuracy of 2% for most proteins.I4

[61

QUANTITATION OF PROTEIN

57

Colorimetric Assays

When measuring protein concentrations it is best to aim in the middle of the range of sensitivity of the assay because the assays sometimes become nonlinear at both very high and very low levels. It is a good idea to also assay your protein solution following a 1 : 1 dilution to ascertain that your sample does indeed lie on the linear portion of the curve. Also, the presence of unknown factors that interfere with the assay may be revealed by an unexpected result. Sometimes the assay can be linearized by plotting the log of the absorbance against the log of the amount of protein. l5 All colorimetric assays have the disadvantage that different proteins produce different absorbances and thus, unless the same protein as that being assayed is used as the standard, the protein values obtained are relative rather than actual values. For this reason it is imperative that when such protein values are reported in the literature, the assay used and the protein used as the standard are identified. Protein standards should be assayed at the same time and in the same solution as the unknowns to take into account interactions of the buffer with the assay reagents, reagent decomposition, time differences, and temperature changes. In many applications, the solution containing the protein contains a mixture of components. To test whether these components will interfere with the protein assay, perform the assay in the usual manner adding the following reagents as samples: water for the blank, the buffer, a protein standard dissolved in water, and the same amount of protein dissolved in the buffer. If the reagent interferes only somewhat with the assay this interference can usually be corrected for by running the standard curve in the presence of the reagent. If the reagent drastically interferes with the assay or the interfering reagent is present in varying amounts, it can be removed as described below. Method 4: Enhanced Alkaline Copper (Lowry) Protein A s ~ a y ' ~ Range: 2 to 100 pg Reagents 2x Lowry concentrate: Dissolve 20 g Na2C03in 260 ml water, 0.4 g CuS04 5H20 in 20 ml water, and 0.2 g sodium potassium tartrate in 20 ml water and mix the solutions to form the copper reagent. Disl5

l6

c. E. Stauffer, Anal. Biochem. 69, 646 (1975). 0 . H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).

58

GENERAL METHODS FOR HANDLING PROTEINS AND ENZYMES

[61

solve 10 g sodium dodecyl sulfate (SDS) in 100 ml water to make a 1% solution. Dissolve 4 g NaOH in 100 ml water to make a 1 M solution. Immediately before use, mix 3 parts copper reagent with 1 part SDS and 1 part NaOH. This reagent is stable for 2 to 3 weeks. If a white precipitate forms warm the solution to 37'; if the precipitate is black, discard the solution 0.2 N Folin reagent: Mix 10 m12 N Folin reagent with 90 ml water. This solution is stable for several months at room temperature if stored in an amber bottle Procedure

1 . To a 400-p1 sample add 400 p1 of the 2 x Lowry concentrate and incubate at room temperature for a minimum of 10 min. 2. Add 200 p1 of the 0.2 N Folin reagent and vortex immediately after each addition. This rapid mixing is important since the reagent decomposes rapidly. Incubate for an additional 30 min at room temperature. Careful timing of each sample is unnecessary providing the samples can be read within 10 min of each other because the color is relatively stable. 3. Turn on the spectrophotometer 15 min before use. Using glass or polystyrene cuvettes read the absorbances at 750 nm. If the absorbances are too high, the absorbances may be read at 500 nm. Figure 2 shows the standard curves for three different proteins.

protein ( j ~ g ) FIG. 2. Enhanced copper protein assay standard curves for three different proteins. Bovine immunoglobulin G (I), bovine serum albumin (B), and gelatin (G) were weighed and solubilized at 2 mglml in water. Aliquots of these solutions were used to produce the standard curves shown above using Method 4.

QUANTITATION OF PROTEIN

Comments. The above technique is modified from the original in order to make it less sensitive to interfering buffer components and more sensitive to low levels of protein. It is similar to the assays reported in Refs. 2 and 17. Despite the improvements, the BCA assay, described below, will probably replace the Lowry since it requires that only one reagent be added to the sample, has less protein-to-protein variability, and is linear over a greater protein range. The critical step for obtaining sensitive and reproducible results is to vortex the samples immediately after the addition of the Folin reagent. This is because the Folin reagent is reactive for only a very short time after addition. Also examine the tubes for thorough mixing. There should be no yellow gradient from top to bottom and the solution should appear homogeneous. At room temperature, the period of time for incubating a soluble protein with the copper solution is not critical and can vary from 5 min to several hours without affecting the final absorbance. Heating the protein with the alkaline copper solution to 100" for 20 min, as suggested in Ref. 18, is reported to equalize the reaction to different proteins. The color decreases for the majority of proteins but for gelatin, a relatively unreactive protein, the color increases by 50% after 10 min of heating. Diluting the Folin reagent to 0.2 N increases the sensitivity of the assay by about 40% over the original procedure.19 An additional 20% increase in sensitivity can be obtained by adding the Folin reagent in two portions, vortexing between each addition.20 The assay can be accelerated by the addition of 100 p1 20 mM dithiothreitol3 min after the addition of the Folin reagent. The assay can be read 5 min after the addition of the thiol with a concomitant 50% increase in sensitivity .?' Special care must be given when the solution is transferred into cuvettes because bubbles may form, producing erroneously high readings. Pipette these solutions gently with large-mouth pipets to reduce this problem. The 2 x Lowry reagent may be diluted with water to accommodate smaller sized samples. Also, to accommodate samples dissolved in NaOH, the Lowry reagent may be made as above, but replacing the

l9

2'

M. A. K. Markwell, S. M. Haas, L. L. Bieber, and N. E. Tolbert, Anal. Biochem. 87,206 (1978). T . E. Dorsey, P. W. McDonald, and 0. A. Roels, Anal. Biochem. 78, 156 (1977). G . M. Oostra, N. S. Mathewson, and G. N. Catravas, Anal. Biochem. 89, 31 (1978). H. H. Hess, M. B. Lees, and J. E. Derr, Anal. Biuchem. 85, 295 (1978). E. Larson, B. Howlett, and A. Jagendorf, Anal. Biochem. 155, 243 (1986).

60

GENERAL METHODS FOR HANDLING PROTEINS AND ENZYMES

[61

NaOH with water, as long as the final NaOH concentration remains the same. An amplification technique is available to increase ~ e n s i t i v i t y . ~ ~ Mechanism. Under alkaline conditions Cu2+forms a complex with the peptide bonds of proteins and becomes reduced to Cu+. The Cu+ as well as the R groups of tyrosine, tryptophan, and cysteine residues then react with the Folin reagent. The reagent reacts by first producing an unstable product which is slowly reduced to become molybdenum/tungsten b l ~ e . ~Certain ~ , ~ predictions ~ , ~ ~ can be made from this mechanism. Agents which acidify the solution (e.g., strong acids or buffers, high ammonium sulfate), chelate the copper (i.e., EDTA), or cause the reduction of copper (e.g., mercaptoethanol, dithiothreitol, phenols) will interfere with the assay. Proteins will produce different color intensities depending primarily on their tyrosine and tryptophan content. Method 5: Bicinchoninic Acid (Smith) Protein Assayz4 Range: 0.2 to 50 p g Reagents Reagent A: 1 g sodium bicinchoninate (BCA), 2 g Na2C03, 0.16 g sodium tartrate, 0.4 g NaOH, and 0.95 g NaHC03 brought to 100 ml with water. Adjust the pH to 11.25 with 10 M NaOH Reagent B: 0.4 g CuS04.5H20 in 10 ml water. Solutions A and B are stable. The solutions are available from Pierce (Rockford, IL) SWR (standard working solution): Mix 100 vol reagent A with 2 vol reagent B. This solution is stable for 1 week and should be green in color Procedure 1. Add 1 ml SWR to the 2 0 - 4 sample and mix. Incubate 30 min at 60". Cool the samples to room temperature and turn on the spectrophotometer. If the samples have been incubated at 60" the readings will be stable for at least 1 hr. 2. Read the absorbance at 562 nm using glass or polystyrene cuvettes. Standard curves for three different proteins are shown in Fig. 3. M. G. Sargent, Anal. Biochem. 163, 476 (1987). G. Legler, C. M. Muller-Platz, M. Mentges-Hettkamp, G . Pflieger, and E. Julich, Anal. Biochem. 150, 278 (1985). 24 P. K. Smith, R . I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartner, M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B. J. Olson, and D. C. Klenk, Anal. Biochem. 150, 76 (1985). 22

23

[61

QUANTITATION OF PROTEIN

61

protein (ug)

FIG.3. Bicinchoninic acid protein assay standard curves for three different proteins. Bovine immunoglobulin G (I), bovine serum albumin (B), and gelatin (G) were weighed and solubilized at 2 mglml and 0.1 mglml in water. Aiiquots of these solutions and were used to produce the standard curves shown above using Method 5.

Comments. Sensitivity of the assay can be increased by incubating the samples for longer periods of time. Conversely, if the color begins to get too dark the heating can be stopped earlier. The assay may also be performed at room temperature. Incubation at temperatures lower than 60" results in diminished sensitivity and greater protein-to-protein variability. This is thought to be a result of the greater reactivity of the peptide bonds with the copper solution at high temperatures. A micromethod is also available in order to measure dilute protein solutions. Five hundred microliters of the reagent is added to 500 p1 of the protein solution. The mixture is incubated at 60" for 1 hr, cooled to room temperature, and the absorbance read at 562 nm. The reagent is made as follows:

Reagent A: 8 g Na2C03.H 2 0 , 1.6 g NaOH, 1.6 g sodium tartrate in 100 ml water and adjusted to pH 11.25 with 10 M NaOH Reagent B: 4 g BCA in 100 ml water Reagent C: 0.4 g CuS04.5H20 in 10 ml water Working solution: Mix 1 vol reagent C with 25 vol reagent B. Then add 26 vol reagent A to the CIB mixture These reagents are available from Pierce as a kit. Mechanism. The BCA assay is related to the Lowry assay in that it depends on the conversion of Cu2+to Cu+ under alkaline conditions (see Mechanism under Method 4). In contrast to the Lowry assay, the BCA

62

GENERAL METHODS FOR HANDLING PROTEINS AND ENZYMES

[61

protein (ug)

FIG.4. Coomassie Blue protein assay standard curves for three different proteins. Bovine immunoglobulin G (I), bovine serum albumin (B), and gelatin (G) were weighed and solubilized at 2 mglml and 0.1 mglml in water. Aliquots of these solutions were used to produce the standard curves shown above using Method 6.

assay utilizes BCA as the Cu+ capture reagent instead of the FolinCiocalteau reagent. This substitution has the advantage that BCA is relatively stable under alkaline conditions (the Folin-Ciocalteau reagent is unstable at alkaline pH). Thus, the BCA can be incorporated into the alkaline copper solution allowing for a one-step procedure. Method 6: Coomassie Blue (Bradford) Protein AssayZ5 Range: 0.2 to 20 pg Dye Reagent. Dissolve 100 mg Serva Blue G (Serva, Westbury, NY) in a mixture of 100 ml85% phosphoric acid and 50 ml95% ethanol. After the dye has completely dissolved, bring the volume to 1 liter with cold water. Procedure 1. Warm up the spectrophotometer 15 min before use. 2. Place 20-p1 samples into the tubes. Add 50 p1 1 M NaOH to the sample (alternatively, the NaOH may be mixed into the reagent itself at 50 pllml to allow one pipetting step). 3. Add 1 ml of the dye reagent and incubate 5 min. 4. Measure the absorbance at 590 nm in glass or polystyrene cuvettes.

Figure 4 shows the standard curves for three different proteins using this assay. 25

M. M. Bradford, Anal. Biochem. 72, 248 (1976).

64

GENERAL METHODS FOR HANDLING PROTEINS A N D ENZYMES

[61

with stirring. After the gold has dissolved rapidly add 1 ml 40 mg/ml trisodium citrate dihydrate with stirring. Boil the solution for 30 min with refluxing. Allow the colloid to cool to room temperature and add 32 pl 25% Tween 20 and mix. Add 400 pl 1 M citric acid to the gold mixture. The final solution should be a clear red. A cloudy solution cannot be used and is usually a result of contaminated glassware or reagents. Store at 4". Procedure 1 . Place 1 ml gold reagent into 1-ml polyethylene microfuge tubes. 2. Make protein dilutions in 0.01% Tween 20 immediately before use. Do not allow standards to sit for prolonged periods of time or freeze them as the proteins will adsorb to their vessels. 3. Add 10 p1 of each protein dilution and unknowns into the dye and vortex immediately. Incubate 15 min. 4. Read the absorbance at 560 nm, using the blank to zero the spectrophotometer. 5. Plot absorbance at 560 nm against nanograms protein. Figure 5 shows the standard curve for three different proteins. Comments. The manufacturer (Janssen, Piscataway, NJ) has changed the formulation of Aurodye which was used in the original publication" so that it can no longer be used for the protein assay. Diversified Biotech (Newton Centre, MA) offers colloidal gold specially formulated for protein assays.

Protein (ng) FIG.5. Colloidal gold protein assay standard curves for three different proteins. Bovine immunoglobulin G (I), bovine serum albumin (B), and gelatin (G) were weighed and solubilized at 2 mglml and diluted in 0.01% Tween 20 in order to produce the above concentrations in a 10-p1 volume. Method 7 was used to produce the standard curves.

161

QUANTITATION OF PROTEIN

65

Overloading the system will produce erroneously low protein values. Such samples have a dark blue or gray color or a visible precipitate. At even higher protein levels the colloid will turn red again. Use greater dilutions in these cases to obtain the most accurate results. Be sure to measure two different concentrations of the same solution to make sure you are on the side of the curve which exhibits proportionate increases in absorbance. Proteins tend to stick to glass and plastic surfaces and at very low concentrations losses from this process can become highly significant. The addition of small amounts of detergents such as 0.001% Tween and for certain proteins, NaCI, decrease protein adsorption. Dilutions of the standards can be easily made on a piece of Parafilm. The gold reagent contains detergent which will produce bubbles upon too vigorous mixing or pipetting. These bubbles will cause inappropriately high absorbance readings. If reagents are present that interfere with the assay, a related, solidphase assay in which the protein is first spotted on nitrocellulose and then the nonprotein components washed away may be used.32 Mechanism. The binding of protein to the colloidal gold causes a shift in its absorbance proportional to the amount of protein added to the assay. The pH of the solution must be acidic in order for the proteins to carry a positive charge and interact with the negatively charged General Instructions

Cuvettes

Glass cuvettes may be used for wavelengths above 320 nm. Although quartz cuvettes may be used for the entire ultraviolet to visible spectrum, they are generally not used for visible wavelength readings because of their cost. Also, in the Coomassie Blue protein assay, the dye sticks to quartz more readily than glass. Disposable styrene cuvettes may be used at wavelengths above 340 nm and acrylic cuvettes may be used at 275 to 350 nm. Disposable cuvettes are especially useful for Coomassie Blue and colloidal gold-based protein assays since the dye-protein complexes stick to cuvette surfaces. When using disposable semimicrocuvettes there may be problems in precision unless a single cuvette is left in place. Cuvettes are fragile and easily scratched. Glass Pasteur pipets should never be inserted into nondisposable cuvettes. Bio-Rad style D polyethyljZ

J . B . Hunter and S. M. Hunter, Anal. Biochem. 164,430 (1987).

'' M. Horisberger and J. Rosset, J. Hisrochem. Cytochem. 25, 295 (1977).

66

GENERAL METHODS FOR HANDLING PROTEINS AND ENZYMES

[61

ene pipets (Cat. #223-9523) are convenient, nonscratching pipets that will fit into standard and semimicrocuvettes. Cuvettes should be washed immediately after use to prevent proteins from adhering to the inside surface of the cuvette. A cuvette washer available at most supply houses will minimize cuvette breakage. Always handle cuvettes by their sides to prevent fingerprints from increasing the absorbance of the sample. If fingerprints do get on the transmitting surfaces these may be wiped away with 70% v/v ethanol using a lintless wiper. Make sure the springs that hold the cuvettes in place are properly adjusted, i.e., the cuvette should not wobble in its holder. One remedy for a high level of variability produced by simply removing the cuvette from its holder and replacing it is to simply leave the cuvette in place and remove and replace solutions with a pipet. Protein Standards Most methods of protein quantitation compare an unknown quantity to a standard curve of a "known" protein amount. Absolute protein quantitation can be tricky for several reasons. First, different proteins will react to varying extents in different protein assays. For example, 10 pg gelatin will produce only 69% as much absorbance as 10 pg BSA in the Lowry assay (Fig. 2). In preparing standards, salts and moisture can add to the weight of a protein powder. Different pH and ionic strengths can alter the UV absorbance of proteins. For these reasons, protein values in many publications are actually relative values and thus it is very important that information be provided about the standard protein and assay used. Commonly, bovine serum albumin is used as a protein standard. This is because it has been used so much in the past that it makes present and future protein determinations readily comparable. It is also easy to handle and inexpensive. Immunoglobulin G is used less frequently, but produces a more average absorbance for both the dye and enhanced copper protein assays. Standards can be purchased from any of the vendors mentioned above. Alternatively, standards can be prepared as follows: the powder should be stored at -20' in a desiccator. Allow the jar to warm up to room temperature before weighing. Weigh out enough protein for accurate measurement. Dissolve in water to make a 2 mg/ml solution. Aliquot into 0.5-1111fractions and store in a freezer at -20". Commercial standards should also be aliquoted and frozen. When possible, the same protein as that being determined should be used as a standard. As a general rule the protein should be extensively dialyzed against deionized, distilled water, lyophilized, and kept desic-

161

67

QUANTITATION OF PROTEIN -

-

- - -

cated. It should be noted that not all proteins are soluble in water. Some require the presence of salts, acid, base, or an organic solvent. The Merck Index34lists some proteins and the methods to solubilize them. Information on the solubility of specific proteins can be obtained from the manufacturer or vendor. Removal of Interfering Substances Neutralization. Strong acids, bases, or buffers should be neutralized or adjusted to the optimal pH of the assay system (Lowry, pH 10; BCA, pH 11; dye, pH 1; gold, pH 3) by the addition of HCl or NaOH. EDTA, which interferes with the copper-based assays by chelating the copper, may be counteracted with the addition of equimolar CaCI2. The CaC12 precipitates in alkaline solutions and must be removed by centrifugation before taking absorbance readings. Thiols, such as dithiothreitol, mercaptoethanol, and cysteine, can be neutralized by the addition of 8 p1 2 M iodoacetic acid dissolved in 2.5 M NaOH.3SMercaptoethanol, by virtue of its volatility, can simply be e v a p ~ r a t e d . ~ ~ Precipitation. Buffers, lipids, reducing agents, and certain detergents can be removed by precipitating the protein using TCA, PCA, or acetone and resuspending the protein in a suitable solution for the chosen assay. The presence of detergents sometimes interferes with protein precipitation depending on the detergent and the procedure used. Certain detergents (e.g., deoxycholate, Triton X-100) precipitate under acidic pH conditions and thus cannot be removed by the addition of TCA or PCA. Acid precipitations result in pellets which are difficult to dissolve unless a coprecipitant is used. Sonication and incubation in 1 M NaOH will promote dissolution of the pellet. These techniques are described in greater detail in Refs. 2, 16, and 37-39. Dialysis and Ultrafiltration. The time required for dialysis is inversely dependent on pore size and volume. Using small volumes and large pore sizes, dialysis can take just a few hours. Ultrafiltration is still faster and can take as little as 30 min to perform. Caution must be used if detergents are present since they can form high-molecular-weight micelles and their capacity to pass through membranes depends on their micellar molecular weight rather than the molecular weight of a single molecule. Dialysis and M. Windholz, "The Merck Index," 10th Ed. Merck & Co., Rahway, New Jersey, 1983. E. Ross and G . Schatz, Anal. Biochem. 54, 304 (1973). l6 H. P. S . Makkar, 0.P. Sharma, and S. S. Negi, Anal. Biochem. 104, 124 (1980). A. Bensdoun and D. Weinstein, Anal. Biochem. 70, 241 (1976). I". Polacheck and E. Cabib, Anal. Biuchem. 117, 311 (1981). " D. Wessel and U. L. Flugge, Anal. Biochem. 138, 141 (1984). '4

Is

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ultrafiltration with and without detergents is discussed in greater detail in Refs. 40-44. Instrumentation

The choice of the spectrophotometer purchased depends on the types of protein assays planned. The most versatile instrument is one with both visible and ultraviolet light sources. The Spectronics 20 spectrophotometer is convenient for routine assays using light in the visible range since Fisher disposable 13 x 100 mm culture tubes can be used as disposable cuvettes. A relatively new, convenient innovation is the microwell plate reader which automates spectrophotometric readings. All of the colorimetric procedures described above have been modified for these plate , ~ ~ B r a d f ~ r d , colloidal ~' gold.48The microwell readers, i.e., L ~ w r yBCA,46 plate reader does not have ultraviolet light capabilities. It should be noted at what point the detector can no longer measure absorbance in a linear fashion. This level is usually about 2. Acknowledgment Supported by the Veterans Administration

" K. C. Retz and W. J. Steele, Anal. Biochem. 79, 457 (1977). A. J. Furth, H. Bolton, J. Potter, and J. D. Priddle, this series, Vol. 104, p. 318. L. M. Hjelmeland and A. Chrambach, this series, Vol. 104, p. 305. 43 A. Helenius, D. R. McCaslin, E. Fries, and C. Tanford, this series, Vol. 61, p. 734. A. J. Furth, Anal. Biochem. 109, 207 (1980). H. J. Fryer, G. E. Davis, M. Manthorpe, and S. Varon, Anal. Biochem. 153,262 (1986). * M. G. Redinbaugh and R. B. Turley, Anal. Biochem. 153, 267 (1986). 47 M. G. Redinbaugh and W. H. Campbell, Anal. Biochem. 147, 144 (1985). T . Ciesiolka and H. Gabius, Anal. Biochem. 168, 280 (1988). 42

"

[71 Concentration of P r o t e i n s and Removal of Solutes By THOMAS POHL

The removal of low-molecular-weight solutes and concentration of protein solutions are the most frequently performed operations in protein purification and analysis. The diversity of applications and the multitude of techniques available today render the selection of the optimal method in a particular case a difficult task for the investigator. There is no generMETHODS IN ENZYMOLOGY, VOL. 182

Copyright O 1990 by Academic Press, Inc. All rights of reproduction in any form resrrved.

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ally advisable way to transfer a given protein from one solution to another. The highly specific characteristics of the individual protein and the requirements of the experiment must be taken into consideration. In most cases concentrating and desalting of a protein solution can be achieved without substantial losses or effects on the biological activity of the protein. The objectives of the procedure may be as diverse as concentrating and desalting bulk quantities of proteins at the initial steps of a purification or the preparation of microgram quantities of a labile enzyme for physical, protein chemical, or biological analysis. Often the composition of protein samples has to be adjusted for a preparative or analytical separation. Electrophoretic techniques, ion-exchange chromatography, and protein sequencing are particularly sensitive to the presence of salts and may additionally require concentrated protein samples as is the case in size-exclusion chromatography. In recent years an ongoing tendency in the field of biochemistry has been to work with minute amounts of proteins. Many of these approaches have become feasible on the basis of advanced methods of protein purification. The advent of modern high-performance liquid chromatography (HPLC) techniques, mass spectrometry, and protein microsequencing has led to admirable progress in the purification and analysis of sensitive proteins on the picomole level. Methods for desalting and concentration of protein solutions have to meet these new requirements. As many of the older techniques, e.g., dialysis, ammonium sulfate precipitation, and lyophilization, possess considerable drawbacks such as low recovery and denaturation of labile proteins, great effort has been applied to the development of new, more efficient and mild methods. Modem technology has provided membranes for ultrafiltration and dialysis allowing the concentration and desalting of proteins with acceptable yields and little denaturing effect. New and improved supports for conventional chromatography have made gel filtration often the method of choice to change the buffer conditions or composition of a protein solution. By far the most difficult problems are encountered in handling dilute protein solutions (i.e., less than 10 pglml). HPLC and FPLC (fast protein liquid chromatography) technology have been shown to be of great value in the preparation of such samples, as they generally give high recoveries and reproducible conditions. Additionally, electrophoretic methods, well known as a preparative tool for protein isolation, have also been employed for the concentration of proteins. A variety of apparatuses and devices for the concentration of proteins and removal of solutes are now commercially available and are being used with varying success. This chapter is meant to provide basic information on the procedures for concentrating and desalting proteins.

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General Considerations

The first parameter to be considered in a concentration or desalting experiment is the initial volume of the protein sample and the volume that is desired after the procedure. In many cases concentration and desalting are interlinked elements between different steps of a protein purification. Whenever possible the order of the individual steps of protein separation should be arranged in a way that concentrating techniques follow those methods leading to dilute protein samples. The combination of gel filtration and ion-exchange chromatography or reversed-phase HPLC is most suitable in this respect. The latter allow the application of large sample volumes and normally yield concentrated protein fractions. Reversedphase chromatography, additionally, can result in complete desalting of the protein if volatile buffer systems are used. This is particularly helpful when preceding techniques such as ion-exchange chromatography and hydrophobic interaction chromatography require the presence of high salt concentrations. An optimal arrangement of the purification procedures saves time and improves the performance of each individual step. Moreover, concentration and desalting of the protein fractions are facilitated and in some cases can even be omitted. Detailed information about the physical and chemical properties of the protein to be concentrated and/or desalted facilitates the choice of the best method. Its molecular weight predetermines the exclusion limit of membranes used for dialysis and ultrafiltration, although the three-dimensional structure of the macromolecule strongly influences the behavior of the individual protein in these techniques. In addition, chromatographic methods for desalting and concentration such as gel filtration, ionexchange chromatography, and reversed-phase chromatography also require some information about the molecular weight of the protein. The average pore size of the support particles and the structure of surface coatings optimal for the particular protein are thereby influenced. For example, pores of gel filtration media should totally exclude the protein of interest to obtain fast and high yield recovery. The choice of a suitable buffer system is dictated by the solubility of the protein and possible effects of the solution on its stability. Otherwise, the protein may be denatured and/or precipitated. Normally, proteins are transferred to a different solvent rather than being entirely desalted or concentrated to dryness. Thus, the composition of the desired solution has to be carefully selected. Appropriate ionic strength, pH, and volume have to be adjusted in order to maintain solubility of the protein. Particular attention has to be paid to the need for essential metal ions and cofactors that may be required to retain the native structure and biological activity of the protein.

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To avoid significant losses of protein during concentration or desalting, the tendency of protein to adsorb to surfaces should also be investigated. Many proteins containing charged or hydrophobic domains show a high affinity toward various surfaces1that may lead to irreversible binding or denaturation. An important decision is whether glass or plastic containers should be used since the adsorptive properties of proteins differ widely on these materials. The strategy to reduce adsorptive loss of protein on surfaces is either based on coating the container ~ u r f a c e or ~,~ modifying the composition of the solution, normally by addition of protein (often albumin), detergents, or salt. The latter approach follows from the observation that proteins are more stable in the presence of a high protein concentration, and this approach appears to be more e f f e ~ t i v eThe . ~ number of methods that can be applied in the case of a highly adsorbing protein is very limited. Those involving the use of semipermeable membranes (dialysis, ultrafiltration, most electrophoretic methods) are commonly more prone to adsorptive losses. Care must be taken to avoid chemical destruction of the protein during concentrating andlor desalting. Oxidative and reductive interaction of reactive chemical compounds may cause considerable modification and damage to sensitive amino acid side-chain groups. Labile sulfhydryl groups, primary amino groups in the unprotonated state, and reactive nitrogen atoms such as N-2 in histidine are easily converted to secondary products that interfere with characterization of the protein or with its native structure. The properties of the solutes are thus of critical importance for maintaining the stability of the protein. Some commonly used buffers or solutes, such as bicarbonate and urea, may contain or generate reactive, mostly electrophilic, compounds that modify proteins. The use of high-purity solvents and chemicals is therefore recommended in any concentrating and desalting procedure. A common problem is modification of the N-terminal amino group, which is known to be particularly r e a c t i ~ eThis . ~ N-terminal blocking renders protein sequencing by Edman degradation impossible. Since modern protein sequence analysis can be performed with picomole amounts of protein, N-terminal blocking is a serious problem. Most of these modifications cannot be removed easily, and the problems imposed can be circumvented only by fragmentation of the protein and subsequent peptide analysis, which is feasible only if milligram amounts of protein are obtainable. I

F. Macritchie, Adu. Protein Chem. 32, 283 (1978). P. L. Felgner and J. E. Wilson, Anal. Biochem. 74, 631 (1976). K. J. Kramer, P. E. Dunn, R. C. Peterson, H. L. Seballos, L. L. Sandburg, and J. H. Law, J . Biol. Chem. 251, 4979 (1976). C. H. Suelter and M. Deluca, Anal. Biochem. 135, 112 (1983). M. J. Walsh. J. McDougall, and B . Wittmann-Liebold, Biochemistry 27, 6867 (1988).

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Another important factor that should be considered is the time required for concentration and desalting of protein solutions. Techniques such as dialysis and lyophilization may take days to be completed. Even when working at low temperatures stability of the proteins can be seriously affected by a number of factors over such a long period of time. The destructive influence of chemical and enzymatic activities present in the protein sample or introduced as impurities due to the solvent and buffer used may become significant. As already discussed, not only is loss of protein a problem, but also modifications of the protein which interfere with the intended purpose. Furthermore, microbial growth has to be prevented by addition of suitable preservatives such as sodium azide (1-5 mM). In order to avoid losses due to such effects, rapid techniques for concentration and desalting should be given preference. The more widely used techniques, with their characteristic benefits and shortcomings are described below. Dialysis

One of the oldest techniques for removal of low-molecular-weight solutes or exchange of buffer is dialysis. The method is based on the properties of a semipermeable membrane separating the protein solution from the dialysis buffer. It allows free passage of molecules below a certain molecular weight, the so-called "molecular weight cutoff," while macromolecules cannot penetrate the pores of the membrane. The process of dialysis is driven by the difference in concentration of the solutes on the two sides of the membrane. As the equilibrium concentration is approached, the diffusion of solutes becomes equal in both directions. Further reduction of the solute concentration in the protein solution can be achieved only by changing the dialysis buffer. Thus, the number of buffer changes is more important than the total volume of dialysis buffer. Dialysis is markedly accelerated by increasing the ratio of membrane area to the volume of the solution. Unfortunately, losses of protein due to adsorption to the membrane increase concomitantly. Hence, a compromise has to be made in the practical case between required dialysis time and optimum protein recovery. The diffusion of solutes is dependent on the temperature and viscosity of the solution. Although a higher temperature increases the rate of diffusion, in most cases the stability of the protein requires dialysis to be conducted at 4-8', normally in a cold room. A protein solution with too high a viscosity (e.g., after PEG precipitation) usually has to be diluted prior to dialysis because the rate of diffusion is drastically reduced with increasing viscosity of the solution.

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Considerable progress has been achieved in the development of dialysis membranes since dialysis techniques were last discussed by McPhie in this series6 Modern dialysis membranes possess a more precise molecular weight cutoff, and show less batch-to-batch variation in terms of average pore size and content of impurities. Membranes are supplied in a variety of sizes in the form of tubing or as membrane sheets for special applications. Molecular weight cutoff ranges from 1000 up to 50,000 allow the selection of the optimal membrane for the individual protein. Dialysis tubing is also available with different forms of pretreatment. Generally, dialysis tubing is purchased in the dry state, but it may also be obtained in a prewashed wet form. For some delicate applications dialysis tubing that has been extensively prewashed and sterilized may be ordered, although its general use is restricted by expense (Spectrum, Los Angeles, CA). Dry dialysis membranes contain significant amounts of sulfurous compounds. Furthermore, they may be contaminated with heavy metals. In some cases, wetting of the membrane and washing with the intended buffer solution may be sufficient as a pretreatment. However, the following procedure to remove impurities is recommended. 1. The entire roll of dry dialysis tubing as supplied (50 ft) is carefully transferred to a 4-liter beaker containing 2 liters of a 100 mM NaHC03, 10 mM Na2EDTA solution adjusted to pH 7.0. 2. The beaker is placed in a shaking water bath, covered, and the temperature is brought to 60". Gentle agitation is continued for 2 hr. 3. The incubation is repeated with fresh solution. 4. The cleaning solution is replaced by 2 liters of double-distilled water and the dialysis tubing is washed for I hr. This step is repeated several times until the solution appears clear. 5. After slowly cooling to 4", the tubing is stored in a fresh volume of double-distilled water including 1 ml chloroform/liter as a preservative. Note: It is recommended that solutions be prewarmed to avoid sudden temperature changes that may cause alteration of the pore size of the membrane.7 Dialysis tubing should be handled with gloves to avoid contamination with proteases. For the simplest method of dialysis, the protein solution is transferred to a length of dialysis tubing with the aid of a small funnel, a pipet, or syringe. The tubing should be sufficiently long to accommodate the volume of the solution, to allow for a possible increase in volume during dialysis, and to permit tight closure at both ends. Leakproof closure of the P. McPhie, this series, Vol. 22, p. 23. 10, p. 870.

' L. C. Craig, this series, Vol.

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dialysis tubing can be achieved by tying knots at both ends of the tubing or, more conveniently, by using plastic clamps (Spectrum, Los Angeles, CA). The filled tubing is then submerged in the dialysis buffer contained in a beaker, Erlenmeyer flask, or most commonly a cylinder. The dialysis buffer is mixed with a magnetic stirrer. Care must be taken to avoid contact of the tubing with the stirring bar, as this can easily damage the tubing. Dialysis can be accelerated by mixing both the protein solution and the dialysis buffer since dissipating the solutes throughout the entire volume prevents the formation of concentration gradients. For this purpose a variety of devices have been d e ~ c r i b e dOne . ~ device, termed a "flat tube microdialyzer" (Hoefer, San Francisco, CA), is commercially available in three different sizes allowing rapid dialysis of volumes from 0.1 to 20 ml. The solution to be dialyzed is contained in dialysis tubing that is flattened out by fastening it to a vertical rod. The arrangement is placed in a cylinder with dialysis buffer, and a built-in magnetic stirring bar produces efficient circulation. An improved solution-to-membrane ratio and the simple but efficient rotating agitation of the sample and dialysis buffer result in more rapid dialysis. Practically, the use of dialysis tubing is limited to volumes of 1-100 ml. For dialysis of smaller volumes (0.01-1 ml) several "microdialyzer" devices are available. They generally consist of two Plexiglas parts separated by a flat sheet of dialysis membrane. A series of vertical, cylindrical wells in the top part accommodates up to 24 samples. The arrangement of corresponding wells in the bottom part is interconnected by narrow channels allowing the flow of dialysis buffer from one cylinder to the other. The device is operated at a flow rate of 1-5 mllmin, thus consuming only limited buffer volume, which can even be recirculated. The continuous flow dialysis provides for maximum difference in concentrations of protein solution and dialysis buffer at all times, resulting in a very fast and efficient dialysis of small samples. Depending on the volume and composition of the protein sample dialysis can be completed within 2-5 hr. A device of analogous design is commercially available for microdialysis of one sample at a time (Dialysis "halve cells," Spectrum, Los Angeles, CA). As already mentioned, the ratio of membrane area to volume of the protein solution determines the time required to achieve eq~ilibrium.~ Semipermeable membranes can be manufactured in the form of hollow fibers with an inner diameter less than 1 mm. Use of these fibers for dialysis either as a single fiber for very small volumes (10 PI) or most K. K. Stewart, Adu. Protein Chem. 31, 135 (1977).

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commonly as bundles of a large number of fibers provides an enormously increased surface area for dialysis. Dialysis time is very short and the system can also be used for concentration of protein solutions since the inner lumen of the fiber can be evacuated. The structure of the single fiber may be isotropic, meaning that the porosity of the membrane is uniform on the inside as well as the outside of the fiber. For this reason the fibers can be used for dialysis in either direction. The fiber arrangements for dialysislconcentration are available with different molecular weight cutoffs, e.g., 6000 and 9000 (Spectrum, Los Angeles, CA). The large surface area may, on the other hand, lead to adsorption of proteins to the membrane. Dialysis of dilute andlor highly purified proteins should therefore be tested with a small aliquot. The processing of different protein solutions imposes problems of cross-contamination which may be difficult to detect and to resolve. Ultrafiltration

It is difficult to draw a line between dialysis and ultrafiltration techniques since both are based on separation of molecules according to size by employing a semipermeable membrane with a defined range of pore sizes. In this chapter ultrafiltration means the use of force, either pressure (mostly provided by pressurized, inert gas), vacuum, or gravitational to drive solutes through a membrane. Ultrafiltration is a very versatile technique with applications ranging from small-scale laboratory concentration and desalting experiments up to industrial ultrafiltration with capacities of hundreds of liters. Much effort has been applied to the development of suitable membranes for ultrafiltration. The optimal membrane has to meet several requirements. It should be a rigid, stable structure able to withstand the working pressures of the technique. This is achieved in many cases by the anisotropic structure of the membrane. Thus, the membrane consists of a compact, spongelike layer coated with an extremely thin "skin" which is the surface that displays the more or less uniformly sized pores exerting the ultrafiltrative properties. The membrane should have the narrowest, "sharp" molecular weight cutoff possible to allow rapid concentration and desalting without loss of protein. The more precisely the molecular weight cutoff is defined, the more applicable ultrafiltration is not only for concentration and desalting but for separation of proteins differing in size. However, present membranes still cannot compete with the resolution of other techniques for size separation of proteins. Another important feature of ultrafiltration membranes is their tendency to adsorb proteins. The membranes currently available consist of

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materials differing widely in their chemical composition. Some, like polysulfone-based membranes, provide excellent flow characteristics and enable rapid processing of large sample volumes. Others, made of regenerated cellulose, show the least tendency to adsorb proteins. The process of manufacturing the surface structure and the chemical composition of individual membranes from different suppliers also have a major effect on protein binding. Additionally, proteins differ in their affinity toward membranes. Thus, several different membrane types should be tested in order to select the optimal one in a particular case. Ultrafiltration is used mainly for concentration of protein solutions. As low-molecular-weight solutes and solvent can pass through the membrane, the composition of the solution is not altered during the experiment. This renders the method one of the most gentle means for the concentration of proteins. The concentrated protein may also be rediluted with a different buffer solution allowing the exchange of buffer conditions or desalting of the protein. The main problem encountered in ultrafiltration is the concentration of proteins in the vicinity of the membrane which may cause decrease flow rates or even lead to solvent flux ceasing entirely. The effect has been termed "concentration polarization," and it is generally more likely to occur as protein solutions become more concentrated in the course of an ultrafiltration e ~ p e r i m e n t .The ~ easiest way to reduce the effect is by constant mixing or stirring of the protein solution by a magnetic stirring device in close proximity to the membrane. These "stirred ultrafiltration cells" can be obtained from several manufacturers (Amicon, Danvers, MA; Spectrum, Los Angeles, CA). In most cases, these are reusable systems, the ultrafiltration membrane being the only disposable part, although entire cells may also be obtained as single-use items to prevent cross-contamination of protein samples (Pharmacia, Uppsala, Sweden). A wide range of membranes are offered with molecular weight cutoffs from 500 to loh. Particularly useful for the processing of small samples are newly developed devices termed Centricon and Centriprep (Amicon, Danvers, MA). They allow ultrafiltrative concentration and desalting of samples from 0.025 to 2 ml and 0.5 to 15 ml, respectively. Both systems consist of small inserts for preparative centrifuge rotors, and use centrifugation to achieve solvent flux through the membrane. They come with two different molecular weight cutoffs (10 K and 30 K). The geometry of the Centriprep has the additional benefit that the direction of solvent flow and centrifugal W. F. Blatt, A. Dravid, A. S. Michaels, and L. Nelson, in "Membrane Science and Technology" (J. E. Flinn, ed.). Plenum, New York, 1970.

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force are opposite, which reduces membrane clogging. Thus, macromolecules, particles, and aggregates are driven away from the membrane by centrifugation rather than concentrated on the membrane surface. Lyophilization The concentration of protein solutions by lyophilization is the drying of materials in the frozen state with the solvent being removed by sublimation.I0The technique has a high capacity and is very easy to perform. The system required for lyophilization (lyophilizer) consists of a sample manifold with standard taper joints, a cooled condensation trap, and a high-capacity vacuum pump. The protein solution is frozen in a suitable container able to withstand the external pressure, the stress of freezing, and with little tendency to adsorb protein. Most commonly used are 25- to 1000-ml round-bottom flasks. To enable rapid removal of the solvent and to prevent additional stress to the container due to expansion during freezing the container should be filled to only one-quarter of its volume. Additionally, a high surface-to-volume ratio is helpful to quickly freeze the liquid. In most cases a mixture of dry ice and organic solvent is used to freeze the samples. Rapid freezing is important for the stability of the proteins to be lyophilized since slow freezing may result in "freezing out" of solution components, leading to concentration gradients and substantial shifts of pH" that can lead to denaturation of the proteins. A device for the concentration of relatively small sample volumes is the Speed Vac concentrator (Savant, Farmingdale, NY) which is of analogous design to the lyophilizer described above. A low-speed centrifuge is used to keep the protein solution at the bottom of the container at all times. Therefore, the samples generally need not be frozen to avoid foaming, boiling, or squirting. Concentration proceeds by connecting the centrifuge to a vacuum system and its rate can be accelerated by a thermostatted heating system which maintains the rotor chamber at 45". Aqueous solutions can be concentrated at approximately 1 mlI100 min. A variety of rotors are available accommodating sample volumes of a few microliters to 50 ml in each tube. However, care is recommended in the use of this device, especially when the heater is on since the physical state of the sample is not easily controlled. It may be frozen, partly frozen (the surface of the sample tends to form a frozen plug), or even in the liquid state, depending on factors like sample composition, heat dissipation, and quality of vacuum. Therefore, requirements for lyophilization may not be J. Everse and F. E. Stolzenbach, this series, Vol. 22, p. 33. 0.P. Chilson, L. A. Costello, and N . 0 . Kaplan, Biochemistry 4, 271 (1965).

lo I'

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fulfilled at all times, and simple evaporation of the solvent may occur which can be detrimental to many labile proteins. Precipitation Methods

A detailed discussion of precipitation techniques is presented in Section VI of this volume. Some methods for the preparation of small amounts of proteins for analytical techniques are presented here. SDSPAGE, for example, requires concentrated and relatively salt-free protein samples. On the other hand, detection methods like silver staining and autoradiography allow analysis of nanogram amounts of proteins by SDS-PAGE (2-10 ng). Thus, precipitation with organic solvents or trichloroacetic acid (TCA) is useful for preparation of small protein samples for electrophoresis. Although these methods can cause denaturation of proteins, samples can be used for SDS-PAGE, which is a denaturing technique itself. These methods provide an efficient and rapid way to concentrate and desalt large numbers of protein solutions. In addition, samples containing substances which interfere with protein assays can be concentrated and the contaminating species removed using this technique. Ethanol and acetone have been used most widely as precipitating agents. Cold acetone (-20") is added to the protein solution to 50% concentration.12 The protein sample is mixed, centrifuged, and the protein sediment obtained is redissolved, precipitated again, and washed once with 50% acetone. Ethanol is used similarly at a final concentration of 66% (vlv). Better recoveries with small amounts of proteins are obtained employing a chloroform/methanol/water system.I3 The protein in solution is precipitated by the subsequent addition of 4 vol of methanol and 1 vol of chloroform. To the sample 3 vol of water is added for phase separation. After centrifugation the protein is present at the interface. The upper phase is removed and three more volumes of methanol are added. Centrifugation yields a solid sediment which is air dried after removal of the supernatant. The method was shown to be applicable for protein amounts of 40-120 mg with recoveries close to 100%. Smaller amounts may lead to significant losses since removal of the upper phase is difficult to achieve without disrupting the interface protein layer. The method is not affected by the presence of detergents, lipids, salts, buffers, or 2-mercaptoethanol. Trichloroacetic acid in concentrations of 5-10% is also used to precipitate proteins.14 Bensadoun and Weinstein introduced sodium deoxychol2

R. L. Hudgin, W. E. Pricer, Jr., and G. Ashwell, J . Biol. Chem. 249, 5536 (1974).

l4

T . P. Bennet, Nature (London) 213, 1131 (1967).

'' D . Wessel and U. I. Fliigge, Anal. Biochem. 138, 141 (1984).

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late as an additive for increasing the yields of precipitation.15 Protein samples are treated with 125 mg/ml sodium deoxycholate and subsequently precipitated with 6% TCA with recoveries approaching 100%. The procedure works satisfactorily for preparation of protein samples for protein determination. SDS-PAGE, however, is very sensitive to pH differences of the sample. Most of the TCA entrapped in the pellet and bound to the proteins can be removed by washing the pellet with 80% aqueous acetone or equivalent organic solvent mixtures, although TCA bound to charged amino groups of the protein (e.g., &-amino group of lysine) is difficult to extract. The strongly acidic pH during precipitation can also cause partial hydrolysis of the protein. The technique was reexamined and improved with respect to the assay of proteins in the presence of interfering substances by Peterson.16 Gel Filtration

In those areas of research where biological or enzymatic activity of proteins is essential, gel filtration is a useful method for desalting protein solutions. Gel filtration media exhibit little protein binding and give high recoveries of even small amounts of proteins. They are compatible with nearly all solvent and buffer mixtures used for chromatography, including buffers containing detergents or denaturing agents. Their improved chemical and physical stability allows reproducible and rapid desalting of labile proteins. Probably the most commonly used gel filtration support for desalting purposes is Sephadex G-25, available in particle grades from coarse to superfine. Macromolecules >5 kD are excluded from the gel pores, and can therefore be effectively desalted. BioGel P-30 is similar in its properties. Sephadex media consist of a polymeric carbohydrate matrix in contrast to the polyacrylamide-based BioGel supports. Therefore, Sephadex normally provides more gentle conditions for the manipulation of proteins than BioGel. The latter, on the other hand, can be manufactured with more precise exclusion limits, which is important for separation of proteins. A suitably sized column should be chosen for a particular experiment in order to reduce adsorptive losses of protein. Optimal removal of salt is achieved with sample volumes not exceeding 20-25% of the column volume. The column is equilibrated with the buffer to which the protein is to be transferred and the sample applied. Choice of the equilibration buffer should be done according to the stability and specific requirements of the protein. Generally, buffers should not be of too low ionic strength l5 l6

A. Bensadoun and D. Weinstein, Anal. Biochem. 70, 241 (1976). G. L. Peterson, Anal. Biochem. 83, 346 (1977).

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[71

since this often results in proteins adsorbing to surfaces. Care must be taken also that neither the sample nor the equilibration buffer is of too high a viscosity, which could lead to a poorer separation. The protein may be eluted with any buffer since the buffer composition of the protein sample obtained after desalting will be identical to the equilibration buffer. Unfortunately, desalting by gel filtration results in a more dilute protein sample. The minimal dilution factor is about 1.1, but practically 1.5-2.0 is more likely to be obtained. Commercially available, prepacked desalting columns, such as Pharmacia PDlO or Bio-Rad 10 DG columns, provide a fast and convenient means for routine desalting of samples volumes up to 2.5 ml. Reversed-Phase HPLC

Reversed-phase (RP) HPLC is one of the most powerful separation techniques for proteins and peptides, and it has also been applied to desalting and concentration of proteins and peptides.17J8 A simple isocratic HPLC system has been described that rapidly and effectively desalts and concentrates proteins and peptides.19 Short reversed-phase columns (3-4 cm) give excellent yields in a very short time (5-10 min). In many cases the behavior of the protein under study in RP-HPLC may already be known from information gained in previous separations. These data can be helpful in choosing the optimal column dimensions and column support for the concentration and desalting experiment. With dimensions ranging from microbore columns (i.d. < 1 mm) to short, preparative columns (i.d. 16-22 mm), processing of protein samples from a few micrograms to as much as 100 mg is feasible in a single experiment. The volume of protein solution may be as little as a few microliters or even as much as 1000 ml. As separation of proteins is not intended, special emphasis can be put on conditions that give maximum recoveries and have the least denaturing effect on the protein. For example, as opposed to separation methods, relatively large particle supports can be used (10-25 pm) for desalting. Additionally, large pore size supports (300- 1000 A) with short alkyl chain reversed-phase coatings (C4) generally increase recovery of proteins and reduce denaturation. Most silica-based reversed-phase supports are stable in the pH range of 2-7, and recently developed supports based on a polymeric matrix of polystyrene feature extended pH stability from 1 to 13 (PLRP columns, I7 l8 l9

Y. C. Pan. J. Wideman, R. Blacher. M. Chang, and S. Stein, J. Chromatogr. 297, 13 ( 1 984). H.-W. Lahm and S. Stein, J. Chromatogr. 326, 357 (1985). T. Pohl and R. M. Kamp, Anal. Biochem. 160, 388 (1987).

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CONCENTRATION OF PROTEINS AND SOLUTE REMOVAL

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Latek, Heidelberg, FRG). This allows the selection of a buffer system at a pH where the protein is most stable. In cases where a volatile buffer system is applicable the protein can be obtained entirely salt free. Furthermore, a variety of organic modifiers are available and several different organic solvents can be used for elution of the protein. Since proteins are eluted by a pulse of organic solvent mixture rather than by a gradient elution, volumes are very small, usually not exceeding 1 ml. Most of the eluate is organic solvent which can be quickly and easily removed by a stream of nitrogen. Hence, for a large number of proteins and peptides in the molecular weight range from lo3 to lo5 suitable conditions can be obtained for the most effective desalting and concentration procedure. Solute-Absorbing Matrices

Dry, water-attracting matrices have been used for the concentration of protein solutions, based on the principle of dialysis.20 The most commonly employed are polyethylene glycol (6000-20,000), carboxy methyl cellulose, and Sephadex G-100. The methods are applicable for protein solutions of a few milliliters to several hundred milliliters. The sample is contained in dialysis tubing and surrounded by a thick layer of dry matrix. Water and low-molecular-weight solutes can penetrate the dialysis tubing and are absorbed by the matrix, while the protein is retained and concentrated in the dialysis tubing. The procedure is relatively slow, involving several changes of the wet matrix and constant monitoring of the degree of concentration. Complete drying of the protein can easily occur and cause losses. The purity of the absorbing matrix is critical since reactive chemicals and low-molecular-weight impurities will diffuse into the protein solution. An elegant method for the concentration of protein solutions has been suggested by Vartak et a1.21Acrylic acid is polymerized at a concentration of 7% (wIv) to form gel rods of 0.6 x 5 cm. The polymer is washed extensively to remove polymerization catalysts and other impurities, then dried at 60". The dried polymer exhibits a water-absorbing capacity of 170 mllg. A sufficient amount of dry sticks is added to the protein solution and, as the matrix is rehydrated, water and low-molecular-weight solutes are absorbed from the solution, proteins being excluded by the small pores of the gel. The procedure seems to work satisfactorily for proteins ranging from 10K to 450K with recoveries of SO%, provided the pH of the protein solution is adjusted to values higher than the pI of the protein.

" W. F. Blatt, this series, Vol. 22, p. 39. 21

H. G. Vartak, M. V . Rele, M. Rao, and V. V. Deshpande, Anal. Biochem. 133,260 (1983).

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GENERAL METHODS FOR HANDLING PROTEINS AND ENZYMES

[71

Otherwise, binding of proteins to the negatively charged matrix occurs. As no pressure, stirring, or other physical impact on the protein is involved, the method may be regarded as relatively gentle. An analogous method for direct absorption of water and low-molecular-weight solutes makes use of the swelling properties of dry gel filtration supports, particularly Sephadex G-25.22It is especially suited for the concentration of small volumes of protein solution (10-500 pl). The protein sample is placed in a 1.5-ml conical plastic tube (Eppendorf, Hamburg, FRG) prepunctured at the bottom and an amount of dry Sephadex G-25 is added that is sufficient to absorb the desired volume of solution. After 10 min the saturated gel is spin dried in a centrifuge at 200 g for 2 min. The concentrated protein solution is thereby transferred to a second vial serving as a receptacle. Recoveries better than 90% of the initial protein have been reported with concomitant 5-fold concentration of the protein solution. Since it is similar to gel filtration chromatography, the method should have little denaturing effect on the protein. Electrophoretic Methods

Electrophoresis, the migration of charged macromolecules under the influence of an electric field, is used mostly for separation and for elution from gels, but it can also be used for the concentration of proteins. Numerous electrophoretic techniques have been d e ~ c r i b e d ~ and ~ - ~ 'several apparatuses for this purpose are commercially available (BIOTRAP, Schleicher and Schiill, Dassel, FRG; Electro Eluter, Bio-Rad, Richmond, CA; Extraphor, Pharmacia-LKB, Uppsala, Sweden). For the most part they involve the use of dialysis tubing or a similar membrane permeable to the electric current to concentrate proteins in a receptacle of the apparatus. The last of the apparatuses mentioned, interestingly, uses a solution of high ionic strength to concentrate and immobilize the proteins, thus avoiding adsorptive losses due to binding to the membrane. Basically, a V-shaped channel connecting the two electrode reservoirs is partially filled with a high salt solution. The protein, migrating out of the gel slice down the elution channel in a low ionic strength solution, is retained when reaching the highly conducting ion solution that provides the major charge transport in the bend of the channel. Most of the procedures are limited to rather small volumes of protein solution, approximately beA. Saul and M. Don, Anal. Biochem. 38, 451 (1984). D. G. Rhodes and D. A. Yphantis, Anal. Biochem. 116, 379 (1981). 24 W. B. Allington, A. L. Cordry, G. A. McCullough, D. E. Mitchell, and J. W. Nelson, Anal. Biochem. 85, 188 (1978). 25 I. Posner, Anal. Biochem. 70, 187 (1976). 22

23

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tween 1 and 50 ml. For microgram amounts of proteins with known electrophoretic properties, they may provide a useful alternative to other methods of concentration. Other

Centrifugation of very large protein molecules, multienzyme complexes, and subcellular organelles in preparative ultracentrifuges can be used for concentration and desalting of these particles. Almost complete recovery is feasible, and depending on the molecular weight of the particle and the viscosity of the solution desalting and concentration can be achieved in a short time. In the case of subcellular particles such as ribosomes it may be the method of choice26(see also [16] in this volume for further details). Unfortunately, the low molecular weight of most proteins precludes its general use. Affinity chromatography is an elegant method to adsorb proteins selectively to a specifically designed solid phase in a reversible manner. Extraordinarily high concentration factors can be achieved, and recovery of biological activity is most often excellent due to the mild, physiological conditions of the procedure.27The main prerequisite is the presence of an individual biological function or chemical structure on which the binding activity is based. The method can provide a very helpful tool for concentration and desalting of protein solutions. A detailed discussion of affinity chromatography techniques is presented in [30] and [3j] in this volume. Acknowledgments Discussions with B. Wittmann-Liebold, J. Spiess, R. B . Mackin, and R. S . Allen are gratefully acknowledged. 26 27

J. Dijk and J. A. Littlechild, this series, Vol. 59, p. 481 J . W. Goding, J . Zmmunol. Methods 13, 215 (1976).

[81 Maintaining Protein Stability By MURRAY P. DEUTSCHER

Proteins are fragile molecules that often require great care during purification to ensure that they remain intact and fully active. Nowadays, many proteins are also purified in small amounts under denaturing conditions by various gel electrophoretic techniques, so that inactive proteins METHODS IN ENZYMOLOGY, VOL. 182

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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are obtained. But even here it is usually advantageous to maintain the protein in an intact form. In the case of enzymes, and other proteins with assayable biological activities, maintenance of activity is generally of prime importance, both for following the protein during purification and for subsequent studies of function. This chapter will focus on the major points to keep in mind with regard to maintaining the stability of a protein during purification and storage. Various chapters in Sections IV, V, and IX describe in detail stabilization procedures for specific biological systems and specific classes of proteins. Causes of Protein Inactivation

Removal of proteins from the cellular environment subjects them to a variety of conditions and processes that can lead to loss of activity or alteration of structure. These include dilution, change in solution conditions, exposure to degradative enzymes, oxygen, heavy metals, and surfaces, and change in physical condition (e.g., freezing and thawing). Awareness that any one of these situations could affect the protein, and knowledge of precautions that could be taken to minimize their effect, will go a long way toward ensuring a successful purification protocol. If the protein of interest is lost or inactivated during the course of any procedure, determination of the reason for this loss will often suggest a simple solution. Thus, if possible, examine whether the loss of activity is accompanied by loss of the protein or changes in its structure, or whether the protein remains but is now inactive. Distinguishing among these different possibilities might indicate what type of process is behind the problem and, thus, what an appropriate solution might be. General Handling Procedures

Obviously, to maintain the stability of a protein avoid treatments that will denature it. Thus, protein solutions should generally not be stirred vigorously or vortexed since this may lead to oxidation or surface denaturation. Protein solutions should not be exposed to extremes of pH, high temperatures, organic solvents, or any other condition that might promote denaturation. Likewise, if one is storing a protein solution for extended periods of time in an unfrozen state, bacterial and fungal growth can become a problem. In these situations sterile solutions and antibacterial or antifungal agents may be necessary. Finally, it is best to make up all solutions that will come in contact with the protein with glass-distilled water, and to store the water in containers that do not have algal growth.

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Concentration and Solvent Conditions

Extraction of proteins from cells inevitably leads to a change in their environment. Since proteins are generally stable in uiuo, the theoretical goal is to try to reproduce the cellular milieu as closely as possible. This would mean very high protein concentrations, close to neutral pH, moderate ionic strengths, reducing conditions, etc. In practice, some of these conditions are compatible with protein purification, and some are not. As a first approximation, it is generally good practice to keep the protein concentration high (> 1 mglml). This would help to maintain protein complexes, possibly minimize the effects of deleterious contaminants, and provide a general stabilizing environment for the protein of interest. It is relatively easy to maintain high protein concentrations early in a purification scheme, but this becomes more difficult as the protein is purified unless one resorts to concentration procedures after each step. Since these latter procedures often have their own problems, one may have to settle for more dilute solutions unless particular stability problems become obvious. It may be helpful to alternate purification steps between ones that concentrate proteins with ones that dilute them. For example, elution of proteins from a column to which they are bound using a batchwise procedure will tend to concentrate the eluted proteins, whereas gradient elution will tend to dilute them. Columns to which proteins bind will tend to concentrate, whereas gel filtration will dilute. By judicious arrangement of purification steps, one may be able to avoid extensive dilution. The solution conditions are also extremely important. Although it is not possible to describe a universal stabilizing solvent applicable to every protein, the addition of certain components is generally helpful. These include a buffer, usually around neutrality, to avoid unnecessary pH changes. Recent work has suggested that careful attention should be given to the buffer anion since in many cases CI- may be harmful.' EDTA is usually added at about 0.1 mM to chelate heavy metal ions that could affect the protein and promote oxidation. A reducing agent such as 2mercaptoethanol or dithiothreitol is often present to counteract oxidative effects, particularly of cysteine residues. The use of dithiothreitol at about 0.1 to 1 mM is preferred because it does not form mixed disulfides with proteins, as 2-mercaptoethanol does.2 Sufficient reducing agent should be present since it can oxidize relatively rapidly. In some cases salts are also

' S. Leirmo, C. Harrison, D. S. Cayley, R. R. Burgess, and M. T. Record, Jr., Biochemistry 26, 2095 (1987).

' W. W. Cleland, Biochemistry 3, 480 (1964).

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added to maintain a certain ionic strength, but only if they are compatible with the next purification or analytical step. Likewise, glycerol at 10 to 20% often helps to maintain stability and is compatible with most purification steps at these concentrations. On occasion, low levels of a detergent are added to prevent aggregation or the sticking of proteins to surfaces, such as glassware. Finally, it is good practice to include protease inhibitors, particularly at early steps (see below). Stability Trials and Storage Conditions

One of the most important studies that can be performed during the course of a new protein purification is a stability and storage study. What this means is that after every step of the purification procedure the stability and storage properties of the protein of interest should be determined. Although rapid purification of a protein is desirable, the situation will often arise, especially during a new purification, when it becomes necessary to keep a protein for some length of time prior to the next step. For this purpose you will need to know how stable it is under different storage conditions. The simplest way to test this is to take small portions of the protein solution, store them under a variety of conditions (e.g., in ice, frozen, at room temperature, with and without different stabilizing agents), and then assay the activity of the protein after different periods of time. Again, keep in mind what the next step in the purification procedure will be. Some storage conditions may be fine for stability, but not useful for further purification. A case in point is storage at -20" in 50% glycerol (vlv). This is often a useful condition for maintaining stability, but terrible if one plans further purification. Sometimes it may be necessary to use such a procedure (the glycerol could be removed by dialysis), but generally it should be a last resort. A different situation arises when one has completed a purification procedure and wants to store the purified protein for long periods of time. Here the primary concern is long-term stability, and many conditions that might be impractical during the course of purification could be used. These might include addition of high concentrations of glycerol, addition of stabilizing substrates, even addition of an extraneous protein such as serum albumin. The choice of storage condition depends on what is effective for stabilization, and what the purified protein will be used for. If one is primarily interested in studying enzyme activity, the presence of serum albumin may not matter. In contrast, one would not want an extraneous protein present if structural studies are planned. If one is unsure, the best course may be to store portions of the protein under a variety of conditions.

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MAINTAINING PROTEIN STABILITY --------

87

Related to the question of storage is the problem of freezing and thawing solutions of purified proteins. One way to avoid repeated cycles of freezing and thawing is to store the purified protein in small portions and to thaw individual samples once, as needed. Alternatively, the protein may be stored under conditions in which it does not freeze, such as high glycerol. If repeated freezing and thawing is necessary, it is best to use quick freezing and thawing procedures. During freezing, solutes are concentrated and the protein could be exposed to unusually harsh conditions. We routinely quick freeze protein solutions in dry ice-ethanol baths to avoid this problem. Likewise, in thawing protein solutions, this should be done rapidly with gentle mixing in lukewarm water until only a small amount of ice is present; the solution is then placed in ice or kept at room temperature during use. The final thawed solution should be mixed gently, or inverted if in a tube, to ensure even distribution. Proteolysis and Protease Inhibitors

Proteolysis is a major problem for the purification of proteins. It is a particularly insidious problem because in many cases the protein of interest is only partially degraded and retains biological activity. This results in erroneous conclusions about the size and structure of the protein. Proteolysis can be a problem at any stage of a purification procedure. Although total proteolytic activity is generally greatest in the initial crude extract since purification will tend to eliminate these contaminating activities, there are also more proteins present that could act to protect the protein of interest. As purification proceeds, even a small contamination with a protease could have a large effect because a larger fraction of the available protein substrate will be the one with which you are working. How can you tell if proteolysis is a problem in your particular situation? The simplest test is to incubate the extract or partially purified protein at a moderate temperature (e.g., 30'7, withdrawing portions at intervals, and assaying for biological activity. Although this method is not foolproof because there may be other reasons for loss of activity, most proteins will not be heat inactivated under these conditions. If activity is lost, the addition of protease inhibitors is recommended since even if proteins are kept at 0-4" throughout purification, some cleavages will occur unless the proteases are inactivated. Cells contain a variety of different types of proteases. Fortunately, a number of protease inhibitors are available that can act on the various proteases. A list of some of the more commonly used inhibitors is presented in Table I. Protease inhibitors useful for particular systems or situations are described in other chapters of this volume. Probably the

88

[81

GENERAL METHODS FOR HANDLING PROTEINS AND ENZYMES

TABLE I COMMON PROTEASE INHIBITORS -

Protease inhibitor

Protease class inhibited

Concentration used

FMSF (phenylmethylsulfonyl fluoride) EDTA and EGTA Benzamidine Pepstatin A Leupeptin Aprotinin Antipain

Serine proteases Metalloproteases Serine proteases Acid proteases Thiol proteases Serine proteases Thiol proteases

0.1-1 mM 0.1-1 mM -1 mM 1 pglml 1 pglml -5 pglml lpglml

-

best approach in working with a new protein is to use a mixture of inhibitors that affect different classes of proteases. Once conditions for maintaining the protein of interest in a stable form are obtained, inhibitors can be removed one by one to determine which are really necessary. Some trial and error will be involved, as well as for deciding which inhibitors, if any, are needed as the purification proceeds. Note that protease inhibitors can be toxic, unstable under certain conditions, etc. They should not be used without first learning their properties. Loss of Activity

The most commonly heard lament during a protein purification is, "I've lost my activity." When this happens a careful analysis of the situation is required to determine the cause. Most importantly, one should have a careful accounting of enzyme units to evaluate the extent of the activity loss. For many purification steps, percentage losses of as much as 50% are not unusual, but of course, these vary with each individual protein. Generally, purification methods that involve binding of a protein to a matrix, and which may require conformational changes during binding, have a greater effect on activity than a procedure such as gel filtration. If activity is totally lost during a particular purification step, other possibilities need to be considered. In some cases proteins may bind very tightly to columns, and require more extreme procedures for elution. Depending on the type of chromatography (see Section VII of this volume), this may require increased ionic strength, use of a chaotropic salt (e.g., KBr), or inclusion of detergent or ethylene glycol in the elution buffer. A second possibility is that more than one component is required for

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the activity of the protein, and these components are separated during the fractionation step. Thus, either component by itself would be inactive, and all have to be present to observe activity. To test for this possibility, all the fractions from the previous step are mixed back together, and activity measured. In some cases it may be necessary to concentrate the mixture back to the original volume in order to observe activity. If mixing of all the fractions results in the appearance of activity, one could then test fractions or regions in a painvise fashion. Often it is found that a little activity remains and that the second component is needed for optimal activity. In this case, one of the required fractions is already known, and the other fractions can then be tested for their stimulating activity. Sometimes activity may be lost between purification steps, such as during dialysis or concentration, or even during storage. In the former situations one should again test for removal of a possible required component. The possibility also exists that the protein has stuck to the dialysis tubing or the concentration membrane. Here, washing the tubing or membrane with buffer containing some detergent may be helpful. Problems of stability during storage have been discussed above. The most frustrating situation is if none of the above possibilities is the cause of the loss of activity. Under these circumstances the most likely explanation is actual inactivation of the protein due to denaturation, proteolysis, etc. If an independent measure for the protein is available (e.g., a Western blot), this can be shown directly. If not, an answer to the inactivation problem may require trial and error experiments to test various conditions. Sometimes, the best solution is simply to avoid that particular purification step.

Section I11 Optimization of Starting Materials

[91 Overproduction of Proteins in Escherichia coli: Vectors, Hosts, and Strategies

The purification of a protein is greatly accelerated if it can be isolated from cells that overproduce it. Our ability to overexpress genes in bacteria has improved enormously during the past few years. The rigor of our understanding of the various aspects of gene expression in Escherichia coli has allowed geneticists not only to engineer increasingly sophisticated expression vectors for maximizing gene expression, but also to create a growing number of host strains for minimizing turnover of gene products. As a consequence, today's molecular biologist is able to achieve a very high-level synthesis of almost any gene product by utilizing and, when necessary, redesigning a minimum number of vectors and host bacteria. This chapter, aimed at beginners of gene expression technology, outlines the general approaches involved and describes selected vectors and strains currently available for high-level expression of cloned genes. It is written with the assumption that the gene of interest has already been cloned and that the investigator is familiar with the standard techniques of gene cloning.' A more detailed account of maximizing gene expression and its practical aspects has been compiled in a separate volume of this series. l a To maximize expression, the cloned gene must be transcribed and translated most efficiently. Moreover, both the mRNA and the protein product must be stabilized as much as possible. Since there is no universal solution applicable to all systems and needs, an investigator will have to deal with each of these steps separately, depending on each particular situation. A comprehensive review of the literature on major aspects of gene expression in E. coli has been compiled by Reznikoff and Gold.2 Optimizing Transcription

Genetic Fusion to Strong Promoters

The level of transcription of a gene depends mostly on the frequency with which the single RNA polymerase initiates tran~cription.~ This in I la

'

S. L. Shelby and A. R. Kimmel, eds., this series, Vol. 152. D. Goeddel. ed., this series, in preparation. W. Reznikoff and L. Gold eds., "Maximizing Gene Expression." Butterworth, Boston, Massachusetts, 1986. W. R. McClure, Annu. Rev. Biochem. 54, 171 (1985).

METHODS IN ENZYMOLOGY, VOL. 182

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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OPTIMIZATION OF STARTING MATERIALS

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turn is a function of the promoter sequence4" that is necessary and sufficient to allow transcription by RNA polymerase. A standard E. coli promoter is an approximately 40 base pair (bp) region having two consensus sequence elements centered around positions - 10 and -35 from the transcription start site.5 The most convenient way to maximize transcription of a cloned gene is to place it downstream from a strong promoter. Numerous plasmid vectors that provide a promoter for transcription of cloned, promoter-less genes have been described; some of these are listed in Table With one exception (pMOB45), each of these vectors listed contains one of several strong, controllable promoters, namely pL, pT7, ptac, and plac. Regulatable promoters are particularly useful if the overexpressed protein is deleterious to the host cell. pL Vectors. These plasmids contain the leftward promoter, pL, of phage lambda (A) which is one of the most active promoters in v i ~ oAn .~~ adjacent operator sequence, oL1, present in these plasmids allows repression of pL with A repressor, the product of the X c I gene.25The repressor protein can be provided by a single-copy, autogenously regulated cI gene present in appropriate bacterial host^.^^'^ Some of the widely used strains (for example, N4830) contain a temperature-sensitive allele of cI, namely, J. Scaife and J. Beckwith. Cold Spring Harbor Symp. Quant. Biol. 31, 403 (1967). P. Youderian, S. Bouvier, and M. M. Susskind, Cell 30, 843 (1982). D. K . Hawley and W. R. McClure, Nucleic Acids Res. 11, 2237 (1983). ' H. Shimatake and M. Rosenberg, Nature (London) 292, 128 (1981). N. Rao and S. Rogers, Gene 3, 247 (1978). A. Levine, P. L. Moreu, S. G. Sedgwick, R. Devoret, M. Gottesman, S. Adhya, and A. Das, Mutat. Res. 50, 29 (1978). lo M. E. Gottesman, S. Adhya, and A. Das, J. Mol. Biol. 140, 57 (1980). l 1 J. Mott, R. Grant, Y. S. Ho, and T. Platt, Proc. Natl. Acad. Sci. U.S.A. 82, 88 (1985). I Z A. Das, B. Ghosh, S. Barik, and K. Wolska, Proc. Natl. Acad. Sci. U.S.A. 82, 4070 (1985). l 3 A. Abeles, J . Biol. Chem. 261, 3548 (1986). l4 W. Whalen, B. Ghosh, and A. Das, Proc. Natl. Acad. Sci. U.S.A. 85, 2494 (1988). l5 S. Tabor and C. C. Richardson, Proc. Natl. Acad. Sci. U.S.A. 82, 1074 (1985). l6 F. W. Studier and B. A. Moffatt, J. Mol. Biol. 189, 113 (1986). " A. H. Rosenberg, B. N. Lade, D. Chui, S. Lin, J. J. Dunn, and F. W. Studier, Gene 56, 125 (1987). F. W. Studier, A. H. Rosenberg, and J. J. Dunn, this series, submitted. l9 C. Yanisch-Peron, J. Vieira, and J. Messing, Gene 33, 103 (1985). H. de Boer, L. J. Comstock, and M. Vasser, Proc. Natl. Acad. Sci. U.S.A. 80,21 (1983). 21 M. Bittner and D. Vapnek, Gene 15, 319 (1981). * B. E. Uhlin, S. Molin, P. Gustafsson, and K. Nordstrom, Gene 6, 91 (1979). 23 B. Ghosh and A. Das, Proc. Natl. Acad. Sci. U.S.A. 81, 6305 (1984). 24 U. Deuschle, W. Kammerer, R. Gentz, and H. Bujard, EMBO J . 5, 2987 (1986). 25 M. Ptashne, A. Jeffrey, A. D. Johnson, R. Maurer, B. J. Meyer, C. 0 . Pabo, T. M. Roberts, and R. T. Sauer, Cell 19, 1 (1980).

TABLE I FOR CONTROLLED, HIGH-LEVEL TRANSCRIPTION OF CLONED GENES SELECTED VECTORS

Plasmid

Repressor origin

Cloning sites

Host

A. Vectors with phage lambda pL promoter controlled by c l repressor pKC30 ColE1 HpaI, BamHI, SalI N4830 (cl,,) or SA1512 (cl+) pRK16F ColE1 EcoRI, BamHI N4830 (cl,,) or SA1512 (cl+) pWW12 ColE 1 EcoRI, Sacl, KpnI, N4830 (cl,,) or SmaI, SalI, HindIII. SAI512 (cl+) BglII B. Vectors with phage T7 promoter controlled by T7 RNA polymerase PT7-1, 2 ColE1 HindIII, SalI, XbaI, HMS273 BamHI, SrnaI, SacI, (pGPI-2) EcoRI pETI-7 ColE 1 BamHI (EcoRI, S ~ U I ) ~BL21 (DE3) C. Vectors with E. coli lac promoter controlled by lac repressor pUC18, 19 ColEIc EcoRI, SacI, KpnI, JMlOl ( 1 a ~ i Q ) ~ BamHI, XbaI, SalI, (AccI. HincII), PstI, SphI, HindIII pDL I9 ColEIc BglII in between JMlOl ( 1 a ~ i Q ) ~ HindIII and SphI of pUC19' D. Vectors with synthetic tac promoter controlled by lac repressor ptacl ColE1 EcoRI JMlOl ( 1 a ~ i Q ) ~ ColE 1 HindIII, XbaI, EcoRI JMlOl ( l a d ) d ptacII E. Vectors with "run-away'' replicon pMOB45 RI (cop,,) EcoRI, BamHI, Hind, DB1443 (hsdR) Cla pMOB48 RI (cop,,) BamHI JMlOl (lacia))

Selection

Ref.

AmpR

7-1 1

AmpR

12-14

AmpR

AmpR

15

AmpR

16-18

AmpR

19

AmpR

AmpR AmpR CmR/TetR CmR

20 20 21-23 21

" Plasmid pWWI2 is a derivative of pWW10,14constructed by W. Whalen (University of Connecticut, Farmington). Downstream of the BglII site, this plasmid contains the Rho-independent terminator TIT2 of the E. coli rrnB operon, followed by the E. coli galK cistron. This plasmid contains an intact nutL site between the pL promoter and the EcoRI site. EcoRI is unique to PET-5, and StuI is present in PET-6 and PET-7.18Cloning at the Stul site will require the growth of vector in dcm mutant hosts, such as BL2l.I8 ' pUC plasmids bear a mutation enhancing plasmid copy number (200-500 copies/cell). An effective down regulation will be achieved by cotransformation with pACYC177-laciQ (see text). pDL19 is a derivative of pCU19,I9 constructed by D. Lazinski (University of Connecticut, Farmington). Its polylinker regionI9 has the following sequence: S'AAGCTTGGAAGATC'ITCCGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTC;the bold sequence represents the 12-mer oligonucleotide inserted in pUC19, generating BglII and XmnI sites. There is one other XmnI site present within the bla gene of the plasmid.

96

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OPTIMIZATION OF STARTING MATERIALS -

-

-

~18.57.Induction of transcription from pL is achieved by shifting the growth temperature from 30 to 42O.I0 Alternatively, if low-temperature growth is desirable (see Ref. 1I), strains with the wild-type cI allele have to be used. Induction of pL in these strains (for example, SA1512) is achieved by treating cultures with SOS-inducing agents, such as nalidixic which mediate the activation of a highly specific protease encoded by the recA gene; the activated RecA protease cleaves, and thereby inactivates, A r e p r e s s ~ r . ~ ~ pT7 Vectors. These plasmids carry the promoter of phage T7 gene 10, which allows selective high-level transcription of cloned genes.15-Is The T7 RNA polymerase specifically recognizes a 23-bp region conserved by T7 late promoters such as that of gene 10.27-2ySelective transcription of cloned genes is carried out in host strains expressing the T7 RNA polymerase. Expression of T7 polymerase in E. coli is lethal if the cell contains a T7 late promoter, presumably because transcription by T7 polymerase is so efficient that it acts as a sink for ribonucleotides, thereby drastically reducing overall transcription by the host polymerase.15 Two different sources for the T7 RNA polymerase have been described. 1. A compatible plasmid (pGPl), containing T7 gene 1 fused to the pL promoter, and a c18.57 gene to control the expression of T7 polymerase15: When transcription of the cloned gene is desired, shifting growth temperature inactivates c1, and allows synthesis of T7 polymerase. This in turn allows transcription from the gene 10 promoter. 2. A A phage recombinant (DE3) containing T7 gene 1, which is fused to the lac operator-promoter16: A strain lysogenic for this phage will allow transcription of the cloned gene from the gene 10 promoter when IPTG is added to the culture to inactivate lac repressor. Vectors with lac and tac Promoters. These vectors contain either of these two promoters controlled by lac repressor. Of these, a widely used set of plasmids represented by pUC18 and pUC19Iycontains the wild-type lac promoter whose optimal functioning in E. coli is dependent on activation by CAMP and its receptor, CRP. The lac promoter can be controlled in two ways. Strains with a coresident compatible plasmid encoding the lac repressor can be induced by the addition of isopropyl thio-galactoside (IPTG). One such compatible plasmid is pACYC177-laciQ(R. Kolodner, Dana Farber Cancer Center, Boston, personal communication), conJ. W. Little and D. W. Mount, Cell 29, 1 1 (1982). M. Charnberlin, J. McGrath, and L. Waskell, Nature (London) 228, 227 (1970). 28 J. J . Dunn and F. W. Studier, J. Mol. Biol. 166, 477 (1983). 29 B. A. Moffatt, J. J. Dunn, and F. W. Studier, J. Mol. Biol. 173, 265 (1984). 26

*'

structed by inserting a lacia fragment within the KanR plasmid pACYC177.30A second control is possible if cya mutant strains are emp10yed.~'These strains are defective in cAMP synthesis and, therefore, defective in transcription of catabolite-sensitive genes such as lac. Transcription of cloned genes in these strains can be induced by the exogenous addition of cAMP to the growth medium.31 In contrast, the tac promoter is a hybrid of trp and lac promoters; its -35 sequence element is derived from the trp promoter and - 10 element from lac.2oThe tac promoter is not dependent on CAMP-CRP for function. However, the presence of a lac operator sequence adjacent to the tac promoter allows down regulation by the laci gene, and activation of a cloned gene by the addition of IPTG.20 It is important to note that an efficient down regulation of tac will require the use of a lacia gene coresident in the vector plasmid or a compatible, high-copy plasmid containing laci gene such as pACYC177-laciQ described above. Increased Gene Dosage A second effective way to increase the amount of specific mRNA in the cell is to amplify the cloned gene. This generally utilizes the gene's own promoter with the gene on a high-copy plasmid, such as pUC.I9 Another method for amplifying genes, utilizes a "run-away" replicon This plasmid maintains about 15-20 derived from plasmid RI drd-19.21.22 copies per cell when cultures are grown at low temperature, but at 40" and above, the copy number is greatly increased (>loo), causing over a 100fold overexpression of cloned genes.23 A better approach to overexpress proteins by increasing gene dosage is to position multiple copies of a gene in tandem downstream of a strong controllable promoter. Concatemeric DNA inserts can be synthesized by a variety of methods utilizing a number of different vectoi-s.32One of these, utilizing the vector pDL19, is described in Fig. 1. pDL19 is a derivative of pUC19 that contains within the polylinker a BglII site in addition to the BamHI site (Fig. 1). Because BamHI and BglII sites are unique and yet generate the same cohesive ends upon restriction, it is possible to easily engineer tandem multimers of a gene with pDL19 (see Fig. 1) and insert the multimer back into pDL19 or other expression vectors with a BamHI, BglII, or BclI sites. A recA mutant host should be used to maintain the concatemeric insert; however, it is not necessary for expression purposes. 'O

"

A. C. Y. Chang and S. N. Cohen, J. Bacreriol. 134, 1141 (1978). F. Warren and A. Das, Proc. Natl. Acad. Sci. U.S.A. 81, 3612 (1984). B. Cohen and G . G . Carmichael, DNA 5, 339 (1986).

98

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OPTIMIZATION OF STARTING MATERIALS

1

' 1

1

Clone ORF at Hincll

I I

iB c : i G

Isolate Bgl-OW-Barn fragment 5 ' GATC:

a

Ligate, resnict with Bgl and B a n and kolate large concatemer

Ic

W-GGAT CCTAGA

CCTAGA

CCTAGA

FIG. 1 . The construction of concatemeric inserts with plasmid pDL19.

General Method for Subcloning and Assessing Degree of Overexpression

1. Choose one of the vectors with pL, pT7, or lacltac promoters listed in Table I, simply on the basis of the presence of convenient sites of cleavage by restriction enzymes.

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MAXIMIZING GENE EXPRESSION IN

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2. Following standard protocols,33isolate both the insert and the vector DNA upon digestion with appropriate restriction enzymes. If necessary, modify the ends of the insert or the vector by adding an appropriate linker.33Ligate the vector and insert fragment under conditions favoring intermolecular joining.33 3. Transform a suitable host listed in Table I using standard protoc o l ~and , ~ select ~ transformants on the basis of resistance to appropriate drugs listed in Table I using agar plates and growth conditions specifically recommended for the respective host-vector system. Determine the frequencies of transformants produced by the religated vector with that of the vector ligated to the target gene fragment. Similar frequencies would suggest that the cloning experiment has gone normally and that the subclone is most likely not a variant of the wild-type gene. Verify the subclone by fine restriction mapping.33 4. Measure overexpression of the cloned gene product by standard gel electrophoresis of the whole cell extract and staining with Coomassie Blue,33by transferring to a nitrocellulose membrane to perform western blots using an antibody raised against the protein,33or by a biochemical assay of the known activity of the protein. Follow the conditions for culturing and induction of gene expression as have been described in detail for each vector.7~11,15~16,20,31 Optimize these procedures, with special emphasis on empirically determining the best condition for overexpression which can vary from one host-vector system to another, and also from gene to gene. Potential Problem with Toxic Genes and Available Methods for Efficient Repression

A given gene product whether foreign or native can be toxic to the cell when expressed in large quantities. Such a toxic gene must be cloned and maintained in the repressed state until its expression is desired for overproduction of the protein. In order to repress transcription from pL and pT7 promoters most efficiently, special vectors and hosts have been designed. These are described below. A single-copy cZ gene resident in the host chromosome is unable to completely down regulate multiple copies of the pL promoter encoded by a standard high-copy vector. To allow down regulation of pL to a minimum basal level, a simple solution is to cotransform the cell with a compatible high-copy plasmid (derivatives of pACYC 1771184 with p l5A ori33

"Current Protocols in Molecular Biology." Wiley, New York, 1987.

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OPTIMIZATION OF STARTING MATERIALS

[91

gin or pNT52 with RSF 1030 origin) that contains the native cZ gene or c I fused to the lac promoter. One such plasmid, pJL23 (constructed by J. Lodge and T. Roberts, Harvard Medical School, Boston, MA), that contains the cZ857 gene, is a derivative of the KanR plasmid ~ A c Y C 1 8 4 . ~ ~ Another solution to this problem is to utilize pL vectors containing the cZ857 gene.34Recently, Bujard and colleague^^^,^^ have engineered plasmids with a synthetic pL promoter that contains the lac operator sequence in the place of oL1. This plasmid system allows for the repression of pL with the lac repressor, the product of laci gene carried by a compatible plasmid. In this case, transcription from pL is induced by treating the culture with IPTG. If the basal level expression from pL continues to be a problem for a highly toxic gene, transcriptional readthrough from minor plasmid promoters must be prevented by placing a strong terminator upstream of pL. The plasmid pWW12 contains a unique ClaI site upstream of the pL promoter. An AccI fragment encoding A tR' terminator14 may be easily inserted at the ClaI site. The correct orientation of the insert can be readily verified by restriction of the plasmid with XmnI which cuts both bla and tR ' once. It is somewhat difficult to completely down regulate the expression of T7 polymerase. However, the activity of T7 polymerase expressed at a basal level could be inhibited by introducing a compatible plasmid pLysE or pLysS.16J8These CmRplasmids, derivatives of pACYC184, encode T7 . ~ ~ expression lysozyme which somehow inhibits T7 RNA p o l y m e r a ~ eThe of lysozyme itself is not toxic to the ce11.18 When transcription is desired, IPTG is added to fully express T7 polymerase. Presumably, a constant amount of lysozyme produced by the coresident plasmid is not sufficient to inhibit fully induced T7 polymerase, and thereby allows efficient expression of the gene cloned downstream of the gene 10 promoter. A new series of repressible T7 promoter vectors has recently been engineered to allow fine tuning of transcription. In these vectors, the lac operator has been placed just downstream of the gene 10 promoter such that the lac repressor protein can bind to this site and block transcription by T7 RNA polymerase. The laci gene has been appropriately placed within the vector plasmid such that this versatile system is most effectively down regulated for expression of the cloned gene. Simultaneous derepression of the two desired promoters is accomplished by the addition of IPTG; T7 polymerase is made, and in turn, the cloned gene is

35

''

M. C. Schmidt and M. J. Chamberlin, Biochemistry 23, 197 (1984). R. Knaus and H. Bujard, EMBO J . 7, 2919 (1988). H. Bujard, this series, submitted. B . A. Moffatt and F. W. Studier, CeN 49, 221 (1987).

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transcribed. Similar plasmids with lac operator controlling both T7 and T3 promoters have also been constructed by Giordano et Solutions to Potential Problems with Premature Termination and mRNA Instability Vectors Allowing Antitermination. The fusion of a cloned gene to a strong promoter may not always lead to a high-level transcription of the cloned gene. Sometimes the DNA insert may contain an intragenic terminator preventing complete, high-level transcription of the cloned gene. A general solution to such a potential problem is offered by vectors which utilize the A antitermination system to overcome transcription stop signals.1° All of the pL vectors described in Table 1 contain the nutL site required for antitermination by A N gene product.39The trans-acting N protein can be supplied by a coresident compatible plasmid, pGE24 (E. Gradzielska and D. Lazinski, University of Connecticut, Farmington) or in limited quantity, by an N+ A prophage carried by a number of host strains.I0 Additional plasmids which contain a nut site downstream of the lac promoter have been r e p ~ r t e d . ~Also, ' it is possible to clone a nut cassette downstream of any promoter tested to date.31,40 The most significant utility of vectors allowing antitermination is in the simultaneous overexpression of multiple genes. There may be instances where a multisubunit protein is to be overproduced by simultaneous expression of several cloned genes from one plasmid. This would be necessary if the stability of one gene product is dependent on the presence of another in the cell .4l Strategies for Enhancing Message Stability. Message instability could be another reason for not achieving high-level accumulation of mRNA of the target gene fused to a strong promoter. Though our knowledge of the genetic basis of mRNA processing and turnover is very limited at this a few general approaches might be taken to attempt to stabilize the mRNA of a cloned gene. First, alterations of the 5' leader sequence T. J . Giordano, U . Deuschle, H. Bujard, and W. T. McAllister, Gene, in press. J . Salstrom and W. Szybalski, J. Mol. B i d . W, 195 (1978). " B. decrombrugghe, M. Mudrij, R. DiLauro, and M. Gottesman, Cell 18, 1145 (1979). 41 H. A. Nash, C. A. Robertson, E. Flamm, R. A. Weisberg, and H. I. Miller, J. Bacreriol. 169, 4124 (1987). 42 M. Deutscher, Cell 40, 731 (1985). 4' D. Kennell. in "Maximizing Gene Expression" (W. Reznikoff and L. Gold, eds.), p. 101. Butterworth, Boston, Massachusetts, 1986. W. P. Donovan and S. R. Kushner, Proc. Null. Acud. Sci. U.S.A. 83, 120 (1986). 45 V. J. Cannistraro, M. N. Subbarao, and D. Kennell, J. Mol. Biol. 192, 257 (1986). G . Braweman, Cell 48, 5 (1987). j9

"

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OPT~M~ZATION OF STARTING MATERIALS

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could dramatically change the overall secondary structure of a message47,48 and, conceivably, the steady-state level of the message. By simple recombinant DNA methods such as trimming or addition of linker sequences,33the junction between the promoter and the coding region could be altered, and their effects on increasing the level of gene product could be rapidly monitored. In addition, the possible stabilizing effect of ompA-like leader sequence element^^^,^^ could be tested easily by insertion of the sequence in between the promoter and the coding region. Second, the possible effect of growth rate on the turnover of the mRNA5' could be easily monitored. A priori, one would not know which growth rate would be favorable for the stability of a given mRNA. Third, the possible internal cleavage of the mRNA by RNase IIP2J3is easily minimized by employing an rnc host. Hosts with mutations in rnb (encoding RNase 11) and pnp (encoding polynucleotide phosphorylase) might also be employed since mRNA turnover appears to involve these exonuc l e a s e ~ .Since ~ ~ , ~transcription ~ from pL, pT7, and plac or ptac can be controlled with a dual plasmid system as described above, a variety of hosts defective in specific RNases could simply be cotransformed with the expression vector and the controller plasmid, and the effect of these RNase mutations on gene expression, if any, can be readily monitored as described above. Finally, the presence of strong terminators at the end of a cloned gene is expected to greatly increase the message half-life.52,55 The vector pWW12, listed in Table I, contains the terminator cluster TI-T2 derived from a ribosomal RNA ~ p e r o nSimilarly, .~~ the T7 promoter vector pET3 contains a T7 terminator. Also, a strong terminator cassette, such as the Rho ( p ) independent terminator tR' present in pWW10,14may be appropriately inserted into other expression vectors in which the gene may already have been cloned.

47

C. Yanofsky, and R. Kolter, Annu. Rev. Genet. 16, 113 (1982).

" H. Masukata and J. Tomizawa, Cell 44, 125 (1986).

J . G. Belasco, G. Nilsson, A. von Gabain, and S. N . Cohen, Cell 46, 245 (1986). M. Baga, M. Goransson, S. Normak, and B. E. Uhlin, Cell 52, 197 (1988). s1 G. Nilsson, J. G. Belasco, S. N . Cohen, and A. von Gabain, Nature (London) 312, 75 ( 1 984). 52 G. Guarneros, C. Montanez, T. Hernandez, and D. Court, Proc. Natl. Acad. Sci. U.S.A. 79, 238 (1982). 53 U. Schmeissner, K. McKenney, M. Rosenberg, and D. Court, J. Mol. Biol. 176, 39 (1984). 54 C. M. Arraiano, S. Yancey, and S. Kushner, J. Bacteriol. 170, 4625 (1988). 55 H. C. Wong and S . Chang, Proc. Natl. Acad. Sci. U.S.A. 83, 3233 (1986). 56 E. A. Morgan, J. Bacteriol. 168, 1 (1986). 49

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MAXIMIZING GENE EXPRESSION IN

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103

Optimizing Translation of Cloned Gene Sequence Determinants for Translation Initiation

The initiation of mRNA translation in E. coli (see Refs. 57 and 58 for reviews) involves a specific ribosome-binding site just upstream of the translation start codon. The start codon is usually AUG but on occasion, GUG, UUG, and AUU are used. The ribosome-binding site, or the Shine-Dalgarno sequence, is complementary to a sequence within the 3' end of 16s rRNA, ACCUCCU. This complementarity plays a crucial role in bringing the 30s ribosome to the initiator codon. From available sethe ' following quence data and genetic evidence, reviewed by S t ~ r r n o , ~ consensus on the optimum sequence requirements has been reached. 1. Efficient initiation will require an optimal Shine-Dalgarno sequence (at least four nucleotides of the sequence AGGAGGU), positioned about seven to nine nucleotides upstream of the initiator codon. 2. The Shine-Dalgarno sequence should not be sequestered in secondary structures, nor should it overlap with a sequence recognized by an RNA-binding protein.58 3. There is a preferred environment in which the initiator codon should be positioned. First, it should not overlap with another initiator codon. Second, the spacer region between the Shine-Dalgarno sequence and the initiator should be mostly A and U , especially an A at -3. Third, the AUG codon should be followed by GCAU or AAAA. Finally, the presence of the sequence UUAA in the fourth or fifth codon would be helpful.

Obviously, many cloned bacterial genes may not have the most optimal translation initiation region. With oligonucleotide mutagenesis techn i q u e ~ ,it~ is~ possible ,~~ to replace the native initiation region of a gene with a synthetic one which meets all the requirements described above.60 More significantly, the cDNA clones of eukaryotic genes will not have the Shine-Dalgarno sequence. It is for this reason that such DNAs and open reading frames (ORFs) in general are cloned into vectors like Agtl 1, which provide a translation start r e g i ~ n . ~ '

-

:

L1-

-

I

G . Stonno, in "Maximizing Gene Expression" (W. Reznikoff and L. Gold, eds.), p. 195. Butterworth, Boston, Massachusetts, 1986. 58 L. Gold, Annu. Rev. Biochem. 57, 199 (1988). r, S. L. Shelby and A. R. Kirnmel, eds., this series, Vol. 152. 1. M. Chernak and H. 0. Smith, Nucleic Acids Res. 17, 1933 (1990). 61 R. A. Young and R. W. Davis, Proc. Natl. Acad. Sci. U.S.A. 80, 1194 (1983).

57

104

[91

OPTIMIZATION OF STARTING MATERIALS (a)

U O G U i R T A T A C A T A!tG GCT AGC ATG ACT GGT GGA CAG CAA ATG GGT CGC G

(b)

AAGCAWLTATACAT ATG GCT AGC ATG ACT GGT GGA CAG CAA ATG GGT CGG G A l X

k)

B T A T A C A T A X GCT AGC ATG ACT GGT GGA CAG CAA ATG GGT C

G

U

W

-37

BglII bla

-+

T7 Promoter

Translation Start

ori

- - w

T7 Terminator

FIG. 2. The cloning of open reading frames with vectors pET3 a, b, and c. Arrows indicate sites of cleavage by restriction enzymes NdeI and BnmHI. Both of these sites are unique to the vector. Cloning at the BamHI site will produce a fusion protein with 11 extraneous amino acids. The NdeI site should be used for the production of proteins with native amino termini, except that there will be a Met residue at the N-terminus of a cloned eukaryotic protein. The terminator present in the vector will prevent the overexpression of 0-lactamase encoded by bla. The numbers above restriction enzyme sites correspond to the position relative to the start site of transcription by T7 polymerase.

Translation Fusion Vectors

There are several transcription vectors described in Table I which are suitable for use as a translation vector as well. Of these, the plasmids pUC18 and pUC19 are most ideal because they allow cloning of ORFs at a site close to the amino terminus of lacZ, and therefore add only a few extraneous amino acids to the target protein. Derivatives of pKC30 containing the initiator region of A cZI gene have been described, which allow translation fusion of ORFs downstream of the pL promoter.62Recently, a series of T7 vectors have been engineered to simplify translation fusion (Fig. 2). These, in addition to the controllable T7 promoter, contain a strong translation start signal originating from T7 gene 10. There are unique cloning sites at or near the initiator codon so that it is possible to engineer fusions having no extraneous amino acids at the amino terminus. Moreover, this system includes sets of plasmids (for example, pET3a, b, and c ; see Fig. 2) that have unique restriction sites in each of three reading frames to which fusions to ORFs can be engineered by simple in-frame insertion of appropriate DNA fragments. 62

A. R. Shatzman and M. Rosenberg, this series, Vol. 152, p. 661.

191

MAXIMIZING GENE EXPRESSION I N

E.

CO/~

105

Potential Problem with Biased Codon Usage Frequencies with which each of the 61 codons are utilized vary from one organism to another, and also from one gene to another within the same organism (see Ref. 63 for a review). Some codons for a given amino acid are used much more frequently than others.64 There seems to be a correlation between the relative abundance of a tRNA isoacceptor and the frequency with which its cognate codon is utilized in E. ~ o l iThere .~~ also seems to be a correlation between the relative abundance of a protein and its pattern of codon usage.6s49Highly expressed genes have a biased codon usage, i.e., they contain mostly the "preferred" codons. In contrast, poorly expressed genes do not show this bias, and they often contain many "rare" codons. Despite these strong correlations, it is not clear whether poor expression of a cloned gene in E. coli could be attributed to its frequent use of rare codon~.~O However, if there is a remarkably high occurrence of rare codons within a cloned gene, a synthetic version of this gene having preferred codons should probably be engineered in order to achieve maximum expression with strong promoters such as pL or pT7. Enhancing the Stability of Protein Products

There is a remarkable difference in the relative stabilities of proteins in the cell. Some proteins are very short lived, and usually they perform physiological functions required only for a short while. Some of the proteases responsible for protein degradation in E. coli have been described, though not all genes encoding these proteases have been identified yet.71,72 Escherichia coli mutants defective in a number of proteases have been i s ~ l a t e d . These ~ ~ - ~ mutant ~ hosts might help minimize proteolysis of some H. A. deBoer and R. A. Kastelein, in "Maximizing Gene Expression" (W. Reznikoff and L. Gold, eds.), p. 225. Butterworth, Boston, Massachusetts, 1986. " R. Grantham, C. Gautier, M. Gouy, M. Jacobzone, and R. Mercier, Nucleic Acids Res. 9, 243 (1981). 65 T. Ikemura, J . Mol. Biol. 146, 1 (1981). T. Ikemura, J . Mol. Biol. 151, 389 (1981). 67 M. GOUY and C. Gautier, Nucleic Acids Res. 10, 7055 (1982). H. Grosjean and W. Fiers, Gene 18, 199 (1982). 69 J . L. Bennetzen and B. D. Hall, J . Biol. Chem. 257, 3026 (1982). 70 M. Robinson, R. Lilley, S. Little, J. S. Emtage, G. Yarranton, P. Stephens, A. Millican, M. Eaton, and G. Humphreys, Nucleic Acids Res. 12, 6663 (1984). 71 A. Goldberg and S. Goff, in "Maximizing Gene Expression" (W. Reznikoff and L. Gold, eds.), p. 187. Butterworth, Boston, Massachusetts, 1986. 72 S. Gottesman, this series, submitted. 73 S. Gottesman and D. Zipser, J . Bacreriol. 1l3, 844 (1978). 63

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OPTIMIZATION OF STARTING MATERIALS

[gl

short-lived proteins. However, since the number of these mutants isolated to date is limited, and since some of these proteases might be very specific for a particular substrate,78it is probably unlikely that they will serve to stabilize every short-lived protein. Use of Protease-Defective Hosts lon and clp Mutants. The lon gene encodes one of the major proteases involved in ATP-dependent degradation of short-lived proteins, nonsense fragments, and missense mutant polypeptides including ts proteins.73 Both deletion and insertion mutations in lon have been isolated.72Strains carrying these mutants have been found to greatly increase the yields of many cloned gene products. There is residual energy-dependent proteolysis in the lon mutant cells, and mutations in a second ATP-dependent protease, clp, may allow further stabilization of the cloned gene product .72,74 htpR Mutant. Proteolysis can be a major problem at high temperature. A number of proteases, in addition to Lon, are thought to be induced in response to heat Induction of these proteases could be prevented by a mutation in htpR, the gene encoding a sigma (a)factor, sigma-32, required for expression of heat shock genes.76 The htpR sigma factor is apparently an essential cellular function. Thus, the viability of a strain bearing a nonsense (amber) allele of htpR is dependent on a coresident amber suppressor. When a temperature-sensitive suppressor allele (sup,,) is used, the level of suppression of htpR,, is sufficiently low even at lowgrowth temperature. Hence, the level of htpR-controlled proteases is greatly minimized in the mutant grown at low temperature. Strains carrying both lon and htpR mutations are available.72 The double-mutant strains appear to further minimize the degradation of foreign proteins, and should be generally applied for overexpression. degP Mutant. Sometimes the intracellular location of a protein can affect its stability. Talmadge and Gilbert79have shown that a variety of insulin antigens are at least 10 times more stable when they are localized --

Y. Katayama, S. Gottesman, J. Pumphrey, S. Rudikoff, W. P. Clark, and M. R . Maurizi, J . Biol. Chem. 263, 15228 (1988). 75 T. Baker, A. Grossman, and C. Gross, Proc. Natl. Acad. Sci. U.S.A. 81,6779 (1984). 76 F. C. Neidhardt and R. A. VanBogelen, in "Escherichia coli and Salmonella typhimurium" ( F . C. Neidhardt et al., eds.), p. 1334, Am. Soc. Microbiol., Washington, D.C., 1987. " K. Strauch and J. Beckwith, Proc. Natl. Acad. Sci. U.S.A. 85, 1576 (1988). M. A. Hoyt, D. M. Knight, A. Das, H. I. Miller, and H. Echols, Cell 31, 565 (1982). 79 K. Talmadge and W. Gilbert, Proc. Narl. Acad. Sci. U.S.A. 79, 1830 (1982). 74

within the periplasmic compartment. It is not clear whether the differential stability of a protein in the periplasmic and cytoplasmic environment is due to a difference in the conformation that the protein might attain in the two environments. However, a group of eight soluble protease activities is known to be differentially distributed in the two subcellular compartments of E. ~ o l i . ~Thus, O it is likely that a protein would be exposed to a different set of proteases when it is present in the cytoplasm compared to when it is secreted in the periplasm. Strauch and B e ~ k w i t hhave ~~ recently described a mutant, degP, which is defective in a periplasmic protease. The protease encoded by degP cleaves and releases the alkaline phosphatase portion of fusion proteins. If this protease is involved in degrading periplasmic proteins, the degP mutant might be useful in stabilizing exported proteins, in general. Method for Testing Protease-Defective Mutant

To test if any of the protease-defective mutants described above help stabilize a cloned gene product, one does not need to reconstruct new host strains. The dual plasmid systems described for each promoter vector can be used to cotransform an existing protease-defective strain, and the potential effect on the stability of the cloned gene product can be readily determined. Stabilization by Specijic Zntracellular Localization or Secretion Localization Vectors. As mentioned above, a protein which is labile in the cytoplasmic conipartment might be comparatively more stable when localized within the periplasm or secreted into the medium. To allow localization of a cloned gene product to extracytoplasmic compartments, "secretion cloning" vectors such as PIN-111-ompA (Fig. 3) have been c o n s t r u ~ t e d . This ~ ' ~ ~vector is most suitable for the expression of proteins, for example, hydrolytic enzymes, that are toxic to the cell when present in the cytoplasm. The plasmid PIN-111-ompA contains a strong, hybrid lpp-lac promoter and the lac operator. Therefore, high-level expression of a cloned, toxic gene can be induced by inactivating lac repressor. The plasmid contains the coding sequence of ompA signal peptide; a DNA fragment encoding the desired protein is fused in frame immediately after the signal peptide. The resultant hybrid (for example, the one made K. H. S. Swami and A. Goldberg, Nature (London) 292, 652 (1981). M . Takahara, H. Sagai, S. Inouye, and M. Inouye, BiolTechnology 6, 195 (1988). 82 C. A. Lunn, M. Takahara, and M. Inouye, this series, Vol. 125, 138. M. Takahara, D. W. Hilber, P. J . Barr, J. A. Gerlt, and M. Inouye, J. Biol. Chem. 260, 2670 (1985).

108

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OPTIMIZATION OF STARTING MATERIALS

Signal Peptide

t

t

A1

TCC AAG CTT GGA TCC GTA GCG CAG GC

GCT GM

TTC CAA GCT TGG ATC

GTA GCG CAG G C GGA ~ ATT CCA AGC TTG GAT

ompA Hybrid lpp-lac promoter-operator

c

cc

A2 A3

laci

I I

Cloning Sites EcoRI, Hidm, BamHI

Cleavage of signal peptide and localization in periplasm

Target Protein FIG. 3. The periplasmic localization of cloned gene product by fusion to the ompA signal sequence. Arrows indicate the fusion sites using EcoRI for cloning. Fusions using Hind111 and BnmHI can be made as well. Al-A3 represent derivatives of PIN-111-ompA with the cloning sites in three different reading frames.

with staphylococcal nuclease A) is overproduced to as much as 40% of total cell protein, and an accurately processed protein (i.e., one with the native amino terminus) is localized into the periplasm to as much as 50%.81 Secretion of Cloned Gene Product. The overexpression and isolation of proteins might be simplified if they were secreted into the medium. Both gram-positive and gram-negative bacteria are known to secrete

some native proteins into the medium (for example, a-amylase by Bacil. ~ ~ there is a wealth of information lus and hemolysin by E. ~ o l i )Though available on the pathways for protein export or localization in E. coli, the genetic mechanism of protein secretion is as yet poorly u n d e r s t o ~ d . ~ ~ , ~ ~ To date, there have been few reports of success in the secretion of cloned, chimeric gene products,8M8and the vectors suitable for protein secretion are only beginning to emerge (see [l I ] in this volume). Use of lky Mutants for Protein Secretion. One potential general approach to protein secretion might be to introduce into E. coli lky mutantsg9 a gene cloned into a periplasmic localization vector. The "leaky" mutants of E. coli and Salmonella typhimurium are known to secrete (or dump into the medium, rather) periplasmic proteins such as alkaline p h o ~ p h a t a s e . ~ ~ Thus, the Iky mutant might secrete any cloned, chimeric protein whose gene has been fused to a periplasmic localization signal such as the one encoded by the PIN-111-ompA vector. Stabilization by Fusion to P-Galactosidase P-Galactosidase Fusion Vectors. A labile protein might be converted ~ ~ ~plasmid ~' to a stable hybrid protein upon fusion to P - g a l a c t o ~ i d a s e . The system pMLB110718 constructed by Berman and colleagues (Molecular Oncology, Inc., Gaithersburg, MD) is ideal for the fusion of an open These plasmids contain the wild-type reading frame to P-galactosida~e.~~ lac promoter-operator region, and a lacZ gene containing the multiple cloning sites of pUC8 and pUC9 at the amino terminus. Using one (or two) of these cloning sites, a DNA fragment encoding an open reading frame may be easily inserted in frame with lacZ. A DNA fragment without the translation start and stop signals will be fused to the lacZ AUG codon producing a chimeric protein whose amino terminus and carboxy terminus are both derived from lacZ. Alternatively, if the native riboI. B. Holland, N . Mackman, and J.-M. Nicaud, BiolTechnology 4, 427 (1986). J. A. Stader and T. J. Silhavy, this series, submitted. K. Nagahari, S. Kanaya, K. Munakata, Y. Aoyagi, and S. Mizushima, EMBO J . 4,3589 (1985). N. Mackman, K. Baker, L. Gray, R. Haigh, J.-M. Nicaud. and I. B . Holland, EMBO J . 6, 2835 (1987). M. Better, C. P. Chang, R. R. Robinson, and A. R. Honvitz, Science 240, 1041 (1988). 89 J.-C. Lazzaroni and R. C. Portaliar, J. Bacteriol. 145, 1351 (1981). 90 H. Shuman, T. Silhavy, and J. Beckwith, J . Biol. Chem. 225, 168 (1980). 91 J. Germino, J. Gray, H. Charbonneau, T. Vanaman, and D. Bastia, Proc. Natl. Acad. Sci. U.S.A. 80, 6848 (1983). 92 T. J . Silhavy, M. L. Berman, and L. W. Enquist, "Experiments with Gene Fusions." Cold Spring Harbor Lab., Cold Spring Harbor, New York, 1984. 84

85

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some-binding site and AUG codon (or a synthetic version) is encoded within the DNA insert, a hybrid p-galactosidase will be produced. Transcription of lac in this plasmid is controlled by lacia encoded by the plasmid itself. Procollagen lac2 Fusion Vector. It is conceivable that the function of a protein may be impaired due to fusion to p-galactosidase, although in some instances the fusion protein is found to retain the biological activities of both moieties of the h ~ b r i d . Toward ~ ~ - ~ ~a solution to this problem, ~ ~ developed a unique fusion vector system, Germino and B a ~ t i ahave namely pJG201 and its subsequent derivatives, which permit site-specific in vitro cleavage to remove the p-galactosidase moiety from the fusion protein. The lacZ gene in these plasmids is already fused to a 60-amino acid peptide derived from chicken pro-a2 collagen (Fig. 4). An open reading frame (without a translation stop signal) placed upstream of the procollagen sequence will lead to the production of a p-galactosidase chimera that can then be digested by collagenase to cleave off the amino terminus, the peptide of interest (Fig. 4). These vectors contain A pR to drive lacZ transcription, and also encode a temperature-sensitive repressor to control the promoter. The hybrid protein is purified by affinity chromatography and subsequently digested with collagenase to cleave the procollagen linker.% To date, there are a variety of vectors developed for the cloning of open reading frames. The procollagen-lacZ fusion vector appears to be most suitable for this purpose because it has the ability to generate a protein that is most similar to its native form. Rapid Purification of p-Galactosidase Fusion Proteins The fusion of a target protein to p-galactosidase provides a convenient method for purification of the fusion protein by affinity chromatography. This is the best approach available for the rapid isolation and purification of a protein whose function is unknown and has never been isolated before. In this approach, the open reading frame encoding the protein is fused in frame to p-galactosidase using one of the fusion vectors described above. To purify the fusion protein, a crude extract of cells producing the protein is first passed through an affinity column able to selectively bind to the 0-galactosidase moiety of the fusion protein, and the ~' bound protein is then eluted with an agent dissociating the c ~ m p l e x . A commercially available affinity matrix for P-galactosidase is p-aminoB. Muller-Hill and J. Kania, Nature (London) 249, 561 (1974). Guarente, G. Lauer, T. M. Roberts, and M. Ptashne, Cell 20, 543 (1980). 95 T . J . Silhavy and J. R. Beckwith, Microbiol. Rev. 49, 398 (1985). % J. Germino and D. Bastia, Proc. Natl. Acad. Sci. U.S.A. 81, 4692 (1984). 93

9" L.

b

~1857

I

a

p~

n ori

pro-collagen

kc2

---------------linker

1

--

Purify by affinity chromatography

Tripartite Protein

1 Target protein

Digest with bacterial collagenase

p -galactosidase

FIG.4. The production of a cleavable, tripartite fusion protein with a procollagen-lacZ vector. Details are described in the text.

phenyl-P-D-thiogalactosidecovalently attached to succinyldiaminohexylSepharose 4B; 0.1 M sodium borate (pH 10) is used to elute the fusion protein selectively retained on the column. Practice and Prospects

The appropriate choice of a vector-host system for the overproduction of a given protein in E. coli will primarily depend on the nature of the cloned gene. For a harmless, stable protein whose mRNA is stable and does not contain many rare codons, any one of a number of vectors listed

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in Table I will suffice for overproduction. For economic reasons and convenience, one might attempt to further amplify the yields of the protein by allowing constitutive expression in E. coli. A high-level constitutive expression of a nontoxic product is possible with high-copy plasmids having a strong promoter as well as a strong terminator when certain permissive hosts are employed. For highly toxic products, the best choice would be to utilize vectors permitting efficient export of the protein to the extracellular medium. However, much remains to be developed in this area. Therefore, at the present state of the art, a more conventional vector should be used and more attention should be given toward minimizing the basal level expression of highly toxic proteins from an efficient expression system. Both pL and pT7 vectors should be utilized, and these should be appropriately redesigned (1) to prevent transcription readthrough from minor unregulated promoters, and (2) to allow maximum repression of the promoter. When a cloned gene is expressed poorly, specifically, to the extent that the protein product is not detected by standard gel electrophoresis and staining techniques, one must be concerned with a number of problems that might cause poor expression. Using appropriate vectors, hosts, and techniques, one has to first determine the rate-limiting step(s), whether it is premature termination, rapid decay of the message, poor translation, or extreme instability of the polypeptide. To maximize expression, one must then attempt to overcome the rate-limiting step(s) by applying one or more of the genetic solutions described above. Acknowledgments The author is indebted to Fred Warren for helpful discussions and to Susan Gottesman and Bill Studier for commanicating unpublished work.

[I01 Overexpression of Proteins in Eukaryotes By MARGARET K. BRADLEY

Why don't you overexpress your protein? This is a common question asked of any researcher holding a gene sequence, and especially since there are so many systems available, both prokaryotic and eukaryotic. The need for overexpression systems is obvious if one wishes to investigate further proteins that are expressed in their host cells at low levels. However, it is important to establish a clear rationale, because the work METHODS IN ENZYMOLOGY, VOL. 182

Copyright O 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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involved is not trivial and requires a firm commitment to a technique and cell culture system. The development of expression systems in eukaryotic cells has been progressing steadily. They include yeast, mammalian, and insect cells, and the vectors available may produce constitutive, regulatable, or transient expression of the gene of interest. These expression systems were designed, for the most part, for investigations into the role of a protein in cell metabolism in situ.' However, the development of overexpression systems has been occurring in tandem, requiring knowledge of eukaryotic promoters and taking advantage of advances in virus research. Why not purify eukaryotic proteins from their original source? The primary consideration may be that sufficient animal tissue is unobtainable and comparable tissue culture systems would involve unacceptable levels of investment in dollars and time. Such costs might be acceptable for single isolations of proteins for gene cloning or gene library constructions, but not for repeated purifications. Even in an acceptable system, the ratio of the concentration of the desired protein to total host cell protein may be low, requiring extensive purification. Overexpression of the protein of interest allows for the use of simple and quick purification schemes. However, though the wild-type protein may be sufficiently well expressed, there may be no way to alter that gene. Engineered expression (or overexpression) allows addition of certain signal sequences, a choice of promoters, as well as mutation of the gene-coding sequences. Every mutant gene product, however, does not warrant the time and effort involved in establishing an overexpressing vector. The above problems might also be addressed by expression in Escherichia coli, which, paired with transient analyses in the mammalian host, has been a satisfactory tool in many cases (e.g., r ~ s ~ -Why ~ ) not . use E. coli? Since the tools for prokaryotic expression of foreign genes are successful and very accessible to researcher^,^ why do we need to investigate the more complex and costly eukaryotic sources? In E. coli, some proteins have only been expressed successfully as fusion peptides, possibly because the gene of interest does not have an acceptable environment for its start codon in prokaryotes. Other difficulties have arisen because the peptide of interest was toxic to E. coli and was degraded on synthesis. B . R. Cullen, this series, Vol. 152, p. 684. L. A. Feig, M. Corbeley, B. T. Pan, T. M. Roberts, and G . M. Cooper, Mol. Endocrinol. 1, 127 (1987). L. A. Feig, B . T. Pan, T. M. Roberts, and G . M. Cooper, Proc. Natl. Acad. Sci. U.S.A. 83, 4607 (1986). L. A. Feig and G . M. Cooper, Mol. Cell. Biol. 8, 3235 (1988). A. Das, this volume [9].

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Modification of the promoter may solve this problem, but may result in a lower level of production that does not satisfy the needs of the investigator. Another significant problem has been that the synthesized peptide is not biologically active or modified appropriately. For example, eukaryotic systems have cellular localization signals, not recognized in bacteria, that may affect posttranslational modifications, and important associations with animal cell proteins may affect the active state of the final product. Finally, overproduction of the foreign gene product in the prokaryotic environment may result in formation of highly aggregated, insoluble material in inclusion bodies within the cells, and no modification of the promoter resolves the problem. These inclusion bodies are thought to contain inappropriately folded protein, and some of these products are rescuable by denaturation-renaturation (see [20] in this volume) or by . ~ is not, however, a problem found exclusively in special t e ~ h n i q u e sThis E. coli, as will be discussed below. Yet some of the above problems might be alleviated in a eukaryotic overexpression system. What is enough protein? Do your needs for purified protein reach milligram levels? It is true that multiple and more complete analyses might be performed with a more abundant reagent. It is also the case that when certain genes are overexpressed constitutively they can be toxic to their native host species. One trick that some researchers have used successfully has been to express modified versions of normally toxic proteins. The development of regulatable promoters and secretion signals as well as the adaptation of lytic viruses as high-level, transient expression vectors provide possible ways to circumvent the toxic effects of foreign gene products. Determine what you will need from an overexpression system, in addition to the large quantities of your protein. Yeast as Host for Overexpression of Foreign Genes

Yeast systems have been used for a number of years, and many investigators have recognized the power of yeast genetics in giving one the potential for manipulating the host genes. Investigators attempting to use this system have appreciated that specific skills are necessary for growing and maintaining yeast culture. With regard to using yeast for overproduction of foreign proteins, there are some differences between it and higher eukaryotes that have just begun to surface that may or may not affect the proper modification of a protein. Counterparts of mammalian genes do exist in yeast, and there has been some success in inactivating the analogous host gene. Perhaps one might be convinced to switch, to a yeast A. Hoss, A. Arthur, and E. Fanning, BiolTechnology 6, 1214 (1988).

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analog of the gene of interest. There are a number of promoters available for yeast expression vectors along with coselection schemes7-lothat will not be discussed here. Many investigators have developed technology for secretion of heterologous proteins as a way to avoid toxicity from accumulated material and to simplify extraction of the protein." Unfortunately, it has been shown that yeast overexpression systems are liable to formation of precipitated aggregates of newly synthesized foreign protein, similar to the inclusion bodies seen in some bacterial overexpression cultures. Modification of the promoter activity did not resolve the problem satisfactorily, leaving the expression level too low for the desired use. l2,I3 Constitutive Expression in Mammalian Cells

Mammalian cell lines that constitutively express a foreign protein can be established by transfection with DNA containing the gene on a strong promoter (e.g., the SV40 early promoter plus its enhancer) recognized by the host in tandem with a selectable marker.14 Expression of the marker gene may confer drug resistance to the host so that nonexpressing cells can be removed and a clonal expressing cell line established (e.g., via G418 resistance). The selectable marker may be on the same or on a separate plasmid from that with which the gene of interest was introduced. In the latter case the selectable marker plasmid is introduced at a lox lower concentration than the primary plasmid so that on selection of expresser cells by drug resistance the statistics favor the coselection of the foreign gene. The levels of expression obtained are dependent on a number of factors including the suitability of the promoter to the host cells, the position of the random integration of the gene, and the possible toxicity of the introduced protein. Efficient integration into the host DNA and significant levels of expression have been achieved with use of the G. A. Bitter, this series, Vol. 152, p. 673. R. A. Smith, M. J . Duncan, and D. T. Moir, Science 229, 1219 (1985). J . M. A. Verbakel, K. A. Dekker, C. A. Ruygers, P. H . Pouwels, and B . E. Enger-Valk, Gene 61, 207 (1987). lo L. D. Schultz, K. J. Hofman, L. M. Mylin, D. L. Montgomery, R. W. Ellis, and J. E. Hopper, Gene 61, 123 (1987). " G. A. Bitter, K . M. Egan, R. A. Koski, M. D. Jones, S . G. Elliot, and J. C. Giffin, this series, Vol. 153, p. 516. R. A. Smith et al., Science 229, 1219 (1985). l 3 L. S . Cousens, J. R. Shuster, C. Gallegos, L. Ku, M. M. Stempien, M. S . Urdea, R. Sanchez-Pescador, A. Taylor, and P. Tekamp-Olson, Gene 61,265 (1987). l4 P. J. Southern and P. Berg, J . Mol. Appl. Genet. 1, 327 (1982).

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retroviral long terminal repeats (LTRs).I5Along with these vectors, there are a few regulatable promoters that may aid in the expression of toxic proteins in cell lines. Two such promoters, metallothionein16and the glucocorticoid-regulated LTR,17have not been completely successful in that there is too high a level of constitutive expression in the host cells. The doubly regulated lac operator from E. coli (negative and positive regulation with repressor and isopropylthio-P-D-galactoside(IPTG), respectively) has been shown to work well in mammalian cells,18 but is limited by toxicity of the inducing reagent.I9 For any of these systems, when a line of cells is selected after transfection, the desired characteristics of the promoter must be coselected from among the isolated clonal cell lines for the optimal off-on ratio. Alternately, secretion of the foreign protein is a way of accumulating high levels of protein from a constitutively expressing cell line.20 It is likely that a mammalian host will modify a mammalian gene product properly, provided that proper tissue and species-specific signals are present or do not interfere. However, production of a protein may be rate limited by the posttranslational machinery. Host cell metabolism may be boosted by coexpression of an oncogene paired with subsequent selection of a transformed cell line expressing high levels of protein. As mentioned above for yeast cell expression, the existence of the host cell counterpart for the protein of interest may cause problems in purification if not in expression. The inserted gene may be engineered to express at sufficiently high levels to overwhelm the host gene or perhaps it can be introduced to complement a defective host.21There have been strategies introduced using LTR-containing plasmids for specifically knocking out host genes by recombination and i n t e g r a t i ~ n .Finally, ~~ it may be extremely important to retrieve the inserted gene for genetic analyses (e.g., to confirm the presence or absence of mutations), and a combination of LTR promoters and other DNA signal sequences has been constructed to produce "shuttle vectors" for excision as well as integration of eukaryotic genes.23 M. Kriegler, C. Perez, C. Hardy, and M. Botchan. Cold Spring Harbor Conf. Cell Proliferation: Cancer Cell 2, 85 (1984). l6 G. N. Pavlakis and D. H. Hamer, Proc. Natl. Acad. Sci. U.S.A. 80, 397 (1983). l7 N. E. Hynes, N. Kennedy, U. Rahmsdorf, and B. Groner, Proc. Natl. Acad. Sci. U.S.A. 78, 2038 (1981). l8 M. Brown, J. Figge, U. Hansen, C. Wright, K.-T. Jeang, G. Khoury, D. M. Livingston, and T. M. Roberts, Cell 49, 603 (1987). l9 J. Figge, C. Wright, C. J. Collins, T. M. Roberts, and D. M. Livingston, Cell 52, 713 (1988). H. Karasuyama and F. Melchers, Eur. J. Immunol. 18, 97 (1988). 21 B . R. Cullen, this series, Vol. 152, p. 684. 22 J. Sedivy and P. A. Sharp, Proc. Natl. Acad. Sci. U.S.A. 86, 227 (1989). 23 C. L. Cepko, B. E. Roberts, and R. C. Mulligan, Cell 32, 1053 (1984). l5

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Mammalian Viruses as Transient Expression Vectors

There are a number of mammalian lytic viruses that have been shown ,~~ to serve as expression vectors. They include SV40,24a d e n o ~ i r u s e spapillomaviruses (BPV2'j), herpesviruses (EBV2'), and poxviruses (vacciThey can be produced in acceptably high titers infecting many different mammalian hosts, and their DNA sequences, including those coding for the gene of interest, can be isolated and analyzed. Vaccinia has 25 kilobases (kb) of nonessential gene sequences allowing substitution and expression of multiple genes at once.29Alternatively, defective viruses carrying foreign genes can be complemented by infection of specific cell lines expressing the required viral g e n e ~ . ~ OViruses ?~' provide transient expression systems with short-term expression of the foreign protein in the host cell, decreasing the probability of toxic effects interfering with foreign gene expression. Furthermore, the infecting virus may stimulate host cell metabolism, increasing production of the virally coded proteins at the expense of cellular proteins. If the protein is synthesized using a late viral gene promoter, it may allow both high levels of protein expression and, most importantly, propagation of the recombinant virus even if expression of the foreign gene is toxic to the host cell. In this system, one must maintain stocks of viable virus containing the correct foreign gene sequences and consider the biohazards associated with infective reagent~.~~ Use of Insect Cells and Viruses for Expression of Mammalian Proteins

The lytic baculovirus-insect cell expression system has been the recent method of choice and will be discussed in detail. Autographica californica nuclear polyhedrosis virus (AcNPV) infects the clonal tissue culture line Sf9 derived from Spodoptera frugiperda cells. Expression of the polyhedrin gene is unnecessary in tissue culture and its strong promoter

" R. C. Mulligan, B. Howard, and P. Berg, Nature (London) 277, 105 (1979).

M. Yamada, J . A. Lewis, and T. Grodzicker, Proc. Narl. Acad. Sci. U.S.A. 82, 3567 (1985). 26 P. Howley, N. Sarver, and M. F. Law, this series, Vol. 101, p. 387. B. Sugden, K. Marsh, and J. Yakis, Mol. Cell. Biol. 5, 410 (1985). 28 A. Piccini, M. E. Perkus, and E. Paoletti, this series, Vol. 153, p. 545. 29 B. Moss and C. Flexner, Annu. Rev.Immunol. 5, 305 (1987). Y. Gluzman, H. Reichl, and D. Scolnick, in "Eukaryotic Viral Vectors," p. 187. Cold Spring Harbor Lab., Cold Spring Harbor, New York, 1982. j1 K. L. Berkner, B. S. Schaffhausen, T. M. Roberts, and P. A. Sharp, J. Virol. 61, 1213 (1987). Consult guidelines and restrictions imposed by your laboratory and by local government concerning biohazardous material.

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can be used for the synthesis of foreign gene products. For the purposes of selection of recombinant virus, the replacement of the polyhedrin gene can be detected by a morphological change in virus plaques, and the recombinant, nondefective virus is produced at very high titers even with large inserts of foreign DNA.33Potential for high levels of expression of the foreign gene has nearly matched that of E. coli (1-100 mg of protein1 liter of culture). The polyhedrin promoter is expressed late in infection as this lytic virus is killing the host cells, giving a reasonable chance for high levels of expression even for certain toxic proteins. There is also potential for a significant decrease in host protein synthesis upon infection by the virus, an advantage for protein purification. We found that the ratio of expression of the protein of interest to total protein extracted was higher in the baculovirus system than in an adenovirus system expressing equal amounts of that protein.34Finally, the Sf9 cells can be grown in suspension, a definite advantage for extraction of large amounts of infected cells. Many posttranslational modifications of proteins are made correctly in the insect cell, although some are not.35 Certain rate-limiting steps in biosynthesis entail posttranslational modification and this bottleneck is not overcome by overproduction of the primary product. Crippling of the host cell at the hands of the infecting virus may also affect the cell's ability to modify the overexpressed protein efficiently. A limiting modifier gene might be coexpressed by insertion into the same virion or by coinfection with a second recombinant virus. A measure of safety has been enjoyed by researchers since AcNPV does not infect vertebrate cells. However, the Sf9 cells are a eukaryotic cell line with specific culture requirements, and the polyhedrin promoter of the virus is restricted to the insect cell system where it requires other virally coded genes for activity. That means that the plasmid vector cannot be checked for expression of its gene sequences in other mammalian cells before going on to construct the recombinant virus. In order to check inserted gene sequences, the plasmid must be sequenced before construction of the recombinant virus. Although the plasmid is large (- 12 kb), we have had no problem sequencing double-stranded constructs using the dideoxy chain-termination method. Importantly, verifying sequences in recombinant viral DNA is not a formidable task since the virus is produced at such high levels.36As for all virus culture, one must maintain a G. E. Smith, M. D. Summers, and M. J. Fraser, Mol. Cell. Biol. 3, 2156 (1983). C. I. Murphy, B. Weiner, I. Bikel, M. K. Bradley, and D. M. Livingston, J. Virol. 62, 2951 (1988). j5 V. A. Luckow and M. D. Summers, BiolTechnology 6,47 (1988). B. Weiner and M. K. Bradley, unpublished observations. 33

34

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viable clone of an identified, recombinant virus in high titer and maintain the cell culture system. Unfortunately, precipitated, newly synthesized proteins, apparently similar to the bacterial inclusion bodies, have been seen in the baculovirus system.37Manipulation of the promoter did not resolve the problem as was the case for yeast. Perhaps some proteins are simply not folded correctly outside their own host or when they are expressed at high levels. On the other hand, there has been successful expression of protein in the insect cell system that was unobtainable as soluble peptide in E. coli expression systems.38 Details for Using Insect Cell-Baculovirus System

The following gives more detailed information on the use of the baculovirus system for overexpression of eukaryotic genes. For further reference there exists a very detailed manual written by Summers and Smith.39 This system requires skills in both virology and eukaryotic cell culture. Reagents Necessary for Construction and Use of Recombinant Baculovirus Plasmids and Recombinant DNA Technology. Gene of interest: It is necessary to have a plasmid containing the cDNA of the coding sequences flanked with one of the linker enzyme sites found in the polyhedrin gene. For results derived in cloning the early genes of SV40 into this expression vector, it appears that the Sf9 cells do not recognize all splicing site signals.40Therefore, it would be wise to remove introns before attempting to express the gene in this system. One strategy is to clone the intron+ gene into a retrovirus vectoF1 and rescue the LTR-bracketed sequences as virus. Although the method is not foolproof, the rescued gene sequences will likely contain a cDNA copy of the original gene. R. E. Lanford, R. C. Kennedy, G. R. Dreesman, J. W. Eichberg, L. Notvall, V. A. Luckow, and M. D. Summers, in "Viral Hepatitis and Liver Diseases" (A. J. Zuckerman, ed.). Alan R. Liss, New York, 1989. C. I. Murphy et a/., J . Virol. 62, 2951 (1988). 39 M. D. Summers and G. E. Smith, "A Manual of Methods for Baculovims Vectors and Insect Cell Procedures," Tex. Agric. Exp. Stn. Bull. No. 1555 (1987). The manual and a startup kit are available by request from M. D. Summers, Department of Entomology. You are requested to identify your use as commercial or noncommercial and to sign an agreement for nonexclusive use of the materials. You will be updated o n any new material that becomes available. K.-T. Jeang, M. Holmgren-Konig, and G. Khoury, J . Virol. 61, 1761 (1987). C. L. Cepko, B. E. Roberts, and R. C. Mulligan, Cell 32, 1053 (1984). 37

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Baculovirus recombinant plasmid: The plasmids available at this time contain pUC8 sequences coding for the origin of replication and ampicillin resistance for growth of the plasmid in E. coli, the promoter and polyhedrin coding sequences of baculovirus including the polyadenylation signals, and several thousand bases of 5' and 3' adjacent viral sequences for efficient recombination with the wild-type virion. There are several plasmids available for insertion of foreign genes having their own ATG: pAc373, pVL941, pVL1393, and pVL1392. The first two pAc373 and pVL941 have only BamHI and Kpn-1 as unique cloning sites downstream of the desired site of insertion. These vectors have been tested extensively and produce recombinant viruses with excellent expression of in3 ~ a~ deletion of the 5' end of serted foreign genes. The plasmid p A ~ 3 7 has the polyhedrin sequence engineered so that the unique BamHI restriction enzyme recognition site in the coding sequence is brought close to the promoter (connecting bases -8 to + 176 with a 10-bp BamHI linker). The latter plasmid pVL941 has an altered polyhedrin ATG (ATT), leaving the 5' sequences intact and potentially allowing more efficient expression of A BamHI site has been engineered the inserted gene (2- to 3-f0ld).~~ starting at nucleotide position +37 in the sequence. The polyhedrin polyadenylation site is active for inserted genes, although addition of a second site attached to the gene of interest is acceptable. The second two plasmids pVL1393 and pVL1392 are new constructions. They include several unique cloning sites in addition to BamHI and the Kpn-1 site is no longer unique. Details on the makeup and use of these plasmids are available-from Dr. Summers. Parallel comparisons of infection of Sf9 cells with recombinant viruses constructed using the two different vector plasmids gave a 2-fold increase in protein production generated from pVL941 over pAc373 as reported by Robert L a n f ~ r d In . ~ our ~ experience a difference is detected only if the cell culture conditions are optimized, since that in itself can produce a 10fold increase in protein yield.45Selection of a clone of virus that produces the highest level of protein (see below) makes a more significant difference than changing the vector. On this basis, we have decided not to exchange any of the recombinant viruses already made using the pAc373 vector. With respect to the question of maximal expression, it may be that a fusion of the inserted gene with the 5' end of the polyhedrin gene might produce the highest expressers. Answers to this question await further study of the molecular biology of the insect cell system. M. D. Summers and G. E. Smith, see ref. 39. 43 V. A. Luckow and M. D. Summers, Virology 167, 56 (1988). R. Lanford, Virology 167, 72 (1988). 45 C. I. Murphy et al., J. Virol. 62, 2951 (1988). 42

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We have successfully inserted a number of gene sequences and constructed recombinant viruses with DNA prepared using a minipreparation procedure for plasmid DNA designed for use in dideoxy ~ e q u e n c i n g . ~ ~ Note that good preparations of RNase A and RNase TI and complete removal of phenol used for extraction of proteins are essential. Keep early stocks of the plasmid vector to ensure against contamination or subsequent loss of necessary sequences. When placing the gene of interest into the pAc or pVL plasmid, consider whether a modified sequence (mutants) will need to be expressed in the future. If so, check to see if a simple replacement of a 3' fragment will leave the 5' end coding sequences intact. Control baculovirus recombinant plasmid: A plasmid is available from Dr. Summers that contains sequences coding for the p-galactosidase (Pgal) gene following the polyhedrin pr~moter.~' This is an invaluable tool for testing the efficiency of your laboratory setup for use of the baculovirus expression system since it can be used to construct recombinant viruses that make colored plaques on Sf9 cell monolayers. Insect Cells, Culture Medium, and the Technology for Eukaryotic Cell Growth and Transfection of Plasmid DNA Insect cells Spodoptera frugiperda ( 5 ' ' ) in culture and reserves in the freezer: Sf9 cells grow in both monolayer and in s ~ s p e n s i o n They . ~ ~ require reasonably rich medium usually containing 10% fetal bovine serum (see below) and are therefore expensive to keep in continual culture. As for all eukaryotic cells, they must be carried in a sterile environment, free from toxic materials, and kept from overgrowing and losing viability. Sf9 cell monolayers are not very adherent and must be treated very gently. In fact no trypsin or EDTA is required to subculture the cells; they are merely resuspended by gentle trituration. Rapping the cell dish or inducing foam in the medium will decrease viability. Check regularly using Trypan Blue exclusion and look for 297% viability. Doubling times of -18-20 hr indicate healthy, log-phase cultures. Cells are seeded into flat or spinner culture at 0.5-1.0 x lo6 cells/ml and grown to 2-2.5 x lo6 cells/ml, usually requiring subculture two to three times per week. Culture volume is limited by the need for aeration (see below). Sf9 cells recover well from having been frozen in 10% dimethyl sulfoxide (DMSO) by 3-4 days. Prepare log-phase cells at 4-10 x lo6 cells/ml by gentle centrifugation at 1000 rpm for 10 min at 4". Prepare a 2x sterile In DNA sequencing information from Boeringer Mannheim, Indianapolis, IN. " G . E. Smith, M. D. Summers. and M. J. Fraser, Mol. Cell. Biol. 3, 2156 (1983). Accession number CRL1711 from the American Type Tissue Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852- 1776. 46

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-

-

-

solution of fresh medium containing 20% DMSO, and add slowly to the cell suspension. Place 1-ml vials of cells in an insulated container at -70" overnight to let them freeze slowly. Move to liquid nitrogen for long-term storage. Recover by immediate thawing to 27" and dilution in fresh medium. Note that as you continue to culture these cells you will run into a number of problems, and it is wise to have stocks of cells put away from a time when culture conditions were optimal. The current conditions of the cell culture will be directly reflected in the production of recombinant virus and the protein of interest. It is advisable to thaw fresh cells for new transfections and plaque purification, since minor contamination with other recombinant viruses shows up as plaques that will be mistaken for the new recombinant. Cell culture medium: Experience and consistency in eukaryotic cell culture are valuable assets to this system. One must plan ahead in supplying the cell culture materials, since optimal growth conditions depend on nontoxic and nondegraded reagents. Preparation of large lots of medium allows consistent culture over a longer period. Grace's medium is the basic component, sufficient for washing and short-term cell culture (1-6 hr), and is available as a dry powder (Gibco). Complete medium (TNMFH) contains filter-sterilized yeastolate and lactalbumin hydrolysate at 3.33 glliter of medium each and 10% fetal bovine serum. It is particularly important to obtain testable lots of fetal bovine serum since some lots are toxic. Note that Sf9 cells may be adapted slowly to some alternate lots of fetal bovine sera, and will eventually grow quite well. Remember to note the serum lot used in a particular freeze-down of cells so that they can be efficiently recovered. There is also the potential for adapting Sf9 cells to non-serum-based medium requiring addition of certain lipids.49There is a nonserum medium available from JR Scientific (EX-CEL 400) that supports spinner cultures well. However, the cells cannot be removed from flat culture without significant loss in viability. Therefore, we recommend its use for laying down cells for plaque assays, for infections in both flat and spinner culture, and for labs that maintain their stock cells in spinner culture. It is expensive and a 2 x medium for agarose-overlay medium is not available as yet. Transfection technology: Calcium phosphate transfection methodology derived from van der EbSOmay be used and one need not change current techniques for Sf9 cells. Transfection efficiency in this system can be checked using the P-Gal-expressing baculovirus plasmid, which produces blue plaques in indicator medium when there is successful recombination and expression. 49 50

B. Maiorella, D. Inlow, A . Shanger, and D. Harano, BiolTechnology 6, 1406 (1988). F. L. Graham and A. J . van der Eb, Virology 52, 456 (1973).

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Autographica californica Nuclear Polyhedrosis Virus (AcNPV) Wild-type AcNPV: The wild-type strain of AcNPV produces occlusion bodies in the infected cell and the area of a plaque in a monolayer of cell is blackened (occlusion-plus virus). Make sure that you have an occlusionplus strain of virus and that you keep an aliquot of the parental strain. Storage of the virus at 4" is the norm since the envelope is subject to breakdown upon freezing and thawing, but long-term storage of backup aliquots at -70" is recommended even with the consequent loss in titer. Virus is propagated by infecting Sf9 cells in log-phase growth with 0.010.1 plaque-forming units (PFU) of virus per cell or a multiplicity of infection (moi) equal to 0.01-0.1. Each successive propagation of virus needs to be titered and labeled in order to be sure that you are working with a known, nondefective reagent. Especially useful for determination of virus titer is the end-point dilution technique using microtiter well plates (10 p11 well from Nunc). Ten replicate infections with diluted virus are monitored for cytopathic effects. Methods for calculating the titer are described by Summers and Smith.51We have adapted this for titering other mammalian cell viruses with much saving in cost and time. Do check your technique with a parallel plaque titer, using basically the standard technique for mammalian viruses (see below). In the plaque assay the occlusion-minus and occlusion-plus viruses can be distinguished. Re diligent in destroying virus on glassware and work surfaces with detergent and sterilization. Most importantly, avoid contaminating uninfected cells and cross-contaminating recombinant virus preparations. Do not work with questionable materials, as it is not worth the effort and expense. DNA prepared from wild-type AcNPV: Since you may need to prepare this only once, keep it in separate aliquots and do not freeze it. From 2100 ml of infected cell culture at -48 hr postinfection (moi = 1) and remove the cells by centrifugation at 2500 rpm for 10 rnin. Pellet the virus from the supernatant fluid by ultracentrifugation at 100,000 g for 30 min at 4". Recover the pellet by resuspending overnight in a small volume of O . l X TE ( 1 mM Tris-HCI, pH 7.5, 0.1 mM EDTA). Layer onto two sucrose gradients (25-56% in 0.1 X TE) and sediment for 90 min in an SW41 rotor. With a Pasteur pipet remove a broad viral band (-113 of the gradient) seen by shining a light from the bottom of the gradient, and repellet as before. Resuspend the virus in 4.5 ml sterile 0.1 M Tris-HC1, pH 7.5, 10 mM EDTA, 0.1 M KC1. Add 200 k g proteinase K and incubate at 68"for 2 hr. Add 0.5 ml of 10% sarkosyl and continue to incubate 2 hr to overnight at 50'. Gently extract twice with phenollchloroformlisoamyl alcohol (25 : 24 : l), avoiding mechanical shearing. Transfer the DNA solu5'

M. D. Summers and G. E. Smith. see ref. 39.

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tion using a wide-mouth pipet. Precipitate with 10 ml of ethanol at -80" for 10 min and pellet at 2500 rpm for 10 min at 4". Wash with cold ethanol and remove. Resuspend carefully in 500 p1 of 0.1 x TE by incubating at 65" for -15 min. Check the suitability of the viral DNA for transfection and recombination using it with the P-galactosidase baculovirus plasmid. Reagents for Detection of Recombinant Viral Genes and Gene Products Specific DNA probe for foreign gene sequence: Preparation of radioactively labeled DNA fragments for hybridization to DNA extracted from cell cultures is described by Label only the gene sequences of interest and not those from the plasmid vector. Specific antibody to gene product: Either polyclonal or monoclonal antibody will serve to detect the expressed gene and quantitate its expression in crude cell extracts. Certain immunochemical methods for early detection of the protein are suggested. This volume contains additional information on these methods. If using monoclonal antibodies, it would be wise to screen with at least two, covering both the N- and C-termini of the peptide of interest in order to avoid selection of a recombinant virus encoding a truncated gene. Test for the enzymatic or biological activity of gene product: One cannot assume that the product of the system will be full sized, fully modified, or folded correctly in this system, although many successes have been so far reported. A test for a known activity will tell whether there is the minimum of protein structure and modification necessary for function in the product. Equipment For Escherichia coli culture: This includes a 37" shaker for liquid cultures and a 37" incubator for agar cultures. Facilities for sterilization of media and glassware are also necessary. For culture and infection of SJ9 cells: As for mammalian tissue culture, a sterile tissue culture hood is necessary and an inverted microscope is used for viewing the growing cells. A 27" incubator with a cooling coil is recommended for culturing Sf9 cells. They do grow at temperatures ranging from 25 to 29", but prediction of cell growth properties is difficult if the environment is variable. Changes in temperature that occur at night versus day and the seasonal changes in air conditioning may stop growth of the Sf9 cells entirely. In healthy, uniform monolayers occluded versus nonoccluded plaques are easily distinguished and therefore recombinant viruses are easily isolated. Predictable efficiency of virus infection and J. Sambrook, E. F. Fritsch, and T. Maniatis, "Molecular Cloning, A Laboratory Manual," 2nd ed., book 2. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989.

51a

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subsequent protein expression depend on optimal cell growth conditions. Simple changes in handling Sf9 cells result in 10-fold higher yields of protein (1-20 pg/ lo6 cells5*). Spinner culture is recommended, even though the highest titers of virus and the highest production of protein have tended to come from flat cultures of cells. However, these data come from extrapolations of small amounts of infected cells, and growth and harvest of large quantities are easier from spinner flasks since slow harvest gives rise to much proteolytic degradation in these cells. Agitation for continual aeration of the growing cells is as necessary as agitation for continual suspension. Without using a sophisticated fermentation device, allowance for a large surface-to-volume ratio of <300-ml cultures will suffice (e.g., use of 2-liter spinner flask). We normally seed the larger flask from a smaller log-phase spinner culture. Methods for culture of large volumes of Sf9 cells have been described by Maiorella et Aliquots of extracts made from infected cells can be kept for extended periods of time below -70°, especially if efforts have been made to extract the cells under conditions that minimize proteolysis and to store the materials with a minimum of desiccation. Cell recovery is best if they are stored below -90". Steps in Procedure for Constructing and Recognizing Recombinant Virus Transfection 1. The importance of transfection eficiency: In our experience, the calcium phosphate system requires controlled pH, clean DNA, and logphase cultures of healthy cells regardless of whether they are mammalian or Sf9 cells. Approximately 10 pg of recombinant plasmid of known sequence in the polyhedrin gene region is transfected together with 1 pg of purified virion DNA that has been pretested with the P-galactosidase recombinant plasmid. A finer precipitate might overlay more cells with the mixture of DNA, and a heavier precipitate may be toxic to the cells. We and others have recovered from 0.1 to 5% recombinant plaques identified by loss of occlusion bodies (see below). 2. Initial recovery after transfection: It is important to realize that the gene is not being directly selected in this procedure. If for some reason its expression is detrimental to virion production, wild-type virus will outgrow the recombinants. Therefore, we suggest that the transfected cells be harvested for virus at -4 days posttransfection or as soon as infection 5Z

C. I. Murphy et al., J . Virol. 62, 2951 (1988).

" B. Maiorella et al., BiolTechnology 6, 1406 (1988).

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is apparent, as noted by enlargement of cells. The AcNPV produces very high titer virus and waiting any longer only requires more dilution as well as risking overgrowth with wild-type virions. Store at 4". Isolation of Recombinant Virus Clones 1 . Recognition by plaque morphology: The wild-type AcNPV produces occluded or blackened plaques if the polyhedrin gene is being expressed. Recombinant virus plaques are either clear or light brown in appearance. If the Sf9 cells are healthy and the monolayers are even, these characteristics are easy to see. Remember that temperature is an important factor. Another factor often overlooked is sufficient dilution of virus recovered after transfection. We urge you to make dilutions from to produce monolayers with
L. P. Villareal and P. Berg, Science 196, 183 (1977).

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bond (FMC products, Rockland, ME) when pouring these overlays. Once the agarose is removed, place a circle of nitrocellulose over the cell monolayer, mark its orientation with respect to the agarose, and blot by overlaying the filter with Whatman 3MM saturated with 0.05 M Tris-HC1, pH 7.4, 0.15 M NaCl. Remove the nitrocellulose filter, cell side up, and place on Whatman paper saturated with 0.5 M NaOH for 2-3 min, repeat with 1 M Tris-HC1, pH 7.4, 0.5 M NaCl to neutralize, and dry on paper towels. Rinse gently in 0.015 M sodium citrate, 0.15 M NaCI, pH 7 (1X SSC) and dry completely. Bake in a vacuum oven at -80' for 20 min and hybridize with a probe specific for the inserted DNA. Align the positive signals with the stored agarose and pick a large area to allow for diffusion. Note that it may be useful to develop a sensitive and direct method for immunochemical detection of expressed protein using the nitrocellulose blot. 4. Plaque purijication of the recombinant virus: Briefly, supernatant fluid from transfected cell cultures is harvested after 4-5 days, cleared of cellular debris by centrifugation or filtration, and diluted to in Grace's medium. Monolayers of Sf9 cells (2 x lo6 cells/60-mm tissue culture dish) are inoculated with 1 ml of virus solution for 1 hr at 27". Low melting point agarose is prepared as a 1.5% sterile solution by autoclaving, cooled to 37-40', and mixed 1 : 1 with 2 x complete medium (Gibco) just before use. Virus inoculum is drained from the plates and 4 ml of agar medium is added gently to the side and rocked to spread. After setting, the plates are sealed with Parafilm to prevent drying and incubated at 27'. In 4-6 days plaques are formed in the monolayer and the occluded (blackened) versus nonoccluded (brown or clear) ones can be distinguished with an inverted microscope or even with the naked eye. Parallel infections with wild type and a recombinant virus will help illustrate the difference. Individual, clearly isolated plaques having the nonoccluded phenotype are picked by insertion of a sterile, cotton-plugged Pasteur pipet through the agar to the plate. The agar plug is extruded into 1 ml of sterile Grace's medium and left at 4" overnight. Reisolate plaques from three sequential infections. The importance of triple plaque purification cannot be emphasized enough. Virus production must not be amplified between sequential plaque isolations. Thereafter, keep close track of the progeny and remernber to always work from identified clones of virus stock. When producing virus use early, low-titer stocks and when infecting cells for protein production use later, high-titer stocks. Recheck virus titers periodically and replaque as necessary, not only to check the virus but to check the continuing competency of the Sf9 cells. Keep different recombinant viruses totally separate and well labeled and resist working with more than one at a time. Always handle the uninfected cells first and always include uninfected controls in the experiments. Remember that

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infected cells are enlarged, so that a simple routine scanning of the culture might reveal abnormalities and contamination with virus. 5. Verijication that a recombinant virus isolate contains certain sequences: After plaque purification of recombinant virus and analysis of the gene product (described below), it may be necessary to confirm that a particular sequence in the gene is present, especially if it is defective in a biochemical assay or if several point mutants of a gene are cloned for expression. Dilutions of a concentrated (lo8 pfulml), recombinant virus solution can be spotted onto nitrocellulose in a dot or slot blot apparatusSS in parallel with wild-type AcNPV. It is recommended that the membrane be preequilibrated with 1 M ammonium acetate and 0.02 M NaOH, the virus applied, and the wells rinsed with the ammonium acetate-NaOH solution.56In order to amplify the signal, the virus can be applied to sterile nitrocellulose paper in a dot blot apparatus and overlaid with Sf9 cells. The paper can then be incubated on growth medium-dampened 3MM paper for 24 to 48 hr before treating with 0.5 M NaOH for lysis of the cells.56aWash the filter briefly in 1x SSC, air dry, and bake in a vacuum oven at 80" for 20 min. Hybridize with a radioactively labeled specific probe for your inserted gene. At this point the gene could also be probed for specific mutant sequences by differential hybridization to an oligonucleotide used to produce the original mutant clone of DNA.57Absolute certainty of sequence, of course, requires purification of the recombinant viral DNA (see above) and recloning of the gene fragment for sequencing. Prepare to purify virus from 50 to 100 ml of culture since the fragment of interest makes up only 2-3% of the viral DNA. Methods for Detection of Protein Encoded by Recombinant Virus 1. Initial detection methods: An immunological procedure can be used to characterize recombinant virus isolates and to help choose highexpressing clones of virus. We have used three relatively simple procedures in initial analyses. All three methods require using part of the viruscontaining solution isolated from a plaque and infecting a small tissue culture well containing Sf9 cells. At the same time, hold the set of filtered recombinant isolates at 4" or proceed with the plaque purification. Studies on the time course of SV40 T-Ag expression indicate that the optimal time for harvesting cell extract is approximately 40 hr postinfection. After that time the viral infection has begun to destroy host cells.58 There is an 55 F. C. Kafatos, C. W. Jones, and A. Efstratiadis, Nucleic Acids Res. 7, 1541 (1979). M. D. Summers and G. E. Smith, see ref. 39. P. R. DeStasio, K. J. Etchberger, V. Paeratakul, and M. W. Taylor, BiolTechniques 442 (1989). 57 M. J. Zoller and M. Smith, this series, Vol. 154, p. 329. s8 R. Lanford, Virology 167, 72 (1988).

7,

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unknown quantity of virus in the plaque isolations and therefore you may need to wait 3 or more days for infection of a majority of cells (note changes in morphology compared to uninfected cells). Proceed then with one of the three following methods using uninfected and wild-type-infected cells as controls. a. For detection of intracellular proteins in situ, permeabilize and fix the infected cell monolayer onto plastic plates by removing the medium and adding acetone : methanol (1 : 1). Incubate the cells for -2 min, then gently wash with phosphate-buffered saline. There are slides available for anchoring suspension cells (AdhesioSlides, from MM Developments in Ontario, Canada). Alternately, the cells may be grown and infected in EX-CELL-400 medium (JR Scientific) mentioned above, which induces adherency. Some such precaution must be made since infected cells are likely to lift off the surface of plastic plates, leaving behind only uninfected cells for analyses. This situation will give a false-negative result when screening for expression of a foreign gene by recombinant virus. Follow with a protocol for immunostaining of cells. Sf9 cells are spherical, small, and have large nuclei compared to their cytoplasm. Proteins localized to the nucleus (e.g., SV40 large T-Ag) or some other cell organelle are easiest to detect. Both uninfected and infected cells will be present. b. Extract infected cells from at least one 60-mm plate and prepare the extract for SDS-acrylamide gel electrophoresis and Western blotting. At early stages, with an uncloned or low-titer virus preparation, concentration of proteins in the cell extract by SlOO centrifugation may be required to detect the protein of interest. One could also immunoprecipitate the protein from a large quantity of extract, thereby enriching the protein for detection by Coomassie Blue staining. c. Alternatively, an infected-cell extract (100 p1 of virus infecting one well of a 96-well microtiter plate of Sf9 cells) can be lysed by freezing and thawing, diluted, and applied to nitrocellulose paper directly in a vacuum dot-blot apparatus. The protein bound to the paper can be detected by common methods for Western blotting on nitro~ellulose.~~ There are two disadvantages in this direct technique. First, the extraction buffer must not interfere with the binding to nitrocellulose (i.e., contain SDS or other detergent unless highly diluted). Second, not all cells are likely to be infected with the recombinant virus. Therefore, the extract may have such a low 59

Western blotting reagents from Promega, Madison, WI.

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concentration of the protein that it is not detectable above background. If one preabsorbs the primary antibody used in these assays in the following manner, the specificity and sensitivity of all three methods of detection will be greatly enhanced: Prepare 200 pl of lightly packed cells. Use any eukaryotic cell source available that does not contain significant amounts of the protein of interest, including HeLa or other tumor cells that are easy to grow in quantity. Transfer to 1.5-ml microfuge tubes for convenience, remove excess fluid, quick freeze, and lyophilize the cell pellet to fix proteins. Store at -20'. To use, rehydrate with water or buffered saline solution, then centrifuge and remove fluid and floating debris. To prepare antisera dilute it 10-' with buffered saline solution, and add to fixed cell pellet. Rock overnight at 4" and recover the diluted and absorbed antibody after centrifugation. This method removes high-affinity, nonspecific antibody from the solution and can be used to remove competing specific antibody with repeated applications. For all immunological methods, proper titration and purity of the second antibody used for detection of the immune antibody is necessary to avoid false positives and null answers in this initial mixed infection (wild type and recombinant viruses). Use a concentration of second antibody that just saturates binding to the first antibody. One last caution must be mentioned. The Sf9 cell cultures have a certain amount of alkaline phosphatase activity that obscures detection of low-expressing clones of virus when using the alkaline phosphatase enzyme-bound second antibody as the detection system. We have used that background as our lower limit of acceptable production, but if you are limited by antibody with low specificity the background reaction can be reduced by using a second antibody bound to radioactive iodine (lZ5I)for the nitrocellulose assays or fluorescein-conjugated second antibody on the fixed cells. Always pretest these reagents; we have found them to be highly variable regardless of the purveyor. 2. Conjirmation that full-length peptide is produced: It is not wise to be satisfied with the presence of immunological reactivity. Direct analyses of the protein are simple after the virus has been cloned, when one can be assured that all cells in the culture can be infected with the same recombinant virus. a. Direct immunoprecipitation and SDS gel electrophoresis or Western blotting of extracts derived from Sf9-infected cells will produce sufficient protein for comparison with authentic protein (the latter may need to be radioactively labeled). When using radioactive methionine for the baculovirus recombinant infection, consider whether you are expressing sufficient protein. Electrophorese an

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aliquot of infected Sf9 extract (containing a known amount of protein) in parallel with immunoprecipitated extract, and Coomassie Blue stain to note how well the protein is expressed relative to other viral proteins. Some, but not all, of the recombinant viruses turn down host protein production and can express their inserted genes at -5% of the infected cell protein. The polyhedrin gene expresses and accumulates up to 25% of the infected cell protein. Ability to achieve high levels of production is strongly affected by the growth conditions of the infected Sf9 cells. b. If there is any question that the gene product is authentic, the peptides can be analyzed by partial proteolysis as compared to that from an original source. V8 protease and N -tosyl-L-phenylalanine chloromethyl ketone treated trypsin (TPCK-trypsin) are commonly used to create a unique pattern of partial digests of radioactively labeled protein. It is also possible to first immunoprecipitate the protein and obtain a specific pattern of proteolytic digestion by antibody p r o t e ~ t i o n . ~ ~ 3. Conjirmation of production of biologically active protein: One way to test your expressed gene for its biological activity is to fuse recombinant virus-infected Sf9 cells to a tissue culture cell type that will allow analysis using polyethylene glycol or Sendai virus. Protein expressed in the Sf9 cells will diffuse into a fused vertebrate cell. The promoter for polyhedrin used for overexpression of the foreign genes apparently requires AcNPV gene expression and AcNPV does not infect vertebrate cells. If an assay for biological activity of the protein can be performed in crude extracts, it may be worthwhile to analyze early extracts of infected cells. Otherwise be prepared to purify the protein for biochemical analyses, since there is no guarantee that the final product will be modified appropriately. Infected suspension cells are easy to extract with a minimum amount of handling, using safeguards against proteolytic degradation in order to extract the protein intact and therefore biologically active. Approximately 1 ml of 50 mM Tris-HC1 or HEPES buffer, pH 9, with 0.5-1% of the nonionic detergent Nonidet P-40 (NP-40) will extract 1-3 x lo6 Sf9 cells without significant proteolytic digestion of SV40 T-Ag produced in Sf9 cells.61 Less buffering capacity allows digestion by acid proteases. If the protein continues to be degraded, rinse the infected cells with medium containing proteolytic inhibitors before addition of extraction buffers containing the inhibitors (e.g., leupeptin and phenylmethylsulfonyl fluoride).

61

M. Schwyzer, Y. Tai, E. Studier, and M. R. Michel, Eur. J. Biochem. 137, 303 (1983). B. Weiner and M. K. Bradley, unpublished observations, 1987.

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High levels of gene expression give many advantages including early and easy detection of recombinant viruses. Protein purification protocols, such as immunoaffinity chromatography, perform best with a high ratio of the protein compared to total protein in the starting material. Do isolate the best expressing recombinant viruses and do handle the Sf9 cells at optimal growth conditions, giving yourself enormous potential for maximal yields of purified protein in high concentration with minimal handling. Acknowledgments I wish to thank Robert Lanford for numerous discussions. He has been working with the baculovirus system in close association with Max Summers and has. as we have, constructed and tested SV40 T-Ag and small t-Ag recombinant insect viruses.62 Others with whom I have shared both good and bad research findings include Beth Weiner, Ellen Fanning, Cheryl Murphy, and Nidhi Williams. 62

R. Lanford, Virology 167, 72 (1988).

[Ill Secretion of Heterologous Proteins in Escherichia coli By I. BARRYHOLLAND, BRENDAN KENNY,BORISSTEIPE,

and ANDREAS PLUCKTHUN Secretion of heterologous proteins from Escherichia coli into the culture medium is a useful means of protein purification that avoids the difficulties associated with renaturation and purification of proteins from inclusion bodies. In principle, secretion to the medium rather than export into the periplasmic space should have some advantages in ease of recovery. In addition, since in many cases export of foreign proteins across the inner membrane via the secA,secY-dependent pathway is rather inefficient, the use of an alternate system may be desirable. The E. coli hemolysin (Hly) secretory process, which involves a completely novel translocation mechanism, and apparently promotes high levels of secretion, is described in this chapter. Secretion from E. coli of heterologous proteins via fusion to the Cterminal Hly signal is still a developing technology and its full potential is still to be explored. This chapter is of necessity, therefore, a presentation of the state of the art technology. The Hly system has the merit of being endogenous to E. coli and as such should be amenable to considerable refinement and exploitation in METHODS IN ENZYMOLOGY, VOL. 182

Copyright D 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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E. coli

A hlyC

hlyA

C

hlyB

EcoRl G AAT C TTA

hlyD

1

pLG609

T A

CT GA

pLG6OQ-1

G

AAT

TTT

Srnal/Xmal CCC C I GGG

pLG600-2

G

AAT

TTT

CCC

pLG60Q-3

G

AAT

TTC

CC

I GG 1G

CTT CAA GAA

AAT

TCT

CTT

GAA

AAT

TCT

CTT

GGA

AAT

TCT

CTT

FIG. 1. Organization of the Hly 2001 cassette. (A) Molecular weights of the Hly proteins are indicated and arrows show probable transcription units. (B) The major restriction sites in hlyA and hlyB referred to in the text: E, EcoRI; D, DraI; P , PvuII; H, HpaI; Hd, HindIII. AS-I, 11, I11 indicates the fragments carrying the HlyA signal domains of 23 kDa, 12 kDa, and approximately 4 kDa (40 terminal amino acids), respectively. ( C ) See text.

the future as basic knowledge of the secretion mechanism itself is acquired. We shall describe in this chapter the essential features of the hemolysin transport process and then the strategies, procedures, and vectors currently available, or under development, for achieving secretion of foreign polypeptides. In addition, some limitations still associated with this system will be discussed. Hemolysin (HlyA) is secreted from certain pathogenic strains of E. coli or from laboratory strains carrying the cloned Hly determinant (see Fig. 1). Secretion occurs continuously throughout most of the growth phase and is not accompanied by cell 1 y ~ i s . Secretion l~~ in this case is dependent on a novel targeting sequence which has been localized to the

' N. Mackman, J.-M. Nicaud, L. Gray, and I . B. Holland, Curr. Top. Microbial. Immunol. 125, 159 (1986). N. Mackman, K. Baker, L. Gray, R. Haigh, J.-M. Nicaud, and I. B. Holland, EMBO J. 6, 2835 (1987).

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last 27-40 amino acids at the extreme carboxy-terminus of HIYA.~ Secretion is independent of the secA protein export pathway, but instead spe~,~ to the mecifically requires the products of hlyB and h l ~ D .Transport dium does not involve any detectable periplasmic intermediate and it is suggested that HlyB and HlyD form part of a specific translocation complex, spanning the inner and outer membranes, allowing direct secretion to the medium. Secretion of the hemolysin toxin normally occurs with a tll2of approximately 2-3 min at 30' (our unpublished data). Secretion levels in E. coli K12 strains carrying a single copy of the wild-type Hly determinant are equal to about 0.02% of total cell p r ~ t e i n However, .~ at least 100-fold higher secretion levels of the C-terminal, 23-kDa fragment alone can be achieved when this peptide is expressed from a lac promoter on a multicopy plasmid (see Fig. 3). The HlyA secretion signal is not processed during translocation. Heterologous proteins bearing the Hly targeting sequence are therefore recovered as fusion polypeptides, although protease-sensitive cleavage sites can be introduced into the constructions. C-terminal fragments of HlyA containing either 210 (AS-I), 120 (AS-11), or 40 (AS-111) amino acid residues have so far been used to promote secretion of heterologous polypeptides. Secretion efficiency is, however, considerably reduced with the shorter targeting fragments. Other structural features upstream of the minimal targeting signal may therefore be important in determining the final level of secretion (see below). General Procedures for Secretion of Fusion Proteins

As illustrated in Fig. 2 the protein of interest is first expressed in E. coli from an appropriate expression vector. Suitable restriction sites may then be selected or engineered to generate a blunt cleavage site at the Cterminus which may subsequently be ligated to the unique SmaIlHpaI fragment encoding the appropriate reading frame of AS-I from the pLG609 series (Fig. IC), generating an in-frame C-terminal fusion. pLG609 [6.1 kilobases (kb)] was constructed by insertion of the EcoRIHind111 fragment of the Hly determinant downstream of the tac promoter of pTTQ18, which carries the laclq gene.5 SmaI linkers were inserted into the EcoRI site, generating fusions in the three reading frames encoded by pLG609-1, 2, and 3, respectively. pLG609 may occasionally give rise to

' L. Gray, K. Baker, B. Kenny, N . Mackman, R. Haigh, and I. B. Holland, J. Cell Sci., Suppl. 11, 45 (1989).

J.-M. Nicaud, N . Mackman, and I. B. Holland, J. Biotechnol. 3, 175 (1985). M. R. J. Stark, Gene 51, 255 (1987).

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AH.., Signal Regio

Target Gene

.E. coli 'Express~on

pLG609 S e r i a l

1

alcut at X

a)cut

SmaIlHpal

b ) d e ~ h o s p h o r y l a t ee n d s

b ) i a o l a t e 0.8-kb fragment

.

I

Ligation

Fusion Site

'Recombinant Plasmid'

FIG.2. Strategy for the construction of hybrid genes bearing the AS-I, HlyA signal domain at the 3' terminus (see also Ref. 2).

internal deletions, eliminating the Hly fragments and generating a smaller, 2.1-kb miniplasmid. In particular, therefore, when preparing plasmid stocks checks should be carried out to ensure that the donor clones do not contain the miniplasmid, which can be readily distinguished from the parent plasmid on agarose gels. In preparing the appropriate DNA fragment from pLG609 it is most convenient to cut with SmaI and HpaI, followed by insertion downstream

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OPTIMIZATION OF STARTING MATERIALS

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of the gene of interest. Alternatively, the SmaI-Hind111 fragment may be used or the SmaI-HpaI fragment in pLG609 can first be subcloned into, for example, pUC18 and then reexcised using a variety of 3' restriction sites. For production of fusions to the 12-kDa (AS-11) signal domain the corresponding DraI fragment (Fig. 1B) can also be removed from the pLG609 vector. In this case alternative reading frames would have to be generated using linkers. Escherichia coli strains carrying the fusions are then transformed with pLG575 (carrying hlyB, hlyD cloned into the tet gene of pACYC1846). Induction of the synthesis of fusion polypeptides is then carried out both in the presence and in the absence of pLG575. Analysis of supernatant fractions should then allow the unequivocal identification of fusion proteins specifically secreted via the Hly system. Confirmation that the secreted protein is the required fusion can be obtained by Western blotting using antibodies to either the protein of interest or to the HlyA signal region. Examples of Use of Hly Secretion System

Secretion of OmpF-AS-I Fusion We have reported previously that fusion of the E. coli outer membrane porin, OmpF, to the HlyA C-terminal 23-kDa signal peptide (AS-I) leads to its efficient secretion in an HlyB, HlyD-dependent manner.2 In this case the fusion protein was expressed from a lac promoter on a multicopy pUC vector, following the addition of IPTG. Secretion of the fusion protein was not accompanied by detectable release of either cytoplasmic or periplasmic markers. In addition, no intracellular hybrid protein was detected when hlyB and hlyD were present, indicating that secretion was extremely efficient. Similar results were obtained when the OmpF porin was fused to the AS-I1 signal peptide. Secretion of Calf Prochymosin Using AS-I and AS-II Signal Peptides Varying portions of calf prochymosin have been fused to AS-I and AS-I1 and successfully secreted in an HlyB,HlyD-dependent manner6a (see Fig. 3). In this case, expression was obtained from a trp promoter carried on the dual-origin, temperature-amplifiable vector described by N. Mackman, J.-M. Nicaud, L. Gray, and I. 9. Holland, Mol. Gen. Genet. 201, 282 (1985). 9. Kenny and B. Holland, submitted.

"

Summary of Quantification Data m M

XCEU

VECrOR 1

23K pUC

5.0

2

23K pUC (a)

0.3

3

23K B/D pUC

8.7

4

800-1

W V

4.4

5

801

W V

5.3

8

802

W V

8.0

7

808

W V

5.2

Prochymosin-Hlya Fusions

B pLG609

(8 and HlyA)

FIG. 3. Secretion levels of the AS-I peptide and different prochymosin-Hly-AS-I, AS-I1 hybrids. Escherichia coli strains (carrying recombinant plasmids) were grown in Difco nutrient medium and induced by addition of IPTG (125 pglml) or temperature shift, at = 0.4 and = 0.7, respectively. Synthesis of the hybrid proteins was continued for 3 hr when the = 3 to 4. Samples of cells and culture supernatants were analyzed by SDS-PAGE (11% acrylamide) and the intensity of the Coomassie Blue-stained bands measured using an LKB Bromma Ultrascan XL laser densitometer. (A) Amounts of elongation factor (Emu), and the intracellular fusion band when present, are expressed as percentage total protein. The level of secreted protein in specific bands is expressed as the percentage of the intracellular form, or of EFTu, or of total cell protein, respectively. The amounts of secreted protein are also given as mglliterlA4sounit. In lines 1-3, the AS-I peptide was expressed from a lac promoter in strain NM522 or JMlOl [strains carrying an F' laclq (Ref. 2)] with export functions provided on plasmid pLG575. Alternatively, as shown in line 3, hlyB,hlyD were incorporated into the same pUC vector. (a) indicates that in this construct the Ser residue at position 996 in HlyA was replaced by Pro in order to disrupt the putative a helix in this region. As indicated, this had no inhibitory effect on secretion. In lines 4-7 E. coli SE50002 canied the dual origin vector (DOV)' and expression was obtained by heat induction. pLG575 provided the export functions hlyB,hlyD. Vectors 800-1, 801, and 802 encoded increasing portions of prochymosin fused to AS-I; 806 encoded approximately the first onethird of prochymosin fused to AS-11. Molecular weights of secreted proteins are given on the right. (B) Details of hybrid constructions indicated above. S, SmaI; E, EcoXI; A, ApaI; B, BalI; D, DraI.

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OPTIMIZATION OF STARTING MATERIALS

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Wright et ~ 1With . ~this system total levels of the expressed hybrid protein can reach 25% of total cell protein. However, most of this protein remains in inclusion bodies within the cells. Nevertheless, secreted levels of fusion proteins equivalent to up to 0.8% of total cell protein have been obtained when the Hly export functions are present. Varying the portion of the calf prochymosin present within the AS-I hybrid protein has little effect upon secreted levels and the largest fusion protein of 60 kDa lacks only the C-terminal three amino acid residues of prochymosin. In this case acidification of culture supernatants containing the fusion protein leads to the formation of a faster running protein in SDS-acrylamide gels,7a suggesting that some autocatalytic proteolysis may be taking place as in the case of authentic prochymosin. Secretion, Purijication, and Dimerization of Variable Domains of Antibody McPC603 Using Hemolysin A Signal To investigate the expression and secretion of antibody domains using the hemolysin export pathway, fusions were constructed between the variable domains of an antibody and the AS-I peptide of HlyA. We used the variable domains VH and VL of McPC603, a particularly well-characterized murine immunoglobulin A which binds phosphorylcholine. It has been shown previously that the FV fragment8,9and the Fab fragmentlocan be secreted to the periplasm of E. coli, using bacterial N-terminal signal sequences, which are correctly processed by the bacterial signal peptidase and that the two chains then associate to form antigen-binding fragments that are fully functional. Construction of Plasmids. The genes for VH and VL were obtained by chemical synthesis and placed under the control of the tac promoter of pAP1O,ll a derivative of pKK223-312 containing the lac14 gene of pJW271.lZaThe AS-I HlyA export signal was obtained as the EcoRIHind111 fragment from pLG609 (Fig. 1). It was connected in frame to the C-terminus of either VLor VHvia a synthetic linker encoding the tetrapep-

' E. M. Wright, G. 0. Humphreys, and G. T. Yarranton, Gene 49, 311 (1986). B. Kenny and B. Holland, unpublished observations. 1038 (1988). A. Pliickthun, A. Skerra, R. Glockshuber, and J. Stadlmiiller, in "Protein Structure and Protein Engineering" (E. L. Winnacker and R. Huber, eds.), p. 123. Springer-Verlag, Berlin, 1988. lo A. Pliickthun and A. Skerra, this series, Vol. 178, in press. A. Pliickthun, R. Glockshuber, I. Pfitzinger, A. Skerra, and J. Stadlmiiller, Cold Spring Harbor Syrnp. Quanr. Biol. 52, 105 (1987). l 2 J. Brosius and A. Holy, Proc. Natl. Acad. Sci. U.S.A 81, 6929 (1984). 12" J. Wang, Harvard University, unpublished observations.

* A. Skerra and A. Pliickthun, Science 240,

"

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139

lac 19

FIG. 4. Plasmids pVL-hly and pVH-hly for the expression of fusion proteins. VL or VH designates the segment coding for the respective antibody variable domain, Xa indicates the factor Xa cleavage site connecting the antibody domain and the secretion signal. 'hlyA is the AS-I fragment of hemolysin A, and hlyB' is the N-terminal region of hemolysin B, both . ~ laclq, rrnBT~Tz,bln, encoded on the 1.6-kb EcoRI-Hind111 fragment from ~ L G 6 0 9ptac, and ori are the tac promoter, the gene coding for its repressor, the rrnB terminator of transcription, the selectable marker, and the origin of replication, respectively, from pAP10.

tide recognition sequence (Ile-Glu-Gly-Arg) for factor Xa,13-ISa highly specific protease of the blood clotting system. This linker allows the cleavage of the variable domain from the fusion protein (Fig. 4). Each of the resulting expression plasmids confers ampicillin resistance. Bacteria harboring the plasmids pVL-Hly and pVH-Hly can be transformed with pLG575 (hlyB, hlyD). Expression and Secretion of VL-Hlyand VxHly Fusions. Both VL ASI and VH AS-I fusions are secreted into the medium in a manner completely dependent on the presence of HlyB and HlyD. The appearance of the fusion proteins in the culture medium is not correlated with cell lysis. l3 l4

l5

K. Nagai and H. C. Thogersen, this series, Vol. 153, p. 461. K. Fujikawa, M. E. Legaz, and E. W. Davie, Biochemistry 11, 4882 (1972). K. Fujikawa, M. E. Legaz, and E. W. Davie, Biochemistry 11,4892 (1972).

140

OPTIMIZATION OF STARTING MATERIALS

1111

FIG.5. Secretion of V,-Hly and VH-Hly fusions: An SDS-PAG stained with Coomassie Brilliant Blue is shown. The culture medium (10 rnl) of the strain SE5000, harboring both pV,-Hly and pLG575 (lanes A and B) or pVH-Hly and pLG575 (lanes D and E), was precipitated with 40 rnl of methanol. Lanes A and D: before induction; lanes B and E: 60 min after induction with 30 pM IPTG; lane C, molecular weight standard.

Some lysis is, however, observed when expression is induced at high levels. The optimal concentration of IPTG in the culture medium was found to be only 30 pM. The level of secretion of the fusion proteins is somewhat strain dependent. Best results were obtained with W31 1016and SE50002as hosts, resulting in levels of about 100 pg of fusion protein per liter of culture medium. The secreted fusion proteins migrate as a single band on SDS-PAGE and no band indicating processing or degradation can be found by staining for total protein or Western blot (Fig. 5). Furthermore, in strains DHl,17 JM109,18 or W3110, no inducible protein is detected in the absence of the export functions. The situation is less clear in strain SE5000, where we could detect some intracellular fusion protein either in the presence or absence of the export functions. l6 l7

B. J. Bachmann, Bacterial. Rev. 36, 525 (1972). D. Hanahan, J. Mol. Biol. 166, 557 (1983). C. Yanisch-Perron, J. Vieira, and J. Messing, Gene 33, 103 (1985).

11 11

SECRETION OF HETEROLOGOUS PROTEINS IN

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141

In all cases, these fusion proteins seem to be targets for rapid degradation by cytoplasmic proteases if they cannot be exported. Thus, we clearly find that secretion into the culture medium protects the fusion proteins. Purification and Cleavage of Antibody Fusion Proteins. Purification of these fusion proteins has proved difficult. They tend to aggregate and can be partially pelleted from the medium by centrifugation. Even though the proteins are hydrophobic, the capacity of phenyl- or octyl-Sepharose columns is limited for these fusion proteins, and bound protein cannot be quantitatively eluted. The fusion proteins can, however, be readily extracted from the medium by stirring with 4% (wlv) CDR [Cell Debris Remover, Whatman (DEAE-derivatized Cellulose)]. Neither 150 m M NaCl nor a pH shift to pH 10 elutes the fusion protein from CDR. The fusion proteins can be efficiently eluted with ionic and nonionic detergents. Unfortunately, detergents that are normally dialyzable cannot be removed by ultrafiltration or dialysis in the presence of the eluted material. This suggests that micelles or other aggregates of a large size are formed. The most convenient way to concentrate the fusion proteins from the medium is by precipitation with (NH4)2S04at 90% of saturation and subsequent dialysis. The fusion proteins are obtained in soluble form, but a large amount of colored material is also present. Both fusion proteins can be cleaved with the highly specific protease factor Xa, yielding bands of the expected molecular weights on Western blot. This cleavage reaction can be carried out directly with the (NH4)2S04precipitate of the culture medium after dialysis against factor Xa reaction buffer (150 mM NaC1, 50 mM Tris-HC1, pH 8.0, 1 mM CaC12). Cleavage is slow and not quantitative, requiring an incubation with high amounts of factor Xa overnight. It remains to be determined whether this reflects the existence of an accessible cleavage site only in a subpopulation of the fusion protein or interference by the high concentration of membrane components still present in the dialysate. While the designed cleavage site is clearly the most susceptible, there is apparently a secondary site within VH that makes it necessary to avoid overdigestion. Optimization of Secretion System

The levels of secretion under given conditions for a number of prochymosin hybrids and the C-terminal AS-I peptide itself are given in Fig. 3. Highest levels of secretion are obtained in rich medium (Difco nutrient broth) and for best results, with minimum amounts of background proteins in the culture supernatants, we recommend that prototrophic strains

142

OPTIMIZATION OF STARTING MATERIALS

L11.1

such as MC41004 be used as hosts. This could avoid or minimize lysis of cells which accompanies the high-level production of the antibody fusions described here. Although in the case of MC4100 this strain is also poorly transformable by conventional methods, high efficiencies are obtained with electroporation. Preliminary results also indicate that higher secretion levels are obtained at 37 than at 30". Other factors including salt concentration, and triethylamine (a membrane perturbant to increase envelope permeability) do not appear to have major effects on the secretion efficiency. In contrast, the positioning of the HlyA signal in the fusion can have substantial effects upon the level of secretion. Thus, placing the signal domain at the N-terminus of prochymosin completely inhibits secretion. On the other hand, while we have shown that internal deletions within the AS-I peptide (leaving intact the terminal 27 amino acids of HlyA) can promote good secretion in certain fusions, studies with other fusions suggest that sequences immediately upstream of this region or between residues 857 and 904 can be important for efficient ~ e c r e t i o n . ~ Further Developments Attempts are being made to increase the efficiency of secretion through, for example, enhanced expression of HlyB, which is probably a limiting factor in the process. Other studies will involve the optimization of the efficiency of the HlyA secretory signal, including the role of sequences at the N-terminus of AS-I and the introduction of mutational changes within the signal region itself. The feasibility of developing a single secretion vector incorporating the export functions is also being explored. To facilitate purification of secreted polypeptides, it would be highly advantageous to reduce the hydrophobic properties of at least some fusion proteins by further shortening of the secretion signal without losing the transport information. Some preliminary data suggest that this problem might be avoided by the use of the AS-I1 signal peptide. The identity of the amino acid sequences within the 23-kDa protein responsible for this phenomenon and purification procedures which might avoid it will also be investigated. Further construction of the fusion proteins for this system may also involve improved designs of the factor Xa site,Ig or cleavage by other enzymatic or chemical methods. This particular problem will have to be addressed individually for each protein. l9

S. Ellinger, R. Glockshuber, G. Jahn, and A. Pliickthun, J. Clin. Microbiol. in press (1989).

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143

Perspectives

The Hly system has now been demonstrated to promote the efficient secretion from E. coli of at least four heterologous polypeptides. Although the precise mechanism of translocation of polypeptides through the cell envelope by the HlyB,HlyD complex is still unclear, these results suggest that a broad range of proteins might be secreted by this means. In other studies, we have observed that the cytoplasmic protein CAT (chloramphenicol acetyltransferase) fused to the AS-I signal region, is also secreted from E. coli. In contrast, P-galactosidase is not secreted and accumulates intracellularly (unpublished data). In this latter respect the Hly system may be subject to the same limitation as the secA,secYdependent export system. In spite of the problems involved, the method holds promise for the secretion of foreign proteins in sufficient quantity and possibly in native conformation after cleavage from the HlyA signal domain. This system may be especially useful if the proteins involved are rapidly degraded in the cytoplasmic compartment. The HlyA secretion mechanism may also be particularly appropriate when expressed in avirulent strains as delivery mechanisms for certain oral vaccines. Acknowledgments This work was supported by SERC Grant No. GRl0182593, to I.B.H. and BMFT Grant BCT0372 to A.P. We are grateful to Richard Haigh for technical assistance and to Rudi Glockshuber for synthesis of the hapten affinity column and for helpful discussions. The authors are also grateful to Joan Warriner for the patience and perserverance in typing this manuscript.

r r r: r:

r r r r r r r r r

r

P

Section IV Preparation of Extracts and Subcellular Fractionation

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PREPARATION OF EXTRACTS FROM PROKARYOTES

147

C 121 Preparation of Extracts from Prokaryotes By MILLARDCULLand CHARLESS. MCHENRY The preparation of lysates is one of the more critical steps in the purification process, since this step influences the total quantity of the desired protein recovered, the biological activity of the protein, the completeness of its primary sequence by avoidance of proteolytic degradation, its association with other cellular components, and the presence of contaminants that may influence subsequent purification steps. A number of variables determine the success of a lysis method. The degree of lysis and retention of biological activity may be influenced by strain differences, choice of growth media, whether the cells are processed immediately or frozen, the presence of protease inhibitors, the choice of buffers, resuspension densities, osmolarity of the resuspension buffer, and growth phase at which the cells are harvested. A trial-anderror approach is often required to optimize lysis conditions.

Enzymatic Lysis

Enzymatic lysis methods minimize denaturation, are scale independent, and allow some selectivity in the release of cellular products. The drawbacks to enzymatic methods include the large number of variables that can influence lysis and the addition of substances that may complicate subsequent purification steps. Enzymatic methods work through digestion of bacterial structural components. In general, enzymatic lysis methods expose the inner, cytoplasmic membrane by degrading the peptidoglycan cell wall with degradative enzymes. The cytoplasmic membrane is breached by solubilization in detergent, osmotic pressure, shear, or by mechanical disruption.

Enzymatic Lysis of Gram-Positive Bacteria Lysis of gram-positive cells is relatively straightforward, with a few exceptions. For example, lysozyme works poorly on Staphylococcus aureus; lysostaphin is used in its place.' J. 0.Cohen, ed., "The Staphylococci." Wiley, New York, 1972.

METHODS IN ENZYMOLOGY, VOL. 182

Copyright D 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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EXTRACT PREPARATION AND FRACTIONATION

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Procedure for Lysing Bacillus subtilis'

1. Resuspend cells 50% ( w / v ) ~in 50 mM Tris-HC1 (pH 7.6), 10% sucrose, 1 mM dithiothreitol. 2. Add lysozyme to 300 pglml, EDTA to 1 mM, and Brij 58 to 0.1% final concentration. 3. Incubate on ice for 1 hr. 4. Centrifuge for 30 min at 40,000 g to remove cellular debris. Enzymatic Lysis of Gram-Negative Bacteria

Degradation of the peptidoglycan in gram-negative cells is made more difficult by the presence of an asymmetric lipid bilayer. The outer membrane is external to the peptidoglycan4 and acts as a permeability barrier to large molecules. Thus, gram-negative bacteria are less susceptible than gram-positive bacteria to lysozyme and detergent^.^ Enzymatic lysis of gram-negative bacteria requires the outer membrane to be permeabilized to expose the peptidoglycan layer to attack. Permeabilizing Outer Membrane. The permeability barrier posed by the outer membrane arises, in part, from the presence of lipopolysaccharide (LPS) on the outer leaf of the lipid bilayer. The polyanionic LPS molecules have strong lateral interactions in the presence of divalent cations, such as Mg2+, which are postulated to neutralize electrostatic repulsion between the LPS m ~ l e c u l e s Substances .~ which permeabilize the membrane and release LPS include chelators of divalent cations (e.g., EDTA), polycationic species, and various small molecules (e.g., Tris). Tris, often used as a buffer in lysis methods, effectively releases LPS and permeabilizes outer membranes.' Treatment of Staphylococcus typhimurium with Tris buffer alone (0.1 M, pH 7.2) releases 20% of the LPS.8 Ammonium glycylglycinate exerts a permeabilizing effect similar to Tris, but TES, HEPES, Bicine, and phosphate buffers do not potentiate lysozyme lysis in Escherichia c o k 5 The permeabilizing effect of Tris can be enhanced by the addition of EDTA; treatment of E. coli cells with EDTA in Tris buffer liberates about one-half of the LPS.5 M. A. Penlava and M. Salsa, Proc. Nail. Acad. Sci. U.S.A. 79, 5522 (1982).

' Throughout this chapter, all concentration percentages are given as weightlvolume unless otherwise noted. Cell weights are given as total packed wet weight. ' H. Nikaido and M. Vaara, Microbiol. Rev. 49, 1 (1985).

L. Lieve, Ann. N. Y. Acad. Sci. 235, 109 (1974). M. A. Asbell and R. G . Eagon, J. Bacteriol. 92, 380 (1966). ' R. T. Irvin, T. J. MacAlister, and J . W . Costerton, J. Bacteriol. 145, 1397 (1981). H. Nikaido and M. Vaara, in "Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology" (J. L. Ingrahm, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger, eds.), p. 14. Am. Soc. Microbiol., Washington, D.C., 1987.

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149 -

Digestion of Cell Wall. Egg-white lysozyme, which hydrolyzes Nacetylmuramide linkages, is most often used to degrade bacterial cell walls. Occasionally, T4 lysozyme will be used on E. coli. T4 lysozyme is more E. coli specific than the egg white lysozyme, but it is difficult to ~btain.~ The activity of lysozyme is influenced by the pH and ionic strength. Hen egg white lysozyme has been shown to be active over a wide pH range (pH 4 to 101, but is most commonly used in the pH range of 6.7 to 8.6.1° Lysozyme requires an ionic strength of at least 0.01 for 50% of maximum activity regardless of the pH. However, the upper range of ionic strength at which lysozyme retains 50% of maximum activity decreases with increasing pH. At pH 6.2, this upper limit occurs at 0.14, decreases to 0.09 at pH 8.0, and to 0.07 at pH 9.2. Lysozyme has close to optimum activity over a wide pH range at an ionic strength of 0.05." Gentle Enzymatic Lysis Method for Escherichia coli. 12.13 All manipulations should be performed in a cold room at 4" using prechilled equipment. The cells used in the described procedure were grown in rich medium, harvested at midlogarithmic growth, resuspended in Tris-sucrose buffer (50%), and poured into liquid N2with a circular motion to give cell clumps the consistency of popcorn. Solutions Tris-sucrose buffer: 50 m M Tris-HC1 (pH 7.51, 10% sucrose Lysis solution: 0.3 M spermidine-HC1, 2 M NaC1, 10% sucrose; adjust to pH 7.5 Procedure

1. Warm 750 ml Tris-sucrose buffer + 75 ml lysis solution (825 ml total volume) to 37' in a 2-liter beaker. 2. Weigh out 600 g of frozen, resuspended E. coli cells that have been at -20" for at least 24 hr (300 g cells total). 3. Quickly add frozen cells to Tris-sucrose-lysis solution prewarmed to 37" while stirring with an overhead stirrer. The temperature quickly drops to around 0". 4. Adjust to pH 7.5 with 2 M Tris base. Monitor pH using narrowrange pH stick. 5. Once a thawed homogeneous mixture is achieved, add 300 mg lysoM. Tsugita, M. Inouye, E. Terzaghi, and G. Streisinger, J. Biol. Chem. 243, 391 (1968). P. Jolles and J. Jolles, Mol. Cell. Biochem. 63, 165 (1984). " R. C. Davies, A. Neuberger, and B . M. Wilson, Biochim. Biophys. Acra 178,294 (1969). l2 W. Wickner and A. Kornberg, J. Biol. Chem. 249, 6244 (1974). l 3 C. S. McHenry, J . Biol. Chem. 257, 2657 (1982). lo

150

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EXTRACT PREPARATION AND FRACTIONATION -

-

-

-

-

zyme dissolved in a minimal amount of Tris-sucrose buffer, rapidly mixing to disperse the lysozyme. Caution: It is important that all ice crystals have melted at this step to achieve efficient lysis. 6. Pour the resulting suspension immediately into centrifuge bottles and place on ice for 1 hr. 7. Then, swirl centrifuge bottles in a large 37" water bath for 4 min, gently inverting every 30 sec. 8. Place immediately on ice. 9. Centrifuge at 23,000 g for 1 hr at 4". 10. Decant and save supernatant. The resulting supernatant should contain 20 mg proteinlml if lysis is complete. If cells are difficult to lyse, a more vigorous lysis can be obtained by adding EDTA to the Tris-sucrose solution (5 to 10 mM final concentration) or by increasing the heating step for up to 15 min. Addition of Freeze-Thaw Step.14A freeze-thaw step is added to some procedures after the incubation with lysozyme; the freeze-thaw procedure is more effective on spheroplasts than whole cells. For the procedure described below, cells were grown to late-logarithmic phase in rich medium, harvested, and resuspended 50% in 50 mM Tris-HC1 (pH 8.01, 10 mM EDTA, 10% sucrose. Resuspended cells were frozen in a dry icelethanol bath and stored at -70". Procedure 1. Thaw 300 g (600 ml) frozen cells on ice. 2. Transfer to centrifuge tubes and adjust to 140 mM NaCl, 1 mM dithiothreitol, and 0.4 mglml lysozyme. 3. Place mixture on ice for 45 min, mixing gently every 15 min. 4. Quick-freeze cells in a dry ice-ethanol bath. 5. Thaw frozen cells in a 37" bath with gentle mixing. 6. Repeat freeze-thaw four times. 7. Centrifuge lysate at 23,000 g for 2 hr to remove cell debris. Simple Vigorous Lysis Method for Lysing Escherichia coli.lSThe first two enzymatic lysis methods are relatively gentle because of the addition of sucrose to stabilize the spheroplasts against osmotic shock. This next method uses osmotic shock to help lyse the cells. Procedure 1. Thaw 300 g of cell paste and resuspend in 300 ml lysis buffer [50

mM Tris (pH 7 3 , 2 0 0 mM NaC1, 5% glycerol (vlv), 1 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride (PMSF)]. l4 l5

E. R. Wood and S. W. Matson, J . Biol. Chem. 263, 15270 (1988). C. I.Pao, T. E. Lee, Y. D. Liao, and C. W. Wu, J . Biol. Chem. 263, 10295 (1988).

1121

151

PREPARATION OF EXTRACTS FROM PROKARYOTES

2. Add lysozyme to 300 pglml and incubate at 4" for 1 hr. 3. Add MgC12 to 10 mM and treat with DNase I at 4" for 30 min. 4. Remove cell debris by centrifuging at 15,000 g for 1 hr. Notes on Enzymatic Lysis Procedures

1. Test the degree of spheroplast formation quickly and easily during the course of the lysis by adding 0.1 ml of the lysis mixture to 2 ml distilled water and measuring turbidity at ODsw in a spectrophotometer.l6 2. Occasionally, inclusion of detergent in the lysis procedure increases the yield of some enzymes.I7 The polyoxyethylene detergents [e.g., Brij 58, Nonidet P-40 (NP-40), and Triton X-100 at ca. 0.5%] are most commonly used in lysis methods because they are nonionic and relatively nondenaturing. l8 3. Some enzymes are sensitive to freezing. In these cases it may be best to resuspend the cell paste immediately upon harvesting with cold buffer, and use the more vigorous lysis procedures. Mechanical Lysis Methods

Mechanical lysis methods have several advantages over enzymatic lysis techniques: ( I ) Mechanical lysis methods do not require the addition of chemicals; (2) strain differences are minimized; and (3) they are economical for large-scale preparations. However, mechanical force is translated into heat which must be carefully controlled, and foaming must be controlled to prevent surface denaturation and oxidation. Mechanical methods, if too rigorous, may form fine cellular debris which may hinder subsequent processing. Mechanical methods release DNA which often must be removed. Mechanical lysis methods fall into two broad categories: Agitation with abrasives and liquid shear methods. Agitation with Abrasives

The bead mills represent the most common example of agitation with abrasives. The Dyno-Mill (manufactured by Willy A. Bachofen AG, Switzerland) is a widely used apparatus. The Dyno-Mill is a horizontal chamber filled to 80-85% with lead-free glass grinding beads. Inside the chamber, a shaft fitted with disks imparts a rotation to the glass beads. The glass beads rupture the cells by a combination of high shear and l6

l7

IS

M. H. Malamy and B. L. Horecker, Biochemistry 3, 1889 (1964). G. N. Godson and R. L. Sinsheimer, Biochim. Biophys. Acta 149, 476 (1967). M. D. Womack, D. A. Kendall, and R. C. MacDonald, Biochim. Biophys. Acra

(1983).

733,210

152

EXTRACT PREPARATION AND FRACTIONATION

1121

impact with the cells. The suspension of broken cells exits the machine but the beads are retained. Thermal control is maintained by pumping coolant through the chamber jacket. Factors which influence the efficiency of lysis include rate of agitation, cell concentration, concentration of glass beads, diameter of the beads, residence time in the chamber, and temperature. All these factors may need to be determined empirically. Starting Conditions for Lysing Escherichia coli in Dyno-Mill Model KDL19

1. Resuspend harvested cells at 30-60% in ice-cold buffer and maintain at 0-5". 2. Glass or stainless steel grinding chambers are available, but the 0.6liter stainless steel chamber is preferred for bacteria because it has better thermal conductance than the glass chamber. Load the chamber to 0.5 liter (80% of the chamber volume) with 0.2-mm-diameter lead-free glass beads. 3. Pump the cell suspension through the machine at a rate of 4 to 6 literslhr. This will give a residence time in the chamber of around 1 to 2 min. 4. Rotational speed of the disks is measured at their tips, and a tip speed of 10 mlsec should give a good agitation of the beads. 5. Refrigerate the jacket coolant to -20". Because of the heat generated by the mechanical action of the beads, it is important that the cell suspension mixture be held as cold as possible. These initial conditions should give an effluent temperature of less than 10" and lysis of 65-85% of the cells. Because small particles may affect purification, multiple passes and long residence times should be avoided. Liquid Shear Lysis

The most common devices using liquid shear to lyse bacteria are the Manton-Gaulin press and the French press. Presses lyse cells by pressurizing the cell suspension and suddenly releasing the pressure. The release of pressure creates a liquid shear capable of lysing the cells. Since the efficiency of lysis is highly dependent on the operating pressure,20high pressures should be utilized to minimize the number of passes required. Manton-Gaulin presses typically operate with feedstream pressures of l9 20

S. Goldberg, Glen Mills, Inc., personal communication. E. Bjurstrom, Chem. Eng. 92, 126 (1985).

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only 6000-8000 psi. Multiple passes are generally required to achieve an adequate ly~is.~O French presses are capable of operating at higher pressures, although for E. coli and S. typhimurium the pressures used are frequently in the range of 7000 to 10,000 psi. High operating pressures requires measures to compensate for a rise in operating temperatures; a pressure differential of 1000 psi raises the temperature by 1 In addition to controlling temperature, care should be taken to avoid inactivating proteins by foaming. In contrast to the bead mill, the efficiency of cell lysis in homogenizers is independent of cell suspension concentration. A typical resuspension density of 50% is used.20 Sonication. Sonication lyses cells by liquid shear and cavitation. Sonication remains a popular technique for lysing small quantities of cells, but is of limited value for cell quantities in the 50-g to 1-kg range because of the difficulty in maintaining low temperatures. The problem of controlling temperature during sonication is addressed by using pulses of 30 to 45 sec in duration, with pauses to reestablish a low temperature in the sonicate. Sometimes lysozyme is added to sonication mixtures in order to minimize exposure of the sample and increase the efficiency of the lysis. Nucleic Acid Removal

The first enzymatic lysis method given for E. coli yields a lysate free of the bulk of chromosomal DNA. This is due to a very gently lysis where holes are created in the membrane large enough for large proteins to escape, but small enough to contain the spermidine-condensed nucleoid. Other lysis methods described in this chapter cause release of nucleic acids. These must often be removed, either because of viscosity problems, interference with assays, or due to their serving as a polyelectrolyte that interferes with subsequent chromatographic steps. Often, RNA and DNA are removed by treatment with RNases and DNases. Alternatively, nucleic acids can be removed by phase partitioning2[ or by treatment of extracts with p~lyethyleneimine.'~

R. Kula, in "Protein Purification: Micro to Macro" (R. Burgess, ed.), p. 99. Alan R. Liss, New York, 1987. 22 R. Burgess and J. Jendrisak, Biochemistry 14, 4634 (1975).

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[I31 Preparation of Extracts from Yeast

This chapter is limited to the genus of budding yeast called Saccharomyces, with which the author possesses direct experience. The fission yeast Schizosaccharomyces pombe has attracted considerable interest recently, and many of the methods that will be discussed can be applied in a rather direct fashion to this organism, although it displays limited susceptibility to lyticase and especially to zymolyase. Why purify proteins from yeast? This organism is exceptionally well suited to genetic analysis, both classical and molecular.' This allows one to proceed from protein to gene and vice versa with extraordinary facility in the analysis of a pathway or process. Transformation of yeast, established in the last decade, makes functional studies in vivo quite apt. There are a wide range of shuttle vectors available that allow a gene to be moved from the yeast to the Escherichia coli environment at will. The functional components of the chromosome have been isolated from yeast. They include replicators, centromeres, and telomeres. In fact, very large, chromosome-sized DNA fragments can be cloned in yeast by the creation of artificial yeast chromosomes with the use of these functional elements. Finally, genes can be directed to homologous sites in the yeast genome, a feat difficult to achieve in higher eukaryotes, providing for gene disruption or replacement. Cell Growth and Harvesting

Commercial Yeast In many applications, the most suitable source of bulk quantities of yeast material are viable yeast sold as pressed yeast cakes in most grocery stores. The lyophilized brands that are encountered more and more frequently are not always a good starting material, because not all enzymatic activities survive in good yield in this form. There are at least four brands known to this author, National, Red Star, Federal, and Fleischmann, all of which are essentially equivalent for the biochemist. There are probably many other regional brands that can be used as well. The use of commercially available yeast is particularly appropriate once a protein has been purified and characterized from a laboratory

' K. Struhl, Nature (London) 305, 391 (1983). METHODS IN ENZYMOLOGY, VOL. 182

Copyright O 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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strain. Then, the same protein can be compared from the commercial source. If all goes well, the commercial yeast may be used for routine large-scale preparations. The availability of this alternative is important, because large quantities of yeast uncontaminated by bacteria can be obtained quickly and at minimal expense in the supermarket. The various fermentation markers that the commercial strains possess are of no consequence in most applications. It should be pointed out that commercial yeasts are not nondescript strains; Fleischmann's yeast, for example, is an ATCC strain. Choice of Yeast Strain

Appropriate yeast strains can be obtained from the Yeast Genetic Stock Center (Berkeley, CA) or from the original author. Proteolysis problems in yeast as in other eukaryotes can be severe. Therefore, the use of strains carrying the genetic markers pep4, prb, and prc, which are deficient in the major yeast proteases, should be used routinely unless special purposes are envisioned. Culture Media

Unless there are other indications, the medium of choice is a rich broth that provides for rapid growth. Two rich culture media are widely encountered in the literature, YPD and YM-1. YPD: 2% peptone 1% yeast extract 2% glucose For adenine-requiring strains, 0.003% adenine sulfate may be added YM-1: 0.7% yeast nitrogen base 0.5% yeast extract 1% peptone 1% succinic acid 0.6% sodium hydroxide 2% glucose In some instances, a particular application will dictate the choice of a different medium. A synthetic minimal medium, called SD, to which various components may be added, has been d e ~ c r i b e dPerhaps .~ the most F. Sherman, G. R. Fink, and J . B. Hicks, "Methods in Yeast Genetics." Cold Spring Harbor Lab., Cold Spring Harbor, New York, 1986.

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widely used medium for radioactive labeling of yeast cells is Wickerham's medium (per liter): Boric acid Potassium iodide Manganese sulfate Zinc sulfate Calcium pantothenate Inositol p-Aminobenzoic acid

500 pg 100 pg 400 pg 400 pg 400 pg 2 mg 200 pg

Riboflavin Ammonium sulfate

200 pg 5g

Magnesium sulfate Calcium chloride

0.5 g 0.1 g

Copper sulfate Ferric chloride Sodium molybdate Biotin Folic acid Niacin Pyridoxine hydrochloride Thiamin hydrochloride Potassium phosphate (monobasic) Sodium chloride

40 Pg 200 Pg 200 Pg 2 Pg 2 Pg 400 I*g 400 pg 400 pg 1g 0.1 g

For low-phosphate medium, replace potassium phosphate with potassium chloride. For low-sulfate medium, substitute ammonium sulfate with ammonium chloride. The carbon source is usually 2% glucose in this medium, and nutritional supplements are added, as necessary. In some cases, nonfermentable carbon sources, such as glycerol or ethanol, are used to induce mitochondria1 functions in a variety of media. Finally, expression of a protein from an inducible promoter may be the goal. An appropriate compound may then be added to the medium. A frequently utilized inducible promoter from which expression of virtually any protein can be engineered is the GAL 1,10 p r ~ m o t e rGalactose .~ (25%) is then included in the medium for induction. (Glucose must be replaced by raffinose to prevent repression.) Culture Vessel

The optimal temperature for culturing yeast cells is 30" unless temperature-sensitive or cold-sensitive mutants are being used. Yeast grow best aerobically. Vigorous aeration can readily be provided on a rotary shaker in an Erlenmeyer flask filled to no more than one-third capacity. Up to about 8 liters of culture, distributed among several flasks, can be cultivated in this way. One or a few cell preparations of this magnitude can provide enough starting material for pilot purifications of many proteins of interest. Large-scale purifications can be carried out starting with material grown in fermenters or with commercial yeast. The description and operM. Johnston and R. W. Davis, Mol. Cell. Biol. 4, 1440 (1984).

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ation of various brands of fermenters lies beyond the scope of this discussion. Antifoam can be used during yeast culture with impunity. Problems can be encountered with bacterial contamination in fermenter runs because bacteria double in number at a rate at least three times higher than do yeast. The antibiotic kanamycin (25 pglml) can be used to ameliorate this problem in fermenter runs. Monitoring Growth The growth of a culture can readily be monitored by determining cell number or the derived measurement of absorbance at 590 to 660 nm. Either a hemocytometer coupled with a light microscope at 400x magnification or a particle counter can be utilized for determining cell number. Obviously, the latter can be more accurate; however, not every laboratory has access to a suitable instrument. For most purposes, a hemocytometer provides sufficient accuracy for the biochemist, provided 100200 cells are counted. With budding yeast, the problem always arises of what to consider an individually countable cell. This issue is always coupled to the problem of cell aggregation. As far as absorbance measurements are concerned, not only accuracy but also relation to actual cell number can pose difficulties. These questions have been discussed in great detail,4 and only the essentials are given here. Yeast cell aggregation is highly dependent on the strain used and on the culture medium. (Contamination with bacteria or other fungi can lead to aggregation as well.) Fortunately, many of the strains the biochemist wishes to utilize pose minimal clumping problems. Brief sonication of most aggregated cultures is sufficient to disperse them, and has little or no effect on cell viability. Remember, the yeast wall is tough, and sonication is of no use in breaking open yeast cells. Sonication detaches cells that have completed cytokinesis. Thus, before performing cell counts in a hemocytometer, it is useful to sonicate the sample. The convention stating that a daughter cell is scored as an individual cell only when separated from the mother is widely applied, and it avoids the subjectivity in determining at what size the bud becomes a separate cell. Absorbance measurements, as a means of following growth, present two major problems. First, the relationship between cell number and absorbance is linear over only a short range. Second, the absolute value of cell number can be derived from the absorbance only under a defined and experimentally determined set of conditions. Generally, unless the spectrophotometer displays a broader linear range, it is good not to take absorbance readings outside the range 0.1 < A < 0.3. This means that it is J. R. Pringle and J.-R. Mor, Methods Cell Biol. 11, 131 (1975).

158

EXTRACT PREPARATION A N D FRACTIONATION

[I31

necessary to make dilutions for most of the measurements taken during cell culture. For every strain and for every set of culture conditions, it is essential to determine the relationship between cell number and absorbance by experiment. Absorbance depends not only on cell number but also on size and shape, all of which can change depending on conditions. For a typical diploid strain, an of 2 might be equivalent to about 1 X lo7 cells/ml, and twice that number for a haploid. Problems with contamination of yeast cultures are infrequent when small volumes of 1 to 2 liters are used, as long as the medium is indeed sterile. However, it is important to be on the lookout for contamination. The best rule to observe is "if the culture doesn't smell as if drinking it chilled would be enjoyable, it is probably contaminated." Visual inspection for molds, coupled to the observation of the speed of settling of the cells and flocculation, are means of identifying contamination. Finally, inspection in the phase-contrast microscope is usually quite informative. Genetics must remain the only resort, if the contamination is with another closely related yeast strain. At what cell density should the cells be harvested? The rule of thumb is mid to late logarithmic phase. A balance is usually struck between vigorous growth in mid-log phase and yields per milliliter of culture in late-log phase. Obviously, there may exist indications to the contrary, depending on the particular application. Mid to late log for a typical diploid strain may be about 5 x lo7 to 1 x lo8 cellslml, and somewhat higher for a haploid. Growth curves should be established for each set of conditions. Harvesting of Cells

The most commonly used method for harvesting yeast is centrifugation. The more rapid the procedure the better. In some cases, it is useful to chill the cells before harvesting; in others, harvesting may or even should be carried out at room temperature. Yeast metabolism is slowed dramatically at 0' in the presence of 10 mM sodium azide and 10 mM sodium fluoride. Centrifugation for 5 min at 3000 g,, is quite sufficient to obtain a yeast pellet. Even at higher speeds the pellets tend to be loose and slide easily down the sides of the centrifuge tube or bottle. Rotors with the capacity for several liters are available (e.g., IEC Centra-8R, 6liter capacity). For processing large volumes, such as those obtained in fermenter runs, the Sharples centrifuge cannot be overrated. In addition to centrifugation, tangential flow and hollow fiber ultrafiltration devices are available (Amicon, Danvers, MA; Millipore, Bedford, MA) for rapid concentration of cells (up to 90%) from large volumes.

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This author has not used these, but mention has been made in the literature .$ Storage of Cells

Once harvested, the cells are usually resuspended in a buffer. Although storage of the cell paste is not normally a problem, immediate resuspension of the harvested cells allows further processing without the necessity of dispersing the cells later. The choice of buffer depends on the method to be used for opening the cells and on the purpose for which the cell extract is being prepared. In any case, cells can be stored frozen at -80" in virtually any application. It is very useful to freeze the cells in the tube or vessel in which they will be subsequently processed after thawing. This can be very important for avoiding shear when attempts are made to remove high-molecular-weight DNA from the extracts. In most instances, the goal is to freeze the cell suspension rapidly. The most rapid procedure is to drip the suspension into liquid nitrogen. This generates small pellets that can be transferred to an appropriate container after pouring off the liquid nitrogen. Slower freezing is obtained by immersing the container with the cell suspension into liquid nitrogen. Slow freezing can be obtained in an insulated container in a -80" freezer or in the vapors over . ~ latter procedure has been used in storing yeast liquid n i t r ~ g e n The spheroplasts for preservation of the integrity of internal organelles after lysis in a freeze-thaw cycle. The final cell density at which cells are resuspended is entirely dependent on the purpose to which they will be applied and on the method of cell lysis. The higher the cell density, the higher the protein concentration will be in the resultant extract. High protein concentration will tend to stabilize the protein-protein interactions present in the cell, and it is generally protective in terms of stabilizing activity. On the other hand, certain adventitious associations can also develop in a concentrated extract. Cell Lysis

Overview Autolysis. This is the oldest procedure for preparing yeast extracts. It is a slow procedure requiring some 24 to 48 hr. But it is simple and cheap. Several milliliters of toluene are added to a yeast suspension, which is G. A. Bauer, H. M. Heller, and P. M. J. Burgers, J . Biol. Chem. 263, 917 (1988). D. Baker, L. Hicke, M. Rexach, M. Schleyer, and R. Schekman, Cell 54, 335 (1988).

160

EXTRACT PREPARATION AND FRACTIONATION

[I31

incubated at room temperature. The toluene serves to prevent bacterial growth and to permeabilize the yeast membrane. The latter releases a wide variety of hydrolases which attack the cell wall. Among these are proteases, which can wreak havoc with the desired activities. Many enzymatic activities can be obtained in this fashion, but they cannot be assumed to be structurally intact or to possess all of the properties of the native enzyme. Needless to say, this procedure is not recommended for the research laboratory. Pressure Cells. A variety of pressure cells that can be used to disrupt yeast are available. These devices are operated at pressures from about 5000 to 20,000 psi to break yeast. They include the French press,' Eaton press,8 and Gaulin hom~genizer.~ Apart from the Gaulin homogenizer, these instruments are generally designed to handle relatively small volumes of cell suspension in one pass. At least in the case of the French press, several passes are needed to achieve reasonable cell breakage. This is cumbersome and ultimately results in heating of the extracts. The equipment is expensive and not available in every laboratory. Abrasives. Disruption of yeast by agitation with glass beads (Braun Instruments, Allentown, PA; Potters Industries, Hasbrouck Heights, NJ; Biospec, Bartlesville, OK; Sigma, St. Louis, MO) is probably the most widely used procedure for obtaining extracts from these cells. Beads ranging in diameter from 0.25 to 0.5 mm have been used. The optimal size falls in the range of 0.4 to 0.5 mm. Beads are prepared by treatment with nitric acid and rinsing thoroughly with deionized water. A batch of beads may be used many times with periodic nitric acid cleaning, if it is washed thoroughly with detergent after each use. The abrasive action of well-agitated glass beads is a very effective method for preparation of yeast extracts. Cell breakage of up to 95% can be obtained, as assessed by phase-contrast microscopy. The effective range of this procedure is from about 25 p1 of cell suspension in a microfuge tube to many liters in a DynoMill apparatus. The procedure is useful for preparing enzymes; however, the shear generated may not preserve the integrity of all protein complexes. This method has also been used for preparation of cell organelles except for nuclei, which are too fragile. Undoubtedly, some breakage and resealing of organelles, such as mitochondria, takes place when glass beads are used. Therefore, enzymatic methods are recommended for this purpose.

' L. F. Bisson and J. Thorner, J. Biol. Chem. 256, 12456 (1981). G. Badaracco, L. Capucci, P. Plevani, and L. M. S. Chang, J . Biol. Chem. 258, 10720 (1983). F. Wyers, A. Sentenac, and P. Fromageot, Eur. J . Biochem. 35, 270 (1973).

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PREPARATION OF EXTRACTS FROM YEAST

161

The real difference from one procedure to the next in which glass beads are utilized is the method used to agitate the beads. The more vigorous this agitation the better the cell breakage. However, this improvement in lysis is coupled to the generation of considerable heat, which must be dissipated in some fashion. The only other difference between the various methods resides in the scale on which they can be applied. The simplest means for agitating the glass beads is with the use of a vortex mixer.6 Several cycles of agitation (30 sec to 1 min) must be interspersed with cycles of cooling on ice. Breakage is variable, but can be well over 50%. This method is most suitable for relatively small volumes. Sonication with glass beads has also been employed.1° The Braun homogenizer has been used with small quantities of cells.11Cooling is provided with liquid C 0 2 in this apparatus. Cell breakage is rather low (25-40%). The Bead Beater (Biospec, Bartlesville, OK) gives good breakage and can be used conveniently with various amounts of cells. The use of this equipment will be described below. For large quantities of cells, the use of a paint mixer1? has been described. Finally, continuous flow can -be achieved using the DynoMill (Impandex, Maywood, NJ) for large-scale preparations.13In all cases, beads are removed either by filtration through several layers of gauze or, after decanting the supernatant, any remaining beads can be removed by centrifugation. Enzymatic Lysis. The yeast cell wall can be removed or weakened by digestion with a variety of enzymatic preparations. These procedures can be used on any scale, and they avoid shear and can be carried out in the cold. They can be used to prepare both dilute and highly concentrated extracts. Spheroplasts can be prepared and purified as an intermediate step or lysis can be carried out directly. In most instances in which cellular organelles are prepared, spheroplasts are first generated with the use of these enzyme preparations. Glusulase, the first such preparation described, is used infrequently in protein purification.14 The use of zymolyase is extensive; on the other hand, lyticase, which is not available commercially, is used less frequently. Procedures utilizing the latter two enzymes will be discussed below. Finally , we have used P-glucuronidase in the preparation of S. DiNardo, K. Voelkel, and R. Sternglanz, Proc. Narl. Acad. Sci. U.S.A. 81, 2616 (1984). G. Schatz, this series, Vol. 10, p. 197. l2 S. G. LaBonne and L. B. Dumas, Biochenzis?ry 22, 3214 (1983). " T. Goto, P. Laipis, and J. C. Wang, J. Biol. Chem. 259, 10422 (1984). l4 L.-C. C. Wu, P. A. Fisher, and J. R. Broach, J. Biol. Chem. 262, 883 (1987). lo

162

EXTRACT PREPARATION AND FRACTIONATION

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spheroplasts, and, presumably this enzyme could be applied in protein purification. Preparation of yeasf extracts by enzymatic digestion of the cell wall is a versatile procedure that has found wide application, although not as wide as disruption with glass beads, in the preparation of enzymes,15 enzyme c ~ m p l e x e sand ' ~ organelles (see below). For the preparation of organelles, and especially nuclei, it cannot be replaced with glass beads. It is certainly the most gentle procedure available. The presence of the protease activity in the lytic enzyme preparation is generally not a handicap, and, if necessary, the activity can be removed. Although it is resistant to most protease inhibitors, the lytic protease is heavily inhibited by polysaccharides such as those in mannan that are present in high concentration in the yeast lysate.17 The use of enzyme preparations adds little to the cost of making the extract. Preparation of Yeast Extracts with Glass Beads The density of the cell suspension plays an important role in the efficiency with which cells are broken. In general, the higher the density the better the breakage achieved with glass beads. Experience in many laboratories indicates that the optimal cell density is approximately 5 x 109/ml for haploid cells (about one-half of this for diploids). This cell density is equivalent to a 1 : 1 (by volume) suspension of a packed cell pellet in an appropriate buffer or, in other words, a 1 : 1 weight by volume suspension of cells. For best results, this suspension is added to an equal volume of beads. The better the agitation of the glass beads the higher the percentage breakage. Any of the methods listed above may be used to achieve this agitation. Here, I will describe the Bead Beater (Biospec), which allows cell suspensions from about 5 to 200 ml to be subjected to agitation at once. lSz0 The Bead Beater is an inexpensive apparatus that consists of a 350-ml chamber, either polycarbonate or stainless steel. The latter is very useful to promote rapid heat exchange because the vigorous blending action provided by the 0.43-hp motor of the Bead Beater base generates considerable heat during cell breakage by the Teflon rotor. A jacket can be used S. M. Jazwinski and G. M. Edelman, J. Biol. Chem. 260, 4995 (1985). S. M. Jazwinski and G. M. Edelman, J. Biol. Chem. 259, 6852 (1984). l7J. H. Scott and R. Schekman, J. Bacterial. 142, 414 (1980). IsA. D. Johnson and I. Herskowitz, Cell 42, 237 (1985). l9 J. Berman, C. Y . Tachibana, and B.-K. Tye, Proc. Natl. Acad. Sci. U.S.A. 83, 3713 (1986). *O T. N . Davis, M. S. Urdea, F. R. Masiarz, and J. Thorner, Cell 47, 423 (1986). l5

l6

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PREPARATION O F EXTRACTS FROM YEAST

163

with the Bead Beater for surrounding the grinding chamber with an appropriate coolant. A small chamber kit, which is composed of a 15-ml and a 50-ml chamber, is also provided. Best results are obtained when beads are added to the chamber in which cell breakage will be carried out to approximately one-half capacity, followed by the cell suspension to fill the chamber. If the procedure is scaled down, these ratios should be maintained. However, it is not a good idea to use a large chamber for a small volume of cell suspension. The Bead Beater should not be used in bursts longer than 1 min. Otherwise, the motor may be damaged and, in any case, the cell suspension may overheat. Cycles of 1 min of blending and 1 min of cooling are recommended. Cell disruption may be performed in the cold room, and ice water is frequently used in the jacket to maintain the cell suspension at a low temperature. We have found a better arrangement for maintaining low temperat~re.~' With the 15- and 50-1111 chambers, we utilize a cold ethanol bath. These chambers are mounted within the stainless steel chamber which is filled with ethanol. This arrangement is placed within the cooling jacket, into which we pour ethanol from a dry ice-ethanol bath. This setup allows us to maintain a temperature of 4 to 6" without any difficulty. The ethanol in the stainless steel chamber prevents freezing of the sample. This arrangement can also be utilized with ethanol as the jacket coolant when the stainless steel chamber is used for cell disruption. It is important to wrap Parafilm around the threads of the 15- and 50-ml chambers when assembling the Bead Beater for use with ethanol as coolant, to prevent leakage into the chambers. Finally, it should be noted that filling Bead Beater chambers with much more than half the volume with glass beads generates considerably more heat during blending. A typical protocol that we have used for preparation of yeast extracts using the Bead Beater is as follows:

1. Yeast cells (strain 20B-12: a pep4-3 t r p l ) are grown overnight on a shaker at 30° to a density of 6 X 107/mlin 3 liters of YPD medium. Cells are harvested by centrifugation at 3000 g, for 5 min at 4" and washed once with deionized water. 2. The cells (1.8 x 10"; 12 g wet weight) are resuspended in ice-cold 0.1 M NaCl-0.05 M Tris-HC1 (pH 8.0) containing 1% dimethyl sulfoxide, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM EDTA, 1 mM EGTA, 1 mM benzamidine, 1.5 pglml leupeptin, and 3 pglml pepstatin A to a volume of 25 ml. 3. Glass beads (0.5 mm) are chilled on ice and added to the 50-ml N. E. Jeansonne and S. M . Jazwinski, unpublished observations.

164 -

EXTRACT PREPARATION AND FRACTIONATION -

-

-

-

[I31

-

chamber of the Bead Beater to a volume of 25 ml. The cell suspension is added to this and stirred briefly with a glass rod to remove air bubbles. The chamber is mounted in the stainless steel chamber of the Bead Beater in ice-cold ethanol. This is placed in the cooling jacket of the Bead Beater, which is filled with ethanol from a dry ice-ethanol bath. 4. Homogenization is carried out in two 1-min bursts, separated by 1 min of cooling. Cell breakage is over 90%. 5. The Bead Beater is disassembled, by which time the glass beads have settled. The supernatant is poured off. The beads are rinsed two times with about 3 ml of buffer each. After combining with the rinses, the lysate is clarified by centrifugation at 100,000 g,, for 1 hr at 4O, to remove unlysed cells, cell debris, and organelles. This yields about 25 ml of extract (=20 mg/ml protein). Preparation of Yeast Extracts by Enzymatic Digestion of Cell Wall

The yeast cell wall is composed primarily of the carbohydrate glucan and mannoprotein, a glycoprotein, with a few percent chitin.22Several enzymatic preparations that digest the wall have been described. The major active component of these preparations is a P-1,3-glucanase. This activity effects the hydrolysis of the cell wall in synergy with proteolytic activity specific for the yeast wall. This hydrolysis is greatly enhanced by either pretreatment with sulfhydryl reagent and/or its presence during hydrolysis. In the absence of the protease, sulfhydryl reagent is essential. Two enzyme preparations are recommended. Zymolyase (produced by Kirin Brewery, Japan) is marketed by Seikagaku Kogyo Company, Inc., and is currently distributed in the United States by ICN (Costa Mesa, CA) in the form of Zymolyase 100T. This is a highly purified preparation. However, some lots may contain traces of nuclease activity. The powder is completely soluble in buffers containing 10% sucrose up to a concentration of at least 5 mg/ml. Thus, removal of insoluble material, described by many, is not necessary when sucrose buffers are used. Furthermore, the extremely high concentrations frequently found in the literature are not necessary to obtain high effective concentrations, when all of the powder is solubilized in the presence of sucrose. The other preparation I have used routinely is lyticase.I7 This preparation is not available commercially. However, the published procedure for lyticase purification is straightforward, and it works exactly as written. A large supply of the enzyme can readily be prepared. In my experience, it is very stable (at C. E. Ballou, in "The Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression" (J. N. Strathem, E. W. Jones, and J. R. Broach, eds.), p. 335. Cold Spring Harbor Lab., Cold Spring Harbor, New York, 1982.

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least 1 year) when stored in the succinate buffer after the Sephadex G-150 purification step in the presence of 10 mM sodium azide. If so desired, one can actually separate the alkaline protease from the P-1,3-glucanase activity, but, except for the most exacting requirements, this is not a necessity. Several points are worth remembering when the enzymatic procedure is used. As already mentioned, the presence of sulfhydryl reagent greatly enhances lysis. A variety of such agents may be used. Most frequently, they are dithiothreitol(2 to 30 mM) or 2-mercaptoethanol(l0 to 140 mM). A brief preincubation with the sulfhydryl reagent prior to addition of the enzyme is often used to augment lysis, with removal of the reagent for the digestion step employed from time to time. Digestion of the cell wall is carried out at room temperature to 37", customarily. It can also be carried out on ice. In this case, sulfhydryl reagent must be added, or, in lieu of this, the cells must be frozen and thawed rapidly prior to digestion. Most probably, this facilitates access of the P-1,3-glucanase to the glucan. The amount of enzyme added to the cell suspension depends on the cell density and on the batch of enzyme. The yeast strain and culture conditions can also make a difference. Stationary phase cells are much more resistant to digestion than logarithmic phase cells. The amount of enzyme to add should be determined empirically in small-scale pilot lysates. With many strains of log-phase cells grown in YPD, 0.25 to 0.5 mg/ ml of Zymolyase lOOT or ~ 2 0 0 0unitslml of lyticase is needed in the absence of sulfhydryl reagent for a cell density of 3 to 9 x 109/ml(frozenthawed cells) at 0" for 30 to 60 min. Zymolyase is active over a broad pH range (pH 5 to lo), although the optimum is about pH 7.5. The preparation is active in KC1 up to at least 1.0 M, and it is not inactivated at temperatures up to 45". Lyticase displays a much narrower pH range with an optimum at pH 8.0; it is also active in high salt (up to at least 1.0 M KCl). If the spheroplasts are stabilized osmotically, they can be washed to remove the enzymes. Usually, 0.8 to 1.2 M sorbitol, which is not metabolized by yeast, is used for this purpose. Potassium chloride at 0.6 M can also be used, but this results in some KC1 carrying over to the subsequent lysate. If spheroplasts are not washed, it is important to keep the presence of the enzymes added to digest the cell wall in mind when evaluating the resultant extracts. Spheroplast lysis can be effected in a variety of ways. A brief heat shock or osmotic shock can be used. The spheroplasts are sensitive to nonionic detergents such as Triton X-100 or Brij 58 at about 0.1%. They can also be disrupted by homogenization in either the Dounce homogenizer or the Potter-Elvehjem glass-Teflon homogenizer. Yeast extracts can be prepared, as stated above, by isolating spheroplasts as an intermediate step (Procotol A) or directly (Protocol B).

166

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Protocol A 1. Yeast cells (strain A364A: a adel ade2 ural his7 lys2 tyrl gall) are grown overnight at 30" in YPD on a shaker, to a density of 2 X lo7 cells/ ml. They are harvested at 4" by centrifugation for 5 min at 3000 g,, . 2. The cell pellet is resuspended in 1 M sorbitol-50 mM Tris-HC1 (pH 7.5)-30 mM dithiothreitol at a density of 1 x lo9 cellslml and incubated for 10 min at 30". 3. The cells are collected by centrifugation as above and resuspended in an equal volume of the same buffer (to about 5 x lo9 cells/ml), except that the dithiothreitol concentration is 2 instead of 30 mM. 4. Zymolyase lOOT (5 mglml in 10% sucrose) is added to a final concentration of 0.5 mglml. The suspension is swirled occasionally while being incubated at 30" for 30 min. Spheroplasting is monitored by phasecontrast microscopy. The spheroplasts appear as small, round, bright, refractile bodies that burst within a few minutes of dilution in water. (Over 95% of the cells become spheroplasts.) 5. The spheroplasts are collected by centrifugation at 1000 g,, for 5 min at 4". (Sedimentation at high g force results in rupture of spheroplasts under their own weight.) The spheroplasts are washed twice with 4 vol of the buffer used for preparation of spheroplasts, to remove the Zymolyase. (Spheroplasts must be resuspended gently to prevent premature lysis.) 6. The final spheroplast pellet is resuspended in 50 mM Tris-HC1 (pH 7.5)-2 mM dithiothreitol at 5 X lo9 spheroplastslml. Lysis is complete after addition of 100 mM EDTA (pH 8.0) to 1 mM, 10% Triton X-100 to 0.2%, and 4 M KC1 to 0.2 M, with gentle mixing. 7. The lysate is centrifuged at 100,000 g,, for 1 hr at 4O, and the supernatant contains =20 mg/ml protein. Protocol B 1. Yeast cells (strain A364A) are cultured and harvested at room temperature, as described in Protocol A above. 2. The cell pellets are drained well and resuspended at 6 x lo9cellslml in 10% sorbitol-50 mM Tris-HC1 (pH 8.0). The pH of the suspension is adjusted to 8.2 with solid Tris base, and the suspension is frozen rapidly in liquid nitrogen in an ultracentrifuge tube. (Freezing and thawing is essential in this procedure.) 3. The suspension is thawed rapidly and placed on ice. To the suspension are added the following: 4 M KC1 to a concentration of 1 M, 100 mM spermidine (pH 7.8) to 5 mM, and either lyticase to ~ 2 0 0 0unitslml or Zymolyase lOOT (5 mglml) to 0.5 mglml. After gentle mixing, the suspension is incubated for 30 min on ice, with occasional swirling. Over 90% of

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the cells are converted to spheroplasts. (Sulfhydryl reagent is absent during the actual lysis, although it is added later, because this improves the yields of DNA replicating activity for which lysis was optimized in this case.) We have carried out this spheroplasting procedure in the presence of several protease inhibitors (EDTA, EGTA, PMSF, benzamidine, pepstatin A, leupeptin) with virtually identical yields.23 4. Brij 58 (5%) is added to a concentration of 0.1% and the spheroplasts are heat shocked for 1 min at 37". For volumes larger than 1 ml, a longer incubation (up to 3 min) is appropriate. The lysate is clarified by centrifugation at 100,000 g,, for 1 hr at 4'. The supernatant contains 15 to 20 mglml protein. Choice of Buffers An appropriate choice of solution in which the cells will be lysed is important. This choice is dictated by compatibility with both the method of lysis and subsequent purification steps. Both the glass bead and the enzymatic methods of yeast lysis are compatible with a broad range of buffers and salts. These lysis procedures will very rarely be the factor limiting the selection. A prime consideration in devising the lysis protocol is stabilization of enzymatic activity. Besides pH and ionic strength, such variables as the presence of enzyme cofactors and divalent cations must be kept in mind. A wide range of specific protein-stabilizing compounds have been employed in yeast extracts. These include glycerol (5 to 20%), ethylene glycol (lo%), dimethyl sulfoxide (I%), sorbitol (lo%), and mannitol(0.25 to 0.4 M). Of course, the single most important stabilizing factor is a high concentration of protein. However, at high concentrations the danger of protein aggregation exists. We have been able to largely avoid this problem in the presence of 0.5 to 1.0 M KCl. A lower concentration of ammonium sulfate can be used to achieve the same ionic strength, and may also prove useful. However, if the salt concentration is too high, protein precipitation may occur. Removal of Nucleic Acids The high-speed centrifugation used to clarify the crude yeast extracts obtained in many different procedures certainly serves to remove large DNA fragments. The glass bead method for obtaining extracts generates considerable shear, resulting in small DNA fragments. When shear is avoided, as in Protocol B above, the enzymatic methods lend themselves 23

N. E. Jeansonne and S . M. Jazwinski, unpublished observations.

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well to removal of DNA by centrifugation. This removal is augmented in high salt and in the presence of spermidine, as described in Protocol B above. There are several other ways to remove nucleic acids from yeast extracts. One procedure involves the slow addition of the polycation polymin P (polyethyleneimine, BASF)24from a 10% solution at pH 7.5 to a final concentration of 0.25 to 0.8%. The extract is stirred for 10 to 30 min, and the precipitate is collected by centrifugation at 10,000 gavfor 15 min. A similar result can be achieved by the addition of 30% polyethylene glycol 8000 to a concentration of 6% with 4 M NaCl to a concentration of 0.4%. With both of these procedures, it is important to keep in mind that the precipitate may contain the activity of interest, particularly when dealing with an enzyme of nucleic acid metabolism. Finally, 2.5% protamine sulfate has been added to yeast extracts to give a final concentration of ~ 0 . 2 % with stirring.2s After 30 min, the nucleic acid precipitate was removed by centrifugation. Residual nucleic acids can be removed from yeast extracts by passage over DEAE-cellulose columns equilibrated with buffers containing 0.3 M KC1 to prevent the retention of proteins. Bed volumes equal to 0.05 to 0.1 the volume of the extract are usually sufficient. With such small columns, there is no dilution of the extract, for all practical purposes. Additional Comments

Two additional comments should be made. First, the concentrated extracts obtained using the procedures described above contain significant lipid material. This can readily be removed by centrifugation of the high-speed supernatants at 40,000 gavfor 10 min at O". The clear amber extract is then removed from below the lipid pellicle. Second, the extracts obtained above are somewhat acidic given the buffering capacities usually employed. The pH can be adjusted by addition of solid Tris base. However, storage at around pH 8.0 for any significant length of time can result in the formation of 2 precipitate. Avoiding Proteolysis

Proteolysis can pose severe problems during fractionation of yeast extracts. Frequently, the problem is only evident at the end of the purification. Many proteins even in a truncated form appear to maintain activ24

25

R. R. Burgess and J. J. Jendrisak, Biochemistry 14,4634, 4639 (1975). G. Badaracco, P. Plevani, W. T. Ruyechan, and L. M. S. Chang, J. Biol. Chem. 258,2022 (1983).

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ity, making it difficult to assess the damage from a knowledge of the pure protein alone. These comments are not meant to indicate that proteolysis is any more of a problem in yeast than in other eukaryotes. One of the best ways of avoiding the problem has already been mentioned; namely, the use of an appropriate protease-deficient yeast strain (pep4-3, prbl, prcl). A detailed treatment of the protease problem and its alleviation has been presented .26 A wide variety of proteases is present in the yeast cell. The major proteases are the vacuolar proteases A, B, and C (or carboxypeptidase Y), which are absent or present at low levels in the protease-deficient strains. Protease A is an acidic endoproteinase that is largely inactive below pH 6. This enzyme is completely inhibited by pepstatin A. Serine protease inhibitors are quite effective with proteases B and C, and metal chelators block the metalloproteases. Fortunately, yeast contain endogenous inhibitors for proteases A, B, and C and, in fact, these enzymes are found as complexes with their inhibitors in crude extracts around neutral pH. On the basis of the known chromatographic properties of the protea ~ e s ,protein *~ fractionation can be designed around ridding the extract of the offending proteolytic activities,' a feat that cannot always be conveniently achieved. Rapid and selective purification steps, such as affinity chromatography, can be very helpful in avoiding proteolysis. Whether or not a given purified protein has suffered from the ravages of protease attack is usually not clear from the properties of the purified protein itself. Once a pure protein is available, an antibody can be prepared. This antibody can be used to identify the polypeptide that corresponds in size to the native protein in rapid, hot, sodium dodecyl sulfate extracts of yeast cells by means of appropriately carried out immunoprecipitations or Western blots. Obviously, cloning of the cognate gene can prove very useful for determining the expected molecular weight of the native protein. Rapid protein extracts of yeast cells for separation by electrophoresis in denaturing polyacrylamide gels or for immunoprecipitation can be prepared in a variety of ways: 1. Yeast cells (1 to 3 x lo8)are collected rapidly by centrifugation and washed once with cold, deionized water. They are resuspended in 100 p1 of cold lysis buffer containing protease inhibitors, as described below [lysis buffer = 50 mM Tris-HC1 (pH 7.5)-1% sodium deoxycholate-1% Triton X-100-0.1% sodium dodecyl sulfate], in a 1.5-ml microfuge tube. An equal volume of glass beads is added (2120 pl) and the suspension is vortexed for 30 sec, followed by chilling on ice for 10 sec, three to five times, until the bulk of the cells is lysed. The lysate is centrifuged in a 26

J. R. Pringle, Methods Cell Biol. 12, 149 (1975).

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microfuge for 15 min in the cold. Samples of the supernatant are applied to polyacrylamide gels or used for immunoprecipitation. 2. Yeast cells are collected and washed as described above. The cells are resuspended in 100 p1 of 50 mM Tris-HC1-1 mM EDTA-1% 2-mercaptoethanol-1% sodium dodecyl sulfate (pH 7.5) and disrupted by vortexing with an equal volume of glass beads, as above. After clarification of the lysate by centrifugation, the supernatant is analyzed by gel electrophoresis. 3. Yeast cells are harvested as described above and resuspended in an appropriate SDS-polyacrylamide gel sample buffer [for example, 2% sodium dodecyl sulfate-10% glycerol-5 mM EDTA-5% 2-mercaptoethanol-0.1 mglml Bromphenol Blue-125 mM Tris-HC1 (pH 6.8)]. The sample is heated immediately at 100" for 3 min and clarified by centrifugation for 15 min in a microfuge, before loading on the gel. Exogenous Protease Inhibitors

As already alluded to above, the great variety of proteases in yeast extracts mandates the use of a selection of protease inhibitors. Some of these inhibitors have already been mentioned, along with the major target enzymes. In general, it is possible to state that the greater the number of different inhibitors used the better. This is largely true regardless of the overlapping specificities of the various compounds. It is most beneficial to add the inhibitors to the cell suspension prior to lysis or as shortly thereafter as possible. If at all feasible, the inhibitors should be present at every stage of the purification. The most widely used protease inhibitor in studies with yeast is phenylmethylsulfonyl fluoride (PMSF). Amounts ranging from 0.1 to 1 mM have been used. I recommend 1 mM. PMSF is not readily soluble in water; however, a 100 mM stock solution in 95% ethanol or dimethyl sulfoxide can be prepared and stored at -20". PMSF possesses a half-life of about 100 min at pH 7, and this is even shorter at higher pH. Due to this hydrolysis, PMSF must be constantly readded to solutions during purification. This compound is compatible with most purification procedures. Similarly, EDTA and EGTA, which are commonly used at 1 mM concentration, are widely compatible. EDTA, in particular, is a widely used inhibitor. The compound 1,lO-phenanthroline at 5 to 10 mM has been used infrequently in place of EDTA but is very effective. Unlike EDTA and EGTA, this substance must be first dissolved in ethanol. Benzamidine is used at 0.5 to 4 mM, and it is soluble in either water or ethanol. It has been used often, and it is for the most part interchangeable with pepstatin A. Both pepstatin A and leupeptin have come into wide use

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recently. These compounds should be dissolved in dimethyl sulfoxide at about 1 mglml each and diluted into the final buffer. Pepstatin A is effective at 1 to 3 pglml. Amounts ranging between 0.4 and 20 pglml have been reported as effective in the case of leupeptin; 0.4 to 3 pglml should be sufficient. Aprotinin (1 to 50 pglml reported) and soybean trypsin inhibitor (at the same final concentrations) are water soluble, but have not been used as often as most of the inhibitors listed above. Finally, the watersoluble compound sodium metabisulfite at final concentrations of 1 to 20 mM has been used from time to time, particularly to prevent histone proteoly sis. Subcellular Fractionation

Nuclei Yeast nuclei have been until recently difficult to prepare in good yield at reasonable purity. This is because this organelle appears to be extremely fragile. Happily, this situation has been remedied with the development of two rapid protocols that are similar in certain details. Protocol A2'

1. Exponentially growing yeast cells are harvested by centrifugation and washed once with deionized water. The cell pellet is resuspended in 1.1 M sorbitol-20 mM KP04 (pH 6.5)-0.5 mM CaC12-0.5% 2-mercaptoethanol at a density of =2 x 109/ml. Zymolyase IOOT is added to a final concentration of 0.1 mglml, and the suspension is rocked gently at 37" for 30 to 60 min, until spheroplasting is complete. 2. The spheroplasts are harvested by centrifugation at 1000 g,, for 5 min. All further manipulations are carried out at 4,and all solutions contain 0.5 mM PMSF. The spheroplast pellet is resuspended gently with a glass rod in 18% Ficoll400 (Pharmacia) dissolved in 20 mM KP04 (pH 6.5)-0.5 mM CaC12 (0.1 ml/lO1Ocells). The spheroplast suspension is diluted with 3 ml of this buffer per 101° cells, and the spheroplasts are broken by homogenization with a loose-fitting Potter-Elvehjem glassTeflon homogenizer. (A Dounce homogenizer with an "A" pestle can also be used.) 3. The nuclear suspension is diluted with an equal volume of 1.0 M sorbitol-0.5 mM CaC12 (pH 6.5), and 2 to 10 ml of this is loaded onto an =30-ml preformed Percoll (Pharmacia) gradient. The gradients are centriI7G. J. Ide and C. A. Saunders, Curr. Genet. 4, 85 (1981).

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fuged at 9500 g, for 20 min in a swinging bucket rotor. The opaque band of nuclei is found near the middle of the gradient. 4. Percoll gradients consist of 32.5% Percoll for haploid nuclei and 35% Percoll for diploid nuclei in 1.O M sorbitol-0.5 mM CaC12 (pH 6.5). Gradients are preformed by centrifugation at 27,000 g, for 50 min in a fixed angle rotor. 5. The nuclei from the gradient are washed by dilution with 2 vol of ,, for 5 min in 1.0 M sorbitol-0.5 mM CaClz (pH 6.5) and pelleted at 4000 g a fixed angle rotor. These nuclei are stored in the buffer used for preparing spheroplasts with or without being washed in this buffer. The nuclei are examined by phase-contrast microscopy. The nuclei show minimal cytoplasmic contamination, as determined by assay of alcohol dehydrog e n a ~ e . ~The ' chromatin in these nuclei displays the "native" pattern of nucleosome phasing, and the histones in these nuclei are intact. Finally, the nuclei are active in transcription, and, in fact, can initiate RNA synthesis. The absence of Mg2+and detergents during the preparation contributes to the native chromatin structure and may facilitate the use of the nuclei in transcription studies. Protocol B2 1. Unless indicated otherwise, all steps are carried out at room temperature. Logarithmic phase yeast cells are harvested and resuspended in 1 M sorbitol-50 mM KP04 (pH 7.8)-0.5 mM PMSF-10 mM MgC1230 mM dithiothreitol to a density of 1 x 109/mland incubated at 30" for 10 min. 2. The cells are collected by centrifugation and resuspended in 1 M sorbitol-10 mM MgC12-2 mM dithiothreitol-0.5 mM PMSF-25 mM KP04 + 25 mM sodium succinate (pH 5.5) at 5 x 109/mland swirled very gently in a thin layer at 30' for 2 min. Zymolyase lOOT is added to a final concentration of 1 mg/ml, and the incubation is continued for 20 to 30 min, until spheroplasting is complete. 3. The spheroplasts are pelleted and the pellet is drained thoroughly. They are resuspended in about the same volume of 40 mM PIPES (pH 6.8) or MES (pH 6.4)-10 mM MgC12-0.5 mM PMSF-0.2% Triton X-100 by gentle stirring with a glass rod. They are finally brought up to about 1 x 109/mlwith the same buffer. 4. The spheroplasts are disrupted by homogenization in a PotterElvehjem homogenizer with a Teflon pestle at 500 rpm. 5. The nuclei are pelleted and resuspended at about 4 x lo8 cell equivalentslml in freshly prepared 50% Percoll-40 mM PIPES (pH 6.8) or MES (pH 6.4)-10 mM MgC12-0.5 mM PMSF-0.05% Triton X-100. The solu-

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tion is distributed into tubes and centrifuged for 35 min at 38,000 g,, in a fixed angle rotor with slow acceleration. 6. Three bands are visible: the top band containing cytoplasmic material, the middle band cell debris, and near the bottom a sharp, white band containing nuclei. The nuclei are withdrawn with a wide-bore Pasteur pipet. The Percoll is removed by diluting the sample with 40 mM PIPES (pH 6.8) or MES (pH 6.4)-10 mM MgC12-0.5 mM PMSF and centrifugation. The identity of the nuclei is confirmed by epifluorescence microscopy after staining with Hoechst 33258 at 5 pg/ml. Nuclei prepared in this fashion have been used as starting material in the preparation of yeast nuclear matrix. l 4 Mitochondria

Several approaches for the preparation of mitochondria from yeast cells have been developed. One well-established procedure for the preparation of intact mitochondria is described below.28 1. Haploid, wild-type yeast cells are grown aerobically to early logarithmic phase on a minimal medium containing (per liter): 3 g yeast extract, 1 g glucose, 1 g KH2P04,1 g NH4C1, 0.5 g CaC12- 2H20,0.5 g NaC1, 0.6 g MgS04 H 2 0 , 3 mg FeC13, and 19.8 g lactic acid, adjusted to pH 5.5 with KOH to induce mitochondria. Cells are harvested by centrifugation, washed once with deionized water, suspended at =5 x 109/ml in 0.1 M Tris-SO4(pH 9.4)-10 mM dithiothreitol, and incubated for 10 min at 30". 2. The cells are washed once with 1.2 M sorbitol and resuspended in 1.2 M sorbitol-20 mM KP04 (pH 7.4) at =1.5 x 109/ml;Zymolyase IOOT is added to a final concentration of 0.1 mglml and the suspension is incubated for 30 to 60 min at 30" with gentle swirling, until spheroplasting is complete. 3. The spheroplasts are harvested by centrifugation at room temperature and washed twice with 1.2 M sorbitol. They are suspended in 0.6 M mannitol-10 mM Tris-HC1 (pH 7.4)-0.1% bovine serum albumin-1 mM PMSF to a concentration of 0.15 g spheroplasts (wet weight)/ml. After chilling on ice, the spheroplasts are homogenized by 10 to 15 strokes of the tight-fitting pestle in a Dounce homogenizer. 4. All subsequent steps are carried out in a cold room and centrifugation is performed at 2". The spheroplast lysate is diluted with 1 vol of the homogenization buffer and centrifuged for 5 min at 1000 g,, . The pellet is rehomogenized as above, and the centrifugation is repeated. 28

G. Daum, P. C. Bohni, and G . Schatz, J . Biol. Chem. 257, 13028 (1982).

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5. The supernatants are combined and centrifuged at 8000 gavfor 10 min. The crude mitochondrial pellet is carefully resuspended in homogenization buffer and centrifuged for 5 min at 1000 gavto remove residual cell debris. The supernatant is centrifuged at 8000 g,, for 10 min. The mitochondrial pellet is washed twice by resuspension and recentrifugation. The final mitochondrial pellet is resuspended in 0.6 M mannitol-10 mM Tris-HC1 (pH 7.4). The purity of the preparation is assessed by the assay of marker enzymes for mitochondria (isocitrate dehydrogenase) and cytosol (glucose-6-phosphate dehydrogenase) using spectrophotometric assays.29 Other Organelles

The preparation and use of translocation- and glycosylation-competent intracellular membranes from yeast have been described.30Similarly, the isolation of the yeast vacuole (the equivalent of the lyso~ome)~' and of coated vesicles from yeast32have been detailed. Acknowledgments The work in the author's laboratory was supported by grants from the National Science Foundation, the Edward G. Schlieder Educational Foundation, and the National Institute on Aging of the National Institutes of Health.

H. U. Bergmeyer, ed., "Methods of Enzymatic Analysis," 3rd Ed. Verlag Chemie, Deerfield Beach, Florida, 1983. J. A. Rothblatt and D. I. Meyer, Cell 44, 619 (1986). 31 T. Stevens, B. Esmon, and R. Schekman, Cell 30, 439 (1982). S . C. Mueller and D. Branton, J . Cell Biol. 98, 341 (1984). 29

[I41 Preparation of Extracts from Plants By PETERGEGENHEIMER

This chapter surveys some of the particular pitfalls of working with plant materials, along with solutions to these problems. Protocols are given for preparation of active cytoplasmic and chloroplastic extracts from a variety of plant tissues. A previous volume in this series,' devoted to plant molecular biology, presented protocols for purification of a variety of plant nuclear, cytoplasA. Weissbach and H. Weissbach, eds., this series, Vol. 118.

METHODS IN ENZYMOLOGY. VOL. 182

Copyright O 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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mic, and chloroplast proteins. Chapters in that volume will provide further information specific to a particular enzyme or class of enzymes. In addition, methods for purification of several chloroplast enzymes are described in the volume edited by Edelman et a1.2 Components of Plant Cells

Cell Walls. Plant cells resemble bacterial cells more than animal cells in that they possess a rigid cell wall. Like animal tissues, however, many plant tissues are fibrous and difficult to macerate. The more fibrous material can be homogenized by freezing and grinding to a dry powder prior to addition of extraction buffer. When intact organelles must be isolated, however, there is a clear trade-off between yield and intactness. Vacuole. The vacuole of a plant cell serves the functions both of a . ~ a mature leaf cell, the vacuole may storage vessel and of a l y s ~ s o m eIn occupy 90% of the cell's volume, the remainder being a "film" of cytoplasm and organelles. The vacuole can be a storage vessel for inorganic ions, tricarboxylic acid (TCA) cycle intermediates, some alkaloids, and sucrose and other sugars. In addition, vacuoles may contain various hydrolases including proteases. The effects of these components are discussed below. Polyphenols. Of prime bedevilment to the plant biochemist is the fact that in many species, and in specialized cells, the vacuoles contain alkaloids and the polyphenolic compounds known as flavonoids and tann i n ~ .These ~ . ~ compounds may inhibit enzyme activity directly or indirectly. They can react with and inactivate proteins by hydrogen bonding with peptide bond oxygens, or by covalent modification of amino acid residues. Susceptible amino acid moieties are hydroxyls, thiols, and primary amines. Phenolics and alkaloids are removed from the crude plant extract by complexing with poly(vinylpyrro1idone) or by gel filtration (as described below). The thorough examination by Loomis4 of phenolics in plant extracts should be consulted for more detailed coverage of this problem. Proteases. Plant tissues contains a diverse assortment of proteases (see Refs. 5-8 for reviews). Seeds are particularly rich in proteases used M. Edelman, R. B. Hallick, and N.-H. Chua, eds., "Methods in Chloroplast Molecular Biology," Chaps. 54-59 and 75-79. Elsevier, Amsterdam, 1982. T. W. Goodwin and E. I. Mercer, "Introduction to Plant Biochemistry." Pergamon, Oxford, 1983. W. D. Loomis, this series, Vol. 31 [54]. C. A. Ryan and M. Walker-Simmons, in "The Biochemistry of Plants" (A. Marcus, ed.), Vol. 6. Academic Press, New York, 1981.

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to hydrolyze storage proteins during the late stages of germination and during embryonic development. An excellent summary of practical information for the use of protease inhibitors during preparation of plant extracts is found in Ref. 7, and general information is provided in Ref. 9. Specific points are described below. Organelles. Plant cells contain, apart from the cytoplasm, four organelles from which enzymes might be purified. These are the nucleus, plastid, mitochondrion, and vacuole. In green tissue the major organelle (other than the vacuole) is the chloroplast, the differentiated photosynthetically active form of the plastid. The abundance of chloroplasts in leaf cells, coupled with the semiautonomous character of their metabolism, presents a special problem. Numerous cytoplasmic and nuclear biochemical pathways are partially duplicated in the chloroplast, such that a given enzyme assay may detect both cytosolic and chloroplastic activities. Techniques are available for isolation of intact chloroplasts, mitochondria, and nuclei from plant cells; the most rigorous purification available for any organelle is that for the chloroplast. We will discuss, below, guidelines for choosing starting material and for preparation of extracts suitable for purification of chloroplast or cytoplasmic enzymes. Choice of Material

For preparation of cytoplasmic, nuclear, or mitochondria1 activities, a tissue devoid of chloroplasts is desirable. Commonly used are roots from dark-sprouted peas, soy or other beans, wheat, or maize. Other useful sources are cauliflower inflorescences and carrot roots; fresh supermarket material has proved adequate for many investigators. Finally, wheat embryo is widely used because it is very active for many enzymes, is convenient to work with, and is available in large amounts. Wheat embryo is available in the form of raw wheat germ. For preliminary trials, wheat germ can be obtained from Sigma (St. Louis, MO). Large (1 to 10 kg) amounts may also be obtained directly from commercial mills. We previously worked with General Mills (Vallejo, CA),1° which no longer supplies wheat germ to the research community. Other sources are Interna-

M. J . Dalling, ed., "Plant Proteolytic Enzymes," Vols. 1 and 2. CRC Press, Boca Raton, Florida, 1986. ' J . C. Gray, in "Methods in Chloroplast Molecular Biology" (M. Edelman, R. B. Hallick, and N.-H. Chua, eds.), p. 1093. Elsevier, Amsterdam, 1982. H. Krauss, Annu. Rev. Plant Physiol. 38, 47 (1987). C. H . Suelter, "A Practical Guide to Enzymology." Wiley, New York, 1985. lo Quality Control Manager, General Mills, Inc., Sperry Division, P.O. Box 32, Vallejo, CA 94590.

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tional Multifoods (Minneapolis, MN) or other commercial mills. In our experience, enzyme activity is unstable unless the raw germ is stored at -70". For purification of chloroplast activities, green leaves free of fibers and low in tannins are best. An ideal source is spinach, which is available in bulk from supermarkets, farmer's markets, or local farms; the best year-round bulk commercial source is a local produce wholesaler. For preparation of labile enzymes, freshly harvested leaves from plants grown hydroponically or in vermiculite are said to give the most consistent results."J2 The freshest possible material should always be used. The use of genetically characterized lines or cultivars is recommended, especially when protein purification is a prelude to isolation of the corresponding gene. Normally, the race or cultivar would be one for which a genomic or cDNA library is available. In the absence of other information, suggested lines include the following: spinach, Spinacea oleracea hybrid No. 424, or nonhybrid lines; peas, Pisum sativum cv. "Laxton's Progress No. 9" (this is a compact gibberellic acid-deficient line); soybean, Glycine max cv. "Williams-82" (others are also suitable); wheat, Triticum aestivum cv. "Yamhill" (many others are also in common use); maize, Zea mays FR9 x FR37 (a cms line); tobacco, Nicotiana tabacum cv. "SR1," "Samsun 6," or "Bright Yellow." Most of these seeds may be obtained from commercial farm supply houses, commercial seed suppliers, or from The Illinois Foundation Seed Company.I3 Most seeds and berries can be planted in trays of soilless potting mix and sprouted in a greenhouse or growth chamber. Peas and beans are planted in trays of vermiculite; peas may be grown to maturity in vermiculite with liquid fertilizer. Other species may be planted densely if they are to be harvested young; otherwise they should be transplanted to soilless potting mix at a density of ca. 1 seedling115 in.2. Typical growth regimens are as follow: spinach, 10-12 hr light (20-22"), 12-14 hr dark (16-18"); peas, 24 hr light (20-22"), or else 16 hr light, 8 hr dark (18"); wheat and maize, 16" hr light (25-28"), 8 hr dark (22"). Experience has shown that some adjustment may be required to optimize these regimens. The organism most suited to genetic and molecular genetic manipulations, and the object of current intensive genetic research, is the miniature It has a streamlined genome consisting crucifer Arabidopsis thaliana.14,'5 E. M. Orozco, Jr., J. E. Mullet, L. Hanley-Bowdoin, and N.-H. Chua, this series, Vol. 118 [IS]. l2 W. Gruissem, perscnal communication. l 3 The Illinois Foundation Seed Co., Champaign-Urbana, IL. l4 R. E. Pruitt and E. M. Meyerowitz, J . Mol. Biol. 187, 169 (1986). E. M. Meyerowitz and R. E. Pruitt, Science 229, 1214 (1985).

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-

of about 7 x lo7 base pairs (bp), containing little repetitive DNA. A genomic library can easily be contained on a single Petri dish. In extreme contrast, the pea genome is about 5 x lo9 bp, containing over 90% repetitive sequences. Although Arabidopsis is not an obvious replacement for leafy plants like spinach, any investigation which may lead to gene isolation or reverse genetics should take into account the suitability of Arabidopsis for these purposes. For cultivation of Arahidopsis, see Ref. 16. Preparation of Extracts

There are several decisions which must be made prior to or during preliminary trials. First, what plant species and tissue is most favorable for purification of the desired activity? This choice has been considered above. Second, if the activity is organellar, can it and should it be purified from isolated organelles, or from a mixed preparation of cytoplasm and lysed organelles? Third, what cofactors, metal ions, and reductants, if any, must be present during purification to yield active enzyme? These considerations are specific to each enzyme and will not be discussed here. Fourth, what substances present in the crude extract inhibit enzyme activity, and what protective agents may be added which are compatible with the necessary "activators" mentioned above? This section discusses guidelines for deciding between a whole-cell and an organellar extract, describes the most common and useful methods for protecting enzyme activity during extract preparation, and presents protocols for preparing extracts containing cytoplasmic and nuclear enzymes from a variety of sources: dry embryos (wheat germ), dry seeds (e.g., soybeans), bulky, fleshy material (e.g., carrots), and whole-cell extracts from leaves. Finally, methods are given for preparation of extracts from isolated chloroplasts. Type of Extract

Consideration must be given to the subcellular location of the activity being sought, and to the trade-offs between speed, yield, and purity. The presence of chloroplasts makes it exceedingly difficult if not impossible to prepare from plant leaves a cytoplasmic extract which is completely free of chloroplast contents. Even the most careful breakage of cell walls will damage some chloroplasts. If enzymes with the desired activity are found both in cytoplasm (or nucleoplasm) and in chloroplasts, the extract must be made from a nongreen tissue, or the purification scheme must be l6

C. R. Sornerville and W. L. Ogren, in "Methods in Chloroplast Molecular Biology" (M. Edelman, R. B. Hallick. and N.-H. Chua, eds.), p. 129. Elsevier, Amsterdam, 1982.

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designed to separate cytosolic from choroplastic activities. Similarly, a purification of chloroplast enzymes may start with isolated chloroplasts or with a total cell homogenate in which the chloroplasts have already been lysed. Plastid purification itself accomplishes a substantial purification of enzyme activity and in some instances removes interfering material. The convenience of bypassing chloroplast isolation must be weighed against the possible difficulty of separating the desired chloroplast enzyme from similar cytoplasmic activities. To determine whether an activity (detected in the chloroplast fraction) is a true chloroplast enzyme or a cytoplasmic contaminant, compare the enzyme's specific activity (per milligram protein or per milligram chlorophyll) in a whole-leaf homogenate with that in the crude chloroplasts and in density gradient-purified intact chloroplasts (both prepared as described below). Enrichment of activity with chloroplast purification is evidence that the activity is located in the chloroplast. Homogenization Buffer

The buffer used for homogenization of plant tissues will typically include high concentrations of reductant, of polyphenol inactivators, and of covalent protease Labile compounds are added to the buffer immediately before tissue homogenization. Covalent inhibitors need be present only during extract preparation; competitive inhibitors should be present at all stages of the purification. Prior to a large-scale purification, trials should be performed, as outlined below, to ascertain what protective compounds are necessary in the homogenization buffer. Their concentration in column buffers can be reduced l O - f ~ l d . ' ~ Reducing Agents. Many plant enzymes, for example those of the chloroplast stroma, require a reducing environment for activity. Typically, the preferred reductants are dithiothreitol (DTT) at 2 to 5 mM, or 2-mercaptoethanol at 14 mM (a 1 : 1000 dilution of the -14 M stock solution). For isolation of chloroplasts, commonly used reductants are the more physiological compounds ascorbate and reduced glutathione. Some chloroplast enzymes are regulated by dithiol reduction, and can be activated only by DTT. DTT: Prepare a 1.0 M stock in water; store at -20" Ascorbate: Prepare a 0.5 M stock solution of sodium ascorbate in 50 mM HEPES-NaOH, pH 7.6; store at -20" Glutathione: Prepare a 0.1 M stock of reduced glutathione plus 5 mM DTT in water; store at -20" l7

J. H. Wong, B. C. Yee, and B. B. Buchanan, J . Biol. Chem. 262, 3185 (1987). L. Pick and J. Hurwitz, J . Biol. Chem. 261, 6684 (1986).

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All these reagents are prone to oxidation. Stocks should be stored in small aliquots and discarded if there is any question about their effectiveness. Polyphenols. Polyphenols are inactivated as follows: (1) by complexing with 1.5% (w/v) insoluble poly(vinylpyrro1idone). Add solid insoluble poly(vinylpyrro1idone) (called polyvinylpolypyrrolidone, PVPP) to the buffer 2 to 24 hr before use; allow to hydrate fully; (2) by maintaining a strong reducing environment to counteract the effect of phenol oxidases. Add dithiothreitol or 2-mercaptoethanol as above; also add 20 mM sodium diethyl dithiocarbamate (preferred) or 10 mM sodium metabisulfite (add solid or from 0.1-0.2 M stocks in water); (3) by using buffers containing borate (which binds cis-diols, including many polyphenols) which are reported to inhibit polyphenol oxidationI9; finally, (4) by using buffer exchange chromatography on BioGel PdDG or Sephadex G-50 (described under Posttreatment), which efficiently removes inhibitory material,20but is practical only when the extract is prepared on a small scale. Preparation of active extracts from plant species with high levels of tannins and other phenolic compounds has been examined for the purpose of routine assay for isozyme marker^.^,^^-^^ Fibrous tissue must be ground to a fine powder after freezing in liquid nitrogen. The most important consideration is the inclusion of PVPP or PVP (2 to 4% soluble PVP, M , 40,00024)in the extraction buffer. Addition of other polyphenol-binding agents (0.2 M sodium tetraborate) or antioxidants [0.25 M sodium ascorbate, 0.02 M sodium metabisulfite, 0.02 M sodium diethyl dithiocarbamate, or 10% (v/v) Me2SO (DMSO)] is necessary only for the most troublesome species. Protease Inhibitors. Serine protease inhibitors: ) Phenylmethylsulfonyl fluoride (PMSF): Prepare a 100 mM ( 1 0 0 ~stock in dry 2-propanol or "anhydrous" ethanol (95% ethanol, 5% 2-propanol, obtained from IBI, New Haven, CT). The stock is stored at room temperature and is stable for at least 6 months. Immediately (within 5 min) before use, the buffer is brought to 1 mM PMSF. The stock solution is added slowly with constant stirring, from a plastic disposable pipet whose tip is submerged in buffer Benzamide: A 0.1 M stock is prepared in ethanol. It is convenient to dissolve benzamide together with PMSF (in 2-propanol or anhydrous ethanol) E. E. King, Phytochemistry 10, 2337 (1971). D. Mills, personal communication. 21 W. A. Kelley and R. P. Adams, Phytochemistry 16, 513 (1977). 22 R. H. Chapman, E. J. Klekowski, and R. K. Selander, Science 204, 1207 (1979). 23 D. E. Soltis, C. H. Haufler, and G. J. Gastony, Sys. Bot. 5, 30 (1980). D. E. Soltis, C. H . Haufler, D. C. Darrow, and G . J. Gastony, Am. Fern J. 7 3 , 9 (1983). l9

20

"

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PREPARATION OF EXTRACTS FROM PLANTS

181

Benzamidine . HCI (or p-aminobenzamidine - 2HC1, a more potent, and more expensive, inhibitor): Prepare an aqueous 0.1 M stock; use at 1 mM E-Amino-n-caproicacid: Prepare a 0.5 M stock in water; use at 5 mM final concentration. We dissolve the benzamidine and caproic acid together to give a lOOx stock Aprotinin (Trasylol): The use of PMSF plus the three competitive inhibitors reduces the need for aprotinin, which is expensive, and we do not routinely use it. It may be prepared as a 1 mglml stock in sterile water. Add to buffer at 1-2 pglml (10-50 kallikrein unitslml) just before use. Since aprotinin contains three disulfide bonds,25it might be inactivated by the high levels of reductants used in most plant extraction protocols. The combination of benzamidine plus benzamide is potentially as effective as aprotinin Cysteine (thiol) prorease inhibitors: Sodium p-hydroxymercuribenzoate [PHMB; the sodium salt of pchloromercuribenzoic acid (PCMB)]: Prepare a 100 m M stock in water; adjust to pH 8 with NaOH or solid HEPES as necessary. Add to homogenization buffer to a final 1 mM The competitive inhibitory proteins antipain and leupeptin (Sigma): These can be added to 1 pglml from 0.1 to 1 mglml stocks in distilled water The covalent inhibitors iodoacetate and N-ethylmaleimide: These are generally not very effective in plant leaf extracts; PHMB is often effective.' These covalent modification reagents are not active, however, in the presence of the reducing agents in the standard homogenization buffer Aspartate (acidic) protease inhibitors: Working at pH 7 or above is probably a reasonable defense against aspartate proteases. Further protection can be afforded by the following: Pepstatin (Sigma), a weak competitive inhibitor: Add to 0.1 mglml from a 10 mglml stock in MeOH Diazoacetylnorleucine methyl ester (DAN): In the presence of Cu(I1) this is a covalent i n h i b i t ~ rThe . ~ requirement for copper ion precludes the simultaneous use of this reagent with chelators used to inhibit metalloproteases. A 100 mM stock of DAN is prepared in methanol; cupric [Cu(II)] acetate is dissolved in water at 100 mM. Each solution is added to 5 mM final concentration 25

B. Kassell and M. Laskowski, Sr., Biochem. Biophys. Res. Commun. 20, 463 (1965).

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EXTRACT PREPARATION AND FRACTIONATION

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Metalloprotease inhibitors: EGTA [ethylene glycol bis(p-aminoethyl ether) N, N,N1,N'-tetraacetic acid]: An efficient chelator of divalent metal cations other than Mg2+ (for which it has a lo3-fold lower affinity26).To prepare a 0.5 M stock, suspend 0.05 mol in 80 ml water, titrate to pH 8 with concentrated or solid NaOH, and dilute to 100 ml. Add to buffers at 10 mM final concentration. Mg2+may be added to these buffers at normal levels if necessary The chelator 1,lO-phenanthroline: ,4 replacement for EGTA. It is toxic, and we have not used it for extract preparations. A 100 mM stock is dissolved in 95% EtOH and used at 5 mM initial Stability Trials To determine the appropriate conditions for protection of the activity one is purifying, it is best to initiate a series of small-scale trials to determine the presence and nature of inhibitory substances. This is important because certain protective regimens are mutually exclusive. Prepare a series of small-volume extracts (1-5 ml) by grinding in a mortar and pestle (see below) in each of the following buffers: (1) homogenization buffer plus inhibitor mix ("complete"), (2) complete minus DTT, plus 2-mercaptoethanol, (3) minus EGTA, plus DAN/Cu(II), (4) minus DTT, plus PHMB, (5) complete plus aprotinin, antipain, pepstatin, and leupeptin. Assay each extract immediately upon preparation and after storage for 1, 2, and 8 to 12 hr at 0 and 20°, and 8 to 12 hr at -70". Protein Determination Many protein determination techniques are inhibited by compounds present in crude plant extracts. The Folin-Ciocalteau reagent, used in the Lowry assay, reacts with tannins and cannot be used in the presence of phenolics unless appropriate modifications are made.27The bicinchoninic acid reagent (BCA; Pierce Chemicals, Rockford, IL) is unreliable at DTT concentrations above 1 mM or at very high chelator concentrations. The Coomassie Blue G-250 dye-binding assayz8gives erroneous results in the presence of tannins bound to protein.27Some phenolics may also interfere with spectrophotometric determinations in the 220- to 280-nm range. We find that standard protein determination techniques are, however, accurate for extracts of wheat germ and of leaves low in tannins. J. S. Blanchard, this series, Vol. 104 [26]. 27 R. L. Mattoo, M. Ishaq, and M. Saleemuddin, Anal. Biochem. 163, 376 (1987). * M. M. Bradford, Anal. Biochem. 72, 248 (1976). 26

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PREPARATION OF EXTRACTS FROM PLANTS

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Pretreatment Washing and Imbibing. Leaves are washed in tap water, rinsed 15 sec in a solution of 0.1% sodium hypochlorite (1 : 50 dilution of household bleach) plus 0.05% nonionic detergent (Brij 58, Brij 35, or Nonidet P-40) in deionized water, and rinsed thoroughly in at least three changes of clean deionized water. The leaves are patted thoroughly dry and large ribs and veins are removed. Dry seeds or beans are surface sterilized by soaking 30 sec in 0.1% sodium hypochlorite and rinsing thoroughly with deionized (distilled quality) water. Seeds are then swollen by soaking overnight at room temperature in sterile deionized water. Organic Flotation. Flotation is used to separate embryo from bran and endosperm (starch-containing vacuoles) in ground seeds or in raw wheat germ preparations. Wheat embryo is pale yellow; bran is brown and starch is white. Embryos purified by flotation have lower levels of nonspecific nuclease, and would be expected to have reduced amounts of vacuolar proteases. An initial trial should be performed to determine whether enzyme stability or yield during purification is affected by this treatment. The flotation is usually omitted in a large-scale preparation. In a fume hood, prepare a solution containing 250 ml CC14 plus 70 ml c y c l ~ h e x a n e .Add ~ ~ . ~125 ~ g raw wheat germ and stir gently with a glass rod. Stop stirring for a minute or so to allow the suspension to separate into floating and nonfloating material. Collect material in the upper 20% of the solvent. Repeat this process twice more, preferably with fresh solvent. Spread the floated wheat germ on a thick filter paper, dry 15 min in the fume hood, and finish drying overnight at 4". Store the dry germ at -70". Cell Breakage Materials Waring Blender (1 liter or 1 gal) Miracloth (Calbiochem, San Diego, CA): Autoclave moist Cheesecloth: Autoclave moist Kitchen spatula Plastic beakers Large plastic funnel to fit beaker Buffers. We use water from 0.2-pm filtered OrganicFree NanoPure I1 (Barnstead, Dubuque, IA) or MilliQ (Millipore, Bedford, MA) cartridge systems. 30

W. Zagbrsky, Eur. J. Biochem. 86, 465 (1978). N. Stange and H. Beier, EMBO J. 6, 281 1 (1987).

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EXTRACT PREPARATION AND FRACTIONATION

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Homogenization buffer 50 mM Tris-HC1 (pH 8.0 at 25") 5% glycerol Monovalent cation [0.1 M KC1 or NH4C1, or 0.05-0.2 M (NH4)2S041 1x Inhibitor mix (see below) Buffer is equilibrated to 0" before use, and should be used as a slurry prepared by allowing buffer to freeze partially until ice starts to form, or by freezing solid in a household ice-cube tray and crushing in a household blender ice-crusher attachment. Mix crushed, frozen buffer with cold liquid buffer to obtain a pourable slurry. Inhibitor Mix Protease inhibitors: 1 mM PMSF, 1 mM benzamide, 1 mM benzamidine - HCl or p-aminobenzamidine 2HC1, 5mM E-amino-n-caproic acid, 10 mM EGTA, 1 pglml antipain, 1 pglml leupeptin, 0.1 mglml pepstatin Reductants: 5 mM DTT; 20 m M sodium diethyl dithiocarbamate Antiphenolic: 1.5% PVPP (insoluble PVP) For isolation of phosphorylated enzymes, add KF to 50 mM. Small-Scale extract^.^' Small amounts of plant material such as wheat germ, leaves, or wet seeds may be homogenized by grinding, with acidwashed siliconized sand if necessary, in a mortar and pestle. To prepare sand, stir (e.g.) 50 g quartz sand (Sigma) into ca. 100 ml chromic acid for 5 min. Allow to settle, decant the acid, and wash the sand exhaustively with tap water. Rinse the sand with distilled water and collect on a Biichner funnel. The sand is oven dried, saturated with 5% dichlorodimethylsilane in CC14, and washed thoroughly in 95% ethanol. Alternately, the damp sand may be treated with an equal volume of aqueous siliconizing reagent (Siliclad, Pierce Chemicals) and rinsed in distilled water. Either treatment is completed by baking at 150". Wheat germ (or other nonleafy plant material), 5 g, is placed in a mortar on ice and ground to a powder with an equal volume of acidwashed, siliconized sand and 2 ml homogenization buffer. One to 3 ml homogenization buffer is added and grinding is continued until a smooth paste is formed. Leaves and other soft material, and all fibrous tissues, are first frozen in liquid nitrogen, then placed into a mortar on dry ice and ground to a fine powder without sand. Homogenization buffer is then added. For either type of extract, the paste is diluted to a pourable consistency with homogenization buffer, transferred to 15- to 50-ml centrifuge P. Gegenheimer, unpublished observations.

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185

tubes, and centrifuged 20 min at 12,000 g . The supernatant is decanted into clean tubes and spun 45 min at 40,000 g . This clarified S40 extract is divided into aliquots, quick frozen, and stored at -70". Cytoplasmic Extracts of Nongreen Tissue. Wheat germ18,32,33: All procedures are carried out in the cold room. Wheat germ (50-500 g) is processed at a ratio of 400 ml homogenization buffer1100 g wheat germ. Suspend 500 g raw wheat germ in 2 liters homogenization buffer (ice cold or a partially frozen slurry) in a 1-gal Waring blender precooled to -20". Larger amounts are processed in 500-g batches. Smaller amounts are scaled down proportionately, and a blender jar is chosen which will be about two-thirds to three-quarters filled. Grind at high speed for four bursts of 15 sec, with 30-sec rests. Be careful to avoid foaming. The use of a foam arrester attachment may prove helpful. Check the pH with pH paper at this point and add solid Tris base or 2 M Tris base if necessary to attain pH 7.0-7.5 at 4". Decant into 500-ml leakproof centrifuge bottles. Rinse the blender jar with 500 ml cold homogenization buffer and add to the extract. Remove debris by centrifugation (at 2") for 30 min at 12,000 g (8500 rpm in the Sorvall GS-3 rotor). Decant the supernatant through one layer of sterile Miracloth into a plastic beaker on ice. Fleshy tissue1' (cauliflower inflorescence, carrot root): Proceed as for wheat germ, but using 1 liter homogenization bufferlkg chopped plant material. Filter the homogenate through four to six layers of cheesecloth and one layer of Miracloth prior to centrifugation. When preparing extracts from ripe fruits or vegetables containing pectin, EGTA must be present to prevent gelation of the extract. Fleshy beans or seeds34:Dry peas or soybeans are washed and imbibed as described above. The beans, at a ratio of 1 liter bufferlkg initial dry weight, are ground five times at low speed in a Waring blender for 30 sec each time with 15- to 30-sec rests. The homogenate will contain svbstantial debris that can be separated in a commercial juice extractor (Oster, or Acme Juicerator). Filter the homogenate through four layers of cheesecloth and one layer of Miracloth. If a juice extractor is unavailable, use six to eight layers of cheesecloth followed by two layers of Miracloth. Whole-Cell Extracts from Leaves. General considerations: Soft, nonfibrous leaves and similar tissue may be ground wet in homogenization buffer. Fibrous leaves, and all leaves which are high in polyphenolic content, are first quick frozen in liquid nitrogen and ground to a powder J . J. Jendrisak and R. R. Burgess, Biochemistty 14, 4639 (1975). P. Gegenheimer, H.-J. Gabius, C. L. Peebles, and J. Abelson, J. Biol. Chem. 258, 8365 (1983). j4 R. A. Bednar and J. R. Hadcock, J. Biol. Chem. 263, 9582 (1988). j2

33

186

EXTRACT PREPARATION A N D FRACTIONATION

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prior to addition of homogenization buffer. This procedure allows more efficient cell lysis, and subjects enzymes to less contact with vacuolar contents and other compartmentalized inhibitory compounds. Freezing plant tissue at -20" is not recommended because during slow freezing intracellular compartments are ruptured with the release of deleterious substances. Nonjibrous leaves: Fresh leaves are washed, dried, and deribbed as described above. Pack the leaves into a Waring blender of appropriate capacity and add ice-cold homogenization buffer at 1.5 liters11 kg leaves. (This ratio may be varied from 1 to 2 literslkg.) Grind for a few 5-sec bursts, packing intermittently with a kitchen spatula to reduce the volume of leaves; then grind for 1.5 to 3 min in 30-sec bursts. The shorter time may be used for soft leaves like those of spinach. Frozen, crushed homogenization buffer may be added to maintain the temperature at 0-4'. Filter the homogenate through six to eight layers of cheesecloth, then through two layers of Miracloth. Remove debris by centrifugation for 15 min at 12,000 g (8500 rpm in the Sorvall GS-3 rotor). When large volumes are being processed, the crude filtrate may be used directly as starting material for ammonium sulfate or acetone precipitation. Fibrous or phenol-rich leaves: Start with washed, deribbed leaves. Pack the leaves into a thick-walled styrofoam box until half full and carefully add liquid nitrogen (LN2) to just cover the leaves. After 30-60 sec, pour off the LN2 into another box of leaves. Transfer the frozen leaves plus a minimal amount of LN2 to a stainless steel Waring blender container (previously chilled to -20 or -70"). Grind the leaves for 1.5 to 3 min at high speed, or until they are reduced to a fine powder. (It might be necessary, with some samples, to complete the process manually with a pestle and a mortar placed on dry ice.) The LN2 should evaporate by the end of the grinding. Add homogenization buffer, 200 m1/100 g initial dry weight of leaves, and mix for 30 sec at high speed. Continue with filtration and centrifugation as for nonfibrous leaves, above. Larger amounts of buffer may be required for maximal yield and activity from fibrous, viscous, or tannin-rich homogenates. Chloroplast Extracts

Chloroplasts are prepared from spinach and pea leaves according to the methods of Orozco et al." and Gruissem et with slight modifications. These protocols, with minor modifications, will work for many species.36For a small-scale preparation, or when initiating a purification 35

36

W. Gruissem, B. M. Greenberg, G. Zurawski, and R. B. Hallick, this series, Vol. 118 [16]. J. D. Palmer, this series, Vol. 118 [Ill.

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PREPARATION O F EXTRACTS FROM PLANTS

187

trial, it is best to use intact plastids purified on Percoll density step gradients. Percoll is preferable to sucrose unless specifically contraindicated. Later, when a chloroplast location has been demonstrated, and if there is minimal interference from cytoplasmic activity, large-scale preparations are done with bulk chloroplasts which have been washed twice to remove cytoplasmic contamination. If desired, a preliminary low-speed spin can be added to remove most nuclei. Mitochondria remain largely in the postchloroplast supernatant if the centrifugation is not prolonged. Materials Waring blender: For chloroplast isolation, the blades of the blender should be replaced with injector razor blades.37 This gives higher yields of intact chloroplasts. Cool the blender to -20" before use Other materials: As for cell breakage Buffers Grinding (GR) buffer (lox concentrate): Sorbitol: 3.3 M (dissolve overnight in 400-450 ml sterile H20, then add other components) HEPES: 500 mM EDTA: 20 mM (up to 100 mM as necessary to prevent clumping of chloroplasts) MgC12: 10 mM MnCI2: 10 mM (optional) Adjust pH to 8.0 with KOH; bring to 500 ml. Do not autoclave or filter. For faster preparation, GR buffer may be made as a 5x concentrate by dissolving the above components in 1000 ml. Hypotonic lysis buffer: HEPES-KOH (pH 8.0): 10 mM DTT: 5 mM EDTA: 1 mM Inhibitor mix: As for homogenization buffer (above); PVPP is normally omitted PBF-Percoll solution (per 100 ml): Percoll: 100 ml PEG 6000: 3.0 g Fico11 400: 1.0 g ( M , 400,000) Autoclave, cool to 4", and add the following: BSA (Pentex grade V): 1.0 g " C . G . Kannangara, S. P. Gough, B. Hansen, J. N. Rasmussen, and D. J. Simpson, Carlsberg Res. Commun. 43, 431 (1977).

188

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EXTRACT PREPARATION AND FRACTIONATION

Percoll gradient solutionsa Final Percoll concentration Components

40%

80%

85%

Either l o x GR buffer Sterile H 2 0 or 5 x GR buffer Sterile H 2 0

PBF-Percoll 1 M DTT 0.1 M glutathione 0.5 M sodium ascorbate Total: a

85 ml 200 p l ( 2 rnM) 300 p1 (0.3 mM) 1.0 ml (5 mM) 100 rnl

This is sufficient to prepare five 22-1111 or two 110-ml gradients. NA, Not applicable.

Percoll gradients : Size of centrifuge tube (ml)

Each gradient contains 1 .O vol 40% gradient solution: 1.2 vol 80 or 85% gradient solution: 0.6 vol chloroplasts in GR buffer:

10 ml 50 ml 80 ml 12 ml 60 ml 96 rnl 6 rnl 30 ml 35 rnl

Dispense the low-density solution first; then underlay it with the highdensity solution carefully dispensed from a syringe connected to a length of plastic tubing. Protocols Determination of chlorophyll content: Following the method of A r n ~ nremove , ~ ~ 500 p1 of diluted chloroplasts, or 50 p1 of a concentrated suspension, and pellet in a polypropylene microcentrifuge tube for 2 min at 10,000g. Remove the supernatant; to the pellet add 1 ml80% acetone in water. Vortex, centrifuge as before, and measure the absorbance of the supernatant at 645 and 663 nm. Calculate chlorophyll concentration of the

'*D. I. Arnon, Plant Physiol. 24, 1 (1949).

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PREPARATION OF EXTRACTS FROM PLANTS

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acetone extract, in pglml, as 20.2(AM5)+ 8.02(A663). Correct for dilution to obtain chlorophyll concentration in the initial suspension. Preparation of crude chloroplasts: Wash and derib leaves as above. Weigh and divide into 100-g batches. Store in the dark overnight at 4" to remove starch. Adequate removal of starch is crucial to obtaining intact chloroplasts; the effectiveness may be judged during purification as described below. All further steps are performed in the cold room. Homogenize leaves in 100-g batches in a 1-liter Waring blender equipped with razor blades, using 200 ml ice-cold grinding buffer (GR)/100 g leaves. (We have used up to 300 ml buffer. Fibrous or tannin-rich leaves, or those giving a viscous extract, may need more buffer.) Larger batches are processed in a 1-gal blender using 2 liters of buffer11 kg leaves. A lower yield of intact chloroplasts may be expected. First chop the leaves with a 2-sec burst followed by packing the leaves with a kitchen spatula. Repeat if necessary so that the buffer covers the leaves. Homogenize for 5 sec at high speed. Filter the homogenate through six layers of cheesecloth placed over two layers of Miracloth in a large funnel resting on a 2- or 4liter beaker placed in a tray of ice. After each batch, squeeze the cheesecloth very gently to wring out residual liquid. After every 100 to 150 g leaves, discard the debris (brei). Pour the filtrate into 500-1111 (or as appropriate) leak-proof centrifuge bottles. Place in a precooled (0 to - 10") rotor and bring to 6000 g . Hold at this speed for 0-30 sec (no more than 30 sec), then turn off. Allow the rotor to come to rest with the brake on. Carefully discard the green supernatant. The pellet will typically consist of a lower white layer of starch plus nuclei, a firmly packed middle layer of intact chloroplasts, and a loosely packed upper layer of broken chloroplasts. Resuspend the pellet in the residual buffer remaining in the centrifuge bottles. Avoid resuspending the starch. The classical utensil is a pointed watercolor paintbrush, although a cotton-wrapped applicator stick or even a rubber policeman may also be used. A gentle painting motion is used to produce a smooth paste. It is crucial that the suspension be uniform and free of clumps. Gradually dilute the pellet with additional GR buffer to about one-tenth the original homogenate volume. Gentle pumping with a wide-bore plastic pipet or large plastic syringe attached to plastic tubing may be used to achieve uniform dispersion. Collect the pellet by centrifugation exactly as before. (A dark green supernatant at this step indicates that the chloroplasts contain starch granules, which are rupturing the plastids during centrifugation. The remedy is to store the leaves in the dark for a longer time prior to homogenization.) Repeat this washing procedure a second time. Lyse the pelleted chloroplasts as described below. Removal of nuclei: The standard protocol for intact chloroplasts gives extracts with little if any detectable nuclear contamination. Occasionally,

190

EXTRACT PREPARATION AND FRACTIONATION

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however, we have observed extremely low levels of nuclear enzyme^.^' Cleaner chloroplasts might be obtained from the supernatant of a preliminary low-speed spin.39The filtered homogenate is centrifuged for 3 min at 200 g; collect chloroplasts from the supernatant by centrifugation for 7 min at 1000 g (or 30 sec at 5000 g). Resuspend the pellet in one-fifth to one-tenth the initial volume of GR mix and wash by successive low- and high-speed spins as before. Preparation of purijied chloroplasts: Prepare a set of Percoll gradients (see above) just prior to starting the homogenization. We use 40 and 80% steps for spinach and pea chloroplasts; other researchers use a high step of 85% or more, depending on the species.ll Prepare bulk chloroplasts exactly as described above, except with only one wash in GR buffer. Resuspend the pellet in the residual buffer and dilute with GR buffer. The final ratio is about 1 ml final volume of resuspensionl4-5 g leaves. The sample volume for each gradient is about 20% of the total volume of gradient plus sample (i.e., about 25% of the gradient volume alone). For 22-ml gradients in 30-ml Corex tubes, 6 ml chloroplast suspension, derived from 25 g leaves, is layered per tube. For 1 10-ml gradients in 150-ml bottles, load 25 ml chloroplasts, prepared from 125 to 150 g leaves, per bottle. Centrifuge the gradients 20 min at 6000 to 8000 g (Sorvall HB-4 rotor; 4000 to 6000 g , Sorvall GSA rotor) and 2". The pellet contains starch and nuclei. Intact chloroplasts are the lower dark green band; broken chloroplasts are in the upper band. Mitochondria are mostly confined to the upper chloroplast band and the region between broken and intact chloroplasts. This region is normally a light green. If it is darker, the gradient has probably been overloaded. Using plastic tubing attached to a 30- to 60-ml plastic syringe, remove all of the gradient contents above the lower green band. With a fresh syringe or pipet, carefully withdraw the lower band of intact chloroplasts. Slowly dilute the intact chloroplasts with 4 vol of icecold GR buffer and collect them by centrifugation for 1 min at 8000 g (2"). Preparation of chloroplast lysate: 1. Hypotonic lysis: Wipe the walls of the tube or bottle with KimWipes. Resuspend the chloroplast pellet in a small amount of hypotonic lysis buffer. Measure the chloroplast volume as the difference between the resuspended volume and the volume of added buffer. Dilute the suspension to a final concentration of 5-7 ml lysis buffer11 ml packed chloroplasts (ca. 7 ml buffer1100 g leaves). We obtain reliable results using this volume ratio, which should result in a chlorophyll concentration of about 0.2 mglml. Other workers have found chloroplasts to lyse at chloro39 J.

Siedlecki, W. Zimmermann, and A. Weissbach, Nucleic Acids Res. 11, 1523 (1983).

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phyll concentrations from 0.05 to 1.5 mglml. The ratio may vary seasonally or with plant maturity. In preliminary trials, the minimum amount of lysis buffer needed may be determined by checking chloroplast lysis in the phase-contrast microscope with an immersion objective. The suspension is held on ice for 10 min. To further hasten plastid rupture, transfer the suspension to a Dounce-type homogenizer vessel and homogenize with several strokes every few minutes. This step may be omitted when large volumes are being processed. 2. Detergent lysis: Alternatively, chloroplasts may be lysed with nonionic detergent. The washed plastid pellet is resuspended in several volumes of GR buffer and diluted with an equal volume of hypotonic lysis buffer containing inhibitor mix and 0.2-1% peroxide-free Triton X-100. Stir on ice 15 min, then spin 15 min at 20,000-30,000 g . Thylakoid Extracts

A preparation of intact thylakoids (thylakoid vesicles) is obtained after vigorous homogenization of leaves in isoosmotic buffer.4w2Chloroplast envelopes are largely disrupted and thylakoids are isolated by differential centrifugation. The thylakoids remain intact as judged by their content of plastocyanin, a lumenal protein.42The thylakoids are extensively washed to remove ribulose-l,5-bisphosphatecarboxylase/oxygenase, the major stromal protein. To obtain unstacked thylakoids perform these washes in buffer containing 10 mM NaC1; if stacked thylakoids are desired the wash buffer should contain 100 mM NaCI. This protocol gives active chloroplast ATP synthase in good yield without protease inhibitors or reducing agents. We recommend addition of protease inhibitors to eliminate any residual proteolysis. Other thylakoid activities may require addition of reductants and protease inhibitors. Materials

As for chloroplast isolation Reagents and Buffers

Thylakoid homogenization buffer: 0.4 M sucrose (or sorbitol) 20 rnM Tricine-NaOH (pH 8.0) NaCl to 10 mM (to prepare unstacked thylakoids) or 100 mM (to prepare stacked thylakoids) 40

4' 42

S. Lien and E. Racker, this series, Vol. 23A [49]. A. Binder, A. Jagendorf, and E. Ngo, J. Biol. Chem. 253, 3094 (1978). M. L. Richter, personal communication.

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Thylakoid wash buffer: 20 rnM Tricine-NaOH (pH 8.0) 10 or 100 mM NaCl (as for homogenization buffer) Protocol. One to 1.5 kg washed, deveined spinach leaves is packed tightly into a l-liter Waring blender. Thylakoid homogenization buffer is added at a ratio of 1.4 literslkg leaves. The mixture is homogenized at high speed for 15 sec after maximum speed is reached. A few initial l-sec bursts alternated with packing may be necessary to achieve a uniform slurry. The homogenate is filtered first through two layers of cheesecloth into a plastic beaker on ice. This filtrate is passed through six layers of cheesecloth and two layers of Miracloth and collected in a beaker in ice. Cell debris is first removed by centrifugation of the filtrate for 10 sec at 2000 g (or 2 min at 300 g); the supernatant is transferred to fresh centrifuge bottles and the thylakoids are pelleted for 10 to 15 min at 3000 to 6000 g . The thylakoid pellets are washed by resuspension in a final total volume of 2 liters ice-cold thylakoid wash buffer and centrifugation as before. This wash is repeated twice. Centrifugation times are prolonged to 30 min for the second wash and 40 min for the third. The final pellet is resuspended in wash buffer. Thylakoids may be lysed by resuspension in hypotonic lysis buffer or hypotonic buffer containing 0.1 to 1% Triton X-100. The lysate is clarified by centrifugation (15 min, 20,000 g). Specialized references should be consulted for further details. Posttreatment of Extracts

PEZ Precipitation

The bulk of the nucleic acids and much of the phospholipids in the extract are removed by precipitation with the synthetic polycation polyethyleneimine (PEI). Further, because polycation treatment (with protamine sulfate) removes ionized polyphenolates and was reported to improve enzyme yields from some plant source^,^ we expect PEI treatment to have a similar beneficial effect (see, e.g., Ref. 18). This step is especially useful for preparation of extracts from dry material like seeds or embryos, in which nucleic acids and lipids are concentrated. PEI treatment is also used as the first step in purification of nucleic acid-binding proteins,34 where it is used both to remove nucleic acids, which would interfere with subsequent chromatographic step^,'^,^^ and also selectively to precipitate acidic DNA-binding proteins.32Thus, a trial extract should be made at a low salt concentration to see whether the activity can be precipitated with PEI and eluted by higher salt from the PEI pellet. Pro-

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tein concentration is determined in the presence of PEI by monitoring absorbance at 280 nm, or by colorimetric procedures after TCA precipitation of protein. Protein fractions are freed of PEI by ammonium sulfate precipitation. Preparation of PEI. A 10% (wlv) solution of PEI is prepared by diluting 10 ml Polymin P concentrate [Miles Laboratories or Sigma; obtained as a 50% (wlv) concentrate] to -20% with sterile water and dialyzing overnight in a half-filled dialysis bag against distilled water. The solution is titrated to pH 8 with HCI, diluted to 50 ml with distilled water, and filtered through one layer of paper or Miracloth. Treatment of the Extract. The extract is brought to 0.1-1.0% (wlv) in PEI by slow addition, with gentle stirring, of e.g., 0.01 vol 10% (wlv) dialyzed PEI. The lowest concentration of PEI required to remove nucleic acids must be determined in preliminary trials. The PET-treated extract is stirred for 10 to 15 min, then centrifuged for 15 min at 12,000 g. The clear supernatant is decanted and used directly for ammonium sulfate fractionation. Gel Filtration

Inhibitory compounds in extracts from plants high in tannins can often be removed by passage over a column of BioGel PdDG or Sephadex G-50. The column resin is prepared by autoclaving in TE buffer [ l o mM Tris-HC1 (pH 8.0), 1 mM EDTA], decanting the supernatant, and adding 0.5 vol sterile TE. A column is prepared containing 4 vol resinlvol crude extract, and equilibrated with homogenization buffer containing 75 m2M KCl. The dimensions of the column are unimportant; a 2 : 1 height :diameter ratio is adequate. The extract is layered on the column bed (or brought to 10% glycerol and layered on the column under the elution buffer), allowed to sink in, and eluted with column buffer. Eluted protein is measured by UV absorbance; the peak can be collected in beaker on ice. The extract is ready for further purification. Acknowledgments I am grateful to those colleagues who shared their protocols, advice, and experience, especially Dr. Bob Buchanan (University of California), and Drs. Mark Richter and Chris Haufler (University of Kansas). Early tutelage in protein purification came from Craig Peebles (University of Pittsburgh) and Hans Gabius (Max-Planck Institut fiir Experimentelle Medizin, Gottingen. FRG). Research conducted in my laboratory is supported by the NSF (DMB 86-09647).

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[ 151 Preparation

[I51

of Extracts from Higher Eukaryotes

By JOHN DAVIDDIGNAM

Successful preparation of crude extracts from eukaryotic cells and tissues for enzyme purification requires that one pay attention to conditions that may alter the activity or native structure of an enzyme, since nonspecific inactivation can result in irreproducibility that may make interpretation of such studies difficult. Whether one is preparing an extract for purposes of enzyme purification or for physiological studies of enzyme activity, using conditions that ensure optimum yield and maintenance of activity and native structure is essential. While developing optimum extraction procedures is largely empirical, there are a number of conditions that, when examined systematically in pilot experiments, make the job of developing optimum conditions much easier. This chapter deals first with some general problems that must be considered when developing an extraction procedure followed by some specific protocols which the reader may find useful; it must be emphasized that no one protocol is appropriate for all cases and I have indicated where some modification of the protocols presented might be required for application to specific problems. General Considerations

Starting Material. In some cases the exact source of an enzyme is not an important consideration, so that one could use material obtained from a slaughterhouse. One would d o well to examine several tissues inasmuch as one might find a tissue to have unexpectedly high levels of the activity. Alternatively, one may find that some tissues, while they may have lower levels of the activity of interest, may also have lower levels of interfering activities or that they are easier to work with than other organs due to lower amounts of connective tissue. In some cases a tissue that otherwise might appear to be inappropriate may in fact be an excellent source of an enzyme; for example, tryptophanyl-tRNA synthetase levels proved to be unexpectedly high in pancreas (approximately 4% of the soluble protein1) and the high levels of protease activity were largely suppressible.* While fresh tissue is preferable, frozen tissue may be used in some cases pro-

' M. *

L. Sallafranque, M. Garret, J.-P. Benedetto, M. Fournier, B. Labouesse, and J. Bonnet, Biochim. Biophys. Acta 882, 192 (1986). L. L. Kisselev, 0. 0. Favorova, and G. K. Kovaleva, this series, Vol. 59, p. 234.

METHODS IN ENZYMOLOGY, VOL. 182

Copyright O 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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vided it is frozen rapidly in small pieces and is not stored for an excessive period. Some deterioration can be expected due to the release of proteases from lysosomes as a result of ice crystal formation, so it is wise to store material below -50". A variety of frozen tissues can be obtained commercially (e.g., Pel-Freez Biologicals, Rogers, AR). In many cases the choice of material is dictated by the organ in which a physiological response is observed and not by practical considerations; this is particularly true of studies of the response of a given cell type to a drug or hormone, where tissue culture cells may be the only source of a pure cell type. While the propagation of tissue culture cells is expensive and time consuming, they can be an excellent source of some enzymes, particularly enzymes that are elevated in rapidly growing cells. Cell Lysis Methods. The method used for lysis of cells depends on the nature of the cells and on whether fibrous proteins such as myosin or collagen are present. Tissue culture cells and cells that can be grown as a suspension in an animal (e.g., Novikoff and Ehrlich ascites) can be lysed readily with a hand-held homogenizer after they have been allowed to swell in a hypotonic buffer; reticulocytes need only be placed in a hypotonic buffer for lysis. Solid tissues may pose more of a problem, although liver and other organs from small animals can be readily homogenized in hand-held or motor-driven homogenizers. This approach is not practical for large quantities of material or for organs from larger animals, where connective tissue and large blood vessels make homogenization difficult. Large quantities of material are generally more easily processed in a blender. Foaming should be minimized, as this can result in inactivation of many enzymes through denaturation of the protein at the surface of bubbles. This problem can be controlled by filling the blender cup as full as possible to reduce the volume of air above the liquid. Skeletal muscle, even from small animals, is difficult to homogenize due to the large quantities of myosin and actin, and must first be cut into small pieces or put through a meat grinder (if large quantities are processed) prior to homogenization in a blender. Some tissues pose unique problems. In our laboratory we found that trying to homogenize the middle silk gland of silkworms is not unlike attempting to homogenize rubber bands and is best accomplished on frozen tissue in a blender. Similarly, Drosophila embryos can be homogenized only after the chorion has been removed. A device with counterrotating blades, a Polytron homogenizer (Kinematica Co., Lucerne, Switzerland), can be quite useful for small amounts of very fibrous tissue, but care should be taken to control foaming and to cool the sample, inasmuch as excessive heat can be generated.

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Most of the methods described above can generate considerable shear forces that disrupt some subcellular organelles, such as lysosomes, with undesirable consequences. It is wise to monitor the extent of cell breakage through enzyme assays or through microscopic examination in pilot experiments so that excessive homogenization can be avoided. A facile way to lyse cells without homogenization is to load them with glycerol3 (by incubation of the cells in a glycerol-containing buffer) followed by suspension in a hypotonic buffer. While this approach is applicable only to cell suspensions and appears to be limited to relatively small quantities of material ( 1 to 20 g), it does avoid the shear forces involved in mechanical homogenization and may be useful in the isolation of particularly labile enzyme complexes. Buffer Components. Although choosing a buffer is important, often little consideration is given to this point; for example, one frequently sees Tris-chloride used at pH values too far from its pK and at an inadequate concentration (see the chapter in this volume4). Since some buffers show marked concentration and temperature dependence, one should always report the pH of the working solution (not of the concentrated stock from which it might be derived) and the temperature at which the pH was measured. In addition, one should consider whether the buffer will interact with the enzyme under study and with other buffer components. For example, some buffers such as phosphate are metabolites and may inhibit an enzyme (through carryover to the assay) or act as an allosteric effector and obscure interesting phenomena; other buffers are known to bind divalent metal ions, reducing their effective concentration. A careful study of the effect of pH on extract preparation may be useful in revealing pHdependent differences in stability or solubility of an enzyme that otherwise might be overlooked. If little is known concerning the effect of ionic strength on the solubility and stability of an enzyme, this parameter should certainly be investigated. For example, many protein synthesis factors and some aminoacyltRNA synthetases remain associated with ribosomes at low ionic strength. In some cases having an enzyme associated with an easily isolatable component is advantageous while in other cases it may interfere with purification. Some enzymes are more stable at higher ionic strength or may be stabilized by specific ionic species (e.g., phosphate or sulfate). Many other components that may be added to buffers to aid in stabilization are listed below. More detailed recommendations for protein stabilization may be found in this v ~ l u m e . ~ M. Jett, T. M. Seed, and G. A. Jamieson, J. Biol. Chem. 252, 2134 (1977). V. S. Stoll and J. S. Blanchard, this volume [4]. M. P. Deutscher, this volume [81.

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1. Thiol compounds are frequently added to protect protein thiols

from oxidation; dithiothreitol is the reagent of choice over Zmercaptoethanol owing to its greater stability, lower redox potential, and lower stench. 2. Chelating agents such as EDTA are useful in protecting enzymes from inactivation by heavy metals sometimes present in reagents and from proteolysis by metalloproteases. 3. Glycerol and other polyols are frequently added to stabilize enzymes and appear to exert their effects by stabilizing protein-protein interactions. 4. Substrates often afford considerable stabilization of enzymes and have the potential to be quite specific. 5. Protease inhibitors with widely differing mechanisms and specificities are available for the suppression of endogenous proteases. Phenylmethylsulfonyl fluoride is effective in inactivating a number of serine proteases, but is unstable under some conditions and must be added to buffers from a stock solution in an organic solvent [ethanol, Zpropanol, or dimethyl sulfoxide (DMSO)]; it should be added to buffers with adequate stirring and one must be certain that the residual organic solvent does not have deleterious effects on the enzymes under study. There are also several proteins (e.g., aprotinin, soybean trypsin inhibitor, a2-macroglobulin) that will inhibit a number of trypsin-like proteases. Some thiol proteases can be suppressed by inhibitors such as antipain. An alternative to using specific inhibitors is to apply the crude extract to hemoglobin agarose or pulverized, formaldehyde-cross-linked gelatin either as a batch procedure or as a column step; this approach removes a number of proteases fairly nonspecifically and may prove more effective if proteases cannot be suppressed with a variety of agents. A more rational approach to the protease problem is to test the effectiveness of various agents with a simple qualitative test designed to detect endoprotease activity (available from Boehringer Mannheim, Indianapolis, IN). Proteolysis can be insidious inasmuch as many enzymes retain activity even after they have been significantly degraded, so that it is possible to isolate an active fragment which may have considerably different properties than the parental molecule. Evaluation of Stability. It is useful to have some rational basis for inclusion of various stabilizing agents in buffers used for preparation of extracts. To this end one can test the effect of various agents on the thermal stability of an enzyme in a crude extract; one usually incubates small aliquots of extract for a fixed time (perhaps 10 min) at various temperatures (5 to 45" is a good range) and then assays for activity. In this fashion one can test a number of conditions simultaneously. The elevated

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temperatures tend to accelerate effects that might be apparent only after days in the cold or that might become apparent only after several purification steps. Removal of Particulate Material. Procedures for preparation of extracts generally have a centrifugation step to remove large organelles (e.g., nuclei, mitochondria), unbroken cells, and insoluble material such as connective tissue and denatured protein. Specific procedures are required to isolate relatively pure organelles (described in this volume6)and may be rather involved, but advantageous if their isolation achieves a substantial purification of the enzyme from other components. If the enzyme is in the soluble fraction, larger components such as nuclei and mitochondria may be removed by a short, high-speed centrifugation step (15,000 g , 30 min, 0-4"), and microsomes may be removed by an ultracentrifugation step (100,000 g, 1 hr or more). If one anticipates purifying an enzyme from large quantities of tissue, using an ultracentrifuge is often impractical because of the large volumes and it is generally more efficient to remove particulate material remaining in an early purification step (e.g., precipitation with ammonium sulfate, fractional precipitation with polyethylene glycol). Experimental Procedures

The protocols that follow are sufficiently general that they may be applied to a number of different tissue and cell types with only minor modifications. All procedures are performed at 0 to 4'. IA. The procedure that follows is an adaptation of a method used in the isolation of an aminoacyl-tRNA synthetase complex from solid tissues. Materials Buffer A: 50 mM Tris-C1 (pH 7.5 at 25"), 2 mM ethylenediamine tetraacetate (EDTA), 150 mM NaC1, 0.5 mM dithiothreitol (DTT) Buffer B: 50 mM Tris-C1 (pH 7.5 at 25"), 10% (v/v) glycerol, 5 mM magnesium acetate, 0.2 mM EDTA, 0.5 mM DTT, 1.0 mM phenylmethylsulfonyl fluoride (PMSF) DTT: A 0.5 M stock solution is prepared in cold water and stored frozen; the reagent is added to cold buffers at the indicated concentrations just prior to use PMSF: A 0.2 M stock solution is prepared in 2-propanol and the reagent is added to cold buffers with adequate stirring just prior to use; the reagent will crystallize from 2-propanol when stored at -20". B. Stonie and E. A. Madden, this volume [161.

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Procedure. After the tissue is excised from the animal it is placed in cold buffer A; if the organ is large, it should first be cut into small pieces to aid in chilling. The tissue is washed several times in buffer A, the excess buffer drained, and the tissue weighed. The tissue is homogenized in 2.5 vol (by weight) of buffer B. Depending on the quantity and nature of the tissue one may use either a blender (1 min at high setting) or a hand-held homogenizer (10 strokes); blender speeds vary as do clearances on homogenizers so that the conditions given should be used only as guides. Large particulate material is removed by centrifugation at 15,000 g for 30 min; smaller particulate material may be removed by additional centrifugation at 100,000 g for 60 min. In either case the supernatant fraction is removed carefully, leaving approximately the last 10 to 15% behind. This material may be used immediately for purification or may be quick frozen as smaller aliquots and stored at -80'. Comments. The original procedure was used quite successfully in isolating a very labile aminoacyl-tRNA synthetase complex from a number of tissues (rabbit and sheep liver, sheep spleen, sheep mammary gland, and rabbit reticul~cytes)~-~ and the slightly modified procedure above has been used in my laboratory in the purification of aminoacyl-tRNA synthetases from rat liver and insect tissues'0." (Bombyx mori ovaries and silk gland). The procedure has worked well on frozen middle silk glands" and frozen mammary gland,8 both of which contain large amounts of fibrous material; in the latter case, some degradation of the aminoacyl-tRNA synthetase complex was observed with frozen mammary gland compared to unfrozen tissue. The original procedure calls for the use of diisopropyl fluorophosphate (DFP) as a protease inhibitor, in addition to PMSF; DFP is extremely toxic and the necessity for using it is questionable. IB. A considerably modified version of the procedure described above was used in the isolation of the aminoacyl-tRNA synthetase complex from reticul~cytes,~ but can also be used on tissue culture cells as outlined below. Materials Phosphate-buffered saline (PBS) Buffer C: 10 mM Tris-CI (pH 7.5 at 25"), 5 mM MgC12, 2 mM PMSF Buffer D: 250 mM Tris-C1 (pH 7.5 at 25'), 0.25 M KC1, 10 mM MgCl?, 5 mM DTT, 5 mM PMSF, 0.5 rnM EDTA, 50% glycerol

'O

'I

0. Kellerman. A. Brevat, H. Tonetti, and J.-P. Waller, Eur. J. Biochem. 99, 541 (1979). 0. Kellermann, C. Viel, and J.-P. Waller, Eur. J. Biochem. 88, 197 (1982). 0. Kellermann, H . Tonetti, A. Brevet, M. Mirande, J.-P. Pailliezz, and J.-P. Waller, J. Biol. Chem. 257, 11041 (1982). S. S. Dignam and J. D. Dignam, J . Biol. Chem. 259, 4043 (1984). S. Viswanathan and J. D. Dignam, J . Biol. Chem. 263, 535 (1988).

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Procedure. Cells are collected by centrifugation (100 g, 10 min) and washed two or three times in 3 vol of cold phosphate-buffered saline (PBS). The cells are suspended completely with a plastic spatula in 3 vol of buffer C; the hypotonic nature of the buffer will cause reticulocytes to lyse spontaneously in 3 to 5 min with gentle stirring while tissue culture cells or cells grown as an ascites in animals will swell considerably. Tissue culture cells are lysed by homogenization in a glass-glass Dounce homogenizer (4 to 10 strokes depending on the cell type). One volume of buffer D is added with gentle stining to the extract and debris and unbroken cells are removed by centrifugation at 15,000 g; depending on the application one may wish to remove smaller particulate material by centrifugation at 100,000 g. Ionic strength of the extract can be adjusted as required for specific applications; for example, the aminoacyl-tRNA synthetase complex from reticulocytes tends to remain associated with ribosomes unless the extract is made 0.3 M with respect to KC1. The extract can be used immediately for purification or quick frozen and stored at -80'. 11. The following procedure was derived from a procedure originally ' ~ ; methods developed for studies of RNA and protein s y n t h e s i ~ ' ~ -the described below have been used extensively in in vitro studies of transcription and mRNA splicing. The procedure allows one to prepare both a cytoplasmic fraction (frequently referred to as an S100) and a nuclear extract.

Materials Phosphate-buffered saline (PBS) Buffer A: 10 mM HEPES-NaOH (pH 7.9 at 25"), 1.5 mM MgCI2, 10 mM KCI, 0.5 mM DTT, 0.5 mM PMSF Buffer B: 0.3 mM HEPES-NaOH (pH 7.9 at 25"), 1.4 M KCI, 30 mM MgC12 Buffer C: 20 mM HEPES-NaOH (pH 7.9 at 25"), 25% (vlv) glycerol, 0.42 M KCI, 1.5 mM MgC12, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF Buffer C': Same as buffer C, but with 0.6 M KC1 Buffer D: 20 mM HEPES-NaOH (pH 7.9 at 25"), 20% (vlv) glycerol, 100 mM KC1, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF Procedure. Cells are collected by centrifugation (1000 g, 10 min) and are washed once in 5 vol of cold phosphate-buffered saline. In studies where the time course of a phenomenon is being studied it may be advanl2 l3 l4

H. Aviv, I. Boime, and P. Leder, Proc. Natl. Acad. Sci. U.S.A. 68, 2303 (1971). G. J. Wu and G . Zubay, Proc. Natl. Acad. Sci. U.S.A. 71, 1803 (1974). G. J. Wu, Proc. Natl. Acad. Sci. U.S.A. 75, 2175 (1978).

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EXTRACT PREPARATION FROM HIGHER EUKARYOTES

20 1

tageous to pour the cell culture over frozen, crushed phosphate-buffered saline to slow metabolic processes prior to washing the cells with PBS as was done in one study of cell cycle-dependent transcription.15 The cells are then suspended in buffer A and allowed to stand on ice for 10 min to allow the cells to swell. The cells are lysed by 10 strokes in a glass-glass Dounce homogenizer using a tight-fitting pestle. Large particulate material, including nuclei, is collected by centrifugation (1000 g , 10 min) and retained for preparation of the nuclear extract. To the supernatant fraction from this step is added 0.1 1 vol of buffer B with gentle stirring followed by centrifugation (100,000 g, 60 min). The supernatant fraction from this step is then dialyzed against 20 vol of buffer C; in some applications the dialysis step may not be necessary or desirable, but was required in in vitro transcription studies to reduce the concentration of endogenous nucleotides. After dialysis, precipitated material is removed by centrifugation (25,000 g, 20 min, 0-4"). The sample may be used immediately for purification or may be quick frozen and stored at -80". This preparation proved to be quite useful for purification of factors required for in vitro transcription of genes transcribed by RNA polymerase 111 (e.g., 5 s and tRNA genes). l6,l7 The crude nuclear pellet obtained can be processed along with the cytoplasmic fraction to produce the nuclear extract18J9;several modifications of the procedure have been made since its original description and these changes are indicated. The nuclear pellet is subjected to an additional centrifugation step (25,000 g, 20 min, 0-4") to remove residual soluble material and to produce a more compact pellet; this pellet is suspended either in buffer C (0.42 M KCI) or buffer C' (0.6 M KCl) at 2.5 m1/109 cells by 10 strokes of a glass-glass Dounce homogenizer and stirred gently (30 min). Particulate material is removed by centrifugation (25,000g , 30 min) and the supernatant is dialyzed for 4 to 5 hr against 50 vol of buffer D. After centrifugation to remove precipitated material (25,000 g, 20 min) the extract may be used immediately or can be quick frozen and stored at -80". Comments. In general, preparing the SlOO and nuclear extract as quickly as possible is important for obtaining maximum transcriptional activity. However, the choice of 30 min as a minimum time for extraction of the nuclei was not arbitrary, because it was observed that a short Is

N. Heintz and R. G. Roeder, Proc. Natl. Acad. Sci. U.S.A. 81, 1713 (1984). P. A. Weil, J . Segall, B . K . Hanis, S.-Y. Ng, and R. G. Roeder, J . Biol. Chem. 254, 6163

l6

(1979). J . Segall, T. Matsui, and R. G. Roeder, J . Biol. Chem. 255, 11986 (1980). l8J . D. Dignam, R. M. Lebovitz, and R. G. Roeder, Nucleic Acids Res. 11, 1475 (1983). l9 J . D. Dignam, P. L. Martin, B. S. Shastry, and R. G. Roeder, this series, Vol. 101, p. 582. l7

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extraction time (10 min or less) was not as effective. In addition, the ionic strength of the solution used for extraction can influence the result depending on the application; in studies of histone gene tran~cription'~ and mRNA ~ p l i c i n g , higher ~ ~ . ~ ~ionic strength (0.6 versus 0.42 M KC1) was found to produce more active extracts. Other considerations are the health, growth rate, and growth conditions of the cells; not surprisingly, healthy, mid-log cells generally give more active extracts. Procedures similar to the one described above, but developed independently, have been used for studies of the transcription of class I11 genes (5sand tRNA) in B. r n ~ r iemploying ,~~ extracts of posterior silk gland and ovaries. Although the procedure described above worked well on tissue culture cells, attempts to make transcriptionally active extracts from tissues (e.g., liver, thymus, placenta) were unsuccessful. An alternate procedure has been used, frequently referred to as a whole-cell extract, which involves making a high-salt lysate; it is simple, reproducible, and appears to give similar results in studies of transcription and mRNA splicing to the procedure described above; this procedure is described in detail elsewhere in this seriesz3A procedure has been developed which circumvents the problems associated with making nuclear extracts from tissues and has been employed in studies demonstrating tissue-specific in vitro transcription from a mouse serum albumin promoter in extracts from rat brain, liver, and spleen.24Key features of the procedure that appear to be important are the isolation of relatively pure nuclei by sedimentation in a viscous, low ionic strength buffer containing both glycerol and sucrose, and the exclusion of divalent cations to minimize degradation of chromatin by endogenous nucleases; the nuclei are extracted using high ammonium sulfate as described by others.2s Concluding Remarks

The procedures described here can be applied to many different cell types and tissues with little or no modification, but the reader should keep in mind that developing extraction conditions for isolating enzymes is empirical; these procedures should be viewed more as a starting place for developing optimum conditions rather than as universally applicable protocols.

" D. L. Black and J. A. Steitz, Cell 46, 687 (1986). 21

D. Morton and K. V. Sprague, Proc. Natl. Acad. Sci. U.S.A. 81, 5519 (1984).

23

J. L. Manley, A. Fire, M. Samuels, and P. A. Sharp, this series, Vol. 101, p. 568. K. Gorski, M. Carneiro, and U. Schibler, CeN 47, 767 (1986). C. S. Parker and J . Topol, CeN 36, 357 (1984).

" E. T. Wilson, D. Larson, L. S. Young, and K . V. Sprague, J . Mol. Biol. 183, 153 (1985).

l4 25

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Acknowledgments I am indebted to my colleagues Sridhar Viswanathan and Susan Dignam for their critical comments and to Valerie Murphy for preparing the manuscript.

[ 161 Isolation of

Subcellular Organelles

By BRIANSTORRIE and EDWARDA. MADDEN Introduction

Cell fractionation and organelle isolation are long-standing topics in biochemistry which have been repeatedly reviewed in this series. The principles described by George Hoogeboom in Volume I of Methods in Enzymology1 are still applicable today. However, appreciable progress has been made in the purification of subcellular organelles, particularly minor organelles, from animal tissue and especially from tissue culture cells. Only recently has the purification of organelles such as mitochondria and lysosomes from tissue culture cells become routine. Many approaches to the isolation of subcellular organelles have been developed over the last 40 years. Most common are those based on size or density differences. In other words, those based on the hydrodynamic properties of an organelle population in a centrifuge. These approaches use generally accessible equipment and may be applied in any laboratory. In recent years, other approaches based on the charge or antigenic properties of organelles have begun to be introduced. These approaches typically require either expensive equipment that is not widespread, such as free-flow electrophoresis apparatus or immunological reagents of limited availability. The aim of this chapter is to detail procedures for the isolation of nuclei, mitochondria, and lysosomes from cultured mammalian cell lines. Emphasis will be given to protocols which are routinely in use by this laborat~ry.~,~ These procedures, although established for suspensioncultured Chinese hamster ovary (CHO) cells, should be equally applicable to other mammalian cell lines and to cell homogenates from animal tissue. I G. Hoogeboom, this series, Vol. 1 , p. 16. %. A. Madden and B . Stonie, Anal. Biochem. 163, 350 (1987). E. A. Madden, J. Wirt, and B. Storrie, Arch. Biochem. Biophys. 257, 27 (1987).

'

METHODS IN ENZYMOLOGY, VOL. 182

Copyright O 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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Preparation of Cell Homogenates for Cytoplasmic Organelle Isolation

Cultured CHO cells are routinely disrupted by low-pressure nitrogen cavitation. The aim of this step is to gently "puff' the cell out so that the cytoplasm may then be stripped from the nucleus by brief homogenization. The appearance of CHO cells after nitrogen cavitation and after homogenization is shown in Fig. 1. A detailed protocol (a modification4of the procedure of Rome et aL5)is given below. All steps are at 4". 1. Harvest 1 to 2 x lo8 cells from complete culture medium by centrifugation. Spin the cell suspension for 5 min at 1200 rpm (380 g,,) in a swinging bucket rotor. Use round-bottom centrifuge bottles such as Corning No. 1260. The cell pellets will concentrate at the bottom of the bottles. All centrifugations are timed from when the rotor reaches running speed. We normally use suspension culture cells at mid-log phase, about 5 x lo5 cells/ml. Two hundred milliliters of cell suspension can be pelleted in one bottle. 2. Resuspend the cell pellet(s) in a total volume of about 2-5 ml of divalent cation-free saline (e.g., PBSa: 0.14 M NaC1, 0.01 M KCl, 0.01 sodium phosphate, pH 7.4). Cell pellets can be readily resuspended in a 1to 2-ml volume of saline using a Pasteur pipet and a rubber bulb. The cell suspension(s) are then brought to a final volume. The cells are then pelleted as before. This step removes divalent cations. 3. Resuspend cells in no more than 2-5 ml of 0.25 M sucrose and pellet as before. This step is to remove monovalent cations. Repeat step 3 once. If turbidity is noticed in the centrifuge supernatant upon repeating step 3, cells should only be washed once in 0.25 M sucrose. Turbidity indicates cell fragility to handling. 4. Resuspend the washed cells in 1-2 ml of 0.25 M sucrose and place in a small nitrogen cavitation bomb. We use a 15-ml bomb from Kontes Glass Company (Millville, NJ). Similar bombs from Yeda (Rehovoth, R. R. Pool, Jr., K. M. Maurey, and B. Stonie, Cell Biol. Znt. Rep. 7, 361 (1983). L. H. Rome, A. J. Garvin, M. M. Allietta, and E. F. Neufeld, Cell 17, 143 (1979).

FIG. 1. Appearance of material during homogenization of CHO cells and preparation of the postnuclear supernatant. (A) Appearance of CHO cells after nitrogen cavitation at 30 psi for 15 min: Note the occurrence of "puffed" cells (P). Most of the cells are in varying degrees of disruption. However, some intact CHO cells (I) are present. (B) Appearance of CHO cells after cavitation followed by homogenization by four strokes of a Potter-Elvehjem homogenizer: Most cells are completely disrupted. However, some puffed cells (P) are still present. Intact nuclei (N) are also present. (C) Appearance of the postnuclear supernatant: No cells or nuclei can be seen in the preparation. The material was viewed with a phase x40 plan achromat objective. The final magnification of the prints was x420. Bar: 50 fim.

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Israel) or Parr Instruments (Moline, IL) should work equally well. Cell concentrations for bombing should be at least 1 x 107/ml.Best results are obtained at a concentration of around 1-2 x 108/ml. 5. Pressurize bomb to 30 psi with N2 and hold cells at pressure for 15 min on ice. Open the outlet at the bottom of the bomb and collect the cell suspension in the tube of a Potter-Elvehjem homogenizer. Close the nitrogen tank. 6. Homogenize the cavitated cells with four strokes of a motor-driven pestle of a Potter-Elvehjem homogenizer. The motor drive is set so that the Teflon pestle almost stops turning as it reaches the bottom of the homogenizer. The bomb and homogenizer are rinsed with about 2 ml of 0.25 M sucrose and this rinse can be used to wash the nuclei and unbroken cells (see below). The bomb is cleaned with dilute detergent and rinsed with distilled water. Do not allow the bomb to dry with 0.25 M sucrose in it. This procedure yields a cell homogenate that exhibits intact organelle membranes (see enzyme latency section). Dounce or Potter-Elvehjem homogenization alone of CHO cells resuspended in 0.25 M sucrose requires 25-30 homogenizer strokes to yield a homogenate in which most of the cells are broken. In these homogenates, the organelle membranes also seem to be broken; enzyme activities are no longer latent. Other investigators have prepared intact CHO cell homogenates using a tight-fitting . ~ and other cells become fragile to pipetball bearing h ~ m o g e n i z e rCHO ting with repeated rinsing in cation-free 0.25 M sucrose. This has been .~ cells, unlike roused by some as a homogenization p r ~ c e d u r e Human dent cells, swell in hypotonic saline. Many laboratories have used a hypotonic swelling step followed by homogenization with a Potter-Elvehjem or Dounce homogenizer to prepare cell homogenates exhibiting a high degree of organelle intactness from cultured human cells (e.g., HeLa; for review see Penman8). Preparation of a Postnuclear Supernatant

For the subsequent isolation of cytoplasmic organelles, nuclei and unbroken cells are rapidly separated from cytoplasmic organelles by differential sedimentation at low centrifugal force. For CHO cells, the steps, all at 4", are as follow: W. E. Balch and J. E. Rothman, Arch. Biochem. Biophys. 240, 413 (1985). E. Harms, H. Kern, and J. A. Schneider, Proc. Natl. Acad. Sci. U.S.A. 77,6139 (1980). S. Penman, in "Fundamental Techniques in Virology" (K. Habel and N. P. Salzman, eds.), p. 35. Academic Press, New York, 1969.

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1. Centrifuge the homogenate for 5 min at 1300 g,, (2300 rpm in a Beckman TH-4 rotor) to pellet nuclei and unbroken cells. 2. Remove supernatant with a Pasteur pipet and save in a separate tube. This and the subsequent supernatants from "washes" of the nuclear fraction, the pellet, constitute the postnuclear supernatant (PNS). 3. Resuspend the nuclear pellet in 2 ml of the 0.25 M sucrose used to rinse the bomb and homogenizer. 4. Centrifuge as in step 1. 5. Pool the resulting supernatant with the previous supernatant. 6. Repeat steps 3-6. Washing the nuclear fraction increases the yield of cytoplasmic organelles in the PNS and in the end, few unbroken cells will be visible by phase microscopy. During these steps the nuclei become fragile to shear forces and may clump as they are resuspended the last time. The yield of cytoplasmic organelles in the PNS should be about 6070% when measured by organelle-specific marker enzyme activity. Significant portions of cytoplasmic organelles are always retained in the nuclear pellet. Presumably this is due to trapping of organelles in the cytoskeleton.9 The PNS is the starting material for later steps in the isolation of cytoplasmic organelles. Often, the degree of organelle purification and yield in later steps are expressed relative to the PNS. The intactness of organelles in the PNS should be 80-95% based on latency measurements (see below). Determination of Organelle Intactness: Latency

Intact organelle membranes prevent substrate access to lumenal enzymes. Hence, organelle-specific enzyme activity is generally measured in the presence of nonionic detergent; comparison of enzyme activity in the presence and absence of detergent provides a quick method to assess organelle intactness. This comparison is normally expressed as percentage latent activity. Latency (%) is defined as [(activity in the presence of detergent - activity in the absence of detergent)/activity in the presence of detergent] x 100. As most enzyme reaction mixtures are hypotonic, sucrose is included at a final concentration of 0.25 M to all latency assays before addition of cellular material. Remember, if the PNS does not show a high latency, there is no point to further fractionation; rather, homogenization problems should be addressed. Generally, this means the use of gentler conditions. K. E. Howell, E. Devaney, and J. Gruenberg, Trends Biochem. Sci. 14, 44 (1989).

208

p

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=1.0408

6%Percoll 5 ml 20.000 rpm

17/35%

F ... -:. ) :-.::.Y+.:.

mitochondria

FIG.2. Schematic of the hybrid Percolllmetrizamide discontinuous density gradient used for the isolation of mitochondria from the postnuclear supernatant (PNS) obtained from CHO cells. Mz, Metrizamide. Interfaces: SIP, sucrose1Percoll; P/17%, Percollll7% metrizamide; and 17135%, 17% metritamide/35% metrizarnide (mitochondria1 preparation). "Top" depicts material found floating on the gradient after centrifugation. Reproduced with the permission of Analytical Biochemistry (Academic Press, Inc.).

Isolation of Mitochondria

Mitochondria are isolated from the PNS in a single 15-min centrifugation using a hybrid Percoll-metrizamide gradient. A schematic presentation of the gradient is given in Fig. 2. All solutions are in 0.25 M sucrose and expressed as weight/volume. Densities are determined by weighing 1-ml aliquots. Gradients are prepared in cellulose nitrate centrifuge tubes for the Beckman SW-40 rotor as follows: 1. Overlay 2 ml of 17% metrizamide (manufacturer: Nyegaard, Oslo, Norway; United States distributor: Accurate Chemical Co., Westbury, NY) on a 2-ml cushion of 35% metrizamide. 2. Onto the 17% metrizamide layer, overlay 5 ml of 6% Percoll. 3. Gently fill the centrifuge tube to within 1 mm of the top with PNS. The volume of PNS should be about 4.75 ml. For the application of overlays, the gradient material is fed by gravity through a syringe fitted with an 18-gauge needle down the side of the centrifuge tube. Ultraclear centrifuge tubes (Beckman) can be made wettable by treatment with poly(viny1 a l c ~ h o l ) . Gradients '~ should be prepared just before use and placed on ice. lo

L. Holmquist, J. Lipid Res. 23, 1249 (1982).

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Centrifugation is for a minimum of 15 min at 50,500 g,, (20,000 rpm) at 4". Use the slowest possible acceleration rate and set the centrifuge brake to slow. At the end of this time, a set of discrete bands at the interfaces should be observed (see Fig. 2). Mitochondria band at the 17135% metrizamide interface. Interfaces are sequentially removed from the top of the gradient with a Pasteur pipet connected to a pipet pump (Bel-Art Products, Pequannock, NJ). About 1-3 ml of material is removed from each interface. Fractions may either be assayed immediately for marker enzyme activity or stored at -20". The 15-min centrifuge time is sufficient. At shorter times, the yield of mitochondria is less. Lengthening the time of centrifugation by as much as 1 hr results in no increase in mitochondria1 yield. Isolation of Lysosomes

For the isolation of lysosomes, the PNS is fractionated in two sequential density gradients. The first is the hybrid Percoll/metrizamide gradient described above. From this gradient, the 6% Percoll/l7% metrizamide interface material is removed, adjusted to 35%, and placed at the bottom of the second gradient. The first gradient separates lysosomes from other organelles except mitochondria, and in the second gradient lysosomes are separated from mitochondria by flotation. The procedure is shown in schematic form in Fig. 3. A detailed protocol is given below. 1. Collect Percollll7% metrizamide interface from the first gradient. All the material from this interface should be taken up in the smallest first gradient : PNS :: ..... .:...:.' . .,:........, , . . .. . . . . .. .. ..,. .. .. ...

second gradient

0.25M

aucrose

Percoll

5%

-

Mz 17% Mz 357. Yz

FIG. 3. Schematic of the two discontinuous density gradients used for the isolation of lysosomes from CHO cells. The starting material for the second, discontinuous metrizamide gradient is the P/17% interface material of the first, hybrid Percoll/rnetrizamide gradient (also see Fig. 2), which was adjusted to 35% metrizarnide and placed in the bottom of the tube. Mz, Metrizamide. Interfaces: S/5%, sucrose/5% metrizamide; 5/17%, 5% metrizamide/l7% metrizamide; 17/35%, 17% metrizamide/35% metrizamide.

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possible volume (i.e., 1.125 ml or less). Other interfaces may also be collected to establish distribution of the other organelles and to calculate the recoveries. 2. Mix 1.125 ml of the material from the Percollll7% metrizamide interface with 0.875 ml of 80% metrizamide (d = 1.4003). This brings the metrizamide concentration to 35% (assuming that the metrizamide concentration in the Percollll7% metrizamide interface preparation is zero). 3. Overlay the 35% metrizamide layer with 2 ml of 17% metrizamide (density = 1.1081) followed by 2 ml of 5% metrizamide (d = 1.0521). Fill the tube with 0.25 M sucrose (d = 1.0317). 4. Centrifuge the second gradient for 15 min at 50,500 g,, (20,000 rpm) as above. 5. Collect interfaces. Lysosomes band at the 5117% metrizamide interface. A second mitochondrial fraction is found at the 17135% metrizamide interface. This mitochondria1 fraction is almost as pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as the mitochondria isolated from the corresponding interface of the first gradient. The sucrosel5% interface is often not detectable due to the narrow difference in metrizamide densities between the layers. For protein analysis, lysosomes and mitochondria can be pelleted by first diluting the material from the appropriate interface to 39 ml with 0.25 M sucrose in a 1 x 3t in. centrifuge tube for the SW-27 or SW-28 rotor, followed by centrifugation at 20,000 rpm (53,000 g,,) for 20 min. Stock metrizamide density solutions can be stored at -20" and repeatedly thawed before use. This reduces microbial contamination, which readily occurs in metrizamide solutions stored at 4". Centrifuge-grade metrizamide can be used although it is more difficult than analytical-grade metrizamide to dissolve to 80% in 0.25 M sucrose. Isolation of Nuclei

In order to minimize the number of unbroken cells, CHO cells (suspended in 0.25 M sucrose) are pressurized to 60 rather than 30 psi for 15 min. Isolation of a nuclear fraction stable to shear forces requires the addition of salt, particularly Mg2+,to the cell homogenate. Hence, after the second wash of the nuclear fraction with 0.25 M sucrose, resuspend the nuclei in 2 ml of 0.25 M sucrose, 10 mM NaC1, 10 mM Tris-HC1, and 1.5 mM MgC12, pH 7.4 (SNTM). SNTM is added after the final wash because organelles aggregate in the presence of SNTM and pellet with the nuclei. Contaminating organelles and whole cells are removed by adding

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' ~ suspension is then vigorously vortexed 0.3 ml of detergent s ~ l u t i o n .The for 3 sec and the nuclei are pelleted at 1300 g,, for 5 min. The detergent solution is prepared by combining one part 10% (wlw) sodium deoxycholate and two parts 10% (wlw) Tween 40. Concentrated Tween 40 should be heated to 60-70" to clarify before preparing the 10% stock. Other nonionic detergents, such as Tween 80 and Nonidet P-40 (NP-40), may be substit~ted.~ Relative to the homogenate, from 0 to about 5% of the organellar marker enzyme activities is recovered in the nuclear preparation. Under phase microscopy, the preparation appears to contain large aggregates of nuclei (Fig. 4). The detergent treatment dissolves the outer nuclear membrane.'* Nuclei prepared in this and other ways are depleted in many nuclear proteins.13 Isolated nuclei also contain altered chromatin (for review see Cook14). General Considerations for the Assay of Organellar Marker Enzymes

The assay conditions described are ideal for the assay of gradient fractions obtained from the PNS of 1 to 2 x lo8 CHO cells (about 10 to 20 mg of protein). Each of the enzyme activities described are stable to at least one freeze-thaw cycle. Prior to fractionation, at least 0.1 ml of PNS is diluted 1 : 10 with 0.25 M sucrose and saved for use as a reference. This dilutes the PNS so that it can be assayed over the same time periods as the gradient fractions. Under normal conditions, the material at the top and the S/5% interface of the second gradient (Fig. 3) does not contain any detectable marker activity. Table I indicates the organelle that each activity is specific for, as well as whether metrizamide inhibits its activity. Note, when making buffers, do not use NaOH or KOH to raise the pH, because NaOH or KOH cause Percoll-containing solutions to become turbid. For many assays we use fluorogenic substrates because a fluorescence assay minimizes turbidity problems with Percoll when an alkaline stop solution is used. Bookkeeping of Organellar Marker Enzyme Activities

The enzymatic assessment of the purity of an organelle preparation requires careful tabulation of marker enzyme recoveries, specific activities, and enrichment (-fold). The purity of the nuclear preparation can be

" S. Penman, J . Mol. Biol. 17, 117 (1966). l3

l4

E. Holtzman, I. Smith, and S. Penman, J. Mol. Biol. 17, 131 (1966). P. L. Paine, C. F. Austerberry, L. J. Desjarlais, and S. B . Horowitz, J . Cell Biol. 97, 1240 (1983). P. R. Cook, J . Cell Sci. 90, 1 (1988).

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FIG. 4. Appearance of the nuclear preparation before (A) and after (B) detergent treatment. Detergent treatment removes presumptive cytoskeletal elements (arrows) with attached organelles. Minor amounts of partially disrupted cells are also removed by this treatment. The preparation was viewed with a phase x 4 0 plan achromat objective. The final magnification of the prints was ~ 4 2 0 Bar: . 50 pm.

inferred from the absence of marker activities for the cytoplasmic organelles and the plasma membrane. Total enzyme activities (sample activitylsample volume x total ml) should be calculated in order to determine the recovery of the marker enzyme activities. The recoveries should range from 80 to 120%. Marker enzyme-specific activities (total activityltotal protein) in each gradient

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TABLE I ACTIVITY AND INHIBITION OF ORGANELLE ENZYMES

Enzyme or substance

Organelle

Cytochrome-c oxidase p-Hexosaminidase p-Galactosidase Horseradish peroxidase Alkaline phosphodiesterase I a-Mannosidase I1 Catalase Lactate dehydrogenase RNA

Mitochondria Lysosome Lysosome Endosomec Plasma membrane Golgi apparatusd Peroxisome Cytosol Rough endoplasmic reticulume -

Protein

Inhibited by metrizamidea

Ref.

No Yes Yes No No Yes No Yes NAf

Determined by assay concentrations of metrizamide up to 7%. References: (I) E. A. Madden and B. Stome, Anal. Biochem. 163, 350 (1987); (2) R. R. Pool, Jr., K. M. Maurey, and B. Storrie, Cell Biol. Znt. Rep. 7, 361 (1983); (3) N. N. Aronson, Jr. and 0. Touster, this series, Vol. 31, p. 90; (4) F. Leighton, B. Poole, H. Beaufay, P. Baudhuin, J. W. Coffey, S. Fowler, and C. de Duve, J. Cell Biol. 37, 482 (1968); (5) L. A. Decker, ed., "Worthington Enzyme Manual," p. 20. Worthington Biochemical Corp., Freehold, New Jersey, 1977. (6) RNA labeling; B. Stome and G. Attardi, J. Biol. Chem. 248,5826 (1973);precipitation with trichloroacetic acid; A. L. Hubbard and Z. A. Cohn, J . Cell Biol. 64, 438 (1975); (7) P. Bohlen, S. Stein, W. Dairman, and S. Udenfriend, Arch. Biochem. Biophys. 155,213 (1973); S. Udenfriend, S. Stein, P. Bohlen, W. Dairman, W. Leimgruber, and M. Weigele, Science 178, 871 (1972). ' CHO cells were pulsed for 10 min with horseradish peroxidase, conditions which label endosomes [C. J. Adams, K. M. Maurey, and B. Stome, J . Cell Biol. 93, 632 (1982)l. The conditions of assay favor a-mannosidase 11, a Golgi apparatus marker [D. R. P. Tulsiani, S. C. Hubbard, P. W. Robbins, and 0. Touster, J . Biol. Chem. 257, 3660 (1982)l. A small portion of the total activity is lysosomal [R. R. Pool, Jr., K. M. Maurey, and B. Stome, Cell Biol. Znt. Rep. 7, 361 (198311. 'Most of the pelletable RNA in the PNS corresponds to bound ribosomes (the rough endoplasmic reticulum). Most of the free ribosomes would fractionate with the cytosol in these gradients. A trace of the RNA would be found in mitochondria. f NA, Not applicable.

a

fraction should be calculated to provide the fold enrichment values (specific activity of the gradient fractionlspecific activity of the PNS). Based on CHO cells, the enrichment of lysosomal marker enzyme activities in the lysosomal preparation should be around 40-fold relative to the PNS. The enrichment of mitochondria1 marker enzyme activity in the mitochondria preparation should be around 43-fold relative to the PNS. The PNS represents 60-70% of the homogenate protein.

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Detailed Enzyme Assay Protocols

A. Cytochrome c Oxidase (EC 1.9.3.1):Mitochondria1 Marker Materials Phosphate buffer: 6.2 mM K2HP04/33.8mM KH2P04,pH 6.2 Reduced cytochrome c (horse heart, Sigma type 111): Dissolve 13.5 mg of cytochrome c in 5 ml of phosphate buffer, then add 60 pl of a 10 mg/ml solution of sodium dithionite (sodium hyposulfite) freshly prepared in phosphate buffer. The color of the cytochrome c solution should change from dark brown to light orange. The resulting ASSo/ A565ratio of the cytochrome c solution when diluted 1 : 10 with phosphate buffer should be between 6 and 9 using phosphate buffer as a blank. The cytochrome c solution should be made fresh and stored in an air-tight container on ice during the assays Lubrol PX (0.244%, wlv) in phosphate buffer: This gives a final assay concentration of 0.2% Lubrol PX. Lubrol PX permeabilizes mitochondria without affecting cytochrome c oxidase activity.15Concentrated Lubrol PX (Sigma) should be stored at 37' to keep it liquid Phosphate buffer for latency determinations: Prepare the phosphate buffer in 0.25 M sucrose. Use this buffer to prepare the cytochrome c solution, and assay plus and minus Lubrol 0.25 M sucrose: This is the background for the PNS and all gradient fractions '

Method 1. To a cuvette add 0.9 ml of 0.244% Lubrol PX in phosphate buffer. Use this buffer to zero the spectrophotometer. 2. Next, add 0.1 ml of reduced cytochrome c. 3. Add the appropriate amount of 0.25 M sucrose to bring the volume up to 1.1 ml (including the volume of sample). 4. Finally, add the sample and mix the contents of the cuvette by inversion. 5. Read the decrease in absorbance at 550 nm at room temperature. Comments. The reaction is linear for decreases in absorbance between 0.01 and 0.1 units. Enzyme activity can be expressed as the decrease in absorbance unitslmin. About 50 p1 of material isolated from the P/17% and 17135% interfaces of the first gradient and from the 17135% interface of the second gradient should be sufficient. For the PNS and the other interfaces, about 100 p1 is sufficient. The sample activities should be l5

J. Rafael, in "Methods of Enzymatic Analysis" (H. U . Bergmeyer, ed.), Vol. 3, p. 266. Verlag Chemie, Weinheim, 1983.

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corrected for the spontaneous oxidation of cytochrome c by air or contaminants in the reagents by subtracting the background oxidation rate. Background is determined by substituting 0.25 M sucrose or gradient media for the sample. Typically, background oxidation of cytochrome c decreases the absorbance 0.005-0.006 A unitslmin. This assay is particularly well suited for software-driven spectrophotometers, which can calculate initial rates and perform baseline adjustments.

B. /?-Hexosaminidase (EC 3.2.1.30) and P-Galactosidase (3.2.1.23): Lysosomal Markers Materials Acetate buffer: 0.4 M sodium acetate, pH 4.4 10% (wlv) Triton X-100 (Sigma) Glycinelcarbonate stop solution: 0.5 M glycine, 0.5 M Na2C03 P-Hexosaminidase substrate solution: 4.0 mM 4-methylumbelliferyl-2acetamido-2-deoxy-p-D-glucopyranoside in H 2 0 p-Galactosidase reaction mixture: 1.0 rnM 4-methylumbelliferyl-PDgalactopyranoside in 0.1 M sodium acetate, pH 4.4 and 0.125% Triton X-100. Methylumbelliferyl derivatives are manufactured by Koch-Light for Research Products International. The stock P-hexosaminidase substrate solution and the p-galactosidase reaction mixture can be stored at -20". Sonication with a sonifier (e.g., Branson Sonic Power Co.) is necessary to dissolve the methylumbelliferyl substrates and to redissolve them after thawing frozen stocks. Alternately, the substrates can be first dissolved in 11100 vol of dimethyl sulfoxide, then added to the final volume of reaction mixture. The reaction mixture should be kept at 37" until use. If kept at cooler temperatures or frozen, it must be sonicated to redissolve the substrate 2.5 M sucrose 0.25 M sucrose Method 1. The P-hexosaminidase reaction mixture is prepared just before use by combining 10 ml substrate solution, 10 ml acetate buffer, 0.5 ml of 10% Triton X-100, and 19.5 ml of water. The p-hexosaminidase substrate decomposes slowly in acidic solutions. Warm to 37". For latency determinations, one-tenth volume of 2.5 M sucrose should be added to the reactioh mixture without Triton X-100. An equal amount of water should be added to the Triton X-100 containing reaction mixture. 2. In a chilled 12 x 75 mm test tube dilute a sample of gradient mate-

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-

rial with 0.25 M sucrose to 0.1 ml. Since metrizamide inhibits p-hexosaminidase and p-galactosidase, no more than 20 p1 of gradient fractions that contain metrizamide should be assayed. For the PNS and the other gradient fractions, 100 p1 should be sufficient. Use 100 pl of 0.25 M sucrose as a blank. 3. Start the assay by adding 0.4 ml of reaction mixture, and incubate at 37" for 45 min to 1 hr. 4. Stop the assay with 1.0 ml of glycinelcarbonate. 5. Zero the spectrofluorometer with the blank, and read the fluorescence at an excitation wavelength of 364 nm and an emission wavelength of 448 nm. With a Perkin-Elmer 650-10s spectrofluorometer both slits should be set at 3 nm. Comments. Good recoveries are possible only when metrizamide concentrations in the assay are less than 1.5%. When determining latency, run the assay for only 5 min or less, because under these assay conditions, substrate penetration gradually occurs. Enzyme activity can be expressed as relative fluorescence unitslmin. C . Horseradish Peroxidase (EC 1.1 1.1.7), Endosomal Marker: Labeling of Endosomal Compartment Materials

Peroxidase, from horseradish (Sigma, type 11) Log-phase CHO cell culture: About 5 x lo5 cellslml in 200 ml of Eagle's minimal essential medium, a-modification without ribonucleotides and deoxyribonucleotides, supplemented with 10% heatinactivated fetal bovine serum (a-MEMFCIO) 0.13 M NaCl, 0.005 M KCI, and 0.001 M MgC12, supplemented with 10% calf serum (NKMClO), 4" Four milliliters of NKMC10 frozen in a pliable polyethylene tube, then crushed gently with a hammer. The crushed frozen NKMC 10 is then placed in the bottom of a 50-1111 conical centrifuge tube (e.g., Corning) Water bath, 37", equipped with a shaker (shaker bath) Method

1. Pellet cells from 200 ml of log-phase culture at 380 g,, (1200 rpm, Beckman TH-4 rotor) for 5 min, wash the cells with 25 ml of a-MEMFC10 at 37", and pellet cells at 24". 2. Resuspend the cells in 9 ml of fresh a-MEMFC10 at 37" in a 25-ml Erlenmeyer flask. The medium is first gassed for 2-3 min with 5% C 0 2 . Incubate the concentrated culture for 10 min at 37" in a shaker bath to temperature equilibrate.

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3. Add 1 ml of a-MEMFCIO containing 10 mg of horseradish peroxidase and continue the incubation for 10 min in the shaker bath. 4. At the end of the uptake period, rapidly pour the culture into the 50-ml centrifuge tube containing 4 ml of crushed frozen NKMC10. Next, fill the tube to the 50-ml mark with ice-cold NKMC10. Finally, pellet the cells for 5 min at 380 g,, at 4". 5. Wash the cells five times with ice-cold NKMCIO, pelleting at 380 g,, for 5 min after each wash. Use a different 50-ml test tube after each wash. 6. Isolate and fractionate the PNS as described above.

Assay of Horseradish Peroxidase Materials 10 mglml o-dianisidine (Sigma) in absolute methanol: Make fresh daily and protect from light 0.3% H202 can be stored for months at 4" in a sealed bottle 0.1 M imidazole-HC1, pH 7.0 10% Triton X-100 0.25 M sucrose Method 1. Prepare reaction mixture by adding 60 p1 of 3% H202, 50 p1 of 10 mglml o-dianisidine, and 68 p1 of Triton X-100 to every 6 ml of 0.1 M imidazole buffer. Use a clean bottle and protect from light. Prepare fresh daily. 2. To a cuvette add 0.9 ml of substrate solution. Use this solution to blank the instrument. 3. Add 0.25 M sucrose to bring the sample volume up to 0.1 ml. 4. Finally, add the sample. Add 50 p1 of material from the top and sucroseIPercoll (SIP) interface of the first gradient, and 100 p1 of the PNS and the other interfaces. Mix the contents of the cuvettes by inversion and read the increase in absorbance at 460 nm for 1-3 min. 5. Prepare a blank sample reaction mixture by adding 0.1 ml of 0.25 M sucrose or blank gradient medium instead of sample. 6. A standard curve can be obtained by adding 0.1 ml of various dilutions (i.e., from 1 to 10 nglml) of horseradish peroxidase. Comments. Enzyme activity can be expressed as an increase in absorbance unitslminute. The activities in the gradient fractions are corrected for background by subtraction. Cuvettes are cleaned by soaking in 0.1 M HCl to remove o-dianisidine deposits.

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D. Alkaline Phosphodiesterase I (EC 3.1.4.1): Plasma Membrane Marker

Materials 10 rnM sodium thymidine 5'-monophosphate, p-nitrophenyl ester (Sigma): Store at -20" 0.1 M Tris-HC1, pH 9.0 0.5 M glycine, 0.5 M Na2C03 0.25 M sucrose Method 1. Prepare the reaction mix by combining one part substrate solution, one part Tris-HC1 buffer, and two parts water. Warm to 37'. 2. In a chilled 12 x 75 mm test tube, dilute a sample of the gradient fraction to 0.1 ml with 0.25 M sucrose. Since the top and SIP interface of the first gradient contain the bulk of the alkaline phosphodiesterase I activity, only 20 to 50 p1 of this material is needed. About 100 p1 of the PNS and material isolated from the other interfaces is needed. As a backgroundlblank use 0.25 M sucrose or gradient medium alone. Prepare two backgroundslblanks. 3. Add 0.4 ml of reaction mixture to the test tubes and incubate at 37". After about 2 hr (TI) the PNS, top, and the SIP-containing reaction mixtures should be yellow. Stop these reactions and one of the blanks with 1.0 ml of glycinelcarbonate, and read their absorbance at 410 nm. From the TI sample values subtract the TI blank. 4. Since less than 1.O% of the total PNS alkaline phosphodiesterase I activity is recovered in the second gradient, the other gradient fractions should be incubated for a long time to provide readings well enough above background. Seal the other test tubes and continue the incubation overnight at 37". After 14 to 18 hr (T2) stop the other reactions and the T2 backgroundlblank with 1.0 ml of glycinelcarbonate. 5. Read the absorbance at 410 nm of the overnight assays and subtract the T2 blank. Comments. Enzyme activity can be expressed as absorbance units1 minute. This reaction is linear to an absorbance of 0.4. For CHO cells, little alkaline phosphodiesterase I activity is found in lysosomes. About 10% of the total alkaline phosphodiesterase I activity has been shown to colocalize with lysosomes purified from rat fibroblasts.I6 l6

J.-P. Draye, P. J. Courtoy, J. Quintart, and P. Baudhuin, Eur. J. Biochem. 170, 405 (1987).

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ISOLATION OF SUBCELLULAR ORGANELLES

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E. a-Mannosidase I1 (EC 3.2.1.24): Golgi Apparatus Marker This assay can be performed exactly as described for the lysosomal marker, /3-hexosaminidase, except for the following: 1. 4-Methylumbelliferyl-a-D-mannopyranoside (4.0 mM) is the sub-

strate solution. This substrate is also produced by Koch-Light and sold by Research Products International. 2. Dulbecco's phosphate-buffered saline (137 mM NaC1, 0.27 mM KC1,0.8 1 m M Na2HP04,0.15 mM KH2P04,0.05 mM MgC12- 6H20,0.09 mM CaC12,pH 7.4) is used instead of sodium acetate buffer. 3. Only about 15 to 30 min of incubation time is necessary. Comments. Enzyme activity can be expressed as relative fluorescence unitslminute. Most of a-mannosidase I1 activity localizes in the top and SIP interface of the first gradient. Thus, only about 20 p1 of these fractions is necessary for good activity. a-Mannosidase I1 activity is inhibited by assay concentrations of metrizamide greater than 1.5%. Hence 20 p1 or less of gradient fractions containing metrizamide should be assayed. A small portion of the pH 7.4 a-mannosidase activity is lysosomal. The extent can be determined by assay in the presence of p-chloromercuriphenylsulfonic acid which inhibits the lysosomal form.4

F . Catalase (EC 1.11.2.6): Peroxisomal Marker Materials

Bovine serum albumin 0.2 M imidazole buffer, pH 7.0 10% (wlv) Triton X-100 30% H202 Stopping solution: In a fume hood, dissolve 2.25 g of TiOS04in 0.3 liter of boiling 2 N &So4. Allow the solution to cool, then filter the solution with a Whatman #42 filter. Add 0.2 liter of 2 N H2S04. Finally, dilute 1 : 1 with water. The stop solution can be stored for weeks at room temperature in a light-proof container Method

1. Prepare the reaction mixture immediately prior to use by combining 50 mg bovine serum albumin, 5 ml 0.2 M imidazole buffer, 1 ml 10% Triton X-100, and 0.1 ml 30% H202in a graduated cylinder. Then bring the volume up to 50 ml with water. 2. In chilled 12 x 75 mm test tubes, place 10 pl of each gradient fraction.

220

EXTRACT PREPARATION AND FRACTIONATION

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3. Prepare a blank reaction mix by adding 10 p1 of 0.25 M sucrose or gradient medium alone to a test tube. 4. Add 0.5 ml of reaction mixture to each test tube and incubate the top and SIP interface of the first gradient for 10 min and the other interfaces and the PNS for 30 min at room temperature. 5. Stop the reaction by adding 2 ml of stop solution. 6. Read the absorbance at 405 nm, using stop solution alone to zero the spectrophotometer. Comments. TiOS04 reacts with H202, forming a yellow complex. Thus, the change in absorbance can be computed by subtracting the sample absorbance from the background absorbance and enzyme activity can be expressed as decrease in absorbance unitslminute. The reaction appears to be linear for changes in absorbance up to 0.9 units. G . Lactate Dehydrogenase (EC 1.I .1.27): Cytosol Marker

Materials 0.2 M Tris-HC1, pH 7.3 6.6 rnM NADH in 0.2 M Tris-HC1, pH 7.3: Prepare fresh daily by adding 0.97 ml of buffer to 5 mg of NADH (Sigma, individual vial pack, 5 mglvial) 30 mM sodium pyruvate in 0.2 M Tris-HC1, pH 7.3 10% (w/v) Triton X-100 Method 1 . Prepare the reaction mix by adding 0.36 ml of 6.6 rnM NADH, 0.36 ml of 30 mM sodium pyruvate, and 0.108 ml of 10% Triton X-100 to 10 ml of buffer. 2. To a cuvette add 0.9 ml of buffer. Use this as a blank to zero the spectrophotometer. 3. Add enough 0.25 M sucrose to bring the sample volume to 0.1 ml. 4. Finally, add the gradient material. Mix the contents of the cuvette and read the decrease in absorbance at 340 nm at room temperature. Comments. Enzyme activity can be expressed as decrease in absorbance unitslminute. Only 5 pl of material from the top of the first gradient is necessary. From 10 to 20 p1 of material from the SIP interface is sufficient. Metrizamide competitively inhibits lactate dehydrogenase activity. Thus gradient fractions containing metrizamide should be diluted at least 1 :5 with 0.25 M sucrose before assay. Lactate dehydrogenase activity is not found below the SIP interface of the first gradient.

[I61

ISOLATION OF SUBCELLULAR ORGANELLES

22 1

H. RNA ([2-I4C]UridineIncorporation into Trichloroacetic Acid-Precipitable Material): Cytosol and Rough Endoplasmic Reticulum Marker Materials

2 pCi [2-14C]uridine(51.2 mCiImmol, ICN Biomedicals) CHO cells and culture: About 2 x loS CHO cells/ml logarithmically growing in 200 ml of culture (Eagle's minimal essential medium, amodification, supplemented with 10% fetal bovine serum) Hybrid Percoll/metrizamide and discontinuous metrizamide density gradients (Fig. 3) Whatman GFIA, 2.4-cm glass microfiber filters 10% trichloroacetic acid (TCA), 4" 90% acetone, -20" Minivials (5 ml) and scintillation cocktail (e.g., Ecoscint, National Diagnostics, Somerville, NJ) Method

1. Add 2 pCi of [2-14C]uridineto a 200-ml spinner culture of logarithmically growing CHO cells at 2 x lo5 cellslml. Incubate the culture overnight (15-18 hr). 2. Harvest the cells, prepare and fractionate the PNS as described above. 3. Label each microfiber filter with India ink, then prewet each filter with ice-cold 10% TCA. Do not add more than 50 p1 of gradient fraction to the filters. About 20 p1 of material from the top and SIP interface is sufficient and about 50 p1 of material from the PNS and the other fractions should be enough. 4. Place the filters in a beaker containing 500 ml of ice-cold 10% TCA and incubate for at least 2 hr on ice. 5. Discard the TCA into radioactive waste and wash the filters twice with 400 ml of 10% TCA at 4". Allow 5 minlwash. 6. Wash the filters once in 90% acetone at -20". 7. Allow the filters to air dry. Place them in minivials, add 5 ml scintillation cocktail, and count. Include two or three blank filters to correct by subtraction for transfer of radioactivity. The blanks should be no more than about 3% of the radioactivity of the hottest filter. The blanks being greater than 3% of the hottest filter would suggest a lack of stirring of the filters in TCA, warmer temperatures, or too small of volume of TCA. Comments. This protocol results in the labeling of free ribosomes, which should localize with lactate dehydrogenase (the cytosol) in the two discontinuous density gradients. The bulk of the pelletable RNA in the

222

EXTRACT PREPARATION AND FRACTIONATION

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PNS is associated with the rough endoplasmic reticulum and about 1020% of the total ribosomes are membrane associated in nonsecretory cells such as HeLa cells.17 A minor proportion of the total rRNA is intramitochondrial. In this two-gradient system, the bulk of the radioactivity localizes at the top and the SIP interface of the first gradient, which is also where lactate dehydrogenase localizes. Metrizamide is soluble in cold 10% TCA and thus does not interfere with scintillation counting. Protein Determination

Materials

1 mg/ml bovine serum albumin in 0.25 M sucrose 0.05 M Na2HP04,pH 8.0 Fluorescamine reagent: Dissolve 30 mg fluorescamine (Sigma) in 100 ml anhydrous acetone (Mallinckrodt, Paris, KY; nanograde). Protect from moisture and light 0.25 M sucrose Method 1. Place a sample of gradient fraction in a 15 x 85 mm test tube. About 50 p1 of material from the top and SIP interface of the first gradient and 200 pl of material from the PNS and the other gradient fractions is sufficient. Prepare a blank by adding 200 ml of 0.25 M sucrose. 2. Prepare protein standards by adding various amounts of the bovine serum albumin solution to each test tube (e.g., 5, 10, 20, 40, and 50 pl). 3. Make the volume constant (i.e., 200 p1) in each test tube by adding 0.25 M sucrose. 4. Add 1.0 ml phosphate buffer and vortex. 5. While vigorously vortexing, rapidly add 0.5 ml of fluorescamine reagent. Rapid mixing of the fluorescamine reagent with the sample is essential because of the rapid hydrolysis of fluorescamine in aqueous solutions. 6. Allow each sample to sit at least 2 min, then zero the spectrofluorometer with the blank at an exciting wavelength of 398 nm and an emission wavelength of 480 nm. The slits should be set at 4 nm. Finally, read the sample fluorescence. Comments

The fluorescence remains stable for over 2 hr, thus a large number of samples can be prepared before reading. Since the slope of the standard B. Attardi, B . Cravioto, and G . Attardi, J. Mol. Biol. 44, 47 (1969).

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ISOLATION OF SUBCELLULAR ORGANELLES

223

curve (relative fluorescence unitslpg) varies slightly from experiment to experiment, a set of standards must be analyzed with each experiment. The readings are linear from 1 pg to 1 mg protein at the lowest sensitivity scale for our Perkin-Elmer 650- 1OS spectrofluorometer. Although, with readings above about 80 fluorescence units, dilutions with phosphate buffer are necessary to stay in the linear range of older model fluorometers such as ours. Electron Microscopy of Cytoplasmic Organelles

Electron microscopy provides an alternate methodology for assessing the homogeneity of organelle preparations. Gradient fractions (e.g., the mitochondria1 and lysosomal preparations) can easily be prepared for electron microscopy. The following is a detailed protocol for the preparation of gradient fractions for electron microscopic examination with the primary goal being preservation of morphology: 1. Carefully remove the interface material with a Pasteur pipet connected to a pipet pump (Bel-Art Products, Pequannock, NJ) or a similar device that does not permit back-flow and place the fraction in a chilled graduate cylinder. 2. Add an equal volume of 5% glutaraldehyde in 0.2 M cacodylate buffer, pH 7.4. Incubate at 4' for 15-18 hr. 3. Next, add an equal volume of 1% 0 ~ 0 in4 0.1 M cacodylate 2nd incubate at 4" for 20 min. 4. Place the gradient material in a 1 x 34411. centrifuge tube for the SW-27 or SW-28 rotor (Beckman) and dilute the material to fill the 39-ml tube with 0.25 M sucrose. 5. Pellet the organelles at 20,000 rpm (53,000 g,,) for 20 min, at 4", with a fast acceleration rate and high brake. 6. Carefully aspirate the supernatant, add 0.5 ml 0.1 M cacodylate, pH 7.4, and transfer the pellet, as clumps, to a "BEEM" embedding capsule (Polysciences, Inc.). 7. Wash the clumps three times with 0.5 ml of 0.1 M cacodylate, pH 7.4. If the pellet disassociates in the buffer, it is necessary to pellet the organelles between each wash. This is accomplished by placing the BEEM capsules "piggyback" into 1.5-ml microcentrifuge tubes and pelleting the small fragments for 30 sec in a microcentrifuge. The BEEM capsules should be pushed tightly into the microcentrifuge tubes. It may be necessary to cut the BEEM capsule out of the microcentrifuge tube. 8. Dehydrate the specimen by 5-min incubations in increasing ethanol concentrations at room temperature. We use 50, 60, 70, 80, 90, and 95% ethanol. As a overnight step, the sample can be left in 70% ethanol at 4". Finally, the sample is incubated twice in 100% ethanol for 10 min each.

224

EXTRACT PREPARATION AND FRACTIONATION

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FIG. 5. Appearance of the mitochondria1 preparation obtained from the "hybrid" Percolllmetrizamide discontinuous density gradient. Material isolated from the 17135% interface of this gradient (Fig. 2) was prepared for electron microscopy as described in the text. The sections on 300-mesh copper grids were poststained with lead citrate and uranyl acetate and were viewed in a Zeiss EM 10-CA electron microscope with an accelerating voltage of 80 kV. The final magnification of the print was X4000. Bar: 5 pm.

Note that during dehydration the sample usually becomes dense enough that centrifugation between steps is no longer necessary. 9. Infiltrate the sample at room temperature with two 15-min incubations in anhydrous acetone, followed by 30 min in 90% acetonello% Spurr low viscosity resin18 (Polysciences, Inc., longer pot life, lower viscosity recipe), 60 min in 40% Spurr's resin, and 1 hr in 90% Spurr's resin. The sample should be incubated in 100% Spurr's resin for 15-18 hr at 4'. Infiltration can be done on a shaker or rotary table. 10. The Spurr's resin should then be removed and replaced with new Spurr's resin. The sample can be packed into the tip of the BEEM capsule by centrifuging the BEEM capsule-microcentrifuge tube assembly for 30 sec, rotating the assembly 90", centrifuging again, and so on about four l8

A. R. Spurr, J. Ultrastruct. Res. 26, 31 (1969).

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PREPARATION OF MEMBRANE FRACTIONS

225

times. The Spurr's resin is polymerized by incubation at 70" for at least 16 hr. Spurr's resin can be made as a stock which can be used for a few weeks provided that it is stored at 4", under an N2 atmosphere. Old resin should be polymerized by heating before disposal. 11. Section the cured blocks, stain the sections (optional) with lead citrate (Reynolds,19 2 min) or uranyl acetate (2 in 50% ethanol, 20 min), and view them in an electron microscope at 60 or 80 kV. Note that mitochondria are more electron dense than other organelles after poststaining (Fig. 5). l9

E. S. Reynolds, J. Cell Biol. 17, 208 (1963).

[17]Preparation of Membrane Fractions

Membrane fractionation may be conveniently divided into several sequential steps: ( I ) selection of the tissue to be investigated, (2) selection of the membrane fraction to be examined, (3) choice of homogenization conditions, (4) preparation of the membrane fraction by differential or density gradient centrifugation, (5) solubilization of membrane proteins, and (6) resolution of the components. This chapter will describe the preparation and characterization of membrane fractions using liver as an example. The choice of liver will depend on the quantity, type, and frequency of isolation of the membrane fraction needed. Since freezing and thawing of isolated vesicles can alter normal membrane architecture, in some instances, fresh preparations may be required. For example, if a small amount of fresh microsomal membrane fraction is needed, then a mouse liver (-2 g) may be a good choice. Rabbit liver (-65 g) is convenient for preparative scale preparations. Rat liver (-10 g) is the most frequently used tissue, since methodology for subcellular fractionation of this tissue has been studied in the most detail.' If the quantity of membrane fractions needed requires a large number of rat livers, then liver of a larger animal should be considered. The choice of animal may depend on the type of packing plant in the area and whether livers are removed within a few minutes after the animal is killed. While proteins from a specific memS. Fleischer and L. Packer, eds., this series, Vol. 31, Part A.

METHODS IN ENZYMOLOGY, VOL. 182

Copyright O 1990 by Academic Press, Inc. All lights of reproduction in any form reserved.

226

EXTRACT PREPARATION AND FRACTIONATION

[I71

brane fraction can be isolated from preparations simply enriched in a particular subcellular fraction, large-scale isolation of pure membrane fractions is still a research project with frequently unpredictable outcomes. It is a common practice to starve animals used for the preparation of liver membranes in order to reduce their glycogen content since glycogen may cause aggregation of organelles and also lead to unnecessary turbidity of solubilized membrane fractions. Livers from fasted rats will weigh about 30% less than those from fed animals. To avoid the increased amounts of connective tissue and associated filaments in the livers of older rats, the use of livers from young animals is preferred. Rats are killed by cervical dislocation or decapitation according to institutional Animal Care Facility guidelines. Before the liver is removed, the gall bladder should be located and its contents removed with a syringe in order to prevent unnecessary contamination with bile salts. Since blood components (e.g., hemoglobin, serum proteases) are adsorbed on the surface of microsomes, and proteases may cause autolysis, immediate perfusion of excised liver with cold 0.25 M sucrose or 0.154 M saline is helpful. All subsequent procedures are carried out in a cold room or at about 5", and as rapidly as possible. After perfusion, the liver is immersed in cold sucrose (0.25 M, neutralized to pH 7.5 with KOH), cut into several large pieces, weighed, and minced into small pieces with a razor blade. Connective tissue and fat are cut away as much as possible. Homogenization is performed in a Teflonglass Potter-Elvehjem or Dounce-type homogenizer in a volume and medium most suited for isolation of a particular subfraction. The homogenization should be done at 5", and be gentle, with minimal generation of bubbles or vacuum, in order to reduce vasiculation and production of small membranes. Plasma membranes can be separated from rnicrosomes only as fragile sheets. Hypertonic sucrose solutions can be used to preserve mitochondria1 morphology, although they inhibit oxidative phosphorylation. Acidic media and divalent cations stabilize nuclei against disruption, but may activate lysosomal proteases. Isotonic sucrose (0.25 M), slightly buffered to pH 7-8 with Tris-HC1 (10 mM), is most commonly used. If large quantities of liver (> 100 g) are processed then a blade homogenizer (Waring type) is used. It is important to avoid overhomogenization of the suspension so that released organelles are not disrupted. In addition to plasma membranes, Golgi vesicles and nuclei are also quite fragile, and their damage releases polybasic nucleoproteins which adsorb to polyanionic membranes and may promote aggregation of particles. When a blender is used, homogenization should be monitored by phase-contrast

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PREPARATION OF MEMBRANE FRACTIONS

227

microscopy, and halted when a maximum of cells and a minimum of nuclei have been disrupted. Differential centrifugation is the most widely used fractionation technique for homogenates. Sequential centrifugation at 600, 10,000, and 105,000 g will give pellets enriched in "nuclear," "mitochondrial," and "microsomal" fractions, respectively. This is most useful as a preliminary step, but the fractions obtained are far from homogeneous. The 10,000 g fraction, for example, contains considerable amounts of lysozymes, peroxisomes, plasma membranes, Golgi vesicles, and endoplasmic reticulum, in addition to mitochondria. The yield of microsomes can be significantly increased by mild resuspension of the first 10,000 g pellet and recentrifugation at 105,000 g. Contaminating membranes of the microsomal fraction include Golgi membranes (1% of the total protein), mitochondria (3%), lysosomes (5%), and plasma membranes (5%). The resolving power of differential centrifugation may be enhanced by layering the homogenate onto a medium of higher density. In densitygradient centrifugation, two methods may be distinguished. In equilibrium density gradient centrifugation the homogenate is layered on top of either a continuous or discontinuous density gradient, and centrifugation is continued until the subcellular particles are in density equilibrium with the surrounding medium. In the second method, rate-zonal centrifugation, sedimentation through a shallow gradient is used and centrifugation is brief, bringing the particles into the gradient in the form of zones whose positions reflect their sedimentation rates. Centrifugation of the 10,000 g supernatant in 0.44 M sucrose layered over 1.3 M sucrose at 105,000 g for about 7 hr, will yield rough microsomes as a pellet and smooth microsomes at the sucrose-sucrose i n t e r f a ~ e . ~ In Volume 31 of this series (Biomembranes, Part A) the isolation of purified subcellular fractions from liver and several other tissues was reviewed. Except for a few modifications this methodology is still applicable today. Isolation of Rat Liver Plasma Membranes3

Reagents and Materials

0.154 M NaCl 0.25 M sucrose-5 mM Tris-HC1, pH 7.2-1 mM MgCI2 (STM), d 1.03 glml ". W. Depierre and G. Dallner, Biochim. Biophys. Acta 415,411 (1975). A. L. Hubbard, D. A. Wall, and A. Ma, J. Cell Biol. 96, 217 (1983).

=

228

EXTRACT PREPARATION AND FRACTIONATION

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1.42 M sucrose-5 mM Tris-HC1, pH 7.2-1 mM MgC12, d = 1.18 glml 2.0 M sucrose-5 mM Tris-HCI, pH 7.2-1 mM MgC12, d = 1.25 glml Dounce homogenizer, type A, with a loose clearance pestle (Kontes Glass Co., Millville, NJ) available in 7-, 1 5 , and 40-ml sizes Abbe 3L refractometer (Bausch and Lomb, or equivalent): Used to adjust the density of sucrose solutions. The measurement is made on a drop of sample Procedure

Young, male Sprague-Dawley rats (130-180 g), fasted overnight, are killed by decapitation. The livers are rapidly excised and perfused with ice-cold 0.154 M NaCl, cut into several large pieces, and weighed. The liver is further diced using a razor blade and homogenized in 5 vol of 0.25 M STM in a 40-1-111 ~ o u n c ehomogenizer. Ten up-and-down strokes are usually sufficient. The homogenate is adjusted to 20% (liver wet weight to total volume) with 0.25 M STM and filtered through four layers of moistened gauze. The fractionation procedure is conveniently carried out with five rat livers. The filtrate is centrifuged (30 m1150-ml tube) at 280 g for 5 min (1 100 rpm in a Beckman TJ-6R rotor). The supernatant is saved and the pellet is homogenized by three strokes in the Dounce homogenizer and resuspended in 0.25 M STM to one-half of the initial homogenate volume. The suspension is again centrifuged at 280 g for 5 min. The first and second supernatant fractions are combined and centrifuged at 1500 g (2600 rpm) for 10 min. The resulting pellets are pooled and dispersed by three strokes in a Dounce homogenizer, in 1 to 2 ml of 0.25 M STMIg of liver. STM (2.0 M) is added to obtain a final density of 1.18 glml(1.42 M), and sufficient 1.42 M STM is added to bring the volume to twice that of the original homogenate. A 35-ml aliquot of this sample is transferred to a cellulose nitrate tube and overlaid with 2 to 4 ml of 0.25 M STM. After centrifugation for 60 min at 82,000 g in a Beckman L5-65 centrifuge (25,000 rpm, SW 28 rotor, without braking), the pellice at the interface is collected with a blunt-tipped Pasteur pipet and resuspended in sufficient 0.25 M STM to obtain a density of 1.05 glml. This suspension is centrifuged at 1500 g for 10 min, and the final pellet. designated plasma membrane (PM) fraction, is resuspended in 0.25 M STM. The purity of PM fractions is characterized by enzymatic assay. 5'Nucleotidase4 and alkaline posphodiesterases activities are markers for C. C. Widnell and J. C. Unkeless, Proc. Natl. Acad. Sci. U.S.A. 61, 1050 (1968). B. Storie and E. A. Madden, this volume, 1161.

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PREPARATION OF MEMBRANE FRACTIONS

229

the PM fraction and should be about 40-fold enriched compared with the homogenate. Cytochrome oxidase5 (mitochondrial marker) P-hexosaminidase and P-galactosidase5activities (lysosomal markers) should show a decrease in their specific activities in the PM compared to those in the initial homogenate. Similar decreases should be observed with the microsomal markers, NADPH-cytochrome-c reductase6 and glucose-6-phosphatase.' Lysosoma1 and mitochondrial contamination of these PM preparations are re.~ ported to be 4.6 and 1.5%, respectively, of the total PM p r ~ t e i n Microsomes (-20% of total protein) are the major contaminating membranes in these PM preparations. Detailed biochemical characterization and morphology of such isolated PM preparations are given in Refs. 8 and 8a. Isolation of Rat Liver Golgi Vesicles9

Reagents and Materials

Sucrose solutions: 1. All are prepared using a special grade of sucrose for density gradients (EM Laboratories, Elmsford, NY) 2. Solutions of sucrose in water used for step gradients or for final suspension of the Golgi fractions are neutralized to pH 7.0 with KOH before use 3. The percentages of sucrose (wlw) for H20-sucrose solutions is adjusted to the desired concentration 50.1% using a Bausch and Lomb Abbe 3L refractometer at room temperature Potter-Elvehjem homogenizer (PEH), 50 ml, with Teflon pestles with clearances of 0.026 in. (loose) and 0.018 in. (tight) and a 1-ml PEH with hand-driven Teflon pestle Spinco L8-70 ultracentrifuge and Beckman 521 centrifuge: The rotors are precooled and centrifugations performed at 6" Procedure

Male Sprague-Dawley rats, 250-300 g, fasted overnight, are killed by decapitation and exsanguinated. Minced livers are homogenized in 3 vol G. Kreibich, P. Debey, and D. D. Sabatini, J. Cell Biol. 58, 436 (1973). A. Leskes, P. Siekevitz, and G. E. Palade, J. Cell Biol. 49, 264 (1971). * A. L. Hubbard and A. Ma, J. Cell Biol. 96, 230 (1983). J. R. Bartles, H. M. Feracci, B. Steiger, and A. L. Hubbard, J. Cell Biol. (1987). B. Fleischer, this series, Vol. 98, p. 60.

105, 1241

230

EXTRACT PREPARATION AND FRACTIONATION

FRACTION

VOLUME

DENSITY

1171

% SUCROSE

(mi)

HOMOGENATE

15

1.20

FIG. 1 . Diagrammatic representation of the fractionation of a rat liver homogenate on sucrose step gradients. The cross-hatched area represents the initial homogenate. Analysis of the fractions from the interfaces is shown in Table I. (Reproduced from Ref. 9, with permission.)

of 52% sucrose containing 0.1 M sodium phosphate, pH 7.1, using the loose PEH. Three full strokes at 1000 rpm are applied. The homogenization is repeated with the tight pestle using three strokes at 1000 rpm. The homogenate is filtered through two double layers of wetted cheesecloth. The filtrate is adjusted to 43.7% sucrose by adding 0.25 M sucrose, as necessary. Fifteen milliliters of the homogenate is pipetted into a Spinco type SW 25.2 centrifuge tube. Cellulose nitrate tubes are preferred so that the interfaces are clearly visible. The homogenate is overlaid sequentially with 10 ml of 38.7%, 10 ml of 36%, 10 ml of 33%, and 12 ml of 28% sucrose solutions. This is best done using 10-ml pipets and running the solutions down the side of the tube. The gradient-containing tubes are centrifuged for 55 min at 24,000 rpm (100,000 g) using a Spinco SW 25.2 rotor. Fractions are collected separately from the 28133% and 33136% sucrose interfaces, diluted with an equal volume of water, and recentrifuged for 10 min at 13,000 rpm (16,000 g ) in a 520 rotor using a 521 Beckman centrifuge. The Golgi fractions are recovered from the supernatants by centrifugation at 40,000 rpm (180,000 g) in a Spinco 42. l rotor for 60 min (Fig. l and Table I). The pellets are suspended in neutralized 0.25 M sucrose using the 1-ml PEH with a hand-driven pestle. The yield of purified Golgi membranes is 0.3 mg of proteinlg wet weight of liver. Golgi-rich membranes are characterized by a high specific activity of galactosyl transferase, lower specific activities of plasma membrane and microsomal enzyme markers, and essentially no mitochondria1 enzyme activities. The functional topology of Golgi membranes has been recently reviewed.1° lo

B. Fleischer, in "Protein Transfer and Organelle Biogenesis" (R. C. Das and P. W. Robbins, eds.), p. 289. Academic Press, San Diego, California, 1988.

1171

r

-

lrc'

r-

23 1

PREPARATION OF MEMBRANE FRACTIONS

TABLE I DISTRIBUTION OF GALACTOSYLTRANSFERASE AND OTHERMARKER ENZYMES AFTER CENTRIFUGATION OF RAT LIVER HOMOGENATE IN H 2 0 - S ~ ~ ~ ~ ~ ~ a Fraction (as In Fig. 1)

Total proteln (rng)

Phosphorus proteln (pglrnl)

Homogenate 1 2 3

1787 1.6 1.5 3.0

29.0 28.3 33.5

a

-

Galactosyltransferase NADH(nmollhr x cytochrome-c mg prote~n) reductaseb 11.7 599 3 14 120

0.385 0.376 0.376 0.994

Succ~natecytochrorne-c 5'-Nucleoreductaseb tldaseb 0.127 0.008 0.010 0.050

0.016 0.100 0.145 0. I 50

Glucose-6phosphataseb 0.075 0.035 0.070 0.115

Reprinted with permission from B. Fleischer, this series, Vol. 98, p. 64. Data given in pmollmin x mg protein.

Isolation and Solubilization of Microsomal Membranes for Preparative Purposes

Fractionation of the 10,000 g supernatant by means of density equilibration in a continuous or discontinuous sucrose gradient is an effective procedure for obtaining rough and smooth microsomal preparations." Because preparative scale sedimentation of microsomal membranes on sucrose gradients is a tedious task, and since such preparations may also contain membrane components other than those present in the endoplasmic reticulum, we use the following procedure to obtain membrane preparations for isolation of microsomal membrane proteins. Reagents and Materials Buffer A: 0.1 M Tris-acetate, pH 7.4, containing 1.O mM EDTA, 0.1 mM dithiothreitol (DTT) Pyrophosphate buffer: 0.1 M sodium pyrophosphate, pH 7.4, 1.0 mM EDTA, 0.1 mM DTT Buffer B: 0.1 M Tris-acetate, pH 7.4, 20% glycerol, 1 mM EDTA, 0.1 mM DTT Buffer C: 5 mM potassium phosphate, pH 7.4, 20% glycerol, 1 mM EDTA, 0.1 mM DTT 10% sodium cholate (Sigma, St. Louis, MO) 50% solution (wlv) of polyethylene glycol 6000 (PEG) (Sigma) 10% solution of Tergitol NP-10 (Sigma) "

G. Dallner, this series, Vol. 52, p. 71.

232

EXTRACT PREPARATION AND FRACTIONATION

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10% Nonidet P-40 (Sigma) 1.5-liter Waring blender Sorvall RC2-B (or equivalent) centrifuge, GS-3 rotor Beckman L8-M ultracentrifuge, Ti-60 rotor Procedure

Male New Zealand rabbits (-2 kg), fasted overnight, are sacrificed by decapitation and exsanguinated. The liver is perfused in situ through the hepatic arteries with cold buffer A. All subsequent steps are performed at 5". The excised liver (-60 g) is chopped in small sections and homogenized in 5 vol of buffer A for two 40-sec intervals. The homogenate is centrifuged at 10,000 g (8000 rpm) in the GS-3 rotor for 30 min. The supernatant fraction is filtered through four layers of wet cheesecloth and sedimented at 36,000 rpm (105,000 g) in a Ti-60 rotor for 90 min. To the resulting pellet, pyrophosphate buffer is added (22 ml per each centrifuge tube). The pellets are dispersed by brief vortexing and recentrifuged at 36,000 rpm for 60 min. The washed microsomes are suspended in enough buffer B to give an approximate protein concentration of 25 mglml. Methods are also available for preparation of microsomes without the use of an ultracentrifuge. Precipitation can be done at low speed in the presence of Ca2+l2 or polyethylene glycol 6000.13 Alternatively microsomes can be isolated using gel filtration.I4 Solubilization of Membrane Proteins

To the glycerol-stabilized membrane preparations, 10% sodium cholate solution is added dropwise with stirring to give a cholate protein ratio of 2.5 : 1. After the addition of cholate, the suspension is stirred for 1 hr. To this partially turbid mixture, 50% PEG is added to a final concentration of 6%. After 30 min, the suspension is centrifuged for 30 min at 8000 rpm in a GS-3 rotor. To the supernatant, 50% PEG is added to give a final PEG concentration of 12%. This mixture is stirred for 45 min and centrifuged for 30 min at 8000 rpm. The 6-12% PEG precipitate is solubilized by the addition of buffer C (50 m1160 g liver), followed by the addition of 2.2 ml of 10% Tergitol NP-10. This mixture is stirred overnight. The 6 to 12% PEG precipitate is very viscous and cannot be readily transferred until it is completely solubilized. Therefore, the solubilization step is performed in the same centrifugation bottle in which the precipitate is obtained. l2 l3

l4

D. L. Cinti, P. Moldeus, and J. B. Schenkman, Biochem. Pharmacol. 21, 3249 (1972). T. A. van der Hoeven, Anal. Biochem. 15, 398 (1981). 0. Taugen, J. Jonasson, and S. Orrenius, Anal. Biochem. 54, 597 (1973).

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PREPARATION OF MEMBRANE FRACTIONS -

-

-

233

-

The solubilized material is fractioned on a DEAE-cellulose column (5 X 12 cm), equilibrated with buffer C containing 0.5% NP-10, or 0.2% Nonidet P-40. Proteins are then eluted using a linear or stepwise gradient of increasing concentrations of KC1 (0-125 mM). The fractions are monitored by SDS-PAGE. The desired fractions are rechromatographed on hydroxylapatite-agarose (HA-Ultrogel, IBF Biotechnics), or CM-cellulose. For example, cytochrome b5 and P-450, form 6, can be obtained in pure form by rechromatography of the DEAE fractions eluted with high salt (after dialysis) on another DEAE column, equilibrated with both cholate (0.5%) and NP-10 (0.2%) at room temperature. The nonionic detergents can be effectively removed from the protein preparations by adsorbing the protein on calcium phosphate gel, extensively washing the gel with detergent-free buffer, and subsequent elution of the protein with high-molarity phosphate buffer. In order to stabilize the membrane proteins, 20% glycerol is included in all chromatography steps. Hydrophobic columns, such as phenyl-Sepharose, can be used to adsorb membranous protein in high salt and they may be eluted with decreasing salt concentrations. For example, fractions eluted from a hydroxyapatite column may be desalted or further purified in such a way.

FIG. 2. SDS-PAGE analysis of rabbit liver microsomal preparations. Lane I: (a) Epoxide hydrase; (b) P-450,form 2; (c) P-450,form 4;(d) P-450,form 6 ;(e) esterases; ( f ) P-450 reductase. Lanes 2, 3, and 4 have increasing concentrations of microsomes. Lane 5: Solubilized 0-6% PEG fractions. Lanes 6, 7, and 8: Solubilized 6 to 12% PEG fractions. Markers indicate the position of various proteins listed in Table 11.

234

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EXTRACT PREPARATION AND FRACTIONATION

TABLE I1 N-TERMINAL SEQUENCE AND MOLECULAR WEIGHTOF RABBITLIVERMICROSOMAL PROTEINS SCALE ISOLATEDON A PREPARATIVE Subunit molecular weight ( X lo3) Band (Fig. 2)

Protein Cytochrome b5 NADH-cytochrome-b5 reductase Epoxide hydrolase Cytochrome P-450, form 1 Cytochrome P-450, form 2 Cytochrome P-450, form x Cytochrome P-450, form x Cytochrome P-450, form 3b Cytochrome P-450, form 3c Cytochrome P-450, form 4 NADPH-reductase, form 2 NADPH-reductase, form 1 Multifunctional proteinh Cytochrome P-450, form 6 Esterase, form 1, glycoprotein Esterase, fonn 2, glycoprotein Rabbit serum albumin precursor NADPH-cytochrome-P-450 reductase

Sequence

SDSPAGE

Sequence

MLLELLLASVLGFVId MAPVVVLVLGLCCLL MEFSLLLLLLAFLAF MDPVVVLPLLLPSAS * MDPVVVLVCGLLVLLb MDLLIILGICLSCVV MDLIFSLETWVLLAAf AMSPAAPLSVTELLL GKKVAIIGAGISGLAC Blocked terminusc SDVLELTDDNFESRIb MVSDFGLPTFISATE HPSAPPVVDTVKGKV QDSASPIRNTHGQVRJ

" J. Ozols, Biochim. Biophys. Acra 997, 121 (1989). Ac, Acetyl. J. O Z O ~(unpublished). S Myr, Myristoyl. J. Ozols, Biochem. Biophys. Res. Commun. 163, 49 (1989). F. S. Heinemann and J. Ozols, J . Biol. Chem. 259, 797 (1984). J. Ozols, F. S. Heinemann, and E. F . Johnson, J. Biol. Chem. 260, 5427 (1985). f D. R. Koop, A. V. Persson, G. E. Tarr, and M. J. Coon, J . Biol. Chem. 256, 10704 (1981). J. OZO~S, J . Biol. Chem. 261, 3965 (1986). The amino acid sequence of this protein is very similar to the cDNA-deduced sequences and subunit molecular weights of rat liver protein disulfide isomerase,' human liver thyroid binding p r ~ t e i np, ~subunit of human prolyl h y d r ~ x y l a s eand , ~ hen oviduct microsomal glycosylation sitebinding protein4: (1) J. C. Edman, L. Ellis, R. W. Blacher, R. A. Roth, and W. J. Rutter, Nature (London) 317, (1985); (2) S. Cheng, Q. Gong, C. Parkison, E. A. Robinson, E. Appella, G. T. Merlino, I. Pastan, J. Biol. Chem. 262, 11,221 (1987); (3) J. Koivu, R. Myllyla, T. Helaakoski, T. Pihlajaniemi, K. Tansanen, and K. I. Kviviriko, J . Biol. Chem. 262, 6447 (1987); (4) M. GeethaHabib, R. Noiva, H. A. Kaplan, and W. J. Lennarz, Cell 54, 1053 (1988). ' G. Korza and J. Ozols, J. Biol. Chem. 263, 3486 (1988). J. Ozols, J. Biol. Chem. 264, 12533 (1989).

f

J

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PREPARATION OF MEMBRANE FRACTIONS

235

Because proteins precipitated in the 6% PEG fraction are difficult to resolubilize, and PEG cannot be easily removed from proteins present in the 12% PEG supernatant, microsomes may also be solubilized without the PEG precipitation step. Solubilization is achieved by adding a mixture of sodium cholate (1 mglmg protein) and the nonionic detergent, Nonidet P-40 (1.3 mg/mg protein) to the membrane preparation. The DEAE column resolution of such an extract, however, will be different from that of one fractionated with PEG. Essentially all proteins will be eluted early, at a lower salt concentration. A good resolution, nevertheless, can be obtained by using an appropriate KC1 gradient of lower molarity. Using the above microsome isolation, solubilization, and resolution procedures, about eight cytochrome P-450 proteins, epoxide hydrolase, several luminal esterases, and reductases have been isolated in pure form (Fig. 2, Table 11). The presence of serum proteins or mitochondria1 proteins in these preparations has, thus far, not been encountered.

Section V Solubilization Procedures

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DETERGENTS: AN OVERVIEW

239

[I81 Detergents: An Overview By JUDITH M. NEUGEBAUER Introduction

The primary role of detergents in protein purification is to effect solubilization. Membrane-bound proteins, in particular, require detergent treatment before they are subjected to separation methods such as chromatography or ultracentrifugation. Detergents are also used as disaggregating agents (e.g., during electrophoresis), as aids to membrane protein reconstitution or recrystallization, and as additives to immunoassay or other protein assay mixtures. A large variety of relatively pure or "protein-grade" detergents are available. While many different detergents can be used for initial membrane solubilization, some of the other techniques mentioned are most successful when certain specific detergents are used. Table I lists some of these detergents along with references to the corresponding techniques. Studies of detergent properties are usually relegated to the domains of colloid science or physical chemistry, not biochemistry; that is, a small genre gap exists. However, there is also a generation gap: While many protein purification techniques were developed and refined relatively recently, techniques for the study of detergents-and many data on detergents-have been around for 30 or more years, leaving the individual who is unfamiliar with the field a deep mine of information to sift through. So, this overview has two aims: to provide a fundamental understanding of detergent properties and to highlight data that are especially relevant to the art of protein purification. Detergent Structure

Detergents are a class of compounds distinguished by their amphiphilic structure (Fig. 1, top). Each molecule contains both hydrophilic and hydrophobic moieties, which gives rise to the phenomenon of surface activity. However, unlike other polar lipids, such as cholesterol or the phospholipids that compose the cell membrane, detergents form micelles and are thus significantly soluble in water. The hydrophobic moieties of detergents are commonly straight- or branched-chain hydrocarbons or steroid skeletons possessing the AIB, CID cis, BIC trans or the A/B cis, BIC, CID trans conformation (Fig. 1, center). Whereas detergents possessing a hydrocarbon chain have an endMETHODS IN ENZYMOLOGY, VOL. 182

Copyright O 19W by Academic Press, tnc. All rights of reproduction in any form reserved.

240

SOLUBILIZATION PROCEDURES

-

[I81

AIR

MONOMER

MICELLE

A/B. C/D cis; B/C transa A/B cis; B/C. C/D transb

A1 R

%.WATER

4 MONOMER

MlCELLE

FIG. 1. Top: Schematic representation of a typical alkyl chain surfactant as monomer, oriented at an air-water interface, and in a spherical micelle. Center: Two possible configurations of steroid-type detergents. a indicates the configuration corresponding to the bile salts; b indicates the configuration corresponding to digitonin. Bottom: Schematic representation of a bile salt detergent as monomer, at an air-water interface, and in a back-to-back micelle. c indicates that for cholate and its derivatives, X = OH; for deoxycholate and its derivatives, X = H.

to-end polarity, those with a steroid skeleton have more of a planar polarity (Fig. 1, bottom). The hydrophilic moieties of detergents are much more varied. They are ionic or nonionic, simple or relatively elaborate. As shown in Fig. 2, there are six primary detergents to which many others are structurally related. Principles of Detergent Aggregation

Whether or not a detergent possesses the classic end-to-end polarity, and regardless of the structure of the hydrophilic head group, it is generally accepted that hydrophobic interactions are the main driving force for

p_

[I81

DETERGENTS: A N OVERVIEW

24 1

TABLE I SPECIFICUSES OF DETERGENTS I N PROTEIN PURIFICATION Technique Electrophoresis and electrofocusing Enzyme immunoassay Liposome preparation Micellar chromatography Protein fractionation by phase separation Protein crystallization Reverse micellar bioseparation Analytical ultracentrifugation in presence of detergent Selective solubilization of membranes

Detergents

Ref.

SDS. NP-40 Tween 20, Triton X-100 Sodium cholate, octylglucoside, sodium deoxycholate Many, e.g., SDS Triton X-114 Octylglucoside, dodecyldimethylamine oxide Aerosol OT Many Triton X-100

" L. M. Hjelmeland and A. Chrambach. Elecrrophoresis I981 2, 1 (1981). 0.L. Goldring, Im~-~rmoassay Technol. 2, 189 (1986).

' F. Szoka, Jr. and 3. Papahadjopoulos, Annu. Rev. Biophys. Bioeng. 9, 467 (1980); R. A. Schwendener, M. Ansanger, and H. G. Weder, Biochem. Biophys. Res. Commun. 100, 1055 (1981). D. W. Armstrong, Sep. Purif. Methods 14, 213 (1985). ' J. G. Pryde, Trends Biochem. Sci. 11, 160 (1986). f J. V. Moller, M. LeMaire, and J. P. Andersen, in "Progress in Protein-Lipid Interactions" (A. Watts and J. J. H. H. M. DePont, eds.), Vol. 2, Chap. 5. Elsevier, New York, 1985. s K. L. Kadam, Enzyme Microb. Technol. 8, 266 (1986); P. L. Luisi and L. J. Magid, CRC Crit. Rev. Biochem. 20, 409 (1986). C. Tanford and J. A. Reynolds, Biochim. Biophys. Acta 457, 133 (1976); J. H. C. Steele, C. Tanford, and J. A. Reynolds, this series, Vol. 48, p. 11. ' A. H. Maddy, in "Red Cell Membranes-A Methodological Approach" (J. C. Ellory and J. D. Young, eds.). Academic Press, London, 1982: S. E. Lux, Nature (London) 281,426 (1979).

rnicellization in aqueous solution. These relatively weak but abundant interactions are also responsible for the spontaneous assembly of lipid bilayers such as the cell membrane. Hydrocarbon chains, when singly dispersed in aqueous solution, are surrounded by structured, cagelike water.' Aggregation of the chains releases some of this structured water, increasing the entropy and thereby decreasing the free energy of the system. Thus, acceptable structures of detergent micelles in aqueous soI

C. Tanford, "The Hydrophobic Effect," 2nd Ed. Wiley, New York, 1980.

242

[I81

SOLUBILIZATION PROCEDURES

-+"aF"

@yoalkyl carboxylate (Sap)

akanoy, sarcosinate

kqy00

-

alky-N,N-dimethylglycine

@*

@@o

tn, c "3

alkyltrimethylammonium

CH,

alkyldirnethylamine oxide pHs

alkylpyridiniurn alkyldimethylamrnoniopropane 1-sulfonate

0

0-8-0d alkyl sulfonate

CHAPS bisblkyl) sulfosuccinate bile salt 0

bile salt OH

OH OH

Big CHAP

FIG. 2. Structures of detergents typically used in protein purification. (Structural relationships are indicated by the heavy lines.) The shorthand used in these structural formulas is as follows: R is a straight-chain alkyl group, B is the bile salt skeleton (see Fig. 1, bottom), and t-R is the tert-octyl group. The following abbreviations are used to refer to nonionic detergents containing polyoxyethylene: alkyl ethoxylates, C,E,; polysorbates, C, sorbitan E m ;p-tert-alkylphenyl ethoxylates, tert-C,+E,. In all cases n is the number of carbons in the alkyl chain and m is the number of oxyethylene groups. For polysorbate 20 and polysorbate 80, m = w + x + y + z = 20.

lution are generally those in which the hydrocarbon portions are in contact with one another rather than with water (Fig. 1, top and bottom). The micellar structure adopted by a particular detergent depends to some extent on the structure of the monomer. A quantity referred to as the packing parameter can be assigned to each monomer, which in many

- -

I181

243

DETERGENTS: AN OVERVIEW

alkylthioglucoside alkylglucoside

OH

alkylmaltoside

wr

%

C-

1

*-

& O / ( ~ ~ 2 ~ ~ 2 ~ ) - ~ alkyl ethoxylate

FIG. 2 . (continued)

cases predicts the micellar structure adopted by the detergent.2The packing parameter, P,, is expressed as follows: Pp = Vclaolc

(1)

where I, is the critical tail length, V , is the volume occupied by the critical tail length, and a, is the optimal surface area per head group. P, < 113 predicts a spherical micelle, while 113 < P, < 112 predicts a cylindrical micelle. Inverted structures or reverse micelles are observed when P, > 1.2 Micellar structure also depends on experimental conditions such as temperature, pressure, pH, ionic strength, and the presence of impurities. For example, many ionic detergents undergo a sphere-to-rod transition at high ionic strength. This effect is predictable from the packing parameter definition, as addition of salt screens electrostatic head-group repulsion, reduces a,, and thereby increases P,. ID. J. Mitchell and B . W. Ninham, J . C . S . Faraday Trans. N 77, 601 (1981); J. N. Mitchell, and B. W. Ninham, J. C. S . Faraday Trans. I1 72, 1525 Israelachvili, D. .I. (1976).

244

SOLUBILIZATION PROCEDURES

[I81

Parameters that Describe Detergent Behavior Besides the packing parameter, several parameters having more practical significance are used to describe detergent behavior. Most of these also depend on experimental conditions and many can be interrelated. The most important with regard to solubilization are as follow: the critical micelle concentration (crnc), the aggregation number (N), the critical micelle temperature (cmt), and, primarily for nonionics, the cloud point (cp) and the hydrophile-lipophile balance number (HLB). The crnc is the concentration at which micelles begin to form, and it is usually determined by measuring some physical property as a function of detergent concentration. Different techniques usually yield slightly different values of crnc for the same detergent. Two models are commonly invoked to describe the phenomenon of ~ . ~the mass-action nod el.^.^ micellization: the phase-separation m ~ d e land According to the phase-separation model, micellization is treated as the formation of a separate phase and monomer concentration is assumed to reach at the crnc its maximum and thereafter constant value. According to the mass-action model, monomers, micelles, and any counterions that may be present are assumed to be in equilibrium, so monomer concentration can change appreciably even above the cmc. The aggregation number (N) is the average number of monomers per micelle. Techniques that are routinely used to determine the size of biological macromolecules are usually also applicable to micelles (with special treatment of data sometimes necessary6). It is important to note that each technique for determining the molecular weight of macromolecules yields a specific type of average molecular weight. This contributes to the disagreement between detergent aggregation numbers determined by different techniques. In addition, some detergents exhibit concentration-dependent growth of micelles,' so aggregation numbers determined at detergent concentration much greater than the crnc may differ from the aggregation number at the cmc. The cmt is the temperature at which a detergentlsolvent system passes

' A. E. Alexander, Trans. Faraday Soc. 38, 54 (1942).

'

D. Attwood and A. T. Florence, "Surfactant Systems: Their Chemistry, Pharmacy, and Biology," p. 98. Chapman & Hall, New York, 1983. E. R. Jones and C. R. Burry, Philos. Mag. 4,841 (1927); R. C. Murray and G . S. Hartley, Trans. Faraday Soc. 31, 185 (1935). J. P. Kratohvil, Hepatology 4,85S (1984): J . P. Kratohvil, J. ColloidInterface Scr. 75,271 (1980); L. H. F'rincen and K. J . Mysels, J. Colloid Interface Sci. l2, 594 (1957); E. W. Anacker and A. E. Westwell, J. Phys. Chem. 68, 3490 (1964). J. P. Kratohvil, W. P. Hsu, M. A. Jacobs, T. M. Aminabhavi, and Y. Mukunoki, Colloid Polym. Sci. 261, 781 (1983).

[I81

DETERGENTS: AN OVERVIEW

245

from a hydrated crystalline state to an isotropic micellar solution. Some detergents apparently lack a cmt, but for such compounds the cmt is simply below the freezing point of the solvent. The Krafft point ( T K ) , specified in some of the literature, is the cmt at the cmc. Some detergents exhibit a lower consolute temperature or cloud point. At the cloud point, a detergent solution passes from an isotropic micellar system to a two-phase system: One phase is depleted of detergent and the other is rich in giant micelles. The usually recorded cloud point is the temperature at which a warmed and cloudy 1% solution clears upon co~ling.~ The HLB of a detergent is an index of its hydrophilicity. It is most simply calculated using one of several empirical equations. For fatty alcohol ethoxylates, HLB

=

El5

(2)

where E is the weight percentage of p~lyoxyethylene.~ For other surfactants, HLB can be calculated from

HLB = 7

+ x(hydrophi1ic group numbers) -

x(lipophi1ic group numbers) (3)

where the group numbers are as listed in Table I1.l0 From both formulas it is evident that the larger the HLB, the more hydrophilic the detergent. Table I11 contains the vital statistics of compounds that are listed as biological detergents in one or more chemical supply catalogs. Cmc values and aggregation numbers are from the literature. Although it was not possible to cite data obtained entirely by a single technique, the values in Table I11 are almost entirely for detergent/HzO solutions at 25" and I atm. Though perhaps not corresponding to typical biological assay conditions, these data can be used to estimate detergent properties under other conditions. For example, the cmc values of anionic and cationic surfactants vary in a regular manner with counterion concentration according to log(cmc)

=

-a log Cc + b

(4)

where a and b are constants for a particular head group and Cc is the total counterion concentration." For sodium dodecyl sulfate at 25O, a = 0.45774 and b = -3.2485." For cetyltrimethylammonium bromide at 25", T . Kuwamura, ACS Symp. Ser. N o 253, p. 27 (1984). W. C. Griffin, J. Soc. Cosmet. Chem. 1,311 (1949); W . C. Griffin, J. Soc. Cosmet. Chem. 5, 249 (1954). lo J. T. Davies, Proc. Int. C o n , ~ rSurf. . Act., 2nd, London 1,426 (1957); P . Becher, Dispersion Sci. Technol. 5, 81 (1984). " M. L. Conin and W. D. Harkins, J . Am. Chem. Soc. 69, 684 (1974).

246

SOLUBILIZATION PROCEDURES

[I81

TABLE I1 HLB GROUPNUMBERS' Groups

Group number

Hydrophilic --SO4-Na+ -COO-Kc -COO-Na+ -N (tertiary amine) Ester (sorbitan ring) Ester (free) -COOH Hydroxyl (free) -0Hydroxyl (sorbitan ring) Lipophilic C H --CH2CH2=CHDerived -(CH2-CH2-0)-(CH2-CHI-CH2-0)a

Reproduced from J. T. Davies, A quantitative theory of emulsion type: I. Physical chemistry of the emulsifying agent. Proc. Int. Congr. Surf. Act., 2nd 1,426 (1957); by permission of the publishers, Butterworth and Co. (Publishers) Ltd.O

a = 0.63 and b = -5.17.12 Approximate values of a and b can be calculated for other ionic detergents if the cmc values at two counterion concentrations are known. Unfortunately, few other precise empirical relationships between detergent properties and experimental conditions have been formulated. Some relationships between physicochemical properties and detergent structure are known. These are useful for predicting the properties of detergents not included in Table 111, in particular members of homologous series for which experimental data are not available. Two empirical relationships between cmc and detergent structure have been formulated, as follow:

l2

B. W. Barry, J. C. Morrison, and G. F. J. Russel, J . Colloid Interface Sci. 33,554 (1970).

248

[I81

SOLUBILIZATION PROCEDURES

TABLE 111 (continued) Detergentlmolecular weight of monomer (synonyms, tradenames)

1-Dodecanesulfonic acid, sodium salt1272 N-Dodecanoylsarcosinate, sodium salt1295 (Sarkosyl NL30, Gardol) Dodecyldimethylamine oxide1229 (LDAO, Ammonyx AO, Empigen OB)

3-Dodecyldimethylammoniopropane 1-sulfonatel336 (Zwittergent 3-12, SB3-12) N-Dodecyl-N,N-dimethylglycine/271 (Empigen BB)

n-Dodecyl-P-D-glucopyranoside/38 1 n-Dodecyl-P-D-maltoside/512 Dodecyl sulfate, sodium salt1288 (sodium dodecylllauryl sulfate, SDS)

Dodecyltrimethylammonium bromidel308 (DTAB) Bis(2-ethylhexyl)sulfosuccinic acid, sodium salt (Aerosol OT, AOT) N,N-Bis-3-D-gluconamidopropylcholamidel850 (BigCHAP) N,N-Bis-3-D-gluconamidopropyldeoxycholamidel833 (deoxyBigCHAP) n-Heptyl-P-D-thioglucoside/294 Hexadecyldimethylammoniopropane 1-sulfonatel392 (Zwittergent 3-16, SB3-16) Hexadecylpyridinium chloride1340

cmc (mM)

Nm

Relevant properties Toxic to humans T , = 32" HLB = 12.3

Zero net charge, pH >7; cationic, pH <3 Zero net charge, pH 2-12 Zero net charge, pH >6; cationic, pH <6

HLB = 40 TK = 16" K+, divalent cation salts insoluble

Zero net charge, pH 2-12

[I81

249

DETERGENTS: AN OVERVIEW

TABLE 111 (continued) Detergent/molecular weight of monomer (synonyms, tradenames)

cmc (mM)

Relevant properties

---

Hexadecyltrimethylammonium bromide1364 Nonanoyl-N-methylglucamide/335 (MEGA 9)

0.92

Ref.a .-

TK = 22O

1, 2, 3, 6, 8 2

Octanoyl-N-methylglucamide/321 (MEGA 8, OMEGA) n-Octyl-P-D-glucopyranoside/292 (octylglucoside, OG) n-Octyl-P-D-thioglucoside/308 (octylthioglucoside, OSG) Taurocholic acid, sodium salt1538 (sodium taurocholate) Taurodeoxycholic acid, sodium salt/ 522 (sodium taurodeoxycholate) Tetradecyltrimethylammonium bromide1336 "eferences: (1) P. Mukejee and K. J. Mysels, NSRDS-NBS 36 (1970); (2) J. Neugebauer, "A Guide to the Properties and Uses of Detergents in Biology and Biochemistry ," Tech. Support Monogr. Calbiochem Brand Biochemicals, Calbiochem Corp., San Diego, California, 1988; (3) A. Helenius and K. Simons, Biochim. Biophys. Acta 415, 29 (1975); (4) J. N . Umbreit and J . L. Strorninger, Proc. Narl. Acad. Sci. U.S.A. 70, 2997 (1973); (5) Y. Kagawa, Biochim. Biophys. Acta 265, 297 (1972); (6) A. C. Newby, in "Brain Receptor Methodology," Part A (P. J. Marangos, ed.). Academic Press, New York, 1984; (7) G. E. Tiller, T. J. Mueller, M. E. Dockter, and W. G. Struve, Anal. Biochem. 141,262 (1984); (8) D. W. Armstrong, Sep. Purif. Methods 14,213 (1985); (9) E. F. Williams, N. T. Woodberry, and J. K. Dixon, J . Colloid Interjace Sci. 12, 452 (1957); (10) S. Saito and T. Tsuchiya, Biochem. J. 222, 829 (1984). 1% solution in water. 3% solution in 1 N NaCI. Absorbance depends on source. pH >8. f In 0.15 M NaCI, pH >8. g 20°, nominal estimate. 27', pH >7. ' 27", pH <3. j 23'. 330". In 0.15 M NaCI. N , aggregation number.

250

SOLUBILIZATION PROCEDURES

1181

where m is the number of carbon atoms in the longest contiguous alkyl chain of the detergent, and A and B are constants for the particular homologous series13;and log(cmc)

= A'

+ B'R

(6)

where A ' and B' are, again, constants for the particular homologous series and R is the number of oxyethylene units in the molecule.14 Besides cmc values and aggregation numbers, Table I11 provides the commonly used abbreviations and synonyms for some detergents. For the nonionics, a few trade names of equivalent products are included. (Many of the trademarked products are heterogeneous mixtures of compounds differing in alkyl chain length or degree of ethoxylation. Note that this means the observed physicochemical parameters will differ from those listed for homogeneous products.15) The column headed "Relevant properties" contains some HLB, cloud point, and cmt values, and, lastly, various bits of information on the unique or anomalous properties of some detergents, e.g., sensitivity to pH variation, precipitation by metal ions, and UV-visible absorbance. The material presented here is truly just an overview. Several comprehensive treatises on detergents have been written, extensive directories and surveys of data have been compiled, and national and international symposia have been chronicled through the years. Each is a precious source for more detailed information on one or several aspects of detergent H. B. Klevens, J . Am. Oil Chem. Soc. 35, 74 (1953). L. Hsiao, H. N. Dunning, and P. B. Lorenz, J . Phys. Chem. 60, 657 (1956). l5Many homogeneous alkylpolyoxyethylenes are available from the Nikko Chemical Co.; distributor: Kouyoh Trading Co., Tokyo. l 6 P. Mukerjee and K. J. Mysels, "Critical Micelle Concentrations of Aqueous Surfactant Systems." NSRDS-NBS 36 (1970). "McCutcheon's Detergents and Emulsifiers, Combined Edition." McCutcheon's Division, MC Publishing Co., Morristown, New Jersey, 1975. 18 "Surfactant Science Series." Vols. 1-26. Dekker, New York, 1966-1987. l9 "Surfactants" (T. F. Tadros, ed.). Academic Press, New York, 1984. K. Shinoda, T. Nakagawa, B. Tamamushi, and T. Isemura, "Colloidal Surfactants: Some Physicochemical Properties." Academic Press, New York, 1963. M. J. Rosen, "Surfactants and Interfacial Phenomena." Wiley, New York, 1978. 22 "Micellization, Solubilization and Microemulsions" (K. L. Mittal, ed.), Vol. 1-2. Plenum, New York, 1977; "Solution Chemistry of Surfactants" (K. L. Mittal, ed.), Vol. 1-2. Plenum, New York, 1979; "Solution Behavior of Surfactants: Theoretical and Applied Aspects" (K. L. Mittal and E. J. Fendler, eds.), Vol. 1-2. Plenum, New York, 1982; "Surfactants in Solution" (K. L. Mittal and B. Lindman, eds.), Vol. 1-3. Plenum, New York, 1984. 23 "Stt-ucture/Performance Relationships in Surfactants" (M. J. Rosen, ed.). Am. Chem. Soc., Washington, D.C., 1984. l3

l4

[I81

DETERGENTS: A N OVERVIEW

25 1

Choosing a Detergent In the process of choosing a detergent, an obvious first move is to eliminate from consideration all those whose solubility or stability cannot be guaranteed under the conditions necessary to preserve the protein in the desired structural or functional state. Here, one should consider the temperature, pH, and ionic strength of the system and, if applicable, the specific ion requirements and hydrolytic abilities of the protein. It may also be necessary to rule out detergents that are difficult to remove,25that interfere with assays, or that are difficult to detect and/or quantify. The next move is not clear: Although some general relationships between hydrocarbon tail length and biological activity have emerged from systematic studies of detergents within homologous series,26general relationships between head-group type and biological activity have been more elusive. This is not surprising if one considers that the tail groups .of detergents are likely to interact with proteins primarily through hydrophobic interactions, while the head groups may be involved in more specific interactions such as hydrogen bonding or ion pairing. Whatever the reason, it is often impossible to foretell which detergent type will be best for a particular application; one must simply try as many as possible within the prevailing practical limits. Given the large variety of detergents available in pure form, it is, in principle, possible to carry out soundly designed, systematic detergent trials27(however, most examples in the literature are not so rigorous). Assuming that the temperature, pressure, pH, and ionic strength of the system are dictated by the protein and are held constant, the factors that are left to be optimized are detergent head group, detergent tail group, and detergent concentration. The array shown in Table IV indexes a variety of detergents by tail group and head group, and may provide a basis for designing systematic detergent trials. For example, to study the effect of tail group, one might start with the sulfobetaine series, the alkylglucoside series, and the alkyl- and alkylphenylpolyoxyethylenes. Should an optimal tail group emerge, one might next experiment with a few different head groups. Naturally, cmc values should be considered when assessing the efficacies of different detergents. If micelles are likely to provide the effect that

" "Reverse Micelles: Biological and Technological Relevance of Amphiphilic Structures in Apolar Media" (P. L. Luisi and B. E. Straub, eds.). Plenum, New York, 1984. L. M. Hjelmeland, this volume [21]. 26 A. T. Florence, I. G . Tucker, and K. A. Walters, ACS Symp. Ser. No. 253, p. 189 (1984). 27 S. N. Deming and S. L. Morgan, "Experimental Design: A Chemometric Approach." Elsevier, New York, 1987. 25

TABLE IV ARRAYOF DETERGENTS FOR SYSTEMATIC TRIALAND ERROR -

- -- -

-

-

-

-

Tail group Head group Anionic

Zwitterionic Cationic Cationiclnonionic Nonionic

CIO Sodium octane sulfonate

Sodium decane sulfonate

Zwittergent 3-08

Zwittergent 3-10

Oct ylglucoside MEGA-9"

Decylglucoside

c 1 2

p-fert-Octylphenyl or p-tert-octylcyclohexyl

Sodium dodecane sulfonate

Steroid skeleton Sodium taurocholate Sodium taurodeoxycholate Sodium cholate Sodium deoxycholate

Sodium dodecyl sulfate Zwittergent 3-12 Empigen BB DTAB LDAO Dodecylglucoside

CHAPS

BigCHAP Deoxy BigCHAP

Octylthioglucoside Decylmaltoside

(C~EIO)~

(CIOEIO)~

Dodecylmaltoside CnEs ClzE9 CIZEIO

Triton X-114 Triton X-100 Triton X-100, reduced Digitonin

a

In indexing the length of the tail group, MEGA-9, nonanoyl-N-methylglucamide, is considered to have eight carbon atoms in its longest contiguous alkyl chain. See structure in Fig. 2. This detergent may not be available from biochemical supply companies.

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SOLUBILIZATION OF NATIVE MEMBRANE PROTEINS

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is sought, then c,,, - cmc, rather that total detergent concentration (c,,,), should be used as the basis for comparing the performance of different detergenk2* Conclusion Detergents have gained an important status in protein biochemistry: They are essential to the isolation and study of membrane-bound proteins, but are also widely used as aids to the study of other kinds of biomacromolecules. The defining characteristics of detergents, amphiphilicity, surface activity, and micellization, are borne by structurally diverse compounds. The specific properties of detergents, cmc, aggregation number, cmt, cloud point, and HLB, depend primarily on the chemical structures of the monomers, but are also affected by experimental conditions such as temperature, pressure, pH, and ionic strength. Although trends in the behavior of simple detergentlsolvent systems can be predicted from theory, no global explanation of the interaction of detergents with biomacromolecules exists. As a result, the task of finding the best detergent for a particular application is usually accomplished by trial and error. If it is practical, a thorough, systematic experimental design involving many detergent types can be used to guide the trial and error process.

* B. Rivnay and H.Metzger, J . Biol. Chem. 257, 12800 (1982).

[I91 Solubilization of N a t i v e M e m b r a n e P r o t e i n s By LEONARD M. HJELMELAND

Introduction Every approach to the purification of a membrane-bound protein unavoidably begins with solubilization. Effective solubilization of mernbrane proteins involves both the selection of a detergent and appropriate solubilization conditions. The compatibility of detergents with specific preparative and analytical methods must be considered, as well as the ease with which detergents may be removed from proteins at the end of purification. This chapter will give a brief consideration of the essential criteria on which a useful choice of detergents may be made, as well as considerations for the selection of solubilization conditions. Previous reMETHODS IN ENZYMOLOGY. VOL. 182

Copyright 0 1990 by Academic Pres%.Inc. All rights o f reproduction in any form reserved.

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SOLUBILIZATION PROCEDURES

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views have dealt extensively with the questions of the physical properties of detergent^,',^ their uses in the solubilization of membrane protein^,^,^ and their specific uses in electrophoresis and isoelectric f o c u ~ i n g The .~ reader is encouraged to rely on these presentations for more in-depth coverage of these topics.

7

-

Criteria for Detergent Selection

In order to make an appropriate choice of a detergent for the solubilization of membrane proteins it is necessary to consider several of their physical properties. In addition to the question of how individual detergents may affect native structures or activities of membrane proteins, the ways in which these same reagents may affect assay or chromatography conditions must also be considered. Nondenaturing Properties of Detergents

The nondenaturing property is the most difficult issue to address in the selection of a detergent. A precise definition of denaturation is usually given in terms of effects on the secondary and tertiary structure of proteins. Most biochemists purifying membrane proteins, however, have differing types of information available with which to make these decisions. Usually, measurements of enzymatic activity or ligand binding are the only data available. These properties may be lost from solubilized proteins for a variety of reasons, including the loss of associated cofactors or subunits, as well as specific interference with the binding of substrates or ligands. These events do not represent true denaturation of the protein structure, but have been included in a broad category of effects loosely referred to as "denaturation." Despite the general lack of precision in defining the loss of activity of membrane proteins upon solubilization, a fairly small number of detergents have emerged as a reasonable set of reagents for a first investigation of the solubilization of a membrane protein. Figure I lists these detergents. The set includes nonionic detergents such as Triton X-100 and octylglucoside, zwitterionic detergents such as CHAPS and Zwittergent 3-14, as well as ionic detergents. I

A. Helenius, D. R. McCaslin, E. Fries, and C. Tanford, this series, Vol. 56, p. 734. A. Helenius and K. Simons, Biochim. Biophys. Acta 415, 29 (1975). L. M. Hjelmeland and A. Chrambach, this series, Vol. 104, p. 305. L. M. Hjelmeland and A. Chrambach, in "Membranes, Detergents, and Receptor Solubilization" (J. C. Venter and L. C. Hanison, eds.), p. 35. Alan R. Liss, New York, 1984. L. M. Hjelmeland and A. Chrambach, Electrophoresis 2, 1 (1981).

--

7

-

-

-

[I91

SOLUBILIZATION OF NATIVE MEMBRANE PROTEINS

CfF

255

0

I

II N-N- 0 MS I II 0

CHAPS

HO*

H

4IOH

9'3

Glu - Gb -Gal

- Gal - Xyl - 0

-H

Digitonin

Triton X-100

O(-

- CH, - CH,)

-

OH

9-10

Lubrd PX

FIG. I . Structural formulas of detergents commonly used for the solubilization of native membrane protein^.^

256

SOLUBILIZATION PROCEDURES

[I91

Spectral Properties

Since routine UV monitoring at 280 nm or higher wavelengths is a necessary part of column chromatography, it is important to know which detergents may interfere with monitoring at these wavelengths. Any of the detergents with aromatic groups such as Triton X-100 or Triton N-101 will have substantial absorbances at 280 nm. The alkyl polyethers such as the Brij and Lubrol series d o not possess the same absorbance properties, and thus do not interfere with UV detection methods. Bile salts, as well as their derivatives, including CHAPS and CHAPSO, also do not interfere with monitoring of columns at 280 nm. Monitoring at 254 nm follows the same general pattern in terms of the absorbance of various detergents, but other considerations must be made for monitoring in the 206- to 210-nm range. At these wavelengths, most functional groups will absorb, and thus alcohols, amides, acids, and bases as part of detergent structures will all interfere with specific monitoring. A practical solution to this problem involves the use of minimal amounts of very clean detergents in all elution buffers, and for this purpose any of the homogeneous synthetic reagents can be recommended. Compatibility with Divalent Cations

Long-chain carboxylic acids have the well-known property of precipitation with divalent cations. For this reason, where calcium or magnesium is absolutely required in a column or assay buffer, consideration should be given to avoid this class of detergents. Problem reagents include N-lauryl sarcosinate, as well as all bile salts and their derivatives bearing a carboxylic acid. Bile salt derivatives such as CHAPS, CHAPSO, and the taurine derivatives of cholic acid and deoxycholic acid do not precipitate with divalent cations. pH Dependence of Solubility and Detergent Properties

Where functional groups are present which may be titratable in a range of pH values being encountered in an experimental protocol, problems can be expected to arise. Carboxylic acid-containing detergents, such as the bile acids and N-lauryl sarcosinate, can be expected to protonate and become insoluble in aqueous media at relatively weakly acidic pH values. Detergents with polar groups containing stronger acids such as the sulfonic acid-containing zwitterions or sulfate esters all have pK values for the acids at pH values of 0-2, and thus are not usually subject to such problems. Detergents containing weak bases such as imidazole derivatives or certain primary amines may also undergo alterations of charge properties if the pH is elevated.

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The experimental conditions under which these considerations may become important are usually related to separation techniques. Isoelectric focusing, where pH values may range from 3 to 11, is a technique that demands that detergent properties be invariant in this range. Similar considerations must be made for ion-exchange resins eluted with pH gradients or chromatofocusing techniques. Temperature Effects

Temperature effects on detergent properties are restricted mostly to a single class of detergents. All nonionic polyoxyethylene ethers such as Triton X-100 and Lubrol PX have a characteristic change of micelle molecular weight with temperature. As temperature increases linearly, the micelle expands in an exponential fashion. This process leads to a separation of detergent as a nonaqueous phase at a discrete temperature known as the cloud point. This effect has been used to advantage in certain procedures for the extraction of membrane proteins, but may cause problems in other techniques. Whenever heat is generated, as is the case in electrophoretic techniques, local areas of heating may change the properties of detergents and thus the results of individual experiments. Adequate and uniform temperature control can always be recommended, but some techniques will always be problematic. Isoelectric focusing, for example, is associated with nonuniform thermal patterns across pH gradients, due to the change in conductance of carrier ampholytes at various pH values. For this reason, other classes of nonionic o r zwitterionic detergents may be more appropriate for this technique. Electrical Properties

Many of the previously discussed properties are directly related to the ionic or electrical properties of the detergent polar group. Both nonionic detergents, and zwitterionic detergents in which the pK values of the two functional groups are widely separated, behave essentially a s uncharged molecules. Such reagents do not move in electrical fields, do not bind to ion-exchange resins, and do not contribute to the net charge of macromolecules to which they are bound. For these reasons, nonionic o r zwitterionic detergents should be used for charge-based preparative procedures including ion-exchange chromatography and preparative electrophoresis. Solubilization of Membrane Proteins

Buffer Selection

The choice of a buffer for the solubilization of membrane proteins has many important implications besides the creation of a stable pH environ-

258

SOLUBILIZATION PROCEDURES

1191

ment. Most of these considerations affect the solubility and stability of the proteins being solubilized. Although a buffer concentration of 25 to 50 mM is normally sufficient, higher concentrations of specific buffers can affect the total solubility of membrane proteins. Phosphate buffer is especially well known for its solubilizing properties. Concentrations of 0.1 to 0.5 M phosphate buffer increase the total solubility of membrane proteins, as noted in a study of endoplasmic r e t i c ~ l u mThis . ~ effect is not entirely due to elevation of the ionic strength of the buffer, and traditional discussions of detergent chemistry often refer to phosphates as "builders" in reference to this p h e n ~ m e n o n . ~ Ionic Strength

Membrane proteins have ionic interactions as well as hydrophobic interactions, and for this reason 150 mM NaCl is usually included in solubilization buffers. Frequently, 150 mM may not be enough, and higher concentrations up to 0.5 M may be used. Urea or Chaotropic Ions

For especially rugged protein-protein interactions, such as those found among structural or cytoskeletal proteins, urea or chaotropic salts such as guanidine hydrochloride may be added to solubilization buffers. In such cases, potential interactions with detergents are an important consideration. Urea forms complexes with all nonionic detergents, and these complexes may affect behavior on chromatography. Many detergents, such as the nonionics and the linear alkyl sulfobetaines, are insoluble in moderate concentrations of guanidine hydrochloride. CHAPS and high concentrations of guanidine have proved especially useful for the solubilization of aggregating proteins, and may be recommended for other difficult solubilization problems. Glycerol

It has long been appreciated that polyols affect the stability of membrane proteins in solution, although no reasonable explanation of this phenomenon has yet been provided. Typical concentrations of glycerol in solubilization buffers range from 5 to 50% (vlv). The inclusion of this reagent substantially affects the viscosity and density of the medium, and thus change the expected behavior of preparations in the ultracentrifuge

'

A. C. Dey. R. Sheilagh, R. L. Rimsay, and I. R. Senciall, Anal. Biochem. 110,373 (1981). P. Becher, in "Nonionic Surfactants" (M. J . Schick, ed.), p. 478. Dekker, New York, 1966.

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SOLUBILIZATION OF NATIVE MEMBRANE PROTEINS

259

and on chromatographic columns. High concentrations of polyols may affect the volume of polymer-based chromatographic resins as well, and care should therefore be exercised before using glycerol with FPLC or HPLC columns. Protease Inhibitors

Almost all initial preparations of proteins are in some way subject to proteolysis. Membrane proteins are no exception and the inclusion of protease inhibitors in initial solubilization buffers is often indicated. Perhaps the single most useful step in inhibiting proteolysis is the addition of sufficient EDTA or EGTA to reduce the free divalent cation or heavy metal concentration in the buffer. Many membranes contain a calciumactivated neutral protease which typical serine protease inhibitors do not affect. These proteases are inhibited by the chelation of free calcium with EGTA or the inclusion of 1-10 pglml leupeptin. A combination of 1 mM EDTA and 1 mM EGTA has often been employed to scavenge both heavy metals and free calcium in an effective fashion. Phenylmethylsulfonyl fluoride at a final concentration of 75 yglml may also be used for the specific inhibition of serine proteases. Finally, if membranes are being solubilized under acidic conditions, the use of pepstatin A at a concentration of 1 to 10 pg/ml may be considered. Solubilization Criteria

All criteria for determining the solubility of membrane proteins in detergent-containing media are by definition operational. The most conventional measures include retention in the supernatant after centrifugation of solutions for 1 hr at 105,000 g, or elution in the included volume from a gel filtration medium with very large pores such as Sepharose 4B. Both methods have inherent pitfalls, which are best explained by considering the process of solubilization. Figure 2 presents a schematic diagram of the process of solubilization of membrane proteins as the ratio of detergent to membrane protein or lipid content is systematically varied. At low ratios of detergent to membrane protein, detergents bind and are incorporated into the intact membranes. As the detergent-to-membrane ratio is increased to 1 : 10, a critical concentration is reached at which membranes are lysed, with the production of large segments of membranes with incorporated detergent molecules. With progressively larger ratios of detergent to membrane protein, 1 : 1, somewhat smaller complexes of membrane lipid and protein with associated detergent are formed. Finally, at ratios of 10 : 1 to 20 : 1 of detergent to membrane protein, individual detergent-protein complexes

260

[I91

SOLUBILIZATION PROCEDURES

Binding Detergent Membranes

--)

Membranes

LvA Membranes

,

-

-

Very Low

~

-

Solubilization

Lysis

Delipidation

Lipid Protein Detergent

Protein + Detergent

Lipid Detergent

Lipid Detergent

-

~

CMC (0.1)

11-2)

-

-

-

-

(10-20)

Detergent to Protein Ratio (wlw)

FIG.2 . Schematic representation of the progressive solubilization of membrane proteins as a function of detergent-to-protein ratio.

and detergent-lipid complexes may be formed. No guarantee can be given, of course, that any particular detergent in combination with any specified membrane preparation will yield these results. Some lipids which are tightly bound may resist extraction by detergents and residual protein-protein complex may remain at the end stage of this process. The importance of these observations lies in understanding experimental results. Often experimentalists assume that the addition of 1% Triton X-100 to a membrane preparation yields single copies of the desired protein stuck into a single Triton-X-100 micelle. This is almost never the case. Usually, large complexes of many proteins with associated lipid and detergent are formed as the result of the initial solubilization. These observations, then, motivate the concern expressed above about the determination of solubility. The inclusion of large amounts of lipid in solubilized complexes may render complexes buoyant due to density factors rather than aggregate size. In a similar way, nonideal interactions between such complexes and chromatographic media might also yield chromatographic results indicating incorrect values for the hydrodynamic radii of the solubilized complexes. Protein and Detergent Concentration

The final subject for consideration is the correct choice of protein and detergent concentrations for the effective solubilization of membranes. As with all preparations of proteins, solutions in which total protein con-

[I91

SOLUBILIZATION OF NATIVE MEMBRANE PROTEINS

26 1

CHAPS (mM) FIG.3. The solubilization of C57BLl6 mouse liver microsomes by CHAPS. Liver microsomes were prepared by differential centrifugation of tissue homogenized in 150 mM KC11 10 mM EDTA, pH 7.25, at 4". For solubilization experiments, the appropriate amount of protein was diluted to give solubilization media with the stated protein and detergent concentrations and with final concentrations of 20% (vlv) glycerol and 0.1 M potassium phosphate, pH 7.25. Aliquots (5 ml) were incubated for 30 min at 25" and then centrifuged at 105,000 g at 25" for 2 hr. 0,1 mglml; A, 3 mglml; 0, 5 mglml. These data represent the amount of cytochrome P-450 remaining in the supernatant at various detergent concentrations; these amounts are expressed a s percentages of the total cytochrome P-450 content of the intact microsomes. The amount of cytochrome P-450 was measured as A450-A4win the reduced vs reduced plus carbon monoxide difference s p e c t r ~ m . ~

centrations are low are subject to losses by surface adsorption. Figure 3 illustrates this effect in the solubilization of cytochrome P-450 from When microsomes are solubilized at 1 mglml, mouse liver rnicro~omes.~ only 80% of the total cytochrome P-450 is recovered. When higher concentrations (3 or 5 mglml) are used, a plateau of recovery of native cytoL. M. Hjelmeland, Proc. Nutl. Acad. Sci. U.S.A. 77, 6368 (1980).

262

[I91

SOLUBILIZATION PROCEDURES

Detergent Concentration (mM)

Detergent protein

):(

FIG.4. The solubilization of [3H]DALAMID-binding activity from NG108 membrane plotted as a function of (A) CHAPS concentration (mM) and (B) CHAPS-to-protein ratio (w/w).

chrome P-450 is reached which is presumably independent of initial protein concentration. At concentrations of membrane protein below 1 mg/ml this effect becomes quite dramatic and thus, whenever possible, protein concentrations in excess of 1 mglml should be used for the preparation of solubilized proteins. When using higher concentrations of protein, however, the ratio of protein to detergent becomes an important experimental variable. As indicated in Fig. 2, this ratio is critical for determining the nature of the soluble species. Figure 4 presents graphical data substantiating this point. In Fig. 4, the solubilization of opiate receptors from the membranes of NGlO8 cells by CHAPS is p r e ~ e n t e d .Experiments ~ were performed in which membranes at three different initial concentrations were solubilized with increasing concentrations of CHAPS. Figure 4A presents these data as a function of CHAPS concentration, and a shifting of the solubilization curve with increasing initial concentrations of membrane protein is seen. In Fig. 4B, the data are plotted as a function of detergent: protein ratio, and a more uniform result can be seen. Figure 4B suggests that optimum recovery of solubilized opiate receptors is associated with a single detergent : protein ratio. W. A. Klee, unpublished observations, 1980.

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SOLUBILIZATION OF NATIVE MEMBRANE PROTEINS

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'These findings taken together suggest that in the process of solubilizing membranes, initial membrane protein concentrations should be kept high (greater than 1 mglml) and constant from experiment to experiment. Alternatively, if protein concentration is to be varied from experiment to experiment, consideration should be given to adjusting the detergent concentration appropriate1y. Protocol for Initial Solubilization Experiment

A final consideration for this chapter is the design of the initial solubilization experiment. The purpose of this procedure is to determine the efficiency of solubilization with a particular detergent, as well as the retention of activity of the membrane protein being studied. It will be assumed for the sake of simplicity that generous amounts of membrane are available for these purposes. I . Stock Membrane Preparation. After a particulate fraction o r purified membrane fraction has been prepared, membranes should be resuspended to a final concentration of 5 mglml in the buffer chosen for solubilization. In addition to the buffer and salt at its final concentration, protease inhibitors should be present at twice their final concentrations in the solubilization medium. 2. Stock Detergent Solution. Preparation of stock detergent solutions will facilitate examining a range of detergent concentrations. For many nonionic detergents, functional concentrations are expressed in terms of percentage (vlv) and as a result, stock solutions of 10% (vlv) may typically be used. These solutions should be made fresh in solubilization buffer. For detergents which have defined molecular weights, it may be more convenient to prepare stock solutions at 0.1 M final concentration in solubilization buffer. 3. Choosing Detergent Concentrations for Solubilization. After identifying a particular detergent for study, it is necessary to choose a set of final detergent concentrations for the experiment. A useful range of concentrations for nonionic detergents includes 0.01% (vlv) to 5% (vlv). For detergents like the bile acids and their derivatives, concentrations from 0.1 to 50 mM should be examined. Dilutions of detergent at twice the final concentration to be used should then be prepared in solubilization buffer. A typical study of the effect of varying concentrations of Triton X- 100 on the solubilization of membranes might examine final concentrations of 0.01, 0.025, 0.050, 0.1, 0.25, 0.5, 1 .O, and 5% (vlv) final concentrations. These concentrations are chosen to give a clear graphical representation of data covering at least two orders of magnitude of detergent concentration.

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SOLUBILIZATION PROCEDURES

[201

4 . Solubilization of Protein. Experimental points should be determined in triplicate. An aliquot of 0.5 ml of the stock membrane suspension is pipetted into each tube at 4". An aliquot of 0.5 ml of the differing detergent concentrations at twice their final concentration to be tested is then pipetted into appropriate tubes. Tubes are maintained at 4" for 1 hr, and then centrifuged at 105,000 g for 1 hr at 4". Tubes may be stirred during the incubation period, but excessive agitation should be avoided since foam formation is associated with the denaturation of proteins. 5. Determination of Solubilized Protein. After centrifugation, the clear supernatant should be removed from any residual pellet. Protein determinations should be made both on the solubilized protein and any pellet after suspension in an equal volume of solubilization buffer. Some pellets are difficult to resuspend, and may require the use of a small tissue homogenizer. Aliquots of the solubilized protein and the resuspended pellet can be assayed for the individual protein or activity being studied. Conclusion

The discussion presented here provides a few relatively simple guidelines for the selection of detergents and their use in the solubilization of membrane proteins in a native state. This is truly a task about which few valid generalizations may be made. For this reason, general recipes concerning the total amount of detergent to be used, or specific suggestions of "nondenaturing" detergents which will be useful in all situations, cannot be made.

[201 Solubilization of Protein Aggregates By FIONA A. 0. MARSTONand DONNAL. HARTLEY Introduction

Major advances in genetic engineering have resulted in the development of bacterial cell systems, especially Escherichia coli, capable of producing high levels of proteins expressed from cloned genes.',= In E. coli, in a majority of cases, the overexpressed protein accumulates intracellularly in an insoluble form, resulting in phase-bright inclusions in

' J . F. Kane and D. L. Hartley, Trends Biotechnol. 6, 95 (1988). F. A. 0. Marston, Biochem. J. 240, 1 (1986).

METHODS IN ENZYMOLOGY, VOL. 182

Copyright O 1990 by Academic Press. Inc. All rights o f reproduction in any form reserved.

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the cyt~plasm.~-~ Solubilization of these protein aggregates is the subject of this chapter. From a purification standpoint, the accumulation of protein in an aggregated form is advantageous. After breaking open the cells and centrifuging the resulting lysate the aggregated protein can be recovered in the pellet fraction about 50% pure, although mostly in an inactive form. The protein in the inclusion bodies can be a mixture of monomeric and multimeric f o r m ~both , ~ reduced and oxidized.? The major problem then becomes one of recovering biologically active protein in high yield. In order to accomplish this the protein in the inclusion bodies must be solubilized, refolded, and purified, in a specific order.2 The common stages in processes designed to recover biologically active, soluble protein from such aggregates include (1) cell lysis, ( 2 ) isolation of inclusion bodies, (3) solubilization of protein in inclusion bodies, and (4) refolding of solubilized protein. In the following sections we first present general approaches used to isolate and solubilize protein aggregates. Upon refolding such methods may yield active soluble protein; however, there are many factors influencing the final yield, each dependent on the nature of the protein itself. The later sections of the chapter illustrate these factors by reference to the processes optimized for specific proteins. General Considerations

Protein inclusion bodies in E. coli are formed during high-level expression of cloned genes.'.2 Exactly why inclusion bodies form is not known, but there are parallels between such cells and cells in which the heatshock system has been The majority of protein contained within these inclusion bodies is in a denatured form, in part due to the reducing environment of the E. coli c y t ~ p l a s m .In ' ~ addition, dimers and higher molecular weight multimers may be present. Hydrophobic interactions between regions of the unfolded protein molecules in the inclusion bodies may also be important. That the protein is not simply precipitated due to a high localized concentration is evidenced by the fact that strong D. C. Williams, R. M. Van Frank. W. L. Muth, and J. P. Burnett, Science 215,687 (1982). R. G . Schoner, L. F. Ellis, and B . E. Schoner, BiolTechnology 3, 151 (1985). L. A. Holladay, R. G. Hammons, Jr., and D. Puett, Biochemistry 13, 1653 (1974). D. F. Marks, S. D. Lu, A. A. Creasey, R . Yamamoto, and L. S. Lin, Proc. Narl. Acad. Sci. U.S.A. 81, 5662 (1984). D. L. Hartley and J. F. Kane, Biochem. Soc. Trans. 16, 101 (1988). W. F. Prouty. M. J. Karnovsky, and A. L. Goldberg, J . Biol. Chem. 250, 1112 (1975). C. A. Schachtele, D. L. Anderson, and P. Rogers, J. Mol. Biol. 33, 861 (1968). '"C. K. Tuggle and J . A. Fuchs, J . Bacterial. 162, 448 (1985). -'

266

SOLUBILIZATION PROCEDURES

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TABLE 1 REAGENTS THATRELEASEEUKARYOTIC POLYPEPTIDES FROM INCLUSION BODIESINTO SOLUTION^' Eukaryotic polypeptide solubilized

Reagent Cuanidine-HCI (5-8 M )

Urea (6-8 M )

SDS

Alkaline pH (>9.0) Acetonitrile/propanol

Insulin A and B chains Bovine growth hormone (bCH) Urokinase Prochymosin Interferon Salmon growth hormone Interferon Interleukin 2 (IL-2) Prochymosin Chicken growth hormone T4 regA protein

Reproduced from Ref. 21.

''solvents" are necessary to solubilize the proteins,* as illustrated in Table I. Once proteins are solubilized, the task becomes one of refolding the protein molecules into their correct conformations. This can be accomplished by removing the solubilizing agent and replacing it with an aqueous buffer (e.g., by dialysis or dilution). The situation is somewhat more complicated in proteins containing disulfide bridges. Oxidation of the reduced protein must occur either prior to or concomitant with refolding. The concentration of protein in the refolding solution also affects the yield of recoverable active protein.ll The most significant loss during refolding of concentrated protein solutions is due to aggregate formation, which is frequently due to covalent modifications of the unfolded protein molecules, such as intermolecular disulfide formation." It is therefore recommended that refolding be carried out at as dilute a concentration as feasible, taking into consideration workable volumes of solutions and possible loss due to high dilution for subsequent purification steps. Finally, purification is usually effected to remove other proteins and contaminating nucleic acids also present in the inclusion body,' as well as to remove incorrectly refolded forms of the protein of interest. Depending on the specific characteristics of the protein to be purified, conditions can often be defined so that an ion-exchange column can effectively remove "

A . Light, BiolTrchnology 3, 298 (1985).

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SOLUBILIZATION OF PROTEIN AGGREGATES

267

nucleic acid contaminants and proteins of very different isoelectric points. Subsequent purification is frequently carried out by HPLC.2," With very low level expression, i.e., very small inclusion bodies, more impurities will be present than when starting with large inclusion bodies,I3 and will undoubtedly have a negative impact on the efficiency of solubization and refolding. This emphasizes the importance of optimizing expression either by genetic manipulations or by amelioration of fermentation and induction parameters, before attempting to purify significant quantities of the expressed protein. General Solubilization Scheme

Preparation of Certain Solubilization Reagents Urea (6 M ) . Ultrapure-grade urea should be used, with deionized distilled water. This solution should be deionized by passage through a mixed bed ion-exchange resin and stored at 4'. Cold storage will reduce the formation of cyanate ions, which can ultimately react with amino groups to form carbamylated derivatives. Guanidine-HCl(6 M ) . Ultrapure grade guanidine is used and the pH of the final solution is adjusted to 7-8 with concentrated HCI. Cell Disruption

There are several ways to recover protein inclusion bodies from pelleted bacterial cells. The most frequently used are ~ o n i c a t i o n ' ~ . and '~.'~ passage through a French pressure ell,'^.'^ but it is also possible to use the lysozyme-detergent treatment. l8 Sonication. Suspend 15 g of cell paste in 50 ml of chilled deionized water. Sonicate two times for 45 sec each at 0°, using 50% power. Keep cell suspension on ice. Y. Saito, Y. Ishi, M. Niwa, and 1. Ueda, J . Biochem. (Tokyo) 101, 1281 (1987). N. Brems, S . M. Plaisted, H. A. Havel, E. W. Kauffman, J . D. Stodola, L. C. Eaton. and R. D. White, Biochemistry 24, 7662 (1985). l4 S. M. Zurawski, T. R. Mosmann. A. Benedik, and G . Zurawski, J . Immunol. 137, 3354 l2

" D.

(1987).

J. Bartholome-DeBelder, M. Nguyen-Disteche, N . Houba-Herin, J . M. Ghuysen, I. N . Maruyama. H. Hara, Y . Hirota, and M. Inouye, Mol. Microbiol. 2, 519 (1988). l6 D. V . Goeddel, D. G . Kleid, F. Bollivar, H . L. Heyneker, D. G . Yansura, R. Crea, T. Hirose, A. Kraszewski, A. Itakura, and A. D. Riggs. Proc. Natl. Acad. Sci. U.S.A. 76, Is

106 (1979).

'' G . Schumacher, D. Sizmann, H. Huag, and A. Bock, Nucleic Acids Res. 14,5713 (1986). l8

J. A. Goliger and J. W. Roberts, J . Biol. Chem. 262, 1 172 1 (1987).

268

SOLUBILIZATION PROCEDURES

[201

French Press. Prechill the French press cell to be used. Suspend 15 g of cell paste in 30 ml of chilled deionized water. Pass the cell suspension through the cell twice at 5000 lblin. pressure. Keep the suspension on ice. Centrifugation and Washing Centrifuge the suspension resulting from cell breakage at 10,000 to 20,000 g for 10 to 20 min at 4". (At this point it is prudent to look at a sample of the supernatant under the microscope to determine if a significant quantity of inclusion bodies remain. This can vary depending on the protein being expressed by the cells. If significant amounts of inclusion bodies remain in the supernatant, recentrifuge at 20,000-25,000 g for 1015 min.) Carefully decant the supernatant and resuspend the pellet in 50 ml of chilled deionized water. Washing Inclusion Bodies The fact that many of the eukaryotic proteins expressed in E. coli are insoluble is an advantage since isolation of inclusion bodies in itself can be a very efficient purification step. However, to a varying degree, protein contaminants do remain after inclusion body isolation by centrifugation coupled with washing procedures. Such contaminants may subsequently interfere with refolding o r may prevent it altogether. Most, if not all, of these contaminating proteins are strongly associated with or entrapped in the inclusion bodies and therefore may require solubilization under the type of conditions listed in Table I to release them. It is frequently advantageous to wash the inclusion bodies with a solution other than water. In this case, the wash step can be carried out as above, usually with the incorporation of some type of detergent in the washing buffer.".I9 It has also been found that treatment of the broken cell pellet with 0.2 mg ml-' lysozymell m M EDTAI1 mg ml-I deoxycholate, while the protein is still insoluble, will remove the majority of nucleic acids, phospholipids, and lipopolysaccharides from bovine somatotropin inclusion bodies, without solubilizing the protein.I9 A similar wash, using lysozyme/EDTA/DNase/Nonidet was also found to be effective in selectively removing contaminants without solubilizing the protein in the inclusion bodies. l5 Denaturants can in some cases be used to solubilize contaminants preferentially. Directly expressed bovine growth hormone (bGH) forms inclusion bodies in E. coli, and a number of contaminants copurify with l9

K. E. Langley, T. G. Berg, T. W. Strickland, D. M. Fenton, T. C. Boone, and J. Wypych. 163, 313 (1987).

Eur. J . Biochem.

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SOLUBILIZATION OF PROTEIN AGGREGATES

269

the aggregated bGH.4 Washing the inclusion bodies with up to 4 M urea only solubilizes the contaminants, but as the urea concentration is increased to 5 M the bGH is partially solubilized as well. Solubilization

Typical classes of reagents which can be used to solubilize proteins from inclusion bodies are listed in Table I. In general a solubilization and refolding protocol for protein aggregates in E. coli will be protein specific. At each stage there are variables which are critical and must be considered. For the solubilization stage these variables are as follow:

1. 2. 3. 4. 5. 6. 7. 8. 9.

pH Incubation temperature Time of exposure to solvent Ionic components of the solvent Concentration of the solubilization agent Concentration of total protein Ratio of solubilization agent to protein Presence or absence of redox agents Derivatization of thiol groups

Having established the optimum reagent for solubilization, the other variables listed must be investigated in conjunction with refolding to determine the most efficient overall process. A recommended strategy is to screen the variables first on a small scale (1- to 2-ml working volume) in order to select a limited number of variables to evaluate on a larger scale. Commonly denaturants are used early in the evaluation of solubilization agents. A typical method might be as follows: Using a clean spatula, remove the washed inclusion body paste to a preweighed glass beaker and determine its weight. Add a stir bar and use a magnetic stir plate on the lowest speed possible. Slowly add a solution of 6 M urea or 6 M guanidine chloride to give a final protein concentration of 1-2 mg/ml. Continue stirring at the lowest speed until the paste is dissolved. It is frequently desirable to add a thiol reagent, such as cysteine or 2-mercaptoethanol, to the urea or guanidine-HC1 solution to reduce all of t6e protein present in the inclusion bodies. For example, it has been estimated that only 80% of the bGH in inclusions is in a reduced form. l9 In order to recover active protein from the remaining oxidized material, it is necessary to reduce it, thus starting with 100% reduced material (i.e., random coil). In contrast, solubilization

270

SOLUBILIZATION PROCEDURES

[201

of prochymosin (in urea or guanidine-HC1) in the presence of thiol reagents has a negative impact on the recovery of active protein.z0 If the solution resulting from solubilization is quite cloudy, it is recommended that a centrifugation step be added here. Centrifuge the protein solution at 20,000-30,000 g for 15 min. Decant the cleared supernatant to a fresh beaker. Refolding

Protein aggregates in E. coli must be first solubilized and then refolded in order to regain active protein. These two processes, solubilization and refolding, are interdependent and their use in recovering proteins from aggregates in E. coli has been re~iewed.~." As for solubilization, there are variables at the refolding stage which are critical and must be considered: 1. pH

2. 3. 4. 5. 6. 7. 8.

Incubation temperature Time Ionic components of the solvent Rate of change from solubilization to refolding solvent conditions Purity of the protein of interest Concentration of the protein of interest Presence or absence of redox agents

It is pertinent in this chapter to consider the key points relating to solubilization which impact on refolding. It is important during both solubilization and refolding to minimize exposure to conditions which result in derivatization of amino acid side chains (e.g., pH values of greater than 9.0). During the refolding stage perhaps the key parameters influencing recovery are purity and concentration of the protein of interest. The concentration of the protein should be such that at the incubation temperature used, intramolecular bonds form in preference to intermolecular bonds. Finally, for proteins which contain cysteine residues and in their native form contain disulfide bonds, the redox conditions during solubilization and refolding may be critical. Strict guidelines cannot be provided since requirements are protein specific. Prochymosin, for example, contains six cysteine residues which form three disulfide bonds in the native protein. If aggregated, prochymosin isolated from E. coli is first fully 20

2'

F. A. 0. Marston, P. A. Lowe, M . T. Doel, J. M. Schoernaker, S . White, and S. Angal, BiolTechnology 2, 800 ( 1984). F. A. 0. Marston, in "DNA Cloning" (D. Glover, ed.), Vol. 3, p. 59. IRL Press, Oxford, 1987.

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SOLUBILIZATION OF PROTEIN AGGREGATES

27 1

reduced, and then solubilized in a strong denaturant (8 M urea). However, little if any activity is recovered upon refolding. The best yields of active protein could be obtained by omitting redox reagents at both the solubilization and refolding stages.'O In contrast, there are examples of proteins which require redox reagents at the solubilization stage, or refolding stage, or both. One such example is interleukin 2 (IL-2) (three cysteine, one disulfide) which is in a fully reduced form in inclusion bodies.22 At the solubilization stage it apparently is essential to maintain the denatured IL-2 in a reduced form.23 In both this study and another in parallel,22it was then clearly demonstrated that oxidative refolding is required to obtain biologically active

Solubilization of H y b r i d P r o t e i n A g g r e g a t e s

Recombinant DNA technology can be used to produce hybrid proteins by fusion of the coding sequence of the gene of interest to one or more other gene sequences. This is a common strategy used to improve expression levels. The mechanism by which expression levels are increased can differ and may be the result of reduced proteolysisZ4or sequestration of the gene product into inclusion bodies.25 Cleavage of the hybrid protein in vitro may be required in order to generate the protein of interest. The cleavage strategy must be considered in advance of gene construction and an appropriate cleavage site engineered immediately before the N-terminus (or after C-terminus) of the coding sequence of the protein of interest. Such a cleavage site must either be absent in the required protein or, if present, must be less reactive than the sequence located at the junction. Essentially, there are two methods of cleavage which can be employed: chemical or enzymatic. When the hybrid protein is aggregated and insoluble such cleavage must be performed under conditions which solubilize the protein and make the cleavage site accessible. Extreme acid conditions can solubilize polypeptides and concomitantly catalyze hydrolysis. Conditions can be adjusted such that hydrolysis occurs at specific amino acid sequences, typically after Asp. For native proteins partial acid hydrolysis is typically performed in 30 mM HCI, 22 23

"

T. Tsuji, R. Nakagawa, N . Sugimoto, and K.-I. Fukuhara, Biochemisrry 26, 3129 (1987). M. P. Weir and J. Sparks, Biochem. J. 245, 85 (1987). K. Itakura, T. Hiroso, R. Crea, A. D. Riggs, H. L. Heyneker, F. Bolivar, and H. W. Boyer, Science 198, 1056 (1977). S.-H. Shen, Proc. Nail. Acad. Sci. U . S . A . 81,4627 (1984).

272

[2 01

SOLUBILIZATION PROCEDURES

CONDITIONS USEDFOR - - - -

TABLE I1 CNBr CLEAVAGE OF CERTAIN AGGREGATED HYBRID PROTEINS

- - --

Hybrid protein Tandem-linked proinsulin or P-galactosidase-proinsulin bcZ-substance P P-galactosidasegrowth hormone

-

-

-

-

Percentage formic acid (v/v)

CNBr : protein (w/w)

Incubation time (hr)

Protein concentration (mg ml-I)

Ref.

70

50

35

-

25

88 44

10 5

12 3

15.0 20.0

29 30

in vacuo at 105" for 20 hr.26AS an example, acid hydrolysis was used to release bGH from fusions to the trpE and trpLE gene product^.^' However, acid conditions alone were not adequate to solubilize aggregated proteins for cleavage. The suspending medium required was 70% formic acid containing the denaturant guanidine hydrochloride at a concentration of 6 M.27The proteins were incubated at concentrations of between 0.85 and 1.0 mg ml-' for 72 hr at 37" to allow cleavage to occur. When the protein of interest lacks Met residues in its sequence, a common strategy is to construct the fusion protein with a Met residue at the junction between the two parts of the hybrid molecule. Then cyanogen bromide (CNBr), which selectively cleaves proteins immediately after Met residues, can be used to cleave the hybrid in vitro. CNBr cleavage is performed under acid conditions (commonly formic acid), and therefore aggregated proteins may be s ~ l u b i l i z e d . ~ ~ Key parameters to consider when establishing hydrolysis conditions include protein concentration, formic acid concentration, ratio of CNBr to protein (wlw), temperature, and time. Table 1129,30illustrates, with a few examples of insoluble hybrid proteins, the wide range of conditions which have been employed. In each of these examples, the acid conditions alone were adequate for the CNBr to gain access to the cleavage site. When cleavage of hybrid proteins is to be catalyzed enzymatically, the R. L. Lundblad and C. M. Noyes, in "Chemical Reagents for Protein Modification," Vol. I. CRC Press, Boca Raton, Florida, 1984. 27 P. R. Szoka, A. B. Schreiber, H. Chan, and J. Murphy, DNA 5, 11 (1986). E. Gross, this series, Vol. 11. p. 27. "T. Kempe, S. B. H. Kent, F. Chow, S. M. Peterson, W. I. Sundquist. J. J. L'ltalien, D. Harbrecht, D. Plunkett, and W. J. DeLorbe, Gene 39, 239 (1985). 'O T. Kempe, F. Chow, S. M. Peterson, P. Baker, W. Hays, G . Opperman, J. J. L'Italien, G. Long. and B. Paulson, BiolTechnology 4, 565 (1986). 26

[201

SOLUBILIZATION OF PROTEIN AGGREGATES

273

hybrid should be purified to >80% homogeneity (see following section) to maximize the efficiency of the proteolytic step. There are two possible proteolytic strategies for aggregated hybrids: I. Solubilize the hybrid proteins and cleave in the presence of the solubilization agent. 2. Refold the solubilized hybrid, removing the solubilization agent, and then cleave. Strategy (1) involves the use of balanced conditions which are severe enough to dissociate aggregates but which do not inactivate the proteolytic enzyme. There are certain proteases which are active in the presence of high levels of denaturant, e.g., clostripain (4 M urea") and carboxypeptidase (5 M urea3*). In an example of this strategy fusion between the bacterial enzyme chloramphenicol acetyltransferase (CAT) and human calcitonin (hCT) was engineered with a -Lys-Arg- cleavage site," which can be cleaved by clostripain. The hybrid CAT-Lys-Arg-hCT was purified by isolation and washing of inclusion bodies. The washed inclusions were then solubilized in 100 m M Tris-HCI, pH 7.8, containing 8 M urea and 0.14 M 2-mercaptoethanol at a final protein concentration of 40 mg ml-I. The suspension was incubated at 37" for 10 min and then diluted 1 : 1 (vlv) with water. Clostripain was then added to a final ratio of protease :fusion protein (wlw) of 1 : 40 and the suspension was incubated for a further 15 min at 37" before the addition of trifluoroacetic acid [5% (vlv), final concentration] to stop the enzymatic reaction. With strategy (2), in which hybrid proteins are solubilized and refolded before cleavage, the potential problem i s that the components of the hybrid may interact and therefore prevent correct folding. However, the strategy has been used successfully with fusions between the hcll gene . ~ ~ fusion protein is isolated in washed inclusion product and P - g l ~ b i nThe bodies that were solubilized in 8 M urea and purified (see below). Then dialysis is used to remove the denaturant and allow refolding. Cleavage of the fusion protein is then catalyzed with blood coagulation factor Xa, approximately 160 mg of fusion protein being cleaved with 5 mg of activated factor Xa. Purification of Solubilized Protein Aggregates Once the typical washing procedures described earlier have been used effectively to purify inclusion bodies, the only method to dissociate the

'' P. A. Lowe, S. K . Rhind, R. Sugrue, and F. A. 0.Marston, Protein Purif.: Micro MacroUCLA Symp. Mol. Cell. Biol. 68, 429 (1987).

" H. M. Sassenfeld and S . J. Brewer, BiolTechnology 2, 76 (1984). 33

K . Nagai, M. F. Perutz, and C. Poyart, Proc. Natl. Acad. Sci. U . S . A . 82, 7252 (1985).

274

[201

SOLUBILIZATION PROCEDURES

7

-

-

-

-

-

-

Stability Chromatographic mode

Matrix

pH

Denaturants

Ion exchange

Mono-S

8 M urea

Ion exchange

Mono-Q

8 M urea

Ion exchange

TSK-DEAE 5PW

Ion exchange

DEAE-Sephacel

Gel filtration

Sepharosc C L

8 M urea, 6 M GuHCl 8 M urea, 6 M GuHCl 8 M urea, 6 M GuHCl

Gel filtration

Superose

8 M urea, 6 M GuHCl

Hydrophobic interaction

Phenyl-Superose

8 M urea

Hydrophobic interaction Reversed phase

TSK Phenyl 5PW

8 M urea, 6 M GuHCl Urea, GuHCl

Reversed phase

Ultrapore short chain C3

PLRP-S

Urea, GuHCl

Detergent

Supplier

Nonionic, anionic, zwitterionic Nonionic, cationic. zwitterionic All

Pharmacia

Pharmacia

Nonionic, anionic

Pharmacia

All (sodium deoxycholate not recommended) All (sodium deoxycholate not recommended) Nonionic, cationic, zwitterionic All

Pharmacia

All

All

Toyo-Soda

Pharmacia

Pharmacia

Toyo-Soda Polymer Laboratories Beckman

remaining contaminating proteins may be total solubilization using the harsh conditions listed in Table I. However, it is still possible to purify the protein of interest using conventional chromatography matrices, which are stable under such extreme conditions (Table 111). The protocol developed to solubilize the XcII-P-globin fusion protein from E. ~ o linvolves i ~ ~ isolation and washing of inclusion bodies, solubilization in 8 M urea, and purification of the denatured protein before refolding and cleavage of the hybrid protein. Isolated inclusion bodies were solubilized in 8 M urea, 25 mM Tris-acetate, pH 5.0, 1 mM EDTA, and 1 mM DTT (urea buffer). The solubilized protein was applied to a CMSepharose column and the fusion protein eluted with a gradient of 0-0.2 M NaCl in urea buffer. The eluted protein was then subjected to gel

-

1201

-;

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SOLUBILIZATION OF PROTEIN AGGREGATES

275

filtration on a Sephacryl S-200 column in 5 M guanidine-HCI, 5 mM TrisHC1, pH 8.0, 1 mM EDTA, 1 mM DTT. The eluate from this column containing the fusion protein was dialyzed to remove denaturant and allow the hybrid protein to refold before cleavage with factor Xa as described in an earlier section. In E. coli over 10 eukaryotic proteins have been expressed fused to AcII via the factor Xa cleavage site.34In each case the process sequence was similar: solubilization (denaturation), purification in a denatured state, refolding, and cleavage. The level of purity required for efficient refolding differed and was found to be protein specific. Interleukin 2 (IL-2) in E. coli is another example of an aggregated protein that has been purified in a solubilized, denatured form before ref~lding.?~ IL-2 contains three cysteine residues and the intramolecular disulfide bond between Cys-58 and Cys-105 in native IL-2 is essential for activity. Partial purification of the solubilized IL-2 facilitated analysis of reoxidation of the molecule during refolding and therefore allowed optimization of refolding conditions to give the maximum yield of correctly oxidized 1L-2. Purification was effected by gel filtration in 6 M guanidineHCI, 50 mMTris-HC1, pH 8.5, 10 mM DTT using Superose-12 (FPLC) on an analytical scale and Sepharose CL-6B on a larger scale. One final purification strategy to consider is that in which gene fusions are designed and constructed to facilitate purification. The principle of this strategy is to fuse the gene of interest to a sequence coding for a polypeptide which is selectively recognized by a matrix-bound ligand. A general approach developed by Sassenfeld and c o - w o r k e r ~was ~ ~ to produce C-terminal polyarginine fusions. Efficient purification of such fusions is possible using two-step cation-exchange chromatography. For example, urogastrone-polyarginine3* expressed in E. coli is insoluble and can be solubilized by sonication of whole cells in 5 M urea, 40 mM Trisacetate, NaOH, pH 9.5. The supernatant from centrifugation of such an extract, adjusted to pH 5.5, was subjected to chromatography on SPSephadex. The fusion protein and other cationic proteins bound and were eluted with a 0-300 mM NaCl gradient. After digestion of the polyarginine tail with carboxypeptidase B, urogastrone does not bind, while the other cationic proteins do. Therefore, the second cation-exchange step yields highly pure urogastrone. The buffer used for both cationexchange steps was 5 M urea, 40 mM Tris-acetate, pH 5.5. There are examples of this fusion protein purification strategy which predate the polyarginine fusion approach (e.g., P-galactosidase3' and

'' K. Nagai, H.-C. Thorgensen, and B . F. Luisi, Biochem. Soc. Tmnu. 16, 108 (1988). " M.

Koenen, U. Ruther, and B . Muller-Hill, EMBO J. 1, 509 (1982).

276

SOLUBILIZATION PROCEDURES

[201

CAT36).However, the conformation of these polypeptide components of the hybrid are essential for selective recognition Sy the affinity ligand. Therefore, when CAT-hCT was found to be insoluble in E. coli and required 8 M urea to effect solubilization, affinity chromatography was not p ~ s s i b l e . ~ ' There have been recent developments in the design of fusion proteins which facilitate purification utilizing maltose-binding protein3' and glutathione S - t r a n s f e r a ~ eHowever, .~~ a central aim of each of these strategies is to obtain a fusion protein which is soluble in the cytoplasm or is secreted to the periplasm. Concluding Remarks

The protocols described in this chapter illustrate the solubilization and refolding requirements of specific proteins. Purification and analysis of the proteins during and after these processes are of importance but are not considered here as they are discussed in detail in later chapters in this volume. However, it is important to emphasize that certain features of the final protein products may be the direct results either of the expression mechanism or the methods used to recover active, soluble protein. These include 1. Authenticity of the N-terminus1C-terminus 2. Lack of posttranslational modification (e.g., glycosylation) 3. Modifications resulting from solubilization conditions (e.g., deamidation at high pH, Met oxidation, and conversion of cysteine to cysteic acid at low pH) 4. Conformational authenticity of the refolded molecule

These features will undoubtedly affect the activity of the final product, and their impact will depend on the application for which the proteins are required.

l6

37

38

A. D. Bennet, S . K. Rhind, P. A. Lowe, and C. C. G . Hentschel. U . K . Pat. No. GB 2140810 B (1983). C. di Guan, P. Li, P. D. Riggs, and H. Inouye, Gene 67, 21 (1988). D. B . Smith and K . S . Johnson, Gene 67, 31 (1988).

1 "

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[2 11

DETERGENT REMOVAL FROM MEMBRANE PROTEINS

277

[211 Removal of Detergents from Membrane Proteins By LEONARD M. HJELMELAND

:

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In addition to the issues of detergent selection and use discussed in [18]-[20] of this volume, it is necessary to consider the topic of detergent removal. The process of the removal of detergent from solubilized membrane proteins may be required at several different stages of both analytical and preparative procedures involving detergents. This chapter will consider the different circumstances under which detergent removal may be advisable, as well as detergent properties which may influence the ease with which detergents may be removed. Finally, a survey of typical removal procedures will be given. Fortunately, this topic has been treated in depth in the published literature, and the reader is advised to consult these references. When to Remove Detergents -

Preparative Procedures

During the course of the purification of a membrane protein, it may be necessary to remove excess detergent at three distinct points in the protocol. The first arises after initial solubilization of membranes. As this step usually requires detergent excess in order to give maximal dispersion of membrane proteins and lipids, removal of excess detergent at this stage usually improves the quality of subsequent chromatographic steps. Often, excess detergent removal at the beginning of a purification protocol is accomplished by the first chromatographic column, and this observation should be taken into account in planning the overall purification strategy. A second point at which detergent may need to be removed occurs during the process of detergent exchange. The detergent initially used for solubilization may not be appropriate for subsequent chromatographic or analytical procedures, and detergent exchange is a common feature of many preparative protocols. The replacement of one detergent with another is

' A. J . Furth, H. Bolton, J . Potter, and J . D. Priddle, this series, Vol. 104, p. 318. A. J. Furth, Anal. Biochem. 109, 207 (1980). N. C. Robinson, D. Wiainton. and L. Talbert. Biochemistrv. 23., 6121 11984). P. C. Adams, F. D. Roberts, L. W. Powell, and J. W. Halliday, J. Chromatogr. 427, 341 (1988). R. Moriyama, H. Nakashima, S. Makino, and S . Koga, Anal. Biochem. 139, 292 (1984).

$3

t

METHODS IN ENZYMOLOGY, VOL 182

Copyright 0 1990 by Academic Press, lnc. All rights of reproduction in any form reserved.

278

SOLUBILIZATION PROCEDURES

[2 11

usually achieved by a chromatographic procedure. The most efficient processes are those which involve adsorption of the detergent-membrane protein complex to a chromatographic support, followed by extensive washing with buffer containing the new detergent. Alternatives involve gel filtration chromatography and equilibrium methods such as dialysis. Finally, detergents are removed at the end of the preparative procedure to facilitate analytical procedures such as amino acid analysis, amino acid sequencing, and analytical high-performance liquid chromatography. Since membrane proteins are not soluble in aqueous media, the goal of these procedures is to replace the detergent-containing aqueous medium with a solvent system which is compatible with the analytical procedure. Methods frequently suggested for the final removal of detergents include adsorption of detergent to hydrophobic media, precipitation with polyethylene glycol, and chromatography on various hydrophobic or reversedphase media. Of these choices, the last mentioned is to be preferred due to the unavoidable losses involved with simple adsorption or precipitation procedures. Analytical Procedures

Many analytical procedures have specific requirements related to the physical properties of the detergents to be used, and thus preparation of samples may necessitate detergent exchange or removal. A frequently encountered example is the preparation of proteins separated by SDSpolyacrylamide gel electrophoresis for subsequent use in sequencing. Another example is the interference that ionic detergents cause in the use of isoelectric focusing or other charge-based electrophoretic methods. A third example would be the interference of an ionic detergent such as cholic acid with analytical high-performance ion-exchange chromatography. Each of these examples requires a slightly different solution to the exchange or removal problem. Excess SDS is usually removed by an electroelution procedure, which is well documented in the literature, and for which commercial instrumentation is available. Excess SDS, which may be present in samples being prepared for isoelectric focusing, can be effectively managed by the addition of excess nonionic detergent such as Triton X-100, and thus a separate exchange or removal step is not necessary. Finally, detergent exchange can often be accomplished along with a specific chromatographic procedure. In the case of a sample in cholic acid to be used for ion-exchange chromatography, initial conditions should be found which will allow the exchange of cholate for an appropriate nonionic or zwitterionic detergent on the column. After the exchange is ac-

[211

DETERGENT REMOVAL FROM MEMBRANE PROTEINS

279

complished by exhaustive washing, membrane proteins can then be eluted with gradients of increasing ionic strength or changes in pH. Types of Detergent Exchange Procedures

Chromatographic Techniques

Chromatographic techniques for detergent removal or exchange come in two basic varieties. The first involves the adsorption of the membrane detergent-protein complex to the chromatographic medium, followed by thorough washing with buffer including the new detergent. As the column is washed, mixed micelles between the molecules of the old and new detergents are formed, leading eventually to complete replacement of detergent. The type of column to be used may involve any specific chromatographic principle. A lectin column would be appropriate, for example, for a plasma membrane receptor with associated carbohydrate structures. Without specific measures of the efficiency of this process, it is usually sufficient to pass 20 column volumes of buffer containing the new detergent over the column to ensure complete replacement of detergent. A second type of procedure involves gel filtration chromatography. This procedure takes advantage of the usual difference in Stokes radius of detergent micelles or detergent-lipid mixed micelles and the Stokes radius of a protein-detergent complex. The column is equilibrated into the target detergent (the detergent into which the protein is being transferred) and then a sample of protein in the initial detergent is chromatographed. After elution of the protein from the gel filtration medium, substantial transfer (usually greater than 90%) has been achieved.

i

Dialysis

Is is possible to replace or exchange detergent by dialysis to equilibrium? It is always necessary to consider whether the desired protein will be soluble at the end of the dialysis. Some membrane proteins, like cytochrome b 5 , will undergo limited aggregation after detergent removal, while most will simply aggregate and precipitate. How effectively detergent may be dialyzed depends on the critical micelle concentration (cmc) and the micelle molecular weight (mMW). The cmc determines free detergent concentration, and this is usually the molecular state in which most detergents will be capable of passing through a typical dialysis membrane. Most often, the mMW is large enough so that detergent micelles are retained along with detergent-protein couples. When very small deter-

280

SOLUBILIZATION PROCEDURES

[2 11

gent micelles are present, such as those found in solutions of the bile acids and their derivatives, micelles may pass directly through the dialysis membrane. The rate of approach of these systems to equilibrium and the final equilibrium state can be favorably affected by placing an adsorptive medium outside the dialysis membrane to capture the detergent. Ionexchange resins may be used for charged detergents such as SDS, and hydrophobic resins are useful for nonionic or zwitterionic detergents. Precipitation

Membrane proteins may be directly precipitated from detergent solution with reagents such as polyethylene glycol. This procedure is especially useful for the removal of nonionic detergents, but suffers from a requirement for substantial amounts of protein for its effective use. As with many preparative procedures, protein concentration should be above 3 mglml in order to minimize protein loss during precipitation. Other precipitants other than PEG may be used, but it is always necessary to examine the solubility of the detergent in the precipitation medium in an independent experiment. Detergents are surprisingly insoluble in many media commonly used to precipitate proteins, such as trichloroacetic acid. Detergent Properties Affecting Ease of Detergent Removal

Often, the success of the detergent removal procedure can be enhanced by a simple consideration of the physical properties of the detergents to be employed. The first critical property is the crnc (critical micelle concentration). Since the crnc is a numerical measure of the hydrophobicity of the detergent, it also serves as a general indicator of the strength of detergent binding to proteins. Detergents with low crnc values, such as the nonionic detergents, are generally more tightly bound to proteins than detergents with high crnc values, such as the bile acids. The strength of binding may have a real effect, for example, on the rate of removal of proteins by procedures such as dialysis. In addition to the cmc, the mMW is also an important physical property of a detergent to be removed from a membrane protein. Detergents with large mMW values will not easily pass through dialysis or ultrafiltration membranes. Most nonionic detergents, and ionic detergents with linear alkyl hydrophobic groups have high mMW values and fall into this category. These observations suggest that when it is very desirable to be able to remove detergent, that the choice of a detergent with a relatively high crnc

[2 11

DETERGENT REMOVAL FROM MEMBRANE PROTEINS

I -?

28 1

and a low mMW be made. Examples of such detergents are the bile acids and their derivatives, and octylglucoside. Conversely, detergents with low cmc values and high mMW values are very difficult to remove. Examples include Triton N-101 and Triton X-100.

A Typical Protocol

Because of the diversity of procedures for the removal of detergents from membrane protein, it is difficult to give any individual protocol which would address all experimental requirements. Instead, a brief example of the exchange of Triton X-100 for CHAPS will be given to illustrate this process.

4

'Cr--

-

Ir:

Preparation of Solubilized Membranes

The preparation of solubilized membranes is explored in [19] of this volume, and we will assume for our purposes here that mouse liver endoplasmic reticulum has been solubilized with Triton X-100 at a final concentration of 2% (vlv) to give a soluble supernatant at approximately 1-3 mglml of soluble membrane protein. A typical buffer composition would be 25 mM Tris-HC1, pH 7.5, 150 mM KCl, 20% glycerol (vlv), and 1 mM DTT. Chromatographic Loading

5

C: 1

The soluble supernatant obtained in step 1 is loaded at a linear velocity of 30 ml/cm2/hron an ion-exchange medium such as DEAE-Sephadex or DEAE-agarose at 4", equipped with a monitor at 280 nm. The monitor output should be set to zero with the initial buffer before the addition of Triton X-100. Due to the absorbance of Triton at 280 nm, a full-scale absorbance should be seen after the membrane protein sample is loaded. Reddish brown cytochrome P-450 should be apparent bound to the top of the column. Detergent Exchange

I

$

A buffer similar to the solubilization buffer, but with the replacement of Triton X-100 by CHAPS, should be prepared [Tris-HCI, 7.5, 20% glycerol (vlv), 1 rnM DTT, and 10 mM CHAPS]. This buffer is then pumped through the column at the same flow rate with monitoring at 280 nm. As the Triton X-100 is removed, absorbance will drop to a baseline value. This process should require 10 to 20 column volumes.

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Elution of Bound Cytochrome P45O Bound cytochrome P-450 can then be eluted with a linear gradient of salt over 5-10 column volumes. The exact elution conditions obviously depend on the exact cytochromes to be purified. Eluted proteins will have essentially no bound Triton X-100 and will be associated with CHAPS. Conclusions

The brief discussion presented here should help to provide a basis for effective planning during the process of purification and analysis of membrane proteins. The ease with which detergents may be removed from proteins is directly related to the physical properties of these reagents and, with these properties in mind, successful removal should always be possible.

Section VI Purification Procedures: Bulk Methods

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[22] Precipitation Techniques

!

By SASHAENGLARD and SAMSEIFTER

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This chapter deals with methods for the precipitation of proteins for preparative purposes. Proteins can be precipitated by causing perturbations in the solvent with respect to pH, ionic strength, and temperature. The properties of the solvent can also be modified by addition of high concentrations of certain salts or of miscible organic solvents. Addition of specific metal cations such as Zn2+, Cd2+,and Ba2+, or of compounds with bulky anions such as picrate, tannate, tungstate, molybdate, trichloroacetate, perchlorate, and sulfosalicylate, can also precipitate proteins at appropriate pH values. Many of the last group of agents, because they also may be denaturing, are used principally for removal of proteins from a solution to allow analysis of nonprotein constituents. In this chapter emphasis is given to perturbants whose use has survived the introduction of more modern methods of protein purification such as column and affinity chromatography. Thus, the use of ammonium sulfate and of ethanol andlor acetone is considered in some detail. Because of limitations of space, and the fact that many excellent reviews already exist,'-lo the theory of protein precipitation is presented only briefly. History

Although the precipitation of proteins from biological fluids had been observed for hundreds of years (e.g., the precipitation of casein from milk S. Schwimmer and A. B. Pardee, Adu. Enzymol. 14, 375 (1953). A. A. Green and W. L. Hughes, this series, Vol. 1 [lo]. M. Dixon and E. C. Webb, Adu. Protein Chem. 16, 197 (1961). P. H. von Hippel and T. Schleich, in "Structure and Stability of Biological Macromolecules" (S. N. Timasheff and G. D. Fasman, eds.), p. 417. Dekker, New York, 1969. R. K. Scopes, in "Techniques in Protein and Enzyme Biochemistry," B101, p. 1. Elsevier1North-Holland, New York, 1978. M. Dixon and E. C. Webb, "Enzymes," 3rd Ed., p. 29. Academic Press, New York, 1979. T. Arakawa and S. N. Timasheff, this series, Vol. 114 [31. 8 R . C. Marshall and A. S. Inglis, in "Practical Protein Chemistry, A Handbook" (A. Darbre, ed.), p. 1. Wiley, New York, 1986. R. K. Scopes, in "Protein Purification: Micro to Macro," UCLA Symp. Mol. Cell. Biol., New Ser., Vol. 68, p. 1. Alan R. Liss, New York, 1987. lo R. K. Scopes, "Protein Purification, Principles and Practice," 2nd Ed. Springer-Verlag. New York, 1987. I

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METHODS IN ENZYMOLOGY. VOL 182

Copyright O 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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by dilute acid), the fractionation and classification of proteins on the basis of solubilities began to receive serious attention only in the middle of the last century. From those studies came a classification of proteins the nomenclature of which persists to this day with modifications in meaning. Thus, globulins were defined as proteins insoluble in water or dilute salt solutions, and albumins as soluble in such media although precipitable by very high concentrations of ammonium sulfate. In a modern sense most of the globulins are in fact globular proteins as determined by physicochemical methods that determine size and shape of molecules, but many of the water-soluble proteins such as hemoglobin and albumin are also globular. Beyond classifying proteins on the basis of solubilities, early biochemists felt compelled to provide a theory for this remarkable precipitating behavior. On the one hand, a great deal of attention was paid to the nature of the salts themselves; this is exemplified in the studies of Hofmeister, whose lyotropic series of electrolytes established an order of differential effects of various ions on solubility of proteins and other properties of biological systems. However, much of the proposed theory of precipitation also came from the then current knowledge of proteins. The theory was developed in an era in which proteins were considered to be colloids of undefined molecular nature with surface electrical charges that could be manipulated either to favor dispersion or to cause precipitation. Much of our current language and some of the concepts of this kind of protein chemistry reflect that historical development: salting in, salting out, isoelectric precipitation, zone of hydration, solvation, and hydrophilic and hydrophobic characters. Proteins were not considered to be in true solution, but rather in colloid dispersion stabilized by such forces as charge repulsion, interaction with solvent, or exclusion of solvent. Destabilization and precipitation consisted of interfering with or neutralizing the stabilizing forces. Eventually, proteins were shown to be molecules of definite molecular weight, size, shape, electrical properties, and amino acid composition, capable of existing in true solution as compared to colloid dispersion. Concurrently, as metabolic pathways were being unraveled and defined, hundreds of new enzymes from a variety of biological sources had to be isolated and purified so that their molecular and catalytic properties could be determined. The art and science of protein fractionation by differential solubility and precipitability reached a pinnacle, and a wide variety of precipitation methods were discovered empirically. Although some adsorbent~such as alumina and calcium phosphate gels were used, most of the methods employed several steps of ammonium sulfate precipitation. The development of column chromatography and its adaptation to affinity chromatography began in earnest in the late 1950s and soon dominated

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the field. From that time on, precipitation methods were supplanted in importance as a means of obtaining proteins in a final state of purity, but found their present place as an adjunct for concentration of proteins and for one or two specific steps on the way to using chromatography. In this more limited capacity, precipitation methods are yet highly significant, and examination of purification procedures for many proteins shows that perhaps 80% include at least one step of precipitation with ammonium sulfate. Theory and Limits of Theory

In a discussion of precipitation of proteins it is customary to begin with the several theories of precipitation and then proceed to a very prosaic description of general methods. One soon learns that the theory is useful only in a broad sense, and that specific procedures applicable to all proteins or even to classes of proteins cannot be given because the subject is almost embarrassingly empirical. Having said this, knowledge of the theory does provide one with a general understanding of what one is doing, and importantly allows an investigator to mark out limits of use of the various agents and conditions being employed. It also sets out precautions to safeguard the structural integrity of particular kinds of proteins and any associated biological activities. The following discussion encompasses theoretical considerations presented in several review^.^^^^^^^ The major forces within a polypeptide chain and between chains that drive a protein to a stable conformation at a state of minimum energy are ion-ion, ion-dipole (including hydrogen bonds),dipole-dipole, and hydrophobic interactions. The structures of proteins in a tissue, whether in solution or associated with insoluble fibers, are influenced by the nature of the aqueous medium as determined by the kinds of other molecules and ions present. Water itself will compete with dipolar interactions in the protein, as may other dipolar substances in the medium. Ions in the medium may competitively disrupt ionic interactions in the polypeptide. Hydrophobic substances in the medium (called lipophilic by organic chemists) may seek out hydrophobic centers in the protein, thus reorganizing the water around the protein. The other macromolecules present may enter into similar interactions with the protein. When the composition of the medium remains constant, at equilibrium the protein arrives at a stable structure that derives from the sum of all the interactions modified by occurrence of covalent cross-links such as disulfide bonds, if any. In many proteins, both of the globular and fibrous (usually coiled coil) kinds, the "interior" of the structure is shielded from the aqueous medium by a concentration of hydrophobic amino acid resi-

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dues. The "exterior" or "surface" of the structure interacts with the medium through side chains of polar and ionic amino acid residues clustered or appropriately distributed on the polypeptide. Small patches of hydrophobic groups may also be located on the surface and cause local exclusion-organization of water; however, the bulk of water will hydrate the remainder of the surface through formation of ion-dipole and dipole interactions with ionic and polar groups of the protein. In all cases, exposed parts of the polypeptide backbone can interact with water, so that even a protein such as elastin, made up primarily of hydrophobic residues, is highly hydrated. Perturbations of the solvent-protein interactions can cause transitions in the structure by disrupting the "old" interactions and promoting formation of new ones. If the new set of interactions seeks its own level of minimum energy in which the "interior" is no longer shielded from solvent, it will probably be expressed as a transition to a condition in which the interior is now unfolded. Perturbations that can cause various conformational transitions include the following. A rise in temperature can weaken the strength of dipolar interactions such as hydrogen bonds and can favor formation of hydrophobic interactions. A decrease in temperature could cause the reverse. Quantitative aspects of the resultant transitions in structure depend on the total numbers of specific kinds of interactions and variation in energies among individual interactions of the same kind. A decrease of pH can cause protonation of certain groups on the surface of the protein and an increase of pH can cause dissociation of protons. A specific pH can be achieved at which a given protein exhibits no net charge because the number of positively and negatively charged groups are equal, that is, the isoelectric point has been reached. At that pH the surface of the protein will be least solvated (hydrated), and the protein, in nondenatured form, usually has minimum solubility. It is to be emphasized that minimum solubility and insolubility are not necessarily equivalent, and some other perturbation of the solvent might have to be introduced to cause the protein to precipitate. The other perturbation could be addition of a large amount of a salt like NaC1, Na2S04,or (NH&S04, or of a water-miscible organic solvent like ethanol or acetone. Salts in solution at low ionic strength relative to that of isotonic saline ( p = 0.15) may represent a perturbation that can cause certain proteins to precipitate from solution. Such a protein probably was originally maintained in solution in the tissue because it was "salted in" at the ionic strength of the medium. Once a protein of this kind is precipitated it can once again be salted in by solutions of suitable ionic strength. On the other hand, salts present in very high concentrations with ionic

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strength much greater than that of tissue media will cause the precipitation of many proteins. Precipitation occurs by neutralization of surface charges by the salt, by reducing the chemical activity of the protein, and by diminishing the effective concentration of the water. This is called "salting out" of proteins. The concentration of any salt necessary to cause precipitation of a particular protein is related to the number and distribution of charges and of nonionic polar groups on the surface of the protein, and to the number and distribution of hydrophobic residues exposed and rendered dominant as the charges are neutralized. Of course, the size and shape of the protein contribute to the relative ease of precipitability. All these factors form the basis of salt fractionation of protein mixtures. The inclusion of water-miscible solvents in the medium in which the protein is dissolved represents a considerable perturbation. Organic solvents used most commonly for precipitation of proteins are methanol, ethanol, butanol, and acetone, all of which have both hydrophobic (lipophilic) and polar domains. In the series of saturated, aliphatic hydrocarbon alcohols, the use of any member is limited by its miscibility with water and its dielectric constant, or its ability to polarize the water in which it dissolves. Through their polar groups, solvents of this type interact with polar groups on the protein in competition with water. In addition, the hydrophobic groups may disrupt the intramolecular hydrophobic interactions stabilizing the protein structure and force the formation of new hydrophobic interactions. Finally, a large volume of organic solvent reduces the effective concentration of the water, leaving less for hydration of the protein. All of these factors cause a decrease in solubility of the protein with resultant precipitation. The concentration of any given organic solvent necessary to effect precipitation of any given protein is related to many of the same intrinsic properties of the protein that regulate precipitation by salts. Once a protein has been purified, its solubility in a specific medium can be expressed in terms of the equation" log S = /3 - K,(I/2) in which S is the solubility of protein in grams per kilogram of water, I is the ionic strength in moles per kilogram of water, and /3 and K , are constants. K , serves as a measure of the slope of the solubility extrapolated to zero ionic strength and P is the logarithm of the solubility at that point. Although this is an important physical relationship, it has very little use in the practical matter of isolating proteins from a biological mixture. R. Czok and T. Bucher, Adv. Protein Chem. 15, 315 (1960).

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Methods

A number of different neutral or slightly acidic salts have been used to solubilize, precipitate, or fractionate proteins in a differential manner. These include NaCl (saturation at 20°, 5.33 M), Na2S04(saturation at 20°, 1.88 M) and NH4(S0J2 (saturation at temperatures ranging from 0 to 25", about 4 M; see footnote to Table I). MgS04 also has been used, KC1, CaC12, and NaCl in varying concentrations and pH have been used to both solubilize and precipitate certain structural proteins such as the myosins,12 collagens,I3 fibrinogens, and keratins. It is not coincidental that these are mostly fibrous coiled coil proteins with rod characteristics; and many have repeating runs of certain specific amino acids or certain types of amino acids, so that often they exhibit a periodic distribution of charged groups on the outside of the coiled coil assembly and hydrophoSaturated solutions of sodium and potassium bic runs on the inside.14~15 phosphates at neutral or near-neutral pH have been used for fractional precipitation of proteins; solutions of potassium phosphates, depending on pH, can be between 3 and 3.7 M with respect to phosphate, but used in such high concentrations they exhibit densities and viscosities considerably greater than the proteins they precipitate, so that sedimentation by centrifugation is difficult. Various potassium and sodium phosphates and sodium acetate can, in selected cases, be used to make an initial separation of a particular protein from a mixture, or to fractionate an already collected protein aggregate. For example, low ionic strength buffers at slightly acidic pH can separate out the C1 complex of complement from serum. For those interested in these procedures, tables that guide one to the preparation of such buffers are a ~ a i l a b l e . ~ As already indicated, ammonium sulfate is the precipitant used most frequently in the salting out of proteins. Its major advantages are (1) at saturation, it is of sufficiently high molarity that it causes the precipitation of most proteins; (2) it does not have a large heat of solution, so that the heat generated is easily dissipated; (3) even its saturated solution (4.04 M at 20") has a density (1.235 g ~ m - that ~ ) is not so large that it interferes with the sedimentation of most precipitated proteins by centrifugation; (4) its concentrated solutions prevent or limit most bacterial growth; and (5) in solution it protects most proteins from denaturation. Because of this last property, one often preserves purified proteins as suspensions in S. S. Margossian and S. Lowy, this series, Vol. 85 [7]. E. J. Miller and R. K. Rhodes, this series, Vol. 82 [2]. l4 A. D. McLachlan and M. Stewart, J. Mol. Biol. 98, 293 (1975). l5 A. D. McLachlan and J . Karn, J. Mol. Biol. 164, 605 (1983). IZ

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TABLE I FINALCONCENTRATION OF AMMONIUM SULFATE: PERCENTAGE SATURATION AT Ooa

Percentage saturation at O0 Initial concentration of ammonium sulfate (percentage saturation at 0") 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 a

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Solid ammonium sulfate (grams) to be added to 1 liter of solution 106 79 53 26 0

134 108 81 54 27

164 137 109 82 55

194 166 139 111 83

226 197 169 141 113

258 229 200 172 143

291 262 233 204 175

326 296 266 237 207

361 331 301 271 241

398 368 337 306 276

436 405 374 343 312

476 444 412 381 349

516 484 452 420 387

559 526 493 460 427

603 570 536 503 469

650 615 581 547 512

697 662 627 592 557

0

27 0

56 28 0

84 56 28 0

115 86 57 29 0

146 117 87 58 29

179 148 118 89 59

211 181 151 120 90

245 214 184 153 123

280 249 218 187 156

317 285 254 222 190

355 323 291 258 226

395 362 329 296 263

436 402 369 335 302

478 445 410 376 342

522 488 453 418 383

0

30 0

60 30 0

92 61 31 0

125 93 62 31 0

159 127 95 63 32

194 161 129 97 65

230 197 164 132 99

268 235 201 168 134

308 273 239 205 171

348 313 279 244 209

0

32 0

66 33 0

101 67 34 0

137 103 68 34 0

174 139 105 70 35 0

Adapted from "Data for Biochemical Research" (R. M. C. Dawson, D. C. Elliott, W. H. Elliott, and K. M. Jones, eds.), 2nd Ed. Oxford Univ. Press, London, 1969. Similar tables prepared for 25" and 20" are found in Ref. 2 (p. 76) and 10 (p. 303), respectively. Saturated solutions of ammonium sulfate are 3.90,4.04, and 4.10 M a t 0°, 20°, and 25", respectively. Appendix A of Ref. 10 also presents a table for the liquid addition of miscible organic solvents or of a saturated solution of ammonium sulfate to raise an initial concentration (%, vlv) to a given higher concentration.

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concentrated solutions of ammonium sulfate. An additional feature of ammonium sulfate solutions is their widespread use in crystallization of protein^.^,'^.'^ A limitation in the use of ammonium sulfate for fine fractionation of a protein is that, in going from one step to the next as one increases the concentration of the salt, the purification achieved is usually only 2 to 5 times over the previous fraction. One should also note that when used with tissue homogenates and extracts, ammonium sulfate up to a concentration of 25% saturation often yields a first cut that contains particulate matter such as ribosomes, membrane fragments, large aggregates of protein, and even denatured proteins.

Bulk Precipitation: Concentrating Protein The purpose of this procedure is to obtain all or most of the protein in a tissue extract or homogenate, or in a bacterial extract or growth medium, in a bulk form that can then serve as the starting material for a fine fractionation. The homogenate or extract should first be clarified by centrifugation. It should be in a buffer at neutral pH unless reasons exist to conduct the precipitation at another pH; in most cases the buffer will have ionic strength close to physiological. (Homogenates made from liver and muscle may tend to become acid if sufficient glycogenolysis-glycolysis occurs, and should be neutralized before use.) Precipitation is usually performed at 0-4", and all solutions should be precooled to that temperature range. From Table I, the weight of solid ammonium sulfate needed to bring the volume of starting material to 80-85% saturation should be determined. Using ammonium sulfate of high purity, usually designated by the manufacturer as suitable for enzyme fractionation, the required amount of salt should be weighed out. The vessel containing the starting material should be equipped with a thermometer and a glass electrode for monitoring pH, and a suitable magnetic or motor-driven stirrer. Ammonium sulfate is added in increments with constant stirring and with adjustment of pH by addition of 1 N NH40H, as required. Each addition of salt is made only after the previously added amount has completely dissolved. When all of the salt has been added, the mixture is stirred for another 15 to 30 min to allow equilibration of the solvent and protein. The mixture is then centrifuged at about lo5 g . mint8(i.e., 10,000 g for 10 min or 3000 g for 30 A. McPherson, this series, Vol. 114 [5]. " A. McPherson, this series, Vol. 114 [7]. l8 See ref. 10, p. 53. l6

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min) in a precooled centrifuge at 0-4". The supernatant fluid is either decanted or drawn off by suction and discarded. The wet precipitate may either be stored in a deep freeze or used immediately for fractionation. In either case one may want first to determine the total amount of protein collected and the biological activity, if any. Notes and Precautions 1. Since all conditions must be capable of reproduction when the method is used subsequently by the experimenter or others, they should be appropriately monitored and recorded. Conditions should be kept as constant as possible. The particular variables to be considered are volume, protein concentration, pH value, temperature, speed of centrifugation, and time used for each step. 2. Any buffers and subsidiary agents used should be as free as possible of trace metals that could catalyze oxidation or binding of susceptible groups, e.g., sulfhydryl groups. The experimenter has the option of using chelating agents such as EDTA if these are compatible with the native conformation and activity of the protein of interest. 3. The starting material may contain protease inhibitors if these had been added during its preparation from the tissue or microorganisms. If the inhibitors are small molecules they usually will be soluble in the nearsaturated ammonium sulfate solution. Some protease inhibitors are proteins and may be precipitated with the bulk protein. 4. If nucleic acids might interfere, they can be removed from the starting extracts by suitable addition of protamine, streptomycin, or MnC12.The resulting nucleic acid complexes can be removed by centrifugation. In rare cases experimenters have the option of using appropriate nucleases. 5. In special cases the experimenter can use appropriate amylases or other carbohydrases to remove glycogens, starches, or any polysaccharides that may be present. Otherwise, these might precipitate with the protein when near-saturated ammonium sulfate is used. 6. Stirring during the solubilization of ammonium sulfate should be regulated to keep frothing to a minimum. Frothing can promote air oxidation of the protein (e.g., of sulfhydryl groups) and surface denaturation.

Fractionation with Ammonium Sulfate

This can be performed by one of four different procedures. First, the bulk precipitate may be extracted successively with solutions of decreasing concentration of ammonium sulfate. Second, the dissolved bulk protein may be precipitated in steps by successive addition of solid ammo-

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nium sulfate and the precipitate removed at each step. Third, a specific series of varying ammonium sulfate concentrations can be achieved by dialysis of precise and known volumes of 3.9 M ammonium sulfate at O" against a known volume of the dissolved bulk protein contained in a dialysis sac. While this method may give sharper cuts than others, it is not used frequently. Fourth, the starting material in a known volume can be successively diluted with precise increasing volumes of 3.9 M ammonium sulfate solution. After each dilution, any precipitate that forms is centrifuged and saved. This method has the advantage that a solution of the salt is being added, but has the disadvantage that greater and greater dilution of the protein occurs as one increases the volume of added reagent. Thus, to achieve 80% saturation, one must add 4 vol of 3.9 M salt to 1 vol of protein solution. Fractionation of Bulk Protein by Successive Extraction with Ammonium Sulfate Solutions of Varying Concentration. This is a procedure not used frequently, but represents an option in purification. The starting material is the protein precipitated at 80-85% saturated ammonium sulfate. A solution of 3.9 M highly purified ammonium sulfate is made at 0'. The pH of the solution is adjusted to 7 with NH40H. From this is prepared a subset of solutions representing saturations of 80, 65, 55, 45, 35, 25, and 15%. Each of these solutions will be used in a volume that is onetenth that of the original volume from which the bulk precipitate was made, although this is strictly an empirical matter. Starting with the highest concentration, each extracting solution is added, and the mixture stirred gently, but thoroughly, for 15 to 30 min with minimum frothing. After each extraction, the mixture is centrifuged, and the supernatant decanted. A final extraction is made with appropriate dilute buffer. The residual protein, if any, may be saved for analysis. All saved materials are then assayed for protein content and biological activity. Fractionation of Bulk Protein or Crude Extract by Using Solid Ammonium Sulfate. Either bulk protein, collected as already described and dissolved in a suitable buffer, or a crude extract of tissue can be the starting material. The volume is an empirical matter, but the protein concentration should usually be between 5 and 30 mglml. Such relatively high protein concentrations favor stability of the protein and minimize denaturation. However, the concentration of protein is a compromise between sharpness of the "cut" being made and yield; concentrated solutions favor yield, but diminish sharpness of the fraction, whereas dilute solutions may not allow full precipitation of the protein being sought since the protein is almost always somewhat soluble, and the amount remaining

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will be proportional to the volume. Thus, only trial and error may indicate the optimum protein concentration to use. If the starting solution or protein is turbid, it may be clarified by centrifugation, or the suspended material may be included in the first fraction and removed from it by centrifugation at that point. Successive fractions may be made by addition, at O" with stirring, of increments of solid ammonium sulfate. The amount of ammonium sulfate to be added can be obtained from Table I. The pH may be monitored and sufficient 1 N NH40H added to keep the pH constant. Most fractionations can be carried out at pH 7, but the experimenter may want to choose another pH value depending on the protein of interest. When all of the ammonium sulfate is dissolved, the mixture is allowed to stand for 15 min and then centrifuged at O". The supernatant fluid is decanted and used as the starting material for the next fractionation. Fractions can be successively precipitated at saturations of 25,40, 50, 60, 80, and 100%. The individual precipitated fraction should be drained, and the side of the vessel wiped free of adhering solution. The material should be dissolved in a small amount of appropriate buffer. Removal of ammonium sulfate, if desired, can be accomplished by gel filtration or dialysis. For dialysis, the material should be transferred quantitatively and dialyzed at 0-4' against several changes of the same buffer, stirring the dialyzing medium constantly. At the end of the dialysis period, the sac should be emptied, the volume noted and adjusted to a standard volume, and suitable assays performed for protein content and biological activity if any. When analyses are completed for all fractions, a table should be constructed, noting the total amount of protein in the fraction, the total amount of activity (if any), and the percentage yields of protein and activity compared to that with which the fractionation was started. Specific activities (total activity in a fraction divided by total protein in the fraction) can be calculated. One may at this point decide whether finer fractionations with ammonium sulfate should be pursued or whether one should go to the next purification procedure. Notes and Precautions 1. If at any point one wants to determine the exact concentration of ammonium sulfate, e.g., to define a cut precisely, one can take an appropriate aliquot and directly assay it for ammonia by distillation in a microKjeldahl apparatus, collecting ammonia in a boric acid mixture, and titrating the ammonia-borate complex with sodium hydroxide. A standard can be prepared from pure ammonium sulfate. It is also possible to adapt the ninhydrin colorimetric method for measuring the amount of ammonium sulfate, using a standard curve made from pure ammonium sulfate. Some

296

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investigators still use the Nessler reagent directly as a colorimetric procedure, a standard curve being constructed at the time of analysis. 2. Methods used for protein determination should be compatible with the amounts of ammonium sulfate present, e.g., even fairly low concenAdvantages trations of salts interfere with the method of Lowry et al.19.20 and disadvantages of different protein assays are given in [ 6 ] . 3. All precautions (control of temperature, pH, conditions of stirring, etc.) noted in the section on bulk precipitation of protein should be observed. 4. All dialysis steps involving removal of ammonium sulfate should be in sacs with enough free volume to accommodate expansion due to osmotic effects. Use of Organic Solvents

Organic solvents, as already indicated, cause precipitation of proteins largely by changing the solvation of the protein with water. The organic solvents used are acetone, ethanol, methanol, and butanol. All are miscible with water, but yield a significant heat of solution. All suffer a volume reduction of about 4 to 5% when mixed with water; this may be ignored for practical purposes. All have a tendency to denature proteins, especially at temperatures above 0". In the use of ethanol for complete fractionation of a biological fluid (e.g., serum) into its individual proteins or classes of proteins, advantage is also taken of the minimum solubility that a protein may exhibit at the isoelectric point. Addition of ethanol (or other organic solvent) to a protein at that pH further diminishes the solubility and causes precipitation. Generally, the farther away from the isoelectric point, the more solvent is required for precipitation of the protein. However, such fractionation to obtain all protein components is rarely the process engaged in by enzymologists. In most cases, one is seeking to separate a single protein, and this process can be carried out at a single pH, and in initial studies one usually favors neutral pH. The protein concentration can be in the range of 5 to 30 mglml, although again this is empiricd. The buffer used can be in the physiological ionic strength range (0.15) and at neutral pH. Because of the utmost importance of controlling the temperature, the reaction is carried out in a vessel that can conduct heat rapidly, stainless steel being used in some commercial operations, but glass in the experi0. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).

G . L. Peterson, Anal. Biochem. 100, 201 (1979).

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297

PRECIPITATION TECHNIQUES -

-

-

-

-

-

-

mental laboratory. Plastic vessels are unsuitable. The temperature is monitored continually with addition of solvent. Mixing can be done by magnetic or motor-driven stirrer. In all cases the stirring should be performed with minimal aeration. Addition of solvent should be dropwise. The solvent temperature should be held below O", sometimes cooled in a dry ice bath. The precipitating mixture should be held at a constant temperature somewhat below 0" even if ice crystals form; the latter will dissolve as organic solvent is added. When addition of solvent is complete, stirring may be discontinued, and the mixture allowed to stand for at least 15 min to allow equilibration. During that time, temperature again must be maintained constant. The material is then sedimented in a centrifuge precooled to the desired temperature; centrifugation is at about 5000 g for 15 min. Most precipitates will sediment rapidly because of the low density of the solvent. Of course, if lipid remains attached to the protein even in the presence of solvent, the time for sedimentation may be longer. The supernatant is decanted carefully, and the sides of the vessel carefully touched with suitable paper to remove excess fluid. The pellet can be frozen at -70" until further use or, if further fractionation by other means is to be performed immediately, the pellet can be dissolved in suitable buffer to give the protein concentration desired. In dissolving the precipitate, the buffer is first added in minimum amount, and stining can be conducted with a glass rod of appropriate size. Precipitates are more readily dissolved or suspended in small volumes; addition of the total amount of buffer at once may make solution difficult. For this process, to minimize denaturation, both pellet and buffer should be at 0". After solution, one should then proceed immediately to the next step of fractionation. In some cases it may be desirable to remove residual solvent from the aqueous solution before proceeding to the next step of fractionation. This can be done by one of several processes, including evaporation under vacuum in a conventional lyophilizing apparatus or by use of a SpeedVac. In the last stages of solvent removal, the temperature may be raised above Vj but this must be done with great caution. Another method that can be used is gel filtration. Dialysis in most cases should not be used because it requires relatively long times of exposure to solvent. In all cases the solvent should be removed without drying the protein, i.e., the protein should be obtained in a hydrated form. To emphasize the extreme care required when organic solvents are ~ ' which he summarized used, we quote from a chapter by K a ~ f m a n , in precautions as follows: 21

S . Kaufman, this series, Vol. 22 [21].

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1. 2. 3. 4.

5.

6. 7. 8. 9.

PROCEDURES: BULK

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Temperature below 0" at all times Adequate rate of stirring during ethanol addition Ethanol to be added is precooled to very low temperature Cooling bath of sufficient volume to ensure large capacity for rapid heat transfer Discrete drop addition of ethanol to minimize local high concentration of ethanol Careful removal of the supernatant solution from the precipitated protein to minimize contamination with excess ethanol Solution of the precipitated protein in sufficient volume of buffer to avoid high final ethanol concentration Equilibration period after the ethanol has been added Temperature during centrifugation is the same as that during equilibration period

The last two precautions are more critical for maximum fractionation than for maximum recovery of activity. Use of Sodium Chloride and Other Univalent-Univalent Salts In the early days of protein chemistry, sodium chloride was used as a precipitant and a solvent for a number of different proteins. However, the lesser ionic strength of saturated NaCl solutions as compared with saturated ammonium sulfate limits the applications of this salt. Nevertheless, certain fibrous proteins, particularly some of those with coiled coil structures, are extractable with dilute acidic solutions that can then be fractionated with sodium chloride or potassium chloride. Such molecules include fibrinogen, un-cross-linked keratin (epidermin), myosin, tropomyosin, and the collagens. Details for the use of KC1 in the preparation of myosin12and of NaCl in the preparation of different types of collagens13are given in other volumes in this series. Heat and pH Denaturative Precipitation as Adjunctive Methods in Purification of Enzymes Because of the great diversity of structure among proteins it follows that enzymes will exhibit a wide range of stabilities to perturbations in heat and pH. Thus, some proteins such as adenylate kinase and trypsin and certain enzymes of thermophilic organisms are relatively heat stable in comparison with the majority of other enzymes. Again, while most enzymes and other proteins undergo denaturative changes in conforma-

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tion when exposed to pH values below pH 5 and above pH 10, some are stable and even biologically active outside that range of pH. Some proteins have isoelectric points in the alkaline range (e.g., cytochrome c and certain lysozymes), while others have isoelectric points in the acid range (e.g., pepsin, albumin, orosomucoid). In seeking to purify a specific protein from a mixture of different proteins, the experimenter therefore has the option to use heat and pH to denature and precipitate the unwanted proteins if the conditions used do not affect the desired protein. Such a step can be a valuable subtractive adjunct in the purification procedure. A classic historical example of the use of pH to effect a gross purification was the preparation of a so-called "pH 5 fraction" of tissue homogenates to obtain a soluble extract containing the enzymes of amino acid activation and transfer to tRNA. Use of Heat Denaturation. If the protein being sought has biological activity, a preliminary test of its heat stability should be made. Small quantities of the extract containing the protein should be distributed into a series of centrifuge tubes. Suitable water baths should be used that allow adjustment of temperature at intervals of 5". Tubes containing the extract should be submerged in the bath, care being taken to mix adequately during the procedure. At the end of a specified time, the tube is removed and rapidly submerged in an ice bath. It is then centrifuged in the cold and the supernatant fluid is decanted and assayed for activity. Protein content may also be determined to evaluate whether the step is really productive. The activity is compared to that of the original extract. Sometimes experiments will be necessary to determine the best time of incubation at a given temperature. From these experiments the appropriate temperature and time of incubation can be determined to allow pretreatment of the total extract before fractionation by other means is undertaken. If preliminary experiments have determined that a heat step is worthwhile, the total extract may then be treated. For this purpose the extract is placed in a suitable large Erlenmeyer flask. Two water baths are prepared. One is kept constant at 90" and the second is held at the predetermined temperature required. With a thermometer held in the extract, the flask is placed in the 90"bath and swirled rapidly until the contents show a temperature about 5" below the temperature desired. The flask is then immediately transferred to the second bath, and the contents brought to the temperature of the bath and held there for the predetermined time. The flask is then rapidly submerged in an ice bath. The mixture is then transferred to suitable centrifuge tubes and sedimented at 0" at about 10,000 g for 10 min. The supernatant fluid is decanted and assayed for activity and protein contents. The material is then kept cold until used for further fractionation.

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Notes and Precautions

I . If proteases are present in the extract, they may digest the protein of interest even during short intervals of heating, especially since some of the proteases themselves may be relatively heat stable. Accordingly, the experimenter may choose to carry out the heat step in the presence of ammonium sulfate since that (and other) salts may inhibit or diminish some protease activities. If that is done, a somewhat higher temperature may be required for denaturation of the unwanted proteins. 2. k specific protection of the enzyme of interest may be achieved by conducting the heat process in the presence of a specific substrate. 3. The pH of the extract and the composition of the buffers in the extract must be known and rigorously controlled, because temperature and rate of denaturation are affected by such possible variables. These precautions are necessary so that subsequent use of the procedure may give reproducible results. Use of pH Denaturation. To achieve specific pH values, the experimenter should avoid the use of strong acids or bases. Tris and acetic acid can be used in the pH range from 5 to 8.5 and lactic acid for pH values below 5. Diethanolamine can be used on the alkaline side to pH 9, and sodium carbonate to achieve a pH of 10.5. A preliminary experiment involving the effect of pH on biological activity of the desired protein should be performed. Aliquots of the initial extract can be placed in suitable centrifuge tubes and submerged in an ice bath. Each tube should be titrated with one of the recommended acids or bases to the specific pH desired. The mixture is allowed to equilibrate for 10 min in the ice bath and then brought to neutral pH to maximize precipitation of the denatured protein. The tubes should then be centrifuged at 0",and the supernatant fluids decanted. These can be assayed for activity and measured for protein content. From these experiments, one can determine whether a step of pH precipitation would be useful in purification of the desired protein. There should be no difficulty in scaling up the method for use with the total extract.

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[231 Precipitation of Proteins with Polyethylene Glycol By KENNETHC. INGHAM

This chapter updates a chapter on protein precipitation using polyethylene glycol that appeared in a previous volume in the series.' The use of nonionic, water-soluble polymers, in particular polyethylene glycol (PEG), for fractional precipitation of proteins was introduced in 1964 by Polson et a1.l Papers appearing over the next two decades provided an improved understanding of the molecular basis of the protein-precipitating action of PEG and additional documentation of the unique advantages of this polymer over other reagents used for this purpose. Although much of the literature on this subject deals with purification of proteins from blood p l a ~ m athe , ~ approach is applicable to any complex mixture. The principles involved have been clarified by studies with purified proteins, and the purpose of this chapter is to summarize briefly these principles with emphasis on practical information enabling the reader to assess the potential applicability of this technique to specific separation problems. Advantages of Polyethylene Glycol

The advantages of PEG as a fractional precipitating agent stem primarily from its well-known benign chemical properties. Unlike ethanol and other organic precipitating agents, PEG has little tendency to denature or otherwise interact with proteins even when present at high concentrations and elevated temperatures. Careful experiments designed to test this principle revealed that PEG 4004 at concentrations up to 30% (wlv) had no detectable effect on the circular dichroic spectrum or thermal denaturaSubsequent studies confirmed this tion temperature of ribonuclea~e.~ result for ribonuclease but suggested that PEG has a destabilizing effect with some proteins at elevated t e m p e r a t ~ r eThis . ~ should be of no con-

' K. C. Ingham, this series, Vol. 104, p. 351. A. Polson, G . M. Potgieter, J. F. Largier, G . E. F. Mears, and F. J. Joubert. Biochim. Biophys. Acta 82, 463 (1964). ". L. Hao, K. C. Ingham, and M. Wickerhauser, in "Methods of Protein Fractionation" (J. M. Curling, ed.), p. 57. Academic Press, New York, 1980. PEG, Poly(ethy1ene glycol), poly(ethy1ene oxide), polyoxyethylene. Chemical formula: HOCH2CH2(CH2CH20),CH2CH20H. PEG 400 and PEG 4000 signify heterogeneous mixtures having nominal average molecular weights of 400 and 4000, respectively. D. H. Atha and K . C. Ingham, J. Biol. Chem. 256, 12108 (1981). L. L. Lee and J. C. Lee, Biochemistry 26, 7813 (1987).

METHODS IN ENZYMOLOGY, VOL. 182

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tern when using the polymer in protein purification where elevated temperatures are seldom employed. The low heat of solution and the relative insensitivity of PEG precipitation curves to minor variations in temperature eliminate the need for controlling temperature during reagent addition. Another advantage of PEG over ethanol or ammonium sulfate is the shorter time required for the precipitated proteins to equilibrate and achieve a physical state suitable for large-scale centrifugation. The advantages of PEG in facilitating the growth of protein crystals is well documented.'

Mechanism of Action

Careful measurements with a variety of purified proteins indicate that their solubilities decrease exponentially with increasing concentration of PEG according to Eq. (I), log S

=

log So - PC

(1)

where S is the solubility in the presence of PEG at concentration C (%, W/V)and So is the apparent intrinsic solubility obtained by extrapolation to zero PEG.SPlots of log S vs [PEG] exhibit striking linearity over a wide range of protein concentration, the slope for a given protein being relatively insensitive to pH and ionic strength, but markedly dependent on the size of the PEG up to about 6000 Da. The slopes also tend to increase with increasing size of the protein, reinforcing the popular notion of a steric exclusion mechanism whereby proteins are concentrated in the extrapolyrner space, eventually exceeding their solubility limit under the given solution conditions. Although a quantitative explanation of this behavior is yet to come, it is clear that, in the absence of specific interactions, the sequence of precipitation of several proteins in a mixture will depend primarily on the ratios of their initial concentrations relative to their respective solubilities in the absence of PEG. Thus, even though larger proteins have steeper slopes, a large protein initially present at high concentration could precipitate later than a small one present at low concentration if the intrinsic solubility of the latter is much less than that of the former. Manipulation of the solution conditions is expected to improve the separation of a given pair of proteins to the extent that their intrinsic solubilities diverge. Selection of PEG

Most workers use material with a nominal average molecular weight in the 4000-6000 range. Polymers larger than this offer no advantage, since

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their solutions are more viscous and the precipitation curves are not much different from those obtained with PEG 6000.235Decreasing the molecular weight below 4000 spreads the precipitation of a mixture over a broader range of PEG concentrations. The improved resolution that might be thus anticipated is partially offset by the shallower slopes obtained for individual proteins. Nevertheless, Honig and Kula8 found the degree of purification of y-glucosidase from yeast extract to be about 2-fold greater with PEG 400 than with PEG 4000 or 6000. That PEG 400 is a liquid at room temperature whose solutions are substantially less viscous than those of the higher polymers, coupled with the potentially greater ease of removing it by molecular sieve methods, indicates a need for further comparisons. Analytical Precipitation Curve

The following simple experiment is designed to quickly overcome ignorance about the amount of PEG required to precipitate a given protein(~)from a complex mixture. The scale of this experiment is dictated by the sensitivity of the assay employed; the availability of a radiolabeled tracer is a definite advantage. One dispenses a fixed amount (0.1-0.5 ml) of the mixture into a series of tubes (preferably in duplicate) to each of which is subsequently added an equal volume of buffer containing increasing amounts of PEG to produce a final concentration of 25-30% in the most concentrated tubes. It is important to buffer the PEG stock solutions to avoid PEG-induced changes in pH.599The increment in PEG concenfration is arbitrary, but 3% (wlv) is adequate for initial screening. The vigor with which one mixes these solutions depends on the extent to which the desired protein(s) can withstand mechanical stress; gentle agitation on a vortex mixer is one approach. After 0.5-1.0 hr of incubation at room temperature or on ice, the samples are centrifuged and the percentage of the desired activity remaining in the supernatant liquid is determined. Inspection of the resulting "analytical precipitation curve" provides an estimate of the maximum concentration of PEG that can be added at one time without precipitating the protein of interest as well as the minimum concentration required to bring it out of solution, parameters that can then be more precisely defined with a second experiment that focuses on the relevant concentration range. With luck, the curve will fall either far to the left or far to the right on the PEG axis, defining a simple

' A. McPherson, Jr., J . Biol. Chem. 251, 6300 (1976). W. Honig and M.-R. Kula, Anal. Biochem. 72, 502 (1976). G. Eichele, D. Karabelnik, R. Halonbrenner, J. N. Jansonius, and P. Christen, J. Biol. Chem. 253, 5239 (1978).

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one-step method for removing a large portion of unwanted macromolecules and/or concentrating the desired activity prior to further processing by other methods. Otherwise, it may be necessary to obtain a "PEG cut" via two precipitation steps utilizing in turn the maximum and minimum concentration of PEG referred to above. It is always possible to manipulate the precipitation curve horizontally along the PEG axis by varying solution conditions. For screening purposes, it is expedient to choose a fixed concentration of PEG that causes approximately 50% precipitation of the desired protein under a given set of solution conditions in order to determine rapidly the extent to which altering conditions such as pH and ionic strength might enhance or inhibit precipitation. The most gratifying result of this approach would be to identify substances or conditions that selectively influence the solubility of the desired protein. This concept is further developed in the following section. Influence of Protein-Protein and Protein-Ligand Interactions

Studies with purified self-associating and heteroassociating proteins have shed some light on the role of protein-protein interactions on solubility in the presence of PEG.5.1@12 Based on the above-mentioned excluded volume considerations, one predicts that conditions that foster protein association should enhance precipitation because of the larger size of the complexes, wheres those that inhibit association would have the opposite effect. This is the case with almost all systems that have been examined. Of particular relevance in the present context was the observationlo that bovine liver glutamate dehydrogenase at 2.8 mg/ml in 0.2 M potassium phosphate at pH 7.0, conditions known to promote extensive self-association, was quantitatively precipitated by PEG 4000 at concentrations above 15% (w/v). Such precipitation was completely inhibited, M even at higher concentration of PEG, by the combined presence of NADH and GTP, cofactors known to reverse the self-association. Similar effects were observed with chymotrypsin, chyrnotrypsinogen, and p-lactoglobulin A, in which cases self-association was manipulated by varying pH and ionic strength, parameters likely to be less selective. Nonspecific electrostatic interactions between oppositely charged proteins such as albumin and lysozyme can also have profound effects on solubility that are most pronounced at low ionic strength at a pH between the pZ of each of the two proteins." While such interactions are frequently viewed as a lo I'

IZ

S. I. Miekka and K . C. Ingham, Arch. Biochem. Biophys. 191, 525 (1978). S. I. Miekka and K . C. Ingham, Arch. Biochem. Biophys. 203, 630 (1980). J . Wilf and A. P. Minton, Biochim. Biophys. Acta 670, 316 (1981).

E7 -

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nuisance, to be minimized by maintaining near-physiological ionic strength, the possibility of using them to advantage in a purification scheme should be kept in mind. A more specific type of heteroassociation of the type that might be exploited in purification is the functional interaction between human plasma fibronectin and denatured collagen, i.e., gelatin. The precipitation curve for the plasma protein in phosphate-buffered saline shifted from 11% PEG to less than 3% PEG upon addition of gelatin, which by itself was not precipitated by PEG under these conditions.I3 Since the complex between the two proteins is very stable, even at high ionic strength, it should be possible to precipitate fibronectin selectively from a complex mixture by this method. The contaminating gelatin could then be removed, e.g., by ion-exchange chromatography in the presence of urea. Although the advantage of this approach over affinity chromatography on immobilized gelatin is debatable, the example serves as an additional illustration of the application of bioaffinity principles to fractional precipitation. Any substance that interacts specifically with the desired protein has the potential to alter its solubility selectively and should thus be tested. Enzymes are ideal candidates for this approach, since they often interact with one or more effectors or cofactors, sometimes with large changes in the state of association. Methods of Removing PEG In many applications, PEG is used early in the purification scheme and is removed during subsequent chromatographic steps on ion-exchange or affinity columns to which PEG has no tendency to absorb. A word of caution is in order regarding the application of PEG-containing solutions to some exclusion columns, the performance of which can be significantly altered owing to osmotic effects of the polymer.I4 Alternative approaches to removing PEG include ultrafiltration15J6and salt-induced phase separation" as reviewed.I8 The latter method is particularly useful for solutions containing relatively high concentrations of PEG and has the potential advantage that the protein may be concentrated in a low-volume, salt-rich phase. For many research purposes it is probably unnecessary to remove l3

K. C. Ingham, S. A. Brew, and S. I. Miekka, Moi. Immunol. 20, 287 (1983).

" K. Hellsing. J . Chromatogr. 36, 170 (1968). l5 l6

I7

Is

T. F. Busby and K. C. Ingham, J . Biochem. Biophys. Methods 2, 191 (1980). K. C. Ingham, T. F. Busby, Y. Sahlestrom, and F. Castino, in "Ultrafiltration Membranes and Applications" (A. R. Cooper, ed.), p. 141. Plenum, New York, 1980. T. F. Busby and K. C. Ingham, Vox Sang. 39, 93 (1980). K. C. Ingham and T. F. Busby, Chem. Eng. Commun. 7 , 315 (1980).

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all traces of polymer from the final product, since it is optically transparent19 and helps prevent loss of protein by absorption of glass. Summary

Polyethylene glycol is a nondenaturing water-soluble polymer whose ability to precipitate protein from aqueous solution can be qualitatively understood in terms of an excluded volume mechanism. The increment in PEG concentration required to effect a given reduction in solubility is unique for a given protein-polymer pair, being insensitive to solution conditions and primarily dependent on the size of the protein and polymer. Selective manipulation of the solubility of specific proteins through control of their state of association or ligand environment can potentially remove some of the empiricism otherwise involved in fractional precipitation. Adequate methods for removing the polymer are available.

l9

The low level of UV absorbance frequently found in some PEG preparations is not inherent to the polymer but is due to a small amount of antioxidant sometimes added by the manufacturer.

Section VII Purification Procedures: Chromatographic Methods

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[241 Ion-Exchange Chromatography By EDWARD F. ROSSOMANDO

This chapter introduces ion-exchange chromatography (1EC) as a method for the purification of proteins and enzymes. While this chapter discusses the general principles for the operation of all types of IEC, the focus will be on the type called open-column IEC, as distinguished from another widely used type, high-performance liquid IEC (HPLC). This latter topic will be discussed elsewhere in this volume (see [32]). When proteins are to be purified by IEC, some problems arise because proteins have unique origins. Proteins are derived from biological sources and their extraction often requires the use of detergents and chaotropic salts for solubilization. One problem is that such solubilizers interfere with the operation of IEC. Another problem is that proteins in cells are compartmentalized and therefore separated from other proteins with proteolytic activity. Extraction results in disruption of these barriers with the exposure of proteins to proteases and the potential for degradation during the course of the purification. A final problem related to source is that, since proteins must be extracted from biological materials, the amount of protein available may be limited, such as when working with embryonic tissue. This necessitates modification of the IEC protocols normally used when larger quantities are available. Problems will also arise because proteins have unique properties. For example, proteins are ampholytes; that is, they contain both positive and negative charges; the former result from the ionization of lysine and arginine residues and the latter from aspartic and glutamic acid residues. Since the ionization of such groups is pH dependent, the net charge on a protein will be a function of the pH of its environment. Also, proteins are often "sticky," adhering to surfaces such as glass or the packing material in the IEC column. Although organic solvents, detergents, and salts can eliminate some adsorption, their addition may precipitate the proteins or, in the case of enzymes, destroy catalytic activity. Some suggestions for dealing with these problems will be discussed in this chapter. IEC is designed specifically for the separation of ionic or ionizable compounds. Similar to other types of liquid chromatography, IEC has both stationary (column packing) and mobile phases. It differs from other types of liquid chromatography in that the stationary phase carries ionizable functional groups, fixed by chemical bonding to the stationary phase. Of course, to satisfy requirements for electrical neutrality, these fixed METHODS IN ENZYMOLOGY. VOL 182

Copyright O 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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charges will carry a counterion of opposite sign. This counterion is not fixed and can be displaced. IEC is named on the basis of the sign of these displaceable charges. Thus, in anion IEC the fixed charges are positive and in cation IEC the fixed charges are negative. Principles of Operation

IEC involves two separate events. These are (1) the binding of the protein to the fixed charges and (2) the elution or displacement of the protein from the fixed charges. Because retention involves an electrostatic interaction between the fixed charges and those of the protein, binding involves replacement of the nonfixed ions by the protein. Elution, in turn, involves displacement of the protein from the fixed charges by a new counterion with a greater affinity for the fixed charges than the protein, and which then becomes the new, nonfixed ion. The ability of counterions (salts) to displace proteins bound to fixed charges is a function of the difference in affinities between the fixed charges and the nonfixed charges of both the protein and the salt. Affinities in turn are affected by several variables, including the magnitude of the net charge of the protein and the concentration and type of salt used for displacement. For additional details on the mechanisms underlying these processes see Refs. 1-3. Types of Ion Exchangers

Widely used solid-phase packings include cellulose, dextrans, agarose, and polystyrene. The exchange groups used include DEAE (diethylaminoethyl), a weak base, that will have a net positive charge when ionized and will therefore bind and exchange anions; and CM (carboxymethyl), a weak acid, with a negative charge when ionized that will bind and exchange cations. Another form of weak anion exchanger contains the PEI (polyethyleneimine) functional group. This material, most usually found on thin layer sheets, is useful for binding proteins at pH values above their pZ. The polystyrene matrix can be obtained with quaternary ammonium G. V. Samsonov, "Ion Exchange Sorption and Preparative Chromatography of Biologically Active Molecules," pp. 99, 105. Consultants Bureau, New York, 1986. C. F. Poole and S. S. Schuette?"Contemporary Practice of Chromatography." Elsevier, Amsterdam. 1984 pp. 304-312. P. J . Schoenmakers, "Optimization of Chromatographic Selectivity." Elsevier, Amsterdam, 1986.

1

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functional groups for strong base anion exchange or with sulfonic acid functional groups for strong acid cation exchange. Intermediate and weak ion-exchange materials are also available. Studies comparing the effectiveness of exchangers of various types have been reported. For example, in one study the effectiveness of a polystyrene resin was compared to cellulose for the purification of a glucoamylase from crude extracts of Aspergillus a ~ a r n o r iBoth . ~ an anionexchange polystyrene resin (Bio-Rad AGl-X4) and DEAE-cellulose were used. The results indicated that the use of the polystyrene resin solved one problem, namely, adsorption of the protein to cellulose. In addition, the resin produced a better separation of this enzyme from another, an amylase. Recently a newer form of anion-exchange system consisting of stacks of thin, noncompressible, microporous poly(viny1 chloride)-silica sheets has been introduced (FASTCHROM, Kontes, Life Sciences Products, Vineland, NJ). When these sheets are coated with PEI they yield a positively charged, hydrophilic surface for the separation of proteins and DNA.5 Preparation and Regeneration of Packing The column packings require washing, swelling, and conversion to the desired "form" prior to use. All these procedures can be performed prior to pouring the column. Washing and Swelling. To allow the column packing to reach a size equilibrium, prepare a slurry by mixing the packing with about 10 times its volume of the buffer to be used for loading the column. The slurry should be allowed to settle for about 1 hr. The top layer of clear solution, containing the "fines," should be decanted and the washing step repeated at least once. This treatment will result in better flow rates for the column. Changing Displaceable Counterion. When obtained from the manufacturer, the fixed charges of the column packing will have associated with them a counterion. This counterion can be changed, a procedure that also should be performed prior to pouring the c ~ l u m nThis . ~ exchange can be accomplished by washing the packing in a salt solution containing the counterion of choice. However, all the packing materials have a "selectivity series" that might be likened to an affinity, and in making the displacement it is necessary to go "up" the selectivity series. For exarnple, the selectivity for a representative cation exchanger might be Rb+ > R. S. Bhella and I. Altosaar, Anal. Biochem. 140, 200 (1984).

' J . J. Piotrowski and M. H. Scholla, BioChromarography 3, 161 (1988). E. L. Johnson and R. Stevenson, "Basic Liquid Chromatography," p. 116. Varian Associates, Palo Alto, California, 1977.

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Cs+ > K + > NH4+ > Na+ > H + > Li+. Such packings are usually supplied in the H+ form. Thus, it is a simple matter to replace the H+ with an ion higher in the series by washing in a 1 M solution of that salt. Similar selectivity series exist for anion exchangers, and the manufacturer's literature should be consulted for this information. Size of Column: Bed Capacity. Although the length of the column is less important, the separation does depend on the amount of column packing available since if this is insufficient, material that should have been retained will flow through. Although the amount of material that the column can bind must eventually be determined by experiment, information provided by the manufacturer on the bed capacity can narrow the quest. The manufacturer provides this information in the form of milliequivalents per dry gram or milliequivalents per milliliter of resin bed. For cation exchangers, supplied in the hydrogen form, this would be milliequivalents of H + that can be exchanged while for anion exchangers supplied in the chloride form this would be milliequivalents of C1-. The columns used for IEC need not be large. For example, columns made from Pasteur pipets have been used for the purification of the peptides derived from proenkephalin.' Used for this purpose, the pipet should be plugged with a small amount of glass wool, the resin prepared, and introduced into the pipet. The volume of the resin in such a column would be on the order of 0.25 cm3. For such columns elution will be a simple matter of washing the column with about 1 ml of each of the eluents. Batch us Open Column. IEC need not be performed using a c o l ~ m n . ~ The alternative, batch IEC, is usually performed with the slurry of the stationary phase in a vessel such as a beaker. In one study, used for the separation of lactate dehydr~genase,~ the essential component of the batch system is the resin (Bio-Rad AG MP-l), which is added to a tube containing the enzyme (isozymal forms of human lactate dehydrogenase). After shaking and equilibration, the resin is separated from the buffer either by centrifugation, filtration, or a combination of the two. The authors note that the separation they obtained was less than desirable, which they attribute to the fact that the batch procedure is both a nonequilibrium and too rapid a process. However, sometimes the speed of the process can be an advantage such as when separations on a larger scale are required.

' S. P. Wilson, J. Neurosci. Methods 15, 155 (1985). D. Reichenberg, in "Ion Exchangers in Organic and Biochemistry" (C. Calmon and T. R. E. Kressman, eds.). Interscience, New York, 1957. M. P. Menon, S. Miller, and B. S. Taylor, J. Chromatogr. 378, 450 (1986).

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If the decision has been made not to use batch IEC, then the alternative is to use open-column IEC for the separation. The essential components of an open-column ion-exchange apparatus include the column, capped at both ends, and a reservoir for the eluants. Usually a pump is required to allow the eluents to be delivered at a constant rate; a detector, coupled to a recorder, to monitor the components in the eluent; and a fraction collector to allow for the recovery of the components. It is useful to have the fraction collector and the recorder coupled with an "event marker" such that each change of the collector is marked on the recorder. This will facilitate the correlation of detector events with the recovery of the components after separation. Optional accessories include a conductance cell to monitor "on line" the concentration of salt in the eluent, and computerized management of data obtained from the various monitors including the detector and fraction collector for graphing purposes. In open-column IEC, the columns are usually transparent and capped at both ends. They can be obtained from a number of suppliers or made from glass or plastic tubing. Each end should have an inlet and outlet port to which tubing is attached. The column should be mounted in a vertical position and, prior to being filled with the slurry, a supporting bed should be placed inside the column against the bottom cap. This bed, the purpose of which is to prevent the resin or cellulose from running through the outlet port at the bottom, can be a plastic mesh, a sintered glass disk, or glass wool. In filling the column, a slurry of the packing is transferred from the beaker in which it had been prepared to the supporting bed. The column packing need not fill the column although it is best that the volume of buffer above the packing be kept small to minimize mixing. The column can be operated (that is, eluted) by pumping the buffer either from the top down or from the bottom up. When operating a column from the bottom to the top, there will be no "head" or layer of buffer between the cap of the column and the packing, minimizing the mixing and dilution of incoming buffers with those already present. Practical Information on IEC Operation

Preparation and Loading Sample. Perhaps one of the most important considerations in purification of proteins is the problem of their degradation, usually as a result of protease activities being brought into contact with them following the rupture of cells and their limiting membranes. Degradation can occur at any time, resulting in inactivation of enzymes or errors in characterization of the components recovered. Many precautions have been included in purification schemes to prevent degradation.

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If the decision has been made not to use batch IEC, then the alternative is to use open-column IEC for the separation. The essential components of an open-column ion-exchange apparatus include the column, capped at both ends, and a reservoir for the eluants. Usually a pump is required to allow the eluents to be delivered at a constant rate; a detector, coupled to a recorder, to monitor the components in the eluent; and a fraction collector to allow for the recovery of the components. It is useful to have the fraction collector and the recorder coupled with an "event marker" such that each change of the collector is marked on the recorder. This will facilitate the correlation of detector events with the recovery of the components after separation. Optional accessories include a conductance cell to monitor "on line" the concentration of salt in the eluent, and computerized management of data obtained from the various monitors including the detector and fraction collector for graphing purposes. In open-column IEC, the columns are usually transparent and capped at both ends. They can be obtained from a number of suppliers or made from glass or plastic tubing. Each end should have an inlet and outlet port to which tubing is attached. The column should be mounted in a vertical position and, prior to being filled with the slurry, a supporting bed should be placed inside the column against the bottom cap. This bed, the purpose of which is to prevent the resin or cellulose from running through the outlet port at the bottom, can be a plastic mesh, a sintered glass disk, or glass wool. In filling the column, a slurry of the packing is transferred from the beaker in which it had been prepared to the supporting bed. The column packing need not fill the column although it is best that the volume of buffer above the packing be kept small to minimize mixing. The column can be operated (that is, eluted) by pumping the buffer either from the top down or from the bottom up. When operating a column from the bottom to the top, there will be no "head" or layer of buffer between the cap of the column and the packing, minimizing the mixing and dilution of incoming buffers with those already present. Practical Information on IEC Operation Preparation and Loading Sample. Perhaps one of the most important considerations in purification of proteins is the problem of their degradation, usually as a result of protease activities being brought into contact with them following the rupture of cells and their limiting membranes. Degradation can occur at any time, resulting in inactivation of enzymes or errors in characterization of the components recovered. Many precautions have been included in purification schemes to prevent degradation.

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Cs+ > K+ > NH4+ > Na+ > H+ > Li+. Such packings are usually supplied in the H+ form. Thus, it is a simple matter to replace the H+ with an ion higher in the series by washing in a 1 M solution of that salt. Similar selectivity series exist for anion exchangers, and the manufacturer's literature should be consulted for this information. Size of Column: Bed Capacity. Although the length of the column is less important, the separation does depend on the amount of column packing available since if this is insufficient, material that should have been retained will flow through. Although the amount of material that the column can bind must eventually be deterinined by experiment, information provided by the manufacturer on the bed capacity can narrow the quest. The manufacturer provides this information in the form of milliequivalents per dry gram or milliequivalents per milliliter of resin bed. For cation exchangers, supplied in the hydrogen form, this would be milliequivalents of H+ that can be exchanged while for anion exchangers supplied in the chloride form this would be milliequivalents of C1-. The columns used for IEC need not be large. For example, columns made from Pasteur pipets have been used for the purification of the peptides derived from proenkephalin.' Used for this purpose, the pipet should be plugged with a small amount of glass wool, the resin prepared, and introduced into the pipet. The volume of the resin in such a column would be on the order of 0.25 cm3. For such columns elution will be a simple matter of washing the column with about 1 ml of each of the eluents. Batch us Open Column. IEC need not be performed using a c o l ~ m n . ~ The alternative, batch IEC, is usually performed with the slurry of the stationary phase in a vessel such as a beaker. In one study, used for the the essential component of the separation of lactate dehydr~genase,~ batch system is the resin (Bio-Rad AG MP-I), which is added to a tube containing the enzyme (isozymal forms of human lactate dehydrogenase). After shaking and equilibration, the resin is separated from the buffer either by centrifugation, filtration, or a combination of the two. The authors note that the separation they obtained was less than desirable, which they attribute to the fact that the batch procedure is both a nonequilibrium and too rapid a process. However, sometimes the speed of the process can be an advantage such as when separations on a larger scale are required.

' S. P. Wilson, J. Neurosci. Methods 15, 155 (1985). D. Reichenberg, in "Ion Exchangers in Organic and Biochemistry" (C. Calmon and T. R. E. Kressman, eds.). Interscience, New York, 1957. M. P. Menon, S. Miller, and B. S. Taylor, J. Chromarogr. 378, 450 (1986).

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These include working at low temperatures (4") to slow proteolytic action and the addition to the "lysing" buffers of a mixture of protease inhibitors. Another problem in the preparation and loading of proteins for IEC is the ampholytic nature of proteins. Proteins contain both positive and negative charges, and for each protein a unique point has been defined, the isoelectric point, or PI,which is that pH at which the net charge on the protein is zero. Although an anion-exchange column would generally be used above the pI, and a cation exchanger below, it is often found that proteins can bind at a variety of pH values because the magnitude of the charge in different regions of the protein may vary. Clearly, in cases where the amino acid composition is unknown, it will be necessary to try both anion and cation exchangers and various pH values to find the best conditions for separation. Also, since a change in pH can result in a change in the charge composition of the proteins, an altered elution profile can be obtained if the pH is not held constant during the elution. Thus, it is advisable not to work with the stationary phase in the H + form and to use buffered salt solutions as eluents to maintain a constant pH during the elution. Finally, loading of IEC columns is a simple matter because samples are introduced onto the column under conditions that promote binding. Loading an IEC colun~ninvolves applying the sample onto the packing. Any volume can be applied as long as the total amount of protein does not exceed the binding capacity of the packing. In fact, IEC can be used for concentration of proteins. For example, proteins can be concentrated by adsorption to an anion-exchange column in low salt and eluted, in a concentrated form, with higher salt concentrations. Composition of Elution Solution. Elution of an IEC column requires a decision on the composition of the elution solution. The components of the elution solution include the buffer, the salt to be used for the displacement, and any components required for solubility and stability. The buffer and its pH should also be chosen on the basis of compatibility with the stability of the protein. Of course, the pH should be one that would allow binding. When dealing with an enzyme, the elution buffer should not result in loss of activity. Further, if the protein is to be located in the fractions on the basis of its activity, a buffer could be chosen which provides optimal conditions for the assay. This problem becomes acute with proteins that require detergents for solubilization, since at the concentrations used for solubilization some may interfere with the determination of activity. However, their removal often results in the precipitation of the protein. Therefore, a compromise often must be made by using

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sufficient detergent to prevent precipitation and sacrificing some loss of sensitivity in the assay. Some consideration of the purpose of the IEC step is necessary when choosing the displacement salt. For example, if IEC is only one step in a multistep purification scheme, then the choice of the buffer and the displacement salt should include some consideration of the subsequent steps in the scheme. For example, if the next step requires the sample from the IEC to be concentrated, then the use of a volatile salt such as ammonium carbonate is recommended. In such solvents, samples can be lyophilized or subjected to rotary evaporation to concentrate the protein without concentrating the salt. Elution Gradients. After the proteins have been adsorbed, their displacement requires the introduction of counterions into the eluent. The counterions, usually added in the form of a salt solution, can be introduced into the system in one of two ways: either discontinuously in a step gradient, or continuously with a linear gradient. Although elution of adsorbed proteins only begins following the introduction of salt, the process of elution of unadsorbed proteins will begin immediately after loading. Therefore, it is best to wash the column first with several volumes of the loading buffer. If the column effluent is monitored continuously during this washing step, it is possible to establish two points: (1) what fraction of the sample adsorbs to the column and (2) how much of the sample did not adhere and just "runs through" with the lowsalt loading buffer. Clearly, if none of the proteins is adsorbed, a change in the IEC conditions is in order. The displacement phase of the elution begins with the introduction of the salt. The salt solution should be introduced at a constant rate and a pump is best used for this purpose. The salt solution can be introduced onto the column from either the bottom up or from the top down. If a step elution is to be used the solution of salt of the next higher concentration in the step should now be introduced and should be maintained for at least two to three column volumes or until there is convincing evidence that this particular salt concentration has achieved equilibrium and has displaced all the protein that can be eluted at this particular concentration. At this point the solution of the next higher concentration can be introduced and the process repeated. The salt concentration is introduced in a stepwise manner until all the protein is eluted. The concentrations of salt in each step are usually determined by trial and error. If the salt is to be introduced as a gradient, two solutions are prepared, one of the low salt and the other of the high salt. With the aid of a mixing device, often two flasks connected by a siphon, the solution is introduced onto the column,

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beginning with the lowest concentration. By the end of the elution the solution of highest salt concentration will have been introduced. It should be noted that the resolution of the components is often affected by the magnitude and number of steps or the form of the gradient. With gradient elution the form of the gradient, steep vs shallow, should be varied to modify the resolution. For both types of elution it is advantageous to have some measure of the salt concentration in the effluent. This can be accomplished with a conductivity meter, which can be installed in line with the other detectors, or measurements of salt concentration can be obtained using a separate instrument that functions like a pH meter. The instrument has a probe which, after calibration, can be inserted into each of the fractions obtained during the column run. With the aid of a calibration curve, the readings obtained on the fractions can be converted to concentration, producing a profile of the salt concentration at each stage of the elution. Although IEC can be performed in the presence of detergents, it is best to use those that are uncharged and that do not have an absorption maximum at the wavelength used for monitoring the column (see below). For example, Triton X-100 absorbs in the ultraviolet (UV) range and will interfere with the detection of proteins at 280 nm. Detergents that do not interfere with UV detection include the type called zwitterions, sold under the name 2-314. However, with detergents, as with all solvents, it is best to test the solubility of the protein in the solvent prior to loading the column. Examples of the use of detergents with IEC have been presented elsewhere . I 0 Of course, when working with proteins, particularly enzymes, in which activity must be maintained, denaturation must be avoided. This requirement often precludes the use of organic solvents as eluents. Graphical Presentation of Data. The elution of proteins from IEC columns can be conveniently followed by monitoring the eluent at 280 nm. This is because most proteins contain aromatic residues (such as tyrosine and tryptophan) which have an absorption maximum in this region. In the absence of such residues, as with the protein collagen, it is possible to monitor the effluent at 210-230 nm, the absorption region of the peptide linkage. The optical density of the effluent can be monitored on line using a spectrophotometer equipped with a flow cell or on samples from each of the fractions. In the latter case, one then plots the absorption vs the fraction number to display the elution profile of the column. The salt concentration monitored either with continuous read-out melo

E. F. Rossomando, "High Performance Liquid Chromatography in Enzymatic Analysis." Wiley, New York, 1987.

1251 -

317

GEL FILTRATION -

-

-

-

-

-

ters or by reading the salt concentration of the fractions is also included on such a plot. If enzyme activity measurements or a specific protein assay are performed, these also should be presented on the column profile. The combination of the specific assay and the protein profile gives a simple visual display of the approximate purification obtained by the column procedure. Acknowledgments I wish to thank Ms. Jane Hadjimichael for suggestions concerning the text and editorial assistance and Ms. Pamela Vachon for typing the manuscript.

[251 Gel Filtration By EARLESTELLWAGEN

Among the chromatographic techniques employed for protein purification, gel filtration is unique in that fractionation is based on the relative size of protein molecules. In contrast to conventional filtration, none of the proteins is retained by a gel filtration column. This feature is at once both the strength and weakness of gel filtration; a strength because the function of fragile proteins is not damaged by binding to a chromatographic support, and a weakness because the absence of such binding limits the resolution of the chromatography. Principle

Gel filtration is performed using porous beads as the chromatographic support. A column constructed from such beads will have two measurable liquid volumes, the external volume, consisting of the liquid between the beads, and the internal volume, consisting of the liquid within the pores of the beads. Large molecules will equilibrate only with the external volume while small molecules will equilibrate with both the external and internal volumes. A mixture of proteins is applied in a discrete volume or zone at the top of a gel filtration column and allowed to percolate through the column. The large protein molecules are excluded from the internal volume and therefore emerge first from the column while the smaller protein molecules, which can access the internal volume, emerge later. The dimensions important to gel filtration are the diameter of the pores that access the internal volume and the hydrodynamic diameter of the METHODS IN ENZYMOLOGY, VOL. 182

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form resewed.

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protein molecules. The latter is defined as the diameter of the spherical volume created by a protein as it rapidly tumbles in solution. Proteins whose hydrodynamic diameter is small relative to the average pore diameter of the beads will access all of the internal volume and are described as being included in the gel matrix. Proteins whose hydrodynamic diameter is comparable to the average pore diameter will access some but not all of the internal volume and are described as being fractionally excluded. Proteins whose hydrodynamic diameter is large relative to the average pore diameter will be unable to access the internal volume and are described as being excluded. This conceptualization has led to the gradual renaming of gel filtration as size-exclusion chromatography. The order of elution of a mixture of proteins from a size exclusion column will then be the inverse of their hydrodynamic diameters. If all the proteins in a mixture are known, or can be assumed to have the same shape, then the order of elution will be the inverse of their molecular weights. This discussion will treat protein dimensions in terms of molecular weight since common usage assumes that protein mixtures contain only globular proteins. However, the reader should bear in mind that hydrodynamic volume is the operative protein dimension and that an asymmetrical protein will appear to elute with an abnormally high molecular weight compared with globular proteins of similar molecular weight. Practice

An elution profile obtained by size-exclusion chromatography is illustrated in Fig. 1A. Zero elution volume is defined as the entry of the sample into the chromatographic support. The elution volume for the excluded component is designated Vo for the void volume, which represents the volume external to the beads. The elution volume for the included component is designated V ,for the total volume, which represents the sum of the external volume and the internal volume within the beads. Elution volumes intermediate between these values are designated V,. A partition coefficient, designated K,, , relating these values is given in Eq. (1):

A semilogarithmic plot of the dependence of the partition coefficient on molecular weight is illustrated in Fig. IB. The separation of proteins based on molecular weight will be greatest in the central linear region of this sigmoidal relationship, spanning K,, values between 0.2 and 0.8. This span is described as the fractionation range of a size-exclusion matrix.

[251

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GEL FILTRATION

Volume (ml)

A,

-vo; 1

1.o 0.8 KAV 0.6 0.4 0.2 0.0 -

3

2

I

I

I

I

-

I

I

I

I

1

10

100

1,000

Molecular Weight

FIG. I . Chromatographic performance of a size-exclusion matrix. (A) illustrates a relatively simple elution profile. The ordinate represents concentration expressed as spectral absorbance at some fixed wavelength, A, and the abscissa represents efRuent volume subsequent to the application of the sample into the column. If the effluent flow rate is constant then the abscissa could be expressed in time. Component 1 is excluded from the matrix and its elution position is denoted as V o . Component 2 is partially excluded and its elution position is denoted as V,. Component 3 is included and its elution position is denoted as V,. The assignment of a component to an elution position is established by application of each component individually to the column. (B) illustrates the sigmoidal dependence of the partition coefficient K,, as defined in Eq. (1) on the logarithm of the molecular weight of a series of components having the same shape.

The steeper the slope of the sigmoidal relationship in the fractionation range the greater the resolving power of a matrix. Accordingly, the best separation among proteins having similar molecular weights will be achieved using a matrix with a narrow fractionation range. Fewer than 10 proteins can be resolved from one another in the effluent from any size exclusion column. This relatively low resolution occurs because none of the proteins is retained by the column during chromatography and because nonideal flow occurs around the beads. Accordingly, prospects for a significant enhancement in purification (-fold) by sizeexclusion chromatography are most promising if the desired protein has a molecular weight either considerably larger or smaller than that of the majority of proteins in a mixture. Since this will generally not be the case, an investigator can anticipate only a modest enhancement in purification (-fold). Accordingly, it is wise to perform size-exclusion chromatography

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relatively late in a purification procedure when the numbers of other proteins are small and when the preceding step has fractionated the protein mixture on the basis of a completely different property. For example, pooled fractions obtained from ion-exchange chromatography will likely contain a mixture of proteins, each having about the same net charge but a range of molecular weights. Matrices

The properties of some conventional and high-performance size-exclusion matrices are given in Tables I through IV. It should be noted that suppliers use a variety of terms and abbreviations to index these products in their catalogs, including gel-filtration chromatography (GFC), gel-permeation chromatography (GPC), and size-exclusion chromatography (SEC).

TABLE I MATRIXPARAMETERS Stability

Name Conventional BioGel A BioGel P Sephacryl HR Sephadex G Sepharose Ultrogel A Ultrogel AcA High performance Protein Pak Shodex Superose SynChropak TSK-SW Zorbax a

Supplier

Chemistryo

Form supplied

Bio-Rad Bio-Rad Pharmacia Pharmacia Pharmacia IBF IBF

Suspension Powder Suspension Powder Suspension Suspension Suspension

Waters Showa Denko Pharmacia SynChrom Toyo-Soda DuPont

Packed Packed Packed Packed Packed Packed

PH

Ternperature ("C)

Bead diameter (fim)

column column column column column column

The following symbols are used to denote the chemical nature of the matrix: AG, cross-linked agarose; PA, cross-linked polyacrylamide; DX, cross-linked dextran; DXIDA, copolymer of ally1 dextran and bisacrylamide; AGIPA, mixture of agarose and polyacrylamide; and S, bonded silica. Individual matrices have narrower ranges.

[251

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TABLE I1 POWDEREDMATRIXPAPAMETERS Hydration time (hr) Name BioGel

Sephadex

a

Code

Fractionation rangea (kDa)

Swollen volume (ml/g)

20"

90"

Linear flow^.^ (cm/hr)

P-60 P- 100 P-200 P-300 G-50 G- 100 G- 1 50 G-200

The values listed are for beads of a medium mesh size.

* The linear flow indicated is appropriate for moderately

high-resolution chromatography. The volume flow in milliliters per hour is obtained by multiplying the linear flow by the cross-sectional area of a column in centimeters squared.

K $

The conventional matrices are distinguished by their relative economy and slow flow rates. These matrices are available in bulk, requiring an investigator to pour columns of any desired dimensions to accommodate the volume of the sample to be chromatographed. The flow rates normally used for chromatography are obtained by multiplying the linear flow rate listed in Table I1 by the cross-sectional area of the column in centimeters squared to yield the flow rate in milliliters per hour. A column can be packed with a flow rate approximately five times that used during chromatography. The high-performance matrices are distinguished by their convenience, rapid flow rates, and expense. These matrices are usually purchased as poured columns which are attached to an existent high-performance chromatograph available to the investigator. The smaller analytical columns, about 8 x 300 mm, are normally loaded with not more than a few milligrams of protein and operated at a flow rate of about 1 mll min. The larger preparative columns generally contain beads having a diameter of 30 pm. The approximately 20 x 300 mm columns can be loaded with between 10 and 100 mg of protein and can be operated at a flow rate of about 5 mllmin while the very large columns can be loaded with up to 2 g of protein and be operated at a flow rate of up to 30 mllmin.

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Sample Preparation

The sample should not have a protein concentration in excess of about 50 mglml and should be clarified by centrifugation, if necessary, in order to prevent particulate matter from slowing the flow rate of the column. The solvent for the protein sample is of little consequence since the protein will advance ahead of the application solvent during chromatography. Chromatographic Solvents

The solvents used to flow through the column have wide latitude, subject only to the pH and temperature constraints listed in Table I. However, the ionic strength of the chromatographic solvent should be at least 0.2 M to minimize the binding of proteins to the matrix by electrostatic or by van der Waals interactions. Most proteins are inherently stable at room temperature and require only low temperatures in order to reduce the rate of peptide hydrolysis catalyzed by any proteolytic enzymes present in the protein sample. However, proteolysis becomes an increasing problem during purification as the desired protein becomes the more abundant substrate for the proteases. In some cases, rather expensive proteolytic inhibitors or effectors need be present in the chromatographic solvent in order to maintain the function of the desired protein. Some economy can be realized by equilibration with only one column volume of the solvent containing the expensive component(s) prior to application of the sample, since the sample advances into the column solvent during chromatography. The solvent following the sample application need not contain the expensive component(s). Columns poured in glass cylinders should be equilibrated with a simple solvent, such as 0.1 M NaCI containing about 0.02% sodium azide, to prevent the growth of microorganisms. Methanol is the preferred storage solvent for columns poured in stainless steel cylinders in order to avoid the corrosion accelerated by the continued presence of salt solutions. Preliminary Screening

In order to optimize the purification (-fold) achieved by size-exclusion chromatography, it is necessary to use a matrix which will best resolve the desired protein from the remaining proteins. Accordingly, a preliminary screening is useful to estimate the molecular weight of the desired protein and the molecular weights over which the remaining proteins are distributed. The elements needed for screening in addition to a protein sample include a size-exclusion column, a fraction collector, an assay for

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total protein, an assay for the desired protein, and a molecular weight calibration mixture. The assay for total protein can either be ultraviolet absorbance or a colorimetric procedure (see [6]). A sufficient concentration of the sample must be applied to the column so that the function of the desired protein can be measured with confidence in the eluate fractions. It should be anticipated that the concentration of the desired protein will be diluted at least an order of magnitude by the chromatography. Molecular weight calibration mixtures, often termed gel filtration standards, can be purchased from several suppliers, including Bio-Rad Laboratories (Richmond, CA), Pharmacia LKB Biotechnology (Piscataway, NJ), and Sigma Chemical (St. Louis, MO). These calibration mixtures contain several identified proteins of known molecular weight as well as components to establish Vo and V,. Alternatively, an investigator can customize a calibration mixture using purified components. Blue dextran and DNA restriction fragments are frequently used to determine Vo. It is important not to use a small aromatic or heterocyclic compound to determine V , since such molecules are particularly prone to reversible adsorption by size-exclusion chromatographic matrices. If a high-performance size-exclusion analytical column and chromatograph is available, the screening is both rapid and simple. The column used for screening should have a broad fractionation range. A guard column should be placed in front of the analytical column to retain any particulate material which has escaped notice. A protein sample containing a minimal volume appropriate for analysis of the desired protein in the column effluent should be injected. The effluent should be monitored for protein concentration using an absorbance flow detector set either at the more sensitive 225 nm, if the solvent absorbance permits, or at the less sensitive 280 nm. Effluent fractions should be collected and analyzed for the total protein, if a flow absorbance detector is not available, and for the desired protein. Finally, a gel filtration standard should be injected into the column and the effluent monitored again at the same wavelength. Comparison of the elution profile for the gel filtration standard with the profiles for the total protein and the desired protein in the sample should facilitate selection of a matrix that will optimize the purification (-fold) achievable by size-exclusion chromatography. If a high-performance analytical column is not available, then the screening must be done with a conventional matrix having a broad fractionation range. It is likely that the matrix selected will have to be poured into a column. Instructions for pouring a column using a conventional matrix are detailed below. Again, a conventional matrix that can optimize the purification (-fold) obtained by size-exclusion chromatography can be

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TABLE I11 SUSPENDED MATRIXPARAMETERS

Name BioGel

Sephacryl

Sepharose Ultrogel

Code

Fractionation range (kDa)

A-O.5m A-1.5m A-5m S-200 HR S-300 HR S-400 HR CL-6B A6 A4 AcA 54 AcA 44 AcA 34 AcA 22 -

a

Linear flowa (cmlhr)

-

-

The linear flow indicated is appropriate for moderately high-resolution chromatography. The volume flow in milliliters per hour is obtained by multiplying the linear flow by the column cross-sectional area in centimeters squared.

selected from the screening based on the elution profiles obtained for the gel filtration standard and for the total protein and the desired protein in the sample. Chromatography Using Conventional Matrix

The volume of a conventional matrix used for protein purification should be 30 to 100 times the volume of the sample to be fractionated. The amount of matrix required to form the column is suspended in the chromatographic solvent and brought to the temperature at which chromatography will be performed. The volume of the suspension should be no more than twice the volume of the column to be made. Fine particles should be removed by gently swirling the suspension and the supernatant removed by suction after about 90% of the beads have settled. Finally, the suspension should be placed under negative pressure to reduce the volume of dissolved air. A filter flask and a laboratory aspirator are useful for this purpose. If the matrix is supplied as a dry powder, the matrix should be swollen in the chromatographic solvent prior to removal of the fine particles. The

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matrix may be swollen at either ambient temperature or at 100°, depending upon the time available to the investigator. As shown in Table 11, swelling of a matrix proceeds much faster at 100" without damage to the matrix. The chromatographic column should be made in a glass or transparent plastic cylinder of either commercial design or laboratory improvisation. The ratio of the length of the cylinder to its diameter may vary from 20 to 100. When improvising, elements of the following procedure can be used. The bottom of the column can be formed from a rubber stopper containing a short length of a thick-walled capillary tube positioned flush with the narrow end of the stopper. The cylinder is oriented vertically and clamped securely in the location in which the chromatography will be performed. The stopper is inserted into the bottom end of the cylinder. A short length of flexible tubing is attached to the protruding glass tube and a clamping device attached to the tubing to control the liquid flow through the cylinder. A nylon or teflon mesh is placed inside the cylinder and pushed to the bottom to fit snugly against the stopper. The clamp is closed and the cylinder filled with the matrix suspension. The excess suspension is placed in a vessel with a bottom exit and stopcock, such as a separatory funnel, and the exit attached to the top of the cylinder with a length of flexible tubing and a one-hole stopper containing a short length of glass tubing. This assembled apparatus should be airtight between the surface of the excess suspension in the separatory funnel and the flexible tubing extending from the bottom of the cylinder. The flow rate is controlled by the height of the separatory funnel relative to the column. The column can be packed using a flow rate about five times greater than that listed in Table 11. Once the desired column height is packed, the clamp and stopcock are closed, the excess matrix suspension removed, and some chromatographic solvent passed through the column using the separatory funnel as the reservoir. A pool of solvent several centimeters in height should be continuously maintained at the top of the column to buffer the impact of the chromatographic solvent as it enters the cylinder so as not to disturb the packing at the top of the column. The packed column should never be allowed to run dry, as it will produce channelling within the column which will severely perturb protein resolution. To apply a sample to the column, the stopcock should be closed, the stopper at the top of the cylinder removed, and the solvent pooled above the column drained through the column until the solvent just dips below the top of the packed column. The clamp is then closed and the sample or standard solution added carefully to minimally disturb the packing at the top of the column. The clamp is then opened and the sample solution allowed to enter the column until it just dips below the top of the column.

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The clamp is then closed and a small amount of chromatographic solvent added with minimal disturbance to the packing at the top of the column. This solvent is then admitted to the column, the clamp again closed, and more chromatographic solvent added to the column to form a pool of desired height. A supply of chromatographic solvent is placed in the separatory funnel and connected by an air-tight seal to the top of the cylinder with the flexible tubing. The height of the separatory funnel is then adjusted to achieve and maintain the desired flow rate. The absorbance of the column effluent can be continuously monitored at a desired wavelength using a flow monitor. It is important that the tubing at the bottom of the column and the flow optical cell in the monitor have a small diameter to prevent convective mixing of the liquid emerging from the column. It is also important that a flexible tubing be used which does not contribute ultraviolet-absorbing material to the chromatography solvent. Alternatively, the column effluent can be directed to a fraction collector and the fractions assayed for both total protein and desired protein. A drop counter is ideal for this purpose. Scaling Upward

Size-exclusion chromatography using conventional matrices can be easily scaled upward by increasing the volume of the column appropriate to the volume of the sample to be fractionated. Very large sample volumes may be best handled with repetitive chromatography as opposed to construction of columns of monumental dimensions. Semipreparative and preparative scale high-performance columns are available as indicated in Table IV and some suppliers will provide bulk material for packing by the investigator. Although these larger high-performance columns can be quite expensive it should be remembered that they represent a considerable saving in investigator time and that the investment can be amortized over many different uses. Trouble Shooting I . Poor Resolution. This is a common lament because size-exclusion chromatography has an inherent low resolution. Nonetheless, changes in some operational parameters may improve resolution. First since flow rate and resolution are inversely related, decreasing the flow rate may improve the resolution. Second, use of a bead size having a smaller diameter should improve resolution. Third, use of a matrix having a narrower fractionation range may be helpful. 2 . Low Flow Rate. This usually results from plugging of the filters or the matrix with particulate material in the samples. The column should be

[251

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TABLE 1V PACKEDCOLUMNMATRIXPARAMETERS

Name -

Protein Pak

Shodex

Superose SynChropak

TSK

Zorbax

Code -

Pore diameter

(A)

Diameter x length (mm)

Fractionation range (kDa)

-

60 125 300 WS 802.5 WS 803 WS804 12 6 60 100 300 500 G2000SW G3000SW G4000SW GF-250 GF-450 -

- -

-

--

" Also 8 x 500 and 20 x 300.

* Also 21.5 X

250.

" Also 21.5 x 600 and 55 x 600.

first washed by reverse flow with a solubilization agent such as a nonionic or ionic detergent, a protein denaturant such as urea or guanidinium hydrochloride, an organic solvent such as methanol or, within the stability of the matrix, brief exposure to a strong acid or base. If this does not succeed for a conventional matrix, then the column should be disassembled, the individual components cleaned, and the column repacked. If this does not succeed for a high-performance matrix, either the column may be sent to Phenomenex or another supplier for cleaning and repacking at a fee or the column may be simply replaced. Laboratories which have facilities for repacking columns at pressure can clean and repack highperformance columns themselves. 3. Skewed Peaks. A primary cause is poor sample application. For a conventional column, the quality of sample application can be observed by placing an inert colored component in the protein sample such as blue dextran or potassium dichromate. If the sample has an irregular appearance in the column it will likely generate an asymmetrical peak in the elution profile. For a high-performance column, the injector can be disas-

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sembled and cleaned. Tailing of peaks generally results from adsorption of proteins to the matrix. This situation can be improved by using a more potent lyotropic salt, such as sodium perchlorate instead of sodium chloride, as the principal ionic component in the chromatographic solvent. In the case of a high-performance column, tailing may indicate the loss of the coating on the silica beads, a situation requiring replacement of the column. Skewed peaks may also result from a reversible equilibrium between different states of polymerization of the protein. For example, hemoglobin can exhibit a dynamic equilibrium between the dimeric and tetrameric forms of the protein. Since polymerization involves a change in molecular weight, the matrix will favor dissociation while chemical equilibrium will favor association. These opposing forces can result in the appearances of a skewed peak characteristic for a dynamic exchange. Changes in the pH, temperature, or chemical composition of the chromatographic solvent may shift the chemical equilibrium such that only one polymeric form is significantly populated. 4. Disappearance of Desired Protein. This may occur for at least two reasons. The desired protein may be moderately adsorbed to the column so that its elution occurs after V , in a very broad peak that is difficult to distinguish from noise in the baseline. If this is the case, a protein solubilization agent such as a nonionic detergent or a modest concentration of a protein denaturant should be added to the chromatographic solvent. A second possibility involves the dissociation of a functional protein complex into discrete proteins of different molecular weight in which none of the dissociated proteins retains the function. Mixing aliquots from different fractions should facilitate complexation of the component proteins and restoration of the function. Further Information

Virtually all the suppliers of size-exclusion matrices and customized chromatographic columns have prepared detailed instructions regarding the use of their products. These instructions are quite helpful and generally free of charge.

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PROTEIN CHROMATOGRAPHY ON

HA COLUMNS

329

[261 Protein Chromatography on Hydroxyapatite Columns*

The use of hydroxyapatite (HA) columns for protein chromatography was introduced by Tiselius et al. in 1956.' Its systematic study was undertaken by B e r ~ ~ a r d who i , ~ , ~examined a variety of proteins using several solvent systems and worked out standard procedures for the operation of HA columns. He also proposed a mechanism for protein adsorption to and desorption from HA, since the principles of ion-exchange chromatography are not applicable to HA chromat~graphy.~.~ In a study extending Bernardi's work,44 a number of additional proteins with isoelectric points ranging from pH 3.5 to 11.0 have been examined, the relation between the ionic state of the HA column and protein retention has been scrutinized, and additional solvent systems have been used to test the effect of the chemical nature of eluants upon the desorption p r o c e ~ s Furthermore, .~ the roles of protein fine structure and specific polar groups have been p r ~ b e dThis . ~ last study included proteins in which individual polar groups were modified specifically, as well as those in which very extensive numbers of amino or carboxyl groups were modified with (1) retention of the same charge, (2) inversion of the charge, or (3) annihilation of the ~ h a r g e As . ~ a result of this work, a mechanism of ,~ was based on three fundaprotein-HA interaction was d e d ~ c e dwhich mental conclusions: 1. Adsorption and elution cannot be regarded as simple reversals of a single process. 2. Amino and carboxyl groups act differently in the adsorption of proteins to HA. 3. Elutions of basic and acidic proteins by different salts follow different mechanisms. * This chapter was previously published in "Methods in Enzymology." Volume 117, pp. 370-380.

' A. Tiselius, S. Hjerten, and 0. Levin, Arch. Biochem. Biophys. 65, 132 (1956).

' G. Bernardi, this series, Vol. 22, p. 325.

G. Bernardi, this series, Vol. 27, p. 471. M. J. Gorbunoff, Anal. Biochem. 136, 425 (1984). M.J . Gorbunoff, Anal. Biochem. 136, 433 (1984). M.J . Gorbunoff, Anal. Biochem. 136, 440 (1984).

METHODS IN ENZYMOLOGY, VOL. 182

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Adsorption of Proteins to Hydroxyapatite Amino groups act in the adsorption of proteins to HA as the result

primarily of nonspecific electrostatic interactions between their positive charges and the general negative charge on the HA column,' when the column is equilibrated with phosphate buffer: This is consistent with the observations that (1) retention of basic proteins (see Table I), polypeptide^,'.^ and amino acids3 is controlled by the ionic state of the column, i.e., its net charge (negative in the phosphate cycle, neutral in the NaCl cycle; positive in the CaC12 or MgC12 cycle); (2) the lower the pH of the equilibrating phosphate buffer, the higher the molarity required for e ~ u t i o n ~and ? ~(3) ; blocking of carboxyls strengthens the binding of both basic and acidic proteins to HA.SThis electrostatic interaction is crucial to the binding of basic proteins. It is sufficient to block 5 out of 19 amino groups in a-chymotrypsin to make its retention marginal, while lysozyme with 7 out of 18 groups blocked is not retained on any column (see Table I). Carboxyl groups act in two ways. First, they are repelled electrostatically from the negative charge of the column. Second, they bind specifically by complexation to calcium sites on the column, forming clusters of This is consistent with the observations that ( I ) the relation between the ionic (net charge) state of the column and retention of acidic amino acids,3 polypeptide^,^ and proteins (see Table I) is a mirror image of that of their basic counterparts; and (2) replacement of COOH by S03H, which does not affect the charge, prevents this complexation (see Table I). Thus acidic proteins must bind almost exclusively by this complexation, since their ability to bind to HA is lost on COOH + S03H replacement. This singularity of COOH-Ca complexation is demonstrated in a particularly striking manner by proteins which have clusters of carboxyls, such as trypsinogen and P-lact~globulin.~ A particularly striking example is trypsinogen (see Table 11). Trypsinogen is a basic protein which displays retention and elution patterns which are a hybrid of those expected for basic and acidic proteins. The isoelectric point of trypsinogen is 9.3. This causes it to behave as a basic protein, except with respect to elution with CaCI2. It contains, however, a cluster of four aspartate residues in the N-

' The surface of HA crystals presents a mosaic of positive (calcium) and negative (phosphate) sites. Since HA columns are normally operated at pH 6.8 after extensive washing with phosphate buffer, the surface of the column can be regarded as negative due to partial neutralization of the positive calcium loci by phosphate ions.

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TABLE I1 ELUTION OF TRYPSINOGEN FROM HYDROXYAPATITE~ Elution molarity Derivative

NaP04 (6.8)

NaF

NaCl

NaC104

CaClz

MgClz

Trypsinogenh Acetyltrypsinogen Trypsinogen-COXc AMS-trypsinogend

0.17 0.004 0.23 0.13

0.23 0.004 0.41 0.18

0.32 0.5 0.32 0.18

0.31

>3.0 >3.0 0.003 0.001

0.1 0.2 0.1 0.001

0.24 0.24

" The loading columns were always in the pH 6.8 phosphate cycle, except for acetyltrypsinogen for which the column was in the chloride cycle. N-terminal peptide, Val-Asp-Asp-Asp-Asp. COX, COOH + CONHNHCONH2. AMS, COOH -+ CONH2CH2S03H.

terminal of the molecule. Their blocking or transformation to S 0 3 H abolishes its inability to be eluted by 3 M CaClz and renders it into a normal basic protein. It is interesting to note that this cluster of carboxyls constitutes one of the Ca2+-bindingsites of this protein. Since retention on the column depends on the cooperative interaction of several carboxyls with HA, a sufficient density of carboxyls is required to generate statistically a cluster complementary to the calcium atoms immobilized in the HA crystals. Therefore, the inability of basic proteins to bind to HA once their positive charge has been abolished must reflect their insufficient density of carboxyls. E l u t i o n of P r o t e i n s from H y d r o x y a p a t i t e

Basic proteins are eluted either as a result of normal Debye-Hiickel charge screening, which operates in the elution by F-, C1-, C104-, SCN-,

and phosphate, or by specific displacement by Ca2+and Mg2+ions which complex with column phosphates and neutralize their negative charges: HAPOI- . . . +NH,-Prot

+ CaCI2-+ HAP04-Ca + C 1 + +H,N-Prot

(1)

The former mechanism is most strikingly illustrated by the separation of five isozymes of lactate dehydrogenase, where elution is related directly to ~ h a r g e . ~ Acidic proteins are eluted by displacement of their carboxyls from HA M. John and J. Schmidt, Anal. Biochem. 141, 466 (1984).

[261 -

PROTEIN CHROMATOGRAPHY ON

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333

calcium sites by ions which form stronger complexes with calcium than do carboxyls, e.g., fluoride or phosphate:

IF

I

+ NaF (or PO,) + HACa-F + Na+ + -0OC-Prot (2) Since the formation constants for CaF or C a p o 4 >> CaOOC, the disHACa-OOC-Prot

placement occurs at a rather low molarity of fluoride or phosphate. The ineffectiveness of C1- as eluant of acidic proteins is due to the fact that it does not form a complex with Ca2+and, thus, cannot compete with the Ca-OOC complexes. The strength of the Capo4 bonds can be taken to be demonstrated by the unusually strong bonding to HA of phosphoprot e i n ~The . ~ behavior of nucleic acids need not contradict this, since they differ from phosphoproteins both in the state of their phosphate groups and their space distribution in the m ~ l e c u l e . ~ The ability of CaClz and MgC12 to strengthen the bonding of acidic proteins to HA is due to the formation of additional bridges between protein carboxyls and column phosphate H A P 0 4 . . . Ca2+ - . . OOC-Prot

b

i

This can be used to advantage to retain acidic proteins on the column since these are eluted neither by 3 M CaC12 nor by 3 M MgC12. The difference in the formation constants of Ca-PO4 and Mg-PO4 provides a useful tool for the discrimination between acidic proteins and neutral proteins, i.e., those with isoelectric points between pH 515.5 and 8, since the last are eluted by MgClz above 1 M, but not by CaC12.

,A'

C P

r r r E .

Guidelines for the Use of HA Columns

The understanding of the principles of protein retention on and elution from HA columns has permitted to establish a set of rules for loading proteins on HA and eluting them from the columns. The choice of column cycle and ionic nature of eluting buffers must be dictated by the charge nature of the protein mixture to be fractionated, as well as the charge (positive, negative, or neutral) of the protein to be isolated. Keeping in mind that (1) basic proteins are not retained by MgC12 or CaClz columns, while acidic proteins are strongly retai led by these columns, as well as by NaCl columns, and (2) basic proteins ire eluted by very low molarities of MgC12 and CaC12, while acidic proterns are not eluted by these solvents and neutral proteins are eluted only by MgC12 at 1 M, (3) that acidic proteins are not eluted by NaCl, while basic proteins are eluted by that solvent at molarities between 0.1 and 0.3, and (4) that acidic proteins are G. Bernardi and W. H. Cook, Biochim. Biophys. Acta 44, 96 (1960).

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eluted by NaF and phosphate solvents at low molarity (-0.1 M), while basic proteins are eluted at molarities similar to those of NaCl, some general guidelines can be proposed. Loading

Use phosphate columns (0.001 M, pH 6.8) for mixtures of predominantly basic proteins, or if it is desired to retain basic proteins on the column, while possibly losing some acidic ones. Use NaCl columns (0.001 M, unbuffered) for mixtures of predominantly acidic proteins (glycoproteins with loose structures in particular), or if in a mixture it is essential that all acidic proteins be retained on the column. Use MgCI2 or CaC12 columns (0.001 M, unbuffered) only for acidic proteins which do not bind to NaCl columns. Washing

Use the same buffer as that on the column. For PO4 columns this assures removal of most denatured proteins. Elution

All elution procedures will give the following order of protein emergence from the column: basic > neutral > acidic. Chromatographic schemes can be developed on the basis of either step or gradient elution. Step elution can be carried out very quickly. The use of gradients, although more time consuming, can provide more refined separation. Since shallow gradients are to be preferred because of better resolution, the use of a gradient in place of step elution might prove at times impractical. Three general schemes can be proposed as points of departure. These will be set up either as a series of steps, gradients, or gradients combined with steps. The choice of a step or gradient will frequently be dictated by the intention of the experiment, whether a certain class of proteins is to be eluted in a batch without resolution, or if some resolution is desired. Replacement of the 1 M MgC12 step in Scheme I by a gradient is not likely to offer any advantages. The use of a gradient in place of the 1 M NaIKCI step is meaningful only in Scheme 11, but not in Scheme 111. Elution with a phosphate gradient should always be preceeded by washing with 0.001 M pH 6.8 phosphate buffer (two column volumes) to remove the salt, at high concentration, left from the preceding step. In the case of NaCl columns one can start with a 0.00110.01 M PO4, pH 6.8 step, which might cause elution of poorly bound acidic proteins. In general one

[261

PROTEIN CHROMATOGRAPHY ON

Step elution

HA

COLUMNS

335

Gradient elution Scheme I

I. 0.005 M MgClz to elute basic proteins 11. 1.0 M MgCI2 to elute proteins with isoelectric points between 515.5 and 8 111. 0.3 M PO4 to elute acidic proteins

0.001 to 0.005 M MgC12 gradient 1.0 M MgCI2in a step 0.01 to 0.3 M PO4 gradient

Scheme I1

I. 0.005 M MgCI2 to elute basic proteins 11. 1 M NaCl or KC1 to elute proteins with isoelectric points of 7.0-7.6 and DNases 111. 0.3 M PO, to elute acidic proteins

0.001 to 0.005 M MgClz gradient 0.01 to 1.0 M NaCl or KC1 gradient 0.01 to 0.3 M PO4 gradient

Scheme 111

I. 1 M NaCl or KC1 to elute basic and neutral proteins, as well as DNases 11. 0.3 M PO4 to elute acidic proteins

1 M NaCl or KC1 in a step

0.01 to 0.3 M PO, gradient

should avoid the use of CaClz eluants, unless very dilute, since these tend to cause plugging of the column. For the purification of individual proteins a phosphate column and an appropriate gradient are sufficient. For basic proteins an NaCl gradient (0.01-0.5 M) is one of choice, since it does not elute most neutral or acidic proteins. The following examples might serve as illustration. 1. The purijication of commercial o ~ o m u c o i d , 'which ~ contains as impurities lysozyme, ovoinhibitor, conalbumin, and ovalbumin. The elution program is shown in Fig. 1 for two samples of the material. The procedure combines an NaCl column, washed with 0.001 M NaCl and stepwise elution: a 0.01 M PO4, pH 6.8 step to elute ovomucoid (a glycoprotein having a loose structure), a 0.5 M NaCl step to remove basic proteins (lysozyme and ovoinhibitor), and a 0.5 M PO4 step to wash off other acidic impurities. 2. The purijication of histone pairs from chromatin1' is shown in Fig. 2. This procedure combined an NaCl step at 0.93 M to elute H2A and H2B, then a gradient to 1.20 M NaCl to wash off residual H2A and H2B, and finally a step at 2 M NaCl to elute the arginine-rich histones, followed by 0.5 M PO4 to remove the DNA from the column. 3. The purijication of commercial 2 x recrystallized papaya lysozyme was carried out in both a phosphate and an NaCl gradient, as shown in lo 'I

M. J. Gorbunoff, J. Chrornarogr. 187, 224 (1980). R. H. Simon and G. Felsenfeld, Nucleic Acids Res. 6, 689 (1979).

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PURIFICATION PROCEDURES: CHROMATOGRAPHIC METHODS

0

50

100 150 Elution Volume ( m l )

200

[261

250

FIG. I . Purification of two samples of commercial ovomucoid on hydroxyapatite columns. Zone I consisted of inactive material with maximal UV absorption at 260 nm; zone I1 contained the ovomucoid factions; zone 111 was lysozyrne; zone IV contained the remainder of the impurities, trypsin- and chymotrypsin-active. (Reproduced from Ref. 10.)

Fig. 3. In both procedures, several basic impurities are eluted first, followed by the main peak at 0.24 M PO4 and 0.25 M C1-. The impurities accounted for a considerable fraction of the total protein and the elution profile of crude, as well as 1 x crystallized papaya lysozyme was identical to the 2 x crystallized material. It is interesting to note that commercial highly purified crystalline soybean trypsin inhibitor was found by this technique to contain up to 20% impurity. 4. The puriJication of tubulin involved the use of an MgCl2 column washed with 0.005 M MgCl2.I2This was used primarily out of considerIZ

L. Grisham, M . J. Gorbunoff, B. Price, and S . N. Tirnasheff, unpublished observations.

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FRACTION NUMBER

FIG.2. Hydroxyapatite column chromatography of chromatin. Chromatin containing 34 rng of DNA in 0.63 M Nr;21,0.1 M potassium phosphate. pH 6.7, was loaded onto a 2.5 x 20 crn column, and eluted in 18-ml fractions at 60 mllhr. The NaCl concentration of the running buffer is indicated by the dotted line. The concentration of potassium phosphate (pH 6.7) was maintained at 0.1 M until tube 79, then stepped to 0.5 M. The optical absorbance of the fractions was determined at 230 nm (solid line) and 260 nm (dashed line). Purified H2A + H2B and H3 + H4 were obtained by pooling fractions 26-42 and 66-69, respectively. (Reproduced from Ref. 11.)

I

Phosphate Grodient

7 ,

E m

C

0

I

I

I

I

I

I

z

(3

0

25

50

75

100

C_

30 ml Elution Volume (mi

FIG.3. Purification of recrystallized papaya lysozyme on hydroxyapatite columns. The protein was loaded on a 1 x 20 cm column in 0.001 M PO4. The appropriate gradient was started after washing with 30 ml of the loading buffer.

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[261

Fraction IT

! Fraction m:

Fraction I

nA

0

10

20

I

30 40 50 Eluted Volume, ml

60

70

80

FIG.4. Purification of Weisenberg tubulin on a hydroxyapatite column. The 1 x 20 cm column was prewashed with 95 ml of 0.005 M MgCI,, 25 mg of protein was loaded on the column and washed with 30 ml of 0.005 M MgC12. A 0.001 to 0.3 M KP04 gradient was applied (50 g of each) and 2-ml fractions were collected. The eluted material was monitored at 275 and 256 nm. All operations were performed at 4" due to protein instability and to prevent aggregation at higher temperatures.

ation of tubulin stability, since this highly labile protein is stabilized by Mg2+ions. The procedure, shown in Fig. 4, involved washing with 0.005 M MgC12, which did not elute any protein, followed by a 0.001 to 0.3 M KP04 gradient. Three peaks were eluted. The first peak, which contained 15% of the protein, was identified as partially denatured tubulin which has lost its GTP. It became bound to HA probably because of the use of MgC12 column. The second peak (85% of the protein) was pure tubulin which contained 2.0 GTP molecules per tubulin dimer. The third peak contained no protein. It was identified as pure GTP. It seems interesting to remark that when the loading column and washing buffer contained no magnesium ions, the protein came out in a single more diffuse peak. This may be due either to the washing through of the denatured tubulin or of the enhanced binding of the native GTP-containing tubulin to the column in the Mg2+cycle, in view of the high affinity of the GTP for divalent cations. NOTE: It has been called to the attention of the author that the chromatographic behavior of commercial hydroxyapatite might differ widely between suppliers. All the studies reported here were performed with hydroxyapatite prepared in the laboratory by the Bernardi procedure2 and

[2 71

HYDROPHOBIC CHROMATOGRAPHY

339

stored in 0.001 M phosphate buffer. This material gave no trouble with flow rates unless it was several months old. Acknowledgment This work was supported in part by NIH Grant GM-14603.

[271 Hydrophobic Chromatography

Hydrophobic interactions are a phenomenon of great biological significance. They are one of the main forces that stabilize the three-dimensional structure of proteins. Hydrophobic interactions are involved in antibody-antigen reactions and enzyme-substrate reactions. They also contribute to the maintenance of the lipid bilayer structure of biological membranes and are involved in the binding of proteins to these membranes. Hydrophobic interactions can be exploited and used as a means of separation. Hydrophobic matrices can be constructed and used for the fractionation of mixtures containing molecules with hydrophobic moieties. We should start by saying something about the nature of hydrophobic interactions. A definition of hydrophobicity is the repulsion between a nonpolar compound and a polar environment such as water. When a single hydrophobic compound is put into water, an energetically unfavorable condition results. The hydrophobic compound forces the surrounding water into an ordered structure as if it were forming a cavity. This process occurs with a decrease in entropy. If two or more nonpolar compounds are put into water, they aggregate spontaneously. This aggregation is due to hydrophobic interactions. Hydrophobic interactions are energetically favorable because of a gain in entropy; that is, there is an increase in disorder when there are less hydrophobic sites exposed to the polar environment. Hydrophobic interactions are neither a binding of hydrophobic groups to each other, nor are they attractive forces per se. Hydrophobic interactions are forced on nonpolar compounds by the polar environment. It is the structure of the water that creates hydrophobic interactions. Given that it is the structure of water that creates hydrophobic interactions, it follows that if one changes the structure of water by dissolving METHODS IN ENZYMOLOGY, VOL. 182

Copyright O 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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salts or organic solvents in it, then hydrophobic interactions would somehow be affected. Generally speaking, increasing ionic strength increases hydrophobic interactions. Both anions and cations can be listed in a series from those that highly favor hydrophobic interactions to those that decrease hydrophobic interactions. For anions, the series is P043- > Sod2> CH3COO- > C1- > Br- > NO3- > Clod- > I- > SCN-, and for cations the series is NH4+ > Rb+ > K+ > Na+ > Cs+ > Li+ > Mg2+> Ca2+ > Ba2+. The strong chaotropic salts disrupt the structure of water and thus tend to decrease the strength of hydrophobic interactions; the antichaotropic salts tend to favor them. Organic solvents are also commonly used to alter the polarity of water. We will, primarily, be discussing the separation of proteins, but keep in mind that most biomolecules have a certain degree of hydrophobic character. The degree of hydrophobicity of a protein is dependent on its amino acid sequence. Certain amino acids are hydrophobic; in order of decreasing hydrophobicity they are tryptophan, norleucine, phenylalanine, tyrosine, leucine, valine, methionine, alanine. Hydrophobic interactions stabilize the tertiary and quaternary structure of proteins. In addition, many hydrophobic amino acids are exposed on the surface and it is these that give a native protein its degree of hydrophobicity. The ability of a protein to undergo hydrophobic interactions in the native state depends on its surface hydrophobic sites and surface hydrophobicity depends on an intact tertiary or quaternary structure. This is to be distinguished from the native hydrophobicity of the protein, which is dependent on its primary structure. When using hydrophobicity as a basis for separation, these differing sources of hydrophobicity in a protein, those which arise from tertiary and quaternary structure and those which arise from primary structure, can be exploited. Several general works are available which explore hydrophobic interactions in depth.',2 Comparison of Hydrophobic Interaction Chromatography and Reversed-Phase Chromatography

Hydrophobic interaction chromatography (HIC) and reversed-phase chromatography (RPC) are two separation methods based on the interactions between the hydrophobic moieties of a sample and an insoluble, immobilized hydrophobic group (i.e., those on the matrix). It is helpful to know the basic differences between HIC and RPC. In hydrophobic interaction chromatography the matrix is hydrophilic and is substituted with C. Tanford, "The Hydrophobic Effect." Wiley, New York, 1980. A. Ben-Naim, "Hydrophobic Interactions." Plenum, New York, 1980.

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HYDROPHOBIC CHROMATOGRAPHY -

34 1

short-chain phenyl or octyl nonpolar groups. The mobile phase is usually an aqueous salt solution. In reversed phase chromatography the matrix is silica that has been substituted with longer n-alkyl chains, usually Cs (octylsilyl)or CI8(octadecylsilyl). The matrix is less polar than the mobile phase. The mobile phase is usually a mixture of water and a less polar organic modifier. In early work a distinction was made between methods on the basis of the polarity of the mobile phase. Normal phase systems were those in which the matrix was silica and the mobile phase was a nonpolar solvent such as hexane. Reversed-phase systems were so called because in these the mobile phase, a water solution, is more polar than the stationary phase, normally a Cs or Clg derivatized silica. Separations on HIC matrices are usually done in aqueous salt solutions, which generally are nondenaturing conditions. Samples are loaded onto the matrix in a high-salt buffer and elution is by a descending salt gradient. Separations on reversed-phase media are usually done in mixtures of aqueous and organic solvents, which are often denaturing conditions. These two methods exploit the different sources of the hydrophobicity of proteins. HIC depends on surface hydrophobic groups and is carried out under conditions which maintain the integrity of the protein molecule. RPC depends on the native hydrophobicity of the protein and is carried out under conditions which expose nearly all hydrophobic groups to the matrix, i.e., denaturing conditions. A study done by Fausnaugh et al. compared the recovery of enzyme activity after HIC and RPC.3 Procedures for Hydrophobic Chromatography

Fortunately, although the mechanics of hydrophobic interactions are complicated, the chromatographic techniques based on hydrophobic interactions are easy to use.

Choice of Gel For an uncharacterized protein, a phenyl-substituted resin is usually the best choice to start, because strongly hydrophobic proteins will not easily be eluted from octyl-substituted resins. The phenyl ligand is intermediate in hydrophobicity between n-butyl and n-pentyl, and will bind to aromatic amino acids through T-.rr interactions. Octyl-substituted resins such as octyl-Sepharose CL-4B can be used for weakly hydrophobic proteins. It is also the medium of choice for use with membrane proteins since it still retains hydrophobic binding properties in the presence of solubilizing concentrations of detergents. J. L. Fausnaugh, L. A. Kennedy, and F. E. Regnier, J. Chromutogr. 317, 141 (1985).

[271

-

Binding. The binding of proteins to hydrophobic gels is influenced by:

-

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Running the Column

1. The hydrophobicity of the ligand: For example, phenyl-Sepharose CL-4B is less hydrophobic than octyl-Sepharose CL-4B. 2. The ionic strength of the buffer: Those salts which cause salting out [e.g., (NH4)2S04]also promote the binding of proteins to hydrophobic ligands. Binding to octyl- and phenyl-Sepharose CL-4B is generally negligible unless high-salt buffer solutions are used. A salt concentration just below that used for salting out the protein is normally used. 3. Temperature: It has been noted that a 20-30% reduction in binding strength is seen when the temperature is reduced from 20 to 4". The strength of the hydrophobic interactions will be lessened, therefore, if the experiment is done in a cold room. To ensure that sample molecules bind to HIC supports, it is usually necessary to add something to the sample that will increase the hydrophobic interactions between the sample and the matrix. In HIC this can be done in several ways. One of the more common strategies is to apply the sample in a high concentration of salt [1.7 M (NH4)2S04,4 M KC!, 4 M NaCl] in a buffered solution, pH range from 6.5 to 8.0. Elution. Once the sample has been applied to the column and the hydrophobic species of interest has bound, unbound proteins are washed through with the starting buffer. Elution of the protein of interest can be done in several ways: 1. Reducing the concentration of salting out ions in the buffer with a negative salt gradient 2. Increasing the concentration of chaotropic ions in the buffer in a positive gradient 3. Eluting with a positive gradient of a detergent (note that the gel must be cleaned afterward due to the hydrophobic nature of detergents) or with a polarity-reducing organic solvent, usually ethylene glycol (up to 75% ethylene glycol has been used) 4. Raising the pH 5. Reducing the temperature Note that the hydrophobicity of the ligand used will affect the ease of desorption. Elution gradients can be either step or linear. Most of the elution strategies are nondenaturing. Use of detergents and lowering the polarity of the eluent are often last-resort methods used to elute a very strongly bound protein since these two procedures often denature proteins. It is often good practice to utilize two or more of these elution tech-

C

-

3$

f-

t

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CHROMATOGRAPHY ON IMMOBILIZED REACTIVE DYES

343

niques simultaneously. The existence of a wide variety of possible elution methods is potentially very valuable for the resolution of complex mixtures. Extensive information on specific applications is available from the manufacturers of hydrophobic media and is not presented here.

C

I:

$

Regeneration and Storage

HIC gels can be reused several times; exactly how many times depends on the quality of the buffers, sample, etc. After every chromatographic run, a wash with 6 M urea will remove tightly bound proteins. The gel can then be equilibrated with starting buffer and is immediately ready for the next run. If detergents have been used on the gel, the cleaning procedure is slightly more complicated. The following procedure is recomrnended by Pharmacia (Piscataway, NJ) for cleaning octyl- and phenylSepharose after use with detergents. Wash the gel sequentially with 1. One bed volume of distilled water 2. One bed volume each of 25, 50, and 95% ethanol 3. Two bed volumes of n-butanol 4. One bed volume of 95, 50, and 25% ethanol 5. One bed volume of distilled water 6 . Reequilibrate the gel with starting buffer to make it ready for the next experiment Chromatography is the most accepted separation tool in modern biochemistry laboratories. Each chromatographic method exploits different physical or biological properties of the molecule as a basis for separation. In this chapter we have explored the uses of hydrophobicity as a basis for two chromatographic methods, HIC and RPC. Most protein purifications require more than one chromatographic step. Hydrophobicity is an often overlooked physiochemical property of the biomolecule which can be exploited in the logical design of a protein purification scheme.

t

1281 Chromatography on Immobilized Reactive Dyes By EARLESTELLWAGEN

Of all the fractionation procedures used in protein purification, only affinity chromatography takes advantage of the property that clearly distinguishes one protein from another, namely its function. The surfaces of virtually all proteins are designed to selectively bind one or a small numMETHODS IN ENZYMOLOGY, VOL. 182

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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ber of molecules with high affinity. Immobilization of such a molecule on a porous inert support should result in the retention of its binding protein from a mixture of proteins. Subsequent elution with the soluble or mobile form of the same molecule should then release the retained protein. Such release results from the competition between the mobile and immobilized forms of the molecule for the binding site on the protein, resulting in an appreciable enrichment of the desired protein. Unfortunately, execution of affinity chromatography is frequently not as simple a s its conceptualization. First, the molecule must be covalently attached to the matrix without significantly reducing its affinity for the desired binding protein, often requiring adroit chemistry. Second, a separate affinity chromatographic matrix must be constructed for each protein to be purified, requiring a substantial fiscal investment if a variety of different proteins are to be purified. Third, affinity chromatography frequently is restricted to a late step in a purification procedure in order to avoid cleavage of the immobilized molecule by degradative enzymes present in crude cellular extracts. Principles and Advantages of Immobilized Dye Chromatography Immobilized reactive textile dyes free the experimentalist from these limitations without compromising purification enhancement. A reactive textile dye in solution can function as a competitive inhibitor for the substrate, coenzyme, or effector of a variety of proteins, often with an affinity greater than that exhibited by the competitive molecule. This affinity likely results from the flexibility of the dye which can assume the polarity and geometry of the surface of a variety of competitive biomolecules. Accordingly, immobilization of a single reactive dye should generate an affinity column useful in the purification of numerous proteins. For example, the immobilized dye reactive blue 2 has been reported' to contribute significantly to the purification of over 60 different proteins. Both free and immobilized reactive dyes are available individually and as kits from a variety of suppliers at modest cost. The chemical structure of reactive dyes facilitates their rapid covalent coupling with porous matrices without recourse to prior chemistry, activation reagents, or spacer arms. An immobilized dye can simultaneously retain a sizable number of different proteins ranging from about 5 to 60% of the protein in a crude .~ results from selective elution by the competcellular e ~ t r a c tPurification itive mobile chemical for the retained protein of interest. Since immobiG . Kopperschlager, H.-J. Bohme, and E. Hofmann, Adu. Biochem. Eng. 25, 101 (1982).

' R . K. Scopes. J. Chromatogr. 376, 13 1 (1986).

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lized reactive dyes are not easily hydrolyzed, they survive exposure to crude cellular extracts facilitating achievement of the full purification enhancement afforded by affinity chromatography. Indeed, many proteins have been purified to homogeneity in a single step using immobilized dye chromatography. In summary, a given immobilized dye can function as an inexpensive stable reagent capable of impressive selectivity and capacity in the purification of a large number of quite different proteins. Getting Started Since the color and hence the structure of each reactive dye is different, each reactive dye will have a somewhat different affinity for a biofunctional site on a given protein. Unfortunately, the affinity of a particular reactive dye for a biofunctional site cannot be predicted with any confidence, necessitating an empirical screening procedure to optimize chromatography. Fortunately, the screening procedure can be completed rather expeditiously, requiring only a small amount of a crude protein extract, a general assay for protein such as the Lowry or Bradford colorimetric procedure, a specific assay for the protein of interest, and a supply of free or immobilized reactive dyes. The simplest screening procedure involves purchase of a kit of immobilized dyes currently available from at least two suppliers, Amicon and Sigma. The characteristics of the immobilized dyes in these kits are given in Table I along with the characteristics of some additional immobilized dyes available individually. The chemical composition of the chromatographic solvent and the crude enzyme extract can be very important to the results obtained by screening. The first consideration must be to maintain the function of the protein of interest. Accordingly, the range in acceptable pH, ionic strength, metal ion composition, temperature, and redox environment will be limited by the inherent properties of the protein. The range in these parameters acceptable for protein function are normally also acceptable for immobilized dye chromatography save one, ionic strength. The affinity of most proteins for immobilized dyes is substantially weakened in solvents having a total ionic strength in excess of 0.2 M. Accordingly, it is well to limit the ionic strength of both the crude extract and the chromatographic solvent to 0.05 M. This may be achieved in the crude extract by simple dilution. If the solvent requirements of the protein are not well known, it is suggested that the initial screening be done in chromatographic solvents containing 50 m M Tris-C1 at pH 7.5 and ambient temperature and that the effects of pH, temperature, ionic strength, and chemical additives (such as EDTA, metallic cations, redox reagents, coenzymes and effectors) be examined systematically.

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TABLE I AVAILABLE IMMOBILIZED DYES" Dye name Group

Generic

1

Yellow 3 Yellow 86 Blue 4 Blue 2

Yellow H-A Yellow M-8G Blue MX-R Yellow MX-3R Blue H-B

Red 120 Brown 10 Green 5 Green 19

Red H-E3B Brown MX-5BR Green H-4G Green H-E4BD

2 3

4 5

Commercialb

Color index number

Supplier Amicon' Matrex gel orange A

-

Matrex gel orange B Matrex gel blue A Matrex gel red A -

Matrex gel green A

Sigmad Yellow 3-Agarose Yellow 86-Agarose Blue 4-Agarose Blue 3GA-Agarose Blue 2-Sepharose Red 120-Agarose Brown 10-Agarose Green 5-Agarose Green 19-Agarose

" Some additional immobilized reactive dyes are available but their group designations are not known. All listed dyes are Procion dyes. Each of the Amicon immobilized dyes listed are included in a kit. Each of the Sigma immobilized dyes listed except for yellow 3 and green 19 are included in a kit.

Such screening can be accelerated using 0.5- to 2.0-ml columns of immobilized dyes placed in disposable columns or simply in a glass woolplugged Pasteur pipet, application of a minimum of crude extract commensurate with good analytical results but not to exceed 20 rng total proteiniml column, and gravitational flow. Alternatively, screening can be done centrifugally using smaller amounts of immobilized dyes in plastic microcentrifuge tubes. The column flow-through as well as three column volumes of wash done with the chromatographic solvent should be analyzed for both total protein and total desired protein and compared to the same quantities in the crude extract. The difference between the total protein applied and recovered and the total desired protein applied and recovered represents the retention by the immobilized dye column. Two extremes should be identified by the screening procedure: the identity of the immobilized dye, which retains a maximum of total protein and a minimum of desired protein, and the immobilized dye, which retains a minimum of total protein and a maximum of desired protein. The former will be termed the negative immobilized dye and the latter the positive immobilized dye. The capacity of both the negative and the positive immobilized dyes for both total protein and desired protein in the crude extract should next

CHROMATOGRAPHY ON IMMOBILIZED REACTIVE DYES

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be estimated by frontal analysis. This simply entails continuous application of crude extract to each column until the concentrations of total and desired protein in the crude extract and in the column effluent are identical. The volume of crude extract applied to each column prior to this equivalence indicates the amount of total protein and desired protein retained by the column. The investigator should now be in a position to rationally decide whether to use a single column of the positive immobilized dye in the development of a purification protocol or whether to use both the negative and positive immobilized dye columns in tandem.3 Examples of single-column and tandem-column protocols in the purification of a protein to homogeneity in a single step are given below. Optimal conditions for elution of the desired protein from the positive immobilized dye should now be screened. Each screening should begin by application of sufficient crude extract to the positive immobilized dye s o as not to overload the column while supplying sufficient desired protein to facilitate reliable quantitative measurement upon its elution. The column should be first screened using a nonspecific eluant such as salt to demonstrate that the retention of the desired protein is reversible and to determine a solvent to conveniently recycle the column. A chromatographic solvent containing 1 M NaCl is usually adequate for this purpose although salts more potent in the lyotropic series may be found n e c e ~ s a r y .If~ retention of the desired protein is reversed by salt, the positive immobilized dye should then be screened to optimize specific elution using a substrate, coenzyme, inhibitor, o r effector. A good starting point might be selection of an anionic molecule at a concentration at least 10 times its dissociation constant from the free enzyme. If the latter is not known, initiate screening with a 10 m M solution and define the minimum concentration required to rapidly elute the desired protein. In some cases it may be necessary to employ combinations of molecules in the elution solvent or to weaken the affinity of the desired protein for the immobilized dye by addition of a modest concentration of salt in order to effect specific The investigator should now be in a position to perform immobilized dye chromatography at a laboratory scale. The volume of the immobilized dye column required will depend upon the features of the desired protein, namely the amount wanted, the amount present in the crude extract, and the fractional recovery. Experience suggests that useful parameters include a column heightldiameter ratio of 2 to 5 , a flow rate of 1 to 5 mllmin,

' Y. Hey and P. D. G . Dean, Biochem. J. 209, 363 (1983). J. B. Robinson, Jr., J . M. Strottmann, and E. Stellwagen, Proc. Natl. Acad. Sci. U.S.A. 78, 2287 (1981).

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and washing and elution with about 1 to five column volumes of chromatographic solvent. In most cases, elution with a molecule at the concentration established during screening will produce a good purification of the desired protein. However, when using a molecule which binds to many proteins, such as ATP, it may be advantageous to employ gradient elution. While each of these parameters can always be improved, it must be recognized that they are all interdependent; that a good purification, yield of protein, and ease of operation are the ultimate criteria on which decisions should be based; and that each purification situation has its own peculiarities. Representative Purification Procedures

A representative protocol employing a single positive immobilized dye column is that used in the purification of Trp-tRNA synthetase (TrptKNA l i g a ~ e ) A . ~ series of 1-ml columns, each having 1 of 32 different reactive dyes covalently attached to Sepharose 6B, were screened for their ability to retain protein in a crude extract of Bacillus stearothermophilus. Each column was first equilibrated with a solvent containing 10 mM phosphate buffer, pH 6.5, 10 mM 2-mercaptoethanol, and 0.1 mM phenylmethylsulfonyl fluoride (PMSF; a protease inhibitor) at 4". One milliliter of crude extract containing 2.5 mg protein was applied to each column followed by 4 ml of buffer and the total protein and synthetase activity in the 5 ml of effluent was measured. The total amount of protein retained varied from 0.1 mg for Procion blue H-ERD to 2.0 mg for Procion orange MX-G. The synthetase activity was totally retained by seven different immobilized dye columns. Three of these columns, Procion brown MX-5BR. Procion green H-E4BD, arrd Procion red H-8BN, were selected for further screening because they retained the least amount of total protein. Each of these columns was next washed with 4 ml of buffer containing 50 mM tryptophan substrate and the effluent analyzed for synthetase activity. Ninety percent of the synthetase activity in the crude extract was eluted by this solvent. These results were then translated to a laboratory-scale purification involving application of about 2 g of protein in a partially purified extract to a 50-ml column of immobilized Procion brown MX-SBR (reactive brown 10) equilibrated with the solvent described above. The enzyme was retained during application and subsequent washing with 10 column volumes of equilibration solvent. The column was then washed with two column volumes of equilibration solvent containing 50 mM tryptophan C. J . Bruton and T. Atkinson, Nucleic Acids Res. 7, 1579 (1979).

[281

T

K C7 K

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and the enzyme located entirely in three 10-ml fractions of eluant. Analysis indicated a 137-fold purification of enzyme in an 88% yield that was homogeneous as determined by SDS-PAGE. A representative protocol using a two-column or tandem arrangement of immobilized dyes is illustrated in the purification of the enzyme alcohol dehydrogenase from Zymononas m o b i l i ~A . ~ total of 45 reactive dyes covalently attached to the Sepharose CL-4B were screened for their retention of total protein and of enzyme. A 2-ml column of each immobilized dye was equilibrated at room temperature with 10 mM MESIKOH buffer, pH 6.5, containing 30 m M NaC1, 2 mM MgC12, 10 m M ascorbate, and 0.5 mM ferrous ammonium sulfate at room temperature. The reagents in addition to the buffer were added to maintain the function of the enzyme and did not interfere with chromatography. About 3 ml of crude extract containing 30 mg of total protein was added to each column followed by 5 ml of buffer. Sixteen immobilized dyes, among them Procion green H-E4BD (reactive green 19), retained no enzyme while 7 immobilized dye columns retained at least 80% of the enzyme. Among the latter immobilized dyes, Procion blue H-4R and Procion blue H-B (reactive blue 2) bound the least enzyme. The enzyme retained by these columns could be quantitatively eluted by addition of either 1 mM NAD or 0.2 m M NADH to the buffer. These observations were translated to tandem chromatography at the laboratory scale by location of a 240-1111 column (15 x 4.5 cm) of immobilized green H-E4BD (1.3 mg dyelg gel) immediately above a 207-ml column (13 X 4.5 cm) of either immobilized blue H-4R (0.9 mg dyelg gel) or immobilized blue H-B. Four hundred milliliters of crude extract containing 3.9 g total protein was applied to the upper green column at a flow rate of 6.7 mllmin and washed with 300-400 ml of buffer. The upper green column was then removed and the lower blue column washed with another 300 ml of buffer and then with 150 ml of buffer containing 1 mM NAD. The eluant fractions containing enzyme were pooled and concentrated. This procedure resulted in a 36-fold purification of enzyme in 80% yield, giving a preparation which was at least 98% homogeneous as judged by SDS-PAGE. Regeneration and Storage of Immobilized Dyes

While washing with 1 M NaCI will usually eluate nearly all the retained protein, a column will become slowly contaminated with retained protein A. D. Neale, R. K. Scopes, J . M. Kelly, and R. E. H. Wettenhall, Eur. J . Biochem. 154, 119 (1986).

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-

-

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which is not removed by this solvent, particularly upon repeated application of crude extracts. Such protein can normally be eluted using several column volumes of either a strong base such as 0.5 M NaOH, a strong denaturant such as 8 M urea, 6 M guanidinium chloride, or 1% SDS, a potent lyotropic salt such as 3 M KSCN, a mixed organic solvent such as chloroform-methanol, or a sequence of these solutions. The column should then be flushed with many column volumes of water to ensure the removal of these agents whose presence would be detrimental to the purification of a functional protein. The washed column should then be stored at 4' in a dilute solution of a microbial growth inhibitor such as 0.02% sodium azide. The initial eluate from a stored immobilized dye column is often colored, indicating that some of the immobilized dye has become mobile on storage. This bleeding results from hydrolysis of the glycosidic linkages of the matrix and not from the reversibility of dye conjugation. Accordingly, a stored column should be washed until the effluent appears clear in order not to contaminate the protein purified with soluble dye-oligosaccharide. Preparation of Immobilized Reactive Dye Columns

As noted above, several investigative groups have utilized immobilized dye columns in addition to those available from suppliers in order to optimize single and tandem immobilized dye chromatography. While this may not be necessary to achieve acceptable purification as opposed to optimal purification, a variety of individual reactive dyes are available from numerous suppliers, some of which are listed in Table 11, as well as a kit of 17 reactive dyes from Polysciences, all at modest cost. The reactive dyes listed in Table 11, as well as 44 others, have been placed in 5 general categories based on their ability to retain total protein from a variety of crude extracts of intracellular protein, with group 1 retaining the most protein and group 5 the least p r ~ t e i nIndividual .~ dyes are designed by a generic name, a commercial name, and a color index (CI) number when known. The interrelationship of these designators is given in the Colour Index published by The Society of Dyers and Colorists in six volumes. The most commonly used reactive dyes are the Procion dyes, produced by Imperial Chemical Industries (ICI) (Manchester, England), and the Cibacron dyes, produced by Ciba-Geigy (Basel, Switzerland). These dyes consist of a polysulfonated chromophore linked to a chlorotriazine group by an aminoether bridge. Bright blue dyes have an anthraquinone chromophore, turquoise dyes have a phthalocyanine chromophore, yellow, orange, and red dyes have an azo chromophore, green dyes have mixed chromophores, while violet, navy, brown, and black dyes are metal com-

[281

CHROMATOGRAPHY ON IMMOBILIZED REACTIVE DYES

35 1

TABLE I1 AVAILABLE REACTIVEDYES

Groupa 1

2

3

Generic name

Commercial name

Blue 140 Orange 16 Violet 5 Yellow 86 Blue 4 Blue 15

Procion turquoise MX-G Remazol brilliant orange 3R Remazol brilliant violet 5R Procion yellow M-8G Procion blue MX-R Cibacron turquoise blue GF-P Procion turquoise H-GF Procion orange H-GR Procion yellow MX-4R Procion scarlet MX-G Procion blue MX-3G Cibacron blue 3G-A Procion blue H-B Remazol brilliant blue R Cibacron brilliant red 4G-E Procion red H-E3B Cibacron brilliant yellow 3G-P Procion yellow H-5G Cibacron brilliant blue BR-P Procion blue H-GR Procion brown MX-5BR Procion orange MX-2R Cibacron brilliant red 3B-A Procion red H-7B Procion red MX-8B Procion violet H-3R Procion blue H-ERD Cibacron brilliant green 4G-A Procion green H-4G Procion green H-E4BD Cibacron brilliant red 4B-E Procion red H-8BN Procion red H-E7B Procion yellow MX-GR

Orange 1 Orange 14 Red 8 Blue 1 Blue 2 Blue 19 Red 120 Yellow 2

4

Blue 5 Brown 10 Orange 4 Red 4

5

Red I I Violet 1 Blue 160 Green 5 Green 19 Red 58 Red 141 Yellow 7

a

Color index number

Supplierh P A A I, P, Si A, G, I, P, Se, Si A P P, Se, Si A G, P A, G , I, P, Se, Si

Based on ability to bind protein from crude extracts with group I binding least and group 5 most. Known commercial suppliers include the following: A, Aldrich Chemical Co. (Milwaukee, WI); G, Gallard-Schlesinger Industries (Carle Place, NY); I, ICN Biomedicals Inc. (Costa Mesa, CA); P, Polysciences Inc. (Wanington, PA); Se, Serva Fine Chemicals Inc. (Westbury, NY); Si, Sigma Chemical Co. (St. Louis, MO).

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CI

so,FIG. 1 . Structures of some reactive dyes: (a) yellow 3; (b) cationic dyez0;(c) red 8; (d) blue 2: ( e )extended blue Z2-'

plexes of hydroxyazo chromophores. Monochlorotriazine dyes are designated H, HE, or P by ICI and Cibacron by Ciba-Geigy, while dichlorotriazine dyes are designed MX by ICI. Some typical structures are illustrated in Fig. 1. Reactive dyes of alternative chemistry are available from other manufacturers, including Farbwerke Hoechst AG (Frankfurt, FRG) Remazol dyes having a sulfatoethyl sulfone as the reactive group,

[28]

CHROMATOGRAPHY ON IMMOBILIZED REACTIVE DYES

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Bayer AG (Leverkusen, FRG) Leavfix dyes having difluorochloropyrimidinyl as the reactive group, and Sandoz, Ltd. (Basel, Switzerland) as Drimarene dyes having trichloropyrimidinyl as the reactive group. Commercial samples of reactive dyes usually contain a series of additives, including phosphate buffer, NaC1, and surfactants to support their use as textile dyes as well as minor amounts of other chromophores generated in the synthesis of the dyes. The salts can easily be removed by precipitation of dye as the potassium salt from aqueous solution. This treatment normally suffices for preparation for an immobilized dye column although the more fastidious investigator may wish to purify the major chromophore by adsorption chromatography." While reactive dyes have been immobilized on a variety of mat rice^,^ cross-linked agarose has been a very satisfactory general purpose matrix. A 100-ml column of Sepharose 4B-CL or 6B-CL containing about 1 to 5 mg dyelg matrix can be prepared from mono- and dichlorotriazine reactive dyes using the following protocol. About 80 g of matrix, which has been washed and air dried on a sintered glass funnel, is suspended in 280 ml of water. Reactive dye (1.2 g) is dissolved in 80 ml of water and added to the matrix suspension followed by 40 ml of 4 M NaCI. Then 0.5 ml of 10 M NaOH is added if a dichlorotriazine dye is being used and the mixture is gently stirred for 4 hr at ambient temperature. If a rnonochlorotriazine dye is used, 4 ml of 10 M NaOH is added and the mixture is stirred for 72 hr at ambient temperature or 16 hr at 55-60". The mixture is then filtered and washed with copious quantities of water, 1 M NaCI, and water again until the filtrate is clear. Any remaining chloro groups on the immobilized dye should be replaced with amino groups by suspension of the dyeSepharose in 2 M ammonium chloride at pH 8.5 for 4 hr at ambient temperature followed by washing in water.9 The amount of immobilized dye can be determined by brief acid hy~ of drolysis followed by a spectrophotometric r n e a ~ u r e m e n t . ~A, 'portion the immobilized dye is added to 0.6 ml of 5 M HCl and maintained at 37" for 5 min. Then 2.4 ml of 2.5 M phosphate buffer, pH 7.5, is added and the absorbance of the solution measured at the A,, for the dye. Immobilized dye concentration is commonly reported as milligrams dye per gram wet weight of matrix. The spectral parameters for several reactive dyes have

' C. R . Lowe and J . C. Pearson, this series, Vol. 104, p. 97.

'O

P. D. G. Dean and F. Quadri. in "Solid Phase Biochemistry" (W. H. Scouten, ed.), p. 79. Wiley, New York, 1983. T. Atkinson, P. M. Hammond, R. D. Hartwell, P. Hughes, M. D. Scawen, R. F. Sherwood. D. A. P. Small, C. J. Bruton, M. J. Harvey, and C. R . Lowe, Trans. Biochem. Soc. 9, 290 (1981). G . K. Chambers. Anal. Biochem. 83, 551 (1977).

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METHODS

TABLE 111 REACTIVEDYEPARAMETERS Visible spectrum Group 1 2 3 4

5

a

Generic name

,A

(nm)

E (mM-' cm-')

Molecular weighto

Ref.

Yellow 3 Blue 4 Blue 2 Red 120 Blue 19 Brown 10 Green 5 Green 19 Red 58 For the acid form.

been reported and are listed in Table III.7,11-14 The visible absorbance maximum and the extinction coefficient for other dyes need be determined by the investigator using the same dye preparation employed in the immobilization reaction. It should be noted that less than 5% of an immobilized dye can retain protein,I5 an observation in keeping with affinity chromatography in general. Nonetheless, this concentration of immobilized dye can facilitate interaction of a retained protein with several immobilized dyes simultaneously. This may result from occupation of more than one of the functional sites on a protein having multiple s u b ~ n i t s l or ~ -from ' ~ weak interactions at other protein sites.'* The occurrence of such multiple interactions will increase the concentration of the competitive biomolecules necessary for elution of the desired protein. Alternative Procedures to Retain Additional Desired Proteins

In spite of the range of reactive dyes available, some proteins of interest are not retained by immobilized reactive dyes. The presence of a relatively low concentration of a metallic cation such as Zn2+, Co2+, Y. D. Clonis, K. Jones, and C. R. Lowe, J. Chromatogr. 363, 31 (1986). S. T. Thompson and E. Stellwagen, Proc. Nail. Acad. Sci. U.S.A. 73, 361 (1976). l 3 P. D. G. Dean and D. H. Watson, J. Chromatogr. 105, 301 (1979). l4 D. Mislovicova, P. Gemeiner, L. Kuniak, and J. Zemek, J . Chromatogr. 194, 95 (1980). l5 Y.-C. Liu, R. Ledger, and E. Stellwagen, J. Biol. Chem. 259, 3796 (1984). l6 P. J. Hogg and D. J. Winzor, Arch. Biochem. Biophys. 240, 70 (1985). l 7 Y.-C. Liu and E. Stellwagen, J. Biol. Chem. 262, 583 (1987). l 8 R. K. Scopes, Anal. Biochem. 165, 235 (1987). l1

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Mn2+,Ni2+, Cu2+, or A13+ in the chromatographic solvent has been found19to facilitate the retention of a number of proteins. Such proteins can be selectively eluted by addition of a chelation agent to the chromatographic solvent in the absence of metal cations. The proteins in crude extracts containing nonionic detergents normally cannot be retained by immobilized dyes because these detergents encapsulate the immobilized dyes in micelles. However, addition of a relatively small concentration of an anionic detergent such as SDS or deoxycholate will often result in the retention of proteins of interest.20It is assumed that anionic and nonionic detergents form mixed micelles that prevent encapsulation of the anionic immobilized dye by charge repulsion. The amount of anionic detergent necessary for retention of a protein of interest must be determined empirically. While most proteins are designed to bind anionic molecules, a number of proteins selectively bind cationic molecules. Such proteins will have anionic groups in their binding sites that will repel the anionic functional ~ catgroups on immobilized reactive textile dyes. R e ~ e n t l y a, ~reactive ionic dye similar in structure to Procion yellow MX-R (Fig. lb) has been synthesized and immobilized on Sepharose 4B-CL. The immobilized cationic dye did not retain proteins such as lactate dehydrogenase, serum albumin, and chymotrypsin, which are retained by immobilized anionic reactive dye, but did retain proteases such as trypsin, thrombin, and carboxypeptidase, which are specific for lysyl and arginyl peptides. Application of a crude pancreatic extract to an 8-ml column of immobilized cationic dye in 0.1 M acetate buffer, pH 5.5, resulted in the retention of all the trypsin activity. After washing in 0.9 M NaC1, the enzyme was eluted with glycine buffer, pH 2.1, in about 80% yield with an 1 I-fold purification to a specific activity characteristic for commercially purified enzyme. k

r % 1

$?

)-

r:

High-Performance Chromatography

The flow detectors present on most HPLC assemblies facilitate continuous monitoring of both total protein, using ultraviolet absorbance, and the desired protein, using a postcolumn functional assay. Since most functional assays involve a change in absorbance, a typical postcolumn assay will only necessitate a second pump to deliver the assay solution and a second absorbance flow detector. In an early report,22Procion blue MX-R (reactive blue 4) was immobilized on preparative grade glycoll9

P. Hughes, C. R . Lowe, and R. F. Sherwood, Biochim. Biophys. Acta 700, 90 (1982).

"

Sci. U.S.A. 77, 5847 (1980). Y. D. Clonis, C. V. Stead. and C. R. Lowe. Biotechnol. Bioeng. 30, 621 (1987). D. A. P. Small, T. Atkinson, and C. R. Lowe, J. Chromatogr. 266, 151 (1983).

" J. B. Robinson, Jr., J . M . Strottmann, D. G . Wick, and E. Stellwagen, Proc. Natl. Acad.

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silylated silica to yield a matrix containing 8 mg dyelg silica. This was packed into a 30 x 0.25 cm column and equilibrated with 10 mM HEPES buffer, pH 7.0, at ambient temperature and a flow rate of 10 mllmin, generating a pressure of about 500 psi. One milliliter of a clarified crude extract of rabbit muscle containing about 14 mg of protein was injected into the column. About 2 mg of homogeneous lactate dehydrogenase was eluted in 80% yield following injection of a 2-ml pulse of 2 mM NADH. The low capacity of this column for dehydrogenase, measured by frontal analysis, was ascribed to the small porosity of the silica employed (70 A). Fortunately, Serva Fine Chemicals, Inc., is now offering granular packings and prepacked columns of silica having either 300- or 500-A pores, appropriate for penetration of most proteins, to which Procion blue MX-R (reactive blue 4), Procion red HE-3B (reactive red 1291, or Cibacron F3GA (reactive blue 2) is immobilized. The latter column is also available from Alltech Associates. A process scale purification of lactate dehydrogenase has recently been described" using a 3.3 1 column of glycol silica containing 2 mg of immobilized Procion blue MX-R (reactive blue 4)/g silica equilibrated with 30 mM phosphate buffer, pH 5.5. A crude rabbit muscle preparation containing 1.8 g of protein was introduced onto the column at a flow rate of 32 llhr. After washing, the column was eluted with a 10-ml pulse of 7 mM solution of NADH to yield 100 mg of the purified enzyme in a single step having an 8.6-fold purification and a 50% yield. It is anticipated that the yield of this promising large-scale HPLC procedure could be further improved. Newer Technologies

The textile dye industry is continuously generating new reactive dyes which may be of interest for protein purification. Ciba-Geigy is now offering a Cibacron F series in which the halogen is a fluorine, thereby providing a better leaving group than chlorine in the immobilization reaction. ICI is now offering a more vivid blue reactive dye, Procion H-EGN, having a larger chromophore than Procion blue H-B (reactive blue 2), which likely will have a greater affinity for protein biofunctional sites and which has already been used to advantage in the purification of 6-phosphogluconate dehydrogena~e.'~ Crystallographic analysis of the orientation of NADH and of Cibacron blue F3GA in the coenzyme-binding site of alcohol dehydrogenase has led Y. Kroviarski, S. Cochet, A . Truskolaski, P. Boivin, and 0. Bertrand, Abstr. Int. Conf. Mod. Aspects Prorein Dye Interact.: Role Downstream Process., I s t , Compiegne, Fr. (1988).

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to the design and synthesis of new reactive dyes outside of the commercial dye industry. It was anticipated that the blue dye could better occupy the coenzyme site if the anthraquinone and the triazine rings were further separated by insertion of an ethylene bridge as shown in Fig. le. This immobilized dye.24in contrast to all other immobilized dyes and biomolecules, is able to resolve the purified alcohol dehydrogenase into two components of different activity, with the low activity form having a covalent modification on a lysine side chain. It is anticipated that additional designed dyes will increase the capability of immobilized dyes in protein purification. The chemistry employed in the synthesis of reactive dyes which is necessary for preparation of designed dyes is described in detail in two t e ~ t s . ~ ~ , ~ ~ Finally, it should be noted that the solid fluorocarbons developed by du Pont de Nemours & Co. (Wilmington, DE) afford a promising new matrix for immobilized dye chromatography. A reactive dye is first subjected to a perfluoralkylation and then the perfluoroalkylated dye is essentially irreversibly adsorbed to a fluorocarbon surface.27Accordingly, this matrix should prevent slow bleeding of dye into proteins purified by immobilized dye chromatography. Most aspects of immobilized reactive dye-protein interaction have been reviewed by several a ~ t h o r s ' . ~ , ' , and * , ' ~the concerned investigator is urged to pursue them for access to more detailed information. C. R. Lowe, S. J. Burton, J. C. Pearson, and Y. D. Clonis, J. Chrotnatogr. 376, 121 (1986). 2s W. F. Beech. "Fibre-Reactive Dyes." Logos Press, London, 1970. 26 K. Venkataraman, "The Chemistry of Synthetic Dyes," Vol. 6. Academic Press, New York, 1972. " J . V. Eveleigh, Absrr. Biotechnol. Microsymp. Macromol. Interact. AfJinity Chromatogr. Techt~ol.,Mogilany, Pol. (1988). 24

[291 Affinity Chromatography: General Methods

Affinity chromatography is one of the most powerful procedures that can be applied to protein purification. Over the years there have been many good books on the subject and many reviews of the theory of affinity chromat~graphy.'~~ This chapter will not be a further review of the I

C

J. Turkova, ed., "Affinity Chromatography." Elsevier, New York, 1978. P. Mohr and K. Pornrnerening, "Affinity Chromatography: Practical and Theoretical Aspects." Dekker, New York, 1985.

METHODS IN ENZYMOLOGY, VOL. 182

Copyright Q 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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theory behind this technique nor will it review all of the proteins that have been separated by this technique since there are so many. Discussion will center on practical ideas and general considerations behind choosing a matrix, coupling a ligand, and some of the more common problems often encountered with the implementation of this procedure. Suggestions in this chapter should allow for the easier and more productive use of affinity chromatography. As the specific purification system for the sample of interest is developed, expect modification of these procedures. This chapter serves only as a guide to performing separations of biomolecules by affinity chromatography. Areas where the manufacturer of the affinity product can be contacted for specific recommendations on the use of the product are indicated throughout. Refer specific questions to the manufacturer. Affinity chromatography as a biospecific technique began only about 20 years ago even though it had been used as an experimental separation procedure for many years.3 This procedure takes advantage of one or more biological properties of the molecule(s) being purified. These interactions are not due to the general properties of the molecule such as isoelectric point (pl), hydrophobicity, or size. This highly specific method of separation utilizes the specific reversible interactions between biomolecules . Some of the biological properties that can be exploited to effect a separation include specific shapes (that "recognize" other molecules such as receptors or enzymes), specific changes in conformation after changes in pH, or certain subareas or regions of the molecule that can interact or bind to other molecules (e.g., epitopes of antibodies). When developing a separation scheme keep in mind that the sample of interest is not the only component in the sample mixture that can be bound to an affinity matrix (gel). One affinity matrix may be specific for the sample of interest while others may be more specific for other components in the mixture (contaminating proteins). Just as the sample of interest can be bound to an affinity matrix, the contaminating proteins may also be specifically bound. An affinity gel could be chosen to bind the contaminating proteins, allowing the sample of interest to pass through the gel in the wash volume. This method of separation could result in a great saving of time. Matrix

Choice of the proper matrix is a very important step in any chromatographic process. A good matrix for affinity chromatography should have the following properties: P. Cuatrecasas and M. Wilchek, Biochem. Biophys. Res. Commun. 33, 235 (1968).

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1. Hydrophilic: Reduce the nonspecific interactions. 2. Large pores: Allow all areas of the matrix to be available to most of the molecules in the mixture. Some matrices allow binding only to the outer surface. This latter type of matrix is useful in separating very large molecules, cells, or viruses. 3. Rigid: The matrix must withstand the pressures of packing and solvent flow during elution or washing. 4. Inert: The matrix should not contribute to the separation. 5. Chemical stability: The matrix must be stable to all solvents used in the separation.

Base the choice of an affinity gel on both the ligand and the sample. There are two major types of affinity gels: group-specific gels and covalent coupling gels. The former are usually supplied ready to use. Table I provides examples of ligands that are group specific in action and can be used to isolate whole families of biomolecules which share common properties. Covalent coupling gels (Table 11) require more chemistry and some specific considerations. First, consider the length and type of the spacer arm; second, the coupling chemistry.

-

TABLE I LIGANDSPECIFICITY

-- - - - - - - -

Ligand

Specificity

NAD, NADP Lectins Poly(U) Poly(A) Histones Protein A Protein G Lysine Arginine Heparin Blue F3G-A Red HE-39 Orange A Benzamidine Green A Gelatin Polymyxin 2',5'-ADP Calmodulin Boronate Blue B

Dehydrogenases Polysaccharides Poly(A) Poly(U) DNA Fc antibody Antibodies rRNA, dsDNA, plasminogen Fibronectin, prothrombin Lipoproteins, DNA, RNA NAD' NADPi Lactate dehydrogenase Serine proteases CoA proteins, HSA, dehydrogenases Fibronectin Endotoxins NADP+ Kinases cis-Diols, tRNA, plasminogen Kinases, dehydrogenases, nucleic acid-binding prote~ns

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TABLE I1 COUPLING CHEMISTRY

Linkage

Ligand POUP

CNBr Thiolpropyl

NHI SH

Thio EPOXY

SH NH2 OH SH NH2 COOH NH2

Tresyl Aminohexyl Carboxylhexyl

Spacer length

Equivalent to about 13 carbons Equivalent to about 11 carbons

6 6

Active PH

Specificity

8-10 9-1 1

Proteins, peptides Sulfhydryls

9-13 9 10 11 8-10

Sulfhydryls Proteins, peptides Carbohydrates Sulfhydryls Proteins, peptides Amino acids, proteins Carboxylic acids

Solvents The solvent system chosen for the entire affinity chromatography separation is also a critical factor to a good separation. The solvent should not degrade the sample. Unfortunately, avoiding denaturing solvents is not always possible. For example, separation of an antibody (1gG)-antigen complex requires some very harsh conditions. Dissociation at a low pH or use of a strong chaotropic agent are the most commonly used methods. Minimizing the time of contact with these agents is vitally important. One method used to reduce the contact time with harsh reagents (e.g., low pH) is to add Tris base (dry) to the collecting tubes. This will rapidly increase the pH and help to protect the sample. Try to choose an elution buffer specific for the sample (e.g., a buffer containing an analog to the sample). The elution buffer should release the sample safely and rapidly. Again, the buffer should not denature the sample, nor cause any change in its specific activity or function. Optimization of sample binding and elution conditions is usually by trial and error. When choosing a buffer system try to avoid using one that has a pK, at or near the p l of the sample. This will help prevent precipitation of the sample on the column. However, when starting a separation read the literature, as it will often provide a good starting point. Even a related separation can serve as a starting point for selecting the separation conditions. Spacer Arms Choosing a gel with or without a spacer arm depends on the ligand, the sample, and the binding chemistry. A spacer arm is used to keep the

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TABLE 111 SPACERLENGTHCONSIDERATIONS Ligand

Protein

Best spacer arm

Small Small Large Large

Small Large Small Large

Short Long None None

ligand away from the matrix so that the active site of the ligand is available to the sample. This is especially important with small ligands. As a general rule if the ligand is large and the sample is small (low molecular weight) this spacing may be unnecessary. With samples of high molecular weight a spacer arm can be used to limit steric hindrance and increase the availability of the active site (Table 111). A wide variety of spacer arm lengths are available. If unsure of the required spacer arm length, start with one that is equivalent to about six carbon atoms. This seems to be a good length for many affinity applicat i o n ~ Shorter .~ arms give less flexibility so the ligand will not "wave" around in the medium. Predicate the spacer arm length on the amount of steric hindrance deemed tolerable. As spacers are evaluated, remember that the spacer molecule itself can cause steric hindrance by blocking adjacent active sites on the gel; thus, longer is not always better. Gel Preparation

After choosing the affinity gel type, prepare the resin (gel) for use. The manufacturer will usually supply the instructions needed to prepare the gel correctly. However, short of those instructions, following these general guidelines will help ensure a successful preparation. First, calculate the amount of gel that needs to be packed into a column (or flask for batch work) by the capacity of the gel for the sample. That is, x units of gel bind y units of sample. Next, calculate the volume: (total sample/sample units) x gel volume per sample unit. This value should be multiplied by a factor of 2-3 and this factored amount of gel should be used. For example, if 1 ml of protein A-Sepharose binds 20 mg IgG and you are using 40 mg of sample (with contaminants), divide the total (40 mg) by the gel capacity (20 mg) and multiply by the gel volume 9

C. R. Lowe, Biochem. J. 133, 499 (1973).

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(1 ml). Multiply this figure by 2-3, giving a final gel volume of 4-6 ml, for best results. If the gel is supplied in a preswollen state, reconstituting the gel is unnecessary to obtain the full swollen volume. A wash is all that is usually required. The swollen gel is typically supplied in glycerin or similar material which is used to help in the gel preparation and to stabilize the ligand or activated coupling complex. Wash on a sintered glass filter of medium grade (#3) or on a membrane-type filter that has a low protein-binding capacity. This allows easy removal of the washed gel with a minimum of loss due to sticking to the filter. A wash ratio of about 200 : 1 (buffer to gel) works well. For the safest wash buffer use either distilled water or the starting buffer (unless otherwise directed). By definition the starting buffer is that buffer used to initially prepare the matrix for the addition of the sample. It creates an environment on the gel so that the sample will bind specifically to the attached ligand. If the gel needs to be swollen to regain full working volume, then use a swelling buffer prior to washing. This buffer is often a low concentration phosphate buffer (0.1 M) at or near neutral pH. Swelling times vary between 15 min and 1 hr. After swelling, wash the gel either in the buffer solution used for swelling, distilled water, or starting buffer. Since washing and swelling buffers are generally pulled through the gel under a low vacuum, it is critical that the gel does not become dry at this stage. Following the reswelling and the wash, the ligand can be bound to the gel or loaded into a column if no ligand is to be added (i.e., groupspecific gels). Coupling or Linkage Chemistry

Before using a covalent coupling gel, the ligand-binding (linkage) chemistry needs to be decided. There are a variety of linking groups available, such as cyanogen bromide (CNBr), tresyl, epoxy, and triazine. The linkage chemistry may be available either in an activated or nonactivated form. Activated means "ready" to use without additional chemical activation steps (washing is still necessary). The nonactivated gels require some additional chemical activation step, such as carbodiimide treatment, to prepare them for binding the ligand. Leaching, or loss of ligand, after binding is inevitable. The trick is to minimize this loss. CNBr-type linkages commonly leach more than do tresyl or epoxy linkages. Other types of ligand linkages are also possible by using C=C, C=O, and other available bonds. Nucleic acids and sugars can also be bound through their amine or hydroxyl groups. Table I1 lists some examples of

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the active groups that can be used to link ligands to the matrix, and the types of sample that can bind to these ligands. Coupling the Ligand Next, couple the ligand to the matrix, assuming that you have a specific ligand that needs to be attached. Consider the steric effects of the ligand, the spacer arm, and the sample. High concentrations of small ligands can block some active sites on the matrix, causing a lower binding efficiency. Large ligands can also block adjacent sites, again resulting in lower binding efficiency. Spacer arms and ligands can also cause some blockage of adjacent sites as the ligand "waves" back and forth. As a general rule use about 10 mg ligandlml of gel. This amount applies also to proteins having an average size of (50 kDa). A lower amount should be applied for larger molecules such as IgG (5 mglml), or IgM (1 mglml), or molecules with low dissociation constant ( K D )values. Mix the ligand and the matrix together using a rotating motion. Avoid magnetic stirrers at all times since they can damage the matrix. The volume ratio of binding buffer (with ligand) to gel matrix should be about 2 : 1 for best results. Carefully control the pH, ionic strength, and ion content during this stage of coupling. Coupling times of 2-4 hr at room temperature or up to 16 hr in the cold (4") are commonly used. The choice of time and temperature is determined by the stability of the ligand and the amount of time available. The time available is important since there should be no processing interruptions from the time the gel is activated until the excess ligand is washed out. This minimizes loss of coupling activity. Once again users should consult the manufacturer's instructions for the optimum coupling conditions. Coupling of the ligand to the matrix can be by a single point or multipoint attachment. An example of single-point attachment is the binding of a single primary amine via CNBr coupling. This type of linkage offers the best flexibility to the ligand and thus the most accessibility of the active site to the sample. Single-point attachment is possible only if secondary and tertiary amines are blocked. Multipoint attachments are stronger than single-point attachments and are less likely to leach during the run. However, this type of coupling often causes blockage of the active site of the ligand. Blocking Unreacted Groups After incubation of the ligand with the matrix, remove the excess ligand and block the unreacted sites on the matrix. When coupling a

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ligand to the matrix some sites on the matrix remain unreacted. These unreacted sites are potential sites for nonspecific interactions with the sample or contaminants in the solution. Blocking these sites is most easily accomplished with reagents that have an opposite charge or can be covalently linked to the matrix. For example, when a carboxyl group (COO-) is used to couple a ligand, such as an amino group (NH2),use a Tris or ethanolamine solution (0.1-1.0 M) as the blocking agent. When NH2 groups are the coupling sites for ligand containing COO-, acetic acid can be used as the blocking agent. The concentration of the blocking agent should be in excess of the total reactive site concentration on the matrix. This assures complete blockage of all unreacted sites. Normally, a 5- to 10-fold excess over the ligand concentration is sufficient. Control of the pH of the blocking agent is another critical factor important to good affinity separations. A pH that is either too high or too low may prevent complete blocking or even destroy the matrix or the bound ligand. The blocking reaction is usually done at room temperature for 2-4 hr, but can also be done in the cold (4") for longer periods of time. Wash out the excess blocking agent and equilibrate the column with 5 to 10 column volumes of starting buffer before sample application. The coupling process can be summarized as follows:

1. 2. 3. 4. 5. 6. 7.

Swell the matrix in swelling buffer (15-60 min). Wash the matrix (200 : 1, buffer : gel). Add ligand and incubate with mixing (2-4 hr, 2 : 1 buffer: gel). Wash out unused ligand and buffer (200 : 1, buffer :gel). Block unreacted sites on the matrix (2-4 hr, room temperature). Wash the matrix (200 : 1, buffer :gel). Use or store the gel under appropriate conditions (4-8").

Monitoring Coupling Efficiency

The extent of ligand coupling determines both the efficiency of the separation and the amount of purified sample that can be prepared. The amount of ligand bound can be determined in several ways: 1. Measure the difference in UV absorption before and after coupling: a. Azsois best for proteins; however, other wavelengths specific for other ligands which can be coupled should be chosen. For example, for heme groups can be used. This technique is best accomplished when the concentration of ligand is not very high, since a small amount of ligand binding will not be detected. In

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addition, avoid buffer components that absorb at this wavelength [e.g., dithiothreitol (DTT)], or high concentrations of Tris buffer. b. Perform a colorimetric protein assay (see [6]). c. Perform a fluorescence detection assay (more sensitive than the colorimetric assays). 2. Dissolve a portion of the gel containing the ligand and do a protein assay, amino acid analysis, or assay for total nitrogen. 3. Perform an activity test (a small binding experiment). 4. Perform a radiolabeled ligand or RIA test (assumes that the radiolabeled ligand binds at the same rate, and to the same extent, as nonradiolabeled ligand). Binding the Sample

The binding of proteins to a ligand, through the carboxyl or the amino groups available, is based on the specific affinity of the protein for a particular ligand. As indicated previously, the ligand should not be coupled in such a way as to block or interfere with the availability of the active site on either the ligand or the protein. Binding between the ligand and the protein is generally noncovalent. Although the binding is specific, the forces involved are general chemical interactions, such as hydrogen bonds. The buffer conditions that are used to load the protein on to the column are often phosphate or Tris buffers (0.1-0.2 M) containing salts such as sodium chloride (0.5 M). The choice of buffer and concentration is predicated on minimizing nonspecific interactions and maximization of the specific attraction between the ligand and the protein. Load the sample in the normal downward direction such that it will bind to the upper half to upper third of the matrix. Flow Rates

Different flow rates are used in the various stages of every affinity chromatography run: (1) the loading of the sample, (2) the wash step to remove nonspecifically bound material, (3) the elution phase, where the protein of interest is removed from the gel, and (4) the regeneration of the matrix for the next run. The flow rate commonly used for loading of the sample is often about 10 cmlhr. The notation cmlhr refers to linear flow rate of the buffer. To calculate the volume flow rate, which is the rate that is used for the run, multiply the linear flow rate by the cross-sectional area of the column. The flow rate used is dependent upon the kinetics of the binding of the

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desired protein to the ligand. Factors such as temperature, concentration, and the KD influence the interaction of the protein and ligand. If the protein has a high affinity for the ligand ( K D< the flow can be faster since the protein still can bind effectively. On the other hand, if the affinity is low, use a much slower flow rate. During the wash step the flow rate can be considerably faster since the wash serves to remove nonspecifically adhering material (assumed for this discussion not to be of interest). The flow rate at this stage can be increased to about 20 to 50 cmlhr in order to effect a rapid cleaning of the matrix. However, if the protein of interest is loosely bound to the ligand, a lower flow rate is better. Perhaps most important is the flow rate during the elution phase. This flow could also be faster then the loading rate. Elution flow rates depend on the strength of the elution buffer as related to the affinity of the sample. The goal is to use a buffer that will easily strip the desired protein from the ligand without damaging it, the ligand, or the matrix. The elution flow rate can be as high as the wash flow, but is always lower than the flow rate used for packing the matrix. The flow rate during reequilibration can also be very rapid. At this point, only the coupled ligand should be left on the matrix. Therefore, flow rates up to the packing rate can be used to save time. Flow rates should not exceed about 80% of the flow rate used to pack the resin. This avoids compression during the chromatography run. Another factor that determines the maximum flow rate is the stability of the matrix. In order to avoid gel compression and deformation of the beads do not exceed the maximum flow rate recommended for the matrix. Also, try to avoid turbulence and high shear rates due to rapid buffer flow in the matrix when loading or eluting. The manufacturer can generally provide information on the best flow rates for all steps. Nonspecific Interactions

Nonspecific interactions, if a problem, can usually be avoided by using a salt concentration between 0.1 to 0.5 M since in this range nonspecific ionic attractions are greatly reduced. These salt concentrations are usually not so high as to make hydrophobic interactions between the protein and the matrix or ligand a problem. As always, the manufacturer of the matrix usually can supply the specific information needed to prevent nonspecific interactions. Other methods that may be used to decrease nonspecific interactions include the addition of agents such as glycerol up to a concentration of about 10% (no higher due to increased viscosity of the buffer, resulting in

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higher back pressures and lower flow rates). Low levels of detergent (below the critical micelle concentration) are also useful in reducing nonspecific interactions. Detergents, however, can interfere with both ligand and protein binding, and not every ligand can be safely used with all detergents. For example, a ligand may dissociate or denature in the presence of detergents and some may interact with the active site of the ligand, lowering its affinity for the protein. Specific vs. Nonspecific Elution Specific elution of the protein of interest is always the best method to use in affinity chromatography (see Table IV). This type of elution is the result of a competitive action of the eluent for the ligand. An eluent is chosen that has a greater affinity for the ligand than the protein so that it will displace the protein from the ligand. The eluent can then be removed by a more stringent cleaning of the matrix. An example of a specific eluent is the use of a-methylglucoside to elute samples from concanavalin A (ConA)-Sepharose. If there are no known specific eluents for the protein of interest then nonspecific elution may be used (e.g., elution using a salt gradient, changing the pH or temperature). Design conditions so that the protein of interest is eluted separately from the majority of contaminating proteins. One procedure is to raise the eluting buffer concentration to a level just below that at which the desired protein starts to be eluted, followed by an

TABLE IV ELUTION CONDITIONS Ligand

Eluent

Protein A

Acetic acid Glycine a-D-Methylmannoside Borate buffer a-D-Methylglucoside Temperature Salt Salt Urea Arginine pH NAD+, NADP+ Salt

ConA

Lysine Blue dye Gelatin 5'-AMP

Specific

Nonspecific

X X

X

X X X X X X

X

x X

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increasing shallow gradient. This results in sharper peaks and greater purity. If there are any proteins remaining bound to the matrix after the protein of interest has been completely eluted, the elution buffer strength can be increased rapidly to remove this remaining material. Elution is most often done in the "forward" direction, i.e., the same direction as sample application. Ideally, the sample should bind to the upper third to half of the column. Molecules with the highest affinity for the ligand will bind near the top of the column, while the remainder will bind further down the column as the affinity decreases. If the protein of interest is bound near the top of the column then the rest of the proteins can be more easily eluted in the forward direction, leaving the protein of interest on the column. Even if this protein moves down during the preliminary elution it will not come off the column. At this point if the eluent flow can be reversed and a strong eluent used, the sample can also be eluted off the top of the column in a sharper peak and in a shorter time, thus limiting exposure of the protein to potentially harsh conditions. Such a flow reversal can be accomplished by turning the column upside down, or using valves to allow the eluent to flow from the bottom of the column to the top. When reversing the direction of flow in a column always make sure that flow adaptor are used to prevent the loss of the matrix through the top of the column. The use of reversed elution to yield a more concentrated sample is valid only in certain situations. These occur when the desired protein binds more strongly than the other proteins and when it is bound to the upper half of the matrix. In all other cases it is still best to elute the sample in the forward direction. Measurement of the elution profile is usually done by monitoring parameters such as the A2S0or fluorescence. Specific assays for the protein of interest, such as enzyme activity, can yield information on the concentration and the condition of the separated material, and should be used whenever possible. Detection of any ligand that has leached off the matrix is usually difficult and requires specific assays. Radioimmunoassays (RIA) for ligand or matrix material are often useful in these situations. Electrophoresis (i.e., SDS-PAGE and immunoelectrophoresis) may also be used to determine protein purity, activity, and the extent of leaching. Regeneration

Thoroughly clean resins prior to their reuse. Regeneration means that any material that remains on the resin must be removed and the gel reequilibrated with starting buffer. If all the material is not removed, and the ligand not properly prepared, the efficiency of the gel will be impaired.

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This will result in less material binding in successive runs and a concomitant loss in resolution. With proper care an affinity resin can be used multiple times. The actual number of uses depends on the sample, the ligand, and the elution conditions. Clean the gel with either a higher concentration of the specific eluent or by using a high concentration of a nonspecific agent such as sodium chloride (e.g., 0.5-1 M). Increasing the salt concentration is usually effective in removing nonspecifically bound material as well as some of the specifically bound sample that may be left on the resin. Take care not to damage the bound ligand or to alter its activity. In some cases high salt levels cause proteins to change their conf~rmation.~ If the ligand is a protein, its active site may be altered, causing it to lose some or all of its binding capacity or affinity for the sample. Some procedures are gentle enough for almost all gels. A general regeneration scheme (recommended by Pharmacia LKB Biotechnology, Piscataway, NJ)6 follows. However, if the manufacturer provides a specific method for regeneration, then follow their advice: 1. Wash with 10 column volumes of Tris-Cl(0.1 M, pH 8.5) containing 0.5 M NaC1. 2. Follow with 10 column volumes of sodium acetate (0.1 M, pH 4.5) containing 0.5 M NaCl. 3. Reequilibrate with 10 column volumes of starting buffer. Any regeneration procedure requires buffer volumes up to 10 times the column volume. This assures that all areas of the resin have been reached and cleaned. Be sure those cations or anions needed for ligand stability are added to the regeneration buffer. These ions are usually present in the start and elution buffers, but are often overlooked in the regeneration buffer. For example, ConA requires Ca2+and Mg2+or Mn2+at concentrations of 110 mM to maintain its tertiary structure. Resins are usually stable to most regeneration buffers, but if in doubt check with the resin manufacturer. Sterilization

If the sample is to be kept sterile, the affinity column and gel must also be sterilized. Take special care throughout the entire process to assure the maintenance of this sterility. The gel can often be sterilized by autoclavP. H. von Hippel and T. Schleich, in "Structure and Stability of Biological Macromolecules" (S. N. Timasheff and G . D. Fasman, eds.). Dekker, New York, 1969. Pharmacia LKB, "Affinity Chromatography: Principles and Methods," p. 88. Piscataway, New Jersey, 1983.

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ing, and is more easily accomplished before ligand attachment. The ligand can be filter sterilized before coupling to the resin. Coupling can be done under aseptic conditions such as with sterile buffers, air, and vessels. Depyrogenation of the matrix with agents such as sodium hydroxide is also best done prior to ligand attachment. If sterilization is necessary after the ligand is attached, take care to avoid altering the ligand or the linkage to the matrix. Autoclaving is not usually feasible at this stage since proteins and most other biological material are denatured under these conditions. Possible solutions to the sterilization of sensitive gels include radiation treatment or chemical sterilization. One gentle method for the sterilization of a sensitive gel-ligand system follows7: 1. Equilibrate the column with 2% chlorhexidine diacetate and 0.2% benzoyl alcohol. 2. Let stand for 4 days. 3. Wash with sterile buffer; a neutral phosphate buffer or the start buffer can be used. 4. Reequilibrate with the chlorhexidine diacetate (2%) and benzoyl alcohol (0.2%). 5. Rewash with the sterile buffer. 6. Store in 0.5% chlorhexidine diacetate and 0.05% benzoyl alcohol. Gel Storage

Storage of the gel after preparation is usually quite easy. The actual conditions used for proper storage are dependent on the ligand that is bound to the matrix. In general, 4" is the preferred temperature. This lowers the possibility of bacterial growth and does not harm either the matrix or the ligand. Avoid freezing since this may rupture the matrix. It is best not to store a gel in the middle of the coupling process. This is especially true with CNBr gels, since they will lose activity rapidly at the pH used for activation. Clean the gel before storage by removing all residual material that is known to adhere to the column. This will allow for easier reuse of the matrix. In general, store the gel at temperatures below 8" but not frozen. Store all affinity resins in the presence of antibacterial agents such as chlorhexidine digluconate (or acetate), sodium azide, 20% ethanol, and thimerosal (do not use this with SH-active ligands, e.g., thiolpropyl-Sepharose). Base the choice of the antibacterial agent on the stability of the ligand to

' S. S. Block, ed., "Disinfection, Sterilization, and Preservation." Lea & Fabiger, Philadelphia, Pennsylvania, 1977.

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-

37 1 -

-

-

that agent and the charge characteristics of the gel-ligand combination. Is it anionic, cationic, or neutral? Is it temperature sensitive? Is it subject to degradation by enzymatic action? To maintain the same level of activity after storage as existed previously, choose a bacteriostatic agent that will not bind to the gel matrix or ligand, and one that is easily washed out when the gels are reused (e.g., ethanol). Carefully remove all of the storage solution prior to reuse to prevent denaturation of the sample. Do not freeze the gel at any time. This will disrupt the matrix and can lead to fine particles that can interfere with the buffer flow. Again, follow the manufacturer's advice for proper storage.

[301 Affinity Chromatography: Specialized Techniques By STEVEN OSTROVE and SHELLY WEISS

This chapter discusses some specialized affinity chromatography techniques: cell affinity chromatography, metal chelate affinity chromatography, covalent affinity chromatography, and other binding techniques and the scaling up of affinity chromatography. It will be a guide in the use of these techniques and give a start in understanding the reasons behind their use. In addition, some of the possible problems and danger areas associated with these techniques are described. Not all of the specific methodologies available for separation by affinity chromatography will be reviewed in this chapter, nor will it provide an exhaustive list of examples for each technique. As you read this chapter, and try to use the techniques, however, you will find new and different ways to accomplish your separation task. Certain assumptions need to be made before we begin: First, that you are aware of general affinity chromatography procedures; second, that you know how some parameters such as temperature, pH, ionic strength, and flow rates affect affinity separations (see [29] in this volume). Cell Affinity Chromatography

Isolating cells by affinity chromatography requires some special considerations due to the size and sensitivities of the living cell. Cells can be separated by affinity chromatography in two ways: either by binding the cell directly to the matrix as one binds a protein, or by binding a protein or METHODS IN ENZYMOLOGY, VOL. 182

Copyright O 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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other ligand that can "recognize" a specific protein or receptor on the cell membrane. The second method is more commonly used. Cells are considerably larger than proteins, and can be even larger than the average bead size used for separating proteins. For this chapter we will assume an average cell size of 50-70 wm. The bead size used as the chromatographic support must be large enough to allow the passage of the cells between the beads when they are packed in a column. A bead diameter of 250-350 p m is usually adequate. The bed support (net) in the column must also be of a size that allows passage of the cells from the column. In general, the mesh size of this bed support should be at least 80 pm (larger than the cells, but smaller than the matrix beads). This allows the cells to pass through without any significant impediment. The matrix must exhibit all the characteristics of a good affinity support (see [29] in this volume) and in addition must be nontoxic to the cells if they are to remain viable. One additional requirement for the resin used to separate cells is the ability to withstand sterilization either by autoclaving or chemical treatment. Cells have many sites on their membrane that can be utilized for their separation. For example, glycoproteins, which are common membrane components, may be selected by using the appropriate lectins. For example, concanavalin A (ConA) will select those proteins containing glucose or mannose, while wheat germ lectin will select those proteins containing N-acetylglucosamine. Other compounds, such as protein A, which binds to the Fc portion of immunoglobulins, can select cells with antibodies on their surface. Consequently, the choice of the affinity ligand is dependent on the cell type being purified. Cells can also be bound directly to the matrix through coupling agents such as cyanogen bromide (CNBr). This reagent binds to amines (preferentially to primary amines) found on cell surface proteins. The process of binding cells to ligands attached to a matrix is very similar to other affinity purifications. The procedure is summarized as follows :

1. Prepare matrix. 2. Wash matrix-ligand complex. 3. Slowly add cell suspension. 4. Wash out nonadhering cells. 5. Add elution buffer (specificity is important). 6. Collect cells. Elution of cells should be accomplished using a specific eluent for the ligand-cell (protein) complex since salt gradients are not recommended due to their osmotic effects on the cells. Special attention should be given

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to controlling pH, osmotic pressure, temperature, shear forces, and sterility. The necessary degree of control depends on the planned use of the cells following their separation. Flow rates for processing cells must also be adjusted so that they do not rupture. Shear forces can act on the cells as they pass through the matrix, causing changes in membrane structure or even some change in cell biochemistry. Thus, there may be alterations in one or more of the cells biochemical pathways as it "adjusts" to the stress of chromatography. For this reason flow rates in cell affinity chromatography are usually slower than in other affinity chromatography procedures. If, after separation, the purified cells are to be grown in culture, then the whole process must be done under aseptic conditions. The ligand, the matrix, and all buffers must be sterilized prior to use. Sterile conditions are not as important if affinity purification is the last step in the process, or if the cells will be used in short-term cultures (those lasting only a few hours), which do not require aseptic handling. Chelation Affinity Chromatography

Immobilized metal affinity chromatography (IMAC),' also known as metal chelate affinity chromatography (MCAC), is a specialized aspect of affinity chromatography applicable to a wide variety of compounds. It ~ approach to was developed over a decade ago by Porath et ~ 1as. a novel protein fractionation. Over the years it has increased in acceptance as a quick, reliable separation technique. At this time, however, its potential has not been fully explored. The principle behind IMAC lies in the fact that many transition metal ions, i.e., zinc3 and copper, can coordinate to the amino acids histidine, cysteine, andtryptophan via electron donor groups on the amino acid side chains. In order to utilize this interaction for chromatographic purposes, the metal ion must be immobilized onto an insoluble support. This can be done by attaching a chelating group to the chromatographic matrix. Most importantly, in order to be useful, the metal of choice must have a higher affinity for the matrix than for the compounds to be purified. The most common chelating group used in this technique is iminodiacetic acid (IDA). It is coupled to a matrix such as Sepharose 6B, via a long hydrophilic spacer arm. The spacer arm ensures that the chelating metal is fully accessible to all available binding sites on a protein. Another popular chelating group for IMAC applications is tris(carboxymethy1)J . Porath and B . Olin, Biochemistry 22, 1621 (1983). J. Porath, J . Carlsson, and I. Olsson et a / . , Nature (London) 258, 598 (1975). D. C. Rijken and D. Collen, J. Biol. Chem. 256, 7035 (1981).

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ethylenediamine (TED). This particular group lends different properties to the gel than IDA. TED gels show stronger retention of metal ions and weaker retention of proteins relative to that of IDA gels. TED gels form a complex (single coordination site) vs a chelate (multiple coordination sites) for IDA gels. The most commonly used metals for IMAC are zinc and copper; however, nickel, cobalt, and calcium have also been used successfully. Theoretically, other heavy metals and transition elements can be utilized, but there is a shortage of information in this area. The basic methodology of IMAC is quite simple. There are three main steps: (1) Charging the gel, (2) binding the proteins, and (3) eluting the proteins. Charging the IMAC gel usually involves passing a solution of the metal salt (ZnC12 or CuS04.5H20) over a column packed with the uncharged chelating matrix. The choice of the best metal is not always predictable. Copper often affords much tighter binding to proteins then does zinc. However, the weaker binding achieved using zinc may be a useful factor in some cases. Unless there are previous data, the appropriate choice of metal is a trial and error process. As with other affinity chromatographic techniques it is not generally recommended to use the full capacity of the gel for the metal, but to use one-third to one-half of the gel's capacity. This is particularly relevant when extremely strong binding of the protein occurS. The most important factor affecting protein binding is the pH. Most protein binding will occur in the range of pH 6-8. At more alkaline pH values binding will most likely be via deprotonated amino groups. The choice of binding buffer is also critical. Avoid buffers containing any type of chelating agent such as EDTA or citrate. Tris, phosphate, and acetate buffers are all suitable for the pH range used for binding. Tris-HCI (but not acetate) may reduce binding4 and should be used only when the metal-protein affinity is quite high. Additional reagents such as urea, salts, or detergents may be added to the binding buffer with either little or no effect on binding. Porath and Olinl have explored this area in detail. Generally accepted, however, is that high concentrations of salt should be present to quench any ionexchange effect. Usually a concentration range of 0.5 to 1 M NaCl is sufficient. Several methods can be employed for elution of biomolecules from a metal chelate affinity column. Each has advantages and the best method for any given separation must be experimentally determined.

' C. A. K. Borrebaeck et al., FEES Lett. 130, 194 (1981).

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Since binding is a pH-dependent function, a pH gradient is the most common method of elution. At the pH required for binding (pH 6 to 8) the groups which coordinate with the metal are deprotonated. Use of a decreasing pH gradient, i.e., pH 7 to 6, will cause protonation and subsequent elution. An alternative elution protocol is the use of a competitive ligand. In the case of IMAC, this involves increasing gradients of imidazole, histamine, glycine, or ammonium chloride. As with other affinity elutions that use competitive ligands, it is best accomplished at a constant pH, and usually the sample application buffer is used. Another elution method is the use of a chelating agent such as EDTA or EGTA. In this case, the metal ions will be completely stripped from the matrix. This may be useful for a quick group-specific elution. However, the disadvantage is that all adsorbed biomolecules will come off together. There will be no resolution of different species. IMAC was first utilized for separating serum protein^.^ Many of the major serum proteins have an affinity for metal ions. Different proteins can be isolated with different chelate columns, sometimes used in series. Presently, there are many applications for this technique, and some of them are listed in Table I.S-10 Interferons have been the subject of intense study for many years. A number of different mammalian interferon species have been purified by IMAC.11-'6Furthermore, Suikowski et al." have used this technique to study the surface topography of various interferon species since the affinity for the metal is dependent on the presence of specific amino acids on the protein surface. They found that human, murine, and hamster interferons can bind to Cu2+chelates. Human interferon can bind to a variety of other metal chelates (Co2+,Ni2+,and Zn2+)as well. They also studied

kx E

i

r r

T. E. Cawston and J. A. Tyler, Biochem. J. 183, 647 (1979). A. R. Torres, E. A. Peterson, W. H. Evans et al., Biochim. Biophys. Acta 576, 385 (1979). H. Kikuchi and M. Watanabe. Anal. Biochem. 115. 109 (1981). L. Sottrup-Jensen, T. E. Petersen, and S. Magnusson, FEBS Lett. 121, 275 (1980). M. F. Scully and V. V. Kakkar, Biochem. Soc. Trans. 9 , 335 (1981). loI. Ohkubo, T. Kondo, and N . Taniguchi, Biochim. Biophys. Acta 616, 89 (1980). E. Bollin, Jr. and E. Sulkowski, Arch. Virol. 58, 149 (1978). l2 K. Berg and I. Heron, Scand. J. Immunol. 11, 489 (1980). l3K. C. Chadha, P. M. Grob, A. J. Mikulski et al., J. Gen. Virol. 43, 701 (1979). l 4 P. C. P. Ferreira, M. Paucker, R. R. Golgher et al., Arch. Virol. 68, 27 (1981). l5J . W. Heine, J . van Damme, M. de Ley et al., J . Gen. Virol. 54, 47 (1981). l6 S. Yonehara, Y.Yanase, T. Sano et a [ . , J . Biol. Chem. 256, 3770 (1981). l7 E. Suikowski, K. Vastola, D. Osezek et al., Proc. 4th Int. Symp. Afinity Chromatogr. Related Techniques Veldhouen, Neth. (T. C. J. Gnbnau, J . Visser, and R. J. F. Nivard, eds.), p. 313. Elsevier, Amsterdam, 1981.

'

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TABLE I REPRESENTATIVE PROTEINS PURIFIED BY MCAC Protein

Metal

Ref.

Plasminogen activator Doiichos bioJlorus lectin Collagenase Lactoferrin Nonhistone proteins a2-Macroglobulin Human fibrinogen Nucleoside diphosphatase

the reversibility of binding. It is even possible to resolve different subclasses of human interferon on a Zn2+chelate c o l ~ m n . ' ~ Andersson and Porath18recently described a method in which immobilized ferric (Fe3+)ions could be used as a group-specific adsorbant to isolate phosphoproteins and phosphoamino acids. In fact, they could distinguish natural amino acids and their phosphorylated counterparts. A large number of amino acids were examined for their affinity to a Fe3+ chelate gel. It was found that the majority had very low affinities while the phosphorylated amino acids were tightly bound. When ovalbumin was used as a model of a phosphoprotein, the matrix was specific enough to resolve the three protein subcomponents (A,, A2, and A3) that differed only in their phosphate content. Covalent Chromatography and Bifunctional Agents

Although binding of most ligands in affinity chromatography is accomplished through the carboxyl or amino groups (especially on proteins), there are other reactive groups that are available under the proper conditions. The use of these groups may make the separation even more specific than if the carboxyl or amino groups were used. Sulfhydryl-containing matrices can be used to couple proteins containing not only sulfhydryl groups, but also compounds containing C=O, C=C, N=N, as well as heavy metals (e.g., Hg) or alkyl and aryl halides (for a more complete review of this chemistry see Ref. 19). The matrix for this type of chromatography is made with an active sulfhydryl group that Is l9

L. Andersson and J. Porath, Anal. Biochem. 154, 250 (1986). P. C. Jocelyn, "Biochemistry of the SH Group. The Occurrence, Chemical Properties, Metabolism and Biological Function of Thiols and Disulphides." Academic Press, New York, 1972.

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will form a covalent disulfide bond with the protein of interest. The active group on the matrix is usually a thiopropyl or glutathione moiety. In this type of chromatography a sample or ligand containing thiol groups is bound to the matrix by the formation of a reversible mixed disulfide bond. The use of covalent chromatography is similar to other affinity chromatography procedures. It is a very powerful method for quickly isolating thiol-containing enzymes or specific blood proteins. The flow rate used for the application of the sample is relatively slow, allowing the formation of the disulfide bond. The extent of binding can easily be monitored at 343 nrn, indicating the formation of 2-thiopyridone. Buffers should not contain reducing agents [e.g., dithiothreitol (DTT)] since these will interfere with the disulfide formation. After the binding step nonreacting proteins are washed out using the start buffer. A buffer containing a reducing agent or L-cysteine(5-20 mM) at pH 8.0 is then added to the buffer to dissociate the disulfide bond that was formed between the protein (ligand) and the matrix. Higher flow rates can be used in these later steps of the process. One method for regeneration of the column is to prepare a solution of 30-40 mglml of 2,2-dipyridyl disulfide in ethanol or 2-propanol. One volume of this solution is mixed with 4 vol of gel in 0.1 M borate buffer, pH 8.0, containing 1 mM EDTA and then refluxed at 80" for 3 hr. The gel is then washed with ethanol and reequilibrated with starting buffer.20Caution must be taken regarding the solubilities of materials used for regeneration since several of the reagents are only sparingly soluble in water. Keep in mind when using resins containing active thiol groups that they are able to bind proteins and other components containing heavy metals (e.g., Hg) so bacteriostatic agents containing these moieties should be avoided. Bifunctional reagents also often employ the use of the disulfide bond. These reagents are useful when the K Dbetween the ligand and the sample is very low or the sample is sensitive to extremes of pH. These agents can be attached to either the ligand or the matrix. The disulfide bond is easily and safely dissociated using reducing agents such as DTT or 2-mercaptoethanol. This allows separation of the sample from the ligand without the use of harsh denaturing agents. Scale-Up

After development of a successful analytical separation, scaling up to preparative levels is often desired to produce a larger quantity of a sub%

"Product Data Sheet for Covalent Chromatography." Pharmacia LKB, Piscataway, New Jersey, 1984.

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[301

stance for further study. Scale-up may mean going from microgram to milligram or from milligram to kilogram quantities. The use of high-capacity affinity resins often simplifies the separation by keeping the increase in column volume, and thus elution volume, to a minimum. The general rule of thumb in scaling up is to scale out, not up. This means that the column diameter should be increased while column height remains constant. This is very important. Increasing the column diameter achieves the larger column volume necessary when larger sample volumes are applied. However, by holding the bed height constant, the kinetics involved in the binding of the sample to a ligand will be unaffected. Further, assuming a constant linear flow rate, the residence time of the sample on the column will remain the same. This becomes particularly important since an increase in residence time may lead to changes in the binding characteristics of the compound, or cell, of interest. Conformational changes in a protein, due to column interactions (e.g., electrostatic or hydrophobic) may change the elution pattern. In the most extreme case, elution may become extremely difficult. Maintaining a constant bed height will also limit exposure of both the sample and the matrix to the harsh conditions often required for elution (e.g., acids or chaotropic agents). Flow rate is the next parameter for consideration when scaling up an affinity separation. The linear flow rate (in centimeters per hour, see [29] in this volume) should be maintained in going from a smaller to a larger column. If the linear flow rate is decreased, the residence time of the sample increases. If the flow rate is increased, the kinetics of binding may be shifted in such a way as to prevent adsorption. This will be especially true in cases of weak binding ( K D to M). The bound molecules are in equilibrium with a small amount of free (unbound) materials. The eluting agent competes with the ligand on the matrix for the free molecules. The rate of elution is limited by the initial dissociation of the bound substance from the gel. Increasing the flow rate will affect this dissociation. Any change in the linear flow rate will change the binding and elution characteristics of a sample with a concomitant change in the resulting separation. The sample is the next parameter to be considered in scaling up. Maintain a constant ratio of sample volume to column volume while maintaining a constant sample concentration. Changes in sample concentration will alter the size and shape of the eluted protein peaks and can have an effect on the binding characteristics (single-point or multipoint attachment) of the sample. Altering the sample-to-column volume ratio can also have an effect in the case of a weak binding interaction. Larger sample

[301

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379

volumes can potentially lead to coelution of sample with nonadsorbed material. The last important factor in scaling up an affinity chromatography separation is the maintenance of the buffer volume to column volume ratio. The effects of variations in this parameter are evident when elution involves the development of a gradient. If the gradient end points are held constant and the gradient length is increased (i.e., a shallow gradient) broader peaks and greater peak separation may result. Decreasing the length of the gradient (steeper gradient) will sharpen the peaks, but may also reduce separation. Either way, the elution pattern will change. As in all chromatographic procedures other factors, such as the mechanical stability of the matrix, must be considered. The actual physical stability of a gel bead is unchanged by the height or width of the column. However, the maximum flow rate in a packed bed is affected by the column size and is generally reduced as the column length increases. In smaller diameter columns, the walls of the column lend considerable support to the gel bed. The degree of support, and thus the flow rate, varies with the column diameter and height. A matrix can tolerate higher flow rates in smaller columns than in larger ones. When very large columns are used (e.g., >30-cm diameter), the walls no longer offer support to the bed. Consequently, the maximum tolerable flow rate is strictly a function of the matrix stability, and thus will be lower in wider columns. Having a clean sample is just as important when the process is scaled up as when it is first developed in smaller columns. It is advisable to "clean up" the sample before application to the column. In small-scale operations, this is not often done since the matrix can either be easily and quickly cleaned, or it can be disposed of and fresh gel used for the next run. In the case of larger columns, disposal is often too costly and cleaning may not be as expeditious. The pretreatment and clean-up steps used in the initial separation should be incorporated into any scale-up protocol. This clean-up may involve delipidation and/or centrifugation to remove particulates. This keeps the sample composition more consistent during the scale-up. It also helps simplify column maintenance procedures.

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[31.1 Chromatofocusing

The electric charge on the surface of protein molecules is one of the intrinsic properties that have been exploited in designing strategies and methods for protein purification. The separation of proteins on the basis of their charge depends ultimately on their acid-base properties, which are largely determined by their amino acid composition and sequence. The electric charge on protein molecules is influenced by the pH of the solution. At lower or acidic pH values many proteins tend to have a positive net charge, whereas at higher or basic pH they carry a net negative charge. Both positively and negatively charged proteins tend to move in an electric field. However, at a certain pH proteins do not carry a net electric charge and do not move when an electric field is applied. This pH value is called the isoelectric pH or isoelectric point (pl). The charge properties of protein molecules also enable counterions present in the buffer to bind to the protein. Similarly, a charged protein will bind to the surface of a resin or any solid support bearing an opposite charge. The charge properties, the movement of charged protein molecules in an electric field, and the electrostatic interactions between protein molecules or between a resin and a protein molecule have given rise to electrophoresis and ion-exchange chromatography as two of the most powerful methods of protein purification and characterization. The isoelectric pH of proteins has been further exploited to separate proteins by isoelectric focusing and chromatofocusing. Isoelectric focusing by electrophoresis has been described elsewhere in this volume (see [35]). Chromatofocusing is the theme of this chapter. Chromatofocusing or isoelectric focusing by ion-exchange chromatogThey proposed that a pH raphy was first described by Sluyterman st gradient could be produced in an ion-exchange column packed with an appropriate ion-exchange resin with good buffering capacity. A pH gradient in a column can be created in a manner similar to that of a salt gradient. If a buffer of one pH is mixed gradually with a volume of buffer of another pH in a mixing chamber and effluent from the chamber introduced into the column, a pH gradient is created. Similarly, a pH gradient can be produced internally in the column by taking advantage of the ~

1

.

~

9

~

' L. A. A. Sluyterman and 0. Elgersma, J. Chrornatugr. 150, 17 (1978). L. A. A. Sluyterman and J . Wijdenes, J. Chromatogr. 150, 31 (1978). Copyright Q 1990 by Academ~cPress. Inc.

METHODS IN ENZYMOLOGY. VOL. 182

All rights of reproduction In any form reserved.

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*

buffering capacity of the resin. If a buffer of a certain initial pH is passed through an ion-exchange column preequilibrated with a buffer of a different pH, a pH gradient is formed in the column just as if two buffers at different pH were gradually mixed in the mixing chamber of a gradient maker. If such a pH gradient is used to elute proteins bound t o the ionexchange resin, the proteins elute in order of their isoelectric points. During this process a focusing effect takes place, resulting in band sharpening, sample concentration, and resolution of peaks corresponding to different pI values of proteins. Mechanism of Chromatofocusing

P

During a chromatofocusing process the individual molecules are continuously changing charged states as the pH gradient develops. Thus, in a descending pH gradient, a single molecular species can change from negative to neutral to positive. When a positively charged column is equilibrated with a starting buffer of high pH, proteins which become negatively charged will initially adsorb to the column. When an elution buffer of low pH is passed through the column, a pH gradient develops and the charge on the protein molecules changes. As the pH gradient moves down the length of the column, proteins are selectively desorbed when the pH is less than or equal to their pZ and they are readsorbed when their pH is greater than their pl. Thus, molecules at the rear of the sample zone are the first to be titrated by the low pH buffer and become desorbed as a result of charge repulsion and are carried rapidly to the front of the sample zone due to the high velocity of the moving buffer. In traveling to the front of the sample zone, the proteins encounter an increase in pH which titrates them from their positive form to neutrality and back to their negative form. Once the molecules become negatively charged, they readsorb to the gel matrix and again fall back to the rear of the sample zone. This exchange of molecules between the front and rear of the sample zone results in "focusing" or a continuous narrowing of this zone until it elutes from the column. At this point the pH of the column effluent is approximately the pZ of the component of interest. Reagents and Equipment

i

Ion-Exchange Resins. In principle, any suitable ion exchanger with appropriate buffering capacity can be used. The chosen ion exchanger should be stable in water, salt solutions, organic solvents, and denaturing agents. The resin should be of homogeneous bead size to allow high flow rates, and be rigid enough to prevent fluctuation in bed volume. It should

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METHODS

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also be stable to sterilization by autoclaving at 110-120", be free of nonspecific interaction with ampholytes, and be of high chemical and mechanical stability at extreme pH values. Generally, Polybuffer exchangers (PBE) 94 and 118, Sepharose CL, QAE-Sephadex A-25 (all from Pharmacia, Piscataway, NJ), DEAE-BioGel A (from Bio-Rad, Richmond, CA), and DEAE-Toyopearl650 M (from Toyosoda, Tokyo, Japan) have been used as anion exchangers in open conventional columns. For FPLC and HPLC, Mono P (from Pharmacia), and SynChropak AX-300 and AX-500 (from SynChrom, Lafayette, IN), respectively, have been used. For ascending pH gradients (starting with low or acidic pH), one can use any appropriate cation exchanger such as SP-Sephadex C-25 (Pharmacia) or CM-BioGel A (Bi~-Rad).'.~ Although ion-exchange capacity information is provided by manufacturers, it is advisable to verify the capacity experimentally by titration in the desired pH range. Buffers. Chromatofocusing requires two buffer solutions, a starting buffer to equilibrate the ion exchanger and the packed column, and an elution buffer (eluent) to elute bound proteins. It is the elution buffer which generates the pH gradient along the length of the column. The starting buffers are usually 20-30 mM amine buffers. The elution buffers, namely polybuffers 74 and 96 (from Pharmacia), which are a cationic and amphoteric class of buffering species, have been used most commonly, either alone or in combination with ampholytes. The most acidic polybuffer binds to basic polybuffer exchanger (PBE) groups on the resin, increasing H+ ions in the vicinity and lowering the pH. This lower pH makes the proteins more positively charged, releasing them from the resin. In descending chromatofocusing the upper limit of the gradient is defined by the pH of the start buffer and the lower limit of the gradient is defined by the pH of the elution buffer. The reverse is true with a cation exchanger in ascending chromatofocusing, which is seldom used. To obtain a linear pH gradient, it is necessary that both buffers have a similar capacity over their working pH range. The pH of the start buffer is normally set 0.4 pH unit above the desired pH to compensate for the fluctuation in pH at the start of the run caused by slight differences in the conductivity of the start buffer and the elution buffer. Table I lists several start buffers and eluents which have been designed exclusively for chromatofocusing in different pH ranges. Columns. A wide range of columns and accessories are available from various suppliers. One can use any open column in the size range of 20 x 1 cm to 60 x 1 cm. Columns of similar sizes, used for other chromatoA. Murel et a / . . J. Chromatogr. 362, 101 (1986).

r31.1

CHROMATOFOCUSING

383

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graphic procedures such as gel filtration and ion exchange, can also be used for chromatofocusing without any noticeable difference in their resolution. Peristaltic Pump. T o maintain a constant flow rate a compact, singlechannel pump without a gradient-forming capability is required. The pump should be able to deliver a s low a flow rate a s possible to obtain good resolution, and back flow should be minimized. UV Monitor. A sensitive, preferably dual-beam, UV monitor can be used for detecting proteins, nucleic acids. and peptides at 280 and 254 nm. The monitor should be equipped with an appropriate flow cell. Alternatively, the absorbance of individual fractions may be monitored. Recorder. Any reliable chart recorder which can be interfaced with the monitor would be appropriate. Factors Influencing Resolution of Chromatofocusing The resolution in chromatofocusing, as in other column techniques, is determined by the width of the zone as it elutes. This is dependent on The most many variables which can be optimized in chromatofoc~sing.~ important factors for optimizing a particular protein separation by chromatofocusing are discussed below. Slope of pH Gradient. A shallow pH gradient gives better resolution. This can be achieved by using low buffer concentrations which give slow and steady pH changes. However, too shallow a gradient can also cause excessive dilution of protein in the eluent. Experimentally, a gradient of 10-15 bed volumes has been found to give good result^.^ Buffers. Most of the separations have been carried out successfully in polybuffers. However, they can also be replaced with appropriate mixtures of conventional buffer component^.^.^ For example, the starting buffer and elution buffer can be identical in their composition, but with two different pH values, representing the upper and lower limits, respectively. Charge o n Ion Exchanger. An optimal charge difference between the ion exchanger and the surrounding medium contributes to zone sharpening in chromatofocusing, just as electric field strength contributes to zone sharpening in isoelectric focusing by electrophoresis. Polybuffer exchanger (PBE) resins, which have a high degree of substitution, give good focusing. Column Packing. Any irregularity in column packing can have a Pharmacia Fine Chemicals, Chromatofocusing 17 (1980).

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PURIFICATION PROCEDURES: CHROMATOGRAPHIC METHODS

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8-6

PBE 94

8-5'

PRE 94

7-6

PBE 94

7-5

PBE 94

7-4

PBE 94

6-5

PBE 94

6-4

PBE 94

pH 8.3, 0.025 M Tris-CHKOOH pH 8.3, 0.025 M Tris-CH1COOH

pH 6.0, Polybuffer 96-CH3COOH pH 5.0, Polybuffer 96 (30%) + Polybuffer 74 (70%)CH3COOH

1 : 13

3.0

9.0

12.0

1 : 10

2.0

8.5

10.5

pH 7.4,0.025 M imidazoleCH,COOH pH 7.4, 0.025 M imidazole-HC1 pH 7.4, 0.025 M imidazole-HC1

pH 6.0, Polybuffer 96-CH,COOH

1 : 13

3.0

7 .0

10.0

pH 5.0, Polybuffer 74-HCI pH 4.0, Polybuffer 74-HCI

1:8

2.5

11.5

14.0

1:8

2.5

11.5

14.0

pH 6.2, 0.025 M histidine-HCl pH 6.2, 0.025 M histidine-HC1

pH 5.0, Polybuffer 74-HC1 pH 4.0, Polybuffer 74-HC1

1 : 10

2.0

8.0

10.0

1:8

2.0

7.0

9.0

Degas all buffers before use. The dilution factor given is not critical, and the best conditions will be found by experience. The figures given for gradient volume are approximate and will vary with the exact conditions chosen. Gradients ending at pH 9 are not recommended since 9 is above the pH of Pharmalyte pH 8-10.5. PBE 118 and Pharmalyte pH 8-10.5 also cover this range, as do PBE 94 and Polybuffer 96. f Mixing gives the best results. Polybuffer 74 works better than Polybuffer 96 when used alone. a

386

1311 -marked effect on resolution. The column should be evenly packed and uniformly equilibrated with start buffer. A certain degree of skill is required to achieve a well-packed column. Ions. Monovalent anions other than C1- can be used as the counterions, but it is critical that the anions have a pK, at least two pH units below the lowest point of the gradient chosen. Bicarbonate ions cause fluctuations in the pH gradient. Therefore, all buffers must be degassed before use. Atmospheric C 0 2 may cause a plateau in the pH region 5.56.5, depending on the conditions. These effects are most apparent with polybuffer 96 in the pH gradient ending at pH 6, and can be avoided by using acetate as the counterion. On the other hand, acetate is not usually recommended as a counterion with polybuffer 74 because of its higher pK,. Length of Column. Although good results have been obtained with short columns, resolution is superior with longer columns having the same internal diameter. However, very long columns, greater than 50 cm, will result in very long running times. Thus, 20- to 30-cm-long columns have been found to be most appropriate. Flow Rate. Theoretically, the flow rate should not have a major impact However, in practice, a significant difference has been on re~olution.',~ noted since a very low flow rate does give increased resolution (Fig. I). A higher flow rate can be used until the component of interest is close to elution. Then the flow rate can be decreased about 10-fold, and after the component has been eluted the flow rate can be adjusted back to its original value. --

PURIFICATION PROCEDURES: CHROMATOGRAPHIC METHODS

Experimental

The proper planning of a chromatofocusing experiment is similar to that of other chromatography techniques. The critical stages in designing a chromatofocusing experiment are described below. Choice of Gel and Buffers. One can choose any anion exchanger and buffers or Polybuffer and Polybuffer exchanger resins designed for chromatofocusing. If the isoelectric point of the protein of interest is known, then pH range of the gradient is chosen so that it elutes after one-third to one-half of the pH gradient in order to obtain optimal resolution. If the isoelectric point of the protein is unknown, it can be determined by isoelectric focusing by electrophoresis5or by a simple test using ion exchangers."' When working with an unknown sample, one can select a range,

'

D. Garfin, this volume [33]. "Ion Exchange Chromatography-Principles and Methods." Pharmacia Fine Chemicals, Uppsala, 1987. G . P. Lampson and A. A. Tytell, Anal. Biochem. 11, 374 (1965).

[3 11

387

CHROMATOFOCUSING

Time (hr)

Time (hr)

FIG. 1. Separation of a standard mixture of proteins at two different flow rates. The conditions include Pharmacia column SR 10/50; bed height: 30 cm; sample: 5 ml elution buffer containing cytochrome c (5 mg), ribonuclease (8 mg), and lentil lectin (10 mg); elution: start buffer 0.025 M triethylamine-HCI, pH 11.0. elution buffer 0.0075 mmol/pH unitlml Pharmalyte pH 8-10.5 equilibrated to pH 8.0. Linear flow rate: (A) 15 cmlhr, (B) 117 cmlhr. (From Pharmacia LKB AB, Uppsala, Sweden.)

such as pH 7-4 for the gradient, since most proteins have p i values in this range.8 If the desired protein has a p I below 4, it will pass through the column and can be recovered easily. On the other hand, if the p l is above pH 7.0, it will bind t o the column and recovery may not be simple. The bound protein has to be eluted with a salt solution, the column reequilibrated with start buffer, and the sample is reequilibrated with a new buffer. Thus, prior information about the p I value of the sample would eliminate these problems. Quantity of Ion Exchanger. The amount of gel used will depend on the amount and nature of the sample and contaminants. For most separations, a bed volume of 20-30 ml is sufficient for a protein sample up to 200 mg. It is important to remember that resolution is compromised with excessive amounts of sample. Preparation ofGel. The ion-exchanger gel should be equilibrated with the start buffer. A list of suitable start buffers is given in Table I. The ionexchanger resin can be equilibrated in a sintered glass funnel before packs E. Gianazza and P. G. Righetti, J. Chromatogr. 193, 1 (1980).

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ing a column o r also in the column after packing. The measured amount of exchanger is poured into the funnel. The start buffer is then passed through the gel at a slow flow rate using aspiration until the pH of the eluent is the same as that of the start buffer. The gel should occasionally be stirred gently t o ensure complete equilibration, which usually requires 10-15 bed volumes of start buffer. With Polybuffer exchangers it is not recommended to speed up the equilibration process by using concentrated buffer followed by one to two bed volumes of start buffer since the high capacity of Polybuffer exchangers can make equilibration to a lower ionic strength difficult. After equilibration the resin can be packed into an appropriate column. Column Packing. It is necessary to have a well-packed column in order to have a successful chromatofocusing experiment. Packing at high flow rates gives the best results. In our laboratories the following method has been used for open columns. 1. The equilibrated gel is dispersed in 150-200 ml of start buffer to make a slurry, and then degassed. 2. The column is mounted vertically. The end of closed outlet tubing is placed approximately 50 cm below the top of the column. 3. Put 3-5 ml of start buffer into the empty column and pour in the gel slurry while mixing it by swirling. If the volume of slurry is greater than that of the column, a packing extension can be used. 4. Open the outlet tubing and allow the gel to settle rapidly. The packing extension can be removed, and the top of the column connected to inlet tubing. 5. Continue to pack the column at a linear flow rate of 100 cmlhr (linear flow rate in centimeters per hour equals milliliters per hour divided by cross-sectional area of the column) until the gel bed has completely settled. Further equilibration can be carried out at a lower flow rate until pH and conductivity of the eluent match that of the start buffer. Any air bubbles should be removed. 6. The column packing can be checked by passing through a colored marker protein with a very high pI value, such a s cytochrome c (pl = 10.5). Use 1 ml of a 2-3 mg/ml solution of cytochrome c in the start buffer. Elute it with the start buffer. This protein should not be adsorbed by the gel and should come through in the void volume. Thus it should come through in an expected elution volume. Sample Preparation and Application. The preparation of a sample depends very much on its nature. Approximately 100 mg of total protein can be applied for every 10 ml of gel bed volume, although this value will vary according to the number of proteins present in the sample. The volume of the sample is not critical, so long as all of the sample is applied

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before the substance of interest is eluted from the column. Nevertheless, it is best if the sample volume does not exceed one-half of the column volume. The sample should not contain salt and should be equilibrated either in start buffer or elution buffer. To ensure even sample application, a layer of 1-2 cm of Sephadex G-25 coarse on the top of the bed is recommended. This serves as a mixing chamber and permits even sample application. The sample should be applied through a syringe or a flow adapter by first running 5 ml of elution buffer, followed by the sample (in elution or start buffer), then switching back to the elution buffer again. In this way sample proteins are never exposed to the extremes of pH. Elution. No special gradient apparatus is required for elution since the gradient is formed in the column automatically. The volume of the gradient is determined by the strength of the elution buffer. The recommendations in Table I are designed to give a gradient volume of approximately 10 column volumes for pH intervals of 3 pH units. Generally, there is a dead volume of 1.5 to 2.5 bed volumes of buffer which passes through the column before the pH in the eluent begins to decrease. Thus, the total amount of buffer required is approximately 12.5 bed volumes. Polybuffer is not suitable for intervals wider than 3 pH units. Flow Rate. From the initial studies'.2it was predicted that in chromatofocusing the separation would be independent of the flow rate. It seems to be true only up to a certain degree. A large variation in flow rate does change the resolution. A linear flow rate of 30-40 cmlhr has been found to give consistently good results in our laboratories. Monitoring. The eluent can be monitored for protein at 280 or 254 nm. Monitoring at 254 nm should be avoided when using Polybuffers because they absorb slightly at this wavelength. The pH of the eluent should also be monitored either by using a pH flow cell electrode or by measuring the pH of the individual fractions soon after they have been collected. Both the UV absorbance and the pH measurement can be recorded with a twochannel Chart recorder. Regeneration. A column can be regenerated and used several times without repacking. The gel should be washed with two or three bed volumes of a 1 M NaCl solution to remove any bound substances. Strongly bound proteins can be removed by washing with 0.1 M HCI. If HCl is used, the gel must be reequilibrated to a higher pH as soon as possible after washing. The column should then be reequilibrated with the start buffer until the eluent pH is the same as that of start buffer. Separation of Polybuffer or Ampholytes from Protein

Although Polybuffer and ampholytes generally do not interfere with enzyme assays, amino acid analysis, or the Coomassie Blue protein as-

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say, they may form a complex with copper ions, thus interfering with other protein determination procedures (see [6] in this volume). Several methods can be used to remove Polybuffer or ampholytes from proteins. Precipitation. The simplest method is precipitation with ammonium sulfate. Solid ammonium sulfate is added to the relevant fractions to a suitable concentration (80% saturation) and the sample is allowed to stand for 1-2 hr or until the protein precipitates. Since the protein is at its pl, it should be fairly simple to precipitate. The precipitate is collected by centrifugation and washed several times with saturated ammonium sulfate. An alternate method would be to place the fractions of interest in dialysis tubing and dialyze against saturated ammonium sulfate. Gel Filtration. Polybuffer or ampholytes can also be removed from most proteins by gel filtration on Sephadex G-75. If the fractions are small enough, prepacked disposable Sephadex desalting columns can also be used. Other chromatography techniques such as hydrophobic interaction chromatography (HIC) and affinity chromatography can also be used to remove Polybuffer and ampholytes. The methods for these techniques are described elsewhere in this v o l ~ m e . ~ J ~ Chvomatofocusing as a One-Step Separation Technique

Chromatofocusing has been used a s an adjunct method to other chromatographic methods, generally as a final purification step. However, a number of papers have described a purification in which chromatofocusing was used as the sole chromatographic method (e.g., Ref. 11). Chromatofocusing in Denaturing Agents

Separation and purification of proteins by chromatofocusing have also been carried out in the presence of dissociating agents such as urea, DMSO (dimethyl sulfoxide), formamide, ethylene glycol, and nonionic detergents such as Nonidet P-40 (NP-40) and Triton X-100 (e.g., Ref. 12). The success of chromatofocusing with any of these dissociating agents depends to a large extent on their interaction with the molecules in the sample. For example, when detergents are used to solubilize proteins, at a concentration approaching the critical micellar concentration (cmc), there may be association of protein molecules. For very hydrophobic protein molecules, dissociation and solubilization may not be as successful as one R . Kennedy, this volume [27]. lo S. Ostrove, this volume [29]. " I. Kalvaria et al., Thromb. Res. 29, 459 (1983). H . Bloemendal and G. Groenewoud, Anal. Riochem. 117, 327 (1981).

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might expect. This may result in a range of molecular complexes which may perturb the focusing. Furthermore, some molecules may be so heavily coated with detergent that the charge-dependent interactions necessary for chromatofocusing are not efficient. Thus, a certain degree of caution would have to be exercised about the concentration of detergents versus the nature of proteins in the sample. High-Performance Chromatofocusing

Although chromatofocusing had proved to be a powerful technique for protein separation, it still requires several hours to complete a single successful experiment. Thus, the demand and needs for protein purification in a short amount of time forced researchers to apply the principles and techniques of chromatofocusing to high-performance liquid chromatography (HPLC) and fast protein liquid chromatography (FPLC). The chromatofocusing principles were successfully integrated into the FPLC concept by Pharmacia.13 Approximately at the same time it was also applied to HPLC by Wagner and Regnier,14 who demonstrated that compared with other HPLC procedures, chromatofocusing was superior based on the number of protein components it resolved in a single run. FPLC chromatofocusing has found widespread application in research as the method of choice for resolving isoenzymes and molecular species with very similar charge characteristics and has proved promising for both analytical and preparative separation of proteins. Chromatofocusing on FPLC does not require any special modification of the regular FPLC ~ystern.~~Jj Concluding Remarks

Chromatofocusing has become very popular as a simple and rapid method for protein purification to attain reasonable purity. The resolution of this method enables one to separate almost identical molecules differing in p l by as little as 0.05 pH unit. This method has advantages of separating and concentrating proteins during the same run. In several instances the resolution of components inseparable by other chromatographic methods has been obtained by chromatofocusing. Although generally a pH gradient range of 10-4 has been suggested, some separation has been achieved even)at a much lower pH range. l3

l4

l5

R. M. Muller and L. Soderberg, Int. Symp. Proteins, Prpt. Polynucleofides, Baltimore. Md. Abtsr. No. 714 (1982). G . Wagner and F. Regnier, Anal. Biochem. 126, 37 (1982). "FPLC Ion Exchange and Chromatofocusing-Principles and Methods." Pharmacia AB, Uppsala, 1985.

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High-performance chromatofocusing has demonstrated advantages over conventional methods particularly with regard to reproducibility of the experimental conditions, speed, and resolution at both the analytical and preparative scales. A wealth of published literature on conventional and FPLC chromatofocusing exists. A compiled reference list can be obtained from Pharmacia LKB Biotechnology, Inc. Acknowledgments I wish to thank my colleagues Drs. David Englert and Bengt Osterlund for reading the manuscript, and Maggie Gustin and John Kubowitz for their help in figure preparation. I would also like to thank Dr. D. A. Hart, Faculty of Medicine, University of Calgary, Canada for sharing his unpublished results with us.

[321 High-Performance Liquid Chromatography: Effective Protein Purification by Various Chromatographic Modes By ROMANM. CHICZand FREDE. REGNIER Introduction

Chromatographic resolution of biological macromolecules in all cases except size-exclusion chromatography is a surface-mediated process, i.e., there is differential adsorption of solutes at the surface of the chromatographic packing material. Optimization of a chromatographic separation is nothing more than a solute adsorption. Structural characterization tells us that biological macromolecules differ physically in their size and shape, charge, hydrophobicity, and arrangement of functional groups within their three-dimensional structure. It is not surprising that the major chromatographic modes by which biopolymers can be fractionated are by size-exclusion chromatography (size and shape discrimination), ion-exchange chromatography (charge discrimination), hydrophobic interaction chromatography (surface hydrophobicity), reversed-phase chromatography (general hydrophobicity), immobilized metal affinity chromatography (surface-available histidines), and bioaffinity chromatography (distribution of specific amino acids at the surface of proteins). It is unlikely that this repertoire of chromatographic fractionation modes will increase to any extent during the next decade. METHODS IN ENZYMOLOGY,VOL. 182

Copyright O 1990 by Academic Press, Inc. AU rights of reproduction in any form reserved.

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Does this mean that improvements in chromatographic methods will diminish or cease? Not at all! Although the discovery of new fractionating modes will diminish, techniques for improving the resolution of protein species within a mode will continue. High-performance liquid chromatography (HPLC) is an example. It was already recognized by Martin in 1941 that the speed and resolution of chromatographic systems would be limited by the time it took large molecules to diffuse into and out of the stagnant mobile phase in the pore structure of chromatographic packing materials.' This limitation has been referred to as the "stagnant mobile-phase mass-transfer problem" for the past four decades. The first carbohydrate gel-type packing materials introduced for the fractionation of biopolymers in the late 1950s and early 1960s were of enormous utility but had serious stagnant mass-transfer limitations arising from the fact that sorbent particle size was <50 pm. Clearly, smaller particle size packing would allow major advances in resolution and speed but at the same time it was not understood how to either make or pack small-particle-diameter chromatographic sorbents. Advances in instrumentation, sorbent synthesis, and packing technology during the 1970s allowed the preparation of columns with 5- to 10-pm particles, which confirmed the enormous gain in resolution and speed possible with microparticulate packing materials. Performance of microparticulate materials is generally 10-60 times better than the chromatographic sorbents of two decades ago. This high-performance liquid chromatographic (HPLC) technology for the separations of biological macromolecules has been referred to in the commercial literature as highspeed chromatography, very high-speed liquid chromatography, fast nucleic acid liquid chromatography, and fast protein liquid chromatography. First, it is important to recognize that these are only trade names and not actually new or unique separation techniques. Second, it is very confusing to refer to separation processes in the scientific literature by the trade name of the instrument or column used to fractionate a mixture. In summary, HPLC is not so much a new technique as an advancement in both the operational theory and fabrication of chromatographic systems. HPLC systems for the separation of biological macromolecules vary from the old soft-gel systems in three ways; (1) the sorbents are of much greater mechanical strength, (2) sorbent particle size has been decreased 5- to 10-fold to enhance adsorption-desorption kinetics and diminish bandspreading, and (3) the columns are operated at 10-60 times higher mobile-phase velocity. The fact that liquid may be forced through columns at a controlled velocity with high-pressure metering pumps, samA. J. P. Martin and R. L. M. Synge, Biochem. J. 35, 1358 (1941).

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ples may be introduced as narrow bands while the system is operating under pressure, the column effluent may be monitored with very highsensitivity spectrophotometers fitted with microflow cells, and data systems manage everything from gradient formation to solute quantitation and fraction collection has elevated chromatography from a slow, laborintensive research technique to a rapid, automated analytical method with semipreparative capability. Instrumentation Pumping Systems

The pumping system is a vital component of any modern HPLC system. Essential features of a pumping system are as follow: 1 . Wettable components of the pumping system must be chemically resistant to common mobile phases. Degradation of materials in pumping systems at seals and joints can lead to leaks that are generally expensive to fix, always a nuisance, and occasionally a hazard. Many of the older HPLC instruments have stainless steel pumping systems that will corrode in the presence of halide under acidic conditions. Above pH 5, many stainless steel systems have been used with halides for years without problems. Although glass is widely perceived as being "biocompatible," it is well known to be unstable above pH 9. However, when the exposed surface is small, as at the walls of a glass column, erosion is too slow to be a problem. Accumulation of eroded materials on columns is perhaps even more serious than damage to the pumping system. Most silica-based and cationexchange sorbents will accumulate heavy metals from stainless steel at their surface. If these materials do not directly denature proteins, they will eventually accumulate sufficiently to alter the chromatographic characteristics of the column. Adsorbed metals may be removed from columns with a chelating agent. Recently, titanium and very resistant plastics have been used to fabricate pumping systems that are stable in both acids and base. These systems are generally more expensive than stainless steel systems but give an added measure of confidence. In view of the fact that pumping systems have a useful life of 5-10 years, the additional expense is justified. 2. Pumping rate must vary by less than 1% to ensure precision and reproducibility. Differences in pumping rate will cause solute retention times to vary between runs. A 1% difference in pumping rate will cause a 12-sec difference in retention time in a 20-min isocratic separation. Similar molecular species can vary in retention time by 15-30 sec in a 20-min separation.

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3. Pumpingpressure should be at least 30 atm but preferably 400 atm for use with microparticulate columns and viscous mobile phases. The pressure ( P I )required to force liquid through a column is a function of mobile phase flow rate ( v ) , sorbent particle diameter ( d p ) ,column length (L),mobile phase viscosity ( T ) , back pressure at the column outlet (Po), and interparticle porosity ( e ) as seen in Eq. (1): PI - Po

=

180 vLq(1 - e)2/d$e2

(1)

As noted above, HPLC columns are commonly packed with 5- to 10pm-diameter particles and eluted with viscous mobile phases such as propanol, ethylene glycol, and 3.0 M ammonium sulfate solutions. Although ion exchange of 5-cm-length columns packed with 10-pm particles may be operated at less than 30 atm, it is common to experience operating pressures in excess of 60 atm with longer reversed-phase and hydrophobic interaction columns. An even more serious consideration is that columns of the future will use <3-pm particle diameter packings. Since operating pressure increases with the inverse square of particle diameter, greater than 100 atm of pressure may be needed to operate these columns at high mobile-phase velocity. 4. Solvent delivery must be pulse free to limit detector baseline noise and damage to columns. Pressure pulsations in solvent delivery cause discontinuous flow through columns and detectors. These rapid flow oscillations produce two problems. The first is detector baseline distortion for flow-sensitive detectors with concomitant loss of sensitivity. Refractive index, electrochemical, and some UV detectors are flow sensitive. Flow sensitivity in UV detectors is a function of flow cell design and may be greatly diminished by exclusion of the turbulent liquid layer at the cell walls from the light path. Pressure pulsations also damage the column packing material. Rapid changes in flow cause a slight movement of particles in the column bed. On an occasional basis this is not a problem. However, several thousand times a day is sufficient to cause particle abrasion. Fine particles generated by this particle abrasion will plug the column filters. 5. Good solvent mixing is required at allflow rates. It is very convenient in modern liquid chromatography instruments to let the instrument prepare mobile phases, i.e., the instrument draws liquid from pure solvent containers and prepares mobile phases of intermediate composition. Since mobile phase velocity may vary from 10 pllmin to 100 mllmin, solvent mixing is necessary to assure constant mobile phase composition at the column inlet. The problem is not in being able to mix solvents, it is the impact of the mixer on column operation. Mixing devices of large volume often cause a significant time delay (also known as "dead time or

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dead volume") between solvent mixing and arrival at the column inlet. The volumetric flow rate and volume of the mixer and tubing connecting the mixer to the column determine the delay time between the start of a gradient program and actual solvent delivery to the head of the column. For example, a dead volume of 6 ml will delay the actual start of a 15-min gradient by 6 min at 1 mllmin mobile-phase velocity. Interlaboratory comparisons of data under these conditions are difficult because retention times have little meaning. The problem is most dramatic in rapid gradient elution separations. 6. It is necessary to have reproducible gradient formation over a 10fold range of mobile-phase velocity. Gradient formation by drawing and mixing liquid from containers of pure solvents is most difficult in the initial and final portions of the gradient, i.e., below 10% and above 90% solvent B. For example, at 99% solvent A the system must accurately draw 99 parts of A and 1 part B from the solvent containers. Obviously this is very difficult, particularly when A and B vary in viscosity, and great accuracy is not to be expected. However, the system should be reproducible. Selecting a pumping system can be a confusing process because there are so many ways to build pumping systems that fulfill these basic requirements. Probably the first consideration is a careful assessment of present and future needs. If they are very simple, there is no need to select a very sophisticated instrument. Table I lists different types of pumps along with some advantages and disadvantages. Detachable pump heads that extend the pumping velocity of systems is one of the attractive features of many new systems. A low-velocity (0.1-5 mllmin) head can be used for analytical applications as opposed to a high-velocity (5-30 mllmin) head for preparative separations. Another attractive feature of some new pumps is a provision for washing the back side of piston seals to prevent seal abrasion by crystallized salts. Solvent sparging with helium or nitrogen to eliminate dissolved air is still another useful addition to a pumping system. Automated valving for column switching will become popular in the future. Fortunately, automated switching valves may easily be installed on any instrument. Solvents

Ideally, HPLC mobile phases should not corrode either pumping systems or columns. This means that high concentrations of halide should not be used in stainless steel systems. Care should be taken to ensure solvent purity. The presence of UV-absorbing species in solvents can be a problem when using absorbance detectors. All solvents (including water, deionized or twice distilled), buffers, and additives (displacing salts, acid

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TABLE I GENERALSUMMARY OF PUMPING SYSTEMS

Advantages No pulsation

Reciprocating piston

Constant pressure

Small stroke (35-400 pl) making solvent changes rapid and accurate; good for gradient elution; flow rate range (0.1- 10 mllmin) No seals; low pulsation; continuous solvent delivery; good accuracy Completely pulseless; inexpensive; precise flow rate; durability

Disadvantages Refilling; compression of liquids disturbs flow; large volume Piston seal failure; pump pulsation

Large dead volume; not efficient for fast gradients; cost Column back pressure, mobile-phase viscosity, and column temperature must remain constant; limited solvent supply; restricted to <2000 psi; inconvenient for gradient elution; limited accuracy

or base for pH adjustment, and displacing agents) should be purified or be of HPLC grade. All solutions used in HPLC analysis should be vacuum filtered through a 0.2-pm filter to remove dust or any other particulate impurities that would plug columns. When buffer solutions are not made fresh daily, refrigerated storage is recommended to prevent microbial growth. Prior to use, all mobile-phase solutions should be deaerated to prevent the formation of gas bubbles inside the instrument. Gas bubbles that lodge in the detector flow cell cause severe baseline drift.

Pulse Dampers Pumping systems that produce discontinuous flow require some type of pulse damping. Two types of mechanical pulse dampers are commonly used. One design incorporates a flexible tube sealed in a container of compressible liquid. As the mobile-phase pulse reaches the pulse damper, the tube expands and the energy is absorbed by the surrounding medium. Another flex-tube design uses 1-3 m of flattened stainless steel tubing which expands with the pulse. The disadvantages of the second design include a relatively large dead volume and continuous flexing of the flattened tube, which causes the brittle steel tube to crack and leak within a

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short time. Electronic pulse damping and pumps with special cams are much more effective ways to provide continuous flow to columns. Mixers

Most HPLC instruments used in protein separations prepare gradients by mixing two or more solvents. The function of the mixer is to mix these solvents without disturbing the gradient. Two types of HPLC mixers are available, dynamic and static. The dynamic mixer uses a magnetic stirring bar within a mixing chamber to mechanically stir the contents together. This mixer is the most versatile, but often has the disadvantage of introducing 1-3 ml of dead volume into the pumping system. Static mixers, on the other hand, consist of a chamber filled with inert materials and operate much like a poor chromatography column with substantial bandspreading. Bandspreading is, in fact, a form of mixing based primarily on eddy diffusion. The advantage of the static mixer is that it can be of lower volume than dynamic mixers. The disadvantage of static mixers is that high-viscosity solutions are not mixed well. Detectors

Ultraviolet-visible (UV-vis) absorbance detectors are popular in HPLC instruments used for protein separations because of their high sensitivity, reliability, broad linear range (lo5), relative insensitivity to changes in refractive index, and, most importantly, nondestructive detection of sample. Variable-wavelength UV detectors allow the operator to choose the optimum detection conditions depending on absorption maxima and extinction coefficients of the sample. For example, if the target protein has a sufficient number of aromatic surface residues, a wavelength near 280 nm can be used to detect eluted protein. In contrast, peptides or proteins with a limited number of aromatic residues may be detected more effectively near 2 12 nm. The diode array detector (DAD) is a particularly attractive version of the UV-vis detector. The DAD is capable of scanning the entire spectrum from 200 to 600 nm in less than a second. Combined with the proper computer support, this detector can acquire and store hundreds of spectra throughout the course of a chromatographic analysis. Chromatograms from a single wavelength or a series of wavelengths can be constructed. Through background subtraction the entire spectrum of any protein peak in the chromatogram can be reconstructed. Using three-dimensional color mapping of elution profiles it is even possible to detect minor impurities in a protein peak. These features provide a method by which on-line structural analysis of proteins can be accomplished.

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Derivative spectroscopy of the complex polypeptide spectrum in the near-ultraviolet region has been used to evaluate the contributions of the three aromatic chromophores in proteins. Small changes in protein solvation or conformation can be detected by shifts in the absorption spectra of these three amino acids. French and Church2 suggested the possibility of applying derivative absorption spectroscopy for the detection of weak absorption bands masked by relatively strong ones. Second derivative spectrophotometry has since been shown to be an effective quantitative method for examining phenylalanine and tryptophan residues in prot e i n ~ .A~ subsequent .~ study reported the determination of tyrosine exposure in proteins, by resolving overlapping bands in the normal spectrum and the greater sensitivity of tyrosine to solvent effeck5 These methods have more recently been applied to examine the conformational changes of proteins eluted from reversed-phase and hydrophobic interaction c01umns.~~~ Chromatographic Modes Size-Exclusion Chromatography Ideally, descriptive names that tell something about the fractionation mechanism should be used to describe the separation processes. Unfortunately, this has not been the case in the chromatographic process by which macromolecules are fractionated on the basis of size. Gel filtration chromatography, gel permeation chromatography, molecular sieve chromatography, and size-exclusion chromatography have all been used to describe size-based chromatographic separations. As will be shown below, size-exclusion chromatography (SEC) is the most suitable name for the technique because filtration, sieving, and the use of gels have little to do with size-based chromatographic separations. Discrimination between molecular species on the basis of size is based on differential permeation into matrices of controlled porosity. It is commonly accepted that small molecules, such as deuterium oxide, totally

* C. S. French and A. B. Church, Carnegie Inst. Wash. Year Book 54, 162 (1955).

' T. Ichikawa and H. Terada, Biochim. Biophys. Acta 494, 267 (1977).

C. Balestrieri, G. Colonna, A. Giovane, G. Irace, and L. Servillo, Eur. J. Biochem. 90, 433 (1978). R. Ragone, G. Colonna, C. Balestrieri, L. Servillo, and G. Irace, Biochernisrry 23, 1871 (1984). S. A. Cohen, K. Benedek, Y. Tapuhi, J . C. Ford, and B. L. Karger, Anal. Biochem. 144, 275 (1985). S.-L. Wu, K . Benedek, and B. L. Karger, J. Chromatogr. 359, 3 (1986).

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permeate the liquid volume within and between the particles in a column and elute from the column in a volume of liquid known as the total permeation volume (Vt). In contrast, macromolecular species that are too large to penetrate the pore matrix of the column packing are excluded from the internal liquid volume of the packing and elute in a column void volume (Vo) equal to the volume of liquid between particles. Subtracting V , from Vo gives the pore volume or internal volume (Vi) of the packing material. All macromolecular species that do not interact with the column packing material elute between these two extremes, the void volume (Vo) and the total permeation volume (VJ. It has been found that the elution volume (V,) of all macromolecular species is described by the simple equation where K , is a size-exclusion distribution coefficient, ranging from zero for excluded macromolecular species to one for small molecules that totally permeate the packing. Fractional values of K , reflect partial access to the liquid volume with the pore matrix of the chromatographic packing. Using macromolecular species of the same shape and known molecular weight, it may be shown that there is a linear relationship between the log of the molecular weight and K , between K, values of approximately 0.15 and 0.9 when there is a narrow distribution of pore diameters in the packing. This linear relationship is deceptive in that it implies SEC determines molecular weight. It does not. The mechanism above clearly shows that size discrimination is based on molecular size or, more precisely, either hydrodynamic volume or radius of gyration. Elongated proteins such as fibrinogen have a larger hydrodynamic volume than globular proteins of the same molecular weight and elute from SEC columns in a smaller elution volume. Pure SEC is based on the assumption that there is no interaction between solutes and the surface of the packing material. This is not always true, particularly when the column is either charged or hydrophobic. Proteins that adsorb weakly to the column matrix through either hydrophobic or electrostatic interactions appear to be smaller than their actual molecular weight. In contrast, solutes of the same charge as the column will be electrostatically repelled from the pore matrix and appear to be larger than their actual molecular weight. Packing Materials. The surface of the ideal SEC packing material must be neutral and hydrophilic to minimize the possibility of adsorption. This has been achieved in several ways. One technique is to fabricate highly cross-linked, mechanically stable, macroporous matrices from neutral, hydrophilic polymers. Both dextrans and agarose have been used to prepare such materials. The problems with this approach are that

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cross-linking agents generally introduce hydrophobic character into the matrix, pore diameter and pore volume can be reduced during crosslinking, the pressure limit of the matrix decreases with increasing porosity, and the upper pressure limit is seldom greater than 30 atm. Other polymeric based supports capable of withstanding pressure limits over 30 atrn and chemically stable in alkaline mobile phase have been devel~ p e d .It~is, ~ only in comparison to the silica-based series of supports that A second technique for preparing the separations seem less SEC packing materials is to coat the surface of macroporous silica with either organosilanes or organic polymer^.'^ The problems with this approach are that silica-based materials are of diminished stability above pH 8.0, coatings often erode during extended use, and the packings generally have some anionic character.I4 At the present time, highest resolution in the SEC mode can probably be obtained on silica-based materials because they are of higher pore volume and smaller particle size.15 Resolution in SEC is determined by three characteristics of the chromatographic packing: pore size distribution, pore volume, and particle size. A small pore-~izedistribution gives the greatest discrimination between solutes on the basis of molecular size. Uniformity of pore distribution can be seen In the slope of log M , vs V , curve, where the slope will approach 2 with extremely homogeneous pore-size materials. Resolution also increases with increasing pore volume. Materials with a Vi/Voratio of 1.5 are approaching the upper limit of useful porosity. Although materials of higher porosity can be made, mechanical strength of the matrix declines rapidly as the Vi/Voratio exceeds 1.6. Stagnant mobile phase mass transfer is the third variable controlling resolution in SEC. As particle size of the SEC packing is decreased, stagnant mobile-phase mass transfer is less of a problem and chromatographic peaks become sharper. Selecting a column with the proper molecular weight fractionating range is another consideration. The pores of the packing must be sufficiently large to allow solute penetration, but not so large that the solute totally penetrates the pore matrix. Fortunately, column manufacturers generally supply tables that show the fractionating range of a given E. Pfannkoch, K. C. Lu, F. E. Regnier, and H. Barth, J . Chromatogr. Sci. 18,430 (1980). P. Roumeliotis and K. K. Unger, J . Chromatogr. 149, 211 (1978). lo F. E. Regnier and R. Noel, J. Chromatogr. Sci. 14, 316 (1976). " T. Hashimoto, H. Sasaki, M. Aiura, and Y. Kato, J . Chromatogr. 160, 301 (1978). IZ M. Okazaki, Y. Ohno, and I. Hara, J. Chromatogr. 221, 257 (1980). l 3 C. T. Wehr and S. R. Abbott, J . Chromatogr. 185, 453 (1979). l4 M. Okazaki, K. Shiraishi, Y. Ohno, and I. Hara, J . Chromatogr. 223, 285 (1981). l5 Y. Kato, K. Komiya, Y . Sawada, H. Sasaki, and T. Hashimoto, J . Chromatogr. 190,305 (1980).

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column. It should be noted, however, that these size-exclusion calibration curves are based on globular proteins. The molecular weight of dextrans, many polynucleotides, and asymmetric proteins will be too high. Columns will foul with particulate matter and denatured proteins as they are used. It is necessary to remove these deposits to extend the life of SEC columns. In the event that the column is stable in base, the column can be washed with 0.1 N base. When columns are fouled with hydrophobic proteins, organic solvents such as propanol may also have to be used. Silica-based SEC columns may be cleaned by eluting them repeatedly with a gradient ranging from aqueous 0.1% trifluoroacetic acid to 0.1% trifluoroacetic acid in 70% propanol. Mobile Phases. A good mobile phase for SEC will have the following properties. First, it will minimize the potential for solute/column interact i o n ~ . ' Since ~ ' ~ hydrophobic and electrostatic interactions are the dominant forces by which solutes adsorb to columns, mobile-phase ionic strength should be sufficiently high (>0.1 M NaC1) to prevent electrostatic interactions, but not high enough (<0.5 M NaCl) to drive hydrophobic interactions. Second, the mobile phase should be noncorrosive. It has already been noted above that high concentrations of halides under acidic conditions corrode stainless steel. Third, it should not alter the threedimensional structure of sensitive solutes such as proteins. Fourth, mobile-phase viscosity should be minimal. Mobile phases containing high concentrations of glycerol, ethylene glycol, or other additives cause severe bandspreading and loss of resolution. Within these limits, a large variety of mobile phases may be used in SEC without altering the chromatographic separation. In some cases, it is desirable to diminish the shape differences between proteins and determine the true molecular weight of an unknown protein. This can be done by adding 0.1% sodium dodecyl sulfate (SDS) or some other denaturant to the mobile phase. There are, however, problems with this technique. First, it is very difficult to elute all of the SDS from a column and return to normal, nondenaturing, use. Second, proteins and columns adsorb so much SDS that the molecular weight fractionation range of the column is diminished with a loss in resolution. Applications. The very attractive feature of SEC is that it may be used with a wide variety of mobile phases at near physiological conditions. This makes it possible to determine molecular weight distribution and l6 l7

l9

D. JOS~C, H. Baumann, and W. Reutter, Anal. Biochem. 142, 473 (1984). C. L. DeLigny, W. J. Gelsema, and A. M. P. Roozen, J . Chromatogr. 294, 223 (1984). T. Arakawa, Anal. Biochem. 144, 267 (1985). S. B. Yan, D. A. Tuason, V. B. Tuason, and W. H. Frey, 11, Anal. Biochem. 138, 137 (1984).

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intermolecular interactions as they might occur in vivo. Separation times of 10-30 min in columns of 30-cm length are common. Little gain in resolution is achieved by reducing the mobile-phase velocity to the point that elution time on'a 30-cm column requires more than 60 min. SEC is much less discriminating than PAGE. A good column is only capable of resolving macromolecular solutes of the same shape that vary 2-fold in molecular weight. When solutes differ in shape or one of the species interacts with the column, it is often possible to do much better. Several unique features of SEC columns should be recognized in their application to separation problems. The first is that solutes eluting in the K , range of 0.05 to 0.2 give much broader peaks than the void volume peak and those eluting later in the separation. This is because the size of these solutes is approaching the pore diameter of the matrix and they are partially trapped in the pore matrix. It should be noted that resolution in SEC is very sensitive to sample viscosity. A phenomenon call "viscous fingering" can be seen in glass columns that are loaded with high-viscosity solutions containing a dye. "Fingers" of dye solution rapidly penetrate larger channels in the column bed and migrate far ahead of the bulk of the solution. It can be seen that this phenomenon drastically compromises the resolution of sample components. The viscous fingering problem is easily solved by diluting the sample with mobile phase. High sample viscosity can either result from the macromolecular solutes themselves or from low-molecular-weight additives (usually sugars or glycerol) that are added to the sample. The low-molecular-weight additive problem is either overcome by dilution or dialysis. There is no solution to the problem of solute viscosity. In fact, solute viscosity largely determines the sample load that may be placed on a column. SEC columns also have a volumetric loading limit that becomes smaller with increasing resolving power of the column. Most columns will show volumetric overloading when sample volume is greater than 10% of the column volume. More precisely, volumetric overloading becomes noticeable when the sample volume is greater than 40 theoretical plate volumes. It has been noted above that cleaning columns is an important part of extending their operation lifetime. When the resolution of an SEC column has declined to 70% of that when it was new, it is time to clean the column. Waiting longer may irreversibly damage the column. Ion-Exchange Chromatography &

Ion-exchange chromatography (IEC) discriminates between proteins on the basis of accessible surface charges and their corresponding electrostatic interaction with the sorbent. The degree of protein retention in IEC,

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like other surface-mediated HPLC modes, is largely dependent on the strength and number of interactions. Three-dimensional structure sterically determines which surface residues may contact the sorbent stationary phase,20while net charge determines which form of IEC should be applied. Considering that even a moderately sized protein (25 to 60 kDa) has hundreds of residues capable of hydrophobic, ionic, or hydrogenbonding interactions with surfaces, it is highly probable that they will be adsorbed at more than one site. Furthermore, such an adsorption process would be cooperative; that is, adsorption at one site would increase the probability of adsorption at other sites. The choice of ion-exchange sorbent is dependent on both the pZ and charge distribution in the protein. As a rule, below its pZ, a protein should have a positive net charge while at conditions above its pZ, the net charge should be negative. Hence, cation-exchange chromatography (CEC) is generally used to separate proteins below their pZ and anion-exchange chromatography (AEC) for separations at pH conditions above their pZ, providing there is a symmetrical charge distribution. Kopaciewicz et al. showed that asymmetrically charged proteins were retained at or beyond their isoelectric point (above their pZ in CEC or below their pZ in AEC), .~' regardless of the mobile-phase pH with respect to the protein ~ 1 This phenomenon was attributed to the role that three-dimensional structure plays in surface recognition. Although all charged surface residues contribute to the net charge and ultimately the isoelectric point of a protein, due to the steric limitations associated with protein tertiary structure all of these residues cannot simultaneously interact with the column stationary phase. In cases where there is charge asymmetry, the protein chromatographic contact area may be focused on the area with an abundance of similar charge and the remaining protein surface charges disregarded. Retention is also dependent on the contact area microenvironment; thus the isoelectric point is not an absolute parameter for choosing AEC or CEC, but rather a guideline in the preexperiment preparation stage. Elution in IEC is generally accomplished at constant pH with increasing mobile-phase ionic strength to displace the protein from the stationary phase. The displacer ion concentration is dependent on the strength of interaction between the protein and the IEC column but is usually <0.5 M. Sodium chloride is the most widely used displacing salt. Mobile phases utilized in IEC normally consist of a buffer and a displacing salt. Providing the protein is stable at the chosen pH conditions, recovery and 20

F. E. Regnier, Science 238, 319 (1987). W. Kopaciewicz, M. A. Rounds, J. Fausnaugh, and F. E. Regnier, J . Chromatogr. 266,3 (1983).

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enzymatic activity for collected fractions consistently exceeds 90%.22 Hence, this chromatographic mode is classified as a nondenaturing method and is suitable for both analytical and preparative scale separations. Stationary Phase. Four types of stationary phase ligands have been used in IEC columns for protein separations: (1) weak anion-exchanging groups consisting of primary, secondary, or tertiary amines, (2) strong anion-exchanging groups consisting of aliphatic quaternary amines, (3) a weak cation-exchanging carboxyl group, or (4) a strong cation-exchanging sulfonic acid. The terms weak and strong do not refer to strength of affmity, but rather to the ligand's state of ionization. Weak IEC columns can be of variable charge density depending on the mobile-phase pH and the pK of the stationary phase. In contrast, strong IEC sorbents arc permanently ionized. Retention is less predictable on weak IEC columns because the charge densities on both the column and the proteins are pH dependent. a nonmechanistic The stoichiometric displacement model retention model, has been applied to ion-exchange23and reversed-phase ~hromatography.~~ It has been shown that chromatographic retention (capacity factor, k t ) is exponentially related to the number of sites (Z) on the protein that interact with the sorbent. This means that small changes in mobile-phase composition during elution produce large changes in retention. This phenomenon has several ramifications. Since elution occurs over such a narrow range of mobile-phase composition, it is necessary to use gradients to resolve complex protein mixtures from chromatography columns. The exponential relationship between k' and the Z number also influences the length of column required for a separation in gradient eluted systems. IEC columns less than 5 cm long generally have more than 80% of the resolving power of 30-cm c o l ~ m n s . ' ~The advantages of smaller columns include (1) less solute dilution upon elution and lower detection limits, (2) lower pressure required for elution, (3) easier and more reproducible packing, and (4) less expense. Mobile Phase. There are two ways to control selectivity in IEC: through pH or the type of salt. Changes in mobile-phase pH will affect ionization in the protein, thus altering the surface charge density and the affinity with which the protein is adsorbed to the column. Because the surrounding microenvironment influences the pK of amino acid side F. E. Regnier, Anal. Chem. 55, 1298A (1983). R. R. Drager and F. E. Regnier, J. Chromatogr. 359, 147 (1986). l4 X. Geng and F. E. Regnier, J. Chromatogr. 332, 147 (1985). 25 G . Vanecek and F. E. Regnier, Anal. Biochem. 109, 345 (1980).

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chains, the titration curve and charge distribution of each protein is unique. Therefore, it should be theoretically possible to find a pH at which any protein has distinctive chromatographic characteristics. It is not possible to predict a priori the optimum pH for resolving any two proteins. However, by constructing pH retention maps, i.e., plots of elution volume versus pH, optimum conditions can be found (Fig. 1).

FIG. 1. Retention maps. Five acidic and five basic proteins were chromatographed on both a strong anion (Mono Q ) and strong cation (Mono S) exchange column. A suitable buffering ion (0.01 M) was chosen for each pH. Proteins were eluted with a 20-min linear gradient from 0 to 0.5 M NaCl at a Bow rate of 1 rnllmin. LYS, Lysozyrne; CYTc, cytochrome c ; RNase, RNase A; CHY, chymotrypsin; CA, carbonate dehydratase; @LAC, Plactamase; STI, soybean trypsin inhibitor; OVA, ovalbumin; aAMY, a-amylase; CON, conalbumin.

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TABLE I1

INFLUENCE OF VARIOUS ANIONSON RETENTION AND RESOLUTION OF SELECTED PROTEINS RS

Relative displacing powerb Anionsa (sodium salt)

OVAc

STIc

CHYd

CYTcd

LYSd

OVAISTI

CYTcILYS

Bromide Phosphate Citrate Chloride Perchlorate Fluoride Bicarbonate Tartrate Sulfate Formate Acetate Propionate

0.70 0.59 1.00 0.60 0.81 0.45 0.61 0.70 0.73 0.54 0.54 0.54

1.00 0.74 0.80 0.88 0.88 nd 0.79 0.69 0.70 0.74 0.59 0.56

0.93 1.00 0.64 0.87 ndC 0.74 nd 0.64 0.70 nd 0.75 nd

0.77 1 .OO 0.44 0.66 nd 0.56 nd 0.56 0.60 nd 0.73 nd

1 .O 0.90 0.51 0.80 nd 0.58 nd 0.51 0.61 nd 0.70 nd

4.1 nd 5.3 5.4 4.3 nd 4.1 4.5 6.1 3.5 5.7 4.2

2.2 3.8 3.6 3.6 nd 6.7 nd 4.4 3.0 nd 4.1 nd

Chromatography was performed at pH 8. The ionic strength of buffer B was 0.5. Unity refers to the strongest displacing salt for that protein; 0 to the least. OVA, ovalburnin; STI, soybean trypsin inhibitor; CHY, chymotrypsin; CYTc, cytochrome c ; LYS, lysozyme. Determined on the Q-300 silica-based strong anion-exchange column. Determined on a Phannacia Mono S strong cation-exchange column. ' nd, Not determined.

Once mobile-phase pH has been selected, the choice of displacing ion type and concentration must be determined. Columns are normally loaded at 0.01 to 0.02 M salt and eluted in a linear gradient at 0.5 or 1.0 M salt. Displacing ions influence IEC retention with the relative displacing power generally following the Hoffmeister series.26Tables I1 and 111 list the influence of various anions and cations on the retention and resolution of various selected protein^.^' Application. Successful isolation does not demand strong protein affinity with the sorbent, but rather discrimination between impurity and target. Therefore, a chromatographic support that retains impurities but allows the desired protein to pass through unhindered is also a valid purification approach. This concept applies equally well to all surfacemediated chromatographic modes and should be considered throughout the purification scheme. The first step when attempting to purify a protein with unknown physical characteristics using IEC is to determine whether J. 0. Moms and P. Monis, "Separation Methods in Biochemistry," p. 228. Wiley (Interscience), New York, 1964. I 7 W . Kopaciewicz and F. E. Regnier, Anal. Biochem. 133, 251 (1983). l6 C.

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TABLE 111 RETENTIONAND RESOLUTION OF FIVE SELECTED PROTEINS

Rs

Relative displacing powerb Cationsa (chloride salt)

OVAc

[321

METHODS

STIc

CHYd

-

-

CYTcd

OVAISTI

LYSd

CYTcILYS

Lithium Sodium Potassium Ammonium Magnesium Calcium Sodiume Magnesiume -

a

f

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Chromztography was performed at pH 8. The ionic strength of buffer B was 0.5. Unity refers to the strongest displacing salt for that protein; 0 to the least. Abbreviations as in Table 11. Determined on the Q-300 strong anion-exchange support. Determined on the Pharmacia Mono S strong cation-exchange column. The acetate salt. nd, Not determined.

AEC or CEC will best resolve the target protein. Load the sample onto a 2-cm3 AEC column (DEAE-cellulose will suffice) with a 0.02 M Tris buffer at pH 8.0. If the target protein does not adsorb, then (1) the process may have retained other impurities, thus improving the sample purity, and (2) the desired protein is of either cationic surface charge or a heterogeneous ioniclhydrophobic surface, in which case either cation-exchange or hydrophobic interaction chromatography may be a more appropriate mode to use. If adsorption did occur, the protein may be eluted from the support with 0.5 to 1.0 M NaCI. An activity assay performed on the collected fraction determines the efficiency of the chromatographic method. Similar evaluation may be performed using a CEC support with the mobile-phase buffer consisting of 0.02 M sodium acetate at pH 5.0. Ultimately, the choice will be based on the column which best discriminates the chromatographic differences between the desired protein and the remaining impurities. If the protein pI is known, then the preexperiment guidelines recommended earlier may be used. Different selectivity is attained on weak and strong ion-exchange columns. It is recommended that strong ion-exchange columns be used during preliminary separations involving unknown proteins, to limit the experimental variables used to optimize resolution.

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Once the appropriate column has been selected, a retention map can be constructed, plotting retention time versus mobile-phase pH. Initial solutions should include the following range of pH buffers: pH 5.0, 6.0, 7.0, and 8.0. Further fine tuning using solutions of intermediate pH can be performed if necessary. From this, the optimum mobile-phase pH can be selected. The caveat of retention map data is that the best resolution does not always occur at the mobile-phase pH where the protein has strongest retention. Mobile-phase pH should be chosen according to the resolution of the target protein from the impurities associated with the sample. Finally, after the retention map is complete and the appropriate pH is chosen the remaining conditions, including salt type, salt concentration, and gradient slope, may be chosen. Careful experimental design incorporating both pH and salt type will provide the mobile-phase conditions for superior separations. Tables I1 and I11 show the influence of various ions on the chromatographic behavior of selected proteins. It is seen that the optimum displacing ion is an experimentally determined variable and cannot be predicted. Salt concentration is determined by the strength of protein retention. Gradient slope is a parameter that also must be adjusted after trial and error determinations. The standard IEC protein gradient is 20 min at a flow rate of 1 mllmin. However, greater resolution usually occurs with longer gradients, until bandspreading begins, thus limiting the separation. Hydrophobic Interaction Chromatography

Proteins contain both charged and neutral external amino acids which are solvent accessible and can interact with chromatographic supports. As indicated above, ion-exchange separations are performed on either AEC or CEC sorbents depending on the pZ and surface charge distribution of the protein. Protein separations based on surface hydrophobicity are accomplished on either hydrophobic interaction or reversed-phase sorbents, depending on the method of elution. When proteins are eluted with organic solvents, the technique is termed reversed-phase chromatography (RPC), while separations achieved with descending salt gradients have been designated hydrophobic interaction chromatography (HIC).28 This section will discuss HIC while the next section will present RPC. ~ protein ~ ~ ~ )sepaHIC was originally introduced (in the early 1 9 7 0 ~for rations using low ligand-density alkyl- or aryl-agarose sorbents and a F. E. Regnier, Science 222, 245 (1983). Z. Er-el, Y. Zaidenzaig, and S . Shaltiel, Biochem. Biophys. Res. Commun. 49,383 (1972). 30 S. Shaltiel and Z. Er-el, Proc. Natl. Acad. Sci. U.S.A. 70, 778 (1973). S. Hjerten, J. Rosengren, and S. Pahlman, J. Chrarnatogr. 101, 281 (1974). 28

Z9

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descending salt gradient. Analogous high-performance silica-based supports were developed employing derivatized polyamine coatings,32 bonded polyether coatings,33and poly(alky1aspartamide) coatings.34One polymeric based support was also developed by introducing phenyl groups with ether linkages into a commercially available TSK gel G5000PW support.35The mechanism of action for all the HIC supports is the same. HIC packings use short hydrocarbon ligands of low density as the stationary phase in an effort to reduce irreversible protein unfolding and binding due to the strong interactions between internal hydrophobic residues and the column. As a result, HIC has been shown to be a nondenaturing chromatographic method.36 Protein samples are induced to bind to these weak hydrophobic ligands through the use of high-ionicstrength mobile phases. The hydrophobic effect, as described by Tanford, is a unique organizing force, based on repulsion by the solvent instead of attractive forces at the site of ~rganization.~' In a polar environment, hydrophobic moieties cause an ordering of the water structure in the immediate vicinity, due to the unfavorable energetics associated with water-hydrocarbon interfaces. Entropy is probably the driving force in HIC. An increase in solvent entropy occurs when hydrophobic regions associate with an expulsion of water and reduction in the exposed surface available for solvation. High-ionic-strength mobile phases drive this association by increasing the surface tension of water, thus decreasing the amount of water molecules available to solvate the hydrophobic regions. This same phenomenon is the basis for the "salting out" of proteins by ammonium sulfate.38 The choice of salt for use in HIC is critical. Recently, the influence of neutral salts on the solubility of proteins, known as the Hoffmeister effect, was investigated and the salts in the series categorized as either kosmotropes (polar water structure makers) or chaotropes (water structure breakers).39Kosmotropes stabilize proteins in solution and chaotropes destabilize proteins. Sulfate was found to be the best kosmotrope. Retention studies have shown selectivity and efficiency

32

J. L. Fausnaugh, E. Pfannkoch, S. Gupta, and F. E. Regnier, Anal. Biochem. 137, 464

(1984). N. T. Miller, B. Feibush, and B. L. Karger, J. Chromarogr. 316, 519 (1985). 34 A. J. Alpert, J. Chromarogr. 359, 85 (1986). 35 Y. Kato, T. Kitamura, and T. Hashimoto, J. Chromatogr. 292, 418 (1984). 36 D. L. Gooding, M. N. Schmuck, and K. M. Gooding, J. Chromatogr. 296, 107 (1984). 37 C. Tanford, Science 200, 1012 (1978). M. Dixon and E. C. Webb, Adv. Protein Chem. 16, 197 (1961). 39 K. D. Collins and M. W. Washabaugh, Q. Rev. Biophys. 18, 323 (1985). 33

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for protein separations in HIC to be most favorable with an ammonium sulfate mobile phase.4w2 Selectivity in HIC arises from variations in stationary phase matrix (both ligand type and density) and mobile phase. These experimental variables determine the affinity of the short-distance hydrophobic forces between the protein surface and the stationary phase. As a result of the high-ionic-strength mobile phases utilized in this mode, sacrifices are made in chromatographic parameters such as detection sensitivity and column efficiency. Detection limits suffer because baseline drift, due to the large change in refractive index between mobile phases of high and low ionic strength, prevents detection in UV absorbance at high sensitivity without sophisticated detectors. Column efficiency suffers as a result of the elution mechanism. The high concentration of (NH4)2S04used increases the mobile-phase viscosity, impeding both protein and mobilephase diffusion into the porous sorbent. Multiple water molecules are needed to solvate both surface residues and the hydrocarbon ligands before elution can occur. Proteins eluting at intermediate ionic strengths undergo more adsorption-desorption steps during elution with a concomBecause there is itant increase in kinetic contributions to band~preading.~~ no direct displacement of protein from the column with a displacing agent, but rather reequilibration or rehydration of both the protein surface and the stationary phase with water molecules, peak shape is inherently broad compared to other chromatographic modes. Other explanations for the unusually broad peaks observed in HIC include heterogeneity in the adsorption sites of the column material,43normal conformational variations ~ ~ .protein ~~ aggregation induced by the in protein tertiary s t r u ~ t u r e ,and high-ionic-strength mobile phase.46 Stationary Phase. HIC media are of much lower surface hydrophobicity compared to RPC sorbents to limit the hydrophobic interactions between solute and stationary phase, thus preserving protein tertiary structure and biological activity. Common ligands used in HIC columns include methyl, propyl, pentyl, and phenyl groups. Selectivity can be enhanced by utilizing a column with a particular ligand. An increase in 40

Y. Kato, T. Kitamura, and T. Hashimoto, J. Chromatogr. 298, 407 (1984).

" J.-P. Chang, Z. El Rassi, and C. Horvath, J . Chromatogr. 319, 396 (1985).

M. N. Schmuck, M. P. Nowlan, and K. M. Gooding, J . Chromatogr. 371, 55 (1986). R. Snnivasan and E. Ruckenstein, Sep. Purif. Methods 9, 267 (1980). P. Strop, J. Chromatogr. 294, 213 (1984). " V. Zizkovsky, P. Strop, S. Lukesova, J. Korcakova, and P. Dvororak, Oncodev. Biol. Med. 2, 323 (1981). 46 M. E. Co-Sarno, M. A. Tapang, and D. G. Luckhurst, J . Chromatogr. 266, 105 (1983). " 43

"

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PURIFICATION PROCEDURES: CHROMATOGRAPHIC METHODS

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alkyl chain length raises the relative hydrophobicity, so pentyl sorbents should be used to separate hydrophilic proteins not retained on the methyl or propyl columns. The phenyl sorbent is particularly attractive in that it may form complexes with accessible aromatic side chains through T-rr interactions, providing yet a different selectivity. Hydration of the packing material is important in HIC, because water must be displaced from the stationary phase as well as from the solute for protein adsorption to occur. Silica-based HIC supports have been prepared by bonding the stationary phase through either a simple organosilane coupling agent or attachment to a hydrophilic polymer film on the silica surface. The polymeric based supports are all of a proprietary nature. However, some designs include the use of hydroxylated polyether as a support materiaL4' This improves the alkaline stability of these supports compared to silica-based sorbents. Mobile Phase. Equally important to HIC selectivity is the mobilephase contribution, which can be manipulated by either changes in salt type or pH.48AS previously mentioned, (NH&S04 has been shown to be the most favorable salt for most protein separations. However, differences in retention behavior have been found as a function of salt type.49 Mobile-phase ionic strengths as high as 2.0-3.0 M are used to drive the hydrophobic interaction. This creates a maintenance problem if the pumping system is not properly cleaned, because salt precipitation on the pistons will diminish the life of pump seals. Protein retention in HIC can be influenced by the mobile-phase pH through indirect contributions of charged surface amino acids. Charged residues will attract water and make the protein more hydrophilic. It was recently discovered that both positively and negatively charged amino acid substitutions in a genetically engineered protein affected protein retention on a HIC column.50 HIC mobile-phase pH was manipulated to separate proteins differing by a single charge substitution. The differences in retention were attributed to preferential water binding for the different substituted residues. Because mobile-phase pH can affect the ionization states of charged surface residues, greatest retention should occur when proteins have minimal surface charge. Changes in chromatographic behavior in the pH range from five to eight are due to the ionization of surface histidine residue^.^' Optimum mobile-phase pH conditions may be determined as previously described in the IEC Mobile Phase section. S. C. Goheen and S. C. Engelhorn. J . Chromatogr. 317, 55 (1984). S. Hierten, K. Yao, K. 0.Eriksson, and B . Johansson, J. Chromatogr. 359, 99 (1986). 49 2. El Rassi and C. Horvath, Chromatographia 19, 9 (1985). 50 R. M. Chicz and F. E. Regnier, J . Chromatogr. 500 (1990), in press. 5' J. Fausnaugh and F. E. Regnier, J. Chromatogr. 359, 1 3 1 (1986). 47

48

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Application. Before attempting HIC separations at least some idea as to the surface hydrophobicity is necessary. Although undocumented, correlation between "salting out" and HIC retention may exist. Perhaps this may be a crude measure for protein surface hydrophobicity. Proteins with little or no hydrophobic surface residues will either pass through an HIC column or need such a high concentration of salt to be retained that further work becomes impractical. A good versatile sorbent to start with would be the propyl column. This ligand has intermediate hydrophobicity and can provide some initial data on whether HIC will be an appropriate purification route for the target protein. If there are known surface-accessible aromatic residues, then the phenyl-linked sorbent should be used as the primary column. Usually, proteins have some amount of aromatic residues near the surface. Hence, the phenyl column is a rather popular choice. Depending on the chromatographic behavior, a more (pentyl) or less (methyl) hydrophobic ligand can be chosen for further separation attempts. Initial HIC separations should be performed using a 20-min descending salt gradient from 2.0 to 0.0 M (NH4)2S04in a 0.01 to 0.05 M phosphate, pH 7.0 buffered solution. Once the proper column has been chosen, mobile-phase optimization can begin. To choose the optimum mobile-phase pH, retention maps similar to those described previously need to be constructed. Again, the optimum pH is not necessarily the condition at which maximum retention is obtained, but where the greatest separation from sample impurities is attained. Once the pH conditions have been determined the length of gradient and type of salt may be established. Excessively long gradient elution times may increase the separation factor, but by design lessen simultaneous elution and promote bandbroadening. Therefore, gradient elution should be limited to a maximum time of 90-120 min from 2.0-3.0 to 0 M (NH4)2S04.40Resolution of proteins by gradient time manipulation must be done experimentally. Although a sample may be sufficiently hydrophobic and need only a relatively low initial concentration of salt to drive the hydrophobic interaction, the best separations occur when initial sample retention is driven with a slightly excessive amount of salt.32,40Perhaps this is due to the indirect dehydration mechanism of both the solute and sorbent. Selectivity may be enhanced by using different salts. Kosmotropic salts are recommended over chaotropic salts because of their stabilizing versus destabilizing nature. It is essential that whatever salt be used is of the highest purity. Due to the excessive concentrations of salt used, and the gradient elution protocol going from 100 to 0% high salt, a large change in refractive index occurs. Providing high sensitivity is not required, this problem may be overcome. However, the addition of inorganic impurities

414

PURIFICATION PROCEDURES: CHROMATOGRAPHIC METHODS

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associated with the manufacture of lower grade salts serves to interfere with protein detection by increasing the baseline noise. Reversed-Phase Chromatography The separation of protein and peptide mixtures on reversed-phase ~ ~ elution , ~ ~ process in RPC is completely supports is widely p r a c t i ~ e d .The different from that in HIC, even though both chromatographic modes utilize hydrophobic interactions to retain proteins. In the RPC of proteins, the weak mobile phase usually consists of an aqueous acidic solution [O.l% trifluoroacetic acid (TFA), 1% phosphate acid, triethylammonium phosphate (TEAP), etc.], while the eluting mobile phase is an organic solvent (methanol, Zpropanol, or acetonitrile). Once the sample is injected onto the column in the weak mobile phase, it is retained until the proper concentration of organic solvent displaces the protein from the support. Unlike HIC, the sample peak shape in an RPC chromatogram is sharp, as a result of this displacement elution. However, because of the acidity and the organic solvents needed to elute proteins from the extremely hydrophobic RPC stationary phase, protein denaturation often occurs. In many cases, biological activity cannot be retrieved after the tertiary structure is disrupted and RPC is not widely used as a preparative technique for proteins of >10 kDa.S4RPC can be used as a preparative technique for proteins that are stable in organic mobile phases, or for proteins that can renature after unfolding occurs during the RPC elution process. This chromatographic mode is, however, a powerful analytical method for both proteins and peptides. Stationary Phase. Alkylsilane-derivatized silica media have been the dominant choice for reversed-phase chromatography of biological macromolecules, although both silica and organic resins have been used. There are four variables to consider when selecting a silica-based RPC sorbent for protein separations. Depending on the ligand length, alkyl residues are generally 7 A apart on a well-coated RPC packing, providing the opportunity for cooperative adsorption of macromolecular solutes.5s Poor recovery of large hydrophobic cyanogen bromide cleavage fragments have been reported for some silica supports.56The combination of too long an alkyl chain length with denaturing mobile-phase conditions may cause irreversF. E. Regnier and K. M. Gooding, Anal. Biochem. 103, 1 (1980). W. S. Hancock and J. T. Sparrow, in "High-Performance Liquid Chromatography" (C. Howath, ed.), Vol. 3, p. 49. Academic Press, New York, 1983. 54 J. L. Fausnaugh, L. A. Kennedy, and F. E. Regnier, J . Chromatogr. 317, 141 (1984). 55 J. L. M. van der Venne, J . P. M. Rindt, G. J. M. Coenen, and C. A. M. G. Cramers, Chromatographia W, 11 (1980). 56 J. Pearson, W. C. Mahoney, M. A. Hermodson, and F. E. Regnier, J. Chromatogr. 207, 325 (1981). 52 53

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ible protein adsorption to the RPC sorbent. The alkyl chain length most effective for proteins is in the shorter range (C4 to CB), while longer lengths (Cs to C18) are normally used for small peptide and sequencing separation^.^^ Nonspecific adsorption may occur through electrostatic interactions between surface silanols and protein amino groups. Exhaustively silylated and end-capped sorbents present the fewest problems, particularly when the sample mixture contains very basic proteins. Another variable associated with RPC sorbents is the support pore diameter. To ensure adequate mass transfer, it is recommended to use macroporous supports with a pore diameter between 300 and 1000 h; for protein separation^.^^ This allows total protein penetration into the pores, thus maximizing available sorbent surface area for protein-support interaction. Nonporous media are acceptable for analytical separations, where speed is a major issue and small samples may be used. The final variable regards particle size. The general practice in analytical separations is to use particles of 5 p m or less to achieve maximum resolution. For preparative applications, 10 to 20-pm media are often used because the resolution of 5-pm sorbents deteriorates quickly when they are overloaded. Macroporous polystyrene-divinylbenzene-based RPC sorbents have also been used in protein separation^.^^.'^ One major advantage of these materials over silica is their alkaline stability. Comparisons of polystyrene- and silica-based supports have shown differences in selectivity, retention, and flow-rate parameter^.^^ Mobile Phase. Selectivity in RPC can be achieved by manipulation of the mobile phase.63The acid component of the mobile phase contributes to retention by (1) influencing the ionic state of the protein, (2) controlling ionization of surface silanols, (3) forming ion pairs between protein cationic surface residues and the acid, and (4) influencing the denaturation of proteins. The mobile-phase pH dictates the ionization states of the protein and surface silanols. Hydrophobic acids, such as TFA64or heptafluorobutyric acid,65increase protein hydrophobicity by forming ion pairs, conseR. V. Lewis, A. Fallon, S . Stein, K. D. Gibson, and S.Udenfriend, Anal. Biochem. 104, 153 (1981). j8 K. A. Tweeten and T. N. Tweeten, J. Chromatogr. 359, 1 1 1 (1986). j9 S. Coppi, A. Betti, C. Bighi, G. P. Cartoni, and F. Coccioli, J. Chromatogr. 442, 97 (1988). R. I. Gregson and A. M. Patch, J. Chromatogr. 242, 349 (1982). 61 W. A. Moats and L. Leskinen, J. Chromatogr. 386, 79 (1987). D. P. Lee, J. Chromatogr. 443, 143 (1988). 63 K. D. Nugent, W. G. Burton, T. K. Slattery, B. F. Johnson, and L. R. Snyder, J. Chromatogr. 443, 381 (1988). M. A. Hermodson, and W. C. Mahoney, this series, Vol. 91, p. 89. 65 H. P. J. Bennett, C. A. Browne, and S . Soloman, J. Liq. Chromatogr. 3, 1353 (1980). j7

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quently increasing retention. A decrease in protein retention, relative to native non-ion-paired protein, is obtained when using a hydrophilic ionpairing agent, such as phosphoric acid.@Other ion-pairing agents such as alkylsulfonic acids and alkylamines have also been used to alter selectivity in RPC columns and increase r e s o l ~ t i o n . ~ ~ Acids are used in the mobile phase to increase both recovery and resolution of many proteins. TFA is the most popular acid because it is an excellent solubilizing agent and allows detection of peptide bonds below 230 nm.@This pairing agent is far less effective with membrane proteins or other very hydrophobic proteins. These applications require formic Gradient elution separations using acid concentrations of 5 to formic acidlwater to formic acid in an organic solvent show substantially higher recovery with equivalent or superior resolution. Organic components such as acetonitrile, methanol, ethanol, and propan01 have been found to contribute little to selectivity in the RPC of proteins.70," The selection of the mobile-phase organic constituent is During the elution process, protein based on solubility and vis~osity.~' solubility may become a problem. After interaction with the stationary phase, protein unfolding often occurs, which exposes the internal hydrophobic residues and propagates protein precipitation. When protein solubility is a problem, 2-propanol is the solvent of choice. Mobile-phase viscosity becomes important when high back pressure is encountered, such as with small pore-diameter microparticulate columns, because the system pressure limits may be exceeded. Another reason for maintaining low viscosity involves protein diffusion and the resulting increase in resolution between separated solutes. Lower viscosity allows faster diffusion rates, therefore protein elution incurs less bandspreading and resolution increases. Acetonitrile is the preferred organic component for separations minimizing viscosity (maximizing resolution). Application. Because RPC usually disrupts protein tertiary structure, it is most often used as an analytical technique rather than for protein purification. However, for those applications where proteins are easily renatured after RPC, the following protocol is suggested. First, polypeptides are not always soluble in RPC mobile phases. Protein solubility G . E. Gerber, R. G . Anderegg, W. C. Herling, C. P. Gray, K. Biemann, and H. G. Khorana, Proc. Natl. Acad. Sci. U.S.A.76,227 (1979). 67 J. L. Meek and Z. L. Rossetti, J . Chromatogr. 211, I5 (1981). K. Karch, I. Sebestian, and I. Halasz, J. Chromatogr. 122, 3 (1976). 69 G. Thevenon and F. E. Regnier, J . Chromatogr. 476,499 (1989). M . J. O'Hare and E. C. Nice, J. Chromatogr. 171,209 (1979). '' S. D. Powers, M. A. Lochrie, and R. 0.Poyton, J . Chromatogr. 266, 585 (1983). 72 M. Rubenstein, Anal. Biochem. 98, 1 (1979).

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dictates the choice for both type and concentration of ion-pairing agent. TFA is the first choice because it has been the most widely used and it does not interfere with optical absorption in the ultraviolet range used for protein detection. Next, the decision on the choice of sorbent should be made. Depending on the characteristics of the protein, either a silica-based short alkylchain column or a polystyrene-divinylbenzene support should be chosen. For example, to perform separations involving extremes in pH, the polystyrene support is recommended. Most other separations have been carried out on macroporous (>300 &, C4to C8 alkylsilane-derivatized silicabased sorbents. Once the column and ion-pairing agent are chosen, the mobile-phase organic component should be selected. If there are no solubility limitations, then the choice of organic component is based on minimizing mobile-phase viscosity. For this reason, acetonitrile is widely used. When solubility is a problem, propanol and 2-propanol are better Once the stationary and mobile phases are chosen, the gradient elution protocol must be determined. As stated in the previous application sections of the different chromatographic modes, gradient protocol is an experimentally determined parameter and cannot always be predicted. Normally a 20-min linear gradient from 100% aqueous buffer to 100% organic solvent is first applied to examine the resolution of the target protein from the impurities. Longer gradient times will usually produce better resolution up to a limit of 60-90 min. Immobilized Metal Affinity Chromatography

r: f

F:

Affinity chromatography is based on the bioaffinity of a protein for an immobilized ligand similar in structure to a natural ligand. The most exploited bioaffinity interactions utilized in HPLC include antigenlantibody binding, pseudo-substrate/enzyme binding, pseudo-cofactorlenzyme binding, and lectin/glycoprotein binding.73One of the disadvantages using these methods is that a different stationary phase must be used for almost every separation. This is both labor intensive and expensive. The advantage of affinity chromatography is that high-yield one-step purifications can be achieved from crude extracts. It is not commonly appreciated in biotechnology that these affinity methods are very poor in discriminating between structural variants of proteins. Because a small region of protein structure determines chromatographic behavior and elution is normally a step gradient, all retained species simultaneously elute. Hence, although

F

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F . E. Regnier, Chromatographia 24, 241 (1987).

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affinity separations are known to be superior for resolving mixtures containing different protein s~ecies,bioaffinity chromatography appears to be a poor choice for exaAning surface-engineered modifications of the same protein. Immobilized metal affinity chromatography (IMAC) is a particularly attractive variation of the affinity concept. The purification of proteins by this approach was introduced in 1975 by Porath et Protein separations are based on the interactions that occur between the electron donor groups (Lewis base) of the surface amino acids and the immobilized metal atom as an electron acceptor (Lewis acid). All proteins carry electrondonating atoms (N, S, 0)in their surface side chains, and as a result many proteins will interact with metal atoms. Because the residues recognized by the support are not necessarily clustered together at one location, IMAC is an affinity method that considers the entire protein surface. Metal chelate separations have several practical advantages over other affinity techniques: 1. Immobilized metals are a group-specific adsorbent. Therefore, a single metal can be used to separate a large number of proteins. Specificity can be maintained by careful design of the elution strategy. 2. Immobilized metal adsorbents have high capacities (up to 100 mg/ ml) and are sturdy. 3. Gradient elution allows discrimination of retained solutes by either direct displacement or by perturbing the coordination complex.

A large number of proteins have been purified using this technique. Selectivity in IMAC is influenced by the amount of accessible electrondonating groups on the protein surface, the type of metal immobilized, and the mobile-phase pH. In an attempt to identify donor group affinity ~~ for an immobilized metal complex, Hemdan and P ~ r a t hinvestigated amino acid interactions on a Ni(I1) support and concluded that His, Trp, and Cys have the greatest affinity among the single residues studied. In an ensuing communication the retention of oligopeptides was explored by vicinal substitutions of charged residues.76 The microenvironment was found to be influenced most by negatively charged amino acids which decreased peptide retention when located adjacent to Trp, Tyr, or His. Stationary Phase. Metal atoms have been immobilized on agarose, silica, and hydrophilic polymeric The most common metals J. Porath, J. Carlsson, I. Olsson, and G . Belfrage, Nature (London) 258, 598 (1975). E. S . Hemdan and J . Porath, J . Chromatogr. 323, 255 (1985). 76 E. S . Hemdan and J. Porath, J . Chromatogr. 323, 265 (1985). 77 J . Porath and B. Olin, Biochemistry 22, 1621 (1983). Z. El Rassi and C. Horvath, J . Chromatogr. 359, 241 (1986). 74

75

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used for IMAC include Cu(II), Ni(II), Ca(II), Zn(II), Fe(II), and Fe(II1). However, other group I11 metals have been ~ t u d i e d . ~The ' sorbent is prepared by first coupling a chelator, such as iminodiacetic acid (IDA), via a spacer arm to the support, followed by chelation with the metal of choice. This interaction is specific and tight binding, due to the nitrogen and two oxygen ligands on the IDA that form the metal coordination complex. To immobilize the metal, 2 ml of 0.2 M metal ion solution in water is injected onto the column immediately after an EDTAIdistilled water wash. Excess metal can be washed out with 5 ml of deionized water. The column must next be equilibrated with I5 ml of the weak buffer followed by a blank gradient before chromatographic analysis is attempted. After the metal is coordinated with the IDA, it has three sites available for specific protein surface electron donor atoms. The chelated metal may be removed from the column using EDTA and replaced with another metal after the column has been washed to remove any remaining EDTA.82IMAC is thus an affinity method in which multiple stationary phases may be manipulated using a single column. Mobile Phase. Sample loading takes place in the presence of salts (0.5-1.0 M NaCl) used to quench nonspecific electrostatic interactions between free IDA and basic surface residues. Elution is carried out by either a continuous decreasing pH gradient (protonation of the electron donor atom on the protein disrupts the coordination complex) or by displacement with a competing electron donor (e.g., i m i d a ~ o l e )A . ~review ~ on the structural requirements underlying metal chelate-protein recognition in lMAC contains a perspective on mobile-phase specification^.^^ It was noted that alkaline pH conditions were usually applied in IMAC separations because protein adsorption only occurred at a pH where electron donor groups were at least partially unprotonated. To accommodate the interaction between histidine nitrogen atoms and the IMAC support, mobile-phase pH should be adjusted above pH 6.0. Because the buffer should not effectively compete with proteins for coordination to IDAMe2+,many biological buffers (BES, MES, Tris) containing either primary or secondary amines should be avoided for use in IMAC. Unfortunately, certain alkaline conditions require a salt with an amino group to operate within the mobile-phase buffering capacity. In another paper, it A. Figueroa, C. Corradini, B. Feibush, and B . L. Karger, J. Chromatogr. 371,335 (1986). Y. Kato, K. Nakamura, and T. Hashimoto, J . Chromatogr. 354, 511 (1986). J. Porath, B. Olin, and B . Granstrand, Arch. Biochem. Biophys. 225, 543 (1983). M. Belew, T.-T. Yip, L. Anderson, and R . Ehrnstrom, Anal. Biochem. 164,457 (1987). 83 Y . Nakagawa, T.-T. Yip, M. Belew, and J. Porath, Anal. Biochem. 168, 75 (1988). 84 E. Sulkowski, Trends Biotechnol. 3, 1 (1985). 79

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was reported that around neutrality, glycine and primary amines not only compete for the metal complex, but actually displace the immobilized Therefore, inorganic salts are metal ions, especially Ni(I1) and ZII(II).~~ recommended for buffer solutions whenever possible. Another condition at which immobilized metal ions are displaced from the IMAC sorbent is at acidic pH, due to ionization of the metal ion. Therefore, elution conditions utilizing pH gradients must be carefully monitored for leaching of metal ligand. Application. As with the previous chromatographic modes, the initial decision regarding protein purification must be the choice of stationary phase. Although the other modes utilize separate columns for each individual ligand, IMAC permits a variety of different metals to be used with a single column. Each metal has its own specificity with respect to protein retention. The choice of metal is protein specific. An examination of standard protein mixtures on IMACg2revealed that the affinity and resolution of the test protein separations for immobilized metal ions followed the order CuZ+> Ni2+ > Zn2+.It is thus recommended, unless existing information regarding an alternate metal and the target protein is available, that initial separations be attempted using an immobilized Cu(I1) column. Basic chromatographic analysis should be performed at neutral to alkaline pH where strongest IMAC retention occurs. Because acidic pH may leach off the immobilized metal, for preliminary investigations it is recommended that the imidazole displacer be utilized. A standard 20-min linear gradient with a flow rate of 1 mllmin using 0.02 M phosphate at pH 7.5 with 0.5 M NaCl from 0.00 to 0.02 M imidazole should be applied. Once satisfactory retention has been established, fine tuning of the chromatographic conditions for optimum separation may begin. Sharpest peak shape and strongest affinity will occur in the more alkaline pH range. However, the disadvantages of prolonged work in this range include (1) deterioration of silica-based supports due to their instability at alkaline pH and (2) variation in immobilized metal-ligand density due to leaching of metal caused by primary amines (certain mobile-phase pH values require the use of amines as buffers). To choose the optimum mobile-phase pH, a retention map should be constructed. As mentioned previously, the greatest separation often does not occur at the pH of maximum retention. A recent IMAC investigation, of single-substitution genetically engineered proteins, revealed that strong retention affinity found at alkaline pH masked the subtle perturbations in surface microenvironments associated with the different amino acids.86The most successJ. Porath, J . Chromatogr. 443, 3 (1988). R. M. Chicz and F. E. Regnier, Anal. Chem. 61, 1742 (1989).

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ful separations occurred at modest acidity, depending on the location and type of amino acid substitution. Nonspecific interactions may interfere with the resolution of proteins in IMAC. Normally, nonspecific electrostatic interactions occur as a result of free IDA contributing negative charge to the stationary p a . As immobilized metal is stripped away from the IDA, the net negative charge of the sorbent increases. For this reason the 0.5 M NaCl is added to the mobile phase. Depending on the surface characteristics of the sample proteins, nonspecific hydrophobic interactions may also ensue. Additional salt, solvent, or surfactant additives may be required to minimize nonspecific binding. Then again, perhaps such nonspecific interactions at the proper pH will provide the extra difference to successfully complete the desired protein purification.

Acknowledgments Part of the work reported here was supported by NIH Grant GM 25413.

Section VIII Purification Procedures: Electrophoretic Methods

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[331 One-Dimensional Gel Electrophoresis

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) is an excellent method with which to identify and monitor proteins during purification and to assess the homogeneity of purified fractions. SDS-PAGE is routinely used for the estimation of protein subunit molecular weights and for determining the subunit compositions of purified proteins. SDS-PAGE can also be scaled up, for use in a preparative mode, to yield sufficient protein for further studies. In addition, twodimensional analysis, combining isoelectric focusing with SDS-PAGE,1.2 is a very high-resolution method for protein fractionation, enabling thousands of polypeptides to be resolved in a single gel. When used in conjunction with blotting method^.^ SDS-PAGE provides one of the most powerful means available for protein analysis. A great many electrophoretic systems have been developed and no attempt is made to summarize them here. In particular, the distinctions between the various "continuous" and "discontinuous" buffer systems are not discussed, nor are alternative support matrices considered. Gradient gels (gels whose pore sizes vary) are also omitted from discussion, since these can be prepared by relatively straightforward adaptation of any of a number of well-known methods for forming gradients. Rather, only the most common (and most reliable) analytical SDS-PAGE procedure"~ described. Those wishing further information on the practical or theoretical aspects of electrophoretic processes can use Refs. 5 through 8 to gain access to the large volume of literature in the field. Some problems may require adoption of alternative procedures,'-" but for most applications the SDS-PAGE method presented here will perform satisfactorily. B. S. Dunbar. H . Kimura. and T. M. Timmons. this volume [34]. S. Dunbar. "Two-Dimension;il Electrophoresis and Immunological Techniques." Plenum. New York. 1987. I T . \I.Timmons and B. S. Dunbar. this volume 1511. ' U. K. Laemmli. .\'~riltrc> (London) 227, 680 (i970). ' A. T . Andrews. "Electrophoresis: Theory, Techniques. and Biochemical and Clinical Applications." 2nd Ed. Oxford Univ. Press, New York. 1986. V. D. Hames. in "Gel Electrophoresis of Proteins: A Practical Approach" (B. D. Hames and D. Rickuood. eds.), p. I . IRL Press. Oxford, 1981. - R. C. Allen. C. '4. Saravis. and H. R. Maurer. "Gel Electrophoresis and lsoelectric Focusing of Proteins: Selected Techniques." d e Gruyter. Berlin. 1984. : B.

METHOUS I N ENZY\lOLOGY. COL. I82

Copyright C 1990 by Acadernlc Prrzs. Inc All r ~ ~ hof t sreproduction in an) form re5erved.

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[331 One-Dimensional Gel Electrophoresis By DAVIDE. GARFIN

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) is an excellent method with which to identify and monitor proteins during purification and to assess the homogeneity of purified fractions. SDS-PAGE is routinely used for the estimation of protein subunit molecular weights and for determining the subunit compositions of purified proteins. SDS-PAGE can also be scaled up, for use in a preparative mode, to yield sufficient protein for further studies. In addition, twodimensional analysis, combining isoelectric focusing with SDS-PAGE,'s2 is a very high-resolution method for protein fractionation, enabling thousands of polypeptides to be resolved in a single gel. When used in conjunction with blotting method^,^ SDS-PAGE provides one of the most powerful means available for protein analysis. A great many electrophoretic systems have been developed and no attempt is made to summarize them here. In particular, the distinctions between the various "continuous" and "discontinuous" buffer systems are not discussed, nor are alternative support matrices considered. Gradient gels (gels whose pore sizes vary) are also omitted from discussion, since these can be prepared by relatively straightforward adaptation of any of a number of well-known methods for forming gradients. Rather, only the most common (and most reliable) analytical SDS-PAGE procedure4 is described. Those wishing further information on the practical or theoretical aspects of electrophoretic processes can use Refs. 5 through 8 to gain access to the large volume of literature in the field. Some problems may require adoption of alternative procedure^,^-" but for most applications the SDS-PAGE method presented here will perform satisfactorily. I

'

B. S. Dunbar, H. Kimura. and T. M. Timmons, this volume [34]. B. S. Dunbar. "Two-Dimensional Electrophoresis and Immunological Techniques." Plenum, New York, 1987. T. M. Timmons and B. S. Dunbar, this volume [51]. U . K . Laemmli, Nature (London) 227, 680 (1970). A. T. Andrews, "Electrophoresis: Theory, Techniques, and Biochemical and Clinical Applications." 2nd Ed. Oxford Univ. Press, New York, 1986. B. D. Hames. in "Gel Electrophoresis of Proteins: A Practical Approach" (B. D. Hames and D. Rickwood, eds.), p. 1. IRL Press, Oxford, 1981. R. C. Allen, C. A. Saravis. and H. R. Maurer, "Gel Electrophoresis and Isoelectric Focusing of Proteins: Selected Techniques." de Gruyter, Berlin, 1984.

METHODS IN ENZYMOLOGY. VOL. 182

Copyright O 1990 by Academic Press. Inc. All rights of reproduction in any form reserved.

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Background

Although the detailed theory of gel electrophoresis is complicated and - ' ~ fundamental concepts are easily underat present i n ~ o m p l e t e , ' ~the stood. Briefly, in an electrophoretic separation, charged particles are caused to migrate toward the electrode of opposite sign under the influence of an externally applied electric field. The movements of the particles are retarded by interactions with the surrounding gel matrix, which acts as a molecular sieve. The opposing interactions of the electrical force and molecular sieving result in differential migration rates for the constituent proteins of a sample. In general, fractionation by gel electrophoresis is based on the sizes, shapes, and net charges of the macromolecules. Systems designed to fractionate proteins in their native configurations cannot distinguish between the effects of size, shape, and charge on electrophoretic mobility. As a consequence, proteins with differing molecular weights can have the same mobility in these systems. Thus, while PAGE methods for native proteins are valuable for separating and categorizing protein mixtures, they should not be used to assess the purity of a preparation or the molecular weight of an unknown. SDS-PAGE overcomes the limitations of native PAGE by imposing uniform hydrodynamic and charge characteristics on all the proteins in a sample mixture. During sample preparation, proteins are treated with hot SDS. The anionic detergent binds tightly to most proteins at about 1.4 mg of SDSImg of protein, imparting a negative charge to the resultant complexes.15 Interaction with SDS disrupts all noncovalent protein bonds, causing the macromolecules to unfold. Concomitant treatment with a disulfide-reducing agent, such as 2-mercaptoethanol or dithiothreitol, further denatures proteins, breaking them down to their constituent subunits. The electrophoretic mobilities of the resultant detergent-polypeptide complexes all assume the same functional relationship to their molecular weights. Migration of SDS derivatives is toward the anode at

A. Chrarnbach, "The Practice of Quantitative Gel Electrophoresis." VCH, Weinheim,

1985. P. J. Blackshear, this series, Vol. 104, p. 237. lo D. M. Neville, Jr., J. Biol. Chem. 246, 6328 (1971); see also D. M. Neville and H . Glossmann, this series, Vol. 32, p. 92. " A. F. Bury, J. Chromatogr. 213, 491 (1981). IZ T. M. Jovin, Biochemistry 12, 871, 879, 890 (1973). l3 A. Chrarnbach and T. M. Jovin, Electrophoresis 4, 190 (1983). l4M. Bier, 0. A. Palusinski, R. A. Mosher, and D. A. Saville, Science 219, 1281 (1983). l5T. B. Nielsen and J. A. Reynolds, this series, Vol. 48, p. 3.

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rates inversely proportional to the logarithms of their molecular SDS polypeptides, thus, move through gels in a predictable weights. manner, with low-molecular-weight complexes migrating faster than larger ones. This means that the molecular weight of a protein can be estimated from its relative mobility in a calibrated SDS-PAGE gel and that a single band in such a gel is a criterion of purity. Most electrophoresis is done in vertical chambers in gel slabs formed between two glass plate^.^,^ The slab format provides uniformity, so that different samples can be directly compared in the same gel. Gel thicknesses are established by spacers placed between the glass plates and sample wells are formed in the gels during polymerization with plastic, comb-shaped inserts. Electrophoresis cells provide means for sealing the assemblies during gel formation and for maintaining contact with the electrode buffers during runs. The better cells provide means for heat dissipation, because uneven heat distribution in the gel slab can cause band distortions. Conventional gels are of the order of 16 to 20 cm long, 16 cm wide, and 0.5-3.0 mm in thickness and can accommodate about 25 samples. Thick gels have greater total protein capacity than thin ones, but are correspondingly less efficient at dissipating electrically generated heat and more difficult to stain and destain. Gel thicknesses of 0.75 or 1 mm are good compromise sizes, combining adequate protein loads and good staining speeds with minimal heat-related distortions. Typical runs take 4-5 hr. Small-format cells (minicells) allow rapid analyses and are adequate for relatively uncomplicated samples. The design of these cells allows analyses to be completed two to three times faster than is possible with conventional cells. The gels are about 7 crn long x 8 cm wide and are very easily manipulated. Each gel can hold up to about 15 samples and a typical run can be completed in less than an hour (not counting set-up and polymerization time). The resolution of complex samples may be better in conventional gels than with minicells, since the separation of protein bands is improved by increasing the lengths of SDS-PAGE gels. 10316,17

Polyacrylamide Gels Polyacrylamide gels are formed by copolymerization of acrylamide monomer, CH2=CH-CO-NH2, and a cross-linking comonomer, l6

l7

A. L. Shapiro, E. Vinuela, and J . V. Maizel, Jr., Biochem. Biophys. Res. Commun. 28, 815 (1967). K. Weber and M. Osborn, J . Biol. Chem. 244, 4406 (1969).

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N,N '-methylenebisacrylamide, CH*=CH-CO-NH-CH2-NHCO-CH=CH2, ( b i s a ~ r y l a m i d e ) .The ~ ~ ,mechanism ~~ of gel formation is vinyl addition polymerization and is catalyzed by a free radical-generating system composed of ammonium persulfate (the initiator) and an accelerator, tetramethylethylenediamine (TEMED). TEMED causes the formation of free radicals from persulfate and these in turn catalyze polymerization. Oxygen, a radical scavenger, interferes with polymerization, so that proper degassing to remove dissolved oxygen from acrylamide solutions is crucial for reproducible gel formation. The sieving properties of a gel are established by the three-dimensional network of fibers and pores which is formed as the bifunctional bisacrylamide cross-links adjacent polyacrylamide chains.I9 Within limits, as the acrylamide concentration of a gel increases, its effective pore size decreases. The effective pore size of a gel is operationally defined by its sieving properties; that is, by the resistance it imparts to the migration of protein molecules. By convention, a given gel is physically characterized by the pair of figures (%T, %C), where %T is the weight percentage of total monomer (acrylamide + cross-linker, in grams per 100 ml), and %C is the proportion of cross-linker (as a percentage of total monomer) in the gel. The practical limits for %T lie between 3 and 30%. The factors governing pore size are complicated, but, in general, the pore size of a gel decreases as %T increases. For any given fixed %T, pore size is at a minimum at about 5% C, increasing at both higher and lower cross-linker

concentration^.^^^'^ The use of high-quality reagents is a prerequisite for reproducible, high-resolution gels. This is particularly true of acrylamide, which constitutes the most abundant component in the gel-monomer mixture. Residual acrylic acid, linear polyacrylamide, and ionic impurities are the major contaminants of acrylamide preparations. Moreover, buffer components should be of reagent grade and only distilled or deionized water should be used for all phases of gel electrophoresis. In SDS-PAGE, the quality of the SDS is of prime importance. Differential protein-binding properties of impurities such as Clo, C I 4 ,and C16 alkyl sulfates can cause single proteins to form multiple bands in gels.20 Even with pure SDS, very basic proteins, very acidic proteins, various glycoproteins, and lipoproteins, because of their unusual compositions, migrate "anomalously" during electroph~resis.~-~

l8 l9

"

A. Chrambach and D. Rodbard, Science 172, 440 (1971). D. Rodbard and A. Chrambach, Proc. Natl. Acad. Sci. U.S.A. 65, 970 (1970). M. M. Margulies and H. L. Tiffany, Anal. Biochem. 136, 309 (1984).

FI sa co hie sta VO

teii the for sl01 for1 tein chlc heig row prot with tizec re t a ~ tive protc pore will 5 be sll prote the T plexe

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Principle of Method

"

The most popular electrophoretic method is the SDS-PAGE system developed by Laemmli.4-7,9This is a discontinuous system consisting of two contiguous, but distinct gels: a resolving or separating (lower) gel and a stacking (upper) gel. The two gels are cast with different porosities, pH, and ionic strength. In addition, different mobile ions are used in the gel and electrode buffers. The buffer discontinuity acts to concentrate large volume samples in the stacking gel, resulting in better resolution than is possible using the same sample volumes in gels without stackers. Proteins, once concentrated in the stacking gel, are separated in the resolving gel. The Laemmli SDS-PAGE system is made up of four components. From the top of the cell downward, these are the electrode buffer, the sample, the stacking gel, and the resolving gel. Samples prepared in lowconductivity buffer (0.06 M Tris-C1, pH 6.8) are loaded between the higher conductivity electrode (0.025 M Tris, 0.192 M glycine, pH 8.3) and stacking gel (0.125 M Tris-C1, pH 6.8) buffers. When power is applied, a voltage drop develops across the sample solution which drives the proteins into the stacking gel. Glycinate ions from the electrode buffer follow the proteins into the stacking gel. A moving boundary region is rapidly formed with the highly mobile chloride ions in the front and the relatively slow glycinate ions in the rear.5-7,9,11,21 A localized high-voltage gradient forms between the leading and trailing ion fronts, causing the SDS-protein complexes to form into a thin zone (stack) and migrate between the chloride and glycinate phases. Within broad limits, regardless of the height of the applied sample, all SDS-proteins condense into a very narrow region and enter the resolving gel as a well-defined, thin zone of high protein density. (The stacking phenomenon is strikingly demonstrated with prestained protein standards, which are mixtures of proteins derivatized with reactive dyes.) The large-pore stacking gel (4% T) does not retard the migration of most proteins and serves mainly as an anticonvective medium. At the interface of the stacking and resolving gels, the proteins experience a sharp increase in retardation due to the restrictive pore size of the resolving gel. (Proteins too large to enter the resolving gel will stop at the interface.) Once in the resolving gel, proteins continue to be slowed by the sieving of the matrix. The glycinate ions overtake the proteins, which then move in a space of uniform pH (pH 9.5) formed by the Tris and glycine. Molecular sieving causes the SDS-polypeptide complexes to separate on the basis of their molecular weights. 2'

M. Wyckoff, D. Rodbard, and A. Chrambach, Anal. Biochem. 78,459 (1977).

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Procedure

Equipment and reagents for SDS-PAGE can be obtained from a variety of suppliers. Electrophoresis cells vary in design, but their operation generally follows the steps outlined below. Since the many available cells differ in size, formulations are presented in conveniently sized units for simplicity. Required volumes can be prepared using multiples of these unit sizes. Except where noted, reagents for SDS-PAGE can be prepared as concentrated stock solutions. Stock Solutions Acrylamide concentrate (30% T, 2.7% C): Dissolve 29.2 g of acrylamide and 0.8 g of bisacrylamide in 70 ml of deionized water. When the acrylamide is completely dissolved, add water to a final volume of 100 ml. Filter the solution under vacuum through a 0.45-pm membrane. Store stock acrylamide at 4" in a dark bottle for no more than 1 month. Caution: Acrylamide monomer is a neurotoxin. Avoid breathing acrylamide dust, do not pipette acrylamide solutions by mouth, and wear gloves when handling acrylamide powder or solutions containing it. For disposal of unused acrylamide, add bisacrylamide (if none is present), induce polymerization, and discard the solidified gel 1.5 M Tris-C1, pH 8.8, concentrated resolving gel buffer: Dissolve 18.2 g Tris base in 4 0 ml of water, adjust to pH 8.8 with HC1, and add water to a final volume of 100 ml. Store at 4" 0.5 M Tris-C1, pH 6.8, concentrated stacking gel buffer: Dissolve 6.1 g Tris base in 280 ml of water, adjust to pH 6.8 with HC1, and add water to a final volume of 100 ml. Store at 4" 10% (wlv) sodium dodecyl sulfate (SDS): Dissolve 10 g SDS in =60 ml of water and add water to a final volume of 100 ml Stock sample buffer (0.06 M Tris-C1, pH 6.8, 2% SDS, 10% glycerol, 0.025% Bromphenol Blue): ,

4.8 ml Water 0.5 M Tris-C1, pH 6.8 1.2 ml 10% SDS 2.0 ml GIycerol 1.0 ml 0.5% Bromphenol Blue (wlv water) 0.5 ml Store at room temperature. SDS-reducing buffer is prepared by adding 50 pl of 2-mercaptoethanol to each 0.95 ml of stock sample buffer before use

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Catalyst 10% ammonium persulfate (APS): Dissolve 100 mg APS in I ml of water. Make the APS solution fresh daily TEMED ( N , N ,N ',N '-tetramethylethylenediamine):Use TEMED undiluted from the bottle. Store cool, dry, and protected from light Electrode Buffer Electrode buffer: 0.025 M Tris, 0.192 M glycine, 0.1% (wlv) SDS, pH 8.3 (0.3 g Tris base, 1.4 g glycine, 1 ml 10% SDS1100 ml electrode buffer). Do not adjust the pH of the electrode buffer; just mix the reagents together and confirm that the pH is near 8.3 (k0.2). Electrode buffer can be made as a 5 x concentrate consisting of 15 g Tris base, 72 g glycine, and 5 g SDSIliter. 5 x electrode buffer concentrate must be stored in glass containers. To use 5 x concentrate, dilute it with four parts water Casting Gels

Thoroughly clean the glass plates, spacers, combs, and upper buffer reservoir of the gel apparatus with detergent and rinse them well. Wear gloves while assembling the equipment. The resolving gel is cast first, then overlaid with the stacking gel. 1. Assemble the casting apparatus and determine the gel volume from the manufacturer's instructions or by calculation. A 1- to 2-cm stacking gel is used above the resolving gel. Determine the height to which the resolving gel is to be poured by inserting a well-forming comb between the glass plates and marking the outer plate 1-2 cm below the teeth of the comb. 2. Prepare the monomer solution for the appropriate resolving gel by combining all of the reagents in Table I except the ammonium persulfate (APS) and TEMED; a disposable, plastic beaker is a convenient mixing vessel. The two gel recipes given in Table I cover the molecular weight ranges usually encountered. Gels of any other acrylamide concentration de~ired~ can , ~be . ~ prepared by adjusting (only) the amounts of 30% monomer stock and water used in the recipes. Deaerate the solution under vacuum (e.g., in a bell jar or desiccator) for at least 15 min. 3. Gently mix the APS and TEMED (Table I) into the deaerated monomer solution. Using a pipet and bulb, add the monomer solution between the gel plates up to the mark delimiting the resolving gel. Immediately overlay the monomer solution with water-saturated 2-butanol or

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TABLE I FORMULATIONS OF SDS-PAGE RESOLVING GELSO

Component

7.5% Tb

12% T c

Water 1.5 M Tris-C1, pH 8.8 10% SDS Acrylamidelbis (30% T, 2.7% C ) 10% ammonium persulfated TEMED

4.85 ml 2.5 ml 0.1 ml 2.5 ml 50 pl 5 PI

3.35 ml 2.5 ml 0.1 ml 4.0 ml 50 pI (0.05%) 5 pl (0.05%)

Any desired volume of monomer solution can be prepared by using multiples of the 10-ml recipes. Combine the first four items and deaerate the solution under vacuum for 15 min. Start polymerization by adding ammonium persulfate and TEMED. For SDS-treated proteins in the approximate molecular weight range between 40K and 250K. " For SDS-treated proteins in the approximate molecular weight range between 10K and 100K. To make 10% ammonium persulfate (APS), dissolve 100 mg APS in 1 ml of water. Make the APS solution fresh daily. a

tert-amyl alcohol to exclude air, which might inhibit polymerization, from the surface of the monomer mixture. Allow the gel to polymerize for 45 min to 1 hr. Polymerization is evidenced by the appearance of a sharp interface beneath the overlay, which will start to become visible in about 15 min. Polymerization is essentially complete in about 90 min, but the stacking gel can be poured after about an hour.22Allow unused monomer to polymerize in the beaker and discard the gel. 4. Prepare 10 ml of stacking gel monomer solution (4% T, 2.7% C ) , by combining

e

1I I

Water 6.1 ml 2.5 ml 0.5 M Tris-C1, pH 6.8 Acrylamide stock solution (30% T) 1.3 ml 10% SDS 0.1 ml Deaerate the monomer solution under vacuum for at least 15 min. 5. Thoroughly rinse the top of the resolving gel with water and dry the area above it with filter paper. Place a well-forming comb between the gel plates and tilt it at a slight angle to provide a way for bubbles to escape.

" Bio-Rad Lab., Bull. No. 1156.

I

;I

1

?-

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6. Add 50 p1 of 10% APS and 10 p1 of TEMED to each 10 ml of

degassed monomer solution and pour the stacking gel solution on top of the resolving gel. Align the comb in its proper position, being careful not to trap bubbles under the teeth. Visible polymerization of the stacking gel should occur in about 10 min. No overlay is required, because the comb excludes oxygen from the surfaces of the wells. Allow the gel to polymerize for 30-45 min. Allow unused monomer to polymerize in the beaker before disposing of it. In some situations, it may be necessary or convenient to let the gel stand overnight before it is used. When this is the case, it is best to pour the stacking gel on the day of the run to maintain the ion discontinuities at the interface between the two gels. For storage, the top of the resolving gel should be rinsed thoroughly and covered with resolving gel buffer (0.375 M Tris-C1, 0.1% SDS, pH 8.8) to avoid dehydration and ion depletion. Also, the tops of the gel sandwiches should be covered with plastic wrap during storage. Sample Preparation

The common biochemical buffers are usually tolerated in SDSPAGE, so that pretreatment of samples is not generally required. Distorted band patterns, such as pinching or flaring of lanes, can be caused by excessive amounts of salt in the samples. These distortions can often be remedied by desalting the samples. I . Prepare the volume of SDS-reducing buffer required for the number of samples to be run by adding 50 pl of 2-mercaptoethanol to each 0.95 rnl of stock sample buffer (to a final concentration of 5% 2-mercaptoethanol). This step may be omitted, if reduction of disulfide bonds is not desired. 2. Dilute samples with at least 4 vol of complete SDS-reducing buffer (although as little as 2-fold dilution may be adequate for some samples). Sample volumes are of the order of 20-50 p1 for conventional gels and 530 pl for minicells, depending on the widths of the wells and the thicknesses of the gels. Detection in gels requires on the order of 1 pg of protein per band for easy visibility when staining with Coomassie Blue R250 or 0.1 pg of protein per band with silver staining (see below). 3. Heat the diluted samples at 95" for 4 min by suspending the sample tubes in hot water. Do not store prepared samples. Electrophoresis

Assemble the electrophoresis cell, fill the upper and lower reservoirs with electrode buffer, and remove the comb from the stacking gel. Load

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-

the prepared samples into the wells in the stacking gel by layering them under electrode buffer using a microliter syringe or micropipet. The glycerol in the samples provides the necessary density for them to sink to the bottoms of the wells and the Bromphenol Blue tracking dye enables the samples to be seen during loading. Finally, attach leads to the unit and connect them to a power supply. The lower electrode is the anode and the upper one is the cathode, in SDS-PAGE. During an electrophoresis run, electrical energy is converted to heat which can cause band distortion and diffusion. In general, electrophoresis should be carried out at power settings at which the run proceads as rapidly as the chamber's ability to draw off heat will allow. In other words, the run should be as fast as possible without exceeding desired resolution and distortion limits. Many of the power supplies which are available allow control of any electrical quantity and the choice is almost a matter of preference. Constant current conditions, as a rule, result in shorter but hotter runs than does constant ~ o l t a g eIn . ~the early stages of a run, the resistance of the gel increases as the chloride ions migrate out of it. Accordingly, voltage will rise or current will fall, depending on whether constant current or constant voltage operation is in use. Small-format minicells, with their thin glass plates, are better able to efficiently dissipate the heat generated by the initially high currents at the beginnings of runs than are standard-sized cells. Thus, the recommendation is that gels should be run under constant current conditions (16-24 mA/mm of gel thickness) in conventional apparatus and at constant voltage (20-30 V/cm of gel length) in minicells. The use of recirculated coolant, where possible, allows higher voltages and currents to be used for shortened run times. Electrophoresis should be started immediately after the samples are loaded and is generally continued until the Bromphenol Blue tracking dye has reached the bottom of the gel. Comments on Method The Laemmli SDS-PAGE ~ y s t e m ~is- ~an* adaptation ~ of an earlier method devised by O r n ~ t e i nand ~ ~ Davis24for fractionation of native serum proteins. The different (discontinuous) buffers used in the stacking and resolving gels are required for the proper functioning of the OrnsteinDavis ~ y s t e m . ' However, ~,~~ inclusion of SDS modifies the rationale of the Omstein-Davis technique in important ways, since the properties of the detergent dominate the s y ~ t e m . ~ . ~ . ~ ' 23 24

L. Ornstein, Ann. N . Y . Acad. Sci. 121, 321 (1964). B . J . Davis, Ann. N . Y . Acad. Sci. 121, 404 (1964).

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The necessary components of the Laemmli SDS-PAGE system are a

Tris-C1 gel buffer, the Tris-glycine-SDS electrode buffer, and the SDSreducing sample buffer. As a consequence of SDS in the system, it is actually not necessary to cast the stacking gels at different pH or ionic strength than the resolving gels. Similar resolution is obtained whether the stacking gel is cast as above or in resolving gel buffer (0.375 M Tris-C1, pH 8.8). This is because the mobilities of SDS-polypeptide complexes are insensitive to pH in this range.' When many gels are being cast at one time for storage and later use, it is convenient to cast the stacking and resolving gels in the same buffer. Total SDS load, on the other hand, has considerable influence on res~lution.~' Inclusion of more than 200 pg of SDS in 30- to 50p1 samples in the minigel configuration can lead to broadening and spreading of protein bands. With dilute, large volume samples, it may prove advantageous to limit the total SDS in the system by dropping the final SDS concentration of the treated sample to about 0.5% and casting the gels without SDS. Because the mobility of SDS is greater than those of proteins, SDS from the electrode buffer quickly overtakes the proteins during electrophoresis. The gel is thus supplied and continuously replenished with SDS from the electrode buffer at a level sufficient to maintain the saturation of the protein^.^

Variations of Method The complete denaturation and dissociation of proteins with the Laemmli SDS-PAGE ~ y s t e m ~are - ~ ,not ~ always desirable. For some analyses, it might be of interest to estimate the molecular weights of particular proteins in their intact, oligomeric forms. In other experiments, interest might center on the biological activities of proteins in their native, nondenatured states. Through selective use of the two denaturants, 2mercaptoethanol and SDS, conditions can be adjusted as needed to separate proteins in the completely denatured, partially denatured, or native states. Covalent associations between protein units can be maintained by omitting 2-mercaptoethanol from the sample buffer. In the absence of the reducing agent, the intra- and interchain disulfide bonds of sample proteins remain intact. The electrophoretic mobilities of the resultant SDSprotein complexes are correspondingly altered relative to those obtained under dissociating conditions. During electrophoresis, the mobilities of oligomeric SDS-proteins are lower than those of their fully denatured SSDS-polypeptide components. Further, the electrophoretic behaviors of single-chain polypeptides can also be affected by reduction. The intra-

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chain disulfide bridges of single-chain proteins can hold them in compact configurations that are more or less retained in the presence of SDS. Thus, some SDS-proteins migrate faster electrophoretically in the absence of Zmercaptoethanol than when in the extended structures brought on by reduction. Proteins often show characteristic, individual responses to reduction, so that comparisons of SDS-PAGE gels run with and without Zmercaptoethanol can be very i n f ~ r m a t i v e . ~ ~ To separate proteins without reduction, carry out the SDS-PAGE procedure described above, omitting Zmercaptoethanol from the sample buffer. Note that oligomeric SDS-protein complexes migrate more slowly than their SDS-polypeptide subunits. It may, therefore, be necessary to use lower concentration (%T) gels than with the fully denaturing method to get oligomers to move adequate distances into the matrices. In addition, nonreduced proteins may not be completed saturated with SDS and, hence, may not bind the detergent in a constant weight ratio. This makes molecular weight determinations of these molecules by SDS-PAGE less straightforward than analyses of fully denatured polypeptides, since it is necessary that both standards and unknown proteins be in similar configurations for valid comparisons. When both SDS and 2-mercaptoethanol are left out of the Laemmli procedure, what remains is the classical Ornstein-Davis PAGE system23,24 for native proteins. This is a high-resolution native PAGE method designed for separation of the full spectrum of serum proteins. Because the system was meant to separate a wide variety of proteins, resolution may not be optimal for some restricted ranges of protein mobilities. Although there are a number of high-resolution native PAGE systems available to meet differing requirement^,^-^ the Ornstein-Davis method should perform adequately for the fractionation of the majority of commonly encountered protein mixtures. Molecular weights are more difficult to determine by native PAGE than by SDS-PAGE, since a single native system cannot distinguish the effects of charge and conformation on protein electrophoretic mobilities.j4 The procedure described here is readily modified for native PAGE. Merely omit 2-mercaptoethanol from the sample buffer and replace the 10% SDS in the recipes for the gel, sample, and electrode buffers with equivalent volumes of water. Follow the procedure as otherwise presented, except for sample treatment. Samples should be diluted in nondenaturing buffer (0.06 M Tris-C1, pH 6.8, 10% glycerol, 0.025% Bromphenol Blue) following the same guidelines as for denaturing gels, but they should not be heated. 25

T. Marshall, Clin. Chem. 30, 475 (1984)

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I I

c

Detection of Proteins in Gels Three of the simplest and most reliable methods for the detection of proteins in SDS-PAGE gels are presented. They should be adequate to cover the requirements of most situations. Coomassie Brilliant Blue R250 is the most common protein stain and is recommended for routine work. Silver staining is the most sensitive method for staining proteins in gels and should be employed when electrophoresis is used to assess the purity of a preparation; e.g., an antigen preparation. Copper staining is a recent development allowing rapid and sensitive staining. Discussions of other detection methods, including radiolabeling and means for quantitating proteins in gels, can be found in Refs. 2, 5-7, and 26. After electrophoresis, remove the gel assembly and separate the glass plates. The gel will probably stick to one of the two plates. Remove the spacers and cut off and discard the stacking gel. Place the glass plate holding the gel into fixative or staining solution and float the gel off of the plate. All of the steps in gel staining are done at room temperature with gentle agitation (e.g., on an orbital shaker platform) in any convenient container, such as a glass casserole or a photography tray. Always wear gloves when staining gels, since fingerprints will stain. Permanent records of stained gels can be obtained by photographing them or by drying them on filter paper using commercially available drying apparatus. Dye Staining with Coomassie Brilliant Blue R-250

This is the standard method of protein d e t e ~ t i o n . ~ -Easy ~ , ~ ' visibility requires on the order of 0.1-1 pg of protein per band.

1. Prepare the staining solution: 0.1% Coomassie Brilliant Blue R-250 (wiv) in 40% methanol (vlv), 10% acetic acid (vlv). Filter the staining solution after the dye has dissolved. The staining solution is reusable. Store it at room temperature. 2. Soak the gel in an excess of staining solution for 30 min. 3. Destain with a large excess of 40% methanol, 10% acetic acid. Change the destaining solution several times, until the background has been satisfactorily removed. The acid-alcohol solutions used in this procedure do not completely fix proteins in the gel. This can lead to losses of some low-molecularweight proteins during the staining and destaining of thin gels. Permanent fixation is obtainable by incubating the gel in 40% methanol (vlv), 10%

" C. R. Meml, this volume [36].

" C. M. Wilson, this senes, Vol. 91, p. 236.

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trichloroacetic acid (wlv) for 1 hr before it is immersed in the staining solution. Silver Staining

This method, developed by Merril and co-workers, can be as much as 100 times more sensitive than dye Bands containing 10-100 ng of protein can be easily seen. The reagents are available in kit form from Bio-Rad Laboratories. Reaction times vary with the thicknesses of the gels. 1. Fix the proteins in the gel in about 400 ml of 40% methanol, 10% acetic acid (vlv) (or 40% methanol, 10%trichloroacetic acid) for 30 min to overnight. 2. Fix twice in 400 ml 10% ethanol, 5% acetic acid (vlv) for 15-30 min. 3. Soak the gel for 3-10 min in 200 ml of fresh oxidizer solution (0.0034 M potassium dichromate, 0.0032 N nitric acid). 4. Wash the gel three or four times for 5-10 min in 400 ml water, until the yellow color has been washed out. 5. Soak the gel in 200 ml fresh silver reagent (0.012 M silver nitrate) for 15-30 rnin. 6. Wash the gel with 400 ml water for 1-2 min. 7. Wash the gel for about 1 min in developer (0.28 M sodium carbonate, 1.85% paraformaldehyde). 8. Replace the developer with fresh solution and incubate for 5 min. 9. Replace the developer a second time and allow development to continue until satisfactory staining has been obtained. 10. Stop development with 5% acetic acid (vlv).

Vertical streaks and sample-independent bands in the 50- to 70-kDa region are sometimes seen in silver-stained gels. These artifacts have been attributed to reduction of contaminants inadvertently introduced into the samples.29 They can be eliminated by adding excess iodoacetamide to sample solutions after treatment with SDS-reducing buffer.30 Copper Staining

Rapid, single-step staining of SDS-PAGE gels is achieved by incubatThe resultant, negatively stained image of ing gels in copper ~hloride.~' C. R. Merril, D. Goldman, S. A . Sedman, and M. H . Ebert, Science 211,1437 (1981); see also C. R . Merril, D. Goldman, and M. L . Van Keuren, this series, Vol. 104, p. 441. 29 D. Ochs, Anal. Biochem. 135, 470 (1983). 'O A. Gorg, W. Postel, J. Weser, S. Giinther, J. R. Strahler, S. M. Hanash, and L. Somerlot, Electrophoresis 8, 122 (1987). C. Lee, A. Levin, and D. Branton, Anal. Biochem. 166, 308 (1987). 28

ONE-DIMENSIONAL GEL ELECTROPHORESIS

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b-

l!-

-

lib

439

the electrophoretogram is intermediate in sensitivity between Coomassie blue and silver staining. 1. Wash the gel briefly in water. 2. Soak the gel in 0.3 M CuClz for 5 min. 3. Wash the gel for 2-3 min in water. The method yields negatively stained gels showing clear protein bands on an opaque, blue-green background. The protein bands can be easily seen and photographed with the gel on a black surface. Proteins are not permanently fixed by this method and can be quantitatively eluted after chelating the ~ o p p e r .The ~ ' electrophoretic pattern is lost when copperstained gels ari dried so they must be photographed, restained kith Coomassie Blue, or stored in water. Marker Proteins

Mixtures of marker proteins are available for calibrating gels. PAGE standards are mixtures of proteins with precisely known molecular weights blended for uniform staining. They are obtainable in various molecular weight ranges. Concentrated stock solutions of the standards are diluted in sample buffer just prior to electrophoresis and treated in the same manner as the sample proteins. These proteins are suitable as reference markers for molecular weight determinations. Prestained SDS-PAGE standards have recently become available. The coupling of dye molecules to the marker proteins changes their molecular weights significantly and unpredictably and they should not be used for molecular weight determinations. However, prestained standards are very useful for following the course of an electrophoretic run and are valuable for assessing the efficiencies of protein transfers when gels are blotted. Molecular Weight Determination

Molecular weights of proteins are determined by comparison of their mobilities with those of several marker proteins of known molecular eight.^-^ After the gel has been run, but before it has been stained, mark the position of the Bromphenol Blue tracking dye to identify the leading edge of the electrophoretic ion front. This can be done by cutting notches in the edges of the gel or by inserting a needle soaked in india ink into the gel at the dye front. After staining, measure the migration distances of each protein (markers and unknowns) from the top of the resolving gel. Divide the migration distance of each protein by the distance traveled by the tracking dye. The normalized migration distances so obtained are called the relative mobilities of the proteins (relative to the dye front) and

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conventionally denoted as Rf. Construct a (semilogarithmic) plot of the logarithms of the molecular weights of the protein standards as functions of the Rf values. Note that the graphs are slightly sigmoid. As long as the extremities of a molecular weight range are avoided, unknown molecular weights can be estimated by linear regression analysis or interpolation from the curves of log M, vs Rf. Keep in mind that the molecular weights obtained using SDS-PAGE are those of the polypeptide subunits and not those of native, oligomeric proteins. Preparative Electrophoresis The most satisfactory way to recover proteins separated by SDSPAGE for further study is to extract them from bands excised from the gels. Many attempts have been made to design continuous elution devices suitable for routine protein purification, in which bands emerging from the bottoms of electrophoresis gels are swept away to fraction collector^.^^^^^^ The scarcity of preparative gel devices is evidence of the disappointing lack of success in developing generally useful instruments. Preparative gel electrophoresis would ideally be capable of yielding high-milligram to gram quantities of individual proteins recovered cleanly with the resolution anticipated from the corresponding analytical gels. In general, though, band distortion and poor elution have limited the resolution attainable with most apparatus so that they have only worked well with relatively simple protein mixtures. The difficulties in scaling gel electrophoresis up to preparative levels has tended to result in devices which are rather cumbersome and which require much technical skill for best results. As a consequence, proteins are usually obtained by extraction from analytical type gels.33 Gels to be run for the isolation of protein^^.^ can be cast using special preparative combs. These combs form wide sample wells spanning the widths of the gels and usually provide a separate, narrow reference well for marker proteins. The maximum amount of sample which can be loaded on a gel ultimately depends on how well the proteins of interest are separated from their neighbors in the sample mixture. Since bands become wider a s the amount of material increases, as sample load is raised, the corresponding loss of resolution will eventually become unacceptable. Protein loads 10- to 50-fold greater per unit of cross-sectional area than are usually run in analytical gels are easily tolerated. Thus, with some large slab gels, proteins can be recovered in tens-of-milligram amounts.

33

A. Chrambach and N. Y. Nguyen, in "Electrokinetic Separation Methods" (P. G. Righrtti, C. J. Van Oss, and I. W . Vanderhoff, eds.), p. 337. Elsevier, Amsterdam, 1979. M. Hamngton, this volume [37].

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Copper staining3' (above) is advisable for the visualization of the bands in preparative SDS-PAGE, since this method does not employ fixative solvents. Desired bands are cut from the gel and destained by incubation in three changes (for 10 min each) of 0.25 M EDTA, 0.25 M Tris-C1, pH 9. After destaining, gel slices are incubated in the appropriate elution buffer. Proteins are often extracted from macerated gel slices by simple diffusion into appropriate buffers or by solubilization of the ge1.5333In the latter method, cross-linkers other than bisacrylamide are copolymerized into the For example, gels cross-linked with N,N1-bisacrylylcystamine (BAC) are dissolvable in 2-mercaptoethanol or dithiothreitol, while both N,N1-dihydroxyethylenebisacrylamide(DHEBA) and N,N l-diallyltartardiamide (DATD) result in gels which can be solubilized with periodic acid. Once gels have been dissolved, proteins must be separated from the large excess of gel material by gel filtration or ion-exchange chromatography. Electrophoretic elution is an efficient method for recovering proteins the . ~simplest versions of this method, proteins are from gel s l i ~ e s . ~In, ~ electrophoresed out of gel pieces into dialysis sacks in the types of apparatus used for running cylindrical gel rods. Devices are available for the rapid recovery of proteins in small volumes with yields of greater than 70% in most cases. Elution takes about 3 hr at 10 mA1tube in 0.025 M Tris, 0.192 M glycine, 0.1% SDS, pH 8.3 (standard SDS-PAGE electrode buffer). SDS can be removed from the eluted samples by dialysis or ionexchange chromatography .34 34

A. J. Furth, Anal. Biochem. 109, 207 (1980).

[341 Protein Analysis Using High-Resolution Two-Dimensional Polyacrylamide G e l Electrophoresis By BONNIE S. DUNBAR, HITOMIKIMURA,and THERESE M. TIMMONS

The term two-dimensional electrophoresis has been used to describe a variety of methods employing separation of molecules in two dimensions. The term high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is now specifically applied to the separation of proteins in the first dimension according to their isoelectric points using isoelectric focusing (IEF) with carrier ampholytes after reduction of disulfide bonds, METHODS IN ENZYMOLOGY. VOL. 182

Copyright D 1990 by Academic Press, Inc. All rights of reproduction in any f ~ r mreserved.

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followed by separation in the second dimension according to their molecular weights using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as defined by O'Farrell.' The history of the major developments in the field of electrophoresis and 2D-PAGE has been summarized in detail e l ~ e w h e r e . ~The - ~ most significant recent advances in this technology have come through the standardization of equipment and reagents and the simplification of equipment for reproducible analysis and large scale a n a l y s e ~ .Because ~.~ large numbers of laboratories are now using these standardized procedures, they are the methods described in this chapter. Since the quality of reagents used is critical for reproducible results, we have listed commercial sources whose reagents are acceptable for these procedures. There are many other sources for most of these reagents, but they should be tested for quality to ensure good results. The use of 2D-PAGE has become increasingly popular during the past decade. Two-dimensional PAGE allows the resolution of a complex protein mixture into more discrete components than ID-PAGE since it separates on the basis of protein charge in addition to molecular weight. The major advantage of large-scale 2D-PAGE is the improvement in reproducibility of protein patterns. This enables the researcher to directly compare the analyses of complex protein mixtures, whether the 2D-PAGE separations are conducted simultaneously or in different experiments. This feature makes 2D-PAGE a versatile and powerful tool in both basic and clinical research. Applications of 2D-PAGE The most common uses of 2D-PAGE are the analysis of complex mixtures of proteins and the analysis of the posttranslational modification of proteins. 2D-PAGE can also provide valuable information about the molecular properties of proteins, including an estimate of the relative isoelectric points (pl) and molecular weights3 of proteins. However, it is generally inadequate to use this as the sole method for the precise determination of these parameters. For example, the disulfide bonds of the P. H. O'Farrell and J. I. Garrels, this series, Vol. 100, p. 411. N . G. Anderson and L. Anderson, Clin. Chem. 28, 739 (1982). B. S . Dunbar, "Two-Dimensional Electrophoresis and Immunological Techniques." Plenum. New York, 1987. B. D. Hames and D. Rickwood, "Gel Electrophoresis of Proteins: A Practical Approach." IRL Press, Washington, D.C.,1981. L. Anderson, "Two-Dimensional Electrophoresis: Operation of the ISO-DALT System." Large Scale Biology Press, Washington, D.C., 1988.

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proteins analyzed by 2D-PAGE are usually reduced so the protein patterns may reflect subunit peptides. The p l and molecular weight values observed may therefore be different from those of the native proteins. One should be careful not to overinterpret data obtained from electrofocusing and 2D-PAGE. Another common use of 2D-PAGE is to rapidly purify a specific protein which can be cut from the gel and used directly to obtain amino acid sequence or to purify antibodies. These antibodies can then be used to immunoaffinity purify the original protein in quantities sufficient for detailed chemical characterization. Immunoblotting using antibodies to detect antigens separated by 2DPAGE also provides an excellent method to analyze antibody specificity and to analyze carbohydrate or other epitopes. Finally, the use of 2D-PAGE with silver staining provides one of the best methods to estimate protein purity. This analysis, in conjunction with one-dimensional analysis of proteins visualized by silver stain (to detect proteins whose pI is outside the pH range of the ampholines), will provide a rigorous estimate of protein purity. Sample Preparation and Solubilization Procedures

The preparation of samples for 2D-PAGE analysis is the most critical step in guaranteeing excellent reproducible results. All tissues and samples should be handled in the cold and stored at -70". It is important that the ratio of solubilization buffer to protein concentration be optimized for each sample. We have found the following ratios to be adequate for most samples: (1) 200-500 pg tissue homogenatel2 ml solubilization buffer, (2) 20-50 p1 cell pellet/300 p1 solubilization buffer, (3) 1 x lo6 cells in tissue culture plate with 500 pl solubilization buffer to solubilize cells directly, and (4) 10-200 pg soluble proteinl30-50 p1 solubilization buffer. Note: 550 p1 of each of the above samples should be adequate for identification of abundant proteins by Coomassie Blue staining or of minor proteins by silver staining in two-dimensional gels. Materials Sodium dodecyl sulfate (SDS) (Bio-Rad, Richmond, CA) Cyclohexylaminoethane (CHES) (Calbiochem, San Diego, CA) Glycerol (Fisher, Pittsburgh, PA) 2-Mercaptoethanol (Bio-Rad) Urea (ultrapure) (Bio-Rad) Nonidet P-40 (nonionic detergent, NP-40) (Accurate Chemical, Westbury, NY)

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Ampholytes [pH 3.5-10: Bio-Rad, LKB, or Pharmacia (Piscataway, NJ): pH 2-11: Serva, Garden City Park, NY]. This wide-range mixture is appropriate for most routine samples. Other pH range or combinations of brands of ampholytes may be used in some instances H20, deionized with mixed bed resin (Continental filter system) or deionized double-distilled H 2 0

Method The two solubilization buffers which can be used for isoelectric focusing are as follow: SDS solubilization solution: 0.05 M CHES, 2% SDS, 10% glycerol, small amount of Bromphenol Blue, pH 9.5. Add 2% 2-mercaptoethanol just before use. Samples should be suspended in SDS solubilization buffer, placed in a tightly capped glass vial, and heated for 510 min in a boiling water bath. (Thick plastic tubes such as microfuge tubes are insulated and interfere with heating.) It may be necessary to solubilize some samples at room temperature for 2-3 hr, with or without heating Urea solubilization solution: 9 M urea, 4% Nonidet P-40. Add 2% 2mercaptoethanol and 2% ampholytes to a small aliquot of solubilization buffer just prior to use. These reagents should be filtered to 0.2 p m with a syringe filter for best results. Samples should be suspended in the urea solubilization solution and incubated at room temperature for 2 hr. Caution: Do not heat, or you will generate charge artifacts Following the incubation, samples are centrifuged to remove nonsolubilized material and nucleic acids that may interfere with focusing or cause streaking in second-dimension protein patterns (100,000-200,000 g for 2 hr is suggested). We recommend using a Beckman Ti-42.2 rotor, which holds 72 tubes. Isoelectric Focusing

Materials

Urea (ultrapure) (Bio-Rad) Ampholytes (LKB, Serva, or Pharmacia recommended); pH will depend on needs of investigator Acrylamide (Bio-Rad) Bisacrylamide (Bio-Rad) Nonidet P-40 (Accurate) Ammonium persulfate (Bio-Rad)

$

t

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N , N , N 1N , '-Tetramethylethylenediamine (TEMED) (Bio-Rad) Sodium hydroxide (Sigma, St. Louis, MO) Phosphoric acid (Fisher) Chromerge (Fisher) Glass pipet tubes (0.2 ml disposable) (American Scientific Products, SIP disposable serological pipets, 0.2 ml, #P4644-2T) Gel electrophoresis apparatus: Any tube gel electrophoresis apparatus can be used if appropriate grommets or corks are prepared to fit small tubes (e.g., Bio-Rad electrophoresis unit model 175 tube gel apparatus). Alternatively, multiple IEF casting systems now available from Pierce Apparatus Branch, Hoefer Scientific, and Integrated Separation Sciences have been optimized for these procedures and are highly recommended Method

To cast IEF gels, add urea (8.25 g) to 6 ml H 2 0 plus 2.0 ml acrylamide stock (30 g acrylamide : 1.8 g bisacrylamide : 100 ml H 2 0 , filtered to 0.2 pm). Dissolve the urea in the H 2 0 by swirling the flask under warm running water. Caution: Do not heat solution. Add ampholytes (0.75 ml) to the mixture of acrylamide, water, and urea, swirl the solution gently to mix,and degas on a lyophilizer. Add 0.3 ml Nonidet P-40, and mix gently. (Hint: A large, plastic Eppendorf pipet tip can be cut off for easier and more accurate pipetting of viscous detergents.) Add ammonium persulfate (70 pl of a 10% solution) and TEMED (10 p1) to the acrylamide solution, and swirl the flask gently to mix. Cast IEF gels to a height of approximately 12 cm using capillary action, by overlayering acrylamide stock with water using a commercial casting apparatus, or a home-made casting chamber prepared from 2- to 50-ml plastic conical centrifuge tubes (as described in Dunbar3). Allow 1 hr for polymerization, and place tubes into the electrophoresis chamber. Prepare upper electrode buffer (0.02 N NaOH degassed thoroughly on lyophilizer) and lower electrode buffer (0.085% phosphoric acid), and add to chamber. Prefocus the gels at 200 V for 1-2 hr. In theory, this will remove ions which may interfere with the focusing. We have frequently omitted this step, however, with no noticeable differences in protein patterns. Load the protein samples (5-50 pl) with a Hamilton syringe under the upper electrode buffer. Carry out isoelectric focusing for 10,000-12,000 V-hr (e.g., 17 hr at 700 V). The optimal conditions will depend on the nature of your sample and the dimensions and volume of your IEF gels. We have found that resolution of proteins is sharper if you focus for a shorter period of time at higher voltage (i.e., 700 V for 16 hr is better than 500 V for 22 hr).

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To remove gel, insert a yellow Eppendorf pipet tip attached to a 3-ml syringe filled with water into the top of the tube, and gently push out the gel. The IEF gels are equilibrated in buffer (0.125 M Tris-base; 2% SDS; 10% glycerol, pH 6.8, plus 0.2-0.8% 2-mercaptoethanol added just before use) for 15 min, to remove arnpholytes and urea and to recoat the proteins with SDS. In some instances, we have equilibrated the gels for as little as 2-5 min with excellent results. Note: The "mercaptan" artifact commonly observed by silver staining which appears as two distinct lines having molecular weights of approximately 50K and 70K can be reduced if little or no 2-mercaptoethanol is used in the equilibration buffer. You should first establish whether the omission of this reducing agent will alter your protein patterns by comparing samples run in its presence or absence. The IEF gel can be frozen at -70" before equilibration, and thawed in equilibration buffer immediately before placing on the surface of the second dimension slab gel. Nonequilibrium pH Gradient Electrophoresis (NEPHGE Gel System) in 2D-PAGE "Nonequilibrium" isoelectric focusing techniques are especially useful for the first dimension separation of basic proteins, which are not well resolved or cannot be resolved by other IEF procedure^.^ Samples must be solubilized in the urea solubilization buffer above. All gel-casting procedures should be carried out as for standard equilibrium IEF, except that the upper and lower buffers are reversed: the upper electrode buffer should contain phosphoric acid, and the lower buffer should contain sodium hydroxide. When attaching the electrodes to the power supply, be sure to attach the upper buffer reservoir to the positive electrode and the lower buffer reservoir to the negative electrode. Finally, the IEF gels should be removed at intervals such as 2000, 4000, 6000, or 8000 V-hr. Total volt-hours will have to be optimized to resolve different proteins of interest, since this is a nonequilibrium system. Casting and Running Individual One-Dimensional Sodium Dodecyl Sulfate-Polyacrylamide Gels for Second Dimension Electrophoresis Materials Acrylarnide (Bio-Rad, Polysciences, Serva Fine Chemicals, or Sigma): Reagents from the latter two sources are less expensive, but require filtering through Whatman #3 filter paper, followed by a 0.2-p MilliP.2.O'FarreU, H. M. Goodman, and P. H. O'Farrell, Cell U,1133 (1977).

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pore filter. These impure reagents may also have contaminants detectable by silver stain. They can easily be distinguished from protein spots in 2D-PAGE, but will make analysis of silver-stained ID-PAGE difficult N,NI-Methylene(bis)acrylarnide (Bio-Rad) Trizma base (Sigma) Glycine (Sigma) SDS (Bio-Rad) 2-Mercaptoethanol (Bio-Rad) Glycerol (Fisher) N,N,N1,N'-Tetramethylethylenediamine(TEMED) (Bio-Rad) see-Butanol (Fisher) Agarose (Bio-Rad) Glass plates and spacers for individual or multiple gel-casting systems: The size plates will depend on the type of electrophoresis chamber that will be utilized (e.g., 18 x 16 cm plates with 1.5-cm spacers are compatible with Bio-Rad or Hoefer electrophoresis units). The recipes in this chapter are for this size gel Electrophoresis chambers: These can be obtained commercially from Bethesda Research Laboratories, Bio-Rad, Hoefer, or can be custom made (Studier apparatus) Gradient maker (double conical style recommended) Multiple electrophoresis gradient gel-casting systems (highly recommended if you d o gels regularly; greatly improves reproducibility !)

Method for Casting Nongradient Gels Prepare stock solutions: Bisacrylamide stock: 30% acrylamide, 0.8% bisacrylamide (filter to 0.2 pm) Gel buffer stock: 1.5 M Trizma base, 0.4%SDS (filter to 0.2 ,um) Ammonium Persulfate: 10 g ammonium persulfate; final volume 100 ml (filter to 0.2 pm). Freeze at -20" in small aliquots to guarantee the consistency of polymerization for as long as the stock lasts Tank buffer: 0.025 M Trizma base, 0.192 M glycine, 0.1% SDS Assemble the gel-casting apparatus. Combine acrylamide, buffer, and HzO; degas (see tabulation below for final acrylarnide concentration). Add TEMED and mix thoroughly but gently by swirling the beaker. Add ammonium persulfate and swirl gently to mix. Pour the mixture down one edge of the spacer of the gel-casting unit using a 25-ml pipet, or a syringe and a large (Isgauge) needle. Fill to within 3 cm of the top of the glass plates. Carefully overlay with water-saturated sec-butanol, and allow to

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-

-

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METHODS

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-

--

polymerize for 45-60 min. (Note: This is the most important step for obtaining good resolution in slab polyacrylamide gels.) When the gel is polymerized, rinse the surface several times with distilled water, and drain well. Final acrylamide concentration Component

7.5%

10%

12.5%

15%

Bisacrylamide stock Gel buffer stock H20 10% ammonium persulfate TEMED

7.1 ml 7.1 ml 14.2 ml 105 p1 15 pl

9.5 ml

11.8 ml 7.1 ml 9.5 ml 105 p1 15 p1

14.2 ml 7.1 ml 7.1 ml 105 p1 15 p1

7.1 ml 11.8 ml 105 p1 15 pl

Method for Casting Gradient Gels If silver staining methods are to be used, all reagents should be filtered to 0.2 pm. Bisacrylamide stock: 30% acrylamide, 0.8% bisacrylamide Second dimension buffer stock: 40 g Trizma base, 20 g Trizma-HCl; final volume 300 ml, pH 8.5-8.6 10% second dimension buffer: Three parts second dimension buffer stock plus five parts H 2 0 20% second dimension buffer: Three parts second dimension buffer stock plus one part glycerol 10% SDS: 10 g SDS; final volume 100 ml Assemble individual slab gel units in casting apparatus. For each gradient gel (approximately 40 ml/volume), prepare the reagents as per the following tabulation:

Gradient mix

Second dimension bisacrylamide stock (ml)

10% second dimension buffer (ml)

20% second dimension buffer (ml)

10% SDS (ml)

10% ammonium persulfate (~1)

TEMED (4)

Place 20% gradient mix in the internal .chamber of the gradient maker and begin mixing with a magnetic stir bar. Place 10% gradient mix in the

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?ji

p-

r

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449

external chamber, start the peristaltic pump, and open the gradient chamber to begin gradient formation. When the gradient is finished, spray the surface of the gel with water-saturated sec-butanol. After polymerization is complete, rinse the surface of the gel with HzO and drain well.

$ .s

i' +

Loading and Running Individual Second Dimension Polyacrylumide Gels

Set the polymerized slab gel on a loading stand and lay the IEF gel on a platform (or sheet of parafilm) and gently straighten out. Allow the gel to slide into place along the surface, being sure not to trap air bubbles. If desired, seal the IEF gel with a small amount of overlay agarose (0.25 M Trizma base, 0.192 M glycine, 0.1% SDS, and 0.5% agarose heated to dissolve thoroughly, and then cooled slightly before overlayering gel). If standard electrophoresis chambers are used (e.g., Studier, Bio-Rad, or Hoefer electrophoresis apparatus), electrophoresis is carried out by placing the electrode buffer in the upper and lower chambers. The slab gels are then placed into these chambers, taking care to avoid air bubbles being trapped at the bottom of the slab acrylamide gel. This can be done by tilting the gel as it is lowered into the chamber and by tilting the chamber so that the buffer will move across the bottom of the gel to remove trapped air bubbles. Electrophoresis can be carried out at 100120 mA/gel (constant amperage) during the day, or as low as 10 mAlgel overnight. Constant voltage or constant power can also be used.

Casting and Running Multiple Gradient Gels

P

-

-

Materials for Second Dimension Electrophoresis

Multiple casting chambers and electrophoresis chambers for running multiple gels are available from Pierce Apparatus Branch or Hoefer. A power supply capable of reaching 1.5 A is also needed. Method for Multiple Gel Casting

Prepare stock solutions (same as those required for ID-PAGE gradient gels). Electrode buffer contains 0.025 M Trizma base, 0.192 M glycine, 0.1% SDS. Prepare glass plates by washing in SDS, followed by rinsing with H20, then ethanol. Load the gel-casting chamber with gel plates and seal chamber. Mix together acrylamide and buffers; degas thoroughly. Add 10% SDS stock. Add ammonium persulfate and TEMED immediately before pouring the gradient. A recipe for casting 10 gradient gels designed for the Pace linear gradient maker and the DALT or MEGA

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casting chambers is shown in the tabulation below. (If the size of the casting chamber or the thickness of the gels or glass plates vary, the total volume will have to be adjusted.)

Gradient mix

Second dimension bisacrylamide stock (ml)

10% second dimension buffer (ml)

20% second dimension buffer (ml)

10% 20%

135.0 270.0

270.0 0.0

0.0 135.0

10% SDS

10% ammonium persulfate

TEMED

(ml)

(111)

(PI)

4.0 4 .O

5 2

130 150

Pour 10% gradient mix into the center of the gradient maker and add a stir bar (choose one which almost completely fills diameter of chamber). Turn on the magnetic stirrer until the surface of the acrylamide starts to funnel downward, taking care that air bubbles do not form. Pour 20% gradient mix into the outer well, open the clamps to the casting chamber, and begin pouring the gradient. Immediately open the gradient chambers to allow 20% gradient mix to combine with 10% gradient mix. The acrylamide solution will gently fill the chamber from the bottom. Rotate the DALT or MEGA casting chamber slowly as acrylamide reaches the top corner of the glass plates. When the chamber is almost filled with acrylamide, switch the feed to an incoming line containing glycerol: water (1 : 1) colored with Bromphenol Blue and fill the remainder of the chamber. This glycerol :dye mixture will prevent the acrylamide from polymerizing in the tubing that connects the gradient former to the casting chamber. Spray the surface of the chamber generously with water-saturated sec-butanol. Allow the gels to polymerize and cool for 1 hr. The gels can then be used immediately or washed and stored at room temperature. For best results, gels should be used soon after casting. (For photographic illustration of this procedure, refer to Ref. 3.) Loading and Running Multiple Second Dimension Polyacrylamide Gels

After polymerization is complete, the gel plates are washed to remove excess acrylamide. Each slab gel is loaded with an IEF gel as described above. (The IEF gels must be sealed on the top of the slab gel with agarose overlay described above.) Electrophoresis chambers for casting and running 10-20 second dimension gels simultaneously are available from Pierce Apparatus Branch,

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Integrated Separation Sciences, Electronucleonics, and Hoefer. Buffer solutions are prepared and added to the tanks. (Note: For convenience, a small volume of buffer may be made from packets of preweighed buffer salts prepared in advance, and the remainder of the volume of water can be added directly to the electrophoresis tanks.) The slab gels are then placed on their sides and are slipped between the rubber spacers. Electrophoresis is carried out for 6-15 hr, depending on the cooling system used, at 70-400 V.

Samples available in only microgram quantity, or those containing only a limited number of components, may be amenable to mini-2DPAGE analysis. Although the small gel size can severely limit resolution of complex mixtures of proteins, the benefits of mini-2D-PAGE sometimes outweigh the disadvantages. First, the sample size can be reduced to a third of that used on standard-sized 2D-PAGE gels. Second, a complete mini-2D-PAGE analysis, including pouring the gels and staining with Coomassie Blue, can be accomplished in 1 day. Third, both isocratic and gradient gels can be prepared, using the same reagents and power supplies. The specialized equipment needed is not prohibitively expensive and the smaller gels are less costly to pour and stain. Another useful application of minigels (ID or 2D) is to quickly estimate the protein content, purity, and composition of a given sample before running it on a standard 2D-PAGE gel. This step can save valuable time and sample, since it will give dependable information about the volume of sample to be loaded per gel and about the appropriate conditions to yield optimal separation of protein components.

-

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Materials The reagents used for mini-2D-PAGE are the same as those for standard 2D-PAGE. Several companies offer minigel equipment, including Hoefer and Bio-Rad. We have been pleased with the performance of the Bio-Rad modular Mini-Protean I1 system, and the methods described here will be based on the use of this system. It includes a main buffer chamber which can be used for SDS-PAGE in addition to IEF, electrophoretic transfer and blotting, and electroelution by interchanging modular units. Several gels can be poured at once using the multigel caster, and they can be stored damp and refrigerated in plastic for several days. For casting gradient gels, the small gradient mixers from MRA Corporation (Clearwater, FL) or Bethesda Research Laboratories work well.

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Sample Preparation

The same methods for sample preparation are used for mini-2D-PAGE and standard 2D-PAGE. However, the sample solubilization buffer should contain extra Bromphenol Blue to help visualize the sample while loading onto the IEF gel and to ensure that no bubbles are trapped between the gel and the sample. When using SDS solubilization, the volume of solubilization buffer should not exceed 5 plIIEF gel, or the sample will smear and may crack the gel. Method for Casting and Running Mini-IEF Gels

Prepare stock solutions: Bisacrylamide stock: 30% acrylamide, 1.8% bisacrylamide (filter to 0.2 pm) Upper electrode buffer: 0.1 N NaOH Lower electrode buffer: 0.06% phosphoric acid The procedure described by the manufacturer can be easily followed, to cast approximately 25 gels at once in capillary tubes, using the following recipe: Urea Ampholytes First dimension bisacrylamide stock Hz0 Nonidet P-40 10% ammonium persulfate TEMED

4.12 g 0.50 ml 1.00 ml 3.00 ml 150 p1 35 p1 5 ~1

Polymerization is complete in 30 min, and the gels should be used within the next 30 min for best results. The directions provided by Bio-Rad can be followed for loading and running mini-IEF gels (up to 16 at a time). The exact amount of sample per gel must be determined experimentally, depending on the composition of your sample. Because of the small gel size and its fragile nature, it is extremely important not to overload the gel. Also, the optimal voltage and time of IEF separation will vary for each sample, and must be determined experimentally. After running, the gels can be easily removed from the tubes, using the syringe adapter from Bio-Rad. They can be ejected directly into a small plastic vial for short-term storage (no longer than 2 weeks, to prevent freezer burn) at -70". If second dimension separation is to be done immediately, each IEF gel can be ejected into 0.5 ml of equilibration buffer (as

I341

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in standard 2D-PAGE) and incubated with gentle shaking at room temperature for 10- 15 min. Method for Casting and Running Mini-SDS-PAGE

Prepare stock solutions. The stock solutions used are the same as those for standard 2D-PAGE. The Bio-Rad individual gel casting stand can be used for either isocratic or gradient gels, between 0.5 and 1.5 mm thick (I .O-mm-thick gels are required for second dimension separation). Polymerization is complete in only 30 min. Teflon combs are available to form 2-15 wells for SDS-PAGE one-dimensional separation, affording a great deal of versatility. The following gel recipes are designed to prepare two 1.0-mm SDSPAGE minigels or one 10-20% gradient minigel in the Bio-Rad MiniProtean I1 casting stand. For separating gels of different height or thickness, volumes must be adjusted. Final acrylamide concentration

-

Component

7.5%

10%

15%

20%

Bisacrylamide stock Running gel buffer Hz0 TEMED 10% ammonium persulfate

3.50 ml 3.48 ml 6.96 ml 6.4 p1 53.0 p1

4.67 ml 3.48 ml 5.80 ml 6.4 p1 53.0 p1

7.00 ml 3.48 ml 3.47 ml 6.4 p1 53.0 p1

9.32 ml 3.48 rnl 1.14 ml 6.4 p1 53.0 p1

Bisacrylamide stock Running gel buffer H2O TEMED 10% ammonium persulfate

10% gradient mix

20% gradient mix

2.33 ml 1.74 ml 2.90 ml 3.2 pI 26.5 pl

4.66 ml 1.74 ml 0.57 rnl 3.2 p1 26.5 pl

To load the minigel, the I E F gel is poured out of its tube after equilibration directly onto the side of the minigel glass plate and excess buffer is blotted up. Using a rounded end spatula, the gel can be gently straightened out, parallel to the top of the gel. From one end, the gel is carefully pushed between the glass plates onto the top of the gel, being sure not to trap any bubbles between the gel surfaces. Excess buffer is again blotted away. After assembling the electrophoresis apparatus with the loaded minigels, they are run using conventional power supplies. The Bio-Rad system

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is run at a constant voltage of 200 V. Two minigels will require approximately 60 min of running time. The gels can be stained using Coomassie or silver stain methods, or electrophoretically transferred onto membranes, just like the larger acrylamide gels. Protein Detection Methods in 2D-PAGE

In general, the same protein detection methods which are used for one-dimensional PAGE can be used for 2D-PAGE gels (see this volume [33] and [36]; see also Refs. 3-5). We have found, however, that the colorbased silver stain first described by Sammons et a1.I and outlined in detail by Dunbar3 is easiest to use and gives optimal protein resolution in 2DPAGE. This stain results in vivid colors (unlike other silver stains which give less color) that are extremely important in identifying proteins and protein families and in comparing protein patterns. It is frequently desirable to first stain gels with the Coomassie Blue method to visualize the most abundant proteins, and then restain the same gel with the colorbased silver stain to visualize the less abundant proteins (after thoroughly destaining). Quantitation of Proteins in 2D-PAGE

Advances in the methods for 2D-PAGE separation of proteins have been accompanied by the development of computer systems to analyze the resulting protein patterns, and to quantitate the individual protein components.&l1Hardware and software are available commercially from a wide variety of sources. These data analysis systems range from simple inexpensive programs for personal computers to more expensive systems which allow the simultaneous analysis and comparison of complex protein patterns in 2D-PAGE gels. The accuracy and precision of the information generated by a computer analysis package depend on both the type of scanning hardware and the quality of the data manipulation soft-

' D. W. Sammons, L. D. Adams, and E. E. Nishizawa, Electrophoresis 3, 135 (1981).

lo

I'

J. I. Carrels, J. T. Farrar, and C. B. Bunvell, in "Two-Dimensional Gel Electrophoresis of Proteins: Methods and Applications" (J. E. Celis and R. Bravo, eds.), p. 38. Academic Press, New York, 1984. L. E. Lipkin and P. F. Lemkin, Clin. Chem. 26, 1403 (1980). D. W. Sammons, L. D. Adams, T. J. Vidmar, C. A. Hatfield, D. H. Jones, P. J. Chubb, and S . W. Crooks, in "Two-Dimensional Gel Electrophoresis of Proteins: Methods and Applications" (J. E. Celis and R. Bravo, eds.), p. 112. Academic Press, New York, 1984. J. Taylor, N . L. Anderson, and N. G. Anderson, Electrophoresis 3, 338 (1983).

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ware. Therefore, it is important to determine what qualitative and quantitative information is needed to interpret the data,8 and to choose the hardware and software most appropriate for that application. If a large number of 2D-PAGE gels will be processed on a routine basis, a sophisticated shared instrumentation computer system which can analyze gels rapidly and accurately and which is "user friendly" is advisable. We have used one such system, the BioImage Visage computer analysis system, and have been pleased with its performance. Before subjecting 2D-PAGE gels to computer analysis there are several points to consider. First, computer quantitation of poor quality gels is of limited value. Therefore, the 2D-PAGE separation of components in a complex protein mixture must be optimized prior to analysis. Second, the information obtained from computer "quantitation" is relative to the method of protein detection used, and thus to the nature of the proteins themselves. For example, the autoradiographic signal generated by [35S]methionine-labeled proteins will be proportional to the number of methionine residues in the protein and not necessarily to the amount of that protein present in the sample. Finally, useful information can be gathered from visual inspection of reproducible, high-quality 2D-PAGE gels without the assistance of computer programs. The lack of a computer system for analysis should not be a major factor in considering the use of 2D-PAGE.

l5r

Ir

Troubleshooting in 2D-PAGE Because of the complex nature of 2D-PAGE methods, there are technical problems that are frequently encountered while conditions are being optimized for a particular ~ a m p l e . ~AJ ~summary J~ of these problems and some suggestions for resolving them are presented in Table I. If the described protocols are followed precisely, only high-quality reagents are used, and care is taken to properly prepare the sample, the 2D-PAGE protein separation and resolution should be excellent. Strategies to Optimize 2D-PAGE Resolution

r I

The 2D-PAGE methods described in this chapter are standard procedures widely used by different laboratories that enable the direct compari-

.

r

l3

J. VanBlerkom, in "Methods in Mammalian Reproduction" (J. C. Daniel, Jr., ed.), p. 67. Academic Press, New York, 1978. R. Bravo, in "Two-Dimensional Gel Electrophoresis of Proteins: Methods and Applications" (J. E. Celis and R. Bravo, eds.), p. 4. Academic Press, New York, 1984.

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TABLE I COMMONPROBLEMS ASSOCIATED WITH 2D-PAGE Problem First dimension Gels do not polymerize

Gels break during focusing

Possible causes

Solutions

Inferior, aged, or improperly prepared catalysts; impure reagents containing contaminants which interfere with polymerization Hole in pH gradient

Start over with fresh, highquality reagents, and check pipetting measurements

Gel is overloaded Gels fall out of tube during focusing Gels will not come out of tubes after focusing Poor separation of proteins

Too much NP-40 in gel

Tubes are not cleaned properly

Improper ampholyte pH range Insufficient protein solubilization Nucleic acid contamination

I E F patterns vary from day to day

Second dimension Irregular gradients

Changing electrofocusing time

Mix ampholytes from two different sources Reduce amount of protein loaded on gel Measure using clipped off pipet tip and wipe outside surface of tip Wash tubes with Chromerge and rinse with water only; do not use methanol or siliconizing solutions Try another pH range Increase ratio of solubilization reagent to protein sample If gels are to be silver stained, increase amount of solubilization reagent and ultracentrifuge at 2 x lo5 g. If proteins are to be detected by autoradiography, add nuclease preparations to sample Standardize voltage-hours

Different lots or sources of ampholytes used Stock reagents are too old Inadequate solubilization

Standardize ampholy te source Prepare fresh reagents Increase amount of solubilization reagent

Improper equipment or technique; acrylamide polymerization is too fast

Decrease slightly the amount of catalyst

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TABLE I (continued) Problem "Fuzzy" protein spots

Streaking

Possible causes

IEF gel was equilibrated too long before loading onto second dimension gel; not optimal acrylamide concentration in SDS gel Nucleic acid contamination of protein sample Improper or incomplete solubilization Gel is overloaded Sample may contain glycoproteins, which often streak in PAGE

Solutions Use a gradient gel, or vary the acrylamide concentration slightly See First Dimension, poor separation of proteins Increase amount of solubilization reagent, or change type of reagent used Reduce amount of protein on gel Reduce amount of sample on gel, or deglycosylate glycoproteins

son of protein patterns independently generated by individual research groups. It is occasionally necessary, however, to modify these procedures for unusual proteins or for a protein which is extensively posttranslationally modified. For example, mixing ampholytes of different ranges may improve resolution of proteins within a particular pH range. Also, protein samples containing an abundance of one protein can alter the pH range of the ampholytes themselves. Therefore, it may be necessary to compensate by adding different ranges of ampholytes to the isoelectric focusing dimension. When carrying out the initial 2D-PAGE analysis of any protein mixture, it is helpful to include a lane for one-dimensional SDS-PAGE separation of the original sample on the same second dimension polyacrylamide gel. This will determine if any of the protein components are outside the range of the ampholytes used for IEF. Depending on the sample and the complexity of the protein pattern, it may be necessary to try different solubilization conditions, different ampholyte ranges, or NEPHGE (nonequilibrium pH gradient electrophoresis) gels to establish the best method to resolve all the proteins of interest. Standardization of 2D-PAGE

The standardization of 2D-PAGE methods has become more important as the need for interlaboratory comparisons of protein patterns has

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IEF

FIG. 1 . Example of cellular proteins resolved by high-resolution two-dimensional polyacrylamide gel electrophoresis using color-based silver stain3.' illustrating the standardized method for presenting 2D-PAGE protein patterns.

increased. As discussed above, the sophisticated equipment now available for running and analyzing two-dimensional polyacrylamide gels has advanced the standardization process. Another factor enabling accurate comparisons among gels is the use of internal standards for both charge and molecular weight separations. (The procedures which measure pH directly in the gels or in gel slices have proved to be totally inadequate and not reproducible.) Proteins which are modified by carbamylation have proved to be excellent charge standard^,^,^ and numerous types of molecular weight standards are commercially available. Publication Format of 2D-PAGE Patterns

The protein patterns obtained by 2D-PAGE can be extremely complicated. However, reproducible protein patterns can easily be recognized if they are presented in a standardized format. Many different laboratories, in conjunction with the International Electrophoresis Society, have agreed on a useful presentation format: the acidic end of the IEF gel at the left, and the basic end at the right; i.e., low pH values at the left, increasing toward the right. The second dimension separation is oriented with low-molecular-weight proteins at the bottom and high molecular weights

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459

at the top, which is standard for ID-PAGE analysis. The presentation of such a gel is illustrated in Fig. 1. This format is now required by the Journal of Electrophoresis and will likely be required by other journals in

Acknowledgments The authors wish to acknowledge the numerous technicians and graduate students who have assisted in the development of these techniques over the years. We thank Drs. N. L. Anderson, S. Tollaksen, and D. Sarnmons for many fruitful discussions, and Ms. Suzanne Mascola for expert secretarial assistance.

[351 Isoelectric Focusing

Proteins, as amphoteric molecules, carry positive, negative, or zero net charges depending on the pH of their local environments. The overall charge of a particular protein is determined by the ionizable acidic and basic side chains of its constituent amino acids and prosthetic groups. Carboxylic acid groups (-COOH) in proteins are uncharged in acidic solutions and dissociate to the anionic form (-COO-) at higher pH values, above about pH 3 . Amines (-NH2) and other basic functions of proteins, such as guanidines, are uncharged at alkaline pH, but are cationic below about p H 10 (e.g., -NH3-). The pH at which individual ionizable side chains actually dissociate is affected by the overall composition of the protein and the properties of the medium. As a result, each individual ionizable group in a protein has a nearly unique dissociation point. The net charge on a protein is the algebraic sum of all its positive and negative charges. There is, thus, a specific pH for every protein at which the net charge it carries is zero. This isoelectric pH value, termed pl, is a characteristic physicochemical property of every protein. If the number of acidic groups in a protein exceeds the number of basic groups, the pZ of that protein will be at a low pH value. If, on the other hand, basic groups outnumber acidic groups, the p l will be high. Proteins show considerable variation in isoelectric points, but p l values usually fall in the range of pH 3 to pH 10. Proteins are positively charged in solutions at pH values below their p l values and negatively charged above their isoelectric points. In electroMETHODS IN ENZYMOLOGY. VOL. 182

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phoresis, the net charge on a protein determines the direction of its migration (electrophoretic mobility). At pH levels below the pZ of a particular protein it will migrate toward the cathode. Conversely, at pH values above its pZ a protein will move toward the anode. A protein at its isoelectric point will not migrate in either direction. Isoelectric focusing (IEF) is a technique that was developed from these concepts to separate proteins on the basis of differences in their p1 values. It is used for both the analysis and preparative isolation of proteins. IEF, generally carried out under nondenaturing conditions, is a highresolution technique. Resolution of proteins differing in their pI values by only 0.02 pH unit, o r less, is common. Because of this high resolution, protein samples which appear to be homogeneous when tested by other means can often be separated into several components by IEF. Such microheterogeneity may be indicative of differences in primary structure, conformational isomers, differences in the kinds and numbers of prosthetic groups, or denaturation. The theoretical and practical aspects of I E F are well documented. Accounts of all aspects of the field, in more detail than can be presented here, can be found in Refs. 1-9. The methods presented in this chapter are simple, effective, and widely applicable for both analytical and preparative IEF. Principle of Method

I E F is an electrophoretic method in which amphoteric molecules are separated a s they migrate through a pH gradient. When a protein is placed in a medium with varying pH and subjected to an electric field, it will initially move toward the electrode with the opposite charge. During migration through the pH gradient, the protein will either pick up or lose P. G. Righetti, "Isoelectric Focusing: Theory, Methodology and Applications." Elsevier, Amsterdam, 1983. R. C. Allen, C. A. Saravis, and H. R. Maurer, "Gel Electrophoresis and Isoelectric Focusing of Proteins: Selected Techniques." de Gruyter, Berlin, 1984. A. T . Andrews, "Electrophoresis: Theory, Techniques, and Biochemical and Clinical Applications. Second Edition." Oxford Univ. Press, New York, 1986. P. G. Righetti, in "Electrokinetic Separation Methods" (P. G. Righetti, C. J. van Oss, and J. W. Vanderhoff, eds.), p. 389. Elsevier, Amsterdam. 1979. A. R. Williamson. in "Handbook of Experimental Immunology" (D. M. Weir, ed.), 3rd Ed., p. 9.1. Blackwell, Oxford, 1978. B. J. Radola, this series, Vol. 104, p. 256. ' B. An der Lan and A. Chrambach, in "Gel Electrophoresis of Proteins: A Practical Approach" (B. D. Hames and D. Rickwood, eds.), p. 157. IRL Press, Oxford, 1981. 0. Vesterberg, this series, Vol. 22, p. 389. P. G. Righetti, E. Gianazza, and K. Ek, J. Chromatogr. 184, 415 (1980). I

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protons. As it does, its net charge and mobility will decrease and the protein will slow down. Eventually, the protein will arrive at the point in the pH gradient equaling its PI. There, being uncharged, it will stop migrating. If a protein at its p l should happen to diffuse to a region of lower pH, it will become protonated and be forced toward the cathode by the electric field. If, on the other hand, it diffuses into a pH higher than its pi, the protein will become negatively charged and it will be driven toward the anode. Thus, in this way, proteins condense, or focus, into sharp bands in the pH gradient at their individual, characteristic p i values. Focusing is a steady-state mechanism with regard to pH. Proteins approach their respective p l values at differing rates but remain relatively fixed at those pH values for extended periods. This type of motion is in contrast to conventional electrophoresis, in which proteins continue to move through the medium until the electric field is removed. Moreover, in IEF proteins migrate to their steady-state positions from anywhere in the system. This means that, unlike other electrophoretic methods, the sample application point is arbitrary. In fact, the sample can be initially distributed throughout the entire separation system. The key to IEF is the establishment of stable pH gradients in electric fields. This is most commonly accomplished by means of commercially available, synthetic carrier ampholytes (amphoteric electrolytes). These compounds are mixtures of relatively small, multicharged, amphoteric molecules with closely spaced pI values and high conductivity. Under the influence of an electric field, carrier ampholytes partition themselves into smooth pH gradients which increase monotonically from the anode to the cathode. The slope of the pH gradient is determined by the pH interval covered by the carrier ampholyte mixture and the distance between the electrodes. Practical Aspects

Format At one time, all IEF was carried out in vertical columns using density gradients of sucrose or glycerol to stabilize the pH gradient against con-~-~ IEF columns are vection and to support separated ~ o n e s . ' . ~However, cumbersome and difficult to operate. Focused zones are inherently unstable (because they are denser than the surrounding medium) and not adequately maintained by density gradients. In addition, the resolution obtained by focusing in columns is usually lost during recovery of the focused materials. As a consequence, density gradients have, for the most part, been replaced by other stabilizing media.

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Most analytical I E F is currently carried out in continuous polyacrylamide gels.lg Polyacrylamide provides the virtually uncharged support matrices required for IEF. Gels are formed with large pores which allow the relatively unimpeded motion of proteins. The most common configuration for I E F is the horizontal gel slab. This configuration provides good cooling efficiency and makes sample application relatively easy. Electrofocusing run in cylindrical tubes7 constitutes the first dimension of the most common two-dimensional gel electrophoresis method.I0 Preparative electrofocusing, too, is a practical reality. Unlike other forms of electrophoresis, the IEF mechanism lends itself to preparative methods. For laboratory-scale protein isolations, density gradients and IEF in beds of granular polyacrylamide or dextran have been ~ s e d . ~ ~ . ~ ~ ~ However, the recently introduced rotating I E F device (the Rotofor C ~ I I ) , described ' ~ ~ ~ ~ below, is becoming the method of choice for laboratory-scale preparative work. Polyacrylamide Gels

Polyacrylamide gels are used for focusing proteins up to about 500,000 Da in size.'"J4 They are formed by copolymerization of acrylamide monomer, CH2=CH-CO-NH2, and a cross-linking comonomer, N,N1-methylenebisacrylamide, CH2=CH-CO-NH-CH2-NHCO-CH=CH2 (bisacrylamide). Polymerization is through a vinyl addition mechanism catalyzed by a free radical-generating system. In IEF, polymerization is initiated by combined use of ammonium persulfate (APS), tetramethylethylenediamine (TEMED), and illuminated riboflavin. The photochemical initiator, riboflavin, is included because the APSTEMED system is inefficient at low pH. The IEF gel matrix must be both nonsieving and mechanically stable. A suitable gel composition for horizontal electrofocusing is 5% (wlv) total monomer (acrylamide + bisacrylamide) with the cross-linker, bisacrylamide, accounting for 3% (w/w) of the total monomer. By convention, this gel composition is denoted by the pair of figures 5% Z', 3% C . Agurose Gels

Agarose gels have much larger pores than polyacrylamide gels. They are used for the separation of large proteins and structures that cannot be B. S. Dunbar, H . Kimura, and T. M . Timmons, this volume [34]. M. D. Frey and B. J. Radola, Electroplloresis 3, 216 (1982). l2 N . B. Egen, W. Thormann, G . E. Twitty, and M. Bier. in "Electrophoresis '83" (H. Hirai, ed.). p. 547. de Gruyter, Berlin, 1984. " M. Bier, U.S. Pat. 4,588.492 (1986). l4 D. E. Garfin, this volume [33].

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readily characterized in polyacrylamide. Molecules larger than 200,000 Da can be separated in 1% agarose gels. The gels are formed by melting an agarose mixture and pouring it onto a glass plate in a manner similar to the methods used in preparing agarose gels for immunoassays and for electrophoresis of DNA. T o minimize e!ectroendoosmotic solvent flows, only agarose prepared specifically for I E F (zero -M,) should be used and the viscosity of the medium should be increased by incorporating sorbitol and glycerol into the gels. Consult Refs. 1-3 and 9 for procedures for carrying out agarose IEF. Apparatus

Horizontal slab gels possess a number of advantages and have become very popular for analytical IEF1-4.9(preparative devices are discussed below). Gels are cast on glass plates or specially treated plastic sheets and run with one face exposed. This allows samples to be applied anywhere desired on the gel surface, and enables pH and voltage measurements to be made directly on the gel surface. With the slab configuration, a number of samples can be compared under identical running conditions, and most apparatuses allow gel lengths and thicknesses to be varied. Horizontal, flat-bed electrophoresis cells can be obtained from a number of manufacturers. The better quality cells have cooling platforms for heat dissipation, condensation control, and movable electrodes that make direct and uniform contact with the gel surface. Most systems also include devices for casting gels. An alternative arrangement for analytical I E F that has recently been rediscovered is the "inverted" gel format in which the gel is run facing downward suspended between two carbon rod electrode^.^.'^ Inverted cells are less expensive and simpler to use, but less versatile, than standard cells. These cells run at lower voltages than standard flat beds and require no active cooling. Resolution is somewhat less than can be obtained with standard horizontal cells. The electrical power supply used with standard cells should be capable of delivering up to 3000 V and 30 W operating power. Inverted cells require only about 500 V and 5 W maximum power. Ideally, the power supply will have a constant power mode of operation. Standard cells require coolant circulation for optimum performance. Casting Gels

Gels are cast containing carrier ampholytes. p H gradients are established during the runs, concurrently with protein separation. The standard l5

Z. L. Awdeh, A. R. Williamson, and B. A. Askonas, Nature (London) 219,66 (1968).

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gel thickness of 0.8 or 1 mm provides easily handled gels with good protein load capacity and good staining speed. Thinner gels, 0.2 or 0.4 mm thick, allow higher voltages to be used for increased resolution and shortened run times. Irregularities in gel surfaces and trapped bubbles must be avoided because they cause local distortions in the electric field. Of the various apparatuses available for preparing gel^,'^,^ the capillary method provides the fastest and easiest method for forming gels for IEF.1."'h.'7A good capillary casting tray1.I6is a worthwhile investment. These units are acrylic trays with precision spacer rails along their edges for establishing gel thicknesses. Gels are formed by introducing catalyst-activated monomer solution into the space between the acrylic tray and a glass plate or treated plastic sheet and allowing the monomer to polymerize. Since the acrylic surfaces are not wetted by monomer solutions, gels do not stick to the trays. Polyacrylamide gels adhere to the glass (or treated plastic) backing plates and are easily lifted from the trays. Carrier Ampholy tes Carrier ampholytes are complex mixtures of synthetic amphoteric buffers that form smooth pH gradients in applied electric fields.'-I~w-8.'8~19 Several varieties of carrier ampholytes are commercially available. The products from different manufacturers are not necessarily interchangeable and may yield different IEF pattern^.^ Exact details of the chemical and physical properties of carrier ampholytes are proprietary. In general, they are mixed polymers (about 300-1000 Da in size) of aliphatic amino and carboxylic acids (polyamino-polycarboxylic acids), although some types contain sulfonic and phosphonic acid residues. Following synthesis, carrier ampholytes are purified and blended by the manufacturers to give smooth and reproducible gradients covering wide or narrow pH ranges. Unknown p l values are estimated with wide-range carrier ampholytes covering 7-8 pH units (e.g.. pH 3 to 10) and more closely established with narrow pH ranges. The proper choice of ampholyte range is very important to the success of a fractionation. Ideally, the pH range covered by the focused carrier ampholytes should be centered on the p l of the proteins of interest to ensure that they focus in the linear part of the gradient while excluding extraneous proteins from the separation zone. Moreover, the resolution obtainable in an I E F run depends on the pH profile in the focused gel; l6

J . F. Monthony, U.S. Pat. 4,246.222 (1981). C. Allen. Elrctrophoresis 1, 32 (1980). W. W . Just, this series, Vol. 91, p. 281. S. Binion and L. S. Rodkey, Anal. Biochem. 112, 362 (1981).

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narrow pH range gradients favor high resolution by spreading out the p I values in the region of interest. The range of carrier ampholytes used in an experiment need not be limited to those commercially available. Almost any range desired can be custom made in the laboratory by the methods of preparative IEF. The concentration of carrier ampholytes is also important. Carrier ampholyte concentrations of about 2% (wlv) should be used. Concentrations of ampholytes below 1% (wlv) often result in unstable pH gradients. Above 3% (wlv), ampholytes are difficult to remove from gels and, since they are stainable, they can interfere with protein detection. Resolution

A goal of both analytical and preparative electrophoresis is to achieve the greatest possible degree of resolution between adjacent protein bands. In this context, resolution refers to separation of protein bands relative to their band widths and is denoted by the difference in pI between clearly distinguishable bands. Two of the factors which enter into successful IEF resolution are under direct experimental control. These are the electric field and the steepness of the pH gradient, as determined by the applied voltage and the pH range of the carrier ampholytes, respectively. According to both theory and experiment, the difference in p l between two resolved adjacent protein IEF bands (ApI) is directly proportional to the square root of the pH gradient and inversely proportional to the square root of the voltage gradient (field strength) at the position of the bands14,6.8.20. Apl

(pH gradientlvoltage gradient)'I2

Thus, narrow pH ranges and high applied voltages give high resolution (small ApI) in IEF. In addition to these two factors, good resolution is favored by substances with low diffusion coefficients and high rates of change of mobility with pH near their isoelectric points. Most proteins satisfy the latter two criteria, but these factors are, of course, not under the control of the experimenter. Changing the interelectrode distance for a given voltage and pH range will change both the pH and voltage gradients to the same extent, so, unless the carrier ampholyte range or applied voltage is also adjusted accordingly, there will be no alteration in obtainable resolution. In addition to the effect on resolution, high electric fields also result in shortened run times. However, high voltages in electrophoresis are accompanied by large amounts of generated heat (Joule heating). Thus, J. C. Giddings and K . Dahlgren, Sep. Sci. 6, 345 (1971).

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there are limitations on the magnitudes of the electric fields which can be applied. This is partly because resolution decreases with increasing temperature (since diffusion coefficients increase with temperature) and partly because gels can actually get hot enough to burn. Because of their higher surface-to-volume ratio, thin gels are better able to dissipate heat than thick ones and are therefore capable of higher resolution. Electric fields used in IEF are generally of the order of 100 Vlcm. Gradient Instability

For most practical purposes, pH gradients are stable once the steady state has been reached. However, during extended focusing runs (longer than about 3 hr under standard analytical conditions), gradients are found to slowly deteriorate.l49' This decay is characterized by a drift of the gradients toward the cathode and is accompanied by acidification at the anode, flattening of the gradient in the neutral pH region, and a loss of alkaline bands. The mechanism of the instability, which has been called "cathodic drift," is not completely understood. Cathodic drift is probably caused by a combination of factors including electroendoosmosis,2' C02 a b ~ o r p t i o n ,and ~ ~ nonzero, pl-dependent electrophoretic fluxes.23 The practical consequence of cathodic drift is that excessively long IEF runs should be avoided. Additives

Many protein samples require the use of detergents for their solubilization. For IEF work, the zwitterionic detergents CHAPS and CHAPSO, or the nonionic detergent octylglucoside at concentrations of 1-2% in the gel are recommended. Consult Refs. 1-4 and 24 for details. Even in the presence of detergents, some samples may have stringent salt requirements. Only if salt is an absolute requirement should it be present in a sample, and substantial band distortions should be expected. Carrier ampholytes contribute to the ionic strength of the solution and can help to counteract a lack of salts. Urea is a common solubilizing agent, especially for those proteins which precipitate at their isoelectric points, even though it denatures proteins.14 Urea (3M) is often found satisfactory for maintaining protein 2'

2'

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H. Rilbe, in "Electrofocusing and Isotachophoresis" ( B . J. Radola and D . Graesslin, eds.), p. 35. de Gruyter, Berlin, 1977. H. DelincCe and B. J. Radola, Anal. Biochem. 90, 609 (1978). R. A. Mosher, W. Thormann, and M. Bier, J . Chromatogr. 351, 31 (1986). L. M. Hjelmeland and A. Chrambach, Electrophoresis 2, 1 (1981).

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solubility, but concentrations up to 8 M urea have been used. Only fresh solutions of urea, treated with a mixed bed ion-exchange resin, should be used in order to prevent carbamylation of amine and sulfhydryl groups of proteins. Experimental Procedure The following protocol describes the use of polyacrylamide slabs for IEF on horizontal flat-bed cells. Gel preparation, sample application, focusing conditions, and detection methods are included. The gel recipe is for 12 rnl of 5% T (3% C) acrylamide, 2% arnpholytes, and 5% glycerol. This is sufficient for casting one standard-size gel of 100 x 125 x 0.8 mm (10 ml) or four 100 x 125 x 0.2 mm gels (10 ml total). The thinner gels can be run at twice the voltage of the thicker ones for increased resolution. Equipment and reagents for IEF are available from many suppliers. For best results, follow the manufacturer's instructions and recommendations, especially when working with high-voltage equipment. Except where noted, reagents for I E F can be prepared a s concentrated stock solutions. All water used should be distilled o r deionized. Stock Solutions Acrylamide monomer concentrate (25% T, 3% C): Dissolve 24.25 g acrylamide and 0.75 g bisacrylamide in about 70 ml of water. Adjust the final volume to 100 ml. Filter through a 0.45-pm filter. Store protected from light at 4" for up to 1 month. Caution: Acrylamide monomer is a neurotoxin. Avoid breathing acrylamide dust, d o not pipette acrylamide solutions by mouth, and wear gloves when handling acrylamide powder o r solutions containing it. For disposal of unused acrylamide, add bisacrylamide (if none is present), induce polymerization, and discard the solidified gel 25% glycerol (w/v): Weigh 25 g glycerol in a beaker. Add about 50 ml of water and mix well. Dilute to 100 ml with water Carrier ampholytes: Use ampholytes undiluted unless instructed otherwise by the manufacturer. Carrier ampholytes are supplied a s aqueous solutions, usually containing 40 o r 20% (w/v) solids. The pH range used will depend on the protein(s) of interest 0.1% (w/v) riboflavin 5'-phosphate (FMN): Dissolve 50 mg riboflavin 5'-phosphate in 50 ml water. Store protected from light at 4" for up to 1 month 10% (w/v) ammonium persulfate (APS): Dissolve 100 mg APS in 1 ml of water. Prepare this solution fresh daily

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TEMED (N,N,Nf,N'-tetramethylethylenediamine): Use TEMED undiluted as supplied. Use only pure, distilled TEMED. Store cool and protected from light Casting Gels

The use of gel support film for polyacrylamide is highly recommended, especially with thin gels, which cannot be easily handled unless supported. Polyacrylamide binds covalently to these sheets of treated polyester,2-25simplifying gel handling in all steps, from running gels through drying and storing them. Although polyacrylamide gels adhere to wellcleaned glass plates and remain bound through the IEF runs, gels will come off of the backing plates during the staining or destaining steps. IEF gels are very difficult to manipulate once they become detached from their backings. Basic ampholytes (pH > 8) may interfere with the adhesion of gels to support films. Increasing the APS concentration in the final gel to 0.7 mglml(84 p1 of 10% APSI 12 ml of the gel solution given below) should alleviate the problem. Prolonged soaking in the acidic staining and destaining solutions can also affect adhesion of polyacrylamide gels to the support films. Do not soak the gels any longer than necessary in the staining and destaining solutions. 1. Place a few drops of water on a clean glass IEF plate and place the hydrophobic side of a gel support film against the plate (water beads on the hydrophobic sides of the films). Roll the support film flat with a test tube or similar object to force out excess water and air bubbles and wipe off excess liquid at the edges. Capillarity is sufficient to hold the supported gel on the plate throughout the run. Place the glass plate on the casting tray with the gel support film facing down. 2. Prepare monomer-ampholyte solution from the stock reagents:

Water Monomer concentrate 25% (wlv) glycerol 40% ampholyte (wlv)

6.6 ml 2.4 ml 2.4 ml 0.6 ml

Adjust the volume of water to accommodate additives and different ampholyte concentrations; e.g., with 20% ampholytes use 6 ml of water and 1.2 ml of ampholyte solution. 3. Deaerate the monomer-ampholyte solution under vacuum for 5 min; e.g., in a bell jar, desiccator, or vacuum flask. 25

B. J . Radola, Electrophoresis 1, 43 (1980).

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4. Add initiators, swirling gently to mix them into the monomerampholyte solution. 0.1%(wlv) FMN 60 p l 10% (w/v) APS 18 p1 TEMED (undiluted) 4 p1 5. Using a pipet and bulb, carefully introduce the monomer solution between the support film-glass plate and the casting tray. Control the flow to prevent air bubbles. If a bubble becomes trapped in the monomer solution, slide the plate sideways until the bubble escapes at an edge, then reposition the plate so that there is a uniform layer of monomer under it. 6. Position a fluorescent lamp directly over the tray about 3-4 cm from the gel. Illuminate the solution for about 45 min. 7. Lift the gel from the tray by gently prying it up with a spatula. Turn the plate over, with the gel upward, and illuminate the gel for a further 20 min to polymerize monomer remaining on the gel surface. 8. The gel may be used immediately or it can be covered in plastic wrap and stored at 4" for several days. Best results are sometimes obtained by letting a gel "cure" (polymerize completely) overnight at 4" before use.

Sample Preparation Protein samples for IEF must be substantially salt free and free of precipitates. Small samples (1 to 10 p1) in typical biochemical buffers are usually tolerated, but better results can be obtained with solutions in deionized water, 2% ampholytes, or 1% glycine. Suitable sample solutions can be prepared by dialysis or gel filtration. Good visualization of focused lanes generally requires a minimum of 0.5 p g of proteinlband with dye staining or 50 ng of proteinlband with silver staining (see below).

Sample Application There are many suitable methods for applying samples to thin-layer polyacrylamide gels. l4 One of the simplest methods is to place filter paper strips impregnated with sample directly on the gel surface. Up to 25 p l of sample solution can be conveniently applied after absorption into 1-cm squares of filter paper. A convenient size for applicator papers is 0.2 x 1 crn, holding 5 p1 of sample solution. There are no fixed rules regarding the positioning of the sample on the gel. In general, samples should not be applied to areas where they are expected to focus. To protect the proteins from exposure to extreme pH

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the samples should not be applied closer than 1 cm from either electrode. Preforming the pH gradient before sample application will also limit the exposure of proteins to pH extremes. Some polypeptides are eluted most efficiently from strips when applied to the anodic side of the gel.'34.26 A good strategy when focusing a protein for the first time is to apply samples to three different areas of the gel, one near each electrode and one near the middle of the gel. This strategy is also valuable in estimating ~ . ~ ~ the patterns obtained on the approach to steady-state f o ~ u s i n g .When applying the sample at opposite ends of the gel become identical, the steady state can be assumed to have been reached. Steady-state conditions should be duplicated when determining the pZ values of particular proteins. It should be noted that samples applied at opposite ends of IEF gels will not always focus into identical patterns. The reason for this is not clear, but may be related to interactions of proteins with differing species of carrier ampholytes during focusing. Focusing

1. Connect the electrophoresis cell to a refrigerated circulator cooled to 4". 2. Wet the cooling stage with a few drops of water or 0.5% glycerol to ensure good thermal contact with the gel backing. Place the gel on the platform (gel upward). Blot any excess liquid from the cooling stage to eliminate possible electrical shorting paths. 3. Cut electrode strips. Thick filter paper or (uncolored) blotter paper work well as electrode strips. More than one thickness can be used. The strips should be about 7 mm wide and cut about 4 mm shorter than the width of the gel (this prevents electrical arcing to the cooling stage). Place the electrode strips on a glass plate and wet them with the appropriate electrolyte solutions. The anode solution is acidic and the cathode solution is basic. a. For most purposes, use 1 N NaOH as catholyte (negative terminal) and 1 N H3PO4as anolyte (positive terminal).I4 b. At high voltages, as in focusing with ultrathin gels (0.2 mm or less), the following electrolytes are r e ~ o m m e n d e d ~ ~ :

Catholyte: 20 mM lysine, 20 mM arginine, 2 M ethylenediamine. Dissolve 0.36 g lysine (free base), 0.34 g arginine (free base), and 13.4 ml ethylenediamine in water to give 100 ml. Two molar ethanolamine (12 m11100 ml) can be substituted for the ethylenediamine. Store at 4". 26

P. G . Righetti and F. Chillemi, J . Chromatogr. 157, 243 (1978).

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Anolyte: 20 mM aspartic acid, 20 mM glutamic acid. Dissolve 0.26 g aspartic acid and 0.29 g glutamic acid in water to give 100 ml of solution. Store at 4". 4. Blot the wetted electrode strips with paper towels until they are slightly moist. Carefully place the strips along the appropriate edges of the gel. Do not allow the strips to extend beyond the sides of the gel. 5. Cut 0.2 X 1 cm pieces of filter paper for sample application. Place the pieces of paper on a glass plate and pipette 5 p1 of a protein sample solution to each piece. Place the applicator papers on the gel 1 cm from the anode (sample application is discussed above). 6. Position the electrodes over the electrolyte strips, making sure that there is good contact between the electrodes and the strips. 7. Set the power supply to constant power and run at 6 W/gel, with a voltage limit of 1500 V for standard size gels (100 x 125 x 0.8 mm). Total run time is about 2 hr.

The actual running conditions will vary with the apparatus, the gel thickness, the sample solution, and the ampholytes. Gels should always be run at the highest voltage compatible with the heat-dissipation capabilities of the electrophoresis cell. Check the manufacturer's recommendations for proper power settings. At the start of a run, when voltage is first applied, the current will be at its highest value, because the carrier ampholytes have not yet focused. As the run progresses, the conductivity of the gel will drop and the current will fall. It is recommended that the run be started in the constant power mode set at the heat-dissipation limit of the cell. The power supply should be maintained in this mode until the current drops to its lowest value and the highest voltage is reached. (Recall that power = voltage x current and current = voltage x conductivity.) Standard-size gels run at 6 W usually plateau at 1200-1500 V in about 1 hr. When the maximum voltage is reached, switch to constant voltage regulation (at the voltage limit) for the remainder of the focusing run. The paper application strips can be removed at the time the switch to the constant voltage mode is made. A good way to monitor the progress of a run is with colored marker proteins. However, not all proteins focus at the same rate, so that for accuracy and reproducibility, it is necessary to predetermine the correct conditions for reaching the steady state for the protein of interest. Initial power settings of 10 W/100 x 125 mm gel are common, and final voltage gradients as high a s 300 Vlcm interelectrode distance have been used with thin gels (0.2 mm). It is customary to characterize the extent of focusing in I E F runs with ~.~~ the time integral of the applied voltage, expressed in v o l t - h o u r ~ . The volt-hour designation is meant as a standard for reproducing focusing

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conditions. The conditions for attaining steady-state focusing, once determined, are reproducible. However, many factors, especially temperature, affect the absolute reproducibility of focusing. Thus, although the volthour quantity is a convenient indicator of the extent of focusing, it is not a definitive measure of the IEF process. High voltages run for short times result in better separations than low voltages and long times. Detection of Protein Bands

Protein staining is the most general method of detection. Discussions of other detection methods and means for quantitating protein bands in gels can be found in Refs. 1-4. Carry out staining and destaining steps at room temperature with gentle agitation (e.g., on an orbital shaker platform) in any convenient container, such as a glass casserole or photography tray. Carefully peel off sample paper strips and electrode wicks (if possible) before beginning the procedure. Standard Procedure. No preliminary fixation step is required with this method. Bands containing proteins in microgram quantities are easily seen.

1. Prepare the staining solution: 0.04% Coomassie Brilliant Blue R-250, 0.05% Crocein Scarlet, 0.5% CuS04 in 27% ethanol, 10% acetic acid: Water CuS04 Ethanol Glacial acetic acid Coomassie Brilliant Blue R-250 Crocein Scarlet

630 ml 5.0 g 270 ml 100 ml 0.4 g 0.5 g

2-Propanol can be substituted for ethanol. Dissolve the cupric sulfate in the water before adding the alcohol. Add the dyes to the solution last. Filter the solution after the dyes have dissolved. The staining solution is reusable. Store it at room temperature. Crocein Scarlet rapidly binds and fixes proteins.27Cupric sulfate enhances stain i n t e n s i t y . ' ~ ~ . ~ ~ 2. Soak gels in staining solution for at least I hr. 3. Destain with a large excess of 12% ethanol. 7% ace'tic acid, 0.5% CuS04 (810 ml H20, 5 g CuS04, 120 ml ethanol, 70 ml acetic acid) until a clear background is obtained. This will require several changes of destaining solution. Grainy precipitates of dye will sometimes settle on the surfaces of gels after the staining solution has been reused several times.

'' A. J. Crowle and L. J. Cline. J . Irnmunol. Methods 17, 379 (1977). 2S

P. G . Righetti and J . W. Drysdale, J.

Chromatogr.

98, 271 (1974).

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These precipitates can be wiped off of the gel with a gloved finger or a moistened tissue while the gel is in the destaining solution. 4. Soak the gel in 12% ethanol, 7% acetic acid to remove the cupric sulfate. Crocein Scarlet can be omitted from the staining solution. If so, gels must be immersed in fixative (4% sulfosalicylic acid, 12.5% trichloroacetic acid, 30% methanol) for at least 30 min prior to staining. If ultra-thin (0.2 mm) gels detach from gel support film during the staining procedure, shorten immersion times, so that the total staining and destaining takes no more than 2 hr. Quick Stain. The following techniquez9 is nearly as sensitive as the above one and requires no destaining (it cannot be used in the presence of detergents): Immerse the gel for 1 hr in 3.5% perchloric acid containing 0.025% Coomassie Brilliant Blue G-250. For intensification, immerse the gel in 7% acetic acid. Silver Stain. Silver staining is 10 to 100 times more sensitive than dye staining. Before beginning the silver staining procedure, IEF gels must first be fixed in 30% methanol, 10% trichloroacetic acid, 3.5% sulfosalicylic acid for 1 hr, followed by at least 2 hr in several volumes of 30% methanol, 12% trichloroacetic acid. The Merril silver staining proto~ o 1 ~ must , ' ~ , be ~ ~modified for IEF gels bonded to gel support film to include two soaks for 5 min each in 400 ml deionized water between the oxidizer and silver reagent steps, and a 1-min wash in 400 ml of water between the silver reagent and developer steps. Preservation of Gels

To dry gels on support film, simply allow them to dry overnight in air in a dust-free location. Alternatively, gels can be carefully dried with a heat gun at a low setting. To dry unsupported gels, first soak them in 7% acetic acid, 5% glycerol for 1 hr, then smooth them on water-wetted filter paper and dry them in a gel dryer. Determining pH Gradients

When focusing is completed, pH gradients can be determined in various ways. The most straightforward method is to base pH profiles on the 29

A. H. Reisner, P. Nemes, and C. ~ u c h o l t zAnal. , Biochem. 64,509 (1975); see also A. H. Reisner, this series. Vol. 104, p. 439. C. R. Merril, D. Goldman, S. A. Sedman, and M. H. Ebert, Science 211, 1437 (1981); see also C. R. Merril, D. Goldman, and M. L. Van Keuren, this series, Vol. 104, p. 441; C. R . Meml, this volume [36].

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positions of focused marker proteins. Extensive, but outdated, tables of protein isoelectric point^^'-^) can be consulted in choosing appropriate markers for a particular experiment. It is much simpler, though, to make use of one of the many commercially available protein mixtures. IEF protein standards are combinations of proteins with well-characterized pl values blended to give uniform staining. The blends often contain naturally colored proteins which allow focusing runs to be continually monitored. They also usually contain proteins that achieve steady-state focusing in relatively short times. Marker proteins usually reach the steady state in about 2500 V-hr. Gels are calibrated with one or two lanes of IEF protein standards. Unknown isoelectric points can be interpolated from graphs of the positions of focused marker proteins plotted as functions of their isoelectric points. pH gradients can also be directly determined with surface electrodes or by elution of ampholytes (before staining). In the latter method, first either slice gels with a blade or punch them into closely spaced pieces with a sharp cork borer. Individually soak each piece of gel in a minimum volume of degassed water or 10 mM KC1 for 1-2 hr and measure the pH of each solution. Regardless of the method used in determining the gradient, what is actually measured is the pH of the focused carrier ampholytes, not the proteins themselves. Ideally, pH measurements should be made at the same temperature as the IEF run. Nevertheless, temperature and solvent effects and interference from absorption of atmospheric C 0 2 are usually neglected in most pH determinations unless accurate p l measurements are required. Discussions of the effects of these factors in Refs. 1, 3.4, 7, and 22 should be consulted. Microheterogeneity and Artgacts. Multiband IEF patterns can arise from molecular interactions and conformation changes as well as from Ampholytes can reversibly inherent isoelectric mi~roheterogeneity.~~ bind directly to proteins, proteins can undergo sequential pH-dependent conformational changes, and proteins can interact with one another. These types of reactions can artifactually alter the pI profiles of proteins. On the other hand, many proteins are inherently heterogeneous, consisting of isoelectric isomers. To distinguish between artifactual and inherent heterogeneity, single focused bands should be cut out and rerun. If a P. G. Righetti and T. Caravaggio. J . Chromutoyr. 127, 1 (1976). D. Malamud and J. W. Drysdale, Anul. Biochem. 86, 620 (1978). 33 P. G . Righetti and G. Tudor, J. Chromatoyr. 220, 115 (1981). j4 J. R. Cann, in "Electrokinetic Separation Methods" (P. G . Righetti, C. J. van Oss, and J. W. Vanderhoff, eds.), p. 369. Elsevier, Amsterdam, 1979; see also J. R. Cann, this series, Vol. 61, p. 142. " 32

[351

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ISOELECTRIC FOCUSING -

single band splits into multiple bands on refocusing, artifact formation is indicated. When rerunning a band, care should be taken to rerun it under the same conditions and from the same position on the gel as the initial sample. Preparative Isoelectric Focusing

Two techniques which are useful for laboratory-scale preparative electrofocusing are IEF in granular beds1-4.6,11,35 and use of the Rotofor ce11.12J3.36 Both methods allow preparative fractionations on the scale of from hundreds of milligrams to grams of protein, with recoveries of greater than 90% possible." Purification levels between 10- and 100-fold place IEF methods intermediate between ion-exchange and ligand-binding chromatographies a s preparative methods. IEF is well suited for use at any stage of a preparative scheme, and is particularly effective in the early stages of purification. In many cases, simple sequential fractionation and refractionation on the same device provides the desired purity. It is not necessary to attain steady-state focusing in preparative IEF, since adequate separations may be achieved before then. IEF in Granulated Gel Beds I

Focusing in beds of granulated polyacrylamide or dextran allows highresolution separation and recovery of relatively large quantities of protein.'-4.6,11.35 Granular polyacrylamide gels are recommended for this procedure because of low residual charge and resistance to enzymatic degradation." Focusing is carried out in ampholyte-containing gel slurries in specially designed trays in standard flat-bed apparatus. The manufacturer's instructions should be followed for proper use of each particular cell. The Rotofor Cell The easiest to use preparative electrophoresis device is the Rotofor cell developed by Egen, Bier, and a s s ~ c i a t e s l ~(available .'~ only from BioRad Laboratories). The principle of the Rotofor cell, I E F in free solution, is similar to that of column methods. However, zone stabilization in the Rotofor cell is achieved not by means of density gradients, but by turning the column on its side. Gravitationally induced convection is inhibited by rotating the column about its (horizontal) axis. The separation column is C. Derneulemester, G. Peltre, D. Panheleux, and B . David, Elecrrophoresis 7,518 (1986). %N. B. Egen, M. Bliss, M. Mayersohn. S. M. Owens, L. Arnold, and M. Bier, Anal. l5

Biochem. 172,488 (1988).

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divided into compartments by means of screens of woven polyester. The screens offer resistance to fluid convection, but do not hinder the flow of current or the transport of proteins. Proteins, which are initially dispersed uniformly throughout the chamber, migrate to the one or more compartments which are at pH values nearest to their isoelectric points. The combined effect of compartmentalization and rotation is superior to either method alone in maintaining the stability of focused zones. The segmentation of the column also facilitates fraction collection. A focusing chamber, capable of holding up to 55 ml of sample, is divided into 20 compartments by a core made up of 19 disks of polyester screen (6-pm pores). A ceramic cooling finger runs through the center of the focusing chamber to dissipate the heat generated during the run. Two electrode assemblies hold the anolyte and catholyte solutions. Appropriate ion-exchange membranes and gaskets isolate the electrolytes from the sample in the focusing chamber while allowing electrical contact with the material in the chamber. The anolyte is usually 0.1 M H3PO4 and the catholyte is usually 0.1 M NaOH, but any other electrolytes which are compatible with IEF can be used. Vent caps provide pressure relief from the gases which build up in the electrode chambers by electrolysis during the run. The entire assembly rotates around the central horizontal axis during the run to inhibit convection, maintain even cooling and efficient electrical contact, and prevent the screens from becoming clogged by precipitated protein. Runs are at 4" at constant power (12 W) for 4 hr. Simple and rapid sample collection is by aspiration through tubing lines connecting the 20 individual compartments with corresponding test tubes in a vacuum chamber. Collection is accomplished in seconds, minimizing remixing of fractions by diffusion. Some remixing of adjacent zones takes place, however, because of the finite dimensions of the fraction compartments. The individual test tube fractions are easily sampled for assay or measured for pH with standard electrodes. Samples for the Rotofor need not be completely desalted before fractionation. Ions in the sample solution will be electrophoresed into the two end compartments in the early stages of the run. Carrier ampholyte (2%, WIV)in the initial sample solution supplies enough ampholyte for refractionation of pooled material. After the tubes containing the protein of interest have been identified, the assay peak can be pooled for a second run. The amount of carrier ampholytes contained in the pooled fractions is adequate for refractionation. The pH range covered on refractionation is determined by the pooled fractions and is generally much narrower than the initial range. Twenty-five-fold purification in a single run and 1000-fold purification by refractionation have been achieved. The ideal sample run on the Rotofor cell would contain only the pro-

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tein mixture, water, and ampholytes. However, p l precipitation may require that 3 M urea be included for solubility. When higher urea concentrations are needed, the Rotofor cell is run at 12". Detergents (1-2%, wlv) may also be added to samples. Zwitterionic detergznts such as CHAPS, CHAPSO, and nonionic octylglucoside are satisfactory. Triton X-100 and NP-40 may be less satisfactory due to their slight charge content.

Remouul c?f'Ampholj~tes from Proteins There are a number of ways to separate ampholytes from proteins.'-4 Electrophoresis, ammonium sulfate precipitation, and gel filtration, ionexchange, and hydroxylapatite chrornatographies have all been used. Dialysis is a simple and effective method for removing ampholytes from solutions of proteins. First, adjust the pooled fractions to I M NaCl to disrupt weak electrostatic con~plexesbetween ampholytes and proteins, then dialyze the solutions into appropriate buffers. Extensive dialysis is required for thorough removal of arnpholytes. There is no good way to demonstrate complete absence of ampholytes in a protein solution, but for many applications they need not be removed.

[361 Gel-Staining Techniques By CARLR. MERRII. Protein Stains

Naturally colored proteins such as myoglobin. hemoglobin. ferritin, and cytochrome c may be directly observed in gels illuminated with light in the visual spectrum, providing that their chromophores are not damaged during electrophoresis.' However, the visualization of most proteins requires the use of dyes or stains. Organic stains were first utilized for the detection of proteins on gels. Recently rnetal-based stains. such as the silver stains. have achieved widespread use because of their increased sensitivity. A number of organic stains have been adapted for the detection of electrophoretically separated proteins, including Bromphenol Blue,I Fast Green (Food Grcen 3) and Amido Black (Acid Black I).' Some of these I

B. D. Davis and E. J . Cohn. Ann. N . Y . A C L I ~Sci. . 39, 209 (1939).

!

W. Grassman and K . Hannig. 2. Pllvsial. Chem. 290, 1 (1952).

' E. L. Durrum, J. Atn. Ckeril. Soc.. 72, 2943 (1950). METHODS IN ENZYMOLOGY. VOL. 182

Cop)r~ghr% 1990 h) Academ~cPress, Inc. All rights of rsproduct~onin any form reserved.

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stains preferentially stain certain classes of proteins: Lipoproteins may be stained by Oil Red 0 , 4while glycoproteins can be detected by a red color that is produced by their oxidation with periodic acid and subsequent reaction with fuchsin sulfurous acid (Schiff s reagent).5 Of the organic stains, Coomassie Blue has proved to be one of the most sensitive. Proteins may also be detected by the use of fluorescent stains. These stains can detect proteins in the nanogram range.6 However, fluorescent stains usually require reaction conditions that are best provided prior to electrophoresis. Furthermore, they may alter the charge of the p r ~ t e i n . ~ However, such charge alteration do not generally present problems for electrophoretic techniques that separate proteins on the basis of molecular weight, such as with sodium dodecyl sulfate (SDS) electrophoresis. Silver staining currently offers the highest sensitivity. These stains generally provide more than a 100-fold increase in sensitivity over that attained by the most commonly used organic protein stain, Coomassie Blue. 899

Coomassie Blue Staining

If one is primarily interested in detection of fairly abundant proteins, and not concerned with the determination of purity or the detection of trace proteins, the Coomassie Blue stains may be useful. They were originally developed as acid wool dyes and they were named "Coomassie dyes" to commemorate the 1896 British occupation of the Ashanti capital, Kumasi or "Coomassie," now in Ghana. Coomassie Blue R-250 (the letter "R" stands for a reddish hue while the number "250" is a dye strength indicator) was the first of these triphenylmethane stains to be introduced. 10.'l Other Coomassie stains, such as Coomassie Blue (3-250 ("G" indicates that this stain has a greenish hue), have augmented the original Coomassie stain. Coomassie Blue (3-250 has a diminished solubility in 12% TCA, permitting its use as a colloidal dispersion which does not E . L. Durrum, M. H . Paul, and E . R. B. Smith, Science 116, 428 (1952). E. Koiw and A. Gronwell, Scand. J . Clin. Lab Invest. 4 , 244 (1952). B. 0. Barger, F. C . White, J . L . Pace, D. L. Kemper, and W . L. Ragland,Anal. Biochem. 70, 327 (1976). ' H . F. Bosshard and A. Datyner, Anal. Biochem. 82, 327 (1977). C . R . Menil, R. C . Switzer, and M. L. Van Keuren, Proc. Natl. Acad. Sci. U . S . A . 76, 4335 (1979). R. C. Switzer, C . R. Merril, and S. Shifrin, Anal. Biochem. 98, 231 (1979). lo S. Fazekas de St. Groth, R . G . Webster, and A. Datyner, Biochim. Biophys. Acta 71,377 (1963). T. S. Meyer and B . L. Lamberts, Biochim. Biophys. Acta 107, 144 (1965).

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penetrate gels. This property permits rapid staining of proteins without an undesired background.I2 Another Coomassie stain, Coomassie Violet R-150, has gained some favor by virtue of its ability to rapidly stain proteins on polyacrylamide gels while not staining carrier ampholytes, and for its ease in destaining.l3-l4Coomassie dyes are no longer made by Imperial Chemical Industries and they are now often sold under a number of different trade names. General Coomassie Staining Method

Gels are stained immediately after electrophoresis in a solution containing 50% (vlv) methanol, 10% (vlv) acetic acid, and 0.25% (wlv) Coomassie Blue for 3 hr. This solution should be filtered (Whatman No. 1) prior to use. Gels are destained overnight in a solution containing 5% (vlv) acetic acid and 10% methanol. The destaining solution must be changed repeatedly, or alternatively it may be pumped continuously through a felt filter. The felt clarifies the destaining solution by binding the Coomassie stain as it diffuses out of the gels. Rapid Coomassie Stain for Isoelectric Focusing Gels

A simple method for staining gels which do not contain sodium dodecyl sulfate utilizes a 6% (wlv) perchloric acid solution containing 0.04% (w/v) of Coomassie G-250. Dense protein bands or spots stain an intense blue and can often be observed within less than a minute. The background stains a pale orange. Less dense proteins can usually be visualized within 90 min. A 3-fold increase in sensitivity can be achieved by placing the gel in 5% (vlv) acetic acid. The background changes to pale blue in the acetic acid. l5 Properties of Coomassie Blue Stains

Coomassie Blue Stain Binding

Coomassie Blue staining requires an acidic medium for the generation of an electrostatic attraction between the dye molecules and the amino groups of the proteins. This ionic attraction, together with van der Waals forces, binds the dye-protein complex together. The binding is fully reversible by dilution under appropriate conditions.1° Polypeptides rich in l2 l3

l4

W. Diezel, G . Kopperschlager, and E. Hofman, Anal. Biochem. 48, 617 (1972). R. Frater, J. Chromatogr. 50, 469 (1970). B. J. Radola, Electrophoresis 1, 43 (1980). A. H. Reisner, this series, Vol. 104, p. 439.

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lysine and arginine are aggregated by Coomassie G dye molecules, suggesting that the dye interacts with the basic groups in the polypeptides.I6 Studies of proteins with known sequences have confirmed these observations and demonstrated a significant correlation between the intensity of Coomassie Blue staining and the number of lysine, histidine, and arginine residues in the protein." Coomassie Blue stains exhibit three times the staining intensity of Fast Green and six times the intensity of Amido Black.I8 The staining intensities of these dyes is approximately proportional to their relative molar absorption coefficients. One milligram of protein will bind 0.17 rng of Amido Black, 0.23 mg of Fast Green, 1.2 mg of Coomassie Blue R-250, and 1.4 mg of Coomassie Blue G-250.19 Since the molecular weights of these dyes vary by only 1.4-fold these 5- to 8-fold variations in dye binding are most likely due to differences in the number of dye molecules bound per protein molecule. The higher staining intensity of Coomassie Blue may also be due to its higher efficiency at forming dye-dye interactions. Secondary binding mechanisms may also occur with Amido Black and Fast Green dyes, although perhaps not at the levels observed with Coomassie Blue R-250, as these dyes display metachromatic effects with certain proteins similar to the metachromasy observed with Coomassie Blue R-250. Amido Black produces blue-green bands with certain histones rather than its characteristic blue-black color, while Fast Green produces a difference in the ratio of blue to green hues.20Collagen and histones often produce redstaining bands or spots with Coomassie Blue.21These metachromatic effects are dependent on temperature, concentrations, and the solvents in the gel. ~~~ Coomassie stains give a linear response up to 20 , ~ g i c m . l OHowever, the relationship between stain density and protein concentration varies for each protein.I0 Preelectrophoretic Fluorescent Stains

Fluorescent stains usually involve the covalent binding of a fluorescent residue to the protein prior to electrophoresis. The advantages of this type of stain include the possibility of performing stoichiometric reactions Ib

l7 l8

l9

" Z'

P. G. Righetti and F. Chillemi, J. Chromatogr. 157, 243 (1978). M. Tal, A. Silberstein, and E. Nusser, J. Biol. Chem. 260,9976 (1985). C. M. Wilson, this series, Vol. 91, p. 236. C. M. Wilson, Anal. Biochem. 96, 236 (1979). R. McMaster-Kaye and J. S . Kaye, Anal. Biochem. 61, 120 (1974). R. C. Duhamel, E. Meezan, and K. Brendel, Biochim. Biophys. Acta 626, 432 (1980).

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GEL-STAINING TECHNIQUES

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with proteins without the diffusion limitations imposed by staining within a gel matrix, the feasibility of following the process of electrophoresis visually with "stained" proteins, and the absence of background problems due to dye trapping or reaction of the dye with the gel. These advantages may be offset in some applications by the alterations in the charge of the proteins, unless an amphoteric stain is employed.' This objection is not of consequence for sodium dodecyl sulfate (SDS) electrophoresis, as the mobility of the protein depends solely on molecular weight with this method, and the dye molecules are usually too small to produce an appreciable size effect. However, the fluorescent stains require ultraviolet light for visualization, and direct quantitation requires fairly sophisticated equipment. Currently fluorescent stains, such as fluorescamine, are the most sensitive preelectrophoretic stains. At room temperature and alkaline pH, fluorescamine can react with the primary amines of the protein to yield a fluorescent derivative. This stain has proved capable of detecting as little as 6 ng of r n y ~ g l o b i n . ~A ~ ,related *~ compound, 2-methoxy-2,4-diphenyl3(2H)-furanone (MDPF), has the same speed and simplicity of reaction as fluorescamine, while its protein derivative is 2.5 times as fluorescent as a fluorescamine-labeled protein. Furthermore, its fluorescent derivative does not fade as rapidly. As little as 1 ng of protein has been detected with MDPF. This stain has a linear response from 1 to 500 ng. As with most other protein stains, a plot of relative fluorescence versus protein concentration reveals a different slope for each protein6 General Fluorescence Staining Method

To label proteins with fluorescamine or MDPF, first add 50 p1 of 0.2 M borate (pH 9.0) buffer to a protein solution containing 50 to 100 pg of protein. Then add, with vortex mixing, 30 p1 of MDPF or fluorescamine stock solution. (The fluorescamine or MDPF stock solution contains 2 mg of stain dissolved in 1 ml of acetone.) Continue the mixing for about 1 min. The proteins will be labeled within this time. There is no need to remove the remaining unreacted stain reagent as it is not fluorescent and it degrades rapidly in water. It also does not interfere with the electrophoretic separation of the labeled proteins. Although maximal fluorescence of the labeled proteins is obtained at pH 8-8.5, fluorescence can be detected over a wider pH range. W. L. Ragland, J. L. Pace, and D. L. Kemper, Anal. Biochem. 59, 24 (1974). J . L. Pace, D. L. Kemper, and W. L. Ragland, Biochem. Biophys. Res. Commun. 57,482 (1974).

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Silver Staining

Studies in which the purity of a protein is critical, or in which there is a need to monitor trace proteins, should employ the highly sensitive silver stains. Silver stain protocols can be divided into three basic categories: the diamine or ammoniacal silver stains, the nondiamine chemical development silver stains, and the photoreduction silver stains. The diamine or ammoniacal silver stains have proved to be particularly good for the staining of proteins separated in gels thicker than 1 mm. The nondiamine chemical development stains are generally more rapid than the diamine stains and they work best with I-mm or thinner gels. The photoreduction silver stains are the most rapid, but they currently lack the sensitivity of the other silver stain methods. Diamine Silver Stains

These stains rely on the stabilization of the silver ions by the formation of silver diamine complexes with ammonium hydroxide. Silver ion concentrations are usually very low in these stains, as most of the silver is bound in the diamine complexes.24In these diamine stains, the ammoniacal silver solution must be acidified, usually with citric acid, for image production to occur. The addition of citric acid lowers the concentration of free ammonium ions, thereby liberating silver ions to a level where their reduction by formaldehyde to metallic silver is possible. The optimal concentration of citric acid results in a controlled rate of silver ion reduction, preventing the nonselective deposition of silver. Diamine Staining Method

Gels are washed for 5 min in deionized water and then placed in a solution containing 5% (vlv) ethanol, 5% (vlv) acetic acid, and deionized water for 3 hr. The deionized water used to make these solutions should have a conductivity of less than 1 mholcm. The gels may be stored in this solution overnight prior to staining. The gels are then washed with deionized water for 5 min and then soaked for 30 min in a 10% (vlv) glutaraldehyde solution. The unreacted glutaraldehyde is removed by five 30-min washes with deionized water. These glutaraldehyde-treated gels are then soaked in an ammoniacal silver nitrate solution for 10 min. The ammoniacal silver nitrate solution is prepared by slowly adding, with stirring, 30 ml of a 1.2 M silver nitrate solution to a solution containing 10 ml of concentrated ammonium hydroxide and 1.5 ml of 10 N sodium hydroxide in 160 ml of deionized water. After the silver nitrate is dissolved the final volume is adjusted to 750 ml. 24

W. .I. H. Nauta and P. A. Gygax, Stain Technol. 26, 5 (1951).

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The gels are removed from the ammoniacal silver nitrate and treated with three 5-min deionized water washes. The image is developed with a solution containing 0.1 g of citric acid and 1 ml of formaldehyde (37% commercial formaldehyde) per liter of deionized water. When the image is sufficiently developed, usually about 3 min, the reaction is stopped by placing the gels in solution containing 5% (vlv) acetic acid. The gels are then washed in a 10% (vlv) ethanol solution. If the gels are left in a solution containing acetic acid some of the trace bands or spots will be lost. Gels are stored in a 7% (v/v) glycerol, 10% (viv) ethanol solution. Diamine stains tend to become selectively sensitive for glycoproteins if the concentration of silver ions is maintained at a low level during image development. This specificity can be minimized by maintaining a sufficient sodium-to-ammonium ion ratio in the diamine solution.25However, in some applications, an emphasis on the specificity of the diamine stain has proved useful, as in the adaptation of a diarnine histological silver stain to visualize neurofilament polypeptides in electrophoretic analyses of spinal cord h ~ m o g e n a t e s . ? ~

Nondiamine Chemical Development Silver Stains These stains are relatively simple and rapid. They rely on the reaction of silver nitrate with protein sites under acidic conditions, followed by the selective reduction of silver ion to metallic silver by formaldehyde under alkaline conditions. Sodium carbonate and/or hydroxide and other bases are used to maintain an alkaline pH during development. Formic acid, produced by the oxidation of formaldehyde, is buffered by the sodium carbonate.

Nondiamine Staining Method Gels are fixed for 20 min in a solution containing 50% (vlv) methanol, 10% (vlv) acetic acid, and deionized water. The gels may be stored in this solution overnight prior to staining. The gels are then washed for 30 min in asolution containing 10% (viv) methanol, 5% (viv) acetic acid, and deionized water. These gels are then soaked in a 3.4 mM potassium dichromate solution containing 3.2 mM nitric acid for 5 min. The gels are then rinsed with deionized water and placed in 12 mM silver nitrate for 20 min. Image development is achieved by rinsing the gels with agitation in 0.28 M sodium carbonate containing 0.5 ml formaldehyde (37% commercial formaldehyde) per liter of deionized water. This step requires at least two changes of the solution to prevent precipitated silver salts from adsorbing

26

R. C. Allen, Electrophoresis 1, 32 (1980). P. Gambetti, L. Autilio-Gambetti, and S. C. H. Papasozomenos, Science 213,1521 (1981).

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-

to the surface of the gel. The pH of the gel is made alkaline so that the formaldehyde can reduce ionic silver to metallic silver. Image development is stopped when a slightly yellowish background appears by placing the gel in a 3% (vlv) acetic acid solution for 5 min. The gels are then washed in a 10% (vlv) ethanol solution. If the gels are left in acetic acid some of the trace bands or spots will be lost. Gels are stored in a solution containing 7% (vlv) glycerol and 10% (vlv) ethanol. Photodevelopment Silver Stains

The use of photoreduction provides for a rapid, simple, staining method for the detection of proteins. It permits the visualization of protein patterns within 10 min after an electrophoretic separation. However, the method currently lacks the sensitivity of the other silver staining methods and it should be reserved for studies of dense protein bands or spot^.^',^^ Photodevelopment stains utilize energy from photons of light to reduce ionic to metallic silver. Photodevelopment Silver Staining Method

Gels are fixed for 5 min in a solution containing 50% (vlv) methanol, 10% (vlv) acetic acid, 2% (wlv) citric acid, and 2% (wlv) sodium chloride in deionized water. The gels are rinsed briefly with deionized water to remove surface chloride and placed in a solution containing 50% (vlv) methanol, 10% (vlv) acetic acid, and 2% (wlv) silver nitrate. The gels immersed in this solution are then transilluminated by placing them 2.5 cm above a uniform fluorescent light source (a 160-W fluorescent grid lamp with a clear Lucite diffusion screen) until an image appears. Image development may be stopped at any time by placing the gel in the dark. Image preservation, which is very good with the other silver-staining methods, is difficult with this photodevelopment silver stain. Archival storage can only be achieved by photographing these gels. Properties of Silver Stains

Silver Stain Reactive Groups

Amino acid homopolymers, individual amino acids, and peptides of known sequence have been studied to gain information about reactive groups that may be involved in the silver-staining reaction^.^^ The consen27

l9

C. R. M e m l and M. G. Harrington, Clin. Chem. 30, 1938 (1984). C. R. M e m l , M. Harrington, and V. Alley, Electrophoresis 5, 289 (1984). C. R. Menil and M. E. Pratt, Anal. Biochem. 117, 307 (1986).

F

-

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sus findings of these studies indicate that the principal reactive groups are the free amines and the sulfur groups contained on the proteins.3G32The importance of the basic and the sulfur-containing amino acids has been corroborated by observations with purified peptides and proteins of known amino acid sequence.29 The importance of the basic amino acids has been further substantiated by evaluations of the relationship between the amino acid mole percentages of proteins and their ability to stain with silver. The best correlations are achieved when comparisons are made between the slope of the staining curve of a denatured protein and that protein's mole percentage of basic amino acids.29 Color Effects with Silver Stains

Ii

r

Most proteins stain with monochromatic brown or black colors. However, silver stains can produce other colors. Lipoproteins tend to stain blue while some glycoproteins appear yellow, brown or red.33This color effect has been demonstrated to be due to the diffractive scattering of light by the microscopic silver grains. A pronounced and reproducible dependence of color and silver-grain size has been observed.34Modifications of the silver-staining procedures, such as lowering the concentration of reducing agent in the image development solution, prolonging the development time, adding alkali, or elevating the temperature during staining, often enhance color formation. Some silver stain protocols have been developed to produce colors that may aid in the identification of certain protein^.^^,^^,^^ Combinations of stains may also be employed for protein identification. In a study of erythrocyte membrane proteins, sialoglycoproteins and lipids were stained yellow with a silver stain, while other membrane proteins counterstained with Coomassie Blue.37 C. Freeman, in "Inorganic Biochemistry" (G. L. Eichhorn, ed.), Vol. I . p. 121. Elsevier, Amsterdam, 1973. 31 J. Heukeshoven and R. Dernick, Electrophoresis 6, 103 (1985). 32 B. L. Nielsen and L. R. Brown, Anal. Biochem. 144, 311 (1984). D. Goldman, C. R. Merril, and M. H. Ebert, Clin. Chem. 26, 1317 (1980). C. R. Menil, M. E. Bisher, M. Harrington, and A. C. Steven, Proc. Natl. Acad. Sci. U.S.A.85,453 (1988). " D. W. Sammons, L. D. Adams, and E. E. Nishizawa, Elecrrophoresis 2, 135 (1981). D. W. Sammons, L. D. Adams, T. J. Vidmar, A. Hatfield, D. H. Jones, P. J. Chuba, and S. W. Crooks, in "Two-Dimensional Gel Electrophoresis of Proteins" (J. E. Celis and R. Bravo, eds.), p. 112. Academic Press, New York, 1984. " J. K. Dzandu, M. H. Deh. D . L. Barratt, and G. E. Wise, Proc. Natl. Acad. Sci. U.S.A. 81, 1733 (1984). 10 H.

"

"

486

PURIFICATION

PROCEDURES: ELECTROPHORETIC

METHODS

[361

Protein-SpeciJic Silver Stains

Silver stains can demonstrate considerable specificity. Stains specific for nucleolar proteins38 and neurofilament polypeptidesz6have been described. Many silver stain protocols detect not only proteins but also lipop01ysaccharides,~~ and polysa~charides.~~ All silver stains do not detect all proteins. It is difficult to stain calmodulin and troponin C with some silver stain protocols. However, pretreatment of these proteins with glutaraldehyde appears to enhance their ability to stain.44Some histones may also fail to stain with silver. Fixation with formaldehyde coupled with the simultaneous prestaining of these proteins with Coomassie Blue has been reported to partially alleviate this problem. However, even with this procedure the sensitivity for histones is reported to be decreased 10-fold when compared with the detection of neutral proteins.45Another example of differential sensitivity has been demonstrated in a study utilizing four different silver stain protocols to stain salivary proteins. Different protein bands were visualized with each of the stains.46 Silver Stain Quenching of Autoradiography

Quenching of 14C-labeled proteins is minimal with most nondiamine silver stains. Even the most intense diamine-stained radioactive proteins can be detected by autoradiography with only a 50% decrease in image density. This loss of autoradiographic sensitivity can generally be compensated for by longer film exposures. However, detection of 3H-labeled proteins is severely quenched by all silver stains. Destaining of silver-stained gel with photographic reducing agents can often permit detection of as much as half of the fluorographic density of 3H-labeled proteins, providing that the initial staining was performed with a nondiamine silver stain. Many diamine stains continue to quench, even after treatment with photographic reducing agents, so that fluorographic detection of 3H-labeled proteins is not feasible with the diamine stains. This impediment to 3H detection with diamine stains is likely to be due to a greater amount of residual silver deposited throughout the gels by these stains, which block the weak /3 emissions from 3H. H. R. Hubbell, L. I. Rothblum, and T. C. Hsu, Cell Biol. Znr. Rep. 3, 615 (1979). L. L. Somerville and K. Wang, Biochem. Biophys. Res. Commun. 10,53 (1981). T. Boulikas and R. J. Hancock, Biochem. Biophys. Methods 5, 219 (1981). 4 ' D. Goldman and C. R. Menil, Electrophoresis 3, 24 (1982). 42 C. M. Tsai and C. E. Frasch, Anal. Biochem. 119, 115 (1982). 43 G. Dubray and G. Bezard, Anal. Biochem. 119, 325 (1982). " M. Schleicher and D. M. Watterson, Anal. Biochem. 131, 312 (1983). 45 S. Irie and M. Sezaki, Anal. Biochem. 134, 471 (1983). 46 R. D. Friedman, Anal. Biochem. U6,346 (1982). 38

39

[361

GEL-STAINING TECHNIQUES

--

487

Residual silver has been demonstrated in gels that have been "cleared" by photographic reducing agents, by the reappearance of a faint silver image of the proteins in "cleared" gels which are dried with heat. Silver has also been demonstrated in these "cleared" gels by electron beam analysis4' Common Staining Artifact and Background Staining

Artifactual bands with molecular weights ranging from 50K to 68K have been commonly observed in silver-stained gels. Evidence has been presented indicating that these contaminating bands are due to keratin skin proteins.48 Background staining has been demonstrated to be due in part to the chemistry of the polyacrylamide gels. Preliminary experiments indicate that alterations in the chemistry of the polyacrylamide gels may result in reduced background staining.49 Quantitation with Protein Stains Quantitation Using Organic Stains

An accuracy of plus or minus 10% in measuring the concentrations of proteins in the range of 0.5-20 pg was reported with the introduction of the Coomassie Blue R-250 stain.I0 However, while individual proteins displayed linear relationships between absorbance and concentration within this protein range, the slopes differed for each protein. This variation in Coomassie Blue staining now appears to be related to the mole percent of the basic amino acids in the protein.'' Therefore, a standard curve must be produced for each protein assayed and quantitative comparisons limited to equivalent protein spots or bands. Quantitation Using Silver Stains

Most silver stain protocols provide a reproducible relationship between silver stain density and protein concentration. The linear portion of this relationship generally extends over a 40-fold range in concentration, Protein concentrations greater than beginning at 0.02 ng/mm2.16.27s50,51 41

M. L. Van Keuren, D. Goldman, and C. R . Merril, Anal. Biochem. 116, 248 (1981).

" D. Ochs, Anal. Biochem. 135,470 (1983). 49

5'

D. F. Hochstrasser, A. Patchomik, and C. R. Menil, Anal. Biochem. 173, 412 (1988). C. R. Menil, D. Goldman, and M. L. Van Keuren, Electrophoresis 3, 17 (1982). C. R. Merril and D. Goldman, in "Two-Dimensional Gel Electrophoresis of Proteins" ( J . E. Celis and R. Bravo, eds.), p. 93. Academic Press, New York, 1984.

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PURIFICATION PROCEDURES: ELECTROPHORETIC METHODS

[371

2 ng/mm2 generally cause saturation of silver images, resulting in nonlinearity above that concentration. In a manner analogous to that observed with the organic stains, the relation between the silver stain densities and the protein concentrations varies for each Protein-specific staining curves have also been observed with most protein assays (see [6] in this volume). The observation that each protein produces a unique density versus concentration curve in these studies illustrates the dependence of the staining reaction on a specific reactive group(s) contained in each protein. The importance of the basic amino acids, particularly lysine and histidine, for both the silver stains and the Coomassie Blue stains indicates the need for a careful choice of "standard protein(~)." If a protein containing an abnormally large number of stainreactive groups is chosen as a standard it will produce a curve which would tend to underestimate the concentration of proteins containing normal numbers of reactive groups." Alternatively, in intergel comparisons only equivalent spots offer valid quantitative comparisons. [Editor's note: See [33] and [35] in this volume for additional information on gel staining.]

[371 Elution of Protein from Gels By MICHAELG . HARRINGTON

Electrophoretic separation of proteins in various types of polyacrylamide gels is employed from the analytical to the preparative scale. After separation, it is frequently necessary to extract, or elute, a specific protein from the gel for further study: this might include amino acid composition or sequence analysis, or partial enzyme or chemical digestion. For optimal efficiency of elution, it is desirable to have a simple technique that successfully extracts all protein from the gel and avoids any additional chemical modification to the protein. The diffusion method of elution involves agitation of the gel fragments in a sodium dodecyl sulfate (SDS) solution. This approach is simple, takes 3-12 hr, but is less efficient than electroelution, and will not be discussed further. Electroelution is more controlled than diffusive elution, and can be performed either during or after electrophoresis. The author's limited experience with elution during electrophoresis precludes further description of what is a less flexible method. The following detailed laboratory procedure pertains to the identification in the gel of a specific protein and the subsequent elution of that Copyright O 1990 by Academic Press, Inc.

METHODS IN ENZYMOLOGY,VOL. 182

All rights of reproduction in any form reserved.

[371

ELUTION OF PROTEIN FROM GELS

489

protein: this has been applied after isoelectric focusing, SDS-polyacrylamide gel electrophoresis (PAGE), and high-resolution two-dimensional electrophoresis (2DE). Quantities ranging from hundreds of picograms to milligrams of varied proteins have been extracted, and studies after elution have included amino acid sequence analysis. In recent years, electrotransfer of proteins that are separated in an electrophoretic procedure onto nitrocellulose or poly(viny1idine difluoride) (PVDF) membranes has become a frequent part of protein research. These membrane-trapped proteins can have contaminant salts and detergents directly removed by washing. While certain procedures can then be performed on the membrane, it may be necessary to recover the protein in solution. Therefore, a brief description of elution from these matrices is also included. Materials and Methods

Chemicals and Equipment. Electrophoresis-grade acrylamide, N,Nfmethylenebisacrylamide, N, N, N, N'-tetramethylenediamine, ammonium persulfate, dithiothreitol, Bromphenol Blue, Coomassie Brilliant Blue R-250, SDS, tris(hydroxymethy1)aminomethane (Tris), 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), urea, glycine, ampholytes, prestained molecular weight and pZ standards were all from Bio-Rad (Richmond, CA), as were the electrophoresis and electroelution cells. Alternative carrier ampholytes can be obtained from Pharmacial LKB (Pleasant Hill, CA), Serva (Garden City Park, NY), or BDH (distributed by Hoefer, San Francisco, CA), and Immobilines from LKB (Pleasant Hill, CA). Alternative electroelution cells can be obtained from Schleicher and Schuell (Elutrap, Keene, NH), Isco (model 1750, Lincoln, NE), or the California Institute of Technology (F. Ostrander, Pasadena, CA). Amido Black IOB, Ponceau S, and Nonidet P-40 were from Sigma (St. Louis, MO); sec-butanol, glycerol, NH4HC03 from Mallinckrodt (Paris, KY); N-ethylmorpholine from Aldrich Chemicals (Milwaukee, WI); trifluoroacetic acid (TFA) (Sequanal quality) and acetonitrile (HPLCISpectro grade) were from Pierce (Rockford, IL). Diacrylylpiperazine was synthesized as described1 or obtained from Bio-Rad. All equipment was washed with warm soap and water, followed by thorough rinsing with distilled water, and all reagent water was deionized and glass distilled. Electrophoresis. Proteins separated after SDS-PAGE, IEF in immobilized pH gradients, and 2DE are described elsewhere in this volume

' D. F. Hochstrasser, A. Patchornik. and C. R. Merril, Anal. Biochem. 173, 412 (1988).

490

PURIFICATION PROCEDURES: ELECTROPHORETIC METHODS

[371

([33]-[35]). Aspects of electrophoresis that particularly influence the successful elution and sequence analysis of proteins include the following: Good purity of reagents is essential for optimal results and the reagents should preferably be purchased from the suppliers listed above. In addition, further contamination of proteins or reagents should be kept to a minimum by the regular use of disposable gloves (rinsed to remove any talc) and lab coats. The time of all protein manipulations must be kept to a minimum to reduce enzymatic digestion and chemical modification. Thus, after preparing the protein mixture or thawing it from storage at -70°, the proteins should be denatured with dithiothreitol(0.2% wlv), and SDS (1% wlv), and heated at 95" for 5 min. This sample, on cooling, should have any other additives combined and be directly applied to the gel for electrophoresis. Gels should be polymerized for at least 12 hr before use and should contain the minimum amount of catalysts, polyacrylamide, and cross-linker that still allows good resolution of proteins. It is important to have a high ratio of protein to gel. For one-dimensional SDS-PAGE, a stacking gel is required in order to allow concentration of any large sample volume. Prerunning the gel with or without sodium glycolate may be considered in order to scavenge free radical^,^.^ but this author has not detected benefit with this additional procedure. A recent procedure for high-resolution 2DE has been described by Hochstrasser et and three points are worth emphasizing. First, the new cross-linker,' diacrylylpiperazine, improved resolution especially of large basic proteins, afforded more resilient gels, and led to an approximately 2-fold increase in yield of amino acid sequence signal compared to the more frequently used bisacry1amide.j Second, with small capillary tube gels (1.5-mm i.d. or less) the transfer of first to second dimension gels can be accomplished with minimal or no SDS equilibration, thus minimizing loss from premature elution of proteins. Third, the temperature of the SDS-PAGE gel buffer should be controlled on both sides of the gel (optimal 10 k 2") to limit diffusion of proteins. Postelectrophoretic Detection of Proteins

One-Dimensional Gels. Analytical electrophoresis allows the initial parameters for separation of the protein of interest to be optimized. Then M. W. Hunkapiller, E. Lujan, F. Ostrander, and L. E. Hood, this series, Vol. 91, p. 227. M. Moos, Jr., N . Y. Nguyen, and T.-Y. Liu, J . Biol. Chem. 263, 6005 (1988). D. F. Hochstrasser, M. G . Hamngton, A. C. Hochstrasser, M. J. Miller, and C. R. Merril, Anal. Biochem. 173, 424 (1988). B. Martin, M. G. Hamngton, D. F. Hochstrasser, A. C. Hochstrasser, and C. R . Meml, in preparation (1989).

[371

ELUTION OF PROTEIN FROM GELS

Analytical Gel

491

Preparative Gel

;k Pi@:;

$?

-

FIG.1 . Strategy for identifying proteins for elution. The analytical SDS-PAGE gel on the left is stained so that locations of specific proteins in lane B are visualized with respect to the prestained molecular weight markers in lanes A and C. Staining can be either a chemical directly on the gel (see [36]), or it can be an immunostain with a specific antibody of electroblotted proteins on a nitrocellulose or PVDF membrane (see [52]), or autoradiography can be performed. The equivalent region of lane B on the preparative gel can be located and excised. The same procedure can be utilized in IEF gels using prestained IEF markers.

a preparative gel is run with prestained markers on both sides of the sample of interest and the protein of interest is excised, without staining, immediately after electrophoresis (Fig. 1). Two-Dimensional Gels. There is minimal, but significant, gel-to-gel variation on 2DE, and it is, therefore, necessary to stain the spot of interest on the preparative gel before excising it. An initial analytical 2DE gel is used as a reference for the spot of interest and its surrounding neighbors. Then one of two methods may be employed: The protein in the preparative gel may be stained with Coomassie Blue (see [36] in this volume) immediately after separation, the spot of interest identified by its relative position to other neighbor proteins in the gel, and excised. Proteins can also be eluted after silver staining in the presence of SDS, but the resultant protein contains material that interferes with subsequent sequence analysis. An alternative approach is to electrotransfer the 2DE gel proteins onto either nitrocellulose or PVDF matrices. This can be performed in a Bio-Rad Transblot cell, with 15 m M Tris, 192 mM glycine, 20% methanol for 3 hr at 200 mA constant current at room temperature. The entire matrix can then be stained very rapidly, and the spot of interest excised for elution. The stain of choice is Ponceau S (0.1% w/v in 1% vlv

492

PURIFICATION

PROCEDURES: ELECTROPHORETIC

1371

METHODS

Frit

Adaptor

1 L

Elutrap

I/

Model 422

1 I

FIG.2. Diagrams of two simple electroelution devices. The Elutrap, seen from above, is placed on a horizontal agarose gel electrophoresis chamber. Outer limits of the trap are defined with disposable membranes at A and G that have a pore size limiting molecule transit to less than 5000 Da. The gel is contained between B and F with a large pore-size membrane, or between C and F if the gel volume is small. The eluted protein collects between F and G (500 pl). The Bio-Rad model 422 elution cell, seen from the side, can trap 500 p1 of protein in the membrane cap: two sizes of dialysis membrane pore size are available, trapping molecular weights above either 3500 or 12,000-15,000. The elution unit can accommodate up to six tubes, each 1 x 6 cm long.

acetic acid) for 30 sec, followed by destaining in water for a second or two. A distinct spot detected in this manner (5-50 pmol) can normally be processed directly for sequence analysis. A 2- to 10-fold greater detection sensitivity can be achieved with Amido Black 10B (0.2% in water, filtered) for 10 min, destained in water for about half a minute. Electroelution. Good results are obtained with all four types of apparatus listed above, but the two most efficient and simple are the Schleicher and Schuell Elutrap6 and the Bio-Rad 422 model (Fig. 2). Elutrap. The apparatus is cleaned and set up as in Fig. 2, and membranes are tested with water for any leaks. The water is removed immediately prior to use. The polyacrylamide gel, containing the protein of interest, is cut into pieces approximately 1-5 mm2 and placed in the elution chamber. A small gel piece can fit into a miniature chamber, between membranes at C and F (Fig. 2), whereas a larger quantity of gel is placed between membranes at B and F (Fig. 2). Elution buffer is added to cover the gel pieces, and to the reservoirs of the electrophoresis chamber, which are filled to the same height as the contents of the Elutrap. The elution buffer composition can be varied, but if one does not know the ease of elution of the protein of interest, then it is best to start with 0.1% SDS in either a volatile basic buffer [50 mM NH4HC03 or 50 mM NE. Jacobs and A. Clad, Anal. Biochem. 154,583 (1986).

ELUTION OF PROTEIN FROM GELS

t i

r: r:

r:

493

ethylmorpholine acetate (pH 8.5)], or in Tris (25 mM)-glycine (192 mM) buffer (pH 8.5). Volatile buffers should be made up freshly, and have the advantage of being more easily removed from the eluted protein. Running conditions vary, being longer for larger proteins (over M, 100,000), thicker gels (over 1.5 mm), and heavier protein loads (milligrams). Two-hundred volts (approximately 70 mA) is usual, for 4 hr at room temperature (20"). At this stage 75-100% of the protein is in the space between F and G and can be removed by direct aspiration immediately after switching off the power. Fresh elution buffer (200 p1) is used to rinse the chamber and is added to the sample. If it is desirable to reduce the amount of SDS in the sample, electrodialysis can be performed as follows: the buffer in the reservoir is removed and replaced with fresh buffer containing 0.001% SDS. This is best run for 2 hr and repeated for another hour with fresh buffer, or run overnight at reduced voltage (100 V). Advantages of this method are that all parts are available commercially at reasonable cost and good quality, the system is simple to use, and very accessible with the open channels on top of the chamber. Particulate matter greater than 2 p m is effectively excluded from the sample collection well by the large pore membrane at position F (Fig. 2). The horizontal array of channels also makes it very unlikely that air bubbles will create any problem, a concern with the vertical units. Bio-Rad Model 422 Electroeluter. The assembly is prepared 1-2 hr before use (Fig. 2). The membrane cap, of pore size appropriate for retaining the protein of interest, is soaked in elution buffer for 1 hr. The glass tube units are assembled by inserting the frit flush with the base of the tube and then inserting the glass tubes in the grommets of the electroelution module. Any open grommet holes are filled with stoppers. The membrane cap is then installed in the bottom of each silicon adaptor and flushed with buffer to remove any air bubbles. The cap and adaptor are connected to the bottom of the glass tube and agitated to remove all air bubbles that tend to collect at the frit. Each tube is filled with elution buffer and checked for leakage of the device. The module is then inserted into the buffer chamber. Elution buffers are selected, as above, with volatile solutions preferred. Again, unless the protein is known to elute well without SDS, it is best to include 0.1% SDS initially, and later to electrodialyze with 0.001% SDS. Elution buffer is added to the lower reservoir to comfortably cover the silicon adaptor, a magnetic stir bar is added, and the apparatus is stirred vigorously to prevent bubbles sticking to the dialysis membrane. The gel with the protein of interest is then cut into approximately 1- to 5-mm2pieces and these are placed in each tube, but not higher than halfway up the tube. Upper buffer is used to cover the tubes and elution is started at 8-10 mA/tube for 4 hr. Most proteins (75-

494

PURIFICATION PROCEDURES: ELECTROPHORETlC METHODS

[371

100%) will elute in this time. Electrodialysis can be performed to reduce SDS exactly as described above. After elution the upper buffer is removed and aspirated from the glass tube down to the level of the frit, taking care not to agitate the protein solution that has collected in the membrane cap. The adaptor and cap are then removed and the liquid carefully aspirated. Fresh elution buffer (200 p1) is used to rinse the cap and this material is added to the sample. Elution from NitrocelluloselPVDF Membranes. The excised protein on a membrane is stained and destained briefly with Ponceau S, as described above, placed in an Eppendorf tube, and sufficient elution solution added to submerge the membrane (about 500 pl). Care is taken to avoid drying of the membrane, which makes subsequent elution incomplete. The selection of elution solution is intended to counteract the ionic bonding between the protein and the membrane. This clearly depends on the matrix, the amino acid sequence, and the size of the protein. Generally, it has been found that elution is more efficient from nitrocellulose than PVDF membranes, and small proteins elute faster than large ones. As in gel elution, nonvolatile and volatile elution solutions may be used. A useful nonvolatile solution is 50 mM Tris-HC1, pH 9.0, containing 2% SDS ( w l v ) and 1% Triton X-100 (v/v).' A volatile cocktail is 30% TFA, 40% acetonitrile. After addition of the eluant, the Eppendorf tube is rotated at room temperature for 1 hr, centrifuged at 10,000 g for 10 min, and the supernatant solution is removed. The tube and membrane are then rinsed with 250 p1 of fresh elution buffer, centrifuged again for 5 min, and the supernatant material retained. Postelectroelution Assay. A small quantity of the eluted protein should be assayed on an analytical gel to assess its purity and concentration. Even if there is only a microgram of protein, 10% of this on a silverstained gel will confirm purity, and give an estimate of quantity. Comments. With care applied to the electrophoresis and elution procedures, the yields mentioned above are quite reproducible for a specific protein, but variation does occur between different proteins. Because of the possibility that a previously unknown protein is resistant to transfer, it is always wise to preserve the gellmembrane until the analytical gel of the eluted protein has been stained. If the result of this demonstrates a poor yield, it may still be possible to try an alternative procedure to salvage a "precious" sample. The use of the cross-linker, diacrylylpiperazine, appears advantageous over bisacrylamide both for separation of proteins and for the sequence yield after 2DE. While the mechanism for these effects is not B. Szewczyk and D. F. Summers, Anal. Biochem. 168,48 (1988).

[371

ELUTION OF PROTEIN FROM GELS

495

clear, it is known that bisacrylamide is unstable and breaks down to formaldehyde, a phenomenon to be avoided. Commercially, diacrylylpiperazine is now available from Bio-Rad as piperazine diacrylamide. Results of elution using either the Isco or Caltech apparatus mentioned above also give a good yield of protein. However, the Isco system has a less stable trap for eluted protein than the two systems described in detail, and it is easy to accidentally lose protein after elution. The Caltech design is quite tricky to set up and operate. As neither system appears to have any advantage over the Elutrap or Bio-Rad eluter, the author does not recommend their use. Amino acid sequence analysis using an automated gas phase sequenato? of both amino terminal segments and products of tryptic digestion of proteins eluted in the manner described above have been successful. Accurate assignments of between 10 and 25 amino acids have been made from quantities ranging from 5-50 pmol of both known and unknown proteins, ranging in size from 3000 to 90,000 Da. It should be recognized that microsequence studies with low microgram quantities are still difficult for certain proteins. Elution directly from the gel may lead to unacceptable purity or poor yield of the protein. In addition to the direct transfer of protein to PVDF membrane, an alternative has recently been described that affords covalent immobilization on glass fiber filters. This has the potential for enabling more rigourous chemistry during sequence analysi~.~ Acknowledgments The author is grateful for the advice and support of D. F. Hochstrasser, B. Martin, and C. R. Merril at NIMH, Bethesda, Maryland, and R. H. Aebersold, G. Pipes. and L. Hood at Caltech, Pasadena, California.

R. M. Hewick, M. W. Hunkapiller, L. E. Hood, and W. J. Dreyer, J. Biol. Chem. 256, 7990 (1981).

R. H. Aebersold, G. D. Pipes, H . Nika, L. E. Hood, and S. 8.H. Kent, Biochemistry 27, 6860 (1988).

Section IX Specialized Purification Procedures

[381

PURIFICATION OF MEMBRANE PROTEINS

499

[381 Purification of Membrane Proteins By THOMASC . THOMAS and MARKG . MCNAMEE

As our interest in molecular mechanisms responsible for cellular function has increased, the purification of membrane proteins has become an important part of many research efforts. The ultimate goal in purifying these proteins is to fully characterize them and determine how they fulfill their unique functional roles in uiuo. Advances in our understanding of membrane proteins have recently culminated in the successful purification, crystallization, and X-ray crystallographic analysis of the photoreaction center from Rhodopseudomonas uiridis.' With continued advances in the field, it may soon be possible to fully characterize other multisubunit, integral membrane proteins, such as the nicotinic acetylcholine receptor and the voltage-dependent sodium channel. The methods available for the purification of membrane proteins are basically the same as those employed to purify water-soluble, nonmembrane-associated proteins. These methods include precipitation, gel filtration, ion-exchange, reversed-phase, and affinity chromatography (described in Section VII of this volume). Several unique characteristics of membrane proteins, however, often make it difficult to apply these methods successfully. In this chapter we will emphasize methods and conditions of purification which are designed to yield functionally active membrane proteins. Functional activity is judged by the ability of proteins to perform tasks such as catalyzing reactions, promoting ion flux, or binding specific ligands. This approach was emphasized in an excellent chapter in . ~ have atthis series by Jos van Renswoude and Christoph K e m ~ fWe tempted to extend the scope of this earlier chapter by emphasizing those areas where advances have been made, both in conventional chromatographic techniques and in newly developed genetic and immunological techniques. It is important to stress that, just as with soluble proteins, there is no way to present a single, precise set of methods for the purification of all membrane proteins. Each membrane protein possesses a unique set of physical characteristics, and conditions which are suitable for the purification of one protein may not be suitable for others. Table I lists a number I

J . Deisenhofer. 0. Epp. K. Miki, R. Huber, and H. Michel, Nuture (London) 318, 618 (1985).

J. van Renswoude and C . Kempf, this series, Vol. 104, p. 329.

METHODS IN ENZYMOLOGY, VOL. 182

Copyright 62 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

Protein (function)

Tissue source

Torpedo californica Nicotinic acetylcholine receptor (ligand-gated cation channel; electric organ synaptic transmission at electric organ and at neuromuscular junction) Nicotinic acetylcholine receptor Chicken brain (ion channel?-specificity not yet known) Muscarinic acetylcholine receptor Pig heart (G-protein-coupled receptor involved in second messenger systems and ion channel activity)

Sodium channel (voltage-sensitive sodium channel in nerve cells; tetrodotoxin sensitive)

Rat brain

Lactose permease (H+: galactoside transporter)

Escherichia coli (IPTG induced)

Growth hormone receptor

Rabbit liver

Atrial natriuretic factor (ANF) receptor (binds ANF and transduces binding via guanylate cyclase; controls fluid volume homeostasis)

Bovine adrenocortical cells

Experimental methods

Detergent

Cmde membranes Affinity chromatography (AffiGel401, ACh analog)

I% sodium cholate

Cmde membranes Immunoaffinity chromatography Enriched membranes WGA-agarose DEAE-agarose Hydroxylapatite Affinity chromatography [3-(2'-aminobenzhydryloxy)tropane agarose] Crude membranes Anion exchange Hydroxylapatite WGA-Sepharose Sucrose density gradient Membrane vesicles Urea preextraction Urealcholate preextraction DEAE-Sepharose Crude membranes Affinity chromatography (recombinant human GH) ANF affinity chromatography GTP-agarose WGA-Sepharose

1.14% Triton X-I00

0.4% digitoninl 0.08% sodium cholate

2.5% Triton X-1001 0.25% PtdCho

1.25% octylglucoside

1% Triton X-I00

1% Triton X-100

Yield (%)

Purification (-fold)

Molecular weight (subunit composition)

Bacteriorhodopsin (light-activated proton pump from Halobacterium halobirrm)

E. coli transfected with

Phospholipase C (membrane form 11) (hydrolysis of phosphoinositides to produce inositol 1.4.5-trisphosphate and t,2-diacylglyceroll

Human platelets

NADPH-cytochrome-c reduclase (substrate oxygenation; detoxification)

Hamster liver

Calmalulin-sensitive adenylale cyclase (CAMP synthesis)

Bovine cerebral cortex

Platelet-derived growth factor (PDGF) receptor (binds PDGF, resulting in mitogenic stimulation; tyrosine kinase activity)

Pig uterus

bacterioopsin gene

E. coli membranes Organic solvent extraction DEAE-Tris ion exchange

Platelet membranes Fast Q-Sepharose ion exchange Heparin Sepharose Zlltrogel AcA-44 gel filtration Mono Q ion exchange Superose 6 1 2 gel filtration Superose 12 gel filtration Microsomal membranes Sequential detergent extraction 2'.5'-ADP awrose affinity chromatography Crude membranes DEAE-Sephacel ion exchangc Calmodulin Sepharose Heptanediamine Sepharose Crude membranes WGA Sepharose Mono Q ion exchange AntiphosphotyrosineSepharose

None (chloroform/ methanollwaterl TEA) 1% Sodium cholate

70.0

0.5

2,740

61K

0.5% Lnhrol PX

14.0

3,000

150K (catalytic subunit)

11

8.0

630

170K

12

2.3% Triton X-100

27K

" PtdCho, Phosphatidylcholine; IPTG, isopropylthiogalactoside; TEA, triethylamine; GH, growth hormone; ACh, acetylcholine. References: (I) 0.T. Jones. J . P. Earnest, and M. G. McNamee, in "Biological Membranes: A Practical Approach" (I. B. C. Findlay and W. H. Evans, eds.), p. 139. IRL Press, Oxford, 1987; (2) P. 1. Whiting and I. M. Lindstrom, Biockemistry25, 2082 (1986); (3)G. L. Peterson, G. S . Herron, M. Yamaki, D. S. Fullerton, and M. 1. Schimerlik, Proc. Natl. Acad. Sci. U.S.A81,4993 (1984): (4) R. P. Hartshorne and W. A. Catterall, J. Biol Chem. 259, 1667(1984); (5) M. 3. Newman, D. L. Foster. T. H. Wilson, and H. K. Kaback, J. Biol. Chem. 256, 11804 (1981); (6) S. A. Spencer, R. G. Hammonds, W. I. Heneel, H. Rodriguez, M. J. Waters, and W. I. Wood, J. Biol. Chem. M ,7862 (1988); (7) R. Takayanagi, T. Inagami, R. M. Snajdar, T. Imada, M. Tamura, and K. S. Misono, J . Biol. Chrm. 262, 12104 (1987): (8) M. S . Braiman, L. J. Stern, B. H. Chao, and H. G. Khorana, J. Biol. Chem. 262,9271 (1987); (9) Y. Banno, Y. Yada, and Y.Nozawa, J. Biol. Chem. 23,I1459 (1988); (10) C. M. Ardies, I. M. Lasker, B. P. Bloswick, and C. S. Lieber, A n d . Biochem. 162,39 (1987); (1 1) R. E. Yeager, W. Heideman, G. 8. Rosenberg, and D. R. Starm, Biochemistry 24,3776 (1985); (12) L. Ronnstrand, M. P. Beckmann, B. Faulders. A. Ostman, R. Ek, and C.-H. Heldin, J . Biol. Chum. 262,ZYZY (1987).

502

SPECIALIZED PURIFICATION PROCEDURES

[381

of well-characterized membrane proteins and the methods used to purify them. The first distinction which must be made is between peripheral and integral membrane protein^.^ Peripheral membrane proteins are predominantly hydrophilic in nature and are mainly associated with the membrane surface through electrostatic interactions. These proteins are easily solubilized and then purified by conventional chromatographic methods. Integral membrane proteins, however, are predominantly amphiphiles with hydrophilic regions exposed to the aqueous environment and hydrophobic regions embedded in the lipid matrix. These proteins must often be associated with lipids, which may differ in their charge and structure, in order to remain functionally a ~ t i v e . ~ Prior to purification, these lipid-associated integral membrane proteins must be extracted from the membrane. This solubilization is typically achieved with amphiphilic detergents, which may also differ in their net charge and structure. As a result, the solubilization of a membrane protein, under conditions that preserve its functional activity, yields a very heterogeneous detergent-lipid-protein micelle. Attempts to chromatographically purify the protein may change the composition of this assembly, thereby inactivating or altering the properties of the protein. Chromatographic methods which provide the highest yield of active, functional protein are those that alter this soluble assembly the least. As a result, the dual goals of preserving functional activity and of attaining high chromatographic resolution are often diametrically opposed.

Preparation of Membranes

Preparation of a suitable membrane fraction is the first stage in the process of isolating and purifying a membrane protein. The methods currently available for isolating enriched membranes are typically of low resolution (2- to 5-fold increase in specific activity), but yields are generally high and significant amounts of contaminating material can be removed. Membrane proteins are most stable while they are embedded in the membrane, and time invested at this stage will improve results during subsequent stages of the purification. The ability to subfractionate membranes is the only way in which membrane proteins offer unique advantages over soluble proteins. The first step is to obtain a tissue in which the protein of interest has a high specific activity. An excellent example is the electric organ of TorS . J. Singer and G. L. Nicolson, Science 175, 720 (1972). 0. T. Jones, J. H. Eubanks, J. P. Earnest, and M. G . McNamee, Biochemistry 27, 3733 (1988).

504

SPECIALIZED PURIFICATION PROCEDURES

[381

mM), which inhibits serine proteases; and N-ethylmaleimide (0.1- 1 mM), which inhibits sulfhydryl proteases (see Ref. 7 for additional protease inhibitors). PMSF has a short half-life in aqueous solution and is prepared as a stock solution (100 mM) in ethanol or 2-propanol. Dithiothreitol (DTT, 0.1-1 mM) is often added to prevent oxidation. Since most of these compounds have the potential to covalently modify and/or alter the activity of membrane proteins, caution is advised when using them.

Purification of Peripheral Membrane Proteins

When preparing a membrane fraction for the purification of peripheral membrane proteins, several additional factors will influence the choice of methods. Membranes intended for this purpose should be prepared under isosmotic conditions (e.g., 0.15 M NaCl). High ionic strength solutions may result in the premature solubilization of peripheral membrane proteins, and low ionic strength buffers may result in nonspecific association of soluble proteins with the membrane. In addition, some proteins exist in both soluble and membrane-associated forms in vivo. l o These proteins can bind reversibly to either the protein or lipid portions of the membrane. Reversible binding may involve conformational changes in the protein induced by changes in cation concentrations or phosphorylation. One example is Ca2+/phospholipid-dependent protein kinase, which binds to the membrane upon activation in a Ca2+-dependentmanner.'' Therefore, the composition of the buffer used during the preparation of a membrane fraction may affect the distribution of these proteins between the soluble and membrane-associated fractions. Peripheral membrane proteins are solubilized by incubating membranes with solutions which interrupt electrostatic and in some cases hydrophobic interactions. Solutions containing one or more of the following compounds are commonly used for this purpose. 1. NaCl or KC1 (>0.15 M)I2 2. Buffers of acidic (3-5) or basic (8-12) pHI3 3. EDTA and EGTA: These are frequently added to destabilize bonds that are enhanced by Mg2+and Ca2+l 4 4. Chaotropic agents (containing I-, Br-, C104-, and SCN- ions)15: lo 'I

" l3

l4

Is

P. Burn, TIBS 13, 79 (1988). J. H. Schwartz, and S . M. Greenberg, Annu. Rru. Neurosci. 10, 459 (1987). H. W. Chang and E. Bock, Biochemistry 16,45 13 (1977). T. Yoshihisa, Y. Ohsumi, and Y. Anraku, J. Biol. Chem. 263, 5158 (1988). 9. J. Bowman, F. Blasco, and C. W. Slayman, J. Biol. Chem. 256, 12343 (1981). V . Bennett, K . Gardner, and J. P. Steiner, J . Biol. Chem. 263, 5860 (1988).

[381

PURIFICATION OF MEMBRANE PROTEINS

505

These agents act by disordering the structure of water. This tends to reduce the strength of hydrophobic bonds, thereby increasing the solubility of partially hydrophobic proteins in the aqueous phase 5. Strong denaturing agents, such as urea and guanidine hydrochlorideI6 6. Diiodosalicylate or s~lfosalicylate'~: These salts may have detergent-like characteristics 7. Detergents, such as deoxycholate or Triton X-100 (discussed late^-).'^.'^ Integral membrane proteins will also be solubilized by these treatments. After incubation for 10 to 60 min on ice, insoluble membranes are removed by centrifugation at 100,000 g for 60 min. The efficiency of solubilization should be monitored by performing activity and protein assays to determine the distribution and specific activity of desired proteins. The distribution of proteins may also be monitored by SDS-PAGE. The solubilized peripheral membrane proteins are then fractionated by methods similar to those applied to soluble proteins. In some cases it may be necessary to include salts and detergents in buffers during chromatography in order to prevent the aggregation and precipitation of proteins.I5 Care must be taken when using these solutions since they all have the ability to inactivate or denature membrane proteins. Solubilization of membrane proteins by one of the first four solutions listed above is commonly used as a method to distinguish between peripheral and integral membrane proteins. In fact, membranes are frequently preextracted with these solutions to remove peripheral membrane proteins prior to the solubilization of integral membrane proteins by detergents. Treatment of membranes with high enough concentrations of these solutions can, however, result in the solubilization of some integral membrane proteins. In one example, a lipid-associated protein was extracted from presynaptic membranes by alkaline extraction.I9 Solubilization of Integral Membrane Proteins

In order to purify integral membrane proteins by chromatographic methods, the proteins must be removed from the lipid bilayer and individually dispersed into solution. This is most effectively accomplished with l6

N. J. Newman, D. L. Foster. T. H. Wilson, and H. R. Kaback. J. Biol. Chem. 256, 11804 (1981).

J . K . Wright and P. Overath, Eur. J. Biochem. 138, 497 (1984). ISW. J. LaRochelle and S. C. Froehner, J. Biol. Chem. 262, 8190 (1987). l9 M. Israel, N . Morel, B. Lesbats, S. Birman, and R. Manaranche, Proc. Narl. Acad. Sci.

l7

U.S.A. 83, 9226 (1986).

[381

PURIFICATION OF MEMBRANE PROTEINS

503

pedo californzca, from which the nicotinic acetylcholine receptor (AchR)

t

I

C

r: r-

has been purified. Due to the high density of receptor-rich synapses in this tissue, only a 50-fold purification is necessary in order to obtain pure AchR.' Another example is the human erythrocyte membrane, from which plasma membrane proteins are easily obtained without contamination by other cell types or by lntracellular membrane protein^.^ While the choice of a tissue or cell source is an important first step in the purification of both soluble and membrane prote~ns,it may be especially important for the purification of membrane proteins. There are very few examples in which membrane proteins of low abundance have been successfully purified. The next step generally entails subcellular fract~onation.Many dlverse methods have been developed, and applications involving both animal and plant cells can be found in two recent reviews7 (see also [I61 in this volume). The most frequently used methods employ a combination of differential centrifugation and sucrose density gradient centrifugation steps. More recently, Percoll has been substituted for sucrose In order to form density gradients which are nearly isosmotic. The best fractionation method to use for enrichment of a particular protein is determined on the basis of a careful balance sheet of yields, changes in specific activity, and the distribut~onsof marker enzymes. Separations using differential centrifugation are rapid and recoveries are high. Sucrose gradients offer improved purification, but they are more time consuming. If the protein of interest is distributed among several gradient fractions then very little improvement in specific activity is seen and yields are low. In these cases, it is common to prepare a crude membrane fraction and use this as the initial source of protein for solubilizatlon. One rapid, high-yield method involves homogenizing the tissue in 10 vol of a 0.25 M sucrose buffer at 4O, followed by centrifugation at 1000 g for 10 min. The supernatant fraction is then centrifuged at 105,000 g for 1 hr at 4" and the crude membrane pellet is r e c ~ v e r e dThis . ~ method yields membranes which are free of whole cells, nuclei, and soluble proteins. During cell disruption harmful proteases may be released. Compounds which have been kery effective at minimizing proteolysis of membrane proteins include EDTA and EGTA (0.1-5 m M ) , which inhib~tdivalent cation-dependent proteases; phenylmethylsulfonyl fluoride (PMSF, 0.1-1

. 3

-

-

R. L. Vandlen. W. C.-S Wu, J . C. Eisenach, and M. A. Raftery, Biochemistry 18, 1845 (1979). G. Fairbanks, T. L. Steck. and D. F. H. Wallach, Biochemistry 10, 2606 (1971). ' W . H . Evans, in "Biological Membranes: A Practical Approach" (J. B. C. Findlay and W. H. Evans, eds.), p. 1 . IRL Press, Oxford. 1987. I. D. Morre. A. 0. Brightman, and A . S. Sandelius. see ref. 7, p. 37. J . Ramwani and R. K. Mishra, J . Biol. Chem. 261, 8804 (1986).

502

SPECIALIZED PURIFICATION PROCEDURES

[381

of well-characterized membrane proteins and the methods used to purify them. The first distinction which must be made is between peripheral and integral membrane protein^.^ Peripheral membrane proteins are predominantly hydrophilic in nature and are mainly associated with the membrane surface through electrostatic interactions. These proteins are easily solubilized and then purified by conventional chromatographic methods. Integral membrane proteins, however, are predominantly amphiphiles with hydrophilic regions exposed to the aqueous environment and hydrophobic regions embedded in the lipid matrix. These proteins must often be associated with lipids, which may differ in their charge and structure, in order to remain functionally a ~ t i v e . ~ Prior to purification, these lipid-associated integral membrane proteins must be extracted from the membrane. This solubilization is typically achieved with amphiphilic detergents, which may also differ in their net charge and structure. As a result, the solubilization of a membrane protein, under conditions that preserve its functional activity, yields a very heterogeneous detergent-lipid-protein micelle. Attempts to chromatographically purify the protein may change the composition of this assembly, thereby inactivating or altering the properties of the protein. Chromatographic methods which provide the highest yield of active, functional protein are those that alter this soluble assembly the least. As a result, the dual goals of preserving functional activity and of attaining high chromatographic resolution are often diametrically opposed.

Preparation of Membranes

Preparation of a suitable membrane fraction is the first stage in the process of isolating and purifying a membrane protein. The methods currently available for isolating enriched membranes are typically of low resolution (2- to 5-fold increase in specific activity), but yields are generally high and significant amounts of contaminating material can be removed. Membrane proteins are most stable while they are embedded in the membrane, and time invested at this stage will improve results during subsequent stages of the purification. The ability to subfractionate membranes is the only way in which membrane proteins offer unique advantages over soluble proteins. The first step is to obtain a tissue in which the protein of interest has a high specific activity. An excellent example is the electric organ of TorS. J. Singer and G . L. Nicolson, Science 175, 720 (1972). 0. T. Jones, J. H. Eubanks, J. P. Earnest, and M. G. McNamee, Biochemistry (1988).

27,3733

504

SPECIALIZED PURIFICATION PROCEDURES

[381

mM), which inhibits serine proteases; and N-ethylmaleimide (0.1-1 mM), which inhibits sulfhydryl proteases (see Ref. 7 for additional protease inhibitors). PMSF has a short half-life in aqueous solution and is prepared as a stock solution (100 mM) in ethanol or 2-propanol. Dithiothreitol (DTT, 0.1-1 mM) is often added to prevent oxidation. Since most of these compounds have the potential to covalently modify and/or alter the activity of membrane proteins, caution is advised when using them.

Purification of Peripheral Membrane Proteins When preparing a membrane fraction for the purification of peripheral membrane proteins, several additional factors will influence the choice of methods. Membranes intended for this purpose should be prepared under isosmotic conditions (e.g., 0.15 M NaCI). High ionic strength solutions may result in the premature solubilization of peripheral membrane proteins, and low ionic strength buffers may result in nonspecific association of soluble proteins with the membrane. In addition, some proteins exist in both soluble and membrane-associated forms in vivo. 'O These proteins can bind reversibly to either the protein or lipid portions of the membrane. Reversible binding may involve conformational changes in the protein induced by changes in cation concentrations or phosphorylation. One example is Ca2+/phospholipid-dependent protein kinase, which binds to the membrane upon activation in a Ca2+-dependent manner." Therefore, the composition of the buffer used during the preparation of a membrane fraction may affect the distribution of these proteins between the soluble and membrane-associated fractions. Peripheral membrane proteins are solubilized by incubating membranes with solutions which interrupt electrostatic and in some cases hydrophobic interactions. Solutions containing one or more of the following compounds are commonly used for this purpose. I . NaCl or KC1 (30.15 M)I2 2. Buffers of acidic (3-5) or basic (8-12) pHt3 3. EDTA and EGTA: These are frequently added to destabilize bonds that are enhanced by Mg2+and Ca2' l 4 4. Chaotropic agents (containing I-, Br-, C104-, and SCN- ions)I5: P. Bum, TIBS 13, 79 (1988). J. H. Schwartz, and S. M. Greenberg, Annu. Reu. Neurosci. 10, 459 (1987). l L H. W. Chang and E. Bock, Biochemistry 16,4513 (1977). I' T. Yoshihisa, Y . Ohsumi. and Y. Anraku, J. Biol. Chem. 263,5158 (1988). l 4 B. J . Bowman, F. Blasco, and C. W. Slayman, J. Biol. Chem. 256, 12343 (1981). V. Bennett, K. Gardner, and J . P. Steincr, J. Biol. Chem. 263, 5860 (1988). lo

"

[381

PURIFICATION OF MEMBRANE PROTEINS

-.

505

These agents act by disordering the structure of water. This tends to reduce the strength of hydrophobic bonds, thereby increasing the solubility of partially hydrophobic proteins in the aqueous phase 5. Strong denaturing agents, such as urea and guanidine hydrochloride16 6. Diiodosalicylate or sulf~salicylate~~: These salts may have detergent-like characteristics 7. Detergents, such as deoxycholate or Triton X-100 (discussed later).I4J8 Integral membrane proteins will also be solubilized by these treatments. After incubation for 10 to 60 min on ice, insoluble membranes are removed by centrifugation at 100,000 g for 60 min. The efficiency of solubilization should be monitored by performing activity and protein assays to determine the distribution and specific activity of desired proteins. The distribution of proteins may also be monitored by SDS-PAGE. The solubilized peripheral membrane proteins are then fractionated by methods similar to those applied to soluble proteins. In some cases it may be necessary to include salts and detergents in buffers during chromatography in order to prevent the aggregation and precipitation of proteins.15 Care must be taken when using these solutions since they all have the ability to inactivate or denature membrane proteins. Solubilization of membrane proteins by one of the first four solutions listed above is commonly used as a method to distinguish between peripheral and integral membrane proteins. In fact, membranes are frequently preextracted with these solutions to remove peripheral membrane proteins prior to the solubilization of integral membrane proteins by detergents. Treatment of membranes with high enough concentrations of these solutions can, however, result in the solubilization of some integral membrane proteins. In one example, a lipid-associated protein was extracted from presynaptic membranes by alkaline extraction.I9 Solubilization of Integral Membrane Proteins

In order to purify integral membrane proteins by chromatographic methods, the proteins must be removed from the lipid bilayer and individually dispersed into solution. This is most effectively accomplished with l6

N. J. Newman, D. L. Foster, T. H. Wilson, and H. R . Kaback, J. Biol. Chem. 256, 11804 (1981).

J. K. Wright and P. Overath, Eur. J . Biochem. 138, 497 (1984). 'W.J. LaRochelle and S. C. Froehner, J. Biol. Chem. 262, 8190 (1987). l9 M. Israel, N. Morel, B . Lesbats, S. Birman, and R. Manaranche, Proc. Natl. Acad. Sri. U.S.A. 83, 9226 (1986). l7

506

SPECIALIZED PURIFICATION PROCEDURES

[381

amphiphilic detergents in aqueous solution, although in a few cases organic solvents have been used. Chapters [18] and [I91 of this volume contain a very thorough discussion of the different types of detergents and the best methods for using them. We will, therefore, provide only a brief overview of detergents and their use as it applies to chromatography of proteins. Detergents may be grouped into two classes, ionic and nonionic. The ionic detergents are either anionic (e.g., sodium dodecyl sulfate and the bile salts, cholate and deoxycholate), cationic (e.g., alkyltrimethylammonium salts), or zwitterionic (e.g., Zwittergent and CHAPS). These detergents are generally very effective at solubilizing integral membrane proteins and dissociating protein complexes. Some ionic detergents (e.g., sodium dodecyl sulfate) are very denaturing and are used only when recovery of functional activity is unimportant. Ionic detergents generally have a high (>1 mM) critical micelle concentration (cmc) and are easily removed by methods such as dialysis. Cholate, deoxycholate, and CHAPS form small micelles (4-6 kDa) which do not interfere with gel filtration. Due to their charge, however, cholate and deoxycholate are not suitable for ion-exchange chromatography. Examples of nonionic detergents include octylglucoside, digitonin, and the polyoxyethylene derivatives (e.g., Triton X-100, Lubrol PX, and the Tween series). These detergents are less effective at dissociating protein complexes, but many proteins are more stable in nonionic detergents than in ionic detergents. Nonionic detergents generally have a low cmc ( < 1 mM) and are difficult to remove without using special resins such as Bio-Rad SM-2 beads.20In addition, the polyoxyethylene derivatives and digitonin form large micelles which may interfere with gel filtration. Triton X-100 absorbs at 280 nm and interferes with the use of ultraviolet absorbance methods to monitor the chromatographic elution of proteins. Octylglucoside is an important exception to the above statements about nonionic detergents. It has a high cmc (23.3 mM), forms small micelles (8 kDa), and does not absorb light at 280 nm. At the present time no detergent has emerged as the best choice for solubilizing a broad range of proteins, but on a practical basis we would suggest screening CHAPS and octylglucoside first. Detergents are screened by preparing membrane fractions at a specific protein concentration and adding extraction solutions with a range of detergent concentrations. Greatest success is achieved when using final protein concentrations of 1 to 10 mglml and detergentlprotein ratios of 0.1 to 10 (wlw). Solutions are incubated at 0-4" for 30 to 60 min (longer incubations may be necessary) and then centrifuged at 105,000 g for 1 hr 20

P. W. Holloway, Anal. Bioclzem. 53, 304 (1973).

[381

PURIFICATION OF MEMBRANE PROTEINS

507

at 4°.21 By definition, solubilized proteins remain in the supernatant solution. Both the specific activity and the yield of soluble, functionally active protein should be determined. The optimum detergentlprotein ratio for solubilization will in most cases be chosen as a compromise between yield and specific activity. Detergents are generally most effective at concentrations above their cmc. In some cases aggregates or assemblies of proteins which have not been fully dissociated are found to be soluble by the above criterion. This is most likely to happen when nonionic detergents are used or when glycerol, sucrose, or urea are added to extraction solutions. These additives will increase the solution density with the effect that some membrane fragments may not sediment. Another method which can be used to determine solubility is gel filtration chromatography. Proteins which elute after the void volume are considered soluble.22Using this method, it was determined that CHAPS is far more effective at solubilizing erythrocyte membrane proteins than reduced Triton X-100 (reduction eliminates absorbance at 280 nm) or Tween 20.22Just as with sedimentation by centrifugation, this definition of solubility is operational and will depend on the fractionation range of the gel used. In some cases it has been found that a mixture of detergents will provide the most effective solubilization of active protein.=' Also, the yield of protein solubilized in an active state can sometimes be improved by including lipid in the solubilization buffer." It is not clear whether the addition of exogenous lipid protects the protein during extraction, thereby allowing more complete solubilization at higher detergent concentrations, or stabilizes the protein after solubilization. In most cases, however, the addition of lipid will simply lower the effective concentration of the detergent. Higher concentrations of detergent will then be needed to achieve the same yield. Organic solvents may sometimes be used as an alternative to deterg e n t ~ . These '~ solvents denature most membrane proteins and only a small percentage of proteins will be aoluble in the organic phase. An additional consideration is that once solubilized in organic solvent, it may be difficult to determine the activity or function of proteins in an aqueous assay system. Those solvent systems which have been used with greatest success have been well described elsewhere.'

" L. M. Hjelmeland and A. Chrambach, in "Membranes, Detergents, and Receptor Solubilization" (1. C. Venter and L. C. Harrison, eds.), p. 35. Alan R . Liss, New York, New York, 1984. R. S. Matson and S. C. Coheen, J . Chromatogr. 359,285 (1986). 23 C. R. Cremo, G. S. Herron, and M. I. Schimerlik, A n d . Biochem. 115, 331 (1981). 24 R. P. Hartshorne and W . A. Catterall, J. Biol. Chem. 259, 1667 (1984).

508

SPECIALIZED PURIFICATION PROCEDURES

[381

Fractionation of Integral Membrane Proteins

Once a suitable preparation of soluble membrane proteins has been prepared, fractionation methods can be employed to isolate a specific protein of interest. These methods include conventional chromatographic techniques (Section VII, this volume) and a few methods specific for membrane proteins. The most widely used chromatographic techniques are gel filtration, affinity, ion-exchange, and reversed-phase chromatography. The following section describes methods for optimizing the use of these techniques with integral membrane proteins. Gel Filtration

This technique is useful for bulk separation of high-, medium-, and low-molecular-weight membrane proteins. High yields of active protein are frequently achieved, but the usefulness of this technique is limited by the low levels of purification which are typically obtained ( < 5 - f 0 l d ) . ~ ~ , ~ ~ The conditions chosen to solubilize active protein will yield a mixture of detergent-protein micelles, detergent-lipid-protein micelles, and possibly small membrane fragment^.^' This heterogeneity can result in a broad distribution for each specific protein. In order to prevent detergent-mediated inactivation of proteins during gel filtration, it is common practice to use detergent in the eluant buffer at a concentration 10- to 100-fold lower than used during the solubilization step.25This practice can promote nonspecific aggregation and further decrease the level of purification which is attained.28In addition, if exogenous lipid has been added to the elution buffer, proteins may be partially reconstituted into vesicles and elute in the void volume. There are several steps which may be taken to increase resolution and , yield, and to prevent protein aggregation. 1. Use large-pore chromatography resins such as Sephacryl S-300 and S-400 (Pharmacia). Detergent-protein complexes may exhibit twice the apparent molecular weight expected for the protein alone, and nondenaturing detergents such as Triton X-100 may not fully dissociate protein complexes or aggregate^.^^ As a result it is common for detergent-solubilized protein complexes to elute with apparent molecular weights between 200,000 and 1,000,000. When using gel filtration resins designed for HPLC A . F. Welton. P. M. Lad. A. C. Newby, H. Yamamura, S. Nicosia, and M. Rodbell, Biochim. Biophys. Acta 522, 625 (1978). 6' E. C. Hulme, C. P. Berrie, T. Haga, N. J . M. Birdsall, A. S. V . Burgen, and J . Stockton, J . Recept. Rrs. 3, 301 (1983). 27 A. Helenius and K . Simons, Biochim. Biophys. Acta 415, 29 (1975). 'R A. C. Newby and A. Chrambach, Biochem. J. 177, 623 (1979).

I

.

25

I

I

[381

PURIFICATION O F MEMBRANE PROTEINS

509

and FPLC systems, the best recoveries will be achieved with larger particle sizes.29 2. Gel filtration protocols should be optimized by determining the resolution and recovery of functionally active proteins at several detergent concentrations. In most cases there will be a minimum detergent concentration which is necessary in the elution buffer in order to achieve maximum resolution. Bacteriorhodopsin from Halobuctrrium required 2.0% (wlw) Triton X-100 (ca. lox cmc) in both the extraction and elution buffers, and 6 hr of incubation, before it was completely dissociated into monomers.30 Analysis of rat liver microsomal proteins determined that the minimum concentration of octaethylene glycol dodecyl ether (C12E8) which was required for optimal resolution was 0.05% (wlw) (lox ~ m c ) . ~ ' These studies clearly indicate that the practice of reducing detergent concentrations in the elution buffer can reduce the level of resolution and purification obtained. The optimal level of detergent in the elution buffer (always > crnc) will be chosen as a compromise between maximal resolution and high recovery of active protein. It should be noted that elution conditions can be rapidly varied and tested on HPLC or FPLC systems. The use of these systems should greatly expand the application of gel filtration to membrane protein purification. 3. Examine the use of detergent mixtures. When adenylate cyclase was solubilized in 1% Lubrol PX (nonionic) and chromatographed in 0.01% Lubrol PX, aggregation occurred. Solubilization of adenylate cyclase in deoxycholate inactivated the enzyme. However, addition of de) oxycholate to the Lubrol PX-containing elution buffer (1 : 3, w / ~ prevented aggregation while maintaining 90% of the activity.28 Gel filtration should be employed as the first step in the purification protocol for two reasons. First, most methods for concentrating membrane proteins are very poor (discussed later), resulting in low yields and aggregation. Since gel filtration requires a concentrated sample of relatively small volume, it is best to extract membranes at a high protein concentration (5-10 mglml) and immediately chromatograph by gel filtration. The dilute sample which is isolated by this procedure can then be purified by either ion-exchange or affinity chromatography since these are suitable methods for use with dilute samples. The second reason is that gel filtration can be used to determine whether or not the protein of interest has been completely solubilized. Optimal solubilization conditions should produce a symmetrical peak of activity which is found com29

"

G. W. Welling, K. Slopsema, and S. Welling-Wester, J . Chromarogr. 359, 307 (1986). R. Pabst, T. Nawroth, and K. Dose, J . Chromatogr. 285, 333 (1984). Y . Kato, T. Kitamura. K. Nakamura, A. Mitsui, Y. Yamasaki, and T. Hashimoto, J . Chromatogr. 391, 395 (1987).

[381

PURIFICATION OF MEMBRANE PROTEINS

509

and FPLC systems, the best recoveries will be achieved with larger particle sizes.29 2. Gel filtration protocols should be optimized by determining the resolution and recovery of functionally active proteins at several detergent concentrations. In most cases there will be a minimum detergent concentration which is necessary in the elution buffer in order to achieve maximum resolution. Bacteriorhodopsin from Halobacterium required 2.0% (w/w) Triton X-100 (ca. l o x cmc) in both the extraction and elution buffers, and 6 hr of incubation, before it was completely dissociated into monomers.30 Analysis of rat liver microsomal proteins determined that the minimum concentration of octaethylene glycol dodecyl ether (Cl2E8) which was required for optimal resolution was 0.05% (w/w) ( l o x c ~ c ) . ~ ' These studies clearly indicate that the practice of reducing detergent concentrations in the elution buffer can reduce the level of resolution and purification obtained. The optimal level of detergent in the elution buffer (always > cmc) will be chosen as a compromise between maximal resolution and high recovery of active protein. It should be noted that elution conditions can be rapidly varied and tested on HPLC or FPLC systems. The use of these systems should greatly expand the application of gel filtration to membrane protein purification. 3. Examine the use of detergent mixtures. When adenylate cyclase was solubilized in 1% Lubrol PX (nonionic) and chromatographed in 0.01% Lubrol PX, aggregation occurred. Solubilization of adenylate cyclase in deoxycholate inactivated the enzyme. However, addition of deoxycholate to the Lubrol PX-containing elution buffer (1 : 3, w / ~ pre) vented aggregation while maintaining 90% of the activity .2s Gel filtration should be employed as the first step in the purification protocol for two reasons. First, most methods for concentrating membrane proteins are very poor (discussed later), resulting in low yields and aggregation. Since gel filtration requires a concentrated sample of relatively small volume, it is best to extract membranes at a high protein concentration (5-10 mglml) and immediately chromatograph by gel filtration. The dilute sample which is isolated by this procedure can then be purified by either ion-exchange or affinity chromatography since these are suitable methods for use with dilute samples. The second reason is that gel filtration can be used to determine whether or not the protein of interest has been completely solubilized. Optimal solubilization conditions should produce a symmetrical peak of activity which is found com8

G. W. Welling, K. Slopsema, and S. Welling-Wester, J. Chromatogr. 359, 307 (1986).

"

Y. Kato, T. Kitamura, K . Nakamura. A. Mitsui, Y . Yamasaki, and T. Hashimoto, J. Chromatogr. 391, 395 (1987).

" R. Pabst, T. Nawroth. and K. Dose, J. Chromutogr. 285, 333 (1984).

510

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pletely within the included volume. If the protein has not been solubilized in a uniform manner, the level of purification attained during subsequent purification steps will also be reduced. Affinity Chromatography

This is by far the most useful and successfully applied method for purification of integral membrane proteins. In fact, it has played an essential role in the purification of large multisubunit receptors. Although nonaffinity methods generally provide less than a 20-fold purification of membrane proteins, affinity chromatography routinely achieves purifications of between 1000- and 10,000-fold. Three types of affinity chromatography will be described. These include the use of specific ligands (e.g., enzyme inhibitors, hormones, neurotransmitters), a general ligand (lectins), and antibodies. Specific affinity ligands immobilize the protein of interest without disturbing the interactions among protein, detergent, and lipid. The immobilized protein can then be eluted by equally gentle methods using a soluble ligand. If the solubilized protein is active in detergent, then it is not necessary to remove the detergent prior to the adsorption step. When a specific ligand is used the major obstacles to attaining a high degree of purification are protein aggregation and nonspecific binding to the column. A level of detergent sufficient to prevent protein aggregation while not inactivating the protein of interest should be included in the buffer. A high ionic strength buffer will reduce nonspecific binding due to electrostatic interactions, and the presence of detergent in the buffer should reduce nonspecific hydrophobic interactions. It is important to note that changing the ionic strength of the buffer, in order to weaken nonspecific interactions, may also affect the strength of specific ligand binding. In some cases the combination of specific and nonspecific interactions may require the simultaneous use of a strong dissociating agent, such as urea, and a soluble ' " addition it is recomligand in order to elute the protein of i n t e r e ~ t . ~In mended that ligands be attached to the column resin by a hydrophilic (rather than hydrophobic) spacer arm.32,33This will increase both the specificity of the binding and the yield of protein. The level of purification achieved may also be improved by using a low ligand density, or by using G. B . Stauber, R. W. Ransom, A. I. Dilber, and R. W. Olsen, Eur. J. Biochem. 167, 125 (1987). " G. Vauquelin, P. Geynet, J. Hanoune, and A. D. Strosberg, Eur. J . Biochem. 98, 543 (1979). E. Sigel, A. Stephenson, C. Mamalaki, and E. A. Barnard, J. Biol. Chem. 258, 6965 (1983). 'la

''

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~ . ~ ~ for preparing and a minimum amount of the affinity r e ~ i n . ?Methods analyzing affinity resins are discussed elsewhere (see [29] in this volume; see also Ref. 35). Although the binding of protein to the affinity ligand should not disturb the interactions among detergent, protein, and lipid, the immobilization of these micelles may create additional problems. Since only the protein portion directly binds to the affinity resin, detergent-containing buffers may extract and elute the lipid portion of these bound complexes. This may inactivate and possibly even elute the protein. For this reason, lipids are frequently included in elution buffers (I-2%, wlv) in order to stabilize active protein^.^ Crude lipid mixtures (e.g., soybean asolectin and bovine brain total lipid extract) or purified lipids (e.g., phosphatidylcholines) may be used. A more general form of affinity chromatography utilizes lectins as the immobilized ligand. Lectins are carbohydrate-binding proteins of nonimmune origin which offer a rapid and mild method to purify plasma membrane glycoproteins. Lectin-glycoprotein interactions are reversible and can be inhibited by simple sugars. Therefore, adsorbed proteins can be eluted from lectin columns using simple sugars without changes in pH and ionic strength and without the use of denaturants. However, this method is unable to achieve significant subfractionation of glycoproteins and therefore yields lower levels of purification than achieved with more specific types of affinity chromatography. Hydrophobic and ion-exchange effects may also cause nonspecific binding. In addition, lectins are very sensitive to treatment with certain types of detergents. Nonionic detergents (up to 2.5%, wlv) have negligible effects on lectin affinity, but ionic detergents such as deoxycholate and SDS significantly reduce the binding capacity of the most commonly used l e c t i n ~ . ~ ~ The saccharides most commonly found attached to animal cell glycoproteins are sialic acid, galactose, mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine. Numerous lectins have been identified which bind to each of these.37 The most widely used lectins are concanavalin A (binds a-D-mannose) and wheat germ agglutinin [binds sialic acid and (/~-D-GIcNAc),].~~,~~ Although different lectins may have "S. A. Spencer, R. G. Hammonds, W. J . Henzel, H. Rodriguez, M. J . Waters, and W. I . Wood, J. Biol. Chem. 263, 7862 (1988). 35 C. R. Lowe, "Laboratory Techniques in Biochemistry and Molecular Biology" (T. S. Work and E. Work. eds.), Vol. 7, Part 2 . North-Holland, Amsterdam, 1979. ' 6 R. Lotan. G. Beattie, W. Hubbell, and G . L. Nicolson, Biochemistry 16, 1787 (1977). " I. J . Goldstein and C. E. Hayes, Adv. Carbohydr. Chem. Biochem. 35, 127 (1978). M. Monsigny, A.-C. Roche, C. Sene. R. Maget-Dana, and P. Delmotte, Eur. J. Biochem. 104, 147 (1980).

5 12

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the same specificity for sugars, their affinities will vary. The binding capacity of lectins for a specific protein can be assessed by determining their ability to precipitate that protein.39 Suitable affinity resins can then be obtained commercially or prepared by the cyanogen bromide meth~d.~~,~~ Some lectins, such as concanavalin A, and lentil and pea lectins require Ca2+and Mnz+(1 mM) for carbohydrate binding. This will affect the choice of buffers and whether or not chelating agents can be used. Adsorption is best performed in 0.15 M buffers of near neutral pH. After the column is washed, proteins are eluted with the same buffer containing an appropriate sugar (0.1-0.5 M). Gradient elution generally does not offer any advantages over step elution. Immunoaffinity ligands (antibodies) have been used with some success in the purification of membrane proteins, especially those of cell surface and viral origin. The major limitation to this technique is the strength with which antibodies bind their target proteins. Most methods of elution are very harsh and proteins isolated by this technique are generally inactive. ',~~ Monoclonal or polyclonal antibodies have both been u ~ e d . ~Antibodies may be linked to CNBr-activated Sepharose (Ref. 40, also commercially available). In some cases, it may be necessary to incubate proteins with the immunoaffinity resin for extended periods of time.4' Nonbound and nonspecifically bound proteins are then preeluted using neutral buffers containing moderately high levels of salt (<0.5 M NaCI) and detergent. Some commonly used elution buffers are as follows: 1. High or low pH buffer: For example, 0.05 M diethylamine (pH Eluted proteins are quickly neutral11.5) or 50 mM citrate (pH 3.0).43,43a ized to minimize loss of functional activity 2. Chaotropic agents, such as 3 M potassium thiocyanate4'

Other methods of elution include the use of high salt concentrations and denaturants such as SDS, urea, and guanidine-HC1. In one case, polyclonal antibodies were produced against a synthetic peptide. The bound protein was eluted with excess peptide and mild buffer conditions which resulted in a 2500-fold p~rification.~~ 39

F. Rieger and M. Vigny, J . Neurochem. 27, 121 (1976).

" S . C. March. 1. Parikh, and P. Cuatrecasas, Anal. Biochem. 60, 149 (1974). J . J . Hubert, D. B . Schenk, H. Skelly, and H. L. Leffert, Biochemistry 25, 4156 (1986). 42 G. Walter, M. A. Hutchinson, T. Hunter, and W. Eckhart, Proc. Natl. Acad. Sci. U.S.A. 79, 4025 (1982). 43 I. S. Trowbridge and M. B. Omary, Proc. Nutl. Acad. Sci. U.S.A. 78, 3039 (1981). 43a P. J . Whiting and J. M. Lindstrom, Biochemistry 25, 2082 (1986).

4'

[381

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Ion Exchange

Purifications on the order of 5-fold are typical in successful applications of this te~hnique.'~,"" High yields of active protein (>go%) have been reported using this method, but there are many cases in which complete loss of activity has also occurred. This has led to the suggestion that some membrane proteins may be irreversibly bound by ion-exchange resins. It is more likely, however, that these proteins are inactivated. Ion-exchange resins not only immobilize proteins, but they also bind charged detergents and lipids. Ion-exchange chromatography, therefore, has the potential to pull apart detergent-lipid-protein complexes. As a first step, only neutral or zwitterionic detergents should be employed. The addition of defined mixtures of neutral and zwitterionic lipids to buffers may also stabilize proteins. In cases of low yields, minimal washing to remove unbound proteins followed by step elution rather than gradient elution may also improve r e s ~ l t s . *Glycerol ~ , ~ ~ (10-20%) is often used to minimize hydrophobic interactions with the resin and to stabilize protein^.^^.^^ Work with rat microsomal membrane proteins indicates that detergent concentrations must be above a certain level, which depends on the detergent, in order to prevent poor resolution and r e ~ o v e r y . ~ Some ' proteins have specific requirements for charged lipids and cofactors which may be removed from the protein by attachment to the ion-exchange resin or by extraction with the elution buffer.47 It may not be possible to recover these proteins in an active state. In cases where high yields of inactive protein are obtained, it may be possible to reactivate these proteins by adding back required factors. For example, delipidated Na+,K+-ATPase can be reactivated by addition of negatively charged ph~sphatidylserine.~' Crude lipid mixtures such as asolectin may also be useful.

Reversed Phuse Reversed-phase chromatography on HPLC columns can achieve highresolution separation of many proteins. Unfortunately, the resins and the organic solvents used with this technique denature most proteins. For this reason, reversed-phase chromatography is used predominantly as an analytical rather than a preparative method for purifying membrane proteins. Highly hydrophobic membrane proteins bind very tightly, and they are

" N . Muto and L. Tan, J. Chrumatogr. 326, 137 (1985). W.-M.HOU, 2.-L. Zhang, and H.-H. Tai, Biot:hint. Biophys. Acta 959, 67 (1988). uiG. W. Wellig, R. van der Zee, and S. Welling-Wester, J. Chromarogr. 418, 223 (1987). " M. 1. DeCaldentey and K. P. Wheeler, Biochem. J. 177, 265 (1979).

45

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SPECIALIZED PURIFICATION PROCEDURES

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recovered in low yields when methods which were developed for use with soluble proteins are employed. In addition, detergents used to solubilize membrane proteins will also bind to reversed-phase resins. Therefore, modified methods have been developed for the successful application of reversed-phase chromatography to the analysis of membrane proteins. Membrane proteins can be directly solubilized into concentrated solutions of trifluoroacetic acid or formic acid, thus alleviating problems associated with the use of detergent^.^^,^^ Samples must be applied directly to columns, since prolonged treatment can lead to esterification of serine and threonine residues, deamination of glutamine and asparagine, and cleavage of Asp-Pro bonds. An alternative is to solubilize proteins with detergent, and then remove the detergent and lipid by chloroform-methano1 e x t r a ~ t i o nProteins .~~ are removed from the water-solvent interface or recovered by centrifugation and then solubilized in acid as above. In some cases, detergent solubilized samples have been applied directly to columns and then eluted with organic solvent^.^' Once bound to the column, proteins are typically eluted with a gradient of acetonitrile or 2propanol. Improved resolution and yield can be obtained by using columns developed for hydrophobic interaction chromatography rather than re~ . ~ ~ ~contain ~ ~ ~ ~ ~ hydrophobic liversed-phase c h r o r n a t ~ g r a p h y . ~These gands of shorter chain length or lowe~hydrophobicitysuch as C3, C4, or phenyl groups. In addition some of these columns have a lower ligand density and a secondary hydrophilic phase (e.g., Bio-Rad BioGel TSK Phenyl-5PW). Proteins can be eluted at lower solvent concentrations if less polar solvents such as 2-propanol are used instead of methanol or a ~ e t o n i t r i l eIn . ~ some ~ cases the use of solvent mixtures such as acetonitrile-propanol have increased r e s ~ l u t i o n .Use ~ ~ of poor ion-pairing agents, such as phosphoric or hydrochloric acid rather than trifluoroacetic acid, will also reduce the strength of hydrophobic binding.46 In addition, when HPLC-suitable columns are used, yields are better with large-pore ~ ' general, steps to reduce the amount of time in columns (100 n ~ n ) . In which proteins remain bound to the column will increase yields. Miscellaneous Chromatographic Methods

Other methods include adsorption on h y d r ~ x y l a p a t i t e ,chromatofo~~ c ~ s i n g isoelectric ,~~ focusing,26 and sucrose or glycerol density gradiM. R. Sussman, Anal. Biochem. 169, 395 (1988). J. Heukeshoven and R. Dernick, J. Chromatogr. 326, 91 (1985). 50 K. R. Brunden, C. T. Berg, and J. F. Poduslo, Anal. Biochem. 164, 474 (1987). j' S . C. Goheen and T. M. Chow, J . Chromatogr. 359, 297 (1986). 52 G. E. Tarr and J . W. Crabb, Anal. Biochem. 131, 99 (1983). " B. R. Aton, 9. J. Litman, and M. L. Jackson, Biochemistry 23, 1737 (1984).

49

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PURIFICATION OF MEMBRANE PROTEINS

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-

e n t ~ . ' The ~ , ~principles ~ involved in attaining high resolution and high yields of functional membrane proteins with these methods are the same as those discussed above.

Phase Separation

Elevated temperatures tend to reduce the solubility of detergents and induce phase separation. The temperature at which phase separation occurs is a characteristic of the detergent used (Triton X-100, 64": Triton X-114, 20").54When phase separation occurs, hydrophilic proteins partition into the aqueous phase and hydrophobic proteins partition into the detergent phase. Therefore, phase separation after solubilization of proteins with Triton X-114 has been proposed as a practical way to separate peripheral from integral membrane proteins. Experience has shown, however, that many proteins do not follow this rule. For example, integral membrane proteins with very large hydrophilic regions, such a s the nicotinic acetylcholine receptor (AchR) and the a subunit of the Na+ channel, partition into the aqueous phase.s5 Phase separation of other detergents (e.g.,Triton X-100 and cholate) can be induced at nondenaturing temperatures by the addition of ammonium sulfate.56 It is important to note that these methods cause a substantial alteration in the composition of the solution and many proteins may not be stable under these conditions.

Transport SpeciJicity Fractionation

Goldin and colleagues have used the calcium oxalate density perturbation method to isolate the synaptosomal ATP-dependent CaZ+ uptake system.s7 Solubilized proteins were reconstituted into oxalate-containing artificial vesicles using an 80-fold excess of exogenous lipid. Under these conditions, each vesicle was expected to contain one protein (or at most a few). The addition of ATP induced the vesicles to take up Ca2+.Calcium oxalate formed and precipitated within the vesicles, thereby greatly enhancing their density. These vesicles were then purified 100-fold by density gradient centrifugation. Although this technique takes advantage of the specific transport properties of ion channels, the number of successful applications is limited. C. Bordier, J. Biol. Chem. 256, 1604 (1981). B. F. X. Reber and W. A. Catterall, J. Biol. Chem. 262, 11369 (1987). 56 C. R . Parish, B . J . Classon, J . Tsagaratos. I . D. Walker, L. Kirszbaum, and 1. F. C . McKenzie, Anal. Biochem. 156, 495 (1986). s7 D. Papazian, H. Rahamimoff, and S. M. Goldin, Proc. Natl. Acad. Sci. U . S . A . 76, 3708 (1979).

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Reconstitution of Integral Membrane Proteins Reconstitution of a functionally active membrane protein involves the incorporation of the solubilized membrane protein into artificial membranes with preservation of the original function of the protein. Many proteins are only functionally active when correctly positioned in a lipid bilayer. The ion flux through channels such as AchR and the Na+ channel can only be measured when these proteins are reconstituted into lipid vesicles of an appropriate composition. Reconstitution is therefore an integral part of many purification schemes. Generally, reconstitution involves the addition of detergent-solubilized lipids to a detergent-solubilized protein sample. Subsequent removal of the detergent results in the formation of lipid bilayer structures and vesicles. It is therefore important that a detergent which is easily removed during reconstitution be used (e.g., cholate. deoxycholate, and octylglucoside). The most common Other method of reconstitution involves removing detergent by dialy~is.~' methods for detergent removal include gel filtration, preferential adsorption, and d i l ~ t i o n . ' ~See , ' ~ E. Racker for a full description of reconstitution methods.58

Renaturation of Functionally Active Proteins after Purification The premise of most of the techniques described so far has been that activity must be maintained at each step in the purification. Nevertheless, solubilization conditions which maintain activity often reduce chromatographic resolution. Mild nonionic detergents such as Triton X-100 often fail to prevent aggregation or to dissociate many protein complexes. On the other hand, harsh ionic detergents such as sodium dodecyl sulfate (SDS) thoroughly disperse proteins into monomers, but cause a loss of functional activity. Hjerten et al. have proposed a different approach to purification of membrane proteins.59 They suggest that proteins should be solubilized and purified in SDS. After purification proteins can then be renatured by removal of SDS. Several membrane-associated proteins, including a 5'-nucleotidase from Acholeplasma laidlawii and a neuramidase from influenza virus, have been successfully purified in SDS and renatured by dialysis against heptaoxyethylene lauryl ether (a neutral detergent). There is additional evidence to support this approach. For example, bacteriorhodopsin solubilized in SDS still possessed approximately 50% 58 59

E. Racker, this series. Vol. 55, p. 699. S. Hjerten, M . Sparrman, and J.-L. Liao, Biochim. Biophys. Acta 939, 476 (1988).

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PURIFICATION OF MEMBRANE PROTEINS

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of its a-helical s t r ~ c t u r e . Even ~ " when bacteriorhodopsin was completely denatured with formic acid-ethanol, it could be renatured by addition of phospholipids, cholate, and retinal. Removal of the cholate by dialysis resulted in functionally reconstituted protein. Whether the purification of integral membrane proteins under denaturing conditions followed by functional reactivation will become a generally applicable method remains to be seen. It may be that this method is limited to relatively small, single-subunit proteins.

Genetic and Immunological Methods

The past decade has seen an explosion in the development of new genetic and immunological techniques. Many of these have direct application to the identification and purification of membrane proteins. The advent of techniques to isolate and sequence full-length cDNA clones of genes has made possible the comparison of primary sequences from important membrane proteins. It appears that many membrane proteins belong to superfamilies sharing a common ancestral gene. Three of these superfamilies include the glycine, GABA, and nicotinic acetylcholine receptors, which are ligand-gated ion channels; the voltage-gated Ca2+,K+, and Naf ion channels; and the P-adrenergic and rnuscarinic acetylcholine receptor^.^' This last family also includes rhodopsin. Identification of the conserved regions which define membership in these superfamilies has provided scientists with a strategy for identifying and isolating new membrane proteins. First, an oligonucleotide probe is synthesized which matches a conserved sequence. This probe is then used in hybridization studies to identify closely related genes. After obtaining the sequence to the gene, a peptide is synthesized (10-20 amino acids) based on this sequence, and then used to prepare polyclonal antiserum. The antibodies from this serum can be used to determine the subcellular localization of the protein and to purify the protein by immunoaffinity methods. As an alternative method, the cloned gene can be attached in frame t o another gene, such as p-galactosidase or protein A, and this fusion gene can be expressed in E. coli. Either of the fusion proteins produced by this method can be easily isolated and used to prepare antisera. In this way, previously unidentified proteins can be isolated based on their homology to known proteins. In a variation of this method, Zinn et al. identified a K.-S. Huang, H. Bayley. M.-J. Liao, E. London, and H. G. Khorana, J . Biol. Chem. 256, 3802 (1981). C. F. Stevens, Nature (London) 328, 198 (1987).

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membrane protein from grasshopper (fasciclin I ) , which is important for neurite outgrowth.62They wished to find a similar protein in Drosophila so that the function of fasciclin could be studied by genetic deficiency analysis. When antibodies against grasshopper fasciclin 1 did not crossreact with Drosophila proteins, this group successfully used an oligonucleotide probe to identify the gene for the putative Drosophila protein. It is now common practice to identify and isolate genes based on their linkage to genetic diseases. Orkin identified a gene believed to be linked to chronic gran~lomatosis.~~ Analysis of the sequence identified several hydrophobic regions and putative glycosylation sites, indicating that the gene product was an integral membrane glycoprotein. Antibodies were prepared against a synthetic peptide and then used to identify the protein as cytochrome b from the NADPH oxidase system. Methods of Sample Concentration and Analysis

Methods of protein concentration, analysis, and quantitation play an important role in every purification protocol. These are general methods which apply to both soluble and membrane proteins. However, the application of these methods to detergent-solubilized membrane proteins can pose numerous problems and these are discussed below. 1 . Methods of concentrating samples: At present there are no concen-

tration methods which are widely applied to integral membrane proteins. In most cases, concentration methods are avoided. Proteins can be solubilized at relatively high concentrations (5-10 mg proteinlml), and methods such as gel filtration or gradient centrifugation, which require small concentrated fractions, may then be used as the initial purification step. Subsequent steps use methods such as affinity chromatography and ion exchange, which can be used with dilute samples. These latter methods also have the effect of concentrating samples. If concentration methods must be used then efforts should be made to avoid dramatic changes in detergent concentrations or protein-detergent ratios. Changes in these conditions can lead to either aggregation or inactivation. Methods of concentration fall into four categories: (a) precipitation, (b) filtration, (c) evaporation, and (d) absorption. a. Precipitation by ammonium sulfate or polyethylene glycol (PEG) is frequently used, but its primary application has been for fractionation rather than concentration. Ice-cold acetone (9 vol/vol sample) or trichloroacetic acid (10-20% wlv) may also be used to precipitate proteins. This harsher method tends to denature proteins. In 62

63

K. Zinn. L. McAllister,

and C . S. Goodman, Cell 53, 577 (1988). S. H. Orkin, TIC 3, 149 (1987).

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addition, precipitation methods d o not work well with protein concentrations below 0.1-1 mglml. b. Samples may also be concentrated by passing aqueous solutes through a filter membrane which retains the proteins of interest. Solute movement is accelerated by pressure (e.g., Amicon stirred cell), vacuum (e.g., Schleicher and Schuell collodion bag apparatus), or centrifugation (e.g., Amicon Centricon). The collodion bag apparatus and the Micro-ProDiCon (Biomolecular Dynamics) are both capable of simultaneously dialyzing and concentrating samples. Detergents with large micelle sizes (e.g., Triton X-100) will be retained by filters or membranes resulting in the concentration of both protein and detergent. A final problem with filtration is that many membrane proteins form irreversible aggregates on the filter. c. Evaporation and lyophilization are rarely used, although they may be applicable to membrane proteins solubilized in organic solvents. d. Finally, two methods of absorption are occasionally used. In the first case, the sample is placed in a dialysis bag which is then placed in a dry bed of NaCI, sucrose, Sephadex, Ficoll, o r PEG. As an alternative, dry Sephadex is added directly to the sample. After the resin has swelled, the concentrated sample is recovered by centrifugation. 2. SDS-PAGE: A widely used method for analysis of chromatographic fractions is polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS).64In addition, methods for obtaining partial amino acid sequences are now sensitive enough to be performed on proteins eluted from electrophoretic gels. This electrophoresis method may require modification when used with some membrane proteins. SDS may not be sufficient to ensure denaturation, in which case 8 M urea should be included in the sample buffer o r in the gel buffer^.^' High concentrations of 2-mercaptoethanol (1-5%) may cause p r o t e ~ l y s i s This . ~ ~ problem is alleviated by using dithiothreitol (1-2 mM). Standard protocols suggest boiling samples for 3 min prior to electrophoresis. However, some membrane proteins, such as the nicotinic ACh receptor, the muscarinic receptor, the P-adrenergic receptor, rhodopsin, H+-ATPase, and lactose permease are aggregated by boiling. An alternative is to incubate samples at 25-60" for 20-90 min. It may be necessary to increase the concentrations of SDS and reducing agent in the sample buffer. Not all proteases will be inactivated by this treatment, and protease inhibitors may need to be 65

U. K. Laemmli, Nature (London)227, 680 (1970). C. H. Siu, K. A . Lerner, and W . F. Loomis, J. Mol. Riol. 116, 469 (1977). J. B. C. Findlay, in "Biological Membranes: A Practical Approach" (J. B. C. Findlay and W. H. Evans, eds.), p. 179. IRL Press, Oxford, 1987.

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added to the sample buffer. It is also possible to electrophorese proteins under nondenaturing conditions by using the Laemmli discontinuous buffer system, but replacing SDS with other detergents such as Triton x-100.6~ 3. Protein Assays: One of the most widely used methods for the quantitation of protein is the Lowry a s ~ a y However, .~ many of the compounds used in purification of membrane proteins interfere with this assay.69 Nonionic detergents such a s Triton X-100 will precipitate in the Lowry protein assay. These detergents can either be removed by centrifugation o r maintained in solution by addition of SDS to the assay.69 Nonionic detergents do not precipitate in the Bradford and BCA protein assays, which have been proposed a s alternatives t o the Lowry However, these assays are also susceptible to interference. For example, the BCA assay is susceptible to interference by some lipids.72 Whichever method is chosen, the influence of detergent- and lipid-containing buffers should be tested on both the assay blank and protein standards (see [61 in this volume for a complete discussion of protein assays). Summary

As stated at the beginning of this chapter, it is not possible to present a single step-by-step protocol for the purification of all membrane proteins. We have discussed many of the individual techniques employed to purify membrane proteins and the problems associated with their application. A successful purification protocol will require the use of a combination of these techniques. It should be clear from the examples given, however, that affinity chromatography is by far the most useful technique available. The greatest single obstacle to performing a successful purification is the ability t o maintain solubilized proteins in fully dispersed monomeric micelles without inactivating the protein. The best combination of techniques and the optimal conditions for their use can be determined only by trial and error. This process should become easier as a greater number of examples become available and a s greater use is made of HPLC and FPLC techniques. C. Bordier, W. F. Loomis, J. Elder, and R. Lerner, J . Biol. Chem. 253, 5133 (1978). 0. H. Lowry, N. J. Rosebrough, A. L. Farr. and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 69 G. L. Peterson, Anal. Biochem. 100, 201 (1979). 'OM. M. Bradford, Anal. Biochem. 72, 248 (1976). 7' P. K. Smith, R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H . Gartner, M. D. Provenzano, E. L. Fujimoto, N. M. Goeke, R . J . Goeke, B . J. Olson, and D. C. Klenk. Anal. Biochem. 150, 76 (1985). 72 R. J . Kessler and D. D. Fanestil, Anal. Riochcm. 159, 138 (1986). 67

[391 Purification of D N A - B i n d i n g P r o t e i n s by Site-Specific DNA Affinity Chromatography By SHLOMO EISENBERG, STEPHENC. FRANCESCONI, CHRISCIVALIER, and SCOTTS. WALKER

The feasibility of using site-specific DNA affinity for the purification of specific DNA-binding proteins was tested by Herrick,' who demonstrated that lac repressor bound more tightly to plasmid DNA containing the lac operator than to a DNA lacking the operator when the DNA was coupled to either a cellulose or a Sepharose matrix. As a result of the tight binding, elution of the protein from the site-specific DNA-cellulose column required buffers with high ionic strength. Consequently, fractionation through the site-specific DNA-cellulose matrix resulted in considerable purification, efficiently separating the lac repressor from other DNAbinding proteins. Further work showed that an even higher ionic strength was required when a specific DNA was present in multiple copies.' Following these initial observations, various procedures for site-specific DNA affinity have been reported. In one, complementary oligodeoxynucleotides corresponding to a specific recognition sequence were chemically synthesized, annealed, and ligated to produce oligomers, and subsequently linked covalently to a Sepharose r n a t r i ~ The . ~ protein of interest was purified by passing a crude fraction through the affinity column in the presence of random DNA, which adsorbs the proteins that bind DNA nonspecifically. Usually sequential passages of the purified fraction are needed to obtain a highly purified protein. Since specific DNA-binding proteins frequently interact with any DNA, albeit at a lower affinity, in order to prevent losses of the protein of interest because of such binding, this procedure requires careful calibration of the amount of competitor DNA added to the crude fraction. Another procedure entails the construction of a plasmid containing the recognition sequence in multiple tandem copies arranged in a head-to-tail direct orientation to maximize the stability of the insert. After plasmid amplification and purification, the DNA is coupled to cellulose to yield the DNA affinity re sir^.^.^ To separate the protein of interest from proteins 1

'

G . Henick, Nucleic Acids Res. 8, 3721 (1980). M. Oren, E. Winocour, and C. Prives, Proc. Nntl. Acad. Sci. U.S.A. 77,220 (1980). J . T. Kadonaga, and R. Tjian, Proc. Narl. Acad. Sci. U.S.A. 83, 5889 (1986). P. J. Rosenfeld, and T. J . Kelly, J . Biol. Chem. 261, 1398 (1986). E. A. O'Neil and T. J. Kelly, J. Biol. Chem. 263, 931 (1988).

METHODS IN ENZYMOLOGY. VOL.

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Copyright O 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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that bind tightly to DNA nonspecifically, a chromatography step involving a nonspecific DNA-cellulose column can be introduced as a prelude t o the fractionation through the site-specific DNA resin.4 A different DNA affinity method, based on the strong biotin-streptavidin interactions, has also been d e ~ e l o p e dIn . ~this procedure a specific biotinylated DNA substrate, prepared in vitro, is incubated with a crude extract containing the specific DNA-binding protein. The proteinbiotinylated DNA complexes formed in solution are then trapped on a streptavidin-Sepharose column by the strong biotin-streptavidin interactions. Proteins are eluted from the column by salt, disrupting the proteinDNA interactions, and leaving the biotinylated DNA bound to Sepharose because of its high affinity to streptavidin. This procedure is useful for analytical identification of specific DNA-binding proteins, but is not well suited to large-scale protein purification. In this chapter we describe a general DNA affinity procedure. This method, in combination with simple, conventional protein chromatography steps, is rapid and suitable for large-scale preparations of specific DNA-binding proteins. The procedure will be illustrated by its application to purification of OBFI, a yeast ARS DNA-binding protein.' General Scheme for the Construction of Plasmid DNA Containing a Multicopy Insert in Direct Head-to-Tail Orientation The diagram in Fig. 1 describes schematically a simple method used for constructing a plasmid with a multicopy insert. The vector utilized in this construction is pUC19 (BglII), which is the canonical pUC19 in which the SphI restriction site, situated in the polylinker, has been converted to a BglII site. The DNA fragment of interest is inserted into the polylinker region of pUC19 (BglII) between the BglII and BamHI sites. Any restriction site present in this polylinker region can be used for the cloning. The insert is then excised with two pairs of enzymes, EcoRIl BglII and HindIIIIBamHI, which generate two fragments, each containing a full copy of the insert, and each terminated at one end by either a BglII or a BamHI restriction site. When these fragments are incubated with ligase in the presence of linear pUC19 (BglII) DNA, terminated by EcoRI and Hind111 sites, the two fragments are joined at their BglI1 and BamHI sites because the two are complementary, and are inserted together into the EcoRI and Hind111 termini of the vector. The result of this reaction is the production of a plasmid that contains a dimer copy of the L. A. Chodosh, R . W . Carthew, and P. A. Sharp, Mol. Cell. Biol. 6,4723 (1986).

' S. Eisenberg, C. Civalier, and B.-K. Tye, Proc. Natl. Acad. Sci. U.S.A. 85, 743 (1988).

FIG.1. Construction of plasmids containing an insert in tandem copies in a direct, headto-tail orientation: The vector used is pUC19 (BgIII) (provided by D. Lazinski of this department) in which the SphI restriction site, situated in the polyl~nker,was converted to a BglII site. The letters designate some of the restriction sites present in the linker: RI (EcoRI), H (HindIlI), Bg (BglII), BH (BamHI). The open bar reprcscnts the polylinker site in the plasmid. The thick arrows represent the insert. The thin lines represent vector DNA. (a) Polylinker site; (b) introduction of insert; (c) excision of insert a s two separate fragments; (d) ligation of fragments and reinsertion into plasmid.

inert in a head-to-tail configuration. This procedure can be repeated successively to generate a plasmid that contains reiterated sequences, situated in a tandem direct orientation. The direct orientation of the insert is necessary to minimize sequences of dyad symmetries, which are prone to recombination in Escherichia c01i.~In order to reduce recombination and thus a high-frequency loss of the multicopy insert, it is also advised to propagate the recombinant plasmid in a RecA- E. coli strain. We have used this scheme to produce a plasmid containing 48 copies of a synthetic nucleotide sequence corresponding to the "footprinted" OBFl region in ARS120.' Specifically, a trimer of the following sequence: CAAGTGCCGTGCATAATGATGTGGGT, prepared as previously den J. Collins, Cold Spring Harbor Symp. Quant. Biol. 45, 409 (1981).

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scribed,' was inserted into the SalI site in the polylinker region of pUC19 (BglII) and a plasmid containing 48 repeats of the above sequence was constructed, using the scheme described in Fig. 1. Plasmid DNA, amplified on a large scale in E. coli, was isolated by the standard alkaline lysis procedure, and the purified DNA was coupled to cellulose by the Alberts and Herrick9 method to yield the DNA affinity matrix.

Procedure for Isolating DNA-Binding Protein (OBF1)

Buffers

Buffer A: 1 M sorbitol, 1 M KCI, 10 mM EDTA, 50 rnM Tris-HC1, pH 7.5 Buffer B: As buffer A, except that sorbitol was omitted Buffer C: 10% glycerol, 1 M KCl, 10 mM EDTA, 20 mM 2-mercaptoethanol, 0.1 mM PMSF (phenylmethylsulfonyl fluoride), and 50 mM HEPES, pH 7.5 Buffer D: 20% glycerol, 20 mM 2-mercaptoethanol, 1 mM EDTA, 0.1 mM PMSF, and 50 mM Tris-HC1, pH 7.5 Buffer E: 20% glycerol, 50 mM KCl, 20 mM 2-mercaptoethanol, 1 mM EDTA, 0.1 mM PMSF, and 50 mM imidazole-HCI, pH 6.9 Buffer F: As buffer E, except that KC1 is replaced by 50 mM NaCl Buffer G: As buffer F, except that NaCl is increased to 0.4 M Growth of Yeast Cells Saccharornyces cerevisiae, strain CYY2 (ura3, ga12, Apep4 ::URA3), provided by Chris Tachibana, was grown in 7 liters of YP medium (1% yeast extract, 2% Bacto-peptone) + 5% dextrose to an ODm of 5.0, in a Maribushi fermenter (MDR-2c3r, B. E. Maribushi Co., Tokyo, Japan). The cells were harvested by pouring the culture onto ice followed by centrifugation for 5 min in a Sorvall centrifuge, GS-3 rotor, at 5000 rpm and 4". The pellets were resuspended in buffer A to a final concentration of 0.5 g of cells/ml. This suspension was frozen in liquid nitrogen and stored at -90". A 7-liter culture yielded about 105 g of wet cell paste.

All operations were carried out at 0-4", unless indicated otherwise. Fraction I: A frozen cell suspension, equivalent to 670 g of wet cell paste, was thawed and diluted by adding one-third volume of buffer B, B . Alberts and G . Henick, this series, Vol. 21, p. 98.

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followed by addition of spermidine chloride, dithiothreitol, and PMSF to 20, 1, and 0.1 mM, respectively. This cell suspension was disrupted batchwise, with 0.5-mm glass beads, in a 350-ml chamber, by 30-sec pulses with an intermittent 30-sec cooling period, using a Bead-Beater (Biospec Products, Bartlesville, OK). Disrupted cells were centrifuged in a Sorvall centrifuge for 60 min. in the GSA rotor at 12,000 rpm. The supernatant material designated fraction I was further fractionated. Fraction 11: Solid ammonium sulfate (0.33 glml) was gradually added to fraction I and the suspension was stirred for 60 min. The resulting precipitate, harvested by centrifugation for 50 min in a Sorvall centrifuge, GSA rotor at 12,000 rpm, was resuspended in buffer C and centrifuged for 60 min in a Beckman ultracentrifuge Ti 45 rotor at 38,000 rpm. Fraction 111: Fraction I1 was dialyzed against buffer D and loaded on a DEAE-cellulose column (30 x 11 cm) equilibrated with the same buffer. After applying fraction 11, the column was washed with 2.5 bed volumes of buffer E followed by 3.5 bed volumes of buffer E + 100 m M KCl. DNA-binding activity, which was collected in 25-ml fractions, was eluted with 3.5 column bed volumes of buffer E + 250 m M KCI. This fraction contained about 50% of the total protein loaded on the column. Fraction IV: Fractions from the DEAE-cellulose column, containing the DNA-binding activity, were pooled (fraction 111) and diluted with buffer F lacking NaCI, until a conductivity equivalent to buffer F was achieved. Diluted fraction I11 was applied to a Bio-Rex 70 column (12 x 4 cm), preequilibrated with buffer F. The column was washed first with three column bed volumes of buffer F followed by a linear 50 to 750 mM NaCl gradient (a total of 10 column bed volumes). Fraction V: Fractions from the Bio-Rex 70 column, active in specific ARS DNA-binding, were pooled (fraction IV) and the conductivity of fraction 1V was adjusted to that of buffer G by adding a 5 M NaCl solution. Fraction IV was then applied to the site-specific DNA-cellulose column, equilibrated with buffer G. The column was washed with buffer G (three bed volumes) followed by a linear 0.4 to 1 M NaCl gradient. Fractions of 10 ml each were collected and those active in DNA binding (fraction V) were frozen in liquid nitrogen and stored at -90". The OBFl protein, prepared by this procedure, was stable for at least 6 months without apparent loss in activity. The effectiveness of the site-specific DNA affinity step is demonstrated in Fig. 2. Figure 2 shows the profile of proteins present after electrophoresis on an SDS-polyacrylamide gel in fraction 1V (lane a), in the flow-through of fraction V (lane b) and in fraction V (lane c), which is the protein eluted from the DNA affinity column by a salt gradient. By Coomassie Brilliant Blue staining of fraction V (lane c) a major protein

SPECIALIZED PURIFICATION PROCEDURES

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0.1

[391

0.2 0.4 0.75 1.5 3.0

Protein (ng) FIG.3. Titration of OBFl in the DNA-binding reaction: The DNA substrate for the binding reactions was prepared by digesting the plasmid pSN1207 (containing the OBFl binding site of ARSIZO) with EcoRI and HitldIII and labeling the termini with [a-3ZP]dATP by reverse transcriptase. The reaction mixtures were as described in the text. Increasing amounts of purified OBFl (fraction V) were added and the products of the reaction were electrophoresed on agarose gel and identified by autoradiography. The bottom band represents the DNA fragment that binds OBFI; the top band represents the remaining portion of the pSN 120 plasmid; the middle band, which appears upon the addition of the OBFl protein, represents the OBFI-DNA complexes.

merous advantages to this approach, especially when large-scale protein preparation are considered. (1) The DNA affinity matrix has a high protein capacity, which is determined by the number of copies of the protein recognition sequence inserted into the plasmid. It is possible to construct a plasmid in which the protein-binding sequences comprise at least 50% of the total DNA content. Once inserted into the plasmid, followed by DNA amplification in E. coli, very large quantities of these sequences could be obtained. Since about 1 mg of DNA can be coupled to 1 g of cellulose powder, it is relatively easy to prepare sufficient quantities of the DNA affinity matrix for a large-scale purification; (2) the protein appears to bind more tightly to the DNA on the column than in solution. We found that in solution the OBFl-DNA complex could not be formed in the presence of

[401

i

1

-..

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NaCl concentrations above 0.2 M, while the binding of ORFl to the multimeric site-specific DNA-cellulose matrix occurred at 0.4 M NaCI. The tight binding to the site-specific DNA-cellulose column ensures effective separation of OBFl from other DNA-binding proteins by simple salt elutions and, when combined with standard ion-exchange chromatography, a highly purified protein may be obtained. We believe that a similar approach may also be utilized and applied toward the purification of other site-specific DNA-binding proteins. Acknowledgments This work was supported by an American Cancer Society Grant MV-368 awarded to S.E. S.C.F.was supported by a USHS US Public Health Service National Research Service Award, GM07407.

[401 Purification of Glycoproteins

Among the various posttranslational modifications which proteins undergo, glycosylation still remains enigmatic in many instances. It is known that glycosylation is involved in protein transport. Glycosylation also appears to affect the half-lives of certain proteins, and in other cases, it may up or down regulate the biologic activity of a hormone or mediator. However, often, no biological change is found upon deglycosylation. Irrespective of the biochemical role of glycosylation, this feature may provide a unique aspect aiding in protein purification, as well as creating problems. In the latter case, chromatographic or electrophoretic heterogeneity of a homogeneous protein can be the result of heterogeneity in its carbohydrate moieties. In this series, methods have been previously presented for the purification of glycopeptides' as well as their analysis and deglyc~sylation.~,~ In this chapter, I will present particular techniques which have proved useful in the purification and analysis of glycoproteins. R. K. Merkle and R. D. Cummings, this series, Vol. 83, p. 27; Vol. 138, p. 232. B. Nilsson and D. Zopf, this series, Vol. 83, p. 46. ' H. T. Sojar, N. T. Thotakura, and 0. P. Bahl, this series, Vol. 138, pp. 341, 350. l

METHODS IN ENZYMOLOGY. VOL. 182

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Copyright 0 1990 hy Academic Press, Inc. All rights of reproduction in any form reserved.

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7 TABLE I LECTINSCOMMONLY USED FOR GLYCOPROTEIN PURIFICATION Lectin

Specificity

Concanavalin A (ConA)

Structures containing alinked mannose such as the N-acetylglucosaminy l core disaccharide in Asnlinked glycoproteins

Lentil lectin

Similar to ConA. narrower specificity in that substitution to the core saccharide may enhance or diminish binding Recognizes Gal(P1-3)GalNAc

Peanut agglutinin

Jacalin

Recognizes Gal@ I3)GalNAc

Wheat germ agglutinin

Recognizes N-acetylglucosamine dimer or trimer structures/chitobiose. Also lower affinity interactions with sialic acid, mannosecontaining structures Reacts with terminal (nonreducing) Gal, less strongly with GalNAc.

Ricinus communis agglutinin I (RCA I)

Soybean agglutinin

Recognizes terminal a- or 6GalNAc.

Comments Broad use due to ubiquitous a-linked mannose containing structures. pH optimum 5.6. As with other mannosebinding lectins, Ca2+and MnZ+are part of the binding site. Buffers may be Ca2+/Mn2+free. Avoid chelators Sometimes employed sequentially with ConA.

Common core structure to many O-linked membrane glycoproteins; however, often requires sialidase treatment to expose binding disaccharide Unlike peanut lectin, this lectin will recognize substituted disaccharide, eliminating the need for sialidase treatment. Specific for O-linked oligosaccharides Widely used for membrane glycoprotein purification, which frequently contain Asn-linked oligosaccharides

May be useful with both N- and O-linked structures. Since these residues are often not terminal, but penultimate, treatment with exoglycosidases (neuraminidase, fucosidase) may uncover binding properties Complementary to RCA 1 lectin

Lectin Affinity Chromatography

Lectins are proteins frequently found in plant sources, although examples exist in many other species as well. In general, these proteins are capable of reversible high-affinity binding to specific carbohydrate sequences (Table I). A solution of impure protein, which may be quite dilute, is allowed to equilibrate with the affinity resin in a buffer such as Tris o r phosphate. In general, the binding is optimal in high ionic strength

[401

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buffers (up to 0.5 M NaCl), so the ion-exchange properties of the matrix are limited. Unbound proteins are washed away and the protein of interest is eluted with specific carbohydrate ligands. Since the resin can be eluted with small volumes of ligand solution, the sample is often concentrated as well as purified. It is important that sufficient time be allowed for equilibration of the ligand with the lectin; an easy way to accomplish this is by batch adsorption to the resin on a rotary o r shaking platform. The binding characteristics must be empirically determined for each protein, but binding is usually complete within several hours at 4'. Mixing of the protein solution with resin must not be too vigorous (i.e., no vortex mixing or magnetic stirrers) for two reasons: First, many supports will fragment or collapse, generating a gel which will not pass buffer when placed into a column; second, many proteins denature when whipped to a foam. The purification of membrane-bound cellular receptors has relied heavily on lectin chromatography and exemplifies some special problems. First, since a typical receptor constitutes a miniscule fraction of solubilized membrane protein, large quantities of cells or tissues are often required to obtain purified protein. Second, it is often found that a significant percentage of glycoprotein does not adsorb to the lectin support. Finally, the presence of detergent may interfere with the chromatography. With regard to the first issue, the methodology may easily be scaled from resin volumes of 1 ml to several hundred milliliters; pilot experiments performed carefully on analytical quantities may be expanded to a preparative model. Therefore, the processing of large volumes of detergent-solubilized protein presents less of a problem. In addition, lectin affinity chromatography often yields 50- 100% recoveries. The problem of incomplete binding to the lectin support is usually due to one of the following: (I) there may be more than one gene product with similar assay properties; (2) the support is overloaded; or (3) there is heterogeneity in the carbohydrate moiety. Sometimes, detergent or solvent effects may interfere with affinity binding. As regards the use of detergents, the following guidelines are useful. Nonionic detergents such as Triton X-100 and NP-40 are compatible with most commercially available lectin supports. Sodium dodecyl sulfate appears compatible in concentrations <0.05% (wlv). The use of sodium deoxycholate is discouraged due to the tendency of this detergent to form viscous gels in the presence of salts. Sodium cholate, however, has been successfully used, although interesting effects may be observed. For example, when dopamine D2 receptors from bovine brain are chromatographed on wheat germ agglutinin-agarose (WGA), the proportion of binding receptors could be increased from 65 to 85% by increasing the concentration of detergent and salt from 0.3% sodium cholate/l .O M NaCl

532

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to 0.35% sodium cholate/l. 16 M NaC1.4 Correspondingly, as the concentration of salt and cholate is reduced to 30% of the initial conditions (0.1% cholate/0.33 M NaCI), <5% of the receptors are bound to the WGAagarose column. Therefore, when dealing with detergents, it is wise to consider the effect of the detergent and the salt concentration, as these are known to affect micelle structure. Finally, two other detergents, CHAPS and digitonin, have been successfully employed with lectin c ~ l u r n n sIn . ~the ~ ~ case of digitonin, two items deserve comment: First, this material is toxic as an inhaled dust and precautions must be taken; second, most "spectral grade" digitonin preparations must be boiled to achieve solubility, and therefore the actual concentration of detergent is in doubt. We have found, however, that the product marketed by Waco Chemical Company (Waco, TX) dissolves at room temperature and remains soluble up to 1 week after preparation of the solution. General Approach to Lectin Chromatography

Choosing a Lectin. Kits are now commercially available (for example, Vector Laboratories, Burlingame, CA) providing limited quantities of common lectins for tests of binding. If the mobility of the protein in question is known, a Western blot of crude SDS-PAGE-resolved material can be performed using a lectin in place of an antibody. Lectin binding can be detected using either iodinated lectin or peroxidase-linked avidin and biotinylated lectins. Test titrations can also be easily performed. In 1.5-ml microcentrifuge tubes one adds varying amounts of glycoprotein preparation to 100-pl portions of a lectin-resin slurry using a batch technique and binding to the gel is measured. This can be done to optimize the time, pH, temperature, salt-detergent concentrations, if applicable, and the binding and elution conditions. For example, in the case of wheat germ agglutinin, binding to Asn-linked oligosaccharides occurs through sialic acid, N-acetylglucose, and N-acetylgalactosaminyl residues. Both high- and low-affinity binding occurs, with the latter apparently dependent on the content of sialic Low-affinity binding sites can be titrated with modest concentrations of N-acetylglucosamine (2.5-30 mM), while concentrations of 100300 mM will elute more avidly binding forms. As another example, the M. N. Leonard, R. A. Williamson, and P. G. Strange, Biochem. J. 255, 877 (1988). E. Hazum, 1. Schvartz, Y. Waksman, and D. Keinan, J. Biol. Chem. 261, 13043 (1986). J. W. Regan, H. Nakata, R. M. DeMarinis, M . G. Caron, and R. J . Lefkowitz, J. Biol. Chem. 261, 3894 (1986). V. P. Bhavandan and A. W. Katlic. J. Biol. Chem. 254, 4000 (1979). J. T. Gallagher, A. Morris, and T. M. Dcxter, Biochem. 3. 231, 115 (1985).

[401

PURIFICATION OF GLYCOPROTEINS

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binding and elution of glycoproteins from concanavalin A-Sepharose is often more effective at room temperature than at 4", binding to wheat germ agglutinin is unaffected by temperature, and binding to soybean or peanut lectin is enhanced at low temperature.' In some instances, glycoproteins may elute from lectin columns with a "trailing" pattern. To avoid this, allow one column volume of elution buffer to enter the column and stop elution for a period of time to allow the ligand to displace the glycoprotein (generally less than I hr will suffice). Scale of Experiment. It is not possible to predict in advance the scale of the chromatographic experiment since each glycoprotein will interact with lectin in a unique way, and many glycoproteins may compare for a limited quantity of lectin. Commercially available supports (Pierce, Pharmacia, Miles, Vector Laboratories) are usually supplied at a content of 110 mg of 1ectinJrnl gel. Typical values for the stoichiometry of binding of purified glycoproteins to immobilized lectins art: less than 20% of theoretical, probably reflecting both inactivation of the lectin by derivitization to the support and steric factors. It is also possible to take advantage of the different binding properties of glycoproteins in a complex mixture. When "overloaded," only the highest affinity proteins will adsorb, while in lectin excess weaker binding forms can also adsorb.I0 Similarly?if a glycoprotein adsorbs weakly to a support, while other components in the mixture bind avidly or not at all, the use of a long, narrow column may resolve the mixture. Commercially prepared lectin columns can be prohibitively expensive for such applications, but such a column can be easily prepared from commercially available reagents for a fraction of the cost. Expectations. Lectin affinity chromatography is rarely sufficient by itself for the purification of a glycoprotein. One should consider this approach as part of a general strategy for protein purification. It has been particularly useful in situations in which a limited amount of protein is available and requires a multistep purification procedure. For example, since the binding of glycoprotein to lectin is not affected by high salt, this technique allows for the simultaneous desalting, concentration, and purification of material following elution from an ion-exchange column, whereas dialysis of the large volumes might result in unavoidable loss of material. Likewise, a glycoprotein eluting in a large volume from a gel filtration column can often be applied directly to a lectin column for concentration purposes. Glycoproteins that have been eluted from an antibody or affinity column under harsh conditions, such as low pH or

lo

R. Lotan, G . Beattie, I. W. Hubbell, and G. L. Nicholson, biochemist^ 16, 1787 (1977). W. L. Adair and S. Kornfeld, J. Biol. Chem. 249, 4696 (1974).

534

SPECIALIZED PURIFICATION PROCEDURES

[401

with denaturants, may simply be diluted and neutralized prior to application to a lectin column. Finally, it should be noted that the sequential use of several lectin affinity columns offers a powerful selection method for glycoproteins. As an example, sialophorin (a major cell-surface glycoprotein of lymphocytes) that is abnormal in the X-linked disorder, Wiskott-Aldrich syndrome, can be purified in such a manner. Preliminary studies suggested that sialophorin did not adsorb to lentil lectin, but bound wheat germ agglutinin with a low affinity via its sialic acid residues. Whereas native sialophorin did not bind peanut lectin, a neuraminidase-treated sample did, suggesting the presence of the Gal@-1-3)GalNac disaccharide." Putting these preliminary studies together, the following purification scheme was developed. Sialophorin could be obtained in milligram quantities from the lymphoblastoid cell line, CEM, by solubilization of 10" cultured cells with NP-40, followed by application to lentil lectin-Sepharose. The nonadherent fraction was then adsorbed and eluted from wheat germ agglutinin-Sepharose and treated with neuraminidase to remove sialic acid residues. This fraction was next adsorbed to peanut lectinSepharose, eluted, and ultrafiltered prior to final purification on Sepharose C L d B . The yield of membrane-bound glycoprotein was 60%.12 Other Affinity Techniques

Antibodies. The interspecies differences in glycosylation patterns of proteins promotes formation of antibodies against these structures; thus reagent-grade mono- and polyclonal antisera can be raised.13 The screen for such determinants relies on the Western blot technique, in which samples are examined before and after chemical or enzymatic deglycos ylation. &tibodies recognizing carbohydrate epitopes may then be used in the affinity purification of glycoprotein; such selection often provides evidence for microheterogeneity in the carbohydrate portion of a glycoprotein. Boronate Af$nity Gels. Insoluble phenyl boronates have been used to purify nonenzymatically glycosylated serum proteins (e.g., hemoglobin AIC) from the serum of diabetics and normals. The basic chemistry is not specific for carbohydrate residues, but rather for vicinal cis-diols which " l2

'3

E. Remold-O'Donnell, D. M. Kenney, R. Parkman, L. Cairns, and F. S. Rosen, J . Exp. M e d . 159, 1705 (1984). E. Remold-O'Donnell, A. E. Davis, 111, D. Kenney, K. R. Bhaskar, and F. S. Rosen, 3. Biol. Chem. 261, 7526 (1986). J. L. Magnani, this series, Vol. 138, p. 484.

PURIFICATION OF GLYCOPROTEINS

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form a moderately stable, five-membered ring with the boronate in neutral pH buffers such as Tris-HCI. The complex can be reversed and the glycoprotein eluted by the addition of 0.2 M sorbitol to the column buffer. The diols must be coplanar for this reaction to proceed spontaneously. This feature imparts limited specificity to this method, since a number and variety of compounds contain this structure, including catechols, ribonucle~cacids, glycoproteins, etc. Following incubation of a sample containing cis-diols with the gel in a neutral buffer, such as phosphate-buffered saline, the gel is washed with buffer and then eluted with buffer containing sorbitol as a displacement molecule. This material is commercially available (Pierce Chemical Co.). Density Gradient Methods

Some of the most unusual eukaryotic glycoproteins contain as much or more mass as carbohydrate than as polypeptide. Such glycoproteins include the blood group substances, proteoglycans, ovarian cyst glycoproteins, and tracheal, gastric, salivary, or reproductive mucus glycoproteins or mucins. In contrast to the asparagine-linked glycoproteins, which possess a limited number of well-defined glycosylation sites (Asn-X-Thrl Ser sequences). these proteins contain the more heterogeneous 0-linked structures and exhibit great microheterogeneity. As a consequence of dense glycosylation with heterogeneous structures, these molecules exhibit extremely high-weight average molecular weights, and are often polydisperse. This feature, however, can be exploited by the technique of equilibrium density gradient centrifugation in solutions of cesium salt^'^.^^ with or without the inclusion of reducing agents, denaturing detergents, or chaotropes. Typical serum proteins band at densities of approximately 1.3 g/cm3and mucin-type glycoproteins at -1.45-1.5 g/cm3. Detection of Glycoproteins

A variety of methods are available for the sensitive and specific analysis of oligosaccharides attached to protein. These include gas chromatography-mass spectroscopy and, more recently, high-performance liquid chromatography. These methods are quantitative and require relatively small amounts of sample, but are technically not practical for the nonspecialist. In cases where the amount of glycoprotein is not limiting, the most reliable method for the nonspecialist would be electrophoresis in polyl4 Ii

J . M. Creeth, K. R . Bhaskar, and J . R. Horton. Biochem. J . 167, 557 (1977). K . K. Bhaskar and L. M . Reid, J . Biol. Chem. 256, 7583 (1981).

536

SPECIALIZED PURIFICATION PROCEDURES

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[401

-

acrylamide gels followed by specific staining. For the detection of oligosaccharide-containing polypeptides, periodate oxidation followed by staining with Schiff's reagent is relatively straightforward. This method is sensitive to approximately 3 pg of glycoprotein. The important points to consider are that false positive results may occur if SDS removal from the gel is incomplete. Because some glycoproteins do not fix readily, the following protocol is recommended. Protocol for PAS Staining oj'Glycoprotein~'~

This protocol requires approximately 7 hr following the completion of electrophoresis. 1. Perform SDS-PAGE in slab gels.

2. The gel is placed in a glass baking dish and submerged in 5% phosphotungstic acid (Sigma) in 2 N HC1. Rock gently for 90 min at room temperature. Drain. 3. Soak gel in 500 rnl 7% methanol-14% acetic acid for 1 hr. Repeat. This step leaches out the SDS. Drain. 4. Oxidize the oligosaccharides with 1% periodic acid in 7% trichloroacetic acid for 1 hr. Drain. 5. Remove excess periodic acid with 250 ml of 0.5% sodium metabisulfite in 0. I N HCI at room temperature. Initially, the gels will be stained with the iodine formed upon reaction of the periodic acid with the reducing agent. This amber color will fade as iodide is formed from iodine in approximately 1 hr. Drain when cleared. 6. Staining with Schiff's reagent (Pierce, Sigma) is performed by submersing the gel in this solution and allowing the pink color to develop in the dark on ice. It is possible to leave the sample at this point in a dark cold room overnight to examine in the morning. Color development is gradual, and stained bands are stable for several days. Alternative Protocol for Thymol-Suljiuric Acid Staining"

This method is slightly easier and is approximately 2-fold more sensitive. The time factor is similar, but since the stain is labile, gels should be photographed within several hours of color development.

1. Perform SDS-PAGE in slab gels. 2. The gel is placed in a glass baking dish and is fixed by submerging in 25% 2-propanol/lO% acetic acid. Rock gently for 2 hr at room ternperature. The gel will shrink significantly in this solvent as it dehydrates. l6

R . S. Leach, J . F. Collawn, and W. W. Fish, Biochemistry 19, 5734 (1980). Rauchsen, Anal. Biochem. 99, 474 (1979).

" P.

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----------p---pp-p-p

Drain. Repeat. The gel may be stored overnight in this solution. SDS and Tris-glycine must be leached from the gel. 3. Immerse the gel in the above solution containing 0.2% (vlv) thymol (Sigma) for 90 min with gentle rocking. The gel may become opaque. Drain.. 4. The gel is immersed in 80% sulfuric acid/20% (vlv) ethanol (just enough to cover gel, approximately 100 ml). The solution is rocked until the gel clarifies and glycoproteins appear as pinklred bands. The color will fade within several hours. 5. The gel is soaked overnight next in 10% acetic acid/lO% (vlv) methanol. The gel may now be counterstained with 0.1% (w/v) Coomassie Blue to detect proteins. The gel will rehydrate to its original size in this solvent.

Lectin Techniques While the aforementioned techniques are specific and direct, they still require relatively large (microgram) quantities of glycoprotein. For the detection of nanogram quantities, indirect lectin techniques are available. In a manner analogous to the Western blot, lectins may be used to detect glycoproteins following gel electrophoresis. The lectin can be biotinylated, and fluorescent avidin complexes used to localize the biotinylated lectin-glycoprotein complex on the gel. Alternatively, the lectin may be linked to horseradish peroxidase or alkaline phosphatase for dye detection. Monoclonal antisera to lectins may also be employed in a "sandwich" technique, with detecti0.n by radiolabel or peroxidase. A kit is commercially available for these applications from Vector Laboratories. Deglycosylation of Glycoproteins

Often the quantity of purified glycoprotein available is so limiting that the amount required to stain for carbohydrate would preclude any other uses (e.g., to obtain a partial sequence for cloning). In these instances, an alternative method to detect oligosaccharide moieties is by the SDSPAGE behavior of the suspected glycoprotein before and after its deglycosylation. In general, the electrophoretic mobility of glycosylated polypeptides is slower than predicted, presumably because of decreased SDS binding to the polypeptide. Analytical deglycosylation with tracer quantities of a putative glycoprotein mandates an enzymatic method, rather than chemical methods. In general, the two major classes of N-linked and 0-linked oligosaccharides are distinguishable by the type and arrangement of sugars.

538

SPECIALIZED PURIFICATION PROCEDURES

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Within the N-linked group, the pattern of complex, high-mannose, or antennary structures are substrates for different glycosidases. The peptide N-glycosidase F (N-glycanase) from Flavobacterium meningosepticum (Genzyme, Boehringer Mannheim), which is distinct from N-acetylglucosaminidase Endo-F, has the broadest substrate specificity and will cleave both complex and high-mannose-type structures. In addition, the product of Endo-F treatment retains a single residue of GlcNAc attached to the asparagine. In contrast, peptide N-glycosidase F removes all carbohydrate and leaves a n aspartic acid residue replacing asparagine. The latter modification may have significant effects on the biochemical properties of the protein. The 0-linked oligosaccharides have less predictable patterns, but often share the Gal(p1-3)GalNAc (0-glycan) core linked to serine or threonine, and this structure is susceptible to endo-Nacetylgalactosaminidase. In addition to these endoglycosidases, exoglycosidases in protease free form have become commercially available. These enzymes, when used sequentially, can frequently provide information on the identity and arrangements of terminal (nonreducing) structures. Neuraminidase (sialidase), which removes terminal sialic acid residues, frequently will alter the electrophoretic mobility of glycoproteins or change the lectin (e.g., wheat germ agglutinin)-binding properties of the preparation. In the analysis of 0-linked glycoproteins, it is generally necessary to prepare the sample for endo-a-N-acetylgalactosaminidasedigestion by removing peripheral sugars substituted on the 0-glycan core disaccharide. The protease contamination problem which complicates the use of many commercial products is inversely related to the cost of the enzyme. For the cheaper products, the inclusion of 0.1 mM phenylmethylsulfonyl fluoride is prudent. Sample Protocols for Deglycosylation Purijied glycoprotein, Asn-linked. When the protein sample is soluble and partially purified, the assay may be performed in 50-250 mM sodium phosphate buffer, p H 8.6. In a final volume of 30 pl, add 0.3-2 U Nglycanase to up to 1 nmol of protein. Incubate at 37' overnight. (Purified N-glycanase from Genzyme requires inclusion of 1,lO-phenanthroline hydrate to 10 mM from a 10x stock in methanol to guard against proteolysis. One unit of enzyme is the release of 1 nmol of oligosaccharide from tritiated dansyl-fetuin glycopeptide per minute at 3 7 O . ) If the sample requires denaturation to expose oligosaccharide, or if it has been purified from membranes and contains detergents, the incubation mixture may contain up to 0.1% SDS. In order to protect the enzyme from denaturation, the SDS-treated sample should be made 1% in Triton X-100 or NP-40

PURIFICATION OF MULTIENZYME COMPLEXES

539

prior to addition of enzyme. For a 20- to 50-pg sample, up to one unit of enzyme is used and incubation is a s above. The treated protein is then subjected to SDS-PAGE. Crude membranes. Crude cellular membranes bearing glycoproteins may be glycosylated in situ using peptide N-glycosidase F (for Asn linkages) with or without neuraminidase and endo-a-N-acetylgalactosaminidase (for potential SerIThr linkages). The following p r o t ~ c o l success'~ fully demonstrated the presence of sialic acid-containing, N-linked oligosaccharides on the rat ovarian lutropin (LH) receptor. Crude membranes (20-30 pg total protein) are suspended in 200 p1 of 100 m M sodium phosphate, pH 7.0, containing 200 p M phenylmethylsulfonyl fluoride. Peptide N-glycosidase F (2 U) and neuraminidase (10 m u ) are added and incubated for 1-3 hr at 30°. (Prolonged incubation of crude receptors frequently activates latent membrane peptidases which degrade receptors. For this reason, buffer controls are mandatory. When working with phagocytic leukocyte membranes, which are among the richest sources of proteolytic activity, the following "inhibitor cocktail" helps": 100 p M PMSF, and 10 pglml each of pepstatin, leupeptin, chymostatin, and soybean trypsin inhibitor. PMSF, pepstatin, and leupeptin are diluted from dimethyl sulfoxide. All stocks are made at lOOx concentration.) The treated protein is then subjected to SDS-PAGE. l8

l9

K. P. Keinanen, Riochrm. J. 256, 719 (1988). N. P. Gerard, M. Hodges, P. F. Weller, J . M. Drazen, and C. Gerard, J. Biol. Chem. 264, 1760 (1989).

14 11 Purification of Multienzyme Complexes By PAULA. SRERE and CHRISTOPHER K. MATHEWS

K r,

r:

The goal of conventional enzyme purification is to obtain a single protein responsible for catalyzing a single (usually simple) chemical reaction. In some instances an apparently simple reaction may be the result of several separate catalytic reactions. In such cases the enzyme itself can be either a multifunctional protein [monomers or oligomers of polypeptide chain(s) with covalently linked active sites] or a multienzyme complex (several active sites, each on distinct polypeptide chains, but forming a complex with a very low dissociation constant). There also are multienzyme complexes of entire metabolic sequences, some of which exist as multifunctional proteins and some as aggregates of METHODS IN ENZYMOLOGY. VOL. 182

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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loosely associated enzymes. In addition, there are variations in the kind of holoenzyme structure that exists for the same overall activity. Thus, the bacterial synthesis of long-chain fatty acids occurs in a series of seven separate enzyme reactions.' To date no interaction between these enzymes has been detected, although this observation does not rule out the existence of a complex in situ. In plants an isolatable complex of separate enzymes has been r e p ~ r t e dwhereas ,~ in yeast and in animal tissues such complexes have all of the active sites covalently linked on multifunctional polypeptide chain^.^ Similar observations have been made for several different enzyme systems. For enzyme complexes that are multifunctional proteins or tight multienzyme complexes, no special protein isolation techniques need be employed. In fact, the unusually large size of these enzymes or complexes usually makes the purification somewhat easier than that for most other enzymes. In the past, several putative multienzyme complexes have been later identified as multifunctional proteins that had been proteolytically nicked during the isolation procedures. However, not all complexes have dissociation constants between their component proteins such that they remain associated under the usual conditions of protein isolation. Thus, a "dynamic" complex is one with a very large dissociation constant between sequential enzymes but with a low dissociation constant for the intermediates.

Special Problems of Isolation of Multienzyme Complexes

Dilution

The factor most responsible for dissociation of weakly associated M ) multienzyme complexes is the dilution of the cell contents (KD = that usually accompanies cell disruption. This dilution factor varies from 5 to 10. Thus, consider a hypothetical protein complex whose KDis M and whose components have a total concentration of 5 X M . In the cell (excluding other effects) 60% of the proteins of this hypothetical system are complexed. After a 5-fold dilution, only about 10% of these proteins are present as a complex (Fig. 1 ) . Disruption of cells without dilution is possible, but the gellike material obtained is difficult to use in ordinary physical separations such as centrifugations and filtrations. P. R. Vagelos, Annu. Rev. Biochem. 33, 139 (1964). L. M. S. Worsham, Z. L. P. Jonak, and M. L. Emst-Fonberg, Biochim. Biophys. Acta

876, 48 (1986).

' J. K. Stoops and S . J. Wakil, J . Biol. Chem. 256, 5128 (1980).

[4 11

PURIFICATION OF MULTIENZYME COMPLEXES

54 1

DILUTION FIG. 1. Computer-generated curves for the dissociation of a bienzyme complex

=El + EZ(data of Dr. G. L. Cottam).

E1E2

k,

k- l

Another consequence of dilution is the reduction of total protein concentration from normal cellular levels (20-30% in the cytosol and 40-50% in mitochondria) to levels that are one-fifth to one-tenth normal. Minton4 has shown that in concentrated protein solutions (20% and greater), a volume-excluding effect causes a change in the activity coefficients of interacting proteins such that complex formation is favored. Thus, dilution would negate this associative force. It is also possible that small cellular molecules (metabolites) have a role in the stability of multienzyme complexes and their dilution could affect the isolatability of the holocomplex. Therefore, to minimize the adverse effects of dilution, cells should be extracted with the smallest possible amount of extraction medium or, perhaps, in the presence of a volume-excluding polymer such as polyethylene glycol.

Cell Disruption The manner of cell disruption can also affect the recovery of enzyme complexes. Sonication, for example, can disrupt enzyme complexes. Several metabolic pathways were stopped when cells were exposed to low sonication levels, but the pathways returned to their normal rates after ' A . P. Minton, Mol. Cell. Biochem. 55, 119 (1983).

542

SPECIALIZED PURIFICATION PROCEDURES

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cessation of sonic oscillati~n.~ In addition, sonic disruption may solubilize some enzymes which are normally bound to membranes. Other methods of cell disruption, usually considered mild, also may pose difficulties. For example, osmotic shock will lead to dilution of the cell contents with the consequent problems discussed above. Freezethaw treatment of a cell or organelle is another disruption method usually considered gentle. However, freeze-thaw procedures are carried out in buffers, and the salt and acid forms of the buffer often have different solubilities in the cold. A relative change in solution concentration of one form over the other during the freeze-thaw procedure can cause drastic changes in pH, with possibly deleterious effects on the protein complexes. Multienzyme Complexes in Nucleotide Biosynthesis

We shall use enzymes of nucleotide metabolism to illustrate procedures for the purification and characterization or multienzyme complexes. Although many multifunctional enzymes and multienzyme complexes have been described in nucleotide m e t a b ~ l i s r n ,only ~ ~ ~ three noncovalently bonded complexes have been highly purified, with yields, enrichment factors, and specific activities reported. These are (1) a complex from vertebrate liver containing two transformylases in purine synthesis and two enzymes of folate coenzyme metabolism that was purified 325-f0ld,~(2) a complex from carrot cell cultures that contains at least three enzymes of thymine nucleotide biosynthesis that was purified 1000fold,9 and (3) a complex from T4 phage-infected Escherichia coli that carries out the synthesis of deoxyribonucleoside 5'-triphosphates (dNTPs) from ribonucleoside diphosphates (rNDPs) or deoxyribonucleoside monophosphates (dNMPs) that was purified 470-f0ld.~The individual reactions catalyzed by the T4 dNTP synthetase complex, with which we have the most experience, is summarized in Fig. 2. Purification of Complexes Even though the three complexes were purified in different laboratories from different starting materials, their fractionation schemes show

'

V. W. Bums, Science 146, 1056 (1964). C. K. Mathews, T. W. North, and G. P. V. Reddy, Adv. Enzyme Regul. 17, 133 (1979). L. K. Moen, M. L. Howell, G . W. Lasser, and C. K. Mathews, J . Mol. Recognition 1,48 (1988). C . A. Caperelli, P. A. Benkovic, G. Chettur, and S. J . Benkovic, J . Biol. Chem. 255, 1885

(1980). I. Toth, G . Lazar, and H. M. Goodman, EMBO J. 6, 1853 (1987).

14 11

PURIFICATION OF MULTIENZYME COMPLEXES

543

ADP GDP UDP CDP

ri

1

d z

i

7 4

1

FIG.2 Reactions of the T4 dNTP synthetase complex.' The structural gene for each enzyme is identified. Two of the proteins are of host origin (adk, adenylate kinase; ndk, nucleoside-diphosphate kinase). The remaining enzymes are encoded by the phage genome. Each enzyme is identified with letters or numbers referring to ~ t structural s gene.'

several similarities. (1) Each of the complexes is amenable to -purification by the standard methods used to fractionate single enzymes-gel filtration, ion-exchange chromatography, affinity chromatography, and ammonium sulfate fractionation. (2) Despite the concerns expressed earlier about lysing cells gently under proper ionic conditions and at low dilution, these complexes can be detected in cells lysed by such classical techniques as sonic oscillation or mechanical rupture of frozen cells. (3) All three fractionation schemes give low recoveries, probably due to the factors mentioned above, especially the dilution effect that invariably accompanies standard protein fractionation schemes. Protein-protein association constants for each complex have not been determined. However, from the semiquantitative considerations pre-

544

SPECIALIZED PURIFICATION PROCEDURES

[41]

Fraction Number FIG.3. Gel filtration of the T4 dNTP synthetase complex on a Sephacryl S-400 c ~ l u m n . ~ Bed volume was 3.5 x 82 cm, and fractions were 4.6 ml each. The lower panel shows elution profiles for three individual enzymes, and the upper profile denotes the activity of a coupled sequence. The inset shows calibration of the column with molecular weight standards.

sented earlier, it is perhaps surprising that all three complexes remain at least partially intact through the dilutions that accompany column chromatography. For example, individual enzymes of the T4 dNTP synthetase complex coelute as symmetrical peaks upon gel filtration on Sephacryl S-400 (Fig. 3). Another unexpected result was the successful use of ammonium sulfate precipitation to concentrate and enrich each of the three complexes. It might be expected that the complexes would be disrupted at the high ionic strength present during ammonium sulfate precipitation. It is also

[411

PURIFICATION OF MULTIENZYME COMPLEXES

545

possible that the high protein concentrations achieved in the protein precipitate would drive together constituent proteins of the complex. Alternatively, proteins of a complex that is dissociated during ammonium sulfate precipitation might reassociate during dialysis of the redissolved ammonium sulfate precipitate. Since such a dissociated-reassociated complex might not be identical to the native complex, multiple independent fractionation approaches must be carried out to verify that a multienzyme aggregate does indeed represent a biologically significant complex. It is also possible, of course, that some complexes are held together primarily by hydrophobic interactions and would not be affected by high ionic strength. Criteria for Existence of Multienzyme Complexes

Cofractionation of Enzyme Activities

The most direct evidence for the existence of a multienzyme complex is the association of multiple activities through several fractionation steps. Obviously, in the isolation of a putative complex several activities must be monitored through the purification steps, and their ratios should remain constant. However, the failure to maintain constant ratios does not necessarily negate the existence of a complex since the proportions of free and complexed enzymes within the cell may vary widely for different proteins of a complex. For example, in the case of nucleoside-diphosphatekinase, an Escherichia coli protein which is a constituent enzyme of the phage T4 dNTP synthetase complex and which also participates in the biosynthesis of ribonucleoside triphosphates, only about 5% of the activity in an extract cosediments through a sucrose gradient with other T4encoded dNTP biosynthetic enzymes. This observation suggests that only a small fraction of this bacterial enzyme interacts with virus-encoded .~ some proteins to form the T4 dNTP synthetase ~ o m p l e xAlternatively, proteins may be less stably associated with a complex and may dissociate during the analytical procedures. Kinetic Coupling

By "channeling" metabolites through reaction pathways, multienzyme complexes are thought to contribute toward efficiency and control of cellular processes. Juxtaposition of catalytic sites of sequential enzymes limits diffusion of intermediates away from these sites. This kinetic behavior has three consequences which can be observed experimentally, and be used to ascertain whether a multimolecular aggregate is in fact a true complex: (1) incomplete equilibration of pools of intermediates, (2)

546

SPECIALIZED PURIFICATION PROCEDURES

[411

reduced transient times, and ( 3 ) reduced steady-state levels of intermediates. First, intermediates that are entrained at the catalytic surface of a complex do not equilibrate with pools that are not so entrained. This can be observed by following the incorporation of radioactivity from a labeled precursor to the product of a reaction sequence and by determining whether an excess of nonradioactive intermediate dilutes the label. Failure of the exogenous unlabeled precursor to dilute the product radioactivity constitutes excellent evidence for channeling by a putative complex. Second, the transient time refers to the interval between starting a multistep reaction pathway by addition of substrate and the time at which the formation of final product reaches its maximal rate. Restricted diffusion of intermediates at active sites enormously reduces the time needed for an intermediate released by one enzyme to bind to the next catalytic site. For the T4 dNTP synthetase complex, we observed transient times in vitro of just a few seconds for a four-step sequence, either dUMP + dTMP -, dTDP + dTTP, or dCTP -, dCMP -.. dUMP + dTMP.6J0 By contrast, simulation of the same sequence catalyzed by noninteracting enzymes predicts transient times of many minutes for the same sequences. Third, restricted diffusion of intermediates greatly increases local concentrations of those intermediates in the vicinity of catalytic sites, but at the same time it greatly decreases average concentrations within an entire system, whether an intact cell or an experimental reaction mixture. For the sequences dUMP -+ dTMP -,dTDP -,dTTP, and dCTP + dCMP + dUMP -, dTMP + dTDP + dTTP, we have observed intermediate concentrations in vitro at least an order of magnitude lower than those predicted from simulations involving the uncomplexed enzyme^.^.^^'^ As noted above, several different enzyme activities should be monitored during purification of a complex. If a convenient assay for kinetic coupling exists, one can simultaneously monitor the presence of each of the constituent activities in a complex and the functional integrity of the whole complex. For example, a particularly convenient assay for purifying the T4 dNTP synthetase complex involve the sequence dCTP -, dCMP + dUMP += dTMP. lo The last reaction, catalyzed by thymidylate synthase, is easily assayed spectrophotometrically. In the assay for coupling, we merely substitute dCTP for the thymidylate synthase substrate, dUMP. After a brief, sometimes unobservable, lag period, the rate of

lo

J. R. Allen, G. W. Lasser, D. A. Goldman, J. W. Booth, and C. K. Mathews, J . Biol. Chem. 258, 5746 (1983).

[411

PURIFICATION OF MULTIENZYME COMPLEXES

547

absorbance increase is the same as when dUMP is provided. This simple assay, therefore, assures that the three enzymes, dCTPase, dCMP deaminase, and thymidylate synthase, are present and in the correct functional relationship. Genetic Evidence With a biological system amenable to genetic analysis, the power of the above approaches is enormously increased. For example, this approach was used in studying a T4 phage strain carrying a temperaturesensitive mutation in gene 42,6which specifies a thermolabile variant form of the enzyme dCMP hydroxymethylase (see Fig. 2). A partially purified preparation of T4 dNTP synthetase complex was analyzed for its ability to catalyze the sequence dUMP + dTMP -, dTDP + dTTP. By the criteria noted above, kinetic coupling was observed at 30°, whereas at 42" kinetic coupling was abolished. Since dCMP hydroxymethylase does not catalyze a reaction in the sequence under investigation, these results indicate that kinetic coupling requires not only proximity of the enzymes that directly catalyze a sequence, but also proper functional relationships among all of the constituent enzymes of a complex. Protein Cross-Linking Bifunctional protein cross-linking reagents can be used to identify neighbor relationships among proteins in a purified complex, to demonstrate that such relationships occur in intact cells, or as an adjunct to purification of a complex. For example, the vertebrate transformylase complex has been analyzed this way after purification.'! Preliminary experiments with uninfected E. coli have suggested that cross-linking will also be useful for further characterization of T4 dNTP synthetase complex in uivo.12 The approach involves treatment of intact bacteria with protein cross-linkers, followed by SDS gel electrophoresis of cell-free extracts and probing of the gels with a monoclonal antibody to the ribonucleotide reductase large subunit. The presence of high-molecular-weight immunoreactive material confirmed that under some conditions ribonucleotide reductase becomes linked to neighbor proteins in viuo. Working with a mandelate metabolizing complex, Kenyon's group13 showed that cross-linking can be carried out in vivo without completely inactivating " G.

K. Smith, W. T. Mueller, G. F. Wasserman, W. D. Taylor, and S. J. Benkovic, Biochemisrry 19, 4313 (1980). l2 C. K. Mathews, B.-M. Sjoberg, and P. Reichard, Eur. J. Biochem. 166, 279 (1987). l3 R. A. Halpin, G . D. Hegeman, and G . L. Kenyon, Biochemistry 19,4633 (1981).

548

SPECIALIZED PURIFICATION PROCEDURES

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the constituent enzymes. Thus, multicomplexes could be fractionated with some assurance that intracellular associations will remain intact. Isolation Procedures for Some Other Multienzyme Complexes A rather complete listing of known multienzyme complexes was provided in a review.I4 Some examples of procedures used for the isolation of multienzyme complexes are listed below:

1. Transketolase, transaldolase, and glyceraldehyde-3-phosphatedehydrogenase from Candida utilis were detected as a complex using Sepharose 6B gel filtration, DEAE-Sephadex chromatography, and polyacrylamide electrophoresis. l5 2. Aminoacyl-tRNA synthetases were shown to exist as a complex in higher eukaryotes and in E. coli. The complex from reticulocytes was isolated by gel filtration on a BioGel A-5m column followed by affinity chromatography on tRNA-Sepharose.I6 Eight synthetases and three unidentified proteins are found in the complex. Isolation of an aminoacyltRNA synthetase complex from rat liver was described using controlledpore glass beads (CPG-10-700B), Sephacryl S-300, and phenyl-Sepharose CL-4B.17 In still another method,18 the complex was prepared as a 24s particle in a 20-40% sucrose gradient. The aminoacyl-tRNA synthetase complex isolated from E. colilg was prepared by using a BioGel A-5m column. Chromatography on Sephadex G-200 was not effective. The size of the complex was larger when cells were disrupted with a freeze-press method (1 X lo6 Da) than when disrupted by sonication (0.4 x lo6 Da). Further purification could be achieved using DEAE-Sephacel chromatography. 3. A functional enzyme complex containing ribose-5-phosphate isomerase, phosphoribulokinase, ribulose-bisphosphate carboxylase/oxygenase, phosphoglycerate kinase, and glyceraldehyde-3-phosphate dehydrogenase was isolated from spinach chloroplast^.^^ The complex was isolated by column chromatography on DEAE-Trisacryl, Sephadex G-200, and hydroxylapatite. This complex was homogeneous in gradient polyacrylamide slab gel electrophoresis and by ultracentrifugal analysis. P. A. Srere, Annu. Rev. Biochem. 56, 89 (1987). T. Wood, C. C. Muzariri, and L. Malaba, Inr. J . Biochem. 17, 1109 (1985). l6 A. M. Pendergast, R . C. Venema, and J. A. Traugh, J. Biol. Chem. 262, 5939 (1987). G . VeUekamp, R. M. Sihag, and M. P. Deutscher, J. Biol. Chem. 260, 9843 (1985). l8 C. V. Dang, B. Ferguson, D. J. Burk, V . Garcia, and D. C. H. Yang, Biochim. Biophys. Acta 829, 3 19 (1985). l9 C. L. Hams, J . Bacteriol. 169, 2718 (1987). B. Gontero, M. L. Cardenas, and J. Kicard, Eur. J. Biochem. 173, 437 (1988). l4

l5

'

PURIFICATION OF MULTIENZYME COMPLEXES

549

4. A multienzyme complex of phosphoribulokinase and glyceraldehyde-3-phosphate dehydrogenase was isolated from the green alga Scenedesmus o b l i q ~ u s Purification .~~ of the complex was accomplished using DEAE-cellulose chromatography followed by Ultrogel AcA 34 and hydroxylapatite chromatography. An overall purification of 60- to 100-fold was obtained for the complex, which was judged to be homogeneous by ultracentrifugal analysis. 5. A complex of the TCA cycle enzymes from E. coli was isolated by gel filtration on Sepharose 4B and by sucrose gradient ~entrifugation.~~ The yield was low, and it was necessary to include 20% glycerol in the buffers to obtain any complex at all. Five enzymes of the TCA cycle were present: fumarase (fumarate hydratase), malate dehydrogenase, citrate synthase, aconitase (aconitate hydratase), and isocitrate dehydrogenase. Similar complexes have been isolated from other bacteria and from rat liver. 6. Preparations of gently disrupted rat liverz3and heart m i t ~ c h o n d r i a ~ ~ have been made which contain all of the TCA cycle enzymes in apparently "open" mitochondria. This is not a simple complex as has been seen for bacteria. It is important to note that its demonstration and isolation depended on gentle sonication of the mitochondria. 7. The two enzyme activities, ATP sulfurylase (sulfate adenylyltransferase) and adenosine 5'-phosphosulfate kinase, copurified 2000-fold from The purification procedure included Sephacryl rat chondr~sarcoma.~~ S-300, DEAE-Sephadex, hydroxylapatite, and ATP-agarose affinity chromatography. 8. A large complex containing glycine decarboxylase [glycine dehydrogenase (decarboxylating)] associated with serine hydroxymethyltransferase (glycine hydroxymethyltransferase) was isolated from pea leaf mito~hondria.~~ Mitochondria were disrupted by using the freeze-thaw procedure. After removal of the membranes by centrifugation, the highmolecular-weight fraction was obtained by filtration on a Diaflo membrane XM300.

In summary, purification of a multienzyme complex is much more difficult than fractionating a single enzyme. Problems stem from the need to keep track of several different enzymes, from the lability of such comS. Nicholson, J. S. Easterby, and R. Powls, Eur. J . Biochem. 162,423 (1987). S . J. Barnes and P. D. J. Weitzman, FEBS Lett. 201, 267 (1986). J . B. Robinson, Jr. and P. A. Srere, J . Biol. Chem. 260, 10800 (1985). J. B. Robinson, Jr., L. Inman, B. Sumegi, and P. A. Srere, J. Biol. Chem. 262, 1786 (1987): D. H. Geller, J . G . Henry, J. Belch, and N. B. Schwartz, J. Biol. Chem. 262,7374 (1987). 26 M. Neuburger, J. Bourguignon, and R. Douce, FEBS Lett. 207, 18 (1986).

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SPECIALIZED PURIFICATION PROCEDURES

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plexes, and from the possibility of being misled by artifacts. In most preparations of multienzyme complexes proteolytic enzyme inhibitors are added. Nevertheless, complexes can be isolated and purified by methods that are not much different from those used to characterize single enzymes. However, a range of kinetic, and where possible, genetic approaches must be used to assure that one is analyzing a true multienzyme complex. Reconstitution of Multienzyme Complexes The analysis of a presumed complex should also include complementary approaches involving reconstitution of part or all of the complex. A complete discussion of techniques for studying protein-protein interactions is beyond the scope of this chapter, but three potentially fruitful approaches will be mentioned. The first is affinity chromatography involving immobilized enzymes. In one case, the T4 gene 32 protein, a singlestranded DNA-binding protein, was immobilized onto an Affi-Gel matrix (Bio-Rad). When extracts of radiolabeled T4 proteins were passed through columns of this material, specific proteins were bound, and these could be eluted and identified by SDS-polyacrylamide gel electrophoresis and radioautography .26 The second approach utilizes polyethylene glycol. One of the factors which may be responsible for multienzyme complex dissociation is the loss of the "crowding" effect of the macromolecular milieu within cells. Ogston2' suggested the use of polyethylene glycol (a volume-excluding polymer) in extraction media for biological material to replace the loss of protein at high concentrations. The enhancement of complex formation in polymer solutions over dilute (with respect to macromolecules) aqueous solutions was illustrated in studies that showed an interaction between the mitochondria1 isozymes of aspartate aminotransferase and malate dehydrogenase in a countercurrent device using volume-excluding polymers28 and in the interaction of citrate synthase and mitochondrial malate dehydrogenase in 14% polyethylene Thus, for complexes which may exist in cells but whose KD values make it difficult to isolate, this second method of study may allow recombination of the isolated individual enzymes in polyethylene glycol. The danger inherent in this approach is that nonspecific interactions may also occur, so that many controls must be run simultaneously. These associa27

A. G. Ogston, Biochem. J . 31, 1952 (1937).

29

L. A. Halper and P. A. Srere, Arch. Biochem. Biophys. 184, 529 (1977).

" L. Backman and G. Johansson, FEBS Lett. 65, 39 (1976).

-

-

PURIFICATION OF MULTIENZYME COMPLEXES

5.5 1

tions are not caused by the polyethylene glycol; rather, weak interactions are enhanced by polyethylene glyc01.~~-~' A third approach involves the assumption that an enzyme in a complex passes its product directly to the enzyme catalyzing the next reaction in a sequence. Srivastava and Bernhard32devised a kinetic test for this supposition and applied it to pairs of glycolytic enzymes. The test involves determining substrate-velocity profiles, first for an ordinary substrate, and then for that same substrate bound to the enzyme which synthesized it in the previous step of a sequence. If the enzyme-intermediate complex is preferred as a substrate to that same intermediate in free solution, then this constitutes evidence for direct transfer of the intermediate from one enzyme to the next. Analysis of this type requires substrate-level quantities of the first of the two enzymes being tested.

im 1

Acknowledgments This work was supported by grants from the Veterans Administration and National Science Foundation (#DCB-8500169) (P.A.S.) and from the National Institute of Health (#R01-AI15145) (C.M.).

J. M. Merz, T. A. Webster, J . R. Appleman, E. K. Manley, H.-A. Yu, A. Datta, B. J. Ackerson, and H. 0.Spivey, Arch. Biochem. Biophys. 258, 132 (1987). S. I. Miekka and K. C. Ingham, Arch. Biochem. Biophys. 203, 630 (1980). D. K. Srivastava and S. A. Bernhard, Science 234, 1081 (1986).

'*

Section X Characterization of Purified Proteins

[421

DETERMINATION OF PURITY

[421 Determination of Purity By DAVIDG. RHODESand THOMAS M. LAUE

In biochemistry there is no means for directly quantitating the purity of a protein sample. Demonstration of purity of a protein preparation always involves an assessment of the quantity of particular types of impurities. It is this concept that is meant whenever the term purity is used. In order to assess the purity of a sample, one must first identify the type of impurity that is to be measured (i.e., nucleic acid, carbohydrate, lipid, other unrelated proteins, isozymes, inactive enzyme), then identify a characteristic property (chemical assay or physical feature) which can distinguish the protein of interest from the putative contaminant(s). Purity is then the demonstration that the sample is free of detectable quantities of contaminant. Clearly, the level of purity will depend on which assay methods are chosen and their sensitivity. Since most isolation procedures are quite good at removing nonprotein contaminants, this chapter will focus on methods for detecting contamination of a protein sample by other proteins. Selective assays for the presence of nucleic acids, lipids, and carbohydrates can be found in other volumes of this series.' There are several sensitive methods that may be used to detect the presence of impurities in a sample (Table I). Each method tests for a different physical property of the molecules. The method of choice depends on the following criteria: (1) the quantity of protein available, (2) the nature of the impurity being tested, (3) the accuracy of the estimate needed, (4) the sensitivity of the test needed, and (5) any peculiarities of the protein or its solvent which might interfere with the use of the techniques. It is easiest and most common to carry out one o r more fractionation procedures and demonstrate that only one component is detectable. A wide variety of fractionation procedures may be used to detect impurities, with the criterion for purity being the presence of a single detectable component. These methods are detailed elsewhere in this volume and will not be reviewed here. Furthermore, several analytical methods for determining molecular weight or molecular size are outlined elsewhere in this v o l ~ r n eWhere .~ appropriate, we will refer to that chapter and will emphasize the variations of those methods which are better suited to detection of contaminant than to quantitative determination of molecular parameI

r

Many t i t l e s w i l l b e found i n t h i s s e r i e s . T. M. L a u e and D. G. R h o d e s . t h i s volume [43].

METHODS IN

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Copyright O 1990 by Academic Press. Inc. All rights of reproduction in any form re\erved.

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CHARACTERIZATION OF PURIFIED PROTEINS

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TABLE I METHODSFOR DETECTING IMPURITIES

Method Electrophoresis Denaturing gel Native gel Isoelectric focusing Chromatography Gel permeation Ion exchange Affinity Sedimentation Velocity Equilibrium

Sample Fractionateu recoveryb Quantity

Easec

Expensed

Property assessed

ng-pg nl3-p.g ng-pg

Chain lengthe,.' Chargelsizef Isoelectric pHI

Pg Pg Pg

Sizeh Net charge' Specific binding*

g' - / Pg

Composition

Variable

Activity

Variable

Mass/si~eJ;~ Mass, associationJk Component content]:' Presence of active sitem

+, Sample fractionated during analysis and separate components may be isolated; 2 , incomplete fractionation, no components isolable; -, no sample fractionation afforded. +, Good recovery of sample following assay; a , sample may be recovered in favorable cases; -, sample destroyed during analysis. +, Simple method; t,somewhat more difficult, special equipment or expertise may be required. +, Equipment and supplies relatively inexpensive; f, some equipment or supplies may be costly; expensive equipment or supplies required. ' D. Garfin, this volume [33]. f T. M. Laue and D. G. Rhodes, this volume [43]. D. Garfin, this volume [35]. G. K. Ackers, in "The Proteins" (H. Neurath and R. L. Hill. eds.), 3rd Ed., Vol. 1, p. 1. Academic Press. New York, 1975. An excellent review of theory and practice of the various forms of column chromatography is contained in a series of booklets published by Pharmacia Biotechnology, Uppsala, Sweden, which are available from their distributors. Their booklet entitled "Gel Filtration Theory and Practice" is recommended. T h e booklet "Ion Exchange Chromatography: Principles and Methods" from Pharmacia Biotechnology is an excellent source of information (see footnote h). j The booklet "Affinity Chromatography: Principles and Methods" from Pharmacia Biotechnology is an excellent source of information (see footnote h). C. H. Chervenka, "A Manual of Methods for the Analytical Ultracentrifuge." Spinco Div., Beckman Instrument, Palo Alto, California, 1970. J. Ozols, this volume 1441; D. Matsudaira, this volume [45]; S. Seifter and S. Englard, this volume (471. R. Biggs, in "Human Blood Coagulation, Hemostasis and Thrombosis" (R. Riggs. ed.), 2nd Ed., p. 722. Blackwell, Oxford, 1976.

a

-.

'

[421

DETERMINATION OF PURITY

557

ters. This chapter will also outline criteria for selecting among these methods, some of their limitations and advantages, and some description of assays for purity that do not involve fractionation. Composition-Based and Activity-Based Analyses

Methods that provide molar quantitation of amino acids, specific prosthetic groups, or of active sites may be used to assess the purity of a sample. In cases where the specific activity of the pure material is known it may be appropriate to determine the unit activity as a measure of relative purity. These methods are secondary in nature, since reference is always made to the quantity of the analysate expected for a sample that is free of contaminants. Typically two measurements are required. The first is the mass of the protein in the sample that will be used in the analysis. The second measurement is the quantitation of the particular analysate or of the known activity. A calculation is then made of the expected quantity of analysate based on the mass of the material used in the analysis. The purity is then expressed a s ratio of the amount measured to the amount expected. Good quantitation has been achieved using end-group analysis3 and quantitative analysis for specific prosthetic groups and for enzymatic a ~ t i v i t yThe . ~ details for this method are the same a s those described in [43] in this volume. Problems and Pitfalls

Composition-based or activity-based estimates are sensitive only to those impurities being assessed, and typically provide little specific information (e.g., size, charge, etc.) concerning the nature of the contaminants. Thus, few clues are provided to help the experimenter in removing the contaminant. Electrophoretic Methods

Electrophoretic methods provide some of the simplest, least expensive, and often most sensitive approaches to determine the number of protein components in a sample. The methods of SDS gel electrophoresis5 and electrophoretic determinations of molecular weight and size2 are described elsewhere in this volume. Any of these approaches could be used J.-Y.Chang, this series, Vol. 91, p. 41. R. Biggs, in "Human Blood Coagulation, Hemostasis and Thrombosis" (R. Biggs, ed.), 2nd Ed., p. 722. Blackwell, Oxford, 1976. D. Garfin, this volume [33].

558

CHARACTERIZATION OF PURIFIED PROTEINS

1421

independently to assess the purity of a sample, depending on which characteristic of the protein is to be tested (Table I). If the expected contaminants differ in molecular weight from that of the desired protein, SDS gel electrophoresis would be the method of choice. On the other hand, molecules of similar molecular weight but different amino acid composition would appear to be identical in SDS gel electrophoresis, but could have different electrophoretic mobilities in native gel electrophoresis. Finally, proteins of almost any molecular weight might be separable using isoelectric focusing techniques. The latter method is detailed elsewhere in this v o l ~ m eand , ~ will not be treated here. Depending on the type of detection method available, nanogram to microgram quantities of sample are required. Since a contaminant constitutes a fixed weight fraction of a given sample, while the detection of a contaminant depends on the total mass of contaminant, the more sample applied to a gel, the better the chances are of detecting the contaminant. However, an upper limit to the quantity of sample that can be applied to a gel is set by sample solubility and by considerations of resolution.' The latter limit is usually more restrictive, with band broadening, hence poorer resolution, occurring at higher sample concentrations and with large sample volumes. Unfortunately, the more sensitive detection methods (requiring the smallest amount of sample), such as the silver stain techniques, do not allow sample recovery. If the protein sample is denatured, or otherwise solubilized under harsh conditions, it is often difficult to recover functional protein. However, if nondenaturing electrophoresis is used, it is possible to recover the sample from the gel by electrophoretic e x t r a c t i ~ n .If~ denaturing electrophoresis is performed, it may not be . ~ success or failure of renaturapossible to recover the native p r ~ t e i nThe tion depends largely on the peculiarities of the protein of interest. Clearly, a large andlor multisubunit protein will pose more problems for recovery than will a small monomeric protein.

Methods The details of sample preparation depend on the nature of the electrophoretic method chosen. The reader is referred to the other chapters for native gel electrophoresis2 and for isoelectric focusing g e k 6 Most frequently, denaturing gels provide the "front-line" method for assessing sample purity. In this case, the methods for sample preparation are described in detail in [33] of this volume. Because purity assessment often is D. Garfin, this volume [35].

' J. Lunney. A. Chrambach, and D. Rodbard, Anul. Biochem. 40, 158 (1971). M. Hanington, this volume [37]. K. Weber and D. J. Kuter, J . Biol. Chem. 246, 4504 (1971).

[421

DETERMINATION OF PURITY

559

not quantitative, one need not (necessarily) be as concerned about such problems as nonlinearity in mobility at protein size extremes, aberrations in mobility due to protein modifications, or nonuniformities in SDS binding that are discussed in 1431 of this volume. On the other hand, one does need to be concerned with consistency and uniformity in sample handling, and the conditions to which the sample is exposed during fractionation. For example, a homogeneous protein preparation in which the reduction of disulfide is not carried out carefully could appear to be heterogeneous. Likewise, misinterpretation is possible if nonuniformity in a gel crosslinking results in mobility differences across the gel. Because one does not normally know the sizes of all protein components within a sample, it is difficult to predict the gel concentration which will give the optimal fractionation of a set of protein components. Gradient gels, or gels of graded porosity, can cover a very wide range of molecular sizes. Although the conditions of a gradient gel are unlikely to be optimal for a particular fractionation, they will cover the widest possible range of conditions, thus maximizing the probability of identifying a contaminant. Ability to resolve a contaminant will depend largely on the range of gel concentrations (wt%) chosen for the gradient. The gradient should be designed so that the protein of interest bands at an intermediate gel concentration. The concentration of acrylamide at the top of the gel (low concentration end) should be sufficiently low that large molecular weight contaminants may enter the gel matrix. The bottom of the gel (high concentration end) should be sufficiently concentrated that small protein is retained in the gel matrix. Most gradient gels begin with a low concentration of approximately 4% acrylamide, although lower concentrations (2%) may be used. Proteins with molecular weights on the order of lo6 should be able to penetrate the 2% gel matrix. The high concentration for most gradient gels generally does not exceed 30% acrylamide, although 40% acrylamide can be used. One must be cautious in dealing with the higher acrylamide concentrations, however, because of the possibilities of heating or shrinkage upon polymerization of the high acrylamide concentrations. Very few polypeptides will penetrate a 30% gel, even after prolonged electrophoresis. The steepness of the cross-linking gradient determines the resolution of the gel. Thus, a gel with a gradient from 2 to 30% will be able to examine the widest range of molecular weights, but will not discriminate between two proteins of nearly identical molecular weights as well as a gel having a lower gradient. The procedure for making gradient gels differs only slightly from that for linear polyacrylamide gels5 or that for making sucrose gradients for sedimentation.I0 The procedure is slightly more difficult than that for 10

J. B. Ifft, this series, Vol. 27, p. 128

560

CHARACTERIZATION OF PURIFIED PROTEINS

1421

linear polyacrylamide gels only because two gels at different acrylamide concentrations must be made up in parallel. The procedure is more difficult than forming sucrose gradients for sedimentation only because one must work within the time constraints of polymerization. Several commercial gradient-forming devices are available, or one may easily be made in the laboratory. Two cylindrical containers of equal size (graduated cylinders with bottoms cut off, large plastic syringe barrels, etc.) should be notched at the base so as to allow a small piece of narrow flexible tubing to be inserted. One cylinder (B) should have two notches at the base on opposite sides and the other (A) should have a single notch. The cylinders are glued onto a flat piece of Plexiglas with flexible tubing joining cylinder A to cylinder B and a second piece of tubing attached to the notch on the opposite side of cylinder B. This arrangement allows known volumes of high and low concentrations of acrylamide to be added to the gradient-forming device. To form the gradient, two solutions of acrylamide, one at the lowest desired acrylamide concentration and one at the highest concentration, are made up to point of catalyst addition. A stir bar is added to cylinder B and the apparatus put on top of a magnetic stirring plate. The effluent tube from cylinder B is inserted into the gel-forming apparatus such that the solution will run down the edge of the gel-forming cavity. Tubing leading to the gel and tubing from cylinder A to cylinder B should be clamped prior to addition of the acrylamide. Catalyst is added to both acrylamide solutions and the high concentration solution is added to cylinder B. The clamps are released slightly so as to allow both lengths of tubing to fill with high-concentration acrylamide. The level of acrylamide in the graduated cylinder should be adjusted (if necessary) to approximately one-half the volume of the final gel. The low-concentration acrylamide is added to cylinder A so as to be approximately the same height as that in cylinder B. (Because of the density difference between the two solutions, solution columns with equal mass will be of different heights in the gradient cylinders. If heights are initially equal, some of the high concentration acrylamide will flow into the bottom of the low-concentration cylinder when the clamp between the two cylinders is released. The result will be a "band" of high-concentration acrylamide at the bottom of the gel.) The clamp between cylinders A and B is released, the stirring plate is turned on, and the clamp leading to the gel apparatus is released. As with any other gradient-forming technique, the concentration of the solution in cylinder B is diluted by that in cylinder A so that the concentration of the effluent goes from the high concentration linearly to the low concentration. The flow must be sufficiently fast that polymerization does not occur before the gradient is complete, but slow enough that one does not get convection in the gel apparatus. Numerous variations on this technique may be found by searching the literature.

[421

DETERMINATION OF PURITY

56 1

Following the formation of the gradient gel, electrophoresis procedures are identical to those used in the analytical Choice of conditions under which the gradient gel will be run depends largely on the researcher's knowledge about the system under investigation. If it is known, for example, that no very small or very large protein is present, one may wish to use a narrower range of concentrations in the gradient or a gel of constant concentration. Likewise, the staining strategy for analysis of the gel will be determined by the expected contaminant. The potential sensitivity of this approach and the information gained through its use can be enhanced by combining it with other techniques to generate a two dimensional electrophoretic analysis. While the second dimension would ordinarily be a gradient SDS-polyacrylamide gel electrophoresis, the first dimension could be a nondenaturing gel electrophoresis, an isoelectric focusing gel, or a denaturing gel under different conditions (e.g., without disulfide reduction). Isoelectric focusing is another technique which can successfully span a wide range of potential contaminants. Ampholines can cover the range of pI = 2 to 10 in the same gel. The procedures for carrying out isoelectric focusing have been described elsewhere in this volume6 and will not be repeated here. Although the wide range of this technique can be a considerable advantage, the sensitivity of the technique to small differences in isoelectric point can be enhanced by covering a narrower range of pl values. In addition, the sensitivity of this technique to contaminants which may differ by properties other than isoelectric point, can be enhanced using two-dimensional electrophoretic methods (as above). Pitfalls and Problems

Because there are so few pitfalls associated with gel electrophoresis, it is usually the method of first choice for purity assessment. There are, however, a few problems that must be borne in mind when using one or the other of these techniques. For denaturing gels, both false-negative and false-positive artifacts are possible. False negatives can result if a comigrating contaminant is present or if a contaminant is unable to penetrate the gel. For this reason, it is important to stain the entire gel, stacking and running portions, and to examine both for protein-staining material. A band of material left behind in the loading well or at the interface between the stacking gel and the running gel is an indication of either a highmolecular-weight contaminant or a contaminant of limited solubility. Likewise, the commonly used protein stains (e.g., Coomassie Brilliant Blue) will bind weakly to fibrous proteins or glycoproteins, resulting in an underestimate of these contaminants. False positives may result when covalent modifications are made during preparation of the sample for

562

CHARACTERIZATION OF PURIFIED PROTEINS

[421

electrophoresis or when the gel is nonuniform or contains residual oxidant. Similar problems may plague nondenaturing gels, with the additional problems caused by uncertainties as to the net charge on the protein of interest or the contaminants (see discussion in [431 of this volume). If the net charge on the contaminants is zero or of opposite sign from that on the protein of interest, the contaminant will not appear in the gel. It is also helpful to run gels over as wide a range of pH as possible. At times, artifacts have been associated with isoelectric focusing. These arise mostly from interactions between the proteins and the polyampholite beads used to create the pH gradient. The results are bands (often smeared) appearing in regions of the gel that may have nothing whatsoever to do with the isoelectric point of the protein of interest. One way to test for this possibility is to isolate protein from one of these bands and to resubmit the isolate to isoelectric focusing. If the original pattern reflects actual heterogeneity in the sample, the isolate will form only its original spot. If, on the other hand, the bands are artifactual, the other bands will be regenerated by the isolate. Gel Filtration Chromatography

Gel filtration chromatography (either HPLC or conventional column chromatography) is one of the simplest methods for the detection of impurities that differ in size from the molecule of interest." The method is nondestructive and rapid. Samples are diluted as they pass through the column, so starting concentrations well above the minimum detectable limit must be used. The exact quantity of material needed will depend on the sensitivity of the assay used to detect for contaminants. Method The method outlined in [43] of this volume for sample and column preparation for the purposes of molecular weight determination should be used for assessing impurities. The only difference is that the method of detection must be sensitive to contaminants as well as to the protein of interest. While in the case of assessing purity it is not necessary to calibrate the column, it is useful to do so for two reasons. First, by using a calibrated column, the experimenter gains some information concerning the size of the impurity. Second, by using a calibrated column, both the test for impurities and the molecular weight determination can be made simultaneously. To do this, one frequently uses two assays. The first is a nonspecific assay for protein (e.g., absorbance at 280 or 220 nm, if possi"

G. K. Ackers, in "The Proteins" (H. Neurath and R. L. Hill, eds.), 3rd Ed., Vol. 1 , p. 1. Academic Press, New York, 1975.

r: i

[421

K

ble) and the second is a specific assay (enzymatic, immunological, etc.) for the desired protein. Impurities are detected as either separate peaks in the chromatogram or as a broadening (e.g., a shoulder) of the elution profile. In principle, the elution profile should be nearly Gaussian (with a very slight skewing of the trailing edge, see Problems and Pitfalls). A strongly skewed peak, a shoulder on the peak, or nonsuperpositioning of the specific and nonspecific assay results suggest that the sample is contaminated.

DETERMINATION OF PURITY

563

Problems and Pitfcllls Gel-permeation chromatography is not as sensitive a method as gel electrophoresis for the detection of size heterogeneity. Typically, a gelpermeation experiment requires larger quantities of material than a gel electrophoresis experiment. Since gel-permeation chromatography usually is conducted under native conditions, it is possible that certain kinds of association behavior will mimic sample heterogeneity. For example, samples containing one type of protein chain that exists in a variety of stable oligomers (e-g., fatty acid synthase) will appear to be heterogeneous by this method. Likewise, a protein that undergoes rapid, reversible self-association can provide anomolous, concentration-dependent elution profiles.'' In both cases, tests are available for distinguishing between heterogeneity and molecular association (e.g., rechromatography), and in both cases the experimenter is presented with some useful information about the molecule of interest. Sedimentation Velocity Sedimentation velocity is a simple, rapid, nondestructive technique for evaluating the purity of a protein. The technique is sensitive to the ratio of the molecular weight to the molecular size. An overview of the topic of sedimentation velocity is provided in [43] of this volume. Typically, when using sedimentation velocity as a test of purity, an experimenter is looking for the presence of more than one sedimentable component. The virtue of the technique is that it can be used to test for a wide variety of materials (especially when using one of the refractive optical systems). Its major limitation is that it is not nearly as sensitive to small differences between molecules as the electrophoretic techniques. This is true even when band sedimentation is used.

Method Except for band, or zonal, sedimentation, details describing- sample preparation, optical systems available, and the interpretation of the ex-

564

CHARACTERIZATION OF PURIFIED PROTEINS

[421

perimental results are provided in [43] of this volume. Consult the manufacturer's instructions for details on how to set up a preparative centrifuge to conduct a rate-zonal sedimentation experiment. For zonal methods in the analytical ultracentrifuge, consult Easonf2or ChervenkaI3 for details. The analysis of the data is analogous to that above describing gel-permeation chromatography. Sedimentation Equilibrium For systems that are suitable for analysis, sedimentation equilibrium provides a sensitive method for the detection of contaminating species that differ in molecular weight. It is a simple, nondestructive, relatively rapid method. Sedimentation equilibrium provides the most powerful method for detecting heterogeneity of a sample with respect to self-association. The basic equations describing sedimentation equilibrium are provided in [43] of this volume, as are the details concerning sample preparation and data handling. Method

Thermodynamic analysis shows that for a single macromolecular component, the concentration dependence of the apparent molecular weight (determined as the local slope from the graph of In c versus r2/2) should be independent of rotor speed, so long as there is no pressure-dependent macromolecular association. If constant molecular weights versus concentration are obtained from a sample spun at two or three different rotor speeds, one can be quite certain that the sample is pure. For samples spanning a wide concentration range, a slight decrease in the apparent molecular weight as a function of concentration indicates solution nonideality, and is insignificant in determining sample purity. If, on the other hand, the molecular weight curves systematically increase at higher concentrations, either heterogeneity o r a mass action equilibrium are indicated. If the curves are systematically displaced to lower apparent molecular at higher rotor speeds, the solution contains a mixture of higher and lower molecular weight species. If, on the other hand, the curves of apparent molecular weight as a function of c are superimposable, a mass action equilibrium is indicated.14 Likewise, for samples containing differIZ

l3

l4

R. Eason, in "Centrifugation, A Practical Approach" (D. Richard, ed.), 2nd Ed., p. 251. IRL Press, Washington, D.C., 1984. C. H. Chervenka, "A Manual of Methods for the Analytical Ultracentrifuge." Spinco Div., Beckman Instruments, Palo Alto, California, 1970. D. E. Roark and D. A. Yphantis, Biochemistry 10, 3241 (1970).

I

[421

K K

ent protein-loading concentrations and examined at a single speed, heterogeneity results in lo~verapparent molecular weights being calculated for the most concentrated solution, whereas self-association results in superimposable curves. Acquisition and analysis of sedimentation data can be automated, so that these analyses can be done quite rapidly. More recently nonlinear least-squares techniques have been used to detect sample heterogeneity.I5 In these methods, the concentration distribution data are fit to equations that include terms describing the main component of interest as well as a second component of either higher or lower molecular weight. The terms returned by the program can be manipulated to provide an estimate on the weight fraction of contaminant. Since the rotor speed can be varied, the method can be made sensitive to contaminants of higher o r of lower molecular weight than the molecule of interest. These same methods may be used to detect heterogeneity in a protein sample in which only a fraction of the material is able to undergo a self-association.16 The reader is directed to the discussion in this reference for details on how the experiments are conducted and in their interpretation.

r: r:

DETERMINATION OF PURITY

565

Problems and Pitfalls

K

r-

It is the slope of the In c versus r 2 / 2 curve that provides information regarding the apparent molecular weight. Because derivatives are being taken, the molecular weight curves tend to be quite noisy, and good data are required to provide smooth, usable curves. For the nonlinear leastsquares methods to work, large quantities of data must be supplied, and specialized programs used in their analysis. This requires that automated data acquisition systems be used if such methods are needed routinely.

l6

M. L. Johnson, J . J. Correia, D. A. Yphantis, and H. R . Halvorson, Bioptivs. J. 36, 575 (1981). D. A. Yphantis, J. C. Correia, M. L. Johnson, and G.-M. Wu, in "Phys~calAspects of Protein Interactions" (N. Castimpoolas, ed.), p. 275. ElsevierINorth-Holland, New York, 1978.

566

CHARACTERIZATION OF PURIFIED PROTEINS

[431

C431 Determination of Size, Molecular Weight, and Presence of Subunits By THOMAS M. LAUEand DAVIDG. RHODES The apparent molecular weight of a given protein is perhaps the most often cited distinguishing characteristic of the molecule. This property is at the basis of many fractionation methods and is an easy-to-use descriptor ("the 30K subunit"). Nevertheless, some care is advisable if one intends to obtain accurate estimates of molecular size. In this chapter, size will refer to the physical dimensions of the protein, as opposed to the molecular weight of the protein, which is related to the mass of the protein. This chapter also considers the asymmetry (or axial ratio) of proteins, as this information is intimately involved in the apparent size of the molecule. Many proteins assemble into larger aggregates, with each constituent chain being considered a subunit.' The concept of a subunit, however, must be defined in context by the individual investigator, taking into account the system at hand and the objectives of the work with the system. For the purposes of this discussion, independent subunits will be defined as those protein moieties which do not have a contiguous polypeptide backbone. Thus, disulfide-linked peptide chains as well as segments associated through noncovalent interactions are considered subunits. The methodological approaches to determine the size or molecular weight of a protein can be categorized into three broadly defined areas: chemical analysis, such as composition analysis or the effect of the molecule on the properties (e.g., vapor pressure, freezing and boiling points) of a solvent, the transport of the molecule in response to some applied force (e.g., electrical, centrifugal, mechanical), and scattering of incident radiation (e.g., light, X rays, neutrons). As they are sensitive to different features of the molecule, selection of appropriate methods depends on what one needs to know about the system and with what accuracy. The capabilities, advantages, and limitations of a number of techniques are outlined in Table I. Beyond these criteria, the protein and the method must be compatible with regard to the quantity of protein available, the attainable level of p ~ r i t ysolvent ,~ requirements, and peculiarities of the

' S. N. Timasheff and G . R. Fasman, (eds.), "Subunits in Biological Systems," Part A. Dekker, New York, 1971. D. G. Rhodes and T. M. Laue, this volume, [42].

METHODS IN ENZYMOLOGY, VOL. 182

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

-

TABLE I

COMPAIUSON OF TECHNIQUES FOR CHARACTER~ZATION OF PROTEINS"

P W

u

Method Chemical Composition Colligative properties Vapor pressure Osmotic pressure Transport Sedimentation Equilibrium Velocity Gel filtration chromatography Electrophoresis SDS Native Viscosity Scattering X-ray diffraction Crystallography Low-angle scattering Powder methods Rayleigh light scattering Electron microscopy - -

-

Size

Shape

M,

Subunits

Primary method

Contaminant sensitivityb

Precision

Accuracy

I

I

-

I

Y

L, H, N

-

-

I I

I

-

I

Y Y

L, N L, N

-

-

+

+

I

+

I

Ease ofuse

Sample size

-

CLg-mg

+ +

mg mg

Ref.

c

2 m "N

I

+-

+

-

-

+

I

I -

+-

+

+

*

+

+ +

+

I

+

+

+

+ + +

+-

-

-

-

-

+

5 2

-

Y Y N N N N

L, N

L

-

Y

-

-

Y

L, H

+

-

Y

H

1

Y

+ +

+-

2

+ +

1-

+

mg mg M-mg

+

f

+

-+

t

+

Pg Fg mg

+

+

-

-

-

-

-

-

-

-

-

2

*

*

+

mg mg mg ng-pg

5 "

9

zu

2 d e

f

g

h

EZ

m

% V)

C

s5

+I

V)

-

I , Not usable; -, not good; 2 ,acceptable; +, good. L, Sensitiye to low M contaminant; H, sensitive to high M contaminant; N, sensitive to nonprotein contaminant. D. W. Kupke, Adv. Protein Chem. 15, 57 (1960). J. T. Yang, Adv. Protein Chem. 16, 63 (1961). J. R. Knox, J. Chenz. Educ. 49, 476 (1972). f 0. Glatter and 0. Kratky, "Small Angle X-Ray Scattering." Academic Press, New York, 1982. g S. N. Timasheff and R. Townend, in "Physical Principles and Techniques of Protein Chemistry." (S. J. Leach, ed.), Part B, p. 147. Academic Press, New York, 1970. U. B. Sletyr, P. Messner, D. Pum, and M. Sara, eds., "Crystalline Bacterial Cell Surface Layers." Springer-Verlag, Berlin, 1988. a

2 4

568

CHARACTERIZATION OF PURIFIED PROTEINS

[431

protein that may interfere with a specific technique. The various approaches are useful probes of other molecular properties as well, and these will be brought out in discussions of individual techniques. In addition, as will be pointed out in the text, many techniques are best used in combination with other techniques (e.g., viscosity and sedimentation, composition and SDS gel electrophoresis). This is not a complete list; many methods not included (such as radiation inactivation and cDNA analysis) may be useful in specific situations. We have selected for detailed discussion those techniques which are most useful and/or most commonly used. Suggested readings for a more complete treatment of other techniques are listed in Table I. Chemical Methods

Composition Overview. Methods that provide molar quantitation of either amino acids and/or specific prosthetic groups may be used to estimate the minimum molecular weight of a protein:

where M ~ is,the molecular weight estimate (glmol) of the protein, m is the mass of the protein used in the analysis, and n is the number of moles of the protein-related component measured in the analysis. Good quantitation has been achieved using amino acid a n a l y s e ~end-group ,~ analy~is,~ and quantitative analysis for specific prosthetic group^.^ The quantity of material required for such composition-based molecular weight analyses is most dependent on the sensitivity of the analytical method being used. Since many newer analytical techniques are sensitive in the nanomole to picomole range, only microgram or even smaller quantities of material may be required. The error in the minimum molecular weight estimate will depend on the errors incurred in both the mass estimate and in the analysis for the particular constituent and, therefore, will depend on the methods chosen for these two measurements. By combining data from analyses for several different constituents, the accuracy can be improved. For this reason, independent estimates based on amino acid analyses, quantitative analysis of the end group, or quantitation of nonprotein cofactors are recommended. J . Ozols, this volume [44]. P. Matsudaira, this volume [45]. S. Seifter and S. England, this volume [47].

[431

SIZE,M , , AND

PRESENCE OF SUBUNITS

569

Because composition analysis provides only a minimum molecular weight, it is very useful to combine these data with results from other approaches to obtain the mass of the protein. An accurate composition analysis can be combined with techniques that provide only low-accuracy total molecular weights to yield high-accuracy results. Perhaps the most often used example of this approach is the combination of amino acid analysis3with SDS gel electrophoresis6(see below). One should, in principle, be able to estimate M by determining the smallest integers that could account for the full amino acid composition. In practice, uncertainty in the concentration of all 20 individual amino acid makes this approach quite unreliable by itself. However, an approximate molecular weight from SDS gel electrophoresis can guide in the selection of the appropriate absolute concentrations from the relative concentrations, indicating to the investigator whether to "round up" or "round down" individual values. Method. An estimate of protein mass is made most accurately from a measurement of dry weight when the protein is present in a volatile buffer (e.g., ammonium bicarbonate). Compensation for nonvolatile buffer components can be made from the difference in mass between dried samples of the protein-containing solution and the buffer alone, but is difficult to do with great accuracy and should not be attempted with partially volatile buffers. Alternatively, protein concentration measurements are often used in place of dry weight estimates (the mass value used for analysis simply being the product of mass-concentration and the volume). Concentration estimates can be made refractometrically, spectrophotometrically, or by chemical assay. The accuracy of such measurements often is limited, even though the precision may be reasonable, because standardization is a major problem. This is especially true for glycoproteins, lipoproteins, or other proteins with unusual compositions where the method of analysis may be sensitive only to certain constituents of the protein (e.g., peptide bonds). Of the methods listed, refractometry (or differential refractometry) is the most accurate means of estimating concentration. Amino acid analysis also provides a suitable means of quantitati0n.l Problems and ~ i r f a l l sComposition-based . minimum molecular weight analyses are sensitive to sample purity. In general, any contaminant that influences either the mass estimation or the quantitative analysis will affect the accuracy of the determination. Since no fractionation of the starting material is afforded by these analyses, contamination or heterogeneity will cause trouble. Therefore, samples must be purified prior to analysis. The degree of purity required depends on the nature of the D.Garfin, this volume [33].

570

CHARACTERIZATION OF PURIFIED PROTEINS

[431

contaminant and the sensitivity of the mass estimate or the analytical method to that contaminant. In all of these methods, it must be assumed that there is only one, or an integral number, of moles of the analyzed component per mole of protein. Since no information concerning quaternary structure is available, only a minimum molecular weight is obtained. For example, quantitative analysis of heme iron in either hemoglobin or myoglobin would yield nearly identical apparent molecular weights, despite the fact that the molecular weight of intact hemoglobin is 4-fold greater than that of myoglobin. Colligative Properties Overview. Dissolution of a solute in a solvent reduces the chemical potential of that solvent and results in a number of observable phenomena known collectively as colligative proper tie^.^ The relationship at the basis of these phenomena is

where pa is the chemical potential of a solute (callmol), P: is pa in the standard state, R is the gas constant (8.3144 X lo7 cal/mol. K), T is the temperature (K), y is the activity coefficient of a , and X, is the mole fraction of a . When X, is less than 1, pa is less than p:. The molecular weight of any solute can, in principle, be determined by the extent to which the solution activity is changed by the presence of a known weight of solute. Thus, the molecular weight of any solute, including proteins, could be determined by measuring freezing point depression, boiling point elevation, osmotic pressure, or vapor pressure. Because changes in freezing temperature or boiling temperature are generally quite small for protein-sized molecules, and occur at extremes of temperature at which the molecules may not be stable, these methods are generally inapplicable for study of protein solutions. On the other hand, both vapor pressure and osmotic pressure have been used for measurement of protein molecular weights, but this will not be discussed in detail here. Vapor pressure osmometers and membrane osmometers are commercially available. The former reliably measure molecular weights in water to approximately 25,000, whereas the latter are used for proteins above 20,000. Protein concentrations on the order of 0.1 to 1 mg/ml and sample volumes of 10 to 200 p1 are required. As with composition methods, accurate determinations of sample mass are necessary for both of these methods. It should also be noted that the molecular weight measured with 'I. Tinoco, K. Sauer, and J . Wang, "Physical Chemistry," p. 117. Prentice-Hall, Englewood Cliffs, New Jersey, 1985.

[431

SIZE,M , ,

AND PRESENCE OF SUBUNITS

57 1

any colligative property is a number average and can be severely affected by contaminating low-molecular-weight species. Additional information can be found in Refs. 7 and 8 and in manufacturer's literature. Transport Methods

Sedimentation Equilibrium Overview. Sedimentation equilibrium provides the single most accurate and powerful method for the determination of the native molecular weight of a protein. It is a simple, nondestructive, relatively rapid method. All parameters that describe sedimentation equilibrium are either readily measured or easily estimated. It has several unique capabilities, and can provide quantitative estimates of molecular weights, stoichiometries, and association constants for a wide range of chemical systems. For example, by sedimenting in neutrally buoyant (nonsedimenting) detergents, the native molecular weight of detergent-solubilized proteins may .~ systems not amenable to analysis by any other techbe m e a ~ u r e dMany nique can often be examined profitably by sedimentation equilibrium. However, because sedimentation equilibrium generally requires sophisticated, expensive equipment, and other methods suitable for many purposes are available, this procedure is used much less frequently than in the past. Nevertheless, equilibrium sedimentation provides one of the most powerful methods for the quantitative examination of protein-protein associations, and is irreplaceable for the study of protein systems with weak to moderate association constants. Method. In an equilibrium sedimentation experiment the purpose is to produce a measurable protein concentration gradient along the radial axis of the centrifuge cell. The length of time needed to achieve sedimentation equilibrium depends on the length of the solution column, the value of cr (see below), and the diffusion constant. Because the time to reach equilibrium depends on the square of the column length, various cells and techniques have been developed to examine short columns (3 or 0.3 mm long).9 Using these cells, equilibrium can be reached in a matter of minutes to a few hours. After sedimenting for this time, the concentration distribution is measured at three or four half-hour or 1-hr intervals. Invariance of the distribution over time indicates that equilibrium has been reached. W. W. Fish, in "Methods in Membrane Biology" (E. E. Korn, ed.), Vol. 4, p. 189. Plenum Press, New York, 1978. C. H. Chervenka, "A Manual of Methods for the Analytical Ultracentrifuge." Spinco Div., Beckman Instruments, Palo Alto, California, 1970.

[431

---

SIZE,Mr, AND

PRESENCE OF SUBUNITS -----

57 1

any colligative property is a number average and can be severely affected by contaminating low-molecular-weight species. Additional information can be found in Refs. 7 and 8 and in manufacturer's literature. Transport Methods

Sedimentation Equilibrium

3

Overview. Sedimentation equilibrium provides the single most accurate and powerful method for the determination of the native molecular weight of a protein. It is a simple, nondestructive, relatively rapid method. All parameters that describe sedimentation equilibrium are either readily measured or easily estimated. It has several unique capabilities, and can provide quantitative estimates of molecular weights, stoichiometries, and association constants for a wide range of chemical systems. For example, by sedimenting in neutrally buoyant (nonsedimenting) detergents, the native molecular weight of detergent-solubilized proteins may be m e a s ~ r e dMany .~ systems not amenable to analysis by any other technique can often be examined profitably by sedimentation equilibrium. However, because sedimentation equilibrium generally requires sophisticated, expensive equipment, and other methods suitable for many purposes are available, this procedure is used much less frequently than in the past. Nevertheless, equilibrium sedimentation provides one of the most powerful methods for the quantitative examination of protein-protein associations, and is irreplaceable for the study of protein systems with weak to moderate association constants. Method. In an equilibrium sedimentation experiment the purpose is to produce a measurable protein concentration gradient along the radial axis of the centrifuge cell. The length of time needed to achieve sedimentation equilibrium depends on the length of the solution column, the value of u (see below), and the diffusion constant. Because the time to reach equilibrium depends on the square of the column length, various cells and techniques have been developed to examine short columns (3 or 0.3 mm long).9Using these cells, equilibrium can be reached in a matter of minutes to a few hours. After sedimenting for this time, the concentration distribution is measured at three or four half-hour or I-hr intervals. Invariance of the distribution over time indicates that equilibrium has been reached. W. W. Fish, in "Methods in Membrane Biology" (E. E. Korn. ed.), Vol. 4, p. 189. Plenum Press, New York, 1978. C. H. Chervenka, "A Manual of Methods for the Analytical Ultracentrifuge." Spinco Div., Beckman Instruments, Palo Alto, California, 1970.

572

CHARACTERIZATION OF PURIFIED PROTEINS

[431

a is the measured quantity in a sedimentation equilibrium experiment.1° It is defined as

where a is the reduced molecular weight, 0 is the protein's partial specific volume (mllg, determined as described for sedimentation velocity), p (glml) is the solution density (as described for sedimentation velocity), and w2 ( s e c 2 ) is the square of the rotor's angular velocity (w = rpm ..rr/ 30). a is most frequently determined as the slope of a graph of the natural logarithm of the concentration as a function of r2/2, where r is the radial position in the rotor. The slope may be calculated at each of several radial positions (or, correspondingly, at each of several concentrations). Molecular weights determined in this fashion are weight-average values. Alternatively, a may be obtained from nonlinear least-squares fitting to equations that describe the concentration distribution (i.e., c as a function of r2/2) that are beyond the scope of this chapter." These equations have been derived from thermodynamic first principles for a variety of models that include association and nonideality of the proteins. The concentration distribution at equilibrium can be determined by any number of means. If a preparative centrifuge1=or an "airfuge" l3 is being used for the measurement, any assay that is proportional to the concentration of protein may be used following fractionation of the content of the cell. However, it is then necessary to perform several experiments for different lengths of time to ensure that equilibrium has been reached. Also, these techniques suffer a loss of precision due to the collapse of the concentration gradient that occurs as the rotor decelerates and as fractionation of the cell contents is performed. The analytical ultracentrifuge alleviates this problem by permitting the solution contents to be examined optically as the centrifuge is operating and the contents of the cell remain at eq~ilibrium.~ Currently, there are two optical systems available for the analytical centrifuge, the absorbance scanner and the Rayleigh interferometer. These two systems provide complementary information, with the selectivity and sensitivity of the scanner being useful for some situations, while the precision and accuracy of the Rayleigh system are needed in others. The radius must be calculated with accuracy. The analytical ultracentrifuge provides reference markers, which along with the optical magnifilo

l2 l3

D. A. Yphantis, Biochemistry 3, 297 (1964). M. L. Johnson, J. J. Correia, D. A. Yphantis, and H . R. Halvorson, Biophys. J. 36, 575 (1981). A. K. Attri and A. P. Minton, Anal. Biochem. 152, 319 (1986). P. Bock and H. Halvorson, Anal. Biochem. 137, 172 (1983).

2 f

%

,

Fk

h2q

cation factor are used to calculate the radial position in the image. The instrument operating manuals provide good information on how to determine the r a d i ~ sWhen . ~ using a preparative centrifuge, the radius must be calculated from geometric considerations. The original articles describe how this is done for various rotors and c e l l ~ . ' ~ , l ~ Problems and Pitfalls. The principal problem in sedimentation equilibrium analysis is obtaining a sufficient quantity of highly purified protein for analysis. This is not as formidable as it seems, since a molecular weight may be measured using the Rayleigh interferometer with just 20 p1 of solution at a concentration of 1 mglml. This is no more material than is often used for gel electrophoresis. In cases where a preparative centrifuge is being used and a sensitive assay is available, less material may be required, but lower precision can be expected. It should also be realized that equilibrium sedimentation does not afford the extent of fractionation that is seen with gel filtration or gel electrophoresis. Even so, the gravitational field does fractionate the solution to some extent, such that very large particles (e.g., dust, flocculant) are removed from observation. This means that the scrupulous cleanliness of samples necessary for scattering techniques (see below) is not needed for analytical centrifugation. Note that buffer components will also be fractionated in the gravitational field. For this reason, buffers that have a zj that is near that of the solvent (Up = 1) are preferred. Thus, buffers such as Tris are good choices for sedimentation, while phosphate buffers can pose problems under some circumstances.I0 Problems of buffer sedimentation are minimized by extensive dialysis and careful filling of the cell. Convection can also be a problem. As with sedimentation velocity analysis, it is usually caused by rotor vibration or thermal gradients, and can be minimized by taking precautions to balance the cells carefully and setting the cooling apparatus appropriately. In addition, convection can be minimized by including a sufficient quantity of a buffer component to provide a small, stabilizing density gradient (e.g., 0.1 M NaC1, or small quantities of sucrose). Convection can often be detected in the concentration gradient as a bump on the ordinarily smooth exponential gradient. Alternatively, a "hook" in the concentration gradient (either positive or negative) may be present near the meniscus. Many times convection can be detected by examining the difference between two concentration curves made at the same speed, but at different times. Thus, the procedure of making several measurements at a given speed serves two purposes, first to ensure that equilibrium has been reached, and second, to check for convection. Under some circumstances, a sample that exhibits convection in the meniscus region may still provide some useful information if that region of the concentration gradient is omitted from the analysis.

574

CHARACTERIZATION OF PURIFIED PROTEINS

[431

Tests are available for detecting impurities in a sample being analyzed by sedimentation equilibrium. One is to generate graphs of the apparent molecular weight (determined as the local slope from the graph of In c versus r2/2) as a function of concentration. If the M ( c ) graphs are independent of rotor speed, one can be quite certain that the sample is pure. More sensitive tests for heterogeneity are outlined in [42] of this volume. Sedimentation Velocity Overview. Sedimentation velocity is a simple, nondestructive technique for the characterization of the hydrodynamic behavior of a protein. It has the advantage over gel chromatography of being a primary technique (not requiring standards) for the determination of hydrodynamic parameters. Because of its roots in first principles, sedimentation analysis can be applied to systems that cannot be analyzed by any other means. The principal result from a sedimentation velocity experiment is the sedimentation coefficient (s), which is a measure of the ratio of the buoyant mass of the protein to its frictional coefficient:

where s is the sedimentation coefficient in Svedberg units (x seccl), No is Avogadro's number, and f is the protein frictional coefficient. The diffusion coefficient, D (cm2/sec), defined in Eq. ( 3 , also is measurable from boundary spreading in measurements of analytical centrifugation experiments: The ratio of slD [Eq. (6)] provides a measure of the molecular weight that is free of any requirements concerning size, shape, or similarity to protein standards: Alternatively, if an accurate molecular weight is known, measurement of the sedimentation coefficient allows the frictional coefficient, f, to be determined using Eqs. (6) and (7). The ratio of flf may then be used to estimate the shape of the protein, where f 0 is defined as the frictional coefficient expected for an anhydrous sphere of equal molecular weight and density. f o is calculated by writing the Stokes equation for this sphere: where

r)

is the viscosity and rsphere is the radius of the equivalent sphere.

1431

SIZE,

Mr, A N D

PRESENCE OF SUBUNITS

575

This ratio f/fO is then compared to standard and theoretical values to estimate the asymmetry and overall shape of the protein (see below). Method. Sedimentation velocity experiments are best performed in an analytical ultracentrifuge, although methods of reasonable accuracy have been devised for preparative centrifuges. The advantage of the analytical machine is its accuracy (+ 1-3%), which is nearly an order of magnitude better than can be expected from techniques using preparative machines. Moreover, data are acquired throughout the experiment so that better diagnostics of unusual behavior is afforded. The advantage of the preparative machines is that sensitive or specific assays may be used so that in cases where the purity of the sample is in doubt, a specific assay can identify the component of interest. Detailed methods for using the analytical ultracentrifuge9and for using preparative centrifuge^'^.'^ are available. As noted before, the principal measurement in any sedimentation experiment is the concentration as a function of radial position. The sedimentation coefficient is determined from the slope of a graph of the natural logarithm of the distance that the molecules have sedimented as a function of time (d In rldt): where r is the distance of the boundary of the molecules from the center of the rotor and t is the time (in seconds) from the start of the experiment (usually taken to be the time at which the rotor is two-thirds its final speed). For experiments where the sample being examined is a thin zone of molecules, r is taken to be the point of maximum concentration. For broad zone or boundary experiments, r is taken to be the midpoint between zero concentration and the plateau concentration. For the schleiren optical system, which provides a measure of dcldr (r), r is usually taken to be the maximum of the schlieren peak. For proper interpretation and comparison, the sedimentation coefficient must be corrected to standard ) conditions of water at 20' and zero protein concentration ( ~ 4 0 , ~using 0 S20,w

= Sobserved

( 1 --

P~O.~&O,~)(% )(vT,~) &O.WPT,~ 720.w ~ T , W

where 0 , p , and 77 are the partial specific volume, density, and viscosity at the experimental temperature (T or 20") and buffer conditions (w, water; b, buffer), respectively. Tabulated values for the viscosity of water are used to correct for the effect of temperature on the sedimentation coefficient. The density is either measured or calculated from tabulated values. l4 l5

D. Freifelder, this series, Vol. 27, p. 140. R.Martin and B. Ames, J . Biol. Chern. 236, 1372 (1961).

576

CHARACTERIZATION OF PURIFIED PROTEINS

[431

-

The effect of protein concentration on s is assessed by determining s at each of several protein concentrations. Typically, there is a slight linear decrease in s as the protein concentration is increased. The diffusion coefficient is determined from the spreading of the boundary as it progresses down the analytical cell. A graph is made of [c/ (dcldr),,,12, where (dcldr),,, is the concentration gradient at the point of maximum gradient, for most cases the midpoint of the boundary9, as a function of time, and the slope of this line is used to estimate D, the diffusion coefficient:

where D is in centimeters squared per second and c is the protein concentration (arbitrary units). The ratio of slD provides the reduced molecular weight, Ma (1 - vr)l R T so determination of M requires other experimental measurements. The solvent density, p , is readily measured or calculable from tabulated data. The partial specific volume of the protein, 0 , can be measured, but more frequently is calculated from its composition using the method of Cohn and Edsall and tabulated values of a for the amino acids.16Values of 0 for carbohydrates may be found in Gibbons1' and Durschlag.18 Values for other common protein-associated components can be found in Steele et al.19 or Reynolds and M c C a ~ l i nFor . ~ ~more accurate work 0 is adjusted for temperature (over the range 4-40") using a coefficient of 4.3 x cm3/g. K, as described by Durschlag.18 The effects of pH, the buffer composition, and preferential hydration on 0 are typically neglected, except in cases where a high concentration of denaturant is used. Special provisions may be made for estimating the isopotential apparent partial specific volume ( n ) of simple proteins in solvents containing 8 M urea and 6 M guanidinium chloride, using the method given by Prakash and Tima~heff.~' Likewise, n may be estimated for simple proteins in solvents containing varying amounts of NaCI, Na2S04, MgS04, glycine, Palanine, a-alanine, betaine, and CH3COONa using the method of Arakawa and T i m a ~ h e f fNeither .~~ estimate is very accurate, however, if the T. L. McMeekin and K . Marshall, Science 116, 142 (1952). R. A. Gibbons, in "Glycoproteins," Part A (A. Gottschalk, ed.), p. 31. Elsevier, Amsterdam, 1972. l8 J. Durschlag, in "Thermodynamic Data for Biochemistry and Biotechnology" (H. Hinz, ed.), p. 46. Springer-Verlag, Berlin, 1986. l9 J. H. C. Steele, C. Tanford, and J. A. Reynolds, this series, Vol. 48, p. 18. 20 J. A. Reynolds and D. R. McCaslin, this series, Vol. 117, p. 47. 21 V. Prakash and S. N. Timasheff, this series, Vol. 117, p. 53. 22 T. Arakawa and S. N. Timasheff, this series, Vol. 117, p. 60. l6

l7

[431

SIZE,

M,,

AND PRESENCE OF SUBUNITS

577

protein contains significant levels (>lo%, wlw) of nonamino acid constituents (e.g., glycoproteins, lipoproteins) or if the axial ratio is greater than 10. Hydrodynamic interpretation of sedimentation data can be made once an accurate molecular weight and v estimate are available. First, one calculates f a using Eq. (7) above. The ratio of the measured frictional coefficient to f O ( f If O ) will be a value greater than one. Note that f must be calculated using ~ 4 0 . ~ : where the various terms are as described above, and s$,, is determined as described in Eq. (9). There are two reasons that f l f will be greater than one. First, proteins are "coated" by a layer of water molecules that move with the protein as it sediments. While these water molecules freely exchange with those in the bulk solvent, the net result of the layer is to increase the effective radius of the protein. As can be seen from Eq. (7), any increase in the effective radius will increase f. Second, the frictional coefficient depends on the surface area of the protein presented to the solvent. For a molecule of given mass and density, a sphere would be the shape that exposes the minimum surface area, so any molecular asymmetry will increase f. The degree of molecular asymmetry is estimated by determining the axial ratio (alb) of the ellipsoids of revolution, prolate (elongated) or oblate (flattened), that would result in an identical value for f l f o . It should be clear that the asymmetry is being modeled using these ellipsoids of revolution, and that the results may not "look" like the molecule at all. However, if one can measure the asymmetry for the isolated subunits of a multimeric protein, as well as for the intact oligomer, it is possible to use changes in alb to distinguish between possible arrangements of the subunit~.*~ Problems and Pitfalls. The use of sedimentation velocity measurements to determine molecular weights has declined in favor of gel electrophoresis, gel chromatography, or the more accurate method of sedimentation equilibrium. However, sedimeniation velocity provides the best and the only primary method for the determination of hydrodynamic parameters available to molecular biologists. Electrophoresis and gel filtration require that standards be used, which places restrictions on the data interpretation. Of all the techniques described in this chapter, the problems and pitfalls of sedimentation analysis are the best documented and most easily overcome. 23

C. R. Cantor and P. R. Schimmel, in "Biophysical Chemistry," Part 11, p. 565. Freeman, San Francisco, California, 1980.

578

CHARACTERIZATION OF PURIFIED PROTEINS

[431

-

The most demanding aspect of sedimentation is the availability of sufficient material for analysis. If the standard optical systems on the current (Beckman model E) analytical ultracentrifuge are used, 0.5 ml of solution with protein concentrations in the range of 0.1-1 mglml is needed to obtain good data. The principal technical difficulty with sedimentation analysis is convection. This problem usually indicates that the drive is vibrating excessively, that the temperature control is not set properly, or that the cell is misaligned or deformed. All of these potential obstacles may be tested for and corrected. The second problem is that the "current" ultracentrifuge is antiquated, and few individuals are well versed in its operation. The techniques are not inherently difficult, but the machinery is ornery. It is expected that a new analytical ultracentrifuge will be available by the 1990s, and that the data acquisition and analysis described above will be automated. The interpretation of sedimentation coefficients for proteins that bind significant levels of buffer components (e.g., detergent-solubilized proteins) is made difficult by the fact that the bound components will contribute to the measured s in four terms: M, 6, p , and f. Of these terms, M, 6, and f are usually the most affected by bound components, and, unlike sedimentation equilibrium (see below), there is no way to "blank out" the contribution of such components. Thus, the measured sedimentation coefficient is for the complex of the protein with the bound component, making it difficult to extract any useful information concerning the protein alone. Another widely used method of estimating molecular weights of proteins is the gradient sedimentation method introduced by Martin and Ames.IS In this method, one creates a linear gradient of sucrose in buffer in a swinging bucket centrifuge tube. The sucrose concentration typically ranges from 5% at the top of the tube to 20% at the bottom; the actual range is less important than the linearity and reproducibility of the gradient. The unknown sample is layered onto a gradient and a set of standard proteins of known molecular weight layered onto an equivalent gradient. The centrifugation proceeds for an appropriate interval (typically 12-24 hr) and the material on the gradients collected as fractions. The protein concentration in these fractions is determined by spectrophotometric, enzymatic, or other assays. The basis of the method is the fact that in a linear sucrose gradient, the distance travelled by a molecule should be a linear function of the time of centrifugation at a specified speed. In addition, the distance will depend linearly on s. The ratio of the distance travelled by an unknown protein to that of a standard will be equal to the ratio of their sedimentation coeffi-

[431

SIZE,

M,,AND

PRESENCE OF SUBUNITS

579

cients, which will, in turn be approximated by the ratio of the molecular weights to the 213 power. This method yields approximate values of s and M, but is simple, requires no specialized equipment, and can be used to estimate s and M for very small amounts of material if a suitable (e.g., enzymatic) assay is available.

Gel Filtration Chromatography Overview. Gel filtration chromatography is one of the most powerful and simplest methods for the estimates of the molecular weight of proteins. Because of the fractionation afforded by the method, and because assays specific for the protein of interest may be used (e.g., enzymatic, immunological), sample purity does not have to be very high. The method is nondestructive, can be fairly rapid, and has moderate accuracy as long as the protein of interest is roughly the same shape as the protein standards used to calibrate the column.23 The determination of a molecular weight by gel chromatography relies on the comparison of the elution volume of the unknown with those of several protein standards whose molecular weights are known. The molecular weight of the unknown is estimated from a graph of the logarithm of the molecular weight as a function of elution volume (or Kav, as described below) made using the data from the protein standards. (It is worth noting that the actual dependence is on the logarithm of the effective hydrated radius, or "Stokes radius" of the protein, and that the fit of standard proteins to this variable is better.) The elution volume (V,) for the standards should cover the range from Vo (the void volume) to V, (where Vi is the included volume). A column-independent measure of the protein behavior, K a v ,is more useful for comparison of results than simply the elution volume:

where K,, is the fraction of the stationary gel volume which is accessible to the protein, and V , is the total volume of the gel bed. Use of K,, is preferred over Ve since, for a given gel type, values of K,, will vary only slightly from column to column. The methods described below can be used for both native and denatured proteins and, therefore, provide a means for establishing the presence of subunits. However, gel filtration in denaturing solvents typically requires more material than denaturing gel electrophoresis and is thus not used as often. Method. The principle of operation and selection of gel media and gel G. K.Ackers, Adv. Protein Chem. 24, 343 (1970).

580

CHARACTERIZATION OF PURIFIED PROTEINS

1431

porosity is described in detail elsewhere in this volume.25 One must choose a gel in which the protein to be examined is partially included. Choice of the gel medium usually is arbitrary, as long as the protein does not bind to the gel matrix. When there is a choice of bead sizes for a given porosity, the smallest bead size should be used, as this improves the column resolution. Check the manufacturer's recommendations for any particular limitations on solvents, but in general, just about any freeflowing aqueous buffer system may be used. It is recommended that buffers of moderate ionic strength be used so that electrostatic interactions between the protein and immobile charges on the gel matrix are minimized. For best results, use a long, narrow column. Preparation of the gel, pouring of the column, and equilibration of the column by washing with buffer should be done according to the manufacturer's specifications. Likewise, flow rates should be chosen in accordance with the manufacturer's specifications. In general, lower flow rates afford better resolution because the solute can fully equilibrate with the gel matrix at all times, but excessive diffusion can limit resolution if a column is run too slowly. For molecular weight estimations, extra care should be exercised in making sure that the "fines" (partially pulverized gel beads) are removed, as these will reduce the column flow rate and reduce the column resolution. All samples should be in the same buffer as that used to equilibrate the column. Sample volumes applied to the column should generally be less than 2% of the column's bed volume. Care should be exercised when applying samples to the column to make sure that the gel bed is not disturbed or that the gel bed does not run dry. In addition, the flow rate of the column should be kept constant throughout all of the analyses, since flow rate dependence of the elution volume can be expected." Elution volume (V,) is the volume eluted from the column, starting once one-half of the sample has penetrated the top of the gel bed and continuing until the maximum (peak) of the protein of interest has eluted. The void volume (Vo)of the column usually can be measured using commercially available, size-graded Blue Dextran (M = 2,000,000), and monitoring the effluent spectrophotometrically at 540 nm (or 280 nm). The included volume can be measured using as a sample some buffer of sornewhat different pH or conductivity (extremes should be avoided), or one that contains a small dye (e.g., Bromphenol Blue). Care should be exercised in the choice of the dye, as many aromatic compounds will stick to gel matrices, resulting in anomalously high K,, and Vi values.

" E. Stellwegen, this volume [25].

[431

-

SIZE,M,, --

A N D PRESENCE OF SUBUNITS -

--

58 1

Protein standards should be used that span the full range of sizes that can be analyzed by the gel medium chosen. For best results, a minimum of four different standards should be used. Kits containing prestained proteins are available. Any convenient assay for detecting the presence of the protein being analyzed may be used. If for any reason the column must be repacked, the calibration must be performed again. Use of column chromatography to estimate the molecular weight of denatured proteins poses special problem^.^ This method relies on the shape of the unknown being identical to that of the standards. This means that the protein must be totally denatured, including reduction of disulfide bonds. Buffers should contain a reducing agent or else the sulfhydryls should be alkylated to prevent reformation of any disulfide bonds. Both the standards and the unknown must be analyzed after the same treatment and the same buffer conditions. Calibration proceeds as described above. Problems and Pitfalls. The greatest source of error in gel chromatography comes from the requirement that the unknown be similar in shape and density to the protein standards. Since the protein standards used are almost universally compact, globular proteins, this means that fibrous proteins, or proteins having fibrous regions, can behave anomalously on gel columns. One indication of such molecular asymmetry is if K,, for the unknown increases when analyzed at decreased flow rates, while those for the standards remain unchanged. Since it is the size of the protein and not the molecular weight that is being assessed by this technique, molecular weight estimates for proteins that are complexed with other molecules (e.g., detergent-solubilized proteins, extensively glycosylated proteins) will be unreliable. Finally, if the protein interacts with the gel matrix (e.g., binds or is repelled), inaccuracies will result. This can be tested by determining the molecular weight using two different types of gel matrix (e.g., Sepharose and acrylamide). If K,, > 1, then the molecule is binding to the column, and a different gel matrix should be used. On the other hand, if K,, < 0, the column is "channeling" and must be repoured and recalibrated.

Electrophoresis Overview. The most widely used method of evaluating the size of a protein molecule is electrophoresis. The method is simple, inexpensive, rapid, and reasonably accurate for a very wide range of proteins. For these reasons it is the method of choice for most protein systems, and almost always included in characterization studies. SDS gel electrophoresis is the most widely used method for determining apparent molecular

582

CHARACTERIZATION OF PURIFIED PROTEINS

[431

weights of denatured proteins (discussed elsewhere in this volume6), but electrophoretic methods for obtaining size, shape, and molecular weight information are not limited to just this approach. Despite its popularity, it is not necessary to include SDS in the gel formulation; native gel electrophoresis of protein samples may be carried out under almost any buffer condition required. In addition, the sensitivity of current staining procedures allows these approaches to be applied to very small amounts of protein.26 Method. The basic procedures are identical to those described earlier6 for electrophoresis of denatured proteins, except that the buffer composition is left to the discretion of the investigator. There are some restrictions; as with gels under denaturing conditions, the buffer in which the gel is cast cannot contain reducing agents. In addition, because the conditions to which the protein is exposed may affect charge, association, or shape, the composition of the buffer in the gel must be controlled carefully. One must, therefore, be especially cautious about relative proportions of catalyst, so that excess oxidant is not left in the gel. It is often easiest to prerun such gels to remove by-products of polymerization. If the geometry of the gel apparatus allows the slab to be exposed (e.g., a horizontal slab or a vertical slab in which one of two glass plates can be removed and later replaced) the gel can be dialyzed against the buffer of choice prior to running. Because slabs are generally quite thin, dialysis for several hours is generally sufficient. This dialysis procedure can also be used to introduce reducing reagents postpolymerization, or to use a set of gels cast together (and thus, presumably, uniform in porosity) with a variety of buffer conditions. The basis of electrophoretic protein size analysis is based on a simple principle: that a charged particle in an electric field is forced through the surrounding medium by a force proportional to the charge on the particle and the strength of the field, and is subject to a frictional force proportional to the velocity, the radius of the particle, and the viscosity of the medium. As with SDS gel electrophoresis, the investigator may control the frictional coefficient by controlling the porosity of the gel matrix. In addition, under nondenaturing conditions, the mobility can be significantly affected by alterations of the intrinsic charge on the protein due to changes of pH at which the electrophoresis is carried out. This distinction is important. In SDS gel electrophoresis, the charge is dominated by the negatively charged SDS associated with the protein so the sample is applied to the cathode end of the gel and the sample always moves toward the anode. In nondenaturing electrophoresis, the direction of migration 26

C. R. Meml, this volume [36].

I

[431

SIZE.

M,,

AND PRESENCE OF SUBUNITS

583

will depend on the buffer pH relative to the pI of the protein. If the pI of the protein is unknown (and quantities are too limited to allow experimental determination with isoelectric focusing), a horizontal electrophoresis apparatus may be used, and the starting wells placed in the center of the gel. Otherwise, a "best guess" might be made by using the p1 of related proteins. The mobility will also be affected by the chosen buffer condition. Beyond association or conformational changes associated with changing buffer conditions, the relative mobilities of various protein at a given pH will usually be fairly constant. The absolute mobility, however, will be strongly affected by the concentrations of counterions. The analysis of native gel electrophoresis mobility data is analogous to that of SDS gels. The only significant difference is that, in the native system, one cannot assume that the proteins are all in the shape of long rods. One must compare the mobility of the sample to that of a set of standard^.^' Ferguson analysis can also be used to identify the feature(s) (size and/or charge) that distinguish two components and to extrapolate to the mobility expected in the absence of sieving effects.28The slope of the Ferguson plot (log of the relative mobility, Rf,vs gel concentration) is proportional to r , , the Stokes radius. Interpolative estimation of an unknown r, is more reliable using this relation than using, for example, the slope of the Ferguson plot with molecular weights of the standards. The analysis simply involves running the unknown and several standards in a set (at least five) of gels of different total concentrations, and plotting log Rf vs the gel concentration for all standards and for the unknown. The slopes for the standards are then plotted as a function of the (known) r , , and the r, of the unknown is derived from this plot and the measured slope. The range of gel concentrations used will depend on the size of the protein under study; at some concentration the protein will be excluded from the gel. A more closely spaced group of gel concentrations covering a lower range should then be used. Problems and Pitfalls. Although electrophoresis under nondenaturing conditions can provide useful information about the physical characteristics of the protein under a number of different conditions, it is important to be aware that this is a zonal method, and that concentration effects (or dilution effects) can be serious. It is probably not generally advisable, for example, to use this approach to study association behavior quantitatively. 27 28

D. Rodbard and A. Chrambach, Anal. Biochem. 40,95 (1971). A. T. Andrews, "Electrophoresis: Theory, Techniques, and Biochemical and Clinical Applications." Clarendon Press, Oxford, 1986.

584 - -

CHARACTERIZATION OF PURIFIED PROTEINS -

- -

-

-

-

-

- -

-

[431

-

This is not a primary technique; it depends on selection of appropriate standards. These are generally globular, unmodified, soluble proteins, so that highly asymmetric proteins or proteins with unusual nonprotein "baggage" may yield erroneous results. The Ferguson analysis helps to account for differences in mobility due to charge differences, but exaggerated charge densities may yield anomalous results. Because the buffer conditions in native gel electrophoresis are selected by the individual investigator, and thus may vary widely, one must be aware of the solution components responsible for carrying current. High ionic strength buffers may result in unacceptably slow protein mobilities, and low ionic strength buffers may run properly at surprisingly low currents. Because of this variability and because protein integrity is more important, one must be particularly aware of power dissipation and cautious about efficient removal of heat generated in the gel. Viscosity

The viscosity of a solution depends on a number of variables (T, P, etc.), including the amount and nature of any solute that might be present. The response of a particle in a fluid under shear will depend on the frictional coefficient of the particle (as the "handle" the solution has on the particle) as well as on the mass of the particle (as this will determine ~ ~ . ~ ~the intrinsic the energy required to attain a given m o ~ e m e n t ) .Because viscosity of a protein depends very strongly on the asymmetry of the molecule, viscosity measurements are sensitive indicators of protein shape. In addition, it is possible to combine viscosity data with sedimentation data to calculate the molecular weight of a protein. Apparatus for rheological measurements vary widely; their use is generally quite simple, but must be performed under rigorously controlled conditions. Scattering Methods

Scattering methods are generally used to obtain radii of gyration, but specific scattering methods can provide diffusion coefficients, molecular weight, and thermodynamic parameter^.^',^^ Details regarding these methods are reviewed elsewhere and will not be treated in depth here.

3' 32

J. T. Yang, Adu. Protein Chem. 16, 63 (1961). J. F. Johnson, J . R. Martin, and R. S. Porter, in "Physical Methods of Chemistry" (A. Weissberger and B. W. Rossiter, eds.), Vol. 1, Part VI. Wiley, New York (1977). S. N. Timasheff and R. Townend, in "Physical Principles and Techniques of Protein Chemistry" (S. 3. Leach, ed.), P a t B, p. 147. Academic Press, New York, 1970. B. Chu, "Laser Light Scattering." Academic Press, New York, 1974.

[431

SIZE,

M,,

AND PRESENCE OF SUBUNITS

585

Electron Microscopy

Electron microscopy is an appealing approach to determining molecular size and shape for large protein molecules or associated complexes of subunits because it is a direct imaging method. Initial work in this area was used to identify the shape of very large complexes like the hemocyanin aggregate^,^^ but more recent work has focused on imaging of crystalline arrays of membrane proteins.34 This method provides very detailed information about the native size and shape of the molecule for a class of proteins that are incompatible with many other analysis methods. The resolution of the transmission electron microscopy approaches utilizing mathematical filtering and reconstruction methods is quite high; domains of specific subunits can often be resolved. Recent work with ultrathin (one to two atomic layers) coating techniques35 has allowed individual proteins as small as 100 kDa (-0.5-nm resolution) to be directly visualized with scanning electron microscopy. The rapidly developing direct imaging technologies seem likely to play an ever-increasing role in analysis of macromolecular size and shape analysis. Presence of Subunits

To determine whether subunits are present generally involves characterization of one or more properties of the system under conditions which favor association, followed by analysis under conditions which are likely to favor dissociation. Clearly, this implies that the investigator has some prior knowledge of the conditions under which association should be expected, or that one is willing to investigate a variety of conditions which have been shown in other cases to result in dissociation. On the other hand, a properly planned "search for subunits" will not only reveal their existence but will yield additional information about the nature of the system. Before initiating a wide-ranging search for associating or dissociating conditions, it is worthwhile using a "brute force" test as a preliminary evaluation. One normally carries out one fractionation procedure under some (often physiological) buffer condition and a second under strongly denaturing conditions. One very simple, rapid approach for searching for subunits is to carry out electrophoresis under nondenaturing conditions, using the buffer in which the protein was isolated, followed by a second dimension under E. J. F. vanBruggen and E. H. Wiebenga, J . Mol. Biol. 4, 1 (1962). U.B. Sletyr, P. Messner, D. Pum, and M. SAra, eds., "Crystalline Bacterial Cell Surface Layers." Springer-Verlag, Berlin, 1988. 35 K.-R. Peters, personal communication.

"

586

CHARACTERIZATION OF PURIFIED PROTEINS

[431 L

dissociating or denaturing conditions. The nature of the denaturing conditions may vary depending on the type of subunit and the association one expects to find. For example, one could run the second electrophoresis at extremes of pH, in the presence of urea or SDS, or in the presence or absence of other buffered components like calcium. Changes in mobility due to changes of buffer condition should be accounted for in analyzing the second dimension. That is, analysis should be based on the apparent size of the molecule under associating and possibly dissociating buffer conditions. If one is investigating the possibility that disulfide-linked subunits are present, SDS gel electrophoresis in the absence of reducing agents may be carried out as the first dimension, and SDS gel electrophoresis in the presence of reducing agents may be carried out in the second dimension. (Note: Because in-gel alkylation of disulfides is difficult, it is recommended that mercaptoacetate be included in the second gel running buffer to avoid reoxidation). Differences in the apparent molecular weight or the appearance of multiple components in the second dimension will indicate that disulfide-linked subunits were present. Differences in apparent molecular weight deduced from nondenaturing electrophoresis compared with the apparent molecular weight based on electrophoresis under completely denaturing conditions (SDS-PAGE) could indicate the presence of subunits, but one must consider the possibility that the electrophoretic behavior of the molecule is anomalous (i.e., extremes of asymmetry or intrinsic charge). In the case of subunits which are in association/dissociation equilibrium, the definitive approach to determining the presence of subunits requires experimental determination of the apparent size of the molecule under conditions where the equilibrium is shifted to either associating or dissociating conditions. Because the dissociation implies that the samples being studied will be very dilute, very sensitive methods must be used. Sedimentation equilibrium is very useful for determining the molecular weight of a native protein, and is therefore useful in determining the stoichiometry of the subunits in the final assembly. This is done by comparison of the native molecular weight with that obtained in separate experiments under denaturing conditions, such as by denaturing gel electrophoresis. If the protein is composed of subunits of a single molecular weight, division of the native molecular weight by the denatured molecular weight will provide the subunit stoichiometry. Likewise, for proteins that contain more than one chain, comparison of the native molecular weight to the sum of the monomer molecular weights often will allow the stoichiometry of the different subunits in the native complex to be determined. In cases where there is a wide discrepancy between the subunit

-

. ,

[441

AMINO ACID ANALYSIS

587

molecular weights or when the native structure contains a large number of monomers, these estimate are imprecise. While the subject requires too much detail to be presented in full here, it is important to note that equilibrium sedimentation provides one of the most powerful means of determining association constants of mass action-driven macromolecular associations." The method of experimentation is essentially as outlined above, except that experiments are done at concentrations that encompass the range where significant mass changes occur due to association. The association constant may be estimated using graphical means, or more accurately using nonlinear leastsquares analyses. " Gel-permeation chromatography can also be useful in determining the stoichiometry of the subunits in the final assembly. Again, this is done by comparison of the native molecular weight determined by gel filtration with that obtained under denaturing conditions, such as by SDS gel electrophoresis. One can test for association and, in principle, determine stoichiometry using the ratio of the native molecular weight and the denatured molecular weight. Gel chromatography is also useful for diagnosing interacting protein systems. If, for example, the protein of interest is undergoing a rapidly equilibrating assembly/disassembly, the peak shape is skewed, with the leading edge being hypersharp and the trailing edge being diffuse. Likewise, if the K,, increases with increasing protein loading concentration, it usually means that such an interaction is occurring, and that more detailed analyses will be required.24

[441 Amino Acid Analysis By JURIS OZOLS

Amino acid analysis provides an important quantitative parameter in the characterization of isolated protein or peptide samples. Because of the availability of highly sensitive instruments for determining amino acids in the picomole range, the preparation of the sample for analysis is of prime importance in obtaining meaningful results. Analysis of a standard mixture of amino acids can be readily achieved reproducibly down to picomole levels, but obtaining accurate results from a microgram of protein after the necessary transfer and hydrolysis steps, without losses or contamination, is far more difficult.

METHODS IN ENZYMOLOGY, VOL. 182

Copyright D 1990 by Academic Press. Lnc. All rights of reproduction in any form reserved.

588

CHARACTERIZATION OF PURIFIED PROTEINS

[441 L

General Information

The first step in the determination of the amino acid composition of proteins is releasing the amino acids quantitatively without concomitant degradation. The most common hydrolysis method uses 6 N HC1 for 20 to 24 hr at 110" under vacuum.' There are several amino acids that will undergo modifications under these conditions. Asparagine and glutamine will be hydrolyzed quantitatively to aspartic acid and glutamic acid. Losses of threonine and serine also occur, and are usually around 5 and lo%, respectively. Destruction of these amino acids increases linearly with time. Tryptophan is not generally recovered in acid hydrolysates, and special methods are required for the determination of this amino acid. Tyrosine can be halogenated during HC1 hydrolysis. Inclusion of 0.1% (w/v) phenol in the 6 N HC1 will prevent modification of tyrosine. Methionine is subject to oxidation to methionine sulfoxide during hydrolysis. Addition of 2-mercaptoethanol to the 6 N HCl prior to hydrolysis will prevent this oxidation. Cysteine cannot be determined directly from acidhydrolyzed samples, but has to be oxidized with performic acid,2carboxym e t h ~ l a t e dor , ~ derivatized with 4-vinylpyridine4 prior to the hydrolysis. During acid hydrolysis of proteins, certain aliphatic amino acids are released more slowly than other amino acids. The Ala-Ala, Ile-Ile, ValVal, Val-Ile, Ile-Val, and Ala-Val bonds are particularly resistant to hydrolysis, and are cleaved only to the extent of about 50 to 70% in 24 hr at 110'. Hydrolysis for 92 or even 120 hr may be necessary to achieve quantitative hydrolysis of these bonds. Extrapolation back to zero time of hydrolysis is then necessary for serine, threonine, and other amino acids that are destroyed during prolonged incubation. Ideally, samples to be hydrolyzed should be dialyzed against water in order to minimize potential interference in the hydrolysis of peptide bonds, and possible destruction or derivatization of amino acids since many contaminants adversely affect precolumn derivatization chemistry or analysis methodology. Practically, it is often impossible to desalt small amounts of sample without risking significant losses. The presence of phosphate salts or divalent metals during the hydrolysis step will give low and variable yields of most amino acids. Volatile buffers, such as ammonium acetate, usually have no negative effects on the hydrolysis. Since it is difficult to predict the effects of residual salts, buffers, or detergents on the hydrolysis or analysis steps, it is recommended that a known quantity of

' S. Moore and W. H. Stein, this series, Vol. 6, p. 819. C. W. H. Hirs, this series, Vol. 11, p. 197.

' C. W. H. Hirs, this series, Vol. 11, p. 199.

Id.A. Raferty and R. D. Cole, J. Biol. Chem. 241,3457 (1966).

-

.

-

AMINO ACID ANALYSIS

r

!, i

[1

'i

E

4

IT-. iL

r, r,

589

standard amino acid mixture be added to the particular buffer system, and the control sample carried through the procedure. Amino acid analysis of blank samples will also indicate the level of contribution of unwanted amino acids arising from solvents, reagents, glassware, and particulates introduced during the handling of the sample. Minimizing the transfer and drying steps will decrease the losses and contamination of the sample to be analyzed. If it is evident that the sample contains substances that interfere with the hydrolysis and analysis steps, then one must rely on the sample preparation methods outlined below. Instrumentation

Because amino acid analyzers are expensive and require considerable expertise to operate, investigators in need of an amino acid analysis frequently depend on a facility equipped to perform such analyses. Currently, about one-half of the amino acid analyzers in shared instrumentation fac~litiesemploy ion-exchange separation of the free amino acids, followed by postcolumn derivatization with ninhydrin, o-phthaldehyde, or fluorescarnine.' The other half of the amino acid analyzers used by the core facilitieq rely on precolumn derivatization with phenyl isothiocyanate, followed by reversed-phase high-performance liquid chromatography (HPLC) separation of the resulting phenylthiocarbamyl (PTC) amino acids.' Most of the shared facilities have both instruments, indicating that there are advantages and disadvantages inherent in both the ion-exchange and the HPLC methodologies. In general, for routine analysis of protein hydrolysates, the ionexchange-ninhydrin method (IE analyzer) is preferred. A typical run is shown in Fig. 1 and calculation of amino acid composition is given in Table I. Precolumn derivati~ationrequires particularly clean samples. The derivatization reactions have a very narrow pH range, and samples containing UV-absorbing compounds, such as mercaptans and detergents, may introduce peaks which interfere with the quantitation. In addition, the performance of reversed-phase columns, even from the same manufacturer, vary. All reversed-phase HPLC columns lose resolution and develop high back pressure much faster than the ion-exchange microcolumns. The sensitivity of the amino acid analyzers are of broad range, depending on the particular instrument and the expertise of the personnel operating the analyzer. Using an IE analyzer, about 200 pmol (5 wg) of a 25-kDa protein is required to obtain an amino acid composition within a t 10% K.R.Williams, R. L. Neice, D. Aterton, A. V. Fowler, R. Kutny, and A. J. Smith, FASEB J . 2,3124 (1988).

590

CHARACTERIZATION OF PURIFIED PROTEINS

- ..tqn.:

[441

(cysteic acid)

FIG.1. Single-column separation of a protein hydrolysate by ion-exchange chromatography using a Beckman model 7300 analyzer. Eight micrograms of cytochrome P-450 was precipitated with HCI-acetone. The pellet was washed with water, centrifuged, and hydrolyzed in HCI vapor in the presence of 2% phenol for 1 hr at 150"under vacuum. One-half of the hydrolyzed material was injected. Expanded hydrolysate I1 program: Na-E/F/D buffers; 25-cm column; sample size, 50 p1; absorbance units full scale, 1.O; attenuation, 32. The results are given in Table I. Proline elution is shown in the lower tracing at 12.7 min.

1441

AMINO ACID ANALYSIS

591

-

TABLE 1 CALCULATION OF AMINO ACIDCOMPOSITION OF 55-kDa PROTEIN FROM CHROMATOGRAM SHOWNI N FIG.1 -

-

-

-

-

-

-

- -

Residueslmole Amino acid

Retention time (min)

Concentration (nmol)

a

b

c

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Ly sine Arginine

" Based on 38 Arg. From sequence data: J . Ozols, F. S. Heinemann, and E. F. Johnson, J . Biol. Chem. 260,5427 (1985).

' Based on an assumed molecular weight of 55,000. This value does not include the molecular weight of the single Trp and the four Cys.

range. Amino acid analysis of PTC derivatives6 by means of HPLC instrumentation (HPLC analyzers) usually is about five times as sensitive as the ion-exchange resolutions. An ion-exchangelo-phthalaldehyde detection7 or a fluorenylmethyl chloroformate8 precolumn derivatization1HPLC approach will provide an additional 10-fold increased sensitivity. Recently, subattomole analysis of fluorescein isothiocyanate derivatives of amino acids was accomplished by combining capillary zone electrophoresis for high-efficiency separation with laser-induced fluorescence for high-sensitivity dete~tion.~

'

B. A. Bidlingmeyer, T. L. Tarvin, and S. A. Cohen, in "Methods in Protein Sequence Analysis" (K. A. Walsh, ed.), p. 229. Humana Press, Clifton, New Jersey, 1986. J.-Y. Chang, R. Knecht, and D. Braun, this series, Vol. 91, p. 41. S . Einarsson, B. Josefsson, and S. Lagerkvist, J . Chromatogr. 282,609 (1983). Y.-F. Cheng and N. J. Dovichi, Science 242, 562 (1988).

592

CHARACTERIZATION OF PURIFIED PROTEINS

[441

Sample Preparation

Concentration and removal of residual salts and various enzyme stabilizers without contamination is probably the most difficult aspect in obtaining a meaningful amino acid composition from small amounts of protein. The methods used for concentrating proteins from dilute solutions include precipitation by organic solvents or by compounds such as trichloroacetic acid (TCA). ,4t the present, there is no universal concentration procedure that is applicable for all dilute protein solutions. If the protein sample can be dialyzed against water containing volatile buffers or dilute organic acids, without partial precipitation of the protein sample, then lyophilization of the dialyzed sample is the preferred method for concentration. A wide range of cellulose filters with molecular weight cut-off values ranging from 1000 to 50,000 are also commercially available. Small samples (up to 2 ml) may be concentrated to 25 to 50 p1 using Centricon membranes in a centrifuge. Adsorption of proteins to dialysis !ubing or membranes. however, is a frequent occurrence. The following methods have been found useful to concentrate protein samples for amino acid or sequence analysis:

'

Acid-Acetone ~ r e c i ~ i t ~ l t i o n ' ~ . ' To a dilute protein soll~tionadd 20 vol of cold (-20") acetone containing 0.2% ?cv!v) HCI. After several hours ar -20". the protein is collected by low-speed centrifugation in a swing-out rotor to collect the pellet at the bottom. The sample is then dried by lyophilization or. more preferably. in a stream of nitrogen. Protein samples in the 1- to 2-pg range, and essentially salt free, may be visible only under magnification. If the sample originally contained quantities of salts, the precipitate is suspended in water and again centrifuged. Membrane proteins uill not redissolve in water after acid-acetone precipitation. but some polar proteins wiil. The precipitated protein sample is then transferred to a hydrolysis tube or sequencer cartridge by using a small amount of 88% formic acid. Some proteins may be difficult to redissolve after precipitation by acid-acetone. In addition to formic acid. other volatile organic acids. such as trifluoroacetic acid (TFA). can 5e used to dissolve the protein for amino acid analysis or sequencing. For electrophoresis purposes the precipitated protein is dissolved in 2 M Tris base prior to the addition of sample buffer. The acid-acetone precipitation procedure removes lipids. nonionic detergents, glycerol. and salts. Complete removal of salts is achieved only

[441

AMINO ACID ANALYSIS

593

after washing the precipitated material with water. TCA, which is often used in protein concentrations, is also removed by this acid-acetone precipitation. Trichloroacetic AcidlDeoxycholate Pre~ipitation"~'~

Although, TCA does not precipitate proteins in the 1- to 5pg range, concentration of microgram quantities of protein from dilute solution by TCA precipitation can be improved by the addition of deoxycholate. A solution (20 mglml) of sodium deoxycholate (>pH 8) is added to the protein sample to give a concentration between 80 and 200 pglml. After 30 min at 4", a 20% solution of cold TCA is added to give a final concentration of 6%. The mixture is allowed to stand on ice for 1 hr and then centrifuged at 2500 g at 4" in a swing-bucket rotor for 45 min. The supernatant is carefully removed by suction. The sample is dissolved in 0.5 ml of 62.5 rnM Tris-HC1, pH 6.8, containing 3% SDS made 0.5 M in NaHC03 (final pH 8.8). The sample is dialyzed overnight against 1 liter of 6.25 mMTris-HC1, pH 6.8, containing 0.3% SDS, and then lyophilized. The detergent is removed as follow;: To the dried sample, 1 ml of extraction solution consisting of acetonelglacial acetic acidltriethylamine (90 : 5 :5, by volume) is added. The sample is transferred immediately to a conical centrifuge tube and pelleted in a desk-top centrifuge at room temperature. The supernatant is removed by suction, the tube is washed with 1 ml of extraction solution, and then with 1 ml of acetone. The remaining solvent is then removed by a stream of nitrogen. Deoxycholate, however, may not yield good protein precipitation with TCA in the presence of detergents such as SDS or digitonin. Before amino acid analysis of a valuable sample is performed, it is imperative that a buffer blank carried through the various precipitation and dialysis steps is hydrolyzed and analyzed. Contamination of the sample by amino acids can arise from many sources, including reagents, airborne particulates, dirty glassware, and poor laboratory practices by the operator. Many organic solvents contain polymers which on acid hydrolysis yield large quantities of glycine. A likely source of sample contamination is from amino acids present on fingers. For example, a single thumbprint on a dry glass surface has been shown to contribute 17 amino acids.14 Such samples have to be diluted some 100-fold in order to be in the recorder range of currently available analyzers. Samples contaminated with mateI'D. Mahuran, P. Clements, M. Carella, and P. M. Strasberg, Anal. Biochem. 129, 513 (1983). " G. L. Peterson, this series, Vol. 91, p. 95. l 4 D. H . Spackman, this series, Vol. I I , p. 3.

594

[441

CHARACTERIZATION OF PURIFIED PROTEINS

rial from fingerprints are typified by very high serine, high glycine, an absence of cystine, and the presence of citrulline and ornithine. Frequent washing of hands, and the use of disposable gloves during the handling of pipets and pipet tips will prevent the introduction of foreign material which may yield large amounts of amino acids upon acid hydrolysis. Reagents and samples are frequently contaminated by use of pipets containing fingerprints. Most polyacrylamide electrophoresis tank buffers contain molar quantities of glycine, and desk tops, glassware, and pipetting devices near these areas can be severely contaminated by glycine. Sample Hydrolysis

Table I1 lists some of the methods available for acid hydrolysis of proteins. In liquid-phase hydrolysis, 6 N HCl containing 0.1% phenol (w/v) and 0.05% 2-mercaptoethanol is added directly to the sample tube, which is then frozen and flame sealed under vacuum (0.2 ml solution1 0.5 mg of protein). This method is used when relatively large amounts of sample are available. The drying of the sample and hydrolysis steps are carried out in preconstricted Pyrex test tubes that have been washed with 6 N HCI, followed by a rinse with HPLC-grade distilled water. The hydro-

TABLE I1 PROTEIN/PEPTIDE HYDROLYSIS METHODS Method 6 N HC + phenol, mercaptans HClIpropionic acid (1 : 1) HCIITFA (2 : 1) HCIITFA (2 : I), 5% (vlv) thioglycolic acid 6 N HCI, 0.5-0.6% (vlv) thioglycolic acid 3 N p-toluenesulfonic acid 3 N mercaptoethanesu~fonicacid

Temperatureltime

Ref.

110"20-24 hr, vacuum 150-160" 15 min or 130"2 hr, vacuum 166"25 min, vacuum 160"25 min

a

c d

110" 24-64 hr, vacuum

e

110"22-72 hr, vacuum 110"24-72 hr, vacuum

'k

b

f

" Moore and Stein, this series, Vol. 6, p. 819. Westall and Hesser, Anal. Biochem. 61, 610 (1974).

" Tsuhita and Scheffler, Eur. J. Biochem. 124, 585 (1982). Yokote, Arai, and Akahane, Anal. Biochem. 152,245 (1986). Matsubara and Sasaki, Biochem. Biophys. Res. Commun. 35, 175 (1969). Liu and Chang, J. Biol. Chem. 246,2842 (1971). Penke, Ferenze, and Kovacs, Anal. Biochem. 60,45 (1974).

[441

AMINO ACID ANALYSIS

595

lysis is performed at 110" for 24, 48, and 92 hr. The 24-hr hydrolysate will have the least loss of sensitive residues, and the 92-hr hydrolysate will give complete cleavage of aliphatic linkages. After hydrolysis, the samples are dried in a clean desiccator containing solid NaOH, under vacuum. When only a few micrograms of sample are available, the preferred method is to transfer the sample to a 6 x 50 mm Pyrex tube (Corning 9820-6), dry, and seal the tube into a larger vessel to which 6 N HC1 has been added at the bottom. This procedure, rather than placing acid in the sample tubes, permits only the acid vapor to come in contact with the sample, and excludes nonvolatile contaminants. Vapor-phase hydrolysis is either done under vacuum, as in the liquid-phase method, or in an environment of argon or nitrogen. The large vial, containing the sample tubes, is equipped with a resealable Teflon closure to allow a vacuum to be applied. Such vials are now commercially available (Pierce). In practice, a volume corresponding to 0.1 to 5 pg of protein is pipetted into a 6 x 50 mm tube marked with a diamond pen. These tubes are placed in the vacuum vial and dried to near dryness. Overdrying of samples before and after hydrolysis should be avoided. The hydrolysis tubes are handled by forceps only, and are not reused. Vacuum should be applied gradually to prevent losses due to "bumping." Hydrolysis of the dried samples is accomplished by pipetting 6 N HCI, containing 2% phenol, into the bottom of the vacuum vial. It is recommended to routinely include a tube containing a "known" protein, and a clean tube without any sample added, to use as controls in case the samples give unusual results. The vial is then evacuated and sealed under vacuum (<70 mTorr). Hydrolysis is performed at 150". A 1-hr hydrolysis at 150"will give results comparable to hydrolysis at 110"for 20 hr. In 90 min at 150°, 90% of lle-Ile bonds will be hydrolyzed, but about 50% of serine and 40% of threonine residues will be lost. The vial is removed from the oven and opened in a hood, while it is still hot, in order to avoid acid condensation in the sample tubes. Extreme caution must be exercised when the hot hydrolysis vials are handled. Protective eye glasses and appropriate gloves must be worn during the transfer of the vial from the oven to the hood. With the protective sash of the hood lowered, the vial is opened very slowly, allowing the HCl vapor to escape gradually. The sample tubes are removed from the vial using gloves and Teflon-coated forceps. The outside of the 6 x 50 mm tubes are wiped to remove acid, and the residual HC1 inside is removed under vacuum. The dried tubes are covered with Parafilm and stored in the freezer. Since hydrolysis of samples requires the use of a dedicated, high-efficiency vacuum pump equipped with coldlacid traps, in addition to meticulous sample handling technique, it is customary for the individual

596

CHARACTERIZATION OF PURIFIED PROTEINS

[441

responsible for operating the amino acid analyzer to perform the sample hydrolysis. The person in charge of the amino acid analyzer usually provides the appropriate sample preparation procedures as well as the hydrolysis reagents compatible with the particular analysis method. However, it is unrealistic and uneconomical to expect the facility personnel to desalt and concentrate samples for hydrolysis and amino acid analysis. The amino acid composition of a protein sample may be expressed as moles per gram or per 100,000 g of protein. In some cases nanomoles per 100 nmol of amino acids are used. If the molecular weight of the protein is known then compositional data may be converted to total micrograms of protein per sample. The latter is then divided by the molecular weight X and a number of representing nanomoles of protein per sample is obtained and used to calculate the number of residues per mole of protein (Table I). The accuracy of minimal molecular weight estimates for microsoma1 proteins by SDS-PAGE is about kg%. If the amino acid sequence of a protein is known, then molar ratios of residues may be calculated based on the content of certain stable amino acid residues. For example, for comparison of the amino acid compositions of liver microsomal proteins, we use the Arg content as the key residue to which other residues are normalized (Table I). Since the future of amino acid analyses lies in the development of protocols for determining the composition of a protein band which has been resolved by electrophoresis and blotted to membrane supports, a procedure that is used in our laboratory for such analysis is included here. Detailed methodology for transblotting proteins for sequence analysis is outlined in [46] in this volume. For transblotting purposes, we use 7.5 or 10% Laemmli gels (16 x 18 cm, 1.5 mm thick) and a five-well comb. After electrophoresis, the gel is rinsed in transfer buffer [lo mM 3-(cyclohexy1amino)-I-propanesulfonic acid, 10% methanol, pH 111 for 5 min to reduce the amount of Tris and glycine. The protein bands are transferred to a polyvinylidene difluoride (PVDF) membrane. If the protein band is to be used for amino acid analysis only, then a nonsequencer-stable membrane such as nitrocellulose or nylon-based supports may also be used. The latter give better recoveries of protein than the sequencer-stable PVDF membrane. Prior to use, the PVDF membrane is rinsed in 100% methanol and stored in the transfer buffer. The electroelution is performed in transfer buffer for 0.5 to 1.5 hr at 145 mA (LKB 2117 Multiphor I1 unit). The PVDF membrane is washed in water for 5 min, stained with 0.1%Coomassie Blue R-250 in 50% methanol for 5 min, and then destained in 50% methanol, 10% acetic acid for 7 min. The membrane is then rinsed in water for 10 min, air dried, and stored at -20". The protein band corresponding to one well (2 cm) is excised and

[441

!

T:

i

r. E

597

AMINO ACID ANALYSIS

extracted with 300 pl of 88% formic acid in a 1.5-ml centrifuge tube. A short sonification of the mixture will increase the protein yield. The extracted membrane is then removed with Teflon forceps and the extract dried. One milliliter of water is then added to the sample, the tube vortexed, and again centrifuged. After decanting the wash water (some 50 p1 usually remains), the protein sample is transferred to a hydrolysis tube with -200 p1 of 88% formic acid. Amino acid analysis of the sample is then performed in HC1 vapor for 1 hr at 150". Because the samples may be readily contaminated during extraction steps, a band from the PVDF membrane of identical size is excised, extracted, hydrolyzed, and analyzed. The most frequent contaminant amino acids encountered in the PVDF membrane extracts are glycine and glutamic acid. Each individual protein elution experiment requires appropriate background contamination measurements for accurate protein compositional analysis. Table I11

TABLE I11 AMINOACIDANALYSES OF PROTEINSAMPLES ELUTEDFROM TRANSBLOTS

r: K r_

r:

Cytochrome P-450

Epoxide hydrolase

Amino acid

Uncorrecteda

Sequenceb

Uncorrectedc

Sequenced

Aspartic acid Threonine Serine Glutamine a c ~ d Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine

42.5 23.8 35.7 61.2 24.8 55.3 28.3 27.5 8.0 24.0 65.0 14.2 40.0 13.5 23.5 (38)

40 26 32 44 27 37 25 29 8 27 66 13 40 14 21 38

35.4 19.6 23.4 63.6 26.0 58.9 20.6 23.9 8.3 21.4 52.9 19.6 25 18.0 28.3 (24)

31 20 28 44 28 36 16 24 11 27 54 22 28 19 27 24

A 2-cm Band of PVDF membrane containing protein was extracted and hydrolyzed as described in the legend of Fig. 1. Values shown represent the result of a single analysis. Residues per mole is based on 38 Arg. Rabbit liver microsomal cytochrome P-450, form 2: F. S. Heinemann and J. Ozols, J . Biol. Chem. 258,4195 (19831. Same as in a. Residues per mole is based on 24 Arg. "Rabbit liver microsomal epoxide hydrolase: F. S. Heinemann and J. Ozols, J . Biol. Chem. 259,797 (1984).

"

598

CHARACTERIZATION OF PURIFIED PROTEINS

[441

shows the results of amino acid analyses of several proteins that have been electrophoresed and transblotted by this procedure. In summary, the first part of amino acid analysis consists of sample preparation, hydrolysis, and quantitation of the amino acids in the sample. This includes analysis of a second aliquot of the sample, either using a different sample preparation procedure or a different loading condition. The second part of an analysis involves the determination of cysteine, tryptophan, and amide content. Performic Acid Oxidation of Cysteine and cystine2

This method converts cysteine and cystine to cysteic acid, and converts methionine to methionine sulfoxide, derivatives that are stable toward acid hydrolysis. Performic acid reagent is made by adding 0.5 ml of 30% H202to 4.5 ml of 88% formic acid and allowing the mixture to stand at room temperature for 1 hr. The solution is then cooled to 4" and used immediately. One hundred microliters of this solution is added to about 20 pg of dried protein sample. After 4 hr at 0",2 ml of water is added and the sample is lyophilized. Performic acid is very corrosive to vacuum pumps, and larger samples must be dried with a rotary evaporator at room temperature. The usual acid hydrolysis of the sample is then performed, preferably in the same tube. Performic acid oxidation will destroy tyrosine and tryptophan. The yield of oxidized methionine and cysteine is around 95%. The molar ratio of cysteic acid residues is best calculated by reference to the molar quantities of the stable amino acids (Ala, Leu, Phe, or Asp). Reduction and Alkylation of cysteine3

Cysteine determination in proteins involves reducing the protein with a low-molecular-weight thiol at slightly alkaline and denaturing conditions (i.e., 8 M urea or 6 M quanidine-HCI). The most frequently used alkylating agents for cysteine are iodoacetic acid and 4-vinylpyridine. SCarboxymethylcysteine is stable to acid hydrolysis and is readily determined in analyzers using ion-exchange methodology. The quantitation, however, may be variable since S-carboxymethylcysteine can undergo intramolecular cyclization under acid conditions to yield a thiazine derivative. This derivative escapes detection by amino acid analysis because it lacks a free amino group. Cyclization to the thiazine derivative varies in individual laboratories, and therefore, analysis of standard proteins such as ribonuclease or P-lactoglobulin A is essential in order to determine the correction factor for recovery of alkylated cysteine. Because carboxymethylation is the most convenient method for introducing a radioactive label into proteins, it is a very commonly used procedure. The classical

[441

AMINO ACID ANALYSIS

599

large-scale preparative carboxymethylation procedure for proteins has been reviewed by H i r ~ The . ~ protocol for carboxymethylation of microgram quantities of protein is performed as described below. Alkylation of cysteine with 4-vinylpyridine is a convenient alternative procedure for quantitating c y ~ t e i n e .S-P-(4-pyridylethy1)cysteine ~ (PEcysteine) elutes from ion-exchange columns in a position immediately after ammonia. This area, however, represents a variable baseline segment in the elution profile of most IE analyzers. PE-cysteine, however, is readily quantitated as the PTH derivative with the HPLC-amino acid analyzer. Carboxymethylation or S-~yridylethylation'~ of Microgram Quantities of Protein

1. Prepare the following stock solutions and filter prior use. a. 1 M Tris-HC1, pH 8.5, containing 4 rnM EDTA b. 8 M guanidine-HC1 (Pierce) c. 10% aqueous 2-mercaptoethanol (Aldrich) d. Iodoacetamide (Sigma), 100 mglml in ethanol 2. Mix the stock solutions of Tris and guanidine-HC1 in a one-to-three ratio to yield the working buffer: 6 M guanidine-HCI buffered with 0.25 M Tris. 3. Dissolve or dilute the sample (1 to 10 pg) in 50 p1 of the above working buffer and add 2.5 p1 of dilute Zmercaptoethanol. Incubate at room temperature in the dark under nitrogen or argon for 2 hr. 4. For carboxymethylation, add iodoacetamide solution to give a 1.5fold molar ratio over total theoretical thiols. Incubate in the dark, at room temperature, for 30 min. Stop reaction by addition of excess mercaptoethanol. For S-pyridylethylation add 2 p1 of 4-vinylpyridine (Aldrich), mix, and incubate at room temperature in the dark for 2 hr. The reagent should be fresh and is stored under argon at -20". 5. Desalt immediately, preferably by chromatography, on a short reversed-phase column or by HC1-acetone precipitation. Tryptophan

Tryptophan is largely destroyed during the HCI hydrolysis, particularly in the presence of carbohydrates. Proteins hydrolyzed with mercaptoethane~ulfonic'~ or methanesulfonic" acids probably give the best recoveries of tryptophan. Alkaline hydroly~is,'~ however, is the method of "User Bulletin," No. 2. Applied Biosystems, Foster City, California, 1988. B. Penke, R. Ferenci, and K. Kovacs, Anal. Biochem. 60,45 (1974). l7 R. J. Simpson, M. R. Neuberger, and T.-Y. Liu, J . Biol. Chem. 251, 1936 (1976). IRT. E. Hugli and S. Moore, J . Biol. Chem. 247,2828 (1972). l6

600

CHARACTERIZATION OF PURIFIED PROTEINS

[441

choice when quantitation of tryptophan is of paramount importance. Unfortunately, none of the currently popular, high-sensitivity amino acid analyzers permits good recoveries of tryptophan. With single-column IE analyzers, tryptophan elutes as a broad peak after ammonia. Recoveries of PTH-Trp on reversed-phase HPLC columns vary with the age of the column. Tryptophan may be recovered in good yield on a cation-exchange column (8 x 9 cm) using sodium citrate buffer, pH 5.4.19 For such analyses the protein (0.2 to 1.0 mg) is hydrolyzed in a vacuum at 110" for 20,48, and 92 hr with 1 ml of 4 M methanesulfonic acid (Pierce). Since methanesulfonic acid is nonvolatile, the hydrolysate is partially neutralized with 3.5 M NaOH. Tryptophan is one of the least frequent amino acids in proteins. Therefore, accurate determination of this amino acid requires relatively large quantities of hydrolyzed protein. Tryptophan in the intact protein may also be determined spectrophotometrically. Goodwin and Morton,20 ~ d e l h o c h , ~ and ' Spande and W i t k ~ pare ~ ~the classical references for this approach. The ratio of the number of tryptophan and tyrosine residues in a protein may be determined from the absorbance at 280 and 288 nm in neutral 6 M guanidine-HC1solution, using the formula23: Trp - Tyr

(Azg8X 1280) - (A280 X 38-51 (AzsoX 4815) - (A288 X 5690)

This method is not applicable when other chromophoric groups are present. Traces of detergents containing aromatic groups, organic contaminants, and turbidity will all contribute to absorbance at 288 and 280 nm. High concentrations of cystine residues also interfere, so analysis on the reduced and carboxymethylated protein are made. The content of tyrosine residues in the protein is determined from the amino acid analysis; hence, the tryptophan content is determined from this value and the ratio of the number of residues of each. The availability of a sensitive spectrophotometer equipped with microvolume cuvettes enables such estimates to be made on submilligram quantities of protein. Amide Residues

Bis(1 ,1-trifluoroacetoxy)iodobenzene(BTI) treatment of a protein, followed by acid hydrolysis, will convert asparagine and glutamine residues l9 20

22

"

S. Moore, D. H. Spackrnan, and W. H. Stein, Anal. Chem. 30, 1185 (1958). T. W. Goodwin and R. A. Morton, Biochem. J. 40,628 (1946). H. Edelhoch, Biochemistry 6, 1948 (1967). T. F. Spande and B . Witkop, this series, Vol. 1 1 , p. 498. G. Allen, "Sequencing of Proteins and Peptides," p. 27. ElsevierINorth-Holland, Amsterdam, 1981.

[441

AMINO ACID ANALYSIS

60 1

to their corresponding diaminopropionic and -butyric acid residues.24In a Pyrex test tube mix 200 pg of protein in 2 ml of 10 mM TFA or 5 M guanidine-HCI with 2 ml of freshly prepared BTI (Pierce) in dimethylformamide (36 mglml) and seal the tube under vacuum. Heat at 60" for 4 hr. Remove excess reagents by dialysis against water (Spectrapor 3 dialysis membrane). Extract the dialyzed sample three times with an equal volume of n-butyl acetate and lyophilize the preparation. Hydrolyze with 6 M HCl under vacuum at 110' for 24 hr. Since 2,3-diaminopropionic acid and 2,4-diaminobutyric acids are normally not resolved from lysine on the IE-amino acid analyzer, Asn and Gln are determined by the difference. The Thr, Met, Cys, Tyr, and His content is also altered. Therefore, the content of these residues must be determined by the usual method of acid hydrolysis. Application of this method to amide analysis of small hydrophobic peptides has been described.*' Frequently, amino acid analysis elution profiles may show the presence of unusual peaks, implying the presence of modified amino acid residues. Most of these are due to artifacts present in buffers used in the protein isolations. Such findings can be confirmed by hydrolysis and amino acid analysis of fractions adjacent to the protein peak. Although acid hydrolysis usually destroys most covalently modified amino acid residues, some modified amino acids will survive the hydrolysis step and may elute as unique peaks. The ion-exchange analysis elution positions of over 30 amino acid derivatives known to exist in proteins has been compiled by Chin.26 Amino acid analysis involves not only placing a sample in an automated amino acid analyzer, but also in the manipulation of the samples and preparation of solutions. Through these steps, the possibility of contaminating the sample is very high. Residual salts, buffers, and detergents can adversely affect the outcome of hydrolysis and derivatization chemistry and may introduce stray peaks, complicating the quantitation of individual amino acids. Therefore, appropriate sample preparation is essential. Moreover, it is highly recommended that a sample of a sequential grade of protein of known amino acid composition (/?-lactoglobulin A, Applied Biosystems) be desalted, concentrated, hydrolyzed, and its amino acid composition determined. The results obtained by such an approach will indicate the accuracy, sensitivity, and reproducibility of the available amino acid analyzer in a particular laboratory.

" L. M. Soby and P. Johnson, Anal. Biochem. 113, 149 (1981). 25

26

J. OZO~S, S . A. Cam, and P. Strittmatter, J. Biol. Chem. 259, 13349 (1984). C. C. Q. Chin, this series, Vol. 106, p. 17.

602

CHARACTERIZATION OF PURIFIED PROTEINS

[451

[451 Limited N-Terminal Sequence Analysis

Identification of the N-terminus of proteins and peptides is important for isolating recombinant DNA clones and for characterizing structural and functional protein domains. The most important step in obtaining an unambiguous N-terminal sequence is to purify suitable quantities of the protein or peptide in a manner compatible with automated or manual Edman degradation procedures. Because the physical and chemical properties of proteins vary widely, there is no simple protocol for purifying protein as there is for purifying DNA. Moreover, if only small amounts (
' G . Allen, in "Sequencing of Proteins and Peptides" (T. S. Work and R. H. Burdon, e d ~ . ) . Elsevier, Amsterdam, New York, 1981.

METHODS IN ENZYMOLOGY, VOL. 182

Copyright O 1990 by Academic Press. Inc. All rights of reproduction in any form reserved.

tridge. The ATZ-amino acid is washed from the reaction cartridge into a conversion flask where it is converted into the PTH-amino acid. The PTH-amino acid is then injected into a high-pressure liquid chromatography (HPLC) column, and the amino acid is detected by UV absorbance and is identified by its elution time. The most sensitive analyzers can detect approximately 1 pmol of PTH-amino acid. The number of residues sequentially identified is dependent on sample amount and on the sequence itself. The efficiency of the sequencing reaction is measured by the repetitive yield (the amount of PTH-amino acid recovered at each cycle). Typical yields range between 85 and 95% for most proteins. Repetitive yields for short peptides are often lower because of sample washout by the solvents used in the sequencing reaction. Requirements for N-Terminal Sequence Analysis

There are several requirements for obtaining sequences from unknown samples. The first is that the sample be relatively pure (>SO%). Mixtures of peptides will generate several PTH-amino acids at each cycle of the Edman degradation procedure. If the peptides are present in comparable quantities, then assigning sequence to a particular peptide is nearly impossible without prior knowledge of the sequence. Also, if the N-terminus of the peptide of interest is blocked, then sequences may arise from contaminating peptides. The second requirement is that the sample be free of contaminants such as Tris, glycine, sodium dodecyl sulfate (SDS), or acrylamide, which will either affect the performance of the sequencing machine or clutter the chromatograms with large artifact peaks. The third requirement is that sufficient quantity of sample be available for analysis. Factors such as sample loss and N-terminal blockage during purification lead to uncertainty in the actual amount of sequenceable sample. Therefore, most sequencing facilities request 10-100 pmol of material for Nterminal sequence analysis. This limit corresponds to 0.5-5 pg of a 50,000-Da protein. If no sequence is obtained from 100 pmol, then one might suspect that the protein or peptide has a blocked N-terminus. With 110 pmol it may be difficult to decide whether the N-terminus is blocked or if there was insufficient material for analysis. Strategies for Obtaining N-Terminal Protein Sequence

There are two general approaches for purifying proteins for sequence analysis. The simplest approach is to purify the protein by SDSpolyacrylamide gel electrophoresis and to sequence the protein from a blot of the gel. An alternative method is to purify the protein by reversed-phase

604

CHARACTERIZATION OF PURIFIED PROTEINS

[451

HPLC and to sequence the fraction containing the protein. The choice between the two approaches depends on the amount and molecular weight of the sample, and the complexity of the purification procedure. Electroblotting from gels is the simplest and fastest approach and the one to try initially. Generally, any protein or peptide that can be resolved on a gel is a good candidate for sequencing from blots. Peptides of < 8000-10,000 Da might not yield extended sequence information because they are more likely to be washed from the membrane during the sequencing reactions than larger proteins: binding to the membrane depends on the number and location of hydrophobic residues in the peptide. The most important consideration is that the band on the gel be composed of a single polypeptide. If this requirement is met, then sequence can be obtained from partially pure preparations from either a spot from a two-dimensional gel or a band from a one-dimensional gel. Reversed-phase HPLC is most useful for handling larger amounts (10 pg- 1 mg) of protein and for purifying peptides from a digest of a protein whose N-terminus is blocked. A disadvantage of this approach is that an HPLC apparatus requires prior training, and often the instrument itself presents a formidable challenge to master. Furthermore, peaks resolved by HPLC may not be composed of a single species and in such cases must be analyzed by independent methods, usually SDS-PAGE. However, because samples eluted from reversed-phase HPLC columns are in volatile buffers, they can be further cleaved with chemicals or proteases with minimal additional manipulation. Protein Purijication by Electrophoresis and Electroblotting

Protein purification from gels can be as easy as cutting a band from the gel. Many methods have been reported in which bands are eluted from the gel into ~ o l u t i o n .However, ~ the samples eluted from gels are usually contaminated with Tris, glycine, and polyacrylamide and must be separated from these contaminants, usually by reversed-phase HPLC, reducing the overall yields in purification. More recent developments are based on electrophoretic transfer of the polypeptide from the gel to a support (membrane or filter) that can be placed directly in the sequencing machine. Initially, glass fiber filters derivatized with aminopropyl groups3or coated with Polybrene4 were used, but proteins on these membranes are diffi-

'

M. W. Hunkapiller and E. Lujan, in "Methods of Protein Characterization" (J. E. Shively, ed.), p. 89. Humana Press, Clifton, New Jersey, 1986. R. H. Aebersold, D. B. Teplow, L. E. Hood, and S. B . H. Kent, J. Biol. Chem. 261,4229 (1986). J . Vandekerckhove, G. Bauw, M. Pyupe, J. Van Damme, and M. Van Montagu, Eur. J. Biochem. 152,9 (1985).

cult to visualize by staining with Coomassie Blue or other charged dyes. Other supports, such as nitrocellulose or nylon, do not withstand the organic solvents, acids, and bases used in automated sequencing machines. Proteins and peptides blotted onto Immobilon-P supports5 (Millipore Corp, Bedford, MA) are readily sequenced with little or no modification of the blotting or sequencing protocols and are the recommended chemicals that would potentially intermembranes for this appli~ation~-'~: fere with the Edman chemistry do not bind to the membrane and are easily washed off. Electrophoresis. Proteins or peptides are separated on 0.5- to 0.75mm-thick gels using standard one-dimensional SDS-PAGE" or twodimensional isoelectric focusing-SDS- PAGE'^ methods. We analyze proteins on fan-cooled minigelsI3 developed at the MRC Laboratory of Molecular Biology (Cambridge, England) and made by Cambridge Electrophoresis, Ltd. (Cambridge, England). Other minigel systems by Owl Scientific, Bio-Rad Laboratories, or Hoeffer Scientific are also suitable. For each of these systems, several gels can be cast at once and stored in the refrigerator until use. Gels should be cast with narrow wells (5 mm) because the band must be smaller than the diameter of the reaction cartridge. Enough protein should be loaded in a well so that at least 10 pmol of sample is in a single band on the blot. Dilute samples can be precipitated with trichlororacetic acid, neutralized with 2 M Tris base, and resolubilized in SDS sample buffer or, alternatively, loaded onto a thicker gel. I do not recommend loading dilute samples into several wells because sequencing is not efficient when multiple bands are loaded into the sequencer reaction cartridge; however, in sequencing instruments modified with a continuous flow reactor,I4 several bands may be sequenced with good yields. Although several reports attribute N-terminal blockage to impure reagents and chemicals, no special precautions are taken in our laboratory to purify reagents and chemicals (e.g., acrylamide or SDS), to age the gels, to preelectrophorese the gels, or to include scavengers of free radicals, such as thiogycolate. M. G. Pluskal, M. B. Kavonian, M. R. Vecoli, and D. A. Hicks, Biotechniques 4, 272 (1982). P. Matsudaira, J. Biol. Chem. 261, 10035 (1987). N. LeGendre and P. Matsudaira, Biotechniques 6, 154 (1988). G. Bauw, M. DeLnose, D. Inze, M. Van Montagu, and J. Vandekerckhove, Proc. Natl. Acad. Sci. U.S.A.84,4806 (1987). Q.-Y. Xu and J. E. Shively, Anal. Biochem. 170, 19 (1988). lo M. Moos, N. Y. Nguyen, and T.-Y. Liu, J. Biol. Chem. 232,6005 (1988). " U. K. Laemmli, Nature (London) 227,690 (1970). IZ P. M. O'Farrell, J. Biol. Chem. 250,4007 (1975). l3 P. T. Matsudaira and D. R. Burgess, Anal. Biochem. 87, 386 (1978). l 4 J. E. Shively, P. Miller, and M. Ronk, Anal. Biochem. 163,517 (1987).

'

606

CHARACTERIZATION OF PURIFIED PROTEINS

[451

Electrotransfer. Electrotransfer to Immobilon-P transfer membrane can be performed following standard immunoblotting p r o t ~ c o l s . ~ ~ ' ~ ~ ' ~

1. While samples are being electrophoresed, a sheet of Immobilon-P membrane is wetted with 100% methanol and equilibrated with CAPS transfer buffer [lo mM 3-(cyclohexy1arnino)-1-propanesulfonic acid, 10% methanol, pH 1 1.01. 2. After electrophoresis is completed, the gel is assembled into a transfer cassette without preequilibration in transfer buffer. The Immobilon-P membrane is usually backed with a sheet of nitrocellulose. 3. Protein is electroblotted for 10-60 min at 0.5 A constant current. The high pH of the CAPS buffer ensures transfer of most proteins. However, excellent results are also obtained with other buffers, including those containing Tris and glycine. Although Tris and glycine will interfere with the sequencing reaction, they are easily washed from the membrane after transfer. Detergents impove elution of proteins from the gel, but detergents in the electroblotting buffer compete with the Immobilon membrane for protein and impede binding to the membrane. As a compromise, we electroblot from gels that have not been equilibrated with transfer buffer, the detergent in the gel being sufficient for elution of most proteins. Gels can be transferred in any apparatus. We use the Hoeffer Mighty Small transfer unit, but have also found semidry blotters to be satisfactory. Since the initial yield of protein sequence from electroblotted samples is most affected by the efficiency of transfer, the transfer time must be determined for each protein; this will vary with the gel thickness, molecular weight of the protein, amperage of transfer, and ionic strength of the buffer. The transfer time can be judged empirically by staining the gel and a nitrocellulose backup sheet after electroblotting: too short a time results in a substantial amount of protein remaining in the gel, and too long a time is detected by protein that has electrophoresed through the Immobilon membrane and is bound to the nitrocellulose backup sheet. The transfer times for proteins smaller than 50,000 Da are 10-30 min and for larger proteins are 30-60 min at 0.5 A constant current. Transfer for longer times with lower currents results in more complete t r a n ~ f e r . ~ Staining

1. The membrane is washed with two changes of transfer buffer and then stained with 0.1% Coomassie Blue in 50% methanol for 1-2 min. Other stains, such as Ponceau S" or Amido Black, can be substituted. l5

H. Towbin, T. Staehelin, and J. Gordon, Proc. Narl. Acad. Sci. U.S.A. 76,4350 (1979).

Ib

J. M. Gershoni and G. E. Palade, Anal. Biochem. W, 396 (1982). 0. Salinovich and R . C. Montelaro, Anal. Biochem. 156, 341 (1986).

I'

2. The blot is destained with 50% methanol and 10% acetic acid (usually 5 min). 3. The membrane is then washed with several changes of HPLCquality H20 for 5-10 min and air dried. The stained blot is stored dry at -20". Coomassie Blue can detect 100 ng of a polypeptide on Immobilon-P membranes, an amount well below the sensitivity of any commercial sequencing instrument; hence, not all bands are sequenceable. An easy test to estimate whether sequenceable amounts of protein are present is to photocopy the blot. If the band is not seen on the copy, then chances are that there is too little material for sequence analysis. The band is cut from the membrane with a clean razor and excess membrane carefully trimmed away to give a 2 x 4 mm segment. Long slits are cut along the band to give a comblike appearance. The band fans out, facilitating access of solvents and reagents with the sample during sequencing. The amount of sequence obtained averages about 25% of the amount of sample loaded on the gel. This number can vary greatly depending on the efficiency of transfer, degree of N-terminal blockage, and the performance characteristics of the sequencer.

HPLC Purification of Proteins and Peptides Although this topic is covered more extensively in other chapters, we use a "standard" HPLC procedure for preparing samples for sequence analysis. A major advantage of HPLC methods, especially reversed-phase separations, is that the protein or peptide is recovered in a volatile buffer that will not interfere with the automated Edman chemistries. Columns for purification of samples by reversed-phase HPLC are available in a variety of sizes. The binding capacities of 4.6-mm i.d. columns are suitable for 100-1000 pg of protein. Small amounts of material should be analyzed on a 2.1- or 1.O-mm i.d. column: a smaller bore column provides increased sensitivity in detection of peptides because of reduced sample dilution. The increased sensitivity is achieved at the expense of the maximum amount of sample that can bind to the c ~ l u m n . ' ~ We - ~usually ~ plan to recover 50% of the starting material from reversed-phase HPLC. The HPLC should be capable of generating a gradient elution profile and detecting proteins and peptides by their absorbance at 214, 230, or R. M. Kamp, in "Advanced Methods in Protein Microsequence Analysis" (B. WittmannLiebold, J. Salnikow, and V. A. Erdmann, eds.), p. 21. Springer-Verlag. Berlin, 1986. l9J. E. Shively, in "Methods of Protein Characterization" (J. E. Shively, ed.), p. 41. Humana Press, Clifton. New Jersey, 1986. 20 R. J. Simpson and E. C. Nice, in "Methods in Protein Sequence Analysis" (K. A. Walsh, ed.), p. 213. Humana Press, Clifton, New Jersey, 1986.

608

CHARACTERIZATION OF PURIFIED PROTEINS

[451

280 nm. Two-channel or diode-array detectors are preferrable because they provide more information about each peak. The pumps should be able to maintain a flow rate of 0.1-1.0 mllmin without generating pulsations or unstable baselines. A fraction collector equipped with peak detection algorithms with a delay line is also desirable. Detergents can interfere with the elution of proteins and peptides from reversed-phase columns and should be removed. See other chapters in this volume for detailed protocols. Sample Preparation

1. Concentrate the sample in a Speed-Vac to a small volume (<100 11.1).

2. Denature and reduce the sample by adding an equal volume of 6 M guanidine-HCl, 0.1 M Tris, pH 8.5, 0.05 M dithiothreitol (DTT). 3. Remove precipitated material by centrifugation (10 min at 10,000 g ) or by filtration through membranes (0.45-pm filters, low protein binding). Because of the potential for sample to adhere to a filter, centrifugation is preferred. 4. Equilibrate a reversed-phase column (C8, 300-.A pore size, 2.1 x 100 mm or 2.1 x 30 mm, Brownlee RP-300 Aquapore, Applied Biosystems, Inc., Foster City, CA) with 0.1% TFA (95% pump A) an 0.085% TFA/80% acetonitrile (5% pump B) mixture. 5. Obtain a baseline by injecting a buffer control and using the following gradient program: 0-5 min, 5% B; 5-45 min, 70% B; 45-50 min, 100% B; 50-55 min, 5% B. The gradient can be modified by using different buffers (formic acid, ammonium acetate, ammonium bicarbonate), organic solvents (methanol or 1-propanol), and gradient programs (lower %B starting condition, shallower or steeper gradients, or slower flow rates). See other chapters in this volume or Refs. 18-20 for additional examples. Sample Elution

1. Inject sample onto the column. 2. Collect peaks by manual or automated fraction collectors equipped with peak-detection algorithms. Fractions are collected in 1.5-ml polypropylene centrifuge tubes. 3. Concentrate fractions in a Speed-Vac without heat. It is best not to concentrate to complete dryness since the dried peptide may be difficult to resolubilize. 4. Remove an aliquot for analysis by SDS-PAGE. 5. Store samples at -20".

The elution profile of the column can be monitored at 214, 230, or 280 nm. The most sensitive setting is at 214 nm, which detects absorbance by the peptide backbone, but because some buffers contain UV-absorbing contaminants it is more convenient to monitor at 230 or 280 nm. Monitoring at 230 nm is approximately 5-fold more sensitive than at 280 nm. However, many peptides do not contain aromatic residues and will be undetected at 280 nm. We monitor absorbance at 214 nm for quantities of protein 4 0 pg. If possible, one can monitor both wavelengths with a dual-channel detector. Peak shape and spectra provide information about sample purity: symmetrical and well-separated peaks are often composed of a single protein or peptide, whereas unsymmetric peaks suggest the presence of a contaminant. Furthermore, with HPLCs equipped with diode-array detectors, comparison of spectra from the front and back halves of the peak indicates the purity of each peak. The length of the peptide cannot be determined from the peak spectrum. With large-bore columns (4.6-mm i.d.) the volume of each peak is 0.1-0.5 ml. Since the glass-fiber filters in the sequenator reaction cartridge absorb a maximum of only 30 p1, fractions from these columns must be concentrated before sequence analysis. Cleavage of Proteins: Strategies for Sequencing N-Terminally Blocked Proteins

If the N-terminus of a protein or peptide is blocked, then it must be cleaved chemically or enzymatically to generate internal peptides with unblocked N-termini. A decision must be made whether to cleave a protein completely at many sites or at a limited number of sites. Complete cleavage at numerous sites produces many peptides which have to be resolved and purified. Although the peptides are often too small or too closely spaced to resolve by SDS-PAGE, they can be separated by reversed-phase HPLC. Cleavage at a few sites simplifies purification because a smaller number of peptides is generated. Chemical or proteolytic cleavage of denatured proteins at rare sites will produce a small number of peptides. Small numbers of large fragments are also produced by limited proteolysis of native proteins. Low ratios (1 : 1000 wlw) of protease achieve efficient limited digestion of native, globular proteins because cleavage tends to occur between compact structural domains; these domains are easily purified by either HPLC or SDS-PAGE. In all cases, cleavage should go to completion because incomplete cleavage reduces the recovery of sequenceable quantities of material. Because of the additional steps involved, one needs at least 5-fold more starting material than is required for simple N-terminal sequencing.

610

CHARACTERIZATION OF PURIFIED PROTEINS

I451

TABLE I CHEMICAL CLEAVAGE REAGENTS Chemical

Sequence cleaved

Cyanogen bromide

Met-Xb

BNPS-skatole

Trp-X

Dilute acid

Asp-Pro

Hydroxylamine

Asn-Gly

a

Cleavage conditionsn lOOx excess CNBr in 70% formic acid, 12 hr in the dark, room temperature, quench by lyophilization 50x excess in 50% acetic acid, 48 hr in the dark, room temperature, quench with tryptophan, and desalt 10% acetic acid, pH 2.5, 24-96 hr at 40°, quench by drying 2 M hydroxylamine in 6 M guanidine-HCI, pH 9.5, 1.5 hr at 45", quench by desalting

All reactions performed in capped tubes under nitrogen gas. X, Any amino acid.

Chemical Cleavage

For a protein purified by HPLC, the protein can be cleaved completely and the fragments separated by HPLC. CNBr cleavage is the method of choice2' since the average number of methionine residues in a protein is relatively low (ca. 2%) and the cleavage goes to completion. In comparison, trypsin digestion of proteins occurs after lysine and arginine residues, which collectively comprise ca. 12% of the amino acids in a protein. Alternative chemical methods for cleavage is either with dilute acid,22 which under mild conditions is selective for Asp-Pro bonds, or with BNPS-skatole [2-(2-nitrophenylsulfenyl)-3-methyl-3'bromoindolenine],23 which cleaves after tryptophan residues. Since aspartic acid and proline residues comprise ca. 5.5 and 4.6%, respectively, of the amino acids in a protein, one would expect based on chance that an Asp-Pro sequence would be present at a frequency of 0.3%. Tryptophan comprises ca. 1% of the amino acids in a protein. In both cases, cleavage is not quantitative and partial fragments are also produced. Table 1 lists chemicals which cleave proteins at specific residues. CNBr Cleavage 1. Purify the protein or peptide by HPLC, as above, and dry in a

Speed-Vac. E. Gross, this series, Vol. 1 1 , p. 238. Landon, this series, Vol. 47, p. 145. A. Fontana, this series, Vol. 25, p. 419.

" N.

23

2. Solubilize the sample in 50 pl of 70% formic acid. 3. Add a small crystal of CNBr and swirl to dissolve. 4. Flush the tube with nitrogen and cap it. 5. Keep the sample in the dark at room temperature for a minimum of 12 hr. 6. Quench the reaction by evaporating the formic acid in a desiccator containing a beaker of NaOH pellets under vacuum or by lyophilization after diluting the formic acid to 7% with H20. 7. Dry the digest completely in a Speed-Vac. 8. Solubilize the digest in 6 M guanidine-HCI, 0.1 M Tris, pH 8.5, 0. I M DTT, and inject onto a reversed-phase column. 9. Collect the peaks as above and process for sequencing. Proteolytic Digestion

Convenient methods for cleavage after lysine, arginine, aspartic acid, and glutamic acid residues rely on highly specific proteolytic enzymes. Table I1 lists proteases that cleave after specific residues. As noted above, cleavage of native proteins in nondenaturing buffers generates fewer peptides than cleavage in denaturing conditions. Complete Proteolytic Cleavage. The digestion is performed under conditions that ensure cleavage at all sites in the protein. The protein is usually denatured and cleaved with high concentrations of protease. Peptides generated by autodigestion of the protease are identified in separate control digests. 1. Protein or peptide is resuspended at 5-20 mglml in the appropriate digestion buffer (usually 1-8 M urea, 50 m M Tris HC1 bicarbonate, 1 mM CaC12,pH 8.0). 2. Protease is added at a 1 : 10-1 : 50 (wlw) ratio and incubated for 12-24 hr at room temperature. 3. The reaction is quenched by injection onto a reversed-phase column. 4. Peaks are collected and stored at -20". Limited Proteolytic Cleavage. It is advisable to set up a small-scale test digest to identify optimal conditions for generating a set of large internal peptides. 1. One to five micrograms of protein (in 50 mM ammonium bicarbonate, 1 mM CaCI2, pH 8.0) is mixed with protease (1 : 100-1 : 1000 wlw) and incubated at room temperature. 2. Aliquots (200 ng) are removed at 1 , 5 , 2 0 and 60 min and the protease is inactivated by boiling in SDS sample buffer. For most serine proteases

612

[451

CHARACTERIZATION OF PURIFIED PROTEINS

TABLE I1 SITE-SPECIFICPROTEASE -

Proteasea

- -

Source

Trypsin

Bovine pancreas

V8 protease

Staphylococcus aureus

Endoproteinase Lys-C Endoproteinase Arg-C Endoproteinase Asp-N

Lysobacter enzymogenes

Chymotrypsin

Porcine pancreas

Mouse submaxillary glands Pseudomonas fragi mutant

-

Sequence cleaved

Comments pH optimum 7.5-8.5, active in 0.1% SDS Active in SDS (0. I%), specific for Glu in ammonium bicarbonate, pH 8.0, or ammonium acetate, pH 4.0; specific for Glu and Asp in phosphate buffer pH optimum 8.0-8.8 pH optimum 8.0-8.5 pH optimum 6.0-8.5, metalloproteinase inhibited by EDTA pH optimum 7.5-8.5, active in 0.1% SDS, inhibited by PMSF

" Obtained from Boehringer Mannheim.

the reaction is quenched with 0.5 p1 of phenylmethylsulfonyl fluoride (0.1 M in 2-propanol). 3. Samples are applied to a minigel. Small amounts of protein (10-50 ng) can be detected on thin polyacrylamide gels by Coomassie Blue staining, which is preferable to more sensitive but slower methods such as silver staining. The protease concentration is adjusted to generate a slowly changing pattern of fragments. At the early time points uncleaved protein should be present, but by 60 min all of the prolein should be cleaved. The gel is examined for the buildup of sequenceable amounts of fragments. A large-scale digest is then performed. The digest is either quenched with SDS sample buffer and the sample applied to a or is quenched by injection onto an HPLC column.

" W. Levoy Bazari, P. Matsudaira, M. Wallek. T. Smeal, R. Jakes, and Y. Ahmed. Proc. Natl. Acad. Sci. U.S.A.85,4986 (1988).

[461

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PEPTIDE MAPPING

Digestion of Proteins Purijied by SDS-PAGE

There are two general strategies for obtaining internal sequence information from proteins purified by SDS-PAGE. In the first approach, the protein band is cut out of a ID or 2D gel. The band is incubated with trppsin or V8 protease and then placed in a well of a second gel.25The peptides are separated by electrophoresis and electroblotted to Immobilon-P membrane. This method has been successful in obtaining sequence from a variety of different Alternatively, if one is trying to "recover" sequence from an electroblotted sample, then the sample can be cleaved on the filter using CNBr'9 or proteases30 and then eluted by organic solvents, acids, or detergenk3' The peptides are then separated by HPLC. The amount of sequence obtained is low because cleavage is inefficient (especially with proteases), elution from the membrane is not complete, and there are additional losses with HPLC separation. Summary

Current methods in protein purification permit sequence analysis of picomole quantities of proteins and peptides. The key to obtaining sequence is to choose as the last step in the purification protocol the simplest (usually the fastest) method for isolating the protein or peptide. D. W. Cleveland, this series, Vol. 96, p. 222. S. Jentsch, J . P. McGrath, and A. Varshavsky, Nature (London) 239, 131 (1987). 27 M. Bodner, J.-L. Castrillo, L. E. Theill, T. Deernick, M. Ellisman, and M. Karin, Cell 55, 25

26

505 (1988). I8T. E. Kennedy, K. Wager-Smith, A. Barzilai, E. R. Kandel, and J . D. Sweatt, Nature (London)366,499 (1988). 29 R. J . Simpson and E. C. Nice, Biochem. Znt. 8,787 (1984). 'OR. H . Aebersold, J . Leavitt, R. A. Saavedra, and L. E. Hood, Proc. Natl. Acad. Sci. U.S.A. 85,6970 (1987). " B. Szewczyk and D. F. Summers, Anal. Biochem. 168,48 (1988).

[461 Peptide Mapping By RALPHC. JUDD

The underlying principle of the peptide mapping process is quite simple. If two proteins have identical primary (lo)sequences, then cleavage of each protein with a site-specific endopeptidic reagent will yield identical peptide fragments. Conversely, if the proteins to be compared differ in lo METHODS IN ENZYMOLOGY, VOL. I82

Copyright O 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

614

CHARACTERIZATION OF PURIFIED PROTEINS

[461

sequence, then the cleavage will yield dissimilar peptides. The degree of similarity or dissimilarity between the peptide fragments reflects the degree of lo structural similarity between the proteins. Peptide mapping can be separated into four processes that are combined to yield primary structural comparisons of proteins: (1) purification of the proteins to be studied, (2) labeling of each protein, and thus the peptide fragments, (3) specific endopeptidic cleavage of the proteins, and (4) the separation and visualization of the resultant peptide fragments. Each process can be accomplished in dozens of different ways. This discussion focuses on several specific methods that have proved to be rapid and reliable. Where appropriate, alternatives are suggested. It is left to the reader to determine which method best suits the needs of their particular research problem. Due to the empirical nature of peptide mapping, reaction times, and concentrations of proteins, peptides, and reagents presented in this discussion may require modification. Protein Purification

A variety of purification procedures are detailed in this volume. Any of these procedures are suitable for peptide mapping. For analytical purposes, the discontinuous buffer, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) procedure is an excellent choice.',2 Most proteins can be separated to adequate purity in a single step, while a Sodium ,~ second SDS-PAGE separation provides exquisite p ~ r i t y . ~ dodecyl sulfate-polyacrylamide gel electrophoresis can be used to separate both soluble and insoluble proteins, either before or after radiolabeling. Moreover, the apparent molecular mass information, and the potential for immunoblotting data provided by SDS-PAGE, help ensure that the proper proteins are being used. Proteins purified by SDS-PAGE can be labeled and cleaved directly in SDS-PAGE gel or they can be electroblotted onto nitrocellulose paper (NCP) (HAHY 00010, Millipore, S. San Francisco, CA) where It is also possible to label labeling and cleavage are more effi~ient.~ proteins prior to SDS-PAGE, either intrinsically8 or e~trinsically.~ In the

' K. C. Judd, Infect. Immun. 54,408 (1986). U. K . Laemmli, Nature (London)227,680 (1970). R. C . Judd, Anal. Biochem. 160, 306 (1987). R. C . Judd, Anal. Biochem. 173, 307 (1988). J . H . Elder, R. A. Pickett, J . Hampton, and R. A. Lerner, J. Biol. Chem. 252,6510 (1977). J . Swanson, Infect. Immun. 23,799 (1979). ' R. C. Judd, Infect Immun. 37,622 (1982). H. D. Caldwell and R. C. Judd, Infect. Immun. 38,960 (1982). M. Monison, this series, Vol. 70, p. 214.

[461

PEPTIDE MAPPING

615

in-gel system of peptide mapping (see below) protein cleavage is accomplished in the stacking gel of an SDS-PAGE gel and the resultant fragments are then directly separated in the gel.'' The most sensitive, efficient, and reproducible purificationlpeptidemapping process I have found is SDS-PAGE followed by electroblotting onto NCP.3 The proteins can then be located by Naphthol Blue Black (NBB) (#484, Allied Chemical Co., New York, NY) staining or India ink (Pelikan, Hannover, West Germany) staining." The bands of interest are then excised, radiolabeled using "'I (#63037, ICN Biomedicals, Irvine, CA), and cleaved on the NCP.3 The resultant peptides, which are released into the supernatant, can then be separated by SDS-PAGE (lowest resolution of pep tide^),^,'^ by two-dimensional thin-layer electrophoresis '~ followed by thin-layer chromatography ( ~ D T L E - T L C ) ~ ~(highest resolution of peptides), or by high-performance liquid chromatography (HPLC)3.'3-'5(good resolution and allows for recovery of the peptides). Peptides can be visualized by Coomassie Briliant Blue2 (CBB) or silver staining16of gels (if adequate amounts of protein are used and the cleavage reagent does not interfere with interpretation), autoradi~graphy'?~*'~~ (SDS-PAGE gels and 2DTLE-TLC), y radiation e m i s ~ i o n ~ . ' ~(HPLC), -" or ultraviolet absorption (HPLC). SDS-PAGE for Peptide Mapping A single SDS-PAGE separation is often adequate to isolate the proteins of interest. Whole-cell lysates, subcellular fractions, or purified proteins can all be separated by SDS-PAGE as a first step in peptide mapping. If radiolabeling is used, a single protein band in a single lane of an SDSPAGE gel is sufficient material for dozens of peptide maps. Ease and efficiency are greatly improved if the protein is electroblotted onto NCP prior to labeling and cleavage. One-Dimensional SDS-PAGE

1. Isolate appropriate amounts of samples to be compared using SDSPAGE. Individual lanes for each sample, or a "preparative" gel where the solubilized sample is loaded over the entire stacking gel,3may be used. D. W. Cleveland, S. G . Fischer, M. W. Kirshner, and U. K. Lammeli, J. Biol. Chem. 252, 1102 (1977). I ' K. Hancock and V. C. W. Tsang, Anal. Biochem. 133, 157 (1983). l 2 W. J. Ritschard, J. Chromatogr. 16, 327 (1964). " R. C. Judd, J. Liq. Chromatogr. 6 , 1421 (1983). R. C. Judd and H. D. Caldwell, J. Liq. Chromatogr. 8, 1109 (1985). R. C. Judd and H. D. Caldwell, J. Liq. Chromatogr. 8, 1559 (1985). I6 C. M. Tsai and C. E. Frasch, Anal. Biochem. 119, 1 15 (1982). lo

616

CHARACTERIZATION OF PURIFIED PROTEINS

[461

-

2. Following electrophoresis, fixation, CBB staining, and destaining, the protein band of interest can be excised and used in the "gel slice" methods (below). It is preferable, however, to immediately overlay the gel with NCP which has been soaked in degassed 20 mM phosphate buffer, pH 8 . 0 , ' ~and to electroblot the proteins to the NCP at 20 V constant current, 0.6 A for 16 hr in 20 mM phosphate buffer, pH 8.0. If protein transfer is not efficient at this pH, adjust pH up or down as needed. 3. The NCP is then removed and the proteins stained with 0.1% NBB in distilled H 2 0 (dH20) for 1 hr.3 Following a brief rinse in H 2 0 , the background will be sky blue and the protein dark blue. If the proteins cannot be located by NBB staining, block unreacted sites on the NCP with 0.05% Tween 20 (#1379, Sigma Chemical Co., St. Louis, MO) in Dulbecco's phosphate-buffered saline (DPBS) for l hr with shaking. Place the NCP into 100 ml of 0.1% India ink in 0.05% Tween 20 in DPBS" for 1 hr. Protein bands will be black, the background whitc. 4. Excise the appropriate protein bands. A 1 x 5 mm band is more than ample. Place the excised strip in a 1.5-ml polypropylene microfuge tube. Wash with H 2 0 until no color is seen in supernatant. The protein is now ready for labeling and cleavage. Note: Do not compare proteins stained with NBB with those stained with India ink. Use the same staining procedure for all proteins to be compared. Two-Dimensional SDS-PAGE

1. Isolate appropriate amounts of samples by SDS-PAGE. Fix, stain with CBB, and destain. 2. Excise the bands of interest. Soak the excised bands in 50% ethanol-50% stacking buffer ( 1 M Tris-HC1, pH 6.8) for 0.5 hr (this shrinks the gel strip, facilitating loading onto a second SDS-PAGE gel). 3. Place the excised band onto a second polyacrylamide gel of a different acrylamide concentration (it is best to go from high concentration in the first gel to a low concentration in the second gel). 4. Fractionate the proteins in a second gel (the CBB runs at the dye front). Proceed with in-gel staining or electroblotting, staining, excision, and washing as above. The protein is now ready for labeling and ~ l e a v a g e . ~ Protein Labeling

While it is possible to observe microgram quantities of peptide fragments by in-gel staining (SDS-PAGE), the ninhydrin reaction (2DTLE"

B. Batteiger, W. J. Newhall, and R. B . Jones, J. Immunol. Methods 55, 297 (1982).

[461

PEPTIDE MAPPING

617

TLC), or UV absorbance (HPLC), radiolabeling allows visualization of significantly less material (pico- to nanogram amounts). Intrinsic labeling with a uniform mixture of I4C-labeledamino acids is effective,*but expensive. Labeling with single amino acids or [35S]Met/[35S]Cys is not appropriate since many of the peptide fragments will not be labeled. Radioiodination with "'I is an excellent method of labeling which is inexpensive, extremely reproducible, and easily visualized by autoradiography or y emission d e t e ~ t i o n . ' ~ The ~ ~utility ' of radioiodination has been confirmed by comparing peptides generated from proteins intrinsically labeled with a uniform mixture of I4C-labeled amino acids and extrinsically labeled with 125~.14These studies demonstrated that only 5 of 66 peptides generated by a-chymotrypsin digestion of a 40,000-Da outer membrane protein were not labeled by lZ51,while all 22 peptides generated by Staphylococcus aureus V8 protease (V8 protease) digestion were labeled with 1251. Radioiodination of Proteins

The chloramine-T (CT) (#C 9887, Sigma)I8 procedure produces extremely high specific activities, but can damage some proteins by cleaving at tryptophan residues,19thus reducing functional properties of the protein (not important for peptide mapping). This can increase the number of peptides generated and, since the cleavage is specific, increase the sensitivity of the procedure (see Ref. 20 for a comparison of peptide maps of CT vs Iodogen-labeled2'proteins). The risk is that small peptides generated by CT cleavage, followed by another enzymatic or chemical cleavage, will be lost during the removal of unbound 1251.Moreover, CT-labeled peptides yield very "noisy" HPLC separations, with many small peaks clustering around the major peaks.I3 In comparison, the 1,3,4,6-tetrachloro-3cu,6c~-glycouril (Iodogen) (#T 9018, Sigma)21procedure is very gentle, minimizing loss of function of the protein, and produces relatively high specific activities. Since the reagent is bound to the reaction vessel, aspiration of the reaction mixture both stops the iodination and separates the protein from the iodination reagent. For these reasons, I prefer this method for radioiodination. It can also be used to specifically label the surface-exposed portions of membrane 1.4,*,15,21

Regardless of the labeling procedure, never use ' 2 5 ~that is over one half-life (60 days) old. Do not increase the amount of older '1 to bring up F. C. Greenwood, W. M. Hunter, and J. S. Glover, Biochem. J. 89, 114 (1963). N. M. Alexander. Biochem. Biophys. Res. Commun. 54,614 (1973). " R. C. Judd. Infect. Immun. 55,273 (1987). M. A. K. Markwell and C. F. Fox, Biochemistry, 112,278 (1978). IS l9

"

618

CHARACTERIZATION OF PURIFIED PROTEINS

1461

the activity as this does not work. Note: Radioemission of lZ51 will be expressed as counts per minute (cpm). This assumes a detector efficiency of 70%. If the detector has a different efficiency, multiply the cpm presented here by 1.43 to determine decays per minute (dpm), then multiply the dpm by the efficiency of the detector. Zodogen Methods Soluble Protein

1. Suspend the protein in 100 to 200 pl of DPBS (any neutral buffer should work) at a concentration up to 1 mglml in a 1.5-ml microfuge tube containing 10 to 20 pg of Iodogen. Iodogen tubes are prepared by placing 10 to 20 pl of chloroform containing 1 mglml Iodogen in the bottom of 1.5-ml polypropylene microfuge tubes and allowing to air dry. Iodogen tubes can be stored at -20' for up to 6 months. 2. Add 100 to 200 pCi '"I (as Na'25~, carrier free, 25 pCiIp1). 3. Incubate at room temperature or on ice for 1 hr. Longer incubation slowly increases the specific activity. 4. Remove the protein-containing supernatant and separate the protein from salts and unbound iodine on a Sephadex G-25 or G-50 (Pharmacia, Inc., Piscataway, NJ) desalting column using H 2 0 as the elution buffer, and lyophilize. It is also possible to remove unbound iodine by dialysis, but this generates large amounts of radioactive liquid waste. One of the easiest ways to separate unbound iodine from the protein is by SDS-PAGE. Solubilize the sample in 2x sample buffer and separate by SDS-PAGE (10 to 20 pgllane). Stop the electrophoresis before the ion front reaches the bottom of the gel. Cut the gel just above the dye front and place the slice in radioactive discard. Unbound iodine will be in this portion of the gel. 5. Either fix, stain, and destain the gel to locate protein or electroblot onto NCP and locate protein by NBB staining or India ink staining, as above. 6. The protein, either as a lyophilized preparation, in a gel slice, or on NCP, is now ready for cleavage. Gel Slice

I. Dry the gel slice in a Speed-Vac concentrator (Savant Instrument, Inc., Farmingdale, NY). Any drying system, such as heat lamps or warm air, will suffice. 2. Place the slice in an Iodogen (10 pg)-coated microfuge tube (see above). 3. Add 50 to 100 p1 of DPBS to the tube plus 100 pCi IZ5I(as N ~ ' ~ ' I , carrier free, 25 pCiIp1).

[461

PEPTIDE MAPPING

619

4. Incubate at room temperature for 1 hr. Longer incubation slowly increases the specific activity. Aspirate the supernatant (use caution; the supernatant is highly radioactive). 5. Remove the gel slice from tube. Soak the gel slice (0.5 to 1 hr) in 1.5 ml H20. Repeat several times. 6. Place 0.5 g Dowex 1x8, 20-50 mesh, anion-exchange resin (#451421, Bio-Rad, Richmond, CA) in the wells of a 24-well microtiter plate. Add 1.5 ml of 15% methanol in H 2 0 to each well. 7. Place the iodinated gel slice in a well with anion-exchange resin. Allow to incubate at room temperature for 16 hr. Unreacted iodine will bind to the beads. 8. Remove the gel slice. Soak in 1.5 ml H 2 0 .Repeat several times. Dry the gel slice in the Speed-Vac. The protein is now ready for cleavage. It is common to have between 2 x lo6 and 4 x lo6 cpm in a gel slice (1 mm x 5 mm). This provides enough material to run 10 to 20 peptide mapslslice. NCP Strip 1. Place the protein-containing NCP strip in an Iodogen tube containing 10 pg Iodogen (see above). 2. Add 100 pl (any neutral buffer should work) and 100 pCi 12'1 (as N ~ ' ~ ' carrier I, free, 25 pCi/pl). 3. Incubate at room temperature for 1 hr. Longer incubation slowly increases the specific activity. Aspirate the supernatant (use caution, the supernatant is radioactive). 4. Remove the NCP strip. Place in a fresh microfuge tube. Wash 3-5 times in 1.5 ml H 2 0 (cpm released into wash should stabilize at less than 10,00O/wash). 5. The protein on the NCP strip is now ready for cleavage. It is common to have between 3 x lo6 and 6 X lo6 cpm for a strip 1 x 5 mm. This provides material to run 15 to 30 peptide mapslstrip. Chloramine-T can also be used to iodinate proteins. Use of chloramineT generally results in higher specific activities, but it can damage some proteins. A step to remove the chloramine-T is also required. Therefore, I recommend the use of Iodogen to label proteins for peptide mapping.

Cleavage Reagents

There are many excellent methods for the cleavage of proteins. If differences in l ostructure are to be accentuted, then a cleavage reagent, or combinations of cleavage reagents which generate many fragments, should be used. However, if similarities in lo structure are to be accentuated, a cleavage agent that generates only a few fragments is desirable.

620

1461

CHARACTERIZATION OF PURIFIED PROTEINS

TABLE I CLEAVAGE REAGENTS Reagent

Site of cleavage

Buffer

-

Chemical Chloramine-Ta (#C 9887, Sigma) Cyanogen bromideb (#C 6388, Sigma) Formic acid (#I-0128, Baker Chem. Co., Phillipsburg, NJ) Enzymesc a-Chymotrypsin (#C 7762, Sigma) Pepsin A (#P 6887, Sigma) Thermolysin (#P 1512, Sigma) Trypsin (#T 1005, Sigma) V8 protease (#P 7664, Sigma)

Carboxy side of Trp

H20

Carboxy side of Met

70% formate

Between Asp and Pro

88% in H 2 0

Carboxy side of Tyr, Trp, Phe, Leu Amino side of Phe, Leu Carboxy side of Leu>Phe Carboxy side of Arg, Lys Carboxy side of Glu, Asp or Carboxy side of Glu

" Chloramine-T is used at 10 mglml in HzO. Cyanogen bromide is used at 1 mglml in 70% formate.

' All enzymes are used at 1 mglml in the appropriate buffer.

There are chemical cleavage reagents and enzymatic cleavage reagents. In general, the enzymatic reagents are the easiest to use, safest, and most reliable. The reagents listed in Table I are very useful. Certainly, many other reagerits are available for protein cleavage, but the list in Table I should provide adequate choices for most comparative situations. Buffers for Cleavage

Volatile buffers are preferred for peptide mapping, particularly when 2D TLE-TLC separation, which is strongly influenced by the presence of salts, is used. For formate and CNBr cleavages, the acid is simply diluted in H 2 0 to 88 or 70% (vlv), respectively. Enzymes requiring basic environments do extremely well in 50 rnM NH4HCOOH adjusted to the appropriate pH with NH40H. The acid peptidase, pepsin A, works well in a simple acetic acid system (add glacial acetic acid to H 2 0 to bring pH to 3.0).

[461

PEPTIDE MAPPING

62 1

Protein Cleavage

Lyophilized Proteins 1. Suspend lyophilized radiolabeled proteins in appropriate buffer at 1 mglml (varying amounts can be used). To 25 p1 of suspended protein add up to 25 p1 of appropriate cleavage reagent (1 mglml). If SDS-PAGE is to be used to separate the peptides, use as little enzyme as possible. 2. Incubate with shaking at 37" for 4 hr. 3. Aspirate the supernatant, place in a microfuge tube, and dry in a Speed-Vac. 4. Wash the sample by adding 50 p1 H 2 0 , vortexing, and drying in the Speed-Vac. Repeat at least four times. Alternate drying systems will work. 5. The sample is now ready for peptide separation. Gel Slice

1. Place the dry gel slice containing radiolabeled protein in a microfuge tube. Determine emission in a y radiation counter. Add 50 p1 of appropriate buffer and 50 p1 cleavage reagent in buffer (1 mg/ml) to the dry gel slice 2. Incubate with shaking at 37" for 4 hr. 3. Aspirate and count the gel slice and supernatant. Enzymes should release 40 to 70% of counts in the slice into the supernatant; CNBr should release S O % . Repeated digestions will release about the same percentage of counts. Generally, one or two digestions are adequate. Only 1 x lo5cpm are necessary to produce a peptide map. Note: If >95% of counts are released in the first enzyme digestion, there may be excess unbound iodine left in sample. This could cause serious problems, as the enzyme may become labeled. The resultant peptide maps will all be identical maps of the enzyme and not the sample. 4. Dry the supernatant in the Speed-Vac. Wash the sample by adding 50 p1 H20, vortexing, and drying in the Speed-Vac. Repeat at least four times. Alternate drying systems will work. 5. The sample is now ready for peptide separation. NCP Strip 1. Place the NCP strip containing radiolabeled protein in a microfuge tube. 2. Block unreacted NCP sites by adding 100 p1 of 0.05% Tween 20dH20. Incubate for 15 to 30 min. Wash gently (2x) with dH2O to remove unbound Tween 20. Excess Tween 20 in the system will interfere with peptide separations.

622

CHARACTERIZATION OF PURIFIED PROTEINS

[461

3. Determine emission in a y radiation counter. Add 50 p1 of appropriate buffer and 50 pl cleavage reagent in buffer ( I mglml) to the NCP strip. 4. Continue as from step 2 for gel slices. Release of counts into the supernatant will be more effective than with gel slices. The same cautions apply Peptide Separation

SDS-PAGE The SDS-PAGE system is generally useful only to separate large peptide fragments (>3000 D ) . A ~ variety of gradient, double cross-linker, or high-concentration gels can be used to resolve the pep tide^.^ Simply boil the peptide fragments in solubilizing solution and load onto an SDS-PAGE gel. It is often advisable to solubilize protein in SDS prior to cleavage since some peptides do not bind SDS well. If autoradiography is to be used, between 5 x lo4 cpm and lo5 cpm should be loaded in each lane. Autoradiography should be performed on unfixed gels. Fixation and staining may wash out small peptides. Place the gel in aplastic bag and overlay with Kodak XAR-5 film (#251891, Kodak, Rochester, NY), place in a cassette with a Lightening Plus (#224156, Du Pont, Wilmington, DE) intensifying screen, and expose for 16 hr at -70". If screens are not available, more sample, or longer exposures, may be used. If CBB or silver staining is employed, load at least 10 pg of proteinllane. If enzyme cleavage has been used to generate the peptide fragments, be sure to run enzyme controls to distinguish protein fragments from enzyme fragments. One-Dimensional Peptide Mapping by SDS-PAGE This technique was pioneered by Cleveland et a1." It results in relatively low resolution of the peptide fragments, but it is fairly easy, requires no special equipment, and can readily be combined with immunoblotting to locate epitopes. Any of the methods described in the Protein Cleavage section can be used to generate peptides. These are then solubilized in SDS and separated by SDS-PAGE. It is also possible to perform the cleavages in the stacking gel and to separate the peptides directly in the separating gel. It is advisable to run several lanes with an increasing concentration of enzyme in each lane to establish the amount of enzyme which gives maximal proteolysis. Failure to do so may result in spurious results. Solubilized Protein 1. Solubilize purified protein sample (- 1 mglml) by boiling for 5 min in solubilizing solution [e.g., 2 ml 10% SDS (wlv) in H 2 0 , 1.0 ml glycerol,

[461

623

PEPTIDE MAPPING -

-

-

0.625 ml 1 M Tris-HC1, pH 6.8, 6 ml H 2 0 , Bromphenol Blue to color]. (Note: Depending on the protein to be cleaved and the enzyme to be used, up to 10% 2-mercaptoethanol may be added to the solubilizing solution.) 2. Place 10 to 20 pl(10 to 20 pg) of protein sample in microfuge tubes. Add the enzyme of choice (range of concentrations for enzymes is 0.005 to 0.5 pglml). Incubate at 37"for varying times (e.g., 0.25,0.5, 1.0, and 2 hr). Stop the reaction by boiling for 5 min. Conversely, the enzyme concentration can be varied while using a constant digestion period. 3. Load 10 to 20 pl(10 to 20 pg) in each well of a gel. Depending on the size of peptide fragments generated, 15% (1.33% N,N-methylenebisacrylamide cross-linker) to 22.5% (2.66% cross-linker) acrylamide gels can be used. 4. Run the gel, fix, stain, destain, or blot to NCP for immunoanalysis. (Note: Different types of blotting paper may be required to bind small peptides.) Solubilized protein can also be loaded directly into wells (10 to 20 pl), overlaid with enzyme solution, and digested in-gel as described below for proteins in gel slices. Protein in Gel Slice. For best results, radiolabeled proteins should be used. 1. Separate the protein of interest by SDS-PAGE. Excise the band, soak in 1 M Tris-HC1, pH 6.8, for 30 min. Push the gel slice into the well of a second gel (acrylamide concentration and cross-linker concentration should be established empirically). It helps to make the second gel slightly wider than the first. Overlay the gel slice with enzyme solution in 0.1% SDS-1 mM EDTA-1% glycerol in 0.1 M Tris-HCI, pH 6.8. Use an enzyme concentration of 1 mglml, and add 1, 5, 10, and 20 p1 to separate wells to determine the correct concentration for maximal digestion. 2. Subject to electrophoresis until the dye front almost reaches the bottom of the stackinggel. Stop the electrophoresis and incubate for 2 hr at 37". Following incubation, complete the separation, fix, stain, and destain, or electroblot onto NCP for immunoanalysis. Once the separation conditions, protein concentrations, and enzyme concentrations have been established, a single digestion lane for each sample can be used for comparative purposes. Two-Dimensional Thin-Layer ElectrophoresisThin-Layer Chromatography

This is the method of choice for l o structural comparisons. With care, peptide maps generated in this manner can be directly overlaid for comparison. Although flat-bed electrophoresis can be used,12it is preferable to use

624

CHARACTERIZATION OF PURIFIED PROTEINS

[461

a Savant TLE 20 electrophoresis chamber (Savant) or its equivalent. Add an extra set of cooling coils which are made by bending $in. i.d. aluminum tubing acquired from a local hardware store. This system allows for the submersion of the thin-layer sheet in a constantly cooled "varsol" (EC123, Savant) cooling solution. Cooling should be supplied by as large a refrigerated bath as possible, such as the Forma 2095 refrigerated cooling bath (Forma Scientific, Marietta, OH). Peptides will migrate based on their charge in the electrophoresis step, and the peptide charge is a function of the pH of the running buffer. If the temperature of the running buffer varies erratically, the pH will vary erratically, and thus the peptide migration will be inconsistent. Cooling is critical for precise peptide mapping. Set cooling bath at 8.5". This should maintain the electrophoresis tank at 13.5". Temperature should not rise more than 1.5" during a run. The 0.1-mm Mylar-backed cellulose sheets of E. Merck (Cat. #5502, MCB Reagents, Gibbstown, NJ) are strongly recommended. For most applications, three peptide maps per 20 x 20 cm sheet is reasonable. However, increased resolution can be obtained by running one or two sampleslsheet. 1. Rehydrate the peptide sample to lo5cpmlpl in H 2 0containing Tyr, Ile, and Asp (1 mg/ml) amino acid markers (any amino acids will do; have fun). 2. Look for machining lines on the back of the Mylar sheet (they are subtle). Always run electrophoresis parallel to machine lines. For two sampleslrun: Divide the sheet along the centerline with a line drawn on the back of the sheet with a laboratory marker. On the back of the sheet, mark two spots 2.5 cm from the end of the sheet and 1.5 cm from the centerline. These spots mark where to load the samples. Turn the sheet face up. Marks will show through the cellulose. For three sampleslrun: Divide the sheet into three sections by drawing lines (parallel to machine lines) 6.7 and 13.4 cm from the left edge of the sheet. Make three spots, each 8 cm from the end of the plate and 1 cm to the right of the left edge and each line. 3. Using a graduated, 1- to 5-p1 capillary pipet, spot 1.5 p1 (-1.5 x 10' cpm) if two samples are used, 1.0 p1 (-lo5 cpm) if three samples are used, 0.5 p1 at a time (dry spot with a hair dryer each time) to one mark on sheet. Repeat for each sample on the other mark(s). You can spot 1 p1 of 1% Methyl Green (Sigma) on the centerline to ensure proper electrophoresis-the Methyl Green should migrate rapidly toward the cathode in a straight line. Any turning indicates a problem. Note: The position of samples on the plate strongly influences peptide migration; therefore, all samples to be compared must be spotted the same distance from the anodal edge of the sheet.

[461 -

PEPTIDE MAPPING -

625

-

4. Spray the plate with TLE buffer (2 liters H20, 100 ml glacial acetic acid, 10 ml pyridine) using a laboratory sprayer (#S 4885, Sigma). Do not overwet. Remove any standing buffer with one paper towel. Always blot the TLE plates in exactly the same manner. 5. Place in the electrophoresis chamber with the samples toward the positive terminal. Separate peptides by electrophoresis at 1200 V (about 20 Wand 20 mA) for 45 min (two sampleslrun) or 31 min (three sampleslrun). 6. Remove from the chamber and immediately dry with a hair dryer. The "varsol" will dry first, then the buffer. Cut the sheet along the lines on the back of the sheet. At the top edge of each piece (the bottom is the edge closest to the sample), score the cellulose to form a moat 0.5 crn wide. 7. Place the sheets in the chromatography chamber containing TLC buffer (260 ml n-butanol, 200 ml pyridine, 160 ml H20, 40 ml glacial acetic acid) that is about 0.5 cm deep. Allow it to chromatograph until buffer reaches the moat. Remove and dry with a hair dryer (best done in hood). 8. Lightly spray the sheet with 0.25% ninhydrin in acetone (do not saturate) and dry with a hair dryer to locate the amino acid markers. [Ninhydrin can also be used to locate peptides if larger amounts of sample are separated (10 to 100 pg). Be sure to run enzyme controls to distinguish sample from enzyme.] 9. Overlay the sheets with X-ray film, place Lightening Plus intensifying screen over the film, and place in a cassette. Expose for 16 hr at -70". Exposure time may need to be increased or decreased. If screens are not available, expose for 4 days at room temperature. You can run more sample but resolution is lost as more sample is added. Develop. Migration of peptides should be consistent enough to directly overlay peptide maps for comparisons. Coordinates of amino acid markers and peptides can be determined and used to compare migration. The labeling procedures described here are precise enough to use emission intensities as a criterion for comparison. High-Performance Liquid Chromatography

High-performance liquid chromatography is a good method for comparison and an excellent method for recovery of pep tide^.^>'^-'' It is best to use the Iodogen method of radiolabeling since there is considerable "noise" produced by CT-generated peptide fragments. Reversed-phase chromatography using a CI8 column (PIN 27324 SIN, Millipore) is an excellent choice for peptide separation. A gradient of H20-0.005% trifluoroacetic acid (TFA) (#T 6220-0, Aldrich Chemical Co., Inc., Milwaukee, WI) to methanol works we1L3 Other gradients using these solvents, or other solvents, such as acetonitrile-0.005% T F A ' ~ - 'and ~

626

CHARACTERIZATION OF PURIFIED PROTEINS

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2-propanol, may be necessary to achieve adequate separation. I use a Beckman model 170, in-line y radiation detector to generate elution profiles, but fractions can be collected and counted. If UV absorbance (280 nm) is to be used, 10 to 100 pg of peptides should be used. The sensitivity of UV roughly parallels that of CBB staining in gels. Chromatograms should be reproducible to 0.05-min retention time for all peaks on repeated separations. This precision allows for the direct comparison of elution profiles of peptides of the proteins to be compared. Rehydrate peptides at 105/p1(this can vary) in H20. Amino acid markers, such as Phe, Trp, and Tyr (1 mglml), can be used to ensure consistent separations. Use between 1.5 x 10' and 1 x lo6 cpmlseparation. Peaks can be collected, washed, dried, and reseparated by SDS-PAGE or 2DTLE-TLC. Acknowledgments I thank Dr. W. 0. Granath and Ms. Robin Pettit for their review of this work. I also thank the Public Health Service, NIH, NIAID (Grants ROI A121236 and KO4 AI00834), and UM Research Program for their continued support.

[471 Analysis for Protein Modifications and Nonprotein Cofactors By SAMSEIFTERand SASHAENGLARD

Two volumes of this series'.' are devoted entirely to posttranslational modifications of proteins. Because of the large diversity of the modifications, and accordingly of the methods for their analysis, a single brief chapter on the same subject, as attempted in the present volume, can have only modest goals. Our aim is to present a selected range of such modifications, and strategies for their identification and isolation as discrete compounds, and some methods for their quantification. In a few instances of newly recognized modifications we shall present more detail; these include the family of membrane-anchoring glycan inositol phospholipids attached

' F. Wold and K. Moldave, eds.. "Posttranslational

Modifications," Part A , this series, Vol. 106. F. Wold and K. Moldave, eds., "Posttranslational Modifications," Part B, this series, Vol. 107.

METHODS 1N ENZYMOLOGY. VOL. 182

Copyright 0 1990 by Academic Press, Lnc. All rights of reproduction in any form reserved.

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to the carboxyl-terminal amino acid residues of certain proteins, and the amidations of carboxyl-terminal residues found in a number of physiologically active peptides. Other modifications acknowledged, but not dealt with in this chapter, include the large groups of glycosylations and phosphorylations. These modifications are mentioned in brief summary sections provided with references. In studying the properties of a newly isolated protein, one is always aware of the possibility that some of its constituent amino acid residues are covalently modified. Such modifications are known to occur on terminal amino or carboxyl groups or on side chains of various residues. These modifications cannot be predicted from the nucleotide sequences of the mRNA of a protein under study, except for the fact that a specific segment of the sequence may encode a polypeptide sequence specifying that a particular residue has the potential for modification by action of a specific enzyme. The hundreds of individual posttranslational modifications now known can be classified in several convenient and useful ways. Tables of modifications have been presented by Uy and wold3and Cohn4in which respective criteria are (1) involvement of a single amino acid residue of one kind without involvement of a second amino acid; (2) involvement of a single kind of amino acid and a prosthetic group; and (3) involvement of two different amino acid residues from among the common 20 amino acids, or of two amino acid residues of which one is not among the common 20. Cohn4 and wold' have also prepared tables of modifications classified by the type of biochemical or chemical reaction involved, e.g., acylation or methylation. These tables are valuable for their overview of a large and complex subject. Primary references are found in two of the reviews4,' and in a review by Kivirikko and M ~ l l y l a . ~ Since the modifications with which we are concerned, except ar-amidation and racemization of aspartyl residues, do not include cleavage of peptide bonds, amino acids retaining the modifications can be isolated after suitable hydrolysis or proteolysis of the protein. Free modified amino acids can then be isolated by various kinds of chr~matograph~'.~ and their structures studied by physical and organic chemical methods, including mass spectrometry . 9

' R. Uy and F. Wold, Science 198, 890 (1977). W. E. Cohn, this series, Vol. 106 [I]. F. Wold, Annu. Rev. Biochem. 50,783 (1981). K. I. Kivirikko and R. Myllyla, this series, Vol. 144 [6]. C. C. Q. Chin, this series, Vol. 106 [2]. K. A. Walsh and T. Sasagawa, this series. Vol. 106 [3]. S. A. Cam and K. Biemann, this series, Vol. 106 [4].

'

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CHARACTERIZATION OF PURIFIED PROTEINS

[471

Amidation of Carboxyl-Terminal Residues

Numerous peptide hormones, hormone-releasing factors, and neurotransmitters are amidated at their carboxyl-terminal residue^.'^-'^ Peptides in this group are generally synthesized with a glycine residue on the carboxyl terminus, as confirmed by the sequence of the specifying DNA or terminal codon of the mRNA. The modification is due to an oxidative cleavage of the peptide bond with formation of glyoxylate and retention of the a-amino group as the amide group of the new carboxyl-terminal amino acid formed from the penultimate residue in the precursor. The function of amidation is not known. Analysis An indication that a given peptide undergoes a-amidation can be gleaned from comparison of the amino acid sequence deduced from coding nucleotide sequences with the amino acid sequence determined from analysis of the protein. If amidation has occurred, the penultimate carboxylterminal residue in the deduced sequence appears as the ultimate residue, in amidated form, in the processed peptide. Carboxypeptidases A and B, if pure, will not cleave the amidated carboxyl-terminal residue, since these enzymes require that the residue be unblocked. However, most preparations of these enzymes are contaminated with amidases; to a variable extent the amidases may unblock a peptide with the consequence that the carboxypeptidases will then cleave the carboxyl-terminal residue. Carboxypeptidase Y will remove carboxylterminal residues that are either esterified or amidated. It has been used successfully in structural analysis of vasopressin and oxytocin, both of which are a-amidated.15 Recently, a publication appeared outlining a sensitive and more specific method of identification of the a-amidated residues on the carboxyltermini of various peptides; final analysis is by high-performance liquid chromatography (HPLC).16 A peptide is cleaved with endopeptidases (e.g., trypsin), or a combination of proteases, to yield a mixture of oligoA. F. Bradbury, M. D. A. Finni, and D. G . Smyth, Nature 298,686 (1982). A. F. Bradbury and D. G . Smyth, Biochem. Biophys. Res. Commun. 112,372 (1983). l2 B. A. Eipper, R. E. Mains, and C. C. Glembotsky, Proc. Natl. Acad. Sci. U.S.A.80,5144 (1983). l 3 K. Sugano, G. W. Aponte, and T. Yamada, J. Biol. Chem. 260, 11724 (1985). l4 B. A. Eipper, V. May, and K. M. Brass, J. Biol. Chem. 263,8371 (1988). l5 R. Hayashi, this series, Vol. 45 [48]. l6 W. E. Schmidt, J. M. Conlon, V. Mutt, M. Carlquist, B. Gallwitz, and W. Creutzfeldt, Eur. J . Biochem. 162,467 (1987). lo

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PROTEIN MODIFICATIONS A N D NONPROTEIN COFACTORS

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peptides, amino acids, and the carboxyl-terminal amidated residue. The digest is treated with phenylisothiocyanate to yield a mixture of phenylthiocarbamoyl derivatives. Because of its greater hydrophobicity, the phenylthiocarbamoyl derivative of the amidated carboxyl-terminal residue can be extracted with organic solvents; in this procedure it is selectively extracted into alkaline butyl acetate. The amidated residue is identified by HPLC using a column of Spherisorb ODS-2. Standard phenylthiocarbamoyl derivatives of 17 a-amidated amino acids have been prepared. Glycoinositol Phospholipid Membrane Anchors

In recent years it has become recognized that a number of proteins (including some enzymes), oriented toward the extracellular space, contain an amphipathic glycolipid that anchors the protein into the cell mernbrane.".l8 The anchor is attached covalently to the carboxyl-terminal residue of the protein through an amide linkage with a phosphoethanolamine residue, and is noncovalently inserted into the membrane by means of the inositol phospholipid portion of the anchor. One may reasonably expect that many enzymes that act pericellularly or extracellularly will be found to be anchored in this way. The structures of two such anchors, taken from Roberts et a1.,I9 are shown in Fig. 1. They have in common an ethanolamine residue whose amine function is in amide linkage with the carboxyl-terminal residue of the protein and whose alcohol function is linked to a phospho residue in phosphodiester linkage to a hexose (e.g., mannose) residue of the glycan portion of the anchor. Other common features are a nonacetylated residue of glucosamine attached glycosidically on the one side to a hexose residue of the glycan and on the other side to the inositol portion of the inositol phospholipid. Variations in this structure include acylation of an inositol hydroxyl group with a palmitoyl residue (shown in Fig. 1 in the acetylcholinesterase of the human erythrocyte), and differences in the substituents on the glycerol moiety of the phospholipid. Thus, the variant surface glycoprotein (variant 117) of Trypanosoma brucei contains a glycerol with two acyl (myristoyl) functions, while the human erythrocyte acetylcholinesterase anchor shown in Fig. 1 has alkyl and acyl substituents on the glycerol moiety. It has been speculated that the nature of the substituents on the glycerol moiety influences the specificity of putative phos" M.

Is

l9

A. J. Ferguson, S. W. Homans, R. A. Dwek, and T. W. Rademacher, Science 239,753 (1988). M. A. J. Ferguson and A. F. Williams, Annu. Rev. Biochem. 57,285 (1988). W. L. Roberts, S. Santikam, V. N. Reinhold, and T. L. Rosenberry, J. Biol. Chem. 263, 18776 (1988).

630

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CHARACTERIZATION OF PURIFIED PROTEINS

0 $ 6 Man o 1-2Man o 1 - 6 M a n a 1-4GlcNH2 o 1 3I

0

~~c-0~-

(t)G a l a l - 2 G a l o l - 6 G a l a 1

HL-oc-

2 I

(tlGalol HO

0

I1

Protein) -CNHCH2 C H 2 0

0.6-o-

I1

0

0

I

;ex-Hex-Hex-GkNH

A-

2y"oLH

- 0 - P - O C H 2 C H 2 NH2 I

OH

OH

HO

HO

w

coc Hcoc fVA=A=A,A=/"\/V

ti2

A.

FIG. 1. Proteins anchored in cell membrane by way of glycolipids. (A) Trypanosome variant surface glycoprotein (VSG); (B) human erythrocyte acetylcholinesterase.

pholipases in the cell membrane that regulate the release of the protein from the surface.20 Analytical Procedures

The first point to be made is that a protein so modified contains a blocked carboxyl-terminal group. Thus, in the case of human erythrocyte acetylcholinesterase, digestion with papain yields the anchor glycan phospholipid linked to a His-Gly d i ~ e p t i d e . ~Digestion ',~~ of the same protein with pronase yields the same anchor in amide linkage with a glycine residue. Is Further analysis of the anchor of that enzyme is shown schematically in Fig. 2, taken from a paper by Roberts et Treatment'of the protein with

" M. G. Low and A. R. Saltiel, Science 239, 268 (1988).

'' W. L. Roberts and T. L. Rosenberry, Biochemistry 25,3091 (1986). 22

23

R. Haas, P. T. Brandt, J. Knight, and T. L. Rosenberry, Biochemistry 25, 3098 (1986). W. L. Roberts, J. J. Myher, A. Kuksis, M. G. Low, andT. L. Rosenberry, J . Biol. Chem. 263, 18766 (1988).

[471

PROTEIN MODIFICATIONS AND NONPROTEIN COFACTORS

63 1

PIPLC methanolysis acetolysis

Deamination

I

Protein-glycan +

HCO- -Acyl

Acyl Phospholipase D

Methanolysis

FIG. 2. Points of cleavage in the glycolipid anchor of human erythrocyte acetylcholinesterase. PIPLC. Phosphatidylinositol phospholipase C.

nitrous acid at pH 4 causes the glucosamine residue to be deaminated and the glycosidic linkage to inositol to be cleaved.24This releases a phosphatidylinositol moiety that may be different in other proteins depending on whether the glycerol has monoalkyl and monoacyl groups or diacyl groups, and whether the inositol has an additional acyl substituent. As indicated, the inositol-containing fragment can be cleaved from the phospholipid by use of phospholipase D. The diacylglycerol or monoalkylmonoacyl glycerol in the several anchors can be cleaved by acidic methanolysis with anhydrous HC1-methanol, by acetolysis with acetic acid-acetic anhydride mixtures, or by treatment with phosphatidylinositol phospholipase C (PIPLC) obtained either from bacteria (e.g., Staphylococcus aureus) or from try pa no some^.^^ Fragments obtained by acetolysis can be subjected to basic methanolysis (with sodium methoxide in methanol/toluene) to cleave off the fatty acids.13Individual components released by these treatments in rational sequence can be identified by gas liquid chromatography (GLC), thin layer chromatography (TLC) and, ultimately, by fast atom-bombardment mass spectroscopy. A statement should be made about the phospholipases that are used to indicate the existence of these kinds of anchor^.^^,^^ Although PIPLCs will release a water-soluble fragment from some of the anchored proteins, they will do so poorly, or not at all, in the case of other such proteins; a case in point is the human erythrocyte acetylcholinesterase whose analysis is described above. However, the inositol phospholipid liberated from the anchor by deamination with nitrous acid becomes more susceptible to cleavage by PIPLC of S. aureus if it is first treated with base to remove the 24

M. A. Ferguson, M. G. Low, and G. A. M. Cross, J. Biol. Chem. 260, 14547 (1985).

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CHARACTERIZATION OF PURIFIED PROTEINS

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palmitoyl substituent on the inositol moiety. Another enzyme containing an anchor resistant to cleavage by PIPLC is 5'-nucleotidase. Carboxyl Methylation of Glutamate and Aspartate Residues This subject is considered in Chapters [28-3 I] of volume 106 of this series .25-28 The chemical methylation of glutamate and aspartate residues in proteins and biological homopolymers was studied many years ago. From other experiments, esters or "ester-like" groups (e.g., cyclic imides of dicarboxylic acids) were indeed inferred to exist in collagens and certain other proteins.29 Only in recent years, however, has carboxyl esterification been shown to have biological significance, such as in chemotaxis and photo taxi^.^^^^ Thus, certain membrane proteins are esterified at the y-carboxyl groups of specific glutamate residues by transfer of methyl groups from S-adenosylmethionine (SAM). In contrast to the enzymes involved in chemotaxis that methylate L-glutamate residues, another class of enzymes known as D-aspartate, L-isoaspartate-methyltransferases catalyze formation of methyl esters of D-aspartate and L-isoaspartate residues. Both of these "unusual" residues are in themselves the products of posttranslational modifications of L-aspartyl and perhaps of L-asparaginyl residues. The mechanisms for the latter posttranslational modifications are not known, although they must involve processes of racemization of L-aspartyl residues on the one hand and deamidation and rearrangement of L-asparaginyl residues on the other. The formation of such residues is considered by some to be a phenomenon of "aging" of proteins at the molecular level.

Analytical Methods and Possibilities In addition to methods described in Vol. 106 of this series, referred to previously, other descriptions can be found in several other publications .31-36

*' S. Kim, this series, Vol. 106 1281. J. B. Stock, S. Clarke, and D. E. Koshland, Jr., this series, Vol. 106 1291. '' M. A. Snyder, J. B . Stock, and D. E. Koshland, Jr., this series, Vol 106 [30]. 26

S. Clarke, P. N. McFadden, C. M. O'Connor, and L. L. Lou, this series, Vol. 106 1311. P. M. Gallop, S. Seifter, M. Lukin, and E. Meilman, J. Biol. Chem. 235, 2619 (1960). 30 S. Clarke, Annu. Rev. Biochem. 54,479 (1985). " E. N. Spudich, C. A. Hasselbacher, and J. L. Spudich, J . Bacterial. 170,4280 (1988). 32 E. N. Spudich, T. Takahashi, and J. L. Spudich, Proc. Narl. Acad. Sci. U.S.A. 86,7746 (1989). " C. M. O'Connor and K. E. Yutzey, J. Biol. Chem. 263, 1386 (1988). 29

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PROTEIN MODIFICATIONS A N D NONPROTEIN COFACTORS -

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633

-

The most sensitive methods for detecting methylated proteins, quantifying the degree of methylation and for identifying residues that are methylated, involve the prelabeling of the proteins with a tritiated methyl group. Cells or fractions of cells containing the methyltransferases are incubated, respectively, with [methyl-3~]methionineor [ m e t h y l - 3 ~ ] ~ adenosylmethionine. The cells or proteins are then treated with 0.1 N NaOH to hydrolyze the methyl groups. The liberated volatile labeled methanol is collected quantitatively and its radioactivity determined. From this one can estimate the extent of methylation. For identification of specific residues that have become methylated, proteins or derived proteolytic fragments can be digested with carboxypeptidase Y. Many preparations of that enzyme are contaminated with small amounts of other proteases; in any event, carboxypeptidase Y will liberate residues containing the methyl function. These can then be traced and analyzed, since the methyl group is tritiated. Carboxypeptidase Y, unlike carboxypeptidases A and B, can cleave ester-blocked carboxyl-terminal residues, and also can cleave D-aspartyl residues; yet it is not fully understood why the enzyme is so relatively efficient in liberating the carboxyl methylated residues. Using suitable standards. one can identify the various amino acid esters by several kinds of chromatography. Carboxyl methyl esters are fairly stable at pH 5, so that some proteolytic digestions can be conducted at that pH. The esters become increasingly sensitive to cleavage as the pH of the medium is increased above neutrality. Complete hydrolysis of peptide bonds in ester-containing proteins with 6 N HCl or with strong alkali will destroy the methyl ester functions. Previous experience with collagens has shown that protein methyl esters can be treated with 2 M hydroxylamine to form hydroxamides. The hydroxamides can be readily converted to dinitrophenylhydroxamates by and heating for 1 min in 0.1 N treatment with 1-fluoro-2,4-dinitrobenzene, NaOH to cause a Lossen arrangement with liberation of dinitrophenol and carbon dioxide. The residual protein now contains residues of diaminobutyric acid in place of methyl esters of glutamic acid and diaminopropionic acid in place of residues of aspartic acid. The protein can be hydrolyzed and the new amino acids identified by chromatography using suitable standards.29To make the method more sensitive in cells that are engaging in protein synthesis, one could use appropriately labeled glutamate or aspartate in the medium followed by isolation of labeled diaminobutyrate P. S. Backlund, Jr. and R. R. Askarnit, J. Bibl. Chem. 263, 15864 (1988). M. O'Connor and B. J . Germain, J. Biol. Chem. 262, 10404 (1987). 36 M. S. Thoelke, H. M. Parker, E. A. Ordal, and G. W. Ordal, Biochemistry 27,8453 (1988). l4

" C.

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CHARACTERIZATION OF PURIFIED PROTEINS

[471

or diaminopropionate. Perhaps trypsin or trypsin-like enzymes would cleave at the sites containing the new diamino acid residues. These methods have not yet been applied to proteins other than collagen. y-Carboxylation of Glutamate Residues

Methods for analysis of y-carboxyglutamic acid (Gla) residues in - ~ ~residues are proteins are discussed in Volume 107 of this ~ e r i e s . ~ 'Gla found in certain plasma coagulation factors, in proteins C, S, and X of serum, and in the abundant bone protein known as osteocalcin or bone matrix Gla protein. Its occurrence in the toxic sleep peptide of the cone snail is of considerable interest.43Undoubtedly, Gla residues will be found in other proteins with specific metal-binding properties since the functions of Gla in proteins are related to its chelation properties. Because proteins with this modification may have multiple Gla residues, under certain conditions one might encounter or produce "undercarboxylated" forms of a given protein; in that case not all of the glutamate residues with potential for carboxylation are in fact carboxylated. The mechanism for carboxylation requires the action of a carboxylase that utilizes the reduced form of vitamin K.44 Analytical Strategies and Methods

A fundamental property of Gla residues is due to the malonic acid-like character of the side chains. As with malonic acid, one of the two carboxyl groups is removed when the material is treated with strong acid; however, the compound is stable to treatment with strong base. Thus, when Glacontaining proteins are hydrolyzed with 6 N HC1, Gla residues are decarboxylated and converted to Glu residues. When treated with strong NaOH, the Gla residues are stable, and carboxyglutamate will be found in the protein hydrolysate. The Gla obtained in that way can be separated and identified by usual amino acid chromatographic analysis or by HPLC techniques. Another method of identification and measurement of Gla residues, especially useful in sequence determination, is based on their chemical G. L. Nelsestuen, this series, Vol. 107 [32]. G. L. Nelsestuen, this series, Vol. 107 [33]. j9 C. M. Gundberg, P. V. Hauschka, J . B. Lian, and P. M. Gallop, this series, Vol. 107 [34]. P. A. Price, this series, Vol. 107 [35]. 41 P. A. Price, this series, Vol. 107 [36]. 42 T. Brody and J. W. Suttie, this series, Vol. 107 [37]. 43 W. R. Gray, B. M. Olivera, and L. J. Cruz, Annu. Rev. Biochem. 57,665 (1988). J. W. Suttie. Annu. Rev. Biochem. 54,459 (1985). ."

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PROTEIN MODIFICATIONS AND NONPROTEIN COFACTORS

635

tritiati~n.~~ Either , ~ ' . ~the ~ Gla-containing protein, or a peptide derived from it by proteolytic cleavage, is treated in lyophilized form with tritiated water to wash out the normal water, and then is evacuated at room temperature. The material, in the presence of tritiated water, is then heated at 110". This causes rnonodecarboxylation of the Gla residues and replacement, in each of the Glu residues produced, of a specific hydrogen atom by an atom of tritium. Thus, wherever a Gla residue had originally existed, a new Glu residue appears with a tritium atom attached to the y-carbon atom. The sample can then be subjected to sequence analysis or to amino acid analysis or both. The binding of certain metal ions to Gla residues in proteins prevents their thermal decarboxylation and, when tritiated water is present, prevents the incorporation of tritium onto the y-carbon atoms. This provides useful methods for probing interactions of Gla residues with metal ions-46-50 P-Carboxylation of Aspartate Residues

Methods for analysis of P-carboxy aspartic acid (Asa) residues are discussed in Volume 107 of this series.'' This posttranslationally produced derivative of aspartyl residues has been found in ribosomal proteins of Escherichia coli. There is some evidence that the carboxylation of aspartyl residues may be vitamin K dependent, as is the carboxylation of glutamate residues. Carboxyaspartate residues, like those of carboxyglutamate, are decarboxylated during acid hydrolysis of proteins, but are stable to base hydrolysis. For analytical purposes the Asa-containing proteins are hydrolyzed by heating at 110" with 2 M KOH for 24 hr in the absence of oxygen. Because perhaps only 1 residue of Asa occurs per 9000 total residues in ribosomal proteins, analysis almost always requires that many of the other amino acids first be removed from the hydrolysate by a prior chromatographic step using an anion-exchange resin. Common amino acids are washed out with water 45

D. J. Liska and J . W. Suttie, Biochemistry 27, 8636 (1988).

" P. M. Tuhy, J . W. Bloom, and K. G . Mann, Biochemistry 18,5842 (1979). P. A. Price, C. Nelson, and M. K. Williamson, Anal. Biochem. 138, 119 (1984). M. R. Lewis, D . W. Deerfield, 11, R. A. Hoke, K. A. Koehler, L. G. Pedersen, and R. G . Hiskey, J. Biol. Chem. 263, 1358 (1988). " S. P. Bajaj, R. Saini, A. Katz, G.-Z. Cai, S. L. Maki, and G. L. Brodsky, J . Biol. Chem. 263, 9725 (1988). G . A. Zapata, P. Berkowitz, C. M. Noyes, J . S. Pollock, D. W. Deerfield, 11, L. G. Pedersen, and R. G . Hiskey, J . Biol. Chem. 263, 8150 (1988). T. H. Koch, M. R. Christy, R. M. Barkley, R. Sluski, D. Bohemier, J . J. Van Buskirk, and W. M. Kirsch, this series, Vol. 107 [38]. 47

"

636

CHARACTERIZATION OF PURIFIED PROTEINS

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and Asa is eluted with HC1. Asa can then be identified and determined using automatic amino acid analysis. For alternative analysis by gas chromatography-mass spectrometry (GC-MS), the fractions containing Asa are flash evaporated and the dry residue treated with diazomethane to form the triester of Asa, and then with acetic anhydride-pyridine to form the acetyl derivative of the amino group. The derivative is volatile and can be measured by GC-MS. NZ-(y-G1utamic)lysineCross-Link This subject is discussed in Volume 107 of this seriess2and is briefly summarized below. The cross-link is an isopeptide bond formed by transglutamination between a glutaminyl residue on one protein chain and the &-aminogroup of a lysine residue in a second protein chain. Extracellular proteins found to contain this cross-link are vertebrate and certain invertebrate fibrins, seminal fluid clots in several vertebrate species, and keratin in skin. Intracellular proteins containing this cross-link include muscle proteins and proteins of plasma and endoplasmic reticulum membranes of certain cells. The introduction of this cross-link between polypeptide chains may diminish the solubility of a protein as it does in the case of fibrin. The frequency of occurrence of this cross-link in the relevant proteins is not large. Analytical Methods

In order to obtain the free amino acid that represents the cross-link, y-glutamyl-NZ-lysine (Glu-Lys), the protein must be digested to completion with proteolytic enzymes. In the case of fibrin, almost 100% hydrolysis occurs with digestion first by pronase and then by leucine aminopeptidase and prolidase. Other proteins may require a different sequence of digestion with a different set of enzymes that may include trypsin, pepsin, chymotrypsin, pronase, and carboxypeptidases. For valid analysis, one should estimate the degree of completion of digestion. Final identification and measurement of the isopeptide can be done variously with column ion-exchange chromatography using a known standard, by thin layer chromatography, or by HPLC reversed-phase chromatography after the isopeptide has been converted into a fluorescent derivative with o-phthalaldehyde. If the parent protein can be synthesized biologically to incorporate ['4C]lysine, a highly sensitive method is available in which a marker isopeptide containing tritium in the glutamyl resi52

A. G . Loewy, this series, Vol. 107 [13].

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due is added to the proteolytic digestion mixture, ultimately allowing a double-label count. Acid hydrolysis of the isolated peptide will yield equimolar amounts of the glutamic acid and lysine. On the other hand, the isolated isopeptide can be dansylated, hydrolyzed with acid, and subjected to thin layer chromatography. The chromatogram would show only dansylglutamic acid and lysine dansylated on its a-amino group.

Many aspects of this rapidly developing topic are considered in great ~ - ~subject ' has also been reviewed detail in Volume 106 of this ~ e r i e s . ~The However, much new information has recently by Ueda and Hayai~hi.~* been obtained concerning the enzymes involved, the functional significance of ADP-ribosylation, and the occurrence of nonenzymatic ADPribosylation with free ADP-ribose. ADP-ribosylationof proteins is a posttranslational process in which the high-energy N-glycosidic bond between the C-l aldehyde function of ADP-ribose and the Nfof oxidized nicotinamide in NAD+ is cleaved with transfer of the ADP-ribose moiety to an acceptor amino acid residue of a protein. Free nicotinamide is liberated. The process is catalyzed by a number of mono(ADP-ribosy1)transferasesfound in both prokaryotic and eukaryotic cells. The transferases are classified according to the amino acid residue that accepts the ADP-ribose group. Thus, mono(ADPribosy1)transferases that ADP-ribosylate arginine residues are classified as A transferases, those that ADP-ribosylate cysteine residues are called C transferases, and those that ADP-ribosylate a posttranslationally modiN. J. Oppenheimer, this series, Vol. 106 [40]. G. Soman, J. F. Miller, and D. J. Graves, this series, Vol. 106 [41]. J. MOSSand M. Vaughan, this series, Vol. 106 1421. 56 C. G. Goff, this series. Vol. 106 1431. " J. MOSSand M. Vaughan, this series, Vol. 106 1441. 'S M. E. Smulson and T. Sugimura, this series, Vol. 106 [45]. j9 M. Miwa and T. Sugimura, this series, Vol. 106 [46]. K. Ueda and 0. Hayaishi, this series, Vol. 106 1471. P. Adamietz and H. Hilz, this series, Vol. 106 [48]. K. Wielckens, R. Bredehorst, and H. Hilz, this series, Vol. 106 [49]. 63 M. K. Jacobson, D. M. Payne, R. Alvarez-Gonzales, H. Juarez-Salinas, J. L. Sims, and E. L. Jacobson, this series, Vol. 106 [SO]. a M. Miwa and T. Sugimura, this series, Vol. 106 [S]]. K. Ueda, J. Zhang, and 0. Hayaishi, this series, Vol. 106 [52]. 66 W. R. Kidwell and M. R. Purnell, this series, Vol. 106 [53]. '' M. E. Smulson, this series, Vol. 106 [54]. 68 K. Ueda and 0. Hayaishi, Annu. Rev. Biochem. 54,73 (1985). '3

''

'

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fied histidine residue called diphthamide (in elongation factor 2) are named D transferases. In addition, a number of different cells, including certain erythrocytes, contain an enzyme that hydrolyzes NAD' to free ADP-ribose, nicotinamide, and a proton. These enzymes are called glycohydrolases. The free ADP-ribose released can react nonenzymatically with a variety of proteins and, in some cases, modify their biological activities. Recently, enzymes have been studied that hydrolyze ADP-ribosyl groups attached to proteins. An enzyme may be specific for the amino acid residue that is ADP-ribosylated, and is named accordingly .69 Many bacterial toxins exert their effects by virtue of their activities as These include cholera toxin, specific mono(~DP-ribosyl)transferases.70-74 E. coli enterotoxin, pertussis toxin, botulinus C2 toxin, and diphtheria toxin. ADP-ribosylation also serves other regulatory functions in microorganisms. Thus, the dinitrogenase reductase of Rhodospirillum rubrum is ADP-ribosylated on an arginine residue by a highly specific mono(ADPribosy1)transferase; simultaneously the enzyme activity is lost.75 Many eukaryotic cells exhibit ADP-ribosyltransferase C activity; that is, they transfer ADP-ribose from NAD' to cysteine, although specific protein receptors in the cells are not known. The ADP-ribose is probably transferred to the -SH group, although with free cysteine the -NH2 group possibly could be ADP-rib~sylated.~~ A number of proteins can be ADP-ribosylated nonenzymatically with ADP-ribose produced by glycohydrolase activity. This is illustrated by mono(ADP-ribosylation) of several different proteins in preparations of submitochondrial particles of rat liver.77Perhaps several different amino acid residues are modified; this is inferred from the fact that the ADPribosylated proteins exhibit differential susceptibilities to hydrolysis of their ADP-ribosyl groups by alkali and/or hydroxylamine. J. Moss, S.-C Tsai, R. Adarnik, H.-C. Chen, and S. J . Stanley, Biochemistry 27, 5819 (1988). S.-I. Tanuma, K . Kawashima, and H. Endo, J. Biol. Chem. 263,5485 (1988). 7' N. Morii, A. Sekine, Y. Ohashi, K. Nakao, H. Irnura, M. Fujiwara, and S. Narurniya, J . Biol. Chem. 263, 12420 (1988). " G. M. Bokoch, C. A. Parkos, and S. M. Mumby, J. Biol. Chem. 263, 16744 (1988). 73 J . Vandekerckhove, B . Schering, M. Barmann, and K. Aktories, J. Biol. Chem. 263,696 (1988). 74 A. Wegner and K. Aktories, J. Biol. Chem. 263, 13739 (1988). 75 R. G. Lowery and P. W. Ludden, J. Biol. Chem. 263, 16714 (1988). 76 S.-I. Tanuma, K. Kawashima, and H. Endo, J. Biochem. (Tokyo) 101,821, 1987. 77 B. Frei and C. Richter, Biochemistry 27,529 (1988). 69

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Analytical Strategies and Aspects

Analysis of ADP-ribosylated proteins must deal with the following questions: (1) In a given mono-ADP-ribosylated protein, how many residues are so modified, and what specific kinds of residues are modified? (2) In a specific case of mono-ADP-ribosylation, what is the nature of the linkage of the ADP-ribose group to a specific amino acid residue? (3) In a given poly-ADP-ribosylated protein, what is the extent of poly-ADPribosylation and is there cross-linking of the ADP-ribose polymers? (4) In a given poly-ADP-ribosylated protein, what amino acid residue initiates the polymer chain, and what is the nature of the bond between the ribose moiety and that residue? All of the above questions are dealt with in detail in the relevant chapters on ADP-ribosylation in Volume 106 of this series. The presence of substituted ribose moieties with vicinal -OH groups on the modified proteins allows for the use of specific column methods for the isolation and purification of the proteins or of peptides derived from them by specific proteolysis. Such methods include the use of boronate chromatography (e.g., with aminophenyl boronate matrices). Of course, other glycosylated proteins may attach to these columns. Analysis of modified proteins and assays of ADP-ribosyltransferases are facilitated by the use of various isotopically labeled substrates. To measure the extent of ADP-ribosylation of a given protein by a specific transferase, one can employ NAD+ labeled with 14C in its nicotinamide carbonyl group. For each ADP-ribose transferred, a labeled molecule of nicotinamide is released; the radioactivity can be measured and enzyme activity determined from the number of nicotinamide molecules released. The protein being modified can be radioactively labeled by use of adenine [U-I4C]NADor [32P]NAD+as a substrate. That allows one to estimate the degree of ADP-ribosylation; it also provides a label useful in isolation of the modified protein and in its degradative analysis. ADP-ribosylated proteins can be converted into peptides that can be subjected to various analyses to identify the residues that are modified and to determine their location in a polypeptide chain. Proteases such as pronase, thermolysin, and carboxypeptidase Y can be used for this purpose. Trypsin can release ADP-ribose from its linkage with carboxyl groups of modified proteins; and since that enzyme has esterase activity, such a result has been interpreted to mean that the carboxylate group is in ester linkage with ADP-ribose. An extremely useful method to study the kinds of linkages involved in ADP-ribosylated proteins is determination of the relative lability of a given bond at neutral, acid, and alkaline pH values, and to treatment with neutral hydroxylamine solutions. In the last instance, one may use 0.5 M hydrox-

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ylamine at pH 7.4, incubating the protein at 37" for different periods of time, and measuring the release of radioactive ADP-ribose. (The assay is done using proteins or other substances that have been labeled biosynthetically with appropriately radiolabeled NAD.) The bond between ADP-ribose and an arginine residue, as occurs in proteins modified by cholera toxin, has the stability characteristic of uredyl and thiouredyl glycosides; thus, it is relatively stable to acid and base and somewhat resistant to attack by hydroxylamine at pH 7.4. In one experiment, for instance, purified platelet actin, ADP-ribosylated at an arginine residue by botulinus C2 toxin, required 4 hr of incubation with neutral hydroxylamine to cause release of about 80% of its ADP-ribose.73 The bond between ADP-ribose and a cysteine residue is considerably more stable to neutral hydroxylamine than the ADP-ribose-arginine bond.77 The most susceptible to hydroxylamine treatment is the ADPribose-glutamate bond as found in poly-ADP-ribosylated histones; this is probably a carboxylate ester bond.70 To measure the number of ADP-ribosylated groups in a modified protein, methods have been devised to prelabel the protein with radioactive ADP-ribose, and then to treat with an appropriate enzyme to release a moiety containing the label. The amount of released radioactivity can be used to estimate the number of ADP-ribosylated groups in the protein. For this purpose one may use, for example, the specific ADP-ribosylarginine hydrolase from turkey erythrocytes.69In a more general method, one may use snake venom phosphodiesterase to release radioactive 5'-AMP. The 5'-AMP can be quantified by suitable chromatography or by a radioimmunoassay using an antibody to 5'-AMP. Finally, very sensitive fluorometric methods63 and radioimm~noassays~~ have been used to quantify ADPribose derivatives. In assays for ADP-ribosyltransferases, one may distinguish between mono- and poly-ADP ribosylation reactions by use of 2'-deoxynicotinamide adenine dinucleotide since that nucleotide appears to be a substrate only for mono(ADP-rib~syI)transferases.'~ N-Myristoylation of Amino-Terminal Residues and Other Acylations

This subject is briefly discussed in Volume 106 of this series.79Myristic acid is a 14-carbon saturated fatty acid that may be found covalently attached to the amino-terminal residue of proteins through an amide linkAlvarez-Gonzalez, J. Moss, C. Niedergang, and F. Althaus, Biochemistry 27, 5378 (1988). 79 A. Aitken and P. Cohen, this series, Vol. 106 [19]. " R.

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age involving the a-amino group of that residue. At present, the number of N-myristoylated proteins has grown to more than 20, and undoubtedly will continue to i n c r e a ~ e . ~In ~ ,several ~' of these cases a common motif of attachment is apparent; that is, the myristoyl group is attached in amide linkage to an amino-terminal glycine residue followed by a sequence of perhaps 6 to 10 residues that are important in determining whether the The enzyme that catalyzes the glycine residue becomes myri~toylated.'~~~' myristoylation reaction, myristoy1CoA:Protein N-myristoyltransferase, has been prepared from yeast and shown to require, in its peptide substrates, a core sequence of six residues that endow the substrates with high affinity for the e n ~ y m e .Towler ~' et ~ 1 .list ~ 'a series of proteins that, on the basis of their amino-terminal sequences, have the potential for Nmyristoylation. N-Myristoylation in muscle proteins has been shown to occur cotranslationally The functions of N-myristoylation remain speculative. Some proteins of this kind attach to plasma membranes, and it has been suggested that the hydrophobic character of the acyl group allows that i n t e r a ~ t i o n .In ~ ~the '~~ case of some intracellular (soluble) myristoylated proteins, it has been postulated that the acyl group allows interactions with internal membranous structures. In a sense, these are membrane-targeting functions. Another possible function is illustrated by the fact that mammalian retrovirus gag polyproteins require N-rnyristoylation for formation and release of mature infectious virus particles from cells.84 Analytical Strategies and Methods Peptides containing the N-myristoyl group can be obtained by digestion with one or more of a variety of proteases, e.g., S. aureus proteinase. The peptides have (additional) hydrophobic character because of the presence of the rnyristoyl group. That allows the use of reversed-phase HPLC for their separation and characterization. Fast atom-bombardment mass spectrometry can then be used to detect the myristoyl group in a specific peptide. Gas chromatography is also useful in identifying myristic acid. Myristic acid is released from myristoylated peptides by the usual

" B. M. Sefton and J. E. Buss, J. Cell Biol. 104, 1449 (1987).

8"

D. A. Towler, S. P. Adams, S. R. Eubanks, D. S. Towery, E. Jackson-Machelski, L. Glaser, and J. I. Gordon. J. Biol. Chem. 263, 1784 (1988). C. WIICOX, J.-S. Hu, and E. N. Olson, Science 238, 1275 (1987). R. 0. Heuckeroth, D. A. Towler, S. P. Adams, L. Glaser, and J . I. Gordon, J. Biol. Chem. 263,2127 (1988). L. E. Henderson, H. C. Krutzsch, and S. Oroszlan, Proc. Natl. Acad. Sci. U.S.A.80,339 (1983).

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hydrolytic procedure for proteins employing 6 N HC1 at 110". The myristic acid can be extracted from the hydrolysate with ether or chloroform. N-Acylation (amides of fatty acids) can be distinguished from Oacylation (esters of fatty acids) by appropriate treatment with 1 M hydroxylamine at pH 7.4 at room temperature. For instance, Wilcox et ~ 1 . ~ ' chemically labeled proteins with tritiated palmitate to form ester derivatives. By treatment with neutral hydroxylamine for 4 hr they showed release of more than 80% of the palmitate. Similar treatment of nascent myristoylated protein chains caused release of only about 8% of the myristic acid. That is consistent with the fact that esters of fatty acids are more readily cleaved by neutral hydroxylamine than are amides of fatty acids. Other acylations of proteins can also occur on the amino-terminal residue of a protein (Na-acylations) or on the side chains of residues (for example, N"-acylations). The former are discussed in Volume 106 of this s e r i e ~ . ~ ~ .N"-acylations ~'-~~; are discussed in Volume 1 0 7 . ' ' ~ ~Methods '~~~ for detecting and/or quantifying posttranslational modifications involving palmitoylation and retinoylation, respectively, are presented in other publications.93.94 Phosphorylations

This subject is considered in two treatise^.^^,^^ Methods and strategies of analysis are given in Volume 107 of this S. Tsunasawa and F. Sakiyama, this series, Vol. 106 [14]. J. E. Dixon and T. A. Woodford, this series, Vol. 106 [IS]. 87 K. L. Redman and P. A. Rubenstein, this series, Vol. 106 [16]. H. Kido, A. Vita, and B. L. Horecker, this series, Vol. 106 [17]. 89 C. E. Deutch, this series, Vol. 106 [18]. 90 P. E. Kolattukudy, this series, Vol. 106 [20]. 91 V. G. Allfrey, E. A. DiPaola, and R. Sterner, this series, Vol. 107 [12]. 92 H. Klostermeyer, this series, Vol. 107 [14]. 93 E. N. Olson, D. A. Towler, and L. Glaser, J. Biol. Chem. 260,3784 (1985). N. Takahashi and T. R. Breitman, J. Biol. Chem. 264,5159 (1989). 95 P. D. Boyer and E. G. Krebs, eds., "The Enzymes," Vol. XVII. Academic Press, Inc., Orlando, Florida, 1986. % P. D. Boyer and E. G. Krebs, eds., "The Enzymes," Vol. XVIII. Academic Press, Inc., Orlando, Florida, 1987. 97 T. M. Martensen, this series, Vol. 107 [I]. 98 J. M. Fujitaki and R. A. Smith, this series, Vol. 107 [2]. M. Brauer and B. D. Sykes, this series, Vol. 107 [3]. 100 P. J. Roach, this series, Vol. 107 [4]. lo' S. Shenolikar and T. S. Ingebritsen, this series, Vol. 107 1:5]. Io2 L. Engstrom, P. Ekman, E. Humble, U. Ragnarsson, and 0. Zetterqvist, this series, Vol. 107 [6]. 86

"

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Phosphorylation of proteins by protein kinases that utilize ATP as the phosphoryl donor is a very common posttranslational modification. Its function is related largely to regulation of the activities of certain enzymes and hormones. Analytical strategies include, where possible, the generation of 32~-labeled proteins by incubation of cells or tissues with 32Pi; the presence of a specific kinase and a specific protein substrate produces labeled phosphorylated amino acid residues that can be traced in analysis. Residues that are labeled include serine, threonine, tyrosine, lysine, arginine, and histidine. Methods for study of phosphohydroxylysine have recently been reported.'03 Suitable proteolytic and hydrolytic procedures may produce free phosphorylated amino acids or limit peptides that can be analyzed by thin layer chromatography, paper electrophoresis, and column chromatography. The phosphorylated amino acids must be distinguished from other compounds such as phosphocholine, phosphoethanolamine, and phosphoinositols. The various phosphorylated amino acids exhibit differential capacities for hydrolysis with base or acid; these differences are useful in analysis.97 Phosphate groups can be cleaved from phosphorylated proteins either by specific or nonspecific phosphatases. Glycosylations

This large and varied subject has been discussed in great detail with respect to analytical procedures in Volumes 83, 138, and 179 of this series. Other pertinent articles have also appeared.'04-106 Cofactors and Prosthetic Groups of Proteins

In this chapter, only a few general comments can be made on this immense subject, and some references provided. Cofactors that have carboxyl groups can form substituted amides with amino-terminal groups or with the amino groups of side-chain lysine residues. Thus, biotin'07 and lipoic acidto8are linked in this way. Retinoic acid appears to be attached to proteins in ester linkage.94Cofactors that have aldehyde groups, including pyridoxal p h o ~ p h a t e and ' ~ retinaL4 can form Schiff bases (adlimines) Y.Urushizaki and S. Seifter, Proc. Natl. Acad. Sci. U.S.A. 82,3091 (1985). R. Kornfeld and S . Kornfeld, Annu. Rev. Biochem. 54, 631 (1985). '05 H. Lis and N. Sharon, Annu. Rev. Biochem. 55,35 (1986). I M M. A. Kukuruzinska, M. L. E. Bergh, and B. J. Jackson, Annu. Rev. Biochem. 56,915

'03 '04

(1987). 'ol

N. H.GOSSand H. G. Wood, this series, Vol. 107 [15].

'08

K. Fujiwara, K. Okamura-Ikeda, and Y. Motokawa, J . Biol. Chem. 261,8836 (1986). Y. Morino and F. Nagashima, this series, Vol. 106 [lo].

'09

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through the interaction of the aldehyde with side-chain N"-groups of lysine residues. Aldimine functions tend to be reversible; for purposes of analysis, then, one can reduce with sodium borohydride. That stabilizes the linkage as a secondary amine. Reduction can be carried out chemically with labeled (tritiated) sodium borohydride; this allows or aids in the detection, isolation, and quantification of modifications even if they occur with low stoichiometry in relation to the other residues in a protein. The aldimine link is also found in the lysine-derived cross links of collagen and elastin, some of which are then biologically converted to stable secondary amine cross-links'10,"'; an example is the formation of lysinonorleucine in elastin. 'I' Phosphopantetheine, the cofactor of the fatty acyl carrier protein, is covalently bound through its phosphate group to a serine residue side chain to form a serine-0-phosphate ester.4 Riboflavin in FAD of various oxidases sometimes is covalently bound; thus, in different enzymes it occurs joined to residues of cysteine, histidine, or tyrosine. 4,113 In cytochrome c, the heme is bound through a vinyl side chain to a cysteine residue of the apoprotein. Dihydrophycocyanobilin is also bound to its protein moiety through a cysteine r e ~ i d u e . ~ The amino acid residue containing the covalently bound prosthetic group can be isolated either in free form or in a limit peptide after suitable hydrolysis either by enzymes or by acid. When, for instance, an aldimine link has been reduced to a secondary amine function, the compound becomes stable to acid hydrolysis. A cysteine addition across a double bond in the cofactor forms a stable thioether. Finally, a brief statement should be made concerning methoxatin or pyrroloquinoline quinone (PQQ), a substance that some think may be a vitamin in mammalian species. PQQ is a cofactor for several oxidizing enzymes, including lysyl oxidase, perhaps dopamine /3-hydroxylase, and Although most PQQ appears to be methylamine dehyd~-ogenase."~-"~ bound noncovalently, some fraction is covalently bound, perhaps in aldimine linkage with the &-aminogroup of a lysine side chain (P. M. Gallop, personal communication). After isolation of any of the modified amino acids, analysis and identification can be performed by one or several of a great number of different "O "I

0.0 . Blumenfeld, P. Gallop, and S . Seifter, Annu. Rev. Biochem. 41, 617 (1972), D.Eyre, this series, Vol. 144 [7]. D. R. Eyre, M. A. Paz, and P. M. Gallop, Annu. Rev. Biochem. 53,717 (1984). W . E. Cohn, this series, Vol. 106 [I]. P. M. Gallop, Physiol. Rev. (in press). J . A. Duine, J . Frank, Jzn, and J . A. Jongejan. Adv. Enzymol. 59, 169 (1986).

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techniques: spectroscopy in the ultraviolet and visual ranges, mass spectroscopy including fast-atom bombardment mass spectroscopy, HPLC, automatic amino acid analysis, the recently described boroxazolidone method,ll6and several enzymatic methods such as deacylation. In most of these methods standards are required; these may be obtained from specific proteins and rigorously characterized, or they may be obtained by chemical synthesis. Nonenzymatic Glycation

This subject, including methods of analysis, is discussed in Volume 106 of this series.ll7 Additional analytical publications have since appeared."8-121 Formerly this modification was called "nonenzymatic glycosylation," but since in many cases a glycosyl group is not transferred, the broader term of "glycation" is now used. Glycation is the covalent addition of a sugar or a sugar derivative to a protein without benefit of enzymatic catalysis. In most cases this occurs with the form of the sugar in which the aldehyde or ketone group is free, and the reaction occurs either with terminal amino groups or with &-aminogroups of internal lysine residues in the protein. The reaction in that case is the formation of aldimine (Schiff base) or ketimine derivatives. The aldimine form can undergo rearrangements, prominent among which is the Amadori rearrangement to form a ketoamine. The chemistry of these reactions is discussed in Volume 106.'l7 The aldimine formation is reversible, but can be fixed by reaction with NaBH4 or NaBH3CN. The ketoamine is stable, but also can be reduced with NaBH4; it is not reduced at neutral pH by N ~ B H ~ C N . ' ~ ~ - " ~ The use of tritiated borohydrides is extremely important in the analysis of these reactions. One of the modified amino acids whose residues are produced in proteins by Amadori rearrangement of the aldimine of glucose and lysine residues is W-fructoselysine (FL). FL can be oxidatively cleaved between C. J. Strang, E. Henson, Y. Okamoto, M. A. Paz, and P. M. Gallop, Anal. Biochem. 178, 276 (1989). "'R. Fliickiger and P. M. Gallop, this series, Vol. 106 [7]. { I 8 E. J. Hindle, G. M. Rostron, and J. A. Gatt, Ann. Clin. Biochem. 22, 84 (398%. R. Fliickiger, T. Woodtli, and W. Berger, Diabetologia 30,648 (1987). ' l o D. A. Arrnbruster, Clin. Chem. 33, 2153 (1987). ''I E. D. Schleicher, R. Mayer, E. M. Wagner, and K. D. Gerbitz, Clin. Chem. 34,320 (1988). P. J. Higgins and H. F. Bunn, J. Biol. Chem. 256,5204 (1981). 123 J . F. Day, R. W. Thornburg, S. R. Thorpe, and J. W. Baynes, J. Biol. Chem. 254,9394 (1979). S. Rogozinski, 0. 0. Blurnenfeld, and S. Seifter, Arch. Biochem. Biophy. 221,428 (1983). [I6

'"

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C-2 and C-3 of the carbohydrate to yield NE-carboxylmethyllysine(CML) and D-erythronic acid.125Cleavage can also occur between C-3 and C-4 of the carbohydrate in F L to yield 3-(W1ysino)lactic acid and D-glyceric acid.126 Fructose can glycate e-amino groups of lysine residues in serum albumin and human ocular lens proteins to produce a fructose-lysine residue.'27This can be reduced with tritiated NaBH4 for purposes of analysis. Iz5

Iz7

M. U.Ahmed, S. R. Thorpe, and J . W. Baynes, J. Biol. Chem. 261,4889 (1986). M. U. Ahrned, J. A. Dunn, M. D. Walla, S. R. Thorpe, and J. W. Baynes, J. Biol. Chem. 263, 8816 (1988). J. D. McPherson, B. H. Shilton, and D. J . Walton, Biochemistry 27, 1901 (1988).

[481 Protein Crystallization By DAVIDOLLISand STEPHEN WHITE

It has been known for many years that proteins can be induced to crystallize. In the past, when methods of protein purification were less sophisticated, a crystallization step was often employed if possible. The availability of crystalline materials eventually allowed the application of diffraction techniques for the purposes of structure determination. Crystallography has subsequently proved to be the most powerful tool for the elucidation of the detailed structure of large biomolecules. The present interest in the crystallization of proteins and nucleic acids is directed almost exclusively toward the production of materials for diffraction studies. Nowadays, most aspects of protein crystallography are highly automated and the major obstacle to determining the structure of a protein of interest is the production of suitable crystals. Automated machines for setting up crystallization experiments have recently a ~ p e a r e d 'and , ~ these should remove a considerable amount of the drudgery involved in setting up a large number of crystallization experiments. This chapter is intended as a practical guide for those who have purified a protein and are now interested in crystallizing it. Many different proteins have now been crystallized and compilations of these are a ~ a i l a b l e .Al~?~ l

J. M. Cox and P. C. Weber, J. Appl. Crystallogr. 20, 366 (1987).

N . D. Jones, J . B. Deeter, J. K . Swartzendruber, and P. W. Landis, Meet. Am. Crystallogr. Assoc., Austin, Tex. Abstr. N o . H 4 (1987). ' A. McPherson, Methods Biochem. Anal. 23,249 (1976). A. McPherson, "Preparation and Analysis of Protein Crystals." Wiley. New York, 1982.

METHODS IN ENZYMOLOGY, VOL. 182

Copyright O 1990 by Academic Press, Lnc. All rights of reproduction in any form reserved.

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though the technique is far from being well understood, certain rules and principles have emerged. Familiarity with the rules such as they are will not ensure success, but will save the investigator valuable protein, much wasted time, and considerable anguish. If the reader is interested in the more theoretical aspects of crystallization, several chapters have appeared in an earlier volume in this series.' The Protein

Probably the person best qualified to crystallize a particular protein is the one who is most intimate with its biochemistry. Information such as cofactors and metal ions required for activity, conformational changes on binding substrates, the purification procedure, etc., are invaluable when designing experiments. Other, less obvious considerations are discussed below. Suitability. Generally, any protein that can be solubilized to a sufficient degree (see below) is suitable for crystallization trials. This chapter will concentrate on water-soluble proteins and the complexes they form with substrates, other proteins, and nucleic acids. For the most part, the methodology is also appropriate for the crystallization of nucleic acids a10ne.~ Recently, much effort has been devoted to the development of techniques for the solubilization and crystallization of integral membrane The structure of the photosynthetic reaction centeri0is stunning evidence of the feasibility and value of high-resolution structural work on this type of protein. Although the techniques employed are similar to those described below, there are important differences and interested readers should consult these articles. It should also be realized from the outset that crystallization trials, when compared to other biochemical procedures, use a great deal of protein. The investigator must be prepared to purify a protein on a regular basis to have a reasonable chance of success. Indeed, the recent revival of protein crystallography is due in part to the increased availability of protein as a result of cloning techniques. H. Wychoff. C. W. H. Hirs, and S. N . Timasheff, this series, Vol. 114 [3-91. A.-C. Dock, B. Lorber, D. Moras, G. Pixa, J.-C. Thierry ,and R. Giege, Biochemie 66, 179 (1984).

' H. Michel and D. Oesterhelt, Proc. Natl. Acad. Sci. U . S . A . 77, 1283 (1980).

'O

R. M. Garavito, J. Jenkins, J. N. Jansonius, R. Karlsson, and J. P. Rosenbusch, J . Mol. Biol. 164, 313 (1983). H. Michel, Trends Biochem. Sci. 8, 56 (1983). J. Deisenhofer, 0.Epp, K. Miki, R. Huber, and H. Michel, Nature (London) 318, 618 (1985).

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Biochemists often will not try to crystallize a particular protein because they feel that its high molecular weight would prohibit structure determination. It is therefore worth stating that high-resolution structures have been obtained for single polypeptides larger than 100,000 ~ a . "Furthermore, low-resolution information has been obtained from molecules as large as the nucleosome," and medium resolution information has been reported for the histone octamer. l2 In cases where the molecular aggregate size is large, but the individual subunits are small and identical (as in an icosahedral virus), high-resolution information can be obtained by the use of molecular replacement. It is the ability to produce suitable crystals that generally restricts the application of modern sophisticated diffraction techniques. Protein Purity. The purity required for crystallization varies for different proteins. Some can accommodate low levels of contamination while, for others, absolute purity is necessary. Proteins that appear as a single band on SDS gel electrophoresis are generally pure enough for initial trials. If initial trials are unsuccessful, difficult to repeat, or produce only tiny crystals, further purification should be considered. In the case of dienelactone hydrolase,13it was observed that conventional chromatography produced protein that crystallized poorly and not reproducibly. Further purification by high-performance liquid chromatography (HPLC) yielded protein that could be crystallized to give large, well-formed crystals that diffract to high resolution. Other workers have used preparative isoelectric focusing to separate closely related protein variants that gave rise to useful

crystal^.'^ Crystals, by definition, consist of identical repeating molecules and every attempt should be made to ensure that the protein preparation is homogeneous. Inhomogeneity can arise for many reasons and, again, an investigator familiar with the protein is best qualified to identify potential problems. Protein Modifications. Certain aspects of protein structure are now known to hinder crystal formation. Generally, compact rigid molecules are more amenable to crystallization than floppy ones, and there have been many examples where crystals have been forthcoming only after removal of regions such as irregular tails and small domains linked to the body of the protein by hingelike regions. Proteolysis or molecular genetics can be "

I' l4

T. J . Richmond, J. T. Finch, B . Rushton, D. Rhodes, and A. Klug, Nature (London) 311, 32 (1984). R . W. Burlingame, W. E. Love, B . C. Wang, R. Hamlin, N.-H. Xuong, and N . Moudrianakis, Science 228, 546 (1985). D. Pathak, K.-L. Ngai, and D. Ollis, J. Mol. Biol. 204, 435 (1988). R. R. Bott, M. A. Navier, and J. L. Smith, J . Biol. Chem. 257, 9883 (1982).

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used to produce stable protein domains, and these frequently have part or all of the activities of the intact protein. Klenow fragment" and A repressor headpiece16 are two interesting domains whose crystal structures have been determined. Proteolysis is always an important factor to consider during crystallization experiments since apparently pure samples of protein can be degraded by an endogenous protease. This may have beneficial effects since it has been observed that the protein fragments can form crystals." In experiments where crystals appear only after many weeks, a check should be made to see whether they are composed of degraded protein. Protein Preparation. Regarding the protein concentration, the solubility limit should be approached and this could be as high as 30 mglml, or even higher. For most proteins a value of around 10 mglml is ideal and crystals rarely form from solutions containing less than 5 mglml. The solubility of proteins can sometimes be enhanced by increasing the ionic strength (usually by the addition of NaC1) and/or shifting the pH of the solution away from the isoelectric point. In addition to the protein, there are a number of other solution components that deserve consideration. The buffer concentration should be sufficiently low (between 5 and 10 mM) that the pH of the solution can be easily adjusted by the addition of a large molar excess of a new buffer in the crystallization trial. A reducing agent such as 2-mercaptoethanol or dithiothreitol should also be added to the solution to protect against the oxidation of cysteines. Finally, proteins are frequently stored in the presence of glycerol, but this should be avoided if the protein stability is not jeopardized. Significant concentrations of glycerol will affect the vapor pressure of solutions and hence interfere with vapor diffusion experiments. Also, there is the possibility of interactions between the protein and glycerol. Often the presence or removal of metal ions is essential for the stability and function of the molecule. Metals frequently present problems in crystallization experiments and a careful choice of buffer and precipitant is required. For example, proteins often require ~ g for~activity, + but this ion is insoluble (and will form crystals) in the presence of common precipitants and buffers such as phosphate. Another problem could arise if citrate is used as a precipitant since it will tend to coordinate ~ g *and + may strip the protein of this metal. The concentration of the metal may also be D. L. Ollis. P. Brick, R. Hamlin, N. G. Xuong, and T. A. Steitz, Nature (London)313,762 (1985). C. 0. Pabo and M. Lewis, Nature (London)298,443 (1982). " D . L. Ollis, P. Brick, S. S. Abdel-Meguid, K. Murthy, J . W. Chase, and T. A. Steitz, J. Mol. Biol. 170,797 (1983). l6

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important. In the case of muconate lactonizing enzyme that needs ~ n for activity, it was found that although the protein crystallized with micromolar levels of Mn2+, much sturdier crystals could be obtained with millimolar level^.'^ Metals may also be needed when not expected, and divalent metal ions in particular are often added to crystallization experiments to facilitate crystal formation. Thioredoxin, which does not require a metal for activity, crystallizes in the presence of Cu2+,which forms part of the crystal lattice. l9 McPherson has notedz0that proteins with an excess of positively or negatively charged residues might be crystallized by the addition of oppositely charged electrostatic cross-linking agents. He produced crystals of RNase A in the presence of oligonucleotides, and the authors have produced crystals of a highly negatively charged restriction enzyme inhibitor in the presence of spermidine (unpublished results). The presence or absence of substrate, product, or cofactors could also affect protein crystallization. If added, the concentrations of these reagents should be carefully considered since their affinity for the protein could diminish as the precipitant levels rise. Protein Crystals

Most biochemists are unfamiliar with protein crystals, and it is worth describing a number of their properties that will help investigators to both identify and manipulate them. Appearance. The most important property of a crystal is its ability to diffract X rays to high resolution. Although this cannot be predicted from a visual inspection, a crystallographer generally looks for single, wellformed prismatic crystals with sharp edges. We emphasize that celebrations should await the definitive test with an X-ray camera. The required size depends on the crystal quality, but bigger is generally better in terms of manipulations and data collection. A size of 0.5 x 0.5 x 0.3 mm is just about ideal. Crystals that grow predominantly in one (needles) or two (plates) dimensions can be difficult to analyze, but several high-resolution structures have been determined from such crystals. Finally, the generally weak nature of protein crystals (see below) can often be used to distinguish them from buffer crystals which may have been inadvertently formed. Poking with a fine glass fiber will generally destroy protein crystals but have little effect on buffer crystals (the so-called click test).

l9

A . Goldman, D. L. Ollis, K.-L. Ngai, and T. A. Steitz, J . Mol. Biol. 182, 353 (1985). A. Holmgren, B.-0. Sodergerg, H. Eklund, and C. I. Branden, Proc. Natl. Acad. Sci.

"

U.S.A. 72,2305 (1975). A. McPherson, G. D. Brayer, and R. D. Monison, J. Mol. Biol. 189,305 (1984).

~

+

[481

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Solvent. The molecules in protein crystals are linked together by a multitude of weak forces, and approximately one-half of the volume consists of solvent. Matthews2' has defined a parameter Vm that reflects the packing density within the crystal. It has units of A 3 / ~ aand , the average value for proteins is about 2.4. Crystals with a lower Vm are more highly packed and generally diffract better, and vice versa. If the crystal space group is known, a value of 2.4 can be used to give an indication of the number of molecules in the asymmetric unit. The high solvent content means that protein crystals must not be allowed to dry out. They are best stored in a stabilizing buffer which has to be determined for each crystal, but which is usually very similar to the growth buffer with, perhaps, a slightly higher concentration of precipitant. Crystals are mounted free from liquid in sealed glass or quartz capillaries containing a small volume of stabilizing buffer to provide an appropriate level of humidity. Soaking Experiments. The high solvent content results in large channels throughout the crystal, and these are useful for diffusing in small molecules. This is the usual method of preparing heavy atom derivatives, but it has also been extensively used for studying protein-substrate interactions. It should be noted that these are sometimes unsuccessful because the crystalline structure may block binding sites or prevent required movements of the molecule. Also, ligand binding is frequently inhibited by the high ionic strength needed for crystallization. This obstacle can sometimes be overcome by cross-linking22the crystals so that they can be transferred to a low ionic strength environment. Finally, many proteins are still active in the crystalline state, and the advent of intense X-ray sources has meant that the mechanisms can be studied using diffraction techniques. Crystallization Procedure

Outline. Protein crystallization is a poorly understood process. It can be divided into three stages: nucleation, postnucleation growth, and cessation of Although quasi-elastic light scattering has been used to monitor nucleation, and optical spectroscopy for determining crystal growth rates,23such techniques are not readily available to the biochemical community. The average researcher is generally restricted to a rather simple set-up in which the soluble protein is slowly moved to a variety of conditions in which the protein is supersaturated. If fortunate, one of these 2'

B. W. Matthews, J. Mol. Biol. 160,659 (1968). R. A. Quiocho and F. M . Richards, Proc. Natl. Acad. Sci. U . S . A . 52,833 (1984). Z . Kam, H. B . Shore, and G. Feher, J. Mol. Biol. l23,539 (1978).

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will promote nucleation and crystal growth. The experiments are examined for crystals using a low-power dissecting microscope. Initially, a wide variety of conditions are investigated and promising results are followed by more finely tuned experiments. Factors Affecting Solubility. There are a number of solution parameters that the investigator can vary to alter the solubility of a protein. These are pH, the type and concentration of precipitant, and the temperature. Reviews of the factors affecting protein solubility have been published e l ~ e w h e r e .In ~ ~general, , ~ ~ the solubility of a protein is at a minimum at its isoelectric point when its net charge is zero. For example, ribosomal proteins that interact with ribosomal RNA usually have an excess of positively charged residues and crystallize around pH The most common precipitants are salts, organic solvents, and polyethylene glycol (PEG). Salt concentration or ionic strength has different effects on the solubility of proteins at the high and low extremes. At low ionic strength, the solubility is increased-a phenomenon referred to as "salting in." However, as the ionic strength increases the solubility decreases and the protein is ultimately "salted out." It is generally thought that the mechanism for salting out involves a competition between protein and ions for water of hydration. At a point, which varies from protein to protein, they are unable to compete and are forced from solution. The ability of different ions to precipitate proteins follows the Hofmeister series and some commonly used ones are ammonium sulfate, sodium chloride, sodium/potassium phosphate, lithium sulfate, and citrate salts. Organic solvents such as ethanol and 2,4-methylpentanediol (MPD) reduce the solubility and cause crystallization of proteins via a mechanism that involves a change in the dielectric constant of the solution. Finally, PEG has become one of the most popular and successful reagents for producing crystals since it was first introduced by ~ c ~ h e r s o Itn .is~ ~ thought to affect protein solubility by competing for water and changing the dielectric properties of the solution. PEG is available in several molecular weight forms (3350, 8000, 20,000), and all can be tested in experiments. A more extensive list of precipitants can be found in Ref. 4. Temperature variation is rarely used as a method for crystallizing proteins, but it can affect protein solubility and hence the results of experiments. It is therefore common practice to set up parallel experiments at a number of temperatures. If the availability of protein is limited, do the L. Blundell and L. N. Johnson, "Protein Crystallography," Academic Press, New York, 1976. T. Arakawa and S. N . Timasheff, this series, Vol. 114, 49 (1985). 26 K. Appelt, S. W. White, and K. S. Wilson, J. Biol.Chem. 258, 13328 (1983). A. McPherson, J. Biol. Chem. 251,6300 (1976). " T.

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experiments at lower temperatures. Whichever temperature is chosen, it is important that it be kept constant. If experiments are performed in a cold room, they should be cushioned from vibration. Search for Conditions. From the previous section, it should be clear that proteins can generally be precipitated by high salt, PEG, or organic solvents and that the required concentrations of these precipitants should increase on either side of the protein isoelectric point. Proteins that require a relatively high ionic strength for solubility can be precipitated by lowering the ionic strength at or near the isoelectric point. Initial trials should involve a concentration dependence of a number of materials over a considerable range of pH. There are a sizeable number of experiments to be done, and given that only a small quantity of protein is generally available, small-scale techniques are preferable. Crystallization experiments involving an increase in precipitant concentration are most readily carried out using a vapor diffusion apparatus. Excluding experiments involving volatile organic reagents, vapor diffusioq implies that the protein solution is placed in an environment of lower vapor pressure and loses water (and becomes more concentrated) until its vapor pressure matches that of its surroundings. The most common way of doing vapor diffusion experiments is the so-called "hanging drop method," which is probably best explained by way of example. A 15-pl aliquot of a 10 mg/ml protein solution is mixed with 15 p1 of a 2.0 M phosphate solution at pH 7.0. The resulting mixture is divided into four parts and each portion is placed as a 7.5-pl drop on an ethanol-washed plastic (or a silica-treated glass) coverslip. The coverslips should be of such a size that they can be inverted and placed over the mouths of wells of a tissue culture dish into which have previously been placed about 1 ml of 1.1, 1.4, 1.7, and 2.0 M phosphate (also pH 7.0) as shown in Fig. 1. The gap between coverslips and the wells is sealed with grease so that the humidity inside the well is dominated by the relatively large reservoir solution at the bottom. The drops will shrink until the phosphate concentration is the same as that in the reservoir. In these experiments the concentrations of the protein and the phosphate gradually increase and crystallization may occur in one or all of the drops. It is not a good idea to use this method to concentrate solutions by more than a factor of approximately 2 as the interface between the coverslip and the solution does not decrease in size and the protein solution may become spread too thinly. If the protein does not crystallize or precipitate at 2 M phosphate, a new experiment can be set up in which the initial concentration of phosphate in the drop is set at, for example, 1.5 M, and the drop is concentrated up to 3.0 M phosphate. Using this approach, it is possible to quickly map out the solubility properties of the protein. This type of experiment should be repeated over a range

654

a

'

u C

[481

CHARACTERIZATION OF PURIFIED PROTEINS

Protein Precipitant (8 buffer)

mix, divide

0 0

b

side view

varying p H

.A. d Invetted coverslip

A'

.

clay to support lid of tray

FIG. 1. Vapor diffusion experiment using the hanging drop technique. (a) Protein and precipitant are mixed in an Eppendorf tube. (b) Equally sized drops are placed on glass or plastic cover slips. If a glass slip is used, it should be treated with a silica solution so as to prevent the drop from spreading. (c) The cover slip is inverted over a well which contains 1 ml of precipitant. (d) The gap between cover slip and well is sealed with grease.

of pH values, as solubility and crystallizability could vary with pH. The pH range to be scanned will depend on the nature of the precipitant and the pH stability of the protein. In the above example, the phosphate acts as both precipitant and buffer and can be replaced in both respects with concentrated citrate. Buffered solutions of (NH4)2S04, NaCI, Li2S04,and other salts can also be used in this type of experiment to bring about crystallization. Frequently a protein will crystallize from a variety of salts, but

1481

PROTEIN CRYSTALLIZATION

655 Lid

Sandwich box

Frc. 2. Vapor diffusion using a "sitting drop." This method is useful for crystallizations involving large volumes of protein solution and solutions that are surface active.

others, such as the Klenow fragment,28show a preference for a particular salt. Although the mechanism of action of organic solvents in crystallization is different from salt solutions, organic solvents can be used with great effect in vapor diffusion experiments. However, organic solvents can reduce the surface tension of water to the point where the hanging drop method is impossible, and in these cases the "sandwich box" arrangement shown in Fig. 2 can be used. PEG is another reagent that works well with vapor diffusion-type experiments. It has been found that most proteins precipitate at between 10 and 20% (wlv) PEG 8000. It is important to note that this molar concentration of PEG in the reservoir is generally lower than that of the buffer typically used to fix the pH of the protein solution. Since vapor pressure is a colligative property of the solution, water will accumulate in the protein drop unless the vapor pressure of the reservoir solution is lowered. This is usually achieved by the addition of buffer or NaCI. For example, if the protein solution contains 10% PEG and 0.1 M buffer, it can be concentrated to 20% PEG by adding 20% PEG and 0.2 M buffer to the reservoir. Proteins can frequently be induced to crystallize at or close to the isoelectric point by lowering the ionic strength of the solution to where the protein becomes insoluble. This approach in generally more successful with relatively insoluble proteins that require a high ionic strength environment (e.g., 0.5 M NaC1) to achieve the protein concentrations necessary for crystallization experiments. Unless the salt in the protein solution is P. Brick, D. Ollis, and T. A. Steitz, J. Mol. Biol. 166,453 (1983).

656

b

I

-

CHARACTERIZATION OF PURIFIED PROTEINS Protein Buffer Salt-may be necessary to keep protein soluble.

[481

side view

dialyze against a solution of lower ionic strength

Well is sealed with grease and cover slip

Tissue culture well with 1 ml low ionic strength buffer

FIG.3. Low ionic strength crystallization is usually used with proteins that are insoluble at low ionic strength but whose solubility is significantly increased by high salt concentrations (say 1 M NaCI). Crystallization is brought about by slowly lowering the salt concentration. A rapid decrease in ionic strength could result in the protein forming an amorphous precipitate. As such, it is desirable to do a number of experiments in which the salt concentration is lowered to different values (e.g., 0.5 M NaCI, 0. I M NaCI, and 0.01 M NaCI). The Eppendorf cap (a) has a hole in its base for the protein solution (d). Salts will diffuse freely across the dialysis membrane (b) which is held tightly against the cap by an 0 ring (c).

volatile (e.g., ammonium acetate), the hanging drop method would result in an increase in drop size due to the lower ionic strength of the reservoir. This may be feasible, but a better method is to use microdialysis methods. Numerous methods for doing microdialysis experiments have been de~ the arrangement used by the authors is scribed in the l i t e r a t ~ r e *and shown in Fig. 3. The protein should be dialyzed against a number of lower salt concentrations and over a small range of pH around the isoelectric point of the protein. An initial scan of a wide range of conditions as described above may not yield crystals. The experimentor could try adding one or a number of reagents to the crystallizing solution. As described in the previous section, metals, cofactors, etc., can often induce crystallization. 29

M. Zeppenzauer, H. Ekland, and E. Zeppenzauer, Arch. Biochem. Biophys. 126, 564 (1968).

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Growth of Large Crystals. Having determined the conditions under which a protein will crystallize, the problem of growing large crystals can be tackled. There are three general methods for doing this: the first is fine tuning the conditions, the second involves increasing the scale of the experiment, and the final method is seeding. Fine tuning simply involves a closer investigation of conditions in the region where the initial crystals were obtained. Early experiments, of necessity, represent a rather crude sampling of parameters, and the initial crystals are usually not obtained at the optimal conditions. All parameters should be tested more rigorously, but the most important are pH, temperature, final precipitant concentration, and, where applicable, the concentrations of metal ion, substrate, cofactor, and inhibitors. It should also be noted that the extent to which the protein concentrates during the experiment often affects the number and size of the crystals produced. This can be controlled by adjusting the initial concentration of the precipitant. If fine tuning yields large, well-diffracting crystals, the crystallization project can stop. Often, a simple increase in the scale of the vapor diffusion experiment is sufficient to prociuce large crystals. This may require the "sitting drop" method to accommodate the larger volumes, and the arrangement shown in Fig. 2 is ideal. If vapor diffusion experiments continue to yield a multitude of very small crystals, dialysis experiments should be tried since these can be arranged to produce a very slow rate of change of parameters. For example, suppose a protein in 1 M phosphate can be crystallized when the phosphate concentration is increased to 2 M. The protein solution in a dialysis bag can be placed at the top of a 100-ml measuring cylinder into which has first been placed 50 ml of 3 M phosphate with another 50 ml of 1Mphosphate carefully layered on top. The dialysis bag should see a very slow increase in the concentration of phosphate as the two layers of phosphate mix. If neither of the above methods is successful, then seeding should be considered. Seeding is generally used when nucleation is critically dependent on the precipitant level and it is difficult to generate just a few nuclei that will grow into large crystals. Seeds represent an attempt to bypass the nucleation step. The general procedure involves bringing the protein solution close to saturation, adding seed crystals, and then adjusting conditions so that the seeds grow without the production of new nucleation centers. The seeds can vary in size from a few microns (microseeding) to crystals easily visible with the naked eye (macroseeding). Small seeds can be produced by simply crushing full-grown crystals with a glass fiber. These seed solutions should be diluted with stabilizing buffer before being added to the protein solution. Ideally, only a few seeds should be transferred and the concentration of microseeds can be determined by using a

658

CHARACTERIZATION OF PURIFIED PROTEINS

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microscope to simply count the number in a small drop of known volume. In macroseeding the object is to allow the crystal growth process to continue by addition of fresh protein. Crystals should be briefly washed in a solution containing a low precipitant level to remove the outer layer and expose fresh surfaces, and then placed in a saturated or supersaturated It should be clear that macroseeding can be repeated protein ~olution.~' several times and could potentially give very large crystals. Crystallization of Complexes

General Considerations. Recently, there has been much success in producing good quality crystals of protein-protein, protein-DNA, and protein-RNA complexes. The resulting structural results have shown how several important macromolecules interact at the molecular level. In general, the methods described above are directly applicable to the crystallization of complexes. However, since only relatively few complexes have been crystallized, it is difficult to make any general conclusions concerning the types of conditions that might be tested. The reader is encouraged to consult some original articles before starting experim e n t ~ . ~There ' , ~ ~are two considerations, however, that have to be noted. First, crystallization conditions that will disrupt the complex should, if possible, be avoided. Second, it is important to know the exact stoichiometry of the individual components in the sample. In several cases, this has been shown to be an important parameter for producing highquality crystals. Protein-DNA Cocrystals. Several years ago, the structures of a number of DNA binding proteins were determined to high resolution. In the absence of bound DNA, it was necessary to resort to model building to investigate the nature of the interaction. This prompted several groups to initiate extensive crystallization trials on protein-DNA complexes. These efforts have recently been rewarded by several high-resolution structures. For our purposes this work has provided an extensive body of information on how to produce such cocrystals. In many crystals of DNA fragments alone, the short helices are stacked end to end to form pseudocontinuous helices.33In protein-DNA cocrystals, it was considered likely that the DNA would be similarly arranged and C. Thaller, L. H. Weaver, G. Eichele, E. Wilson, R. Karlsson, and J. N. Jansonius, J. Mol. Biol. 147,465(1981). " R. A. Maruizza, D. L. Jankovic, G. Boulot, A. G. Amit, A. Saludjian, A. LeGuern, J. C. MaziC, and R. J . Poljak, J . Mol. Biol. 170, 1055 (1983). D. Moras, M. B. Comarmond, J . Fischer, R. Weiss, J . C. Thierry, J . P. Ebel, and R. Giege, Nature (London) 288,669 (1980). 33 A. J.-H. Wang, G. J. Quigley, F. J. Kolpak, J. L. Crawford, J . H . van Boom, G. van der Marel, and A. Rich, Nature (London) 282,680 (1979). 'O

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that the bound protein would be attached to this framework. The diffraction patterns from subsequently obtained cocrystals supported this idea34-37 and the eventual structures confirmed it.38,39Not surprisingly, therefore, the length of the DNA fragment and the nature of the ends (i.e., blunt or overlapping) are extremely important parameters in crystallization trials. Early successes did suggest some trends in these parameters (see, for example, Ref. 34), but it is now clear4' that this is not the case and that different DNA fragments have to be tested. Other requirements of the DNA fragment are more clear. Obviously, the fragment should contain the sequence that is recognized by the protein and the amount of flanking DNA should be minimal to avoid unbound floppy ends. Also, the sequence may require some modification to prevent self-annealing of individual strands36and the formation of hairpin loops . ~ ~ the fragment should be highly purified. and cruciform s t r ~ c t u r e sAlso, Most experiments have employed vapor diffusion using the hanging drop technique. Success has been achieved with a whole variety of precipitants, including different PEGS, MPD, ammonium sulfate, and sodium chloride. It has even been shown to be difficult to extrapolate the results from one DNA fragment to a n ~ t h e r . Finally, ~' as regards the stoichiometry, it appears that an excess of DNA is preferable and the authors suggest a 2: 1 ratio of DNA duplex: bound protein species for initial trials. Conclusion

The theory of protein crystallization is poorly understood and techniques for monitoring the process are in their infancy. The physical properties of proteins cover a very broad spectrum and it is difficult to make general statements about crystallization. The biochemist is left with a systematic trial of a wide variety of parameters in order to determine the correct conditions. Most proteins will crystallize under some conditions although, initially, the crystals are rarely suitable for diffraction studies. The difficult part of the investigation is to find conditions for the growth of large crystals.

" J. Anderson, M. Ptashne, and S. C. Hanison, Proc. Natl. Acad. Sci. U.S.A. 81, 1307 (1984). R. Jordan, T. V. Whitcombe, J. M. Berg, and C. 0. Pabo, Science 230, 1383 (1985). 6' R. G . Brennan, Y. Takeda, J. Kim, W. F . Anderson, and B. W. Matthews, J. Mol. Biol. 188, 115 (1986). " J. Grable, C. A. Frederick, C. Samudzi, L. Jen-Jacobson, D. Lesser, P. Greene, H. W. Boyer, K. Itakura, and J. M. Rosenberg, J. Biomol. S~ruct.Dyn. 1, 1149 (1984). * D. L. Ollis and S. W. White, Chem. Rev.87,981 (1987). 39Z. Otwinowski, R. W. Schevitz, R.-G. Zhang, C. L. Lawson, A. Joachimiak, R. Q. Marmorstein, B. F. Luisi, and P. B. Sigler, Nature (London)335, 321 (1988). "A. Joachimiak, R. N. Marmorstein. R. W. Schevitz, W. Mandecki, J. L. Fox, and P. B. Sigler, J. Biol. Chem. 262,4917 (1987). " S.

Section XI Immunological Procedures

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PREPARATION OF POLYCLONAL ANTIBODIES

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[491 Preparation of P o l y c l o n a l A n t i b o d i e s By BONNIES. DUNBAR and ERICD. SCHWOEBEL

The term "polyclonal antibody" is defined as the total population of antibodies present in animal serum. This complex population contains different antibody subclasses including IgG, IgM, IgE, IgA, and IgD. Each antibody represents the secretory product from a single stimulated lymphocyte and its clonal progeny. A complex antigen such as a protein, glycolipid, etc., may contain many distinct antigenic determinants or epitopes, each of which is specifically recognized by antibodies from a single lymphocyte clone. These epitopes may be composed of amino acid sequences, conformational determinants, or molecular structures derived from posttranslational modification (e.g., glycosylation, phosphorylation, or acetylation). The term "immunogen" refers to any molecule that is capable of eliciting an immune response. Immunogenicity therefore refers to the level of immune response elicited. It is advised that basic texts on introductory immunology'-2be consulted prior to initiating studies utilizing antibodies. It is important to first decide whether the investigators' studies are amenable to the use of polyclonal antibodies. It is often desirable to develop banks of both monoclonal and polyclonal antisera such that the advantages of each can be exploited. The major advantages and disadvantages of polyclonal antisera are outlined in Table I. A complementary table describing monoclonal antibodies is given elsewhere in this text.3 Antigen Purification and Preparation

It is first necessary to obtain sufficient quantities of the highly purified immunogen that will be used to immunize the animal. In a protein preparation that contains a s little as 1% contamination, the majority of antibodies may recognize that contaminant if it is highly immunogenic. Both the level of immune response (immunogenicity) and the type of epitopes recognized (conformational vs sequential) can be modulated by the method chosen for immunogen purification. Immunogens purified by nondenaturing bio-

' B. Benacerraf and E. R .

Unanue, "Textbook of Immunology." Williams & Wilkins, Baltimore, Maryland, 1979. W. R. Clark, "The Experimental Foundation of Modem Immunology." Wiley, New York, 1986. B. S. Dunbar and S. M. Skinner, this volume 1501.

METHODS IN ENZYMOLOGY, VOL. 182

Copyright B 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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TABLE I AND DISADVANTAGES OF POLYCLONAL ANTIBODIES ADVANTAGES Major advantages Multiple subclass and high-affinity antibodies are present in antibody population Multiple specificities of antibodies are likely to recognize sequential as well as conformational antigenic determinants Antibodies should recognize multiple determinants specific for a protein (important if screening gene expression libraries!) Highly specific polyclonal antisera may easily be developed by immunization with proteins purified in a single step by 2D-PAGE Amenable to experiments involving immunoprecipitation Major disadvantages Immunogen must be highly purified to obtain desired specificity Individual domains of complex antigens are difficult to study because multiple antigenic determinants are recognized by the polyclonal antisera Quantities of antibodies are limited to the life of the immunized animal Different bleedings have to be characterized individually due to changes in antibody affinity, specificity, and subclass

chemical methods tend to elicit a strong response toward the conformational epitopes present in the molecule. Most of these methods do not, however, purify proteins to homogeneity. Procedures which denature the immunogen tend to yield antisera reactive toward sequential determinants. In many instances this is desirable since antibodies made against denatured proteins are usually best for immunoblotting of SDS-PAGE separated proteins. The use of two-dimensional gel electrophoresis has revolutionized the ability to easily isolate denatured proteins or peptides which are sufficiently purified to generate specific polyclonal a n t i b ~ d i e s . ~ , ~ However, even though acrylamide may act as a "carrier" and help to enhance the immune response in some instances, denatured proteins are generally less immunogenic than are native proteins. It may therefore be necessary to use greater concentrations of denatured protein than the native protein or to enhance the immunogenicity of these proteins. Exceptions include the highly conserved proteins actin and tubulin, which are not immunogenic unless d e n a t ~ r e d . ~ Two other methods are also commonly used to increase the immune response to an antigen. The first method is to combine the immunogen with B. S. Dunbar, H. Kinura, and T. M. Timmons, this volume [34]. B. S. Dunbar, "Two-Dimensional Electrophoresis and Immunological Techniques." Plenum, New York, 1987. S. L. Brenner, R. L. Pardue, L. Wible, L. Reading, and B. R. Brinkley, in "Laboratory Methods Manual for Hormone Action and Molecular Endocrinology" (W. T. Schrader and B. W. O'Malley, eds.), p. 12-1. Houston Biol. Assoc., Houston, Texas, 1987.

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an adjuvant, a broad term encompassing several substances that enhance the overall immune response. Some examples of adjuvants include alum, dextran sulfate, and large polymeric anion^.^ Two of the most effective .~ adjuvants are the water and oil emulsions developed by F r e ~ n dImmunogen is slowly released from the oil, provoking local inflammation and prolonging the immune response. The addition of bacterial antigens (Freund's complete adjuvant) further stimulates lymphocyte infiltration and response. A second method to enhance immune response is to modify the immunogen itself. The immunogen can simply be polymerized, or it may be conjugated to other carrier proteins or synthetic antigens. These modifications are particularly useful for highly conserved proteins or unusually small immunogens (e.g., steroids, prostaglandins, or cyclic nucleotide^).^.'' In addition to these methods, the immune response is also enhanced by choosing a species that is phylogenetically removed from the species that is the source of antigen. Other factors affecting choice of animal species are outlined in Table 11. Antibody Preparation: Polyclonal Antibodies

Because rabbits are the most commonly used animal for the production of polyclonal antibodies, this procedure is given in detail (similar methods are used for other animals). (Note: Before initiating animal experiments, it will be necessary to have protocols approved by the animal welfare committee of the institution.) Since the quality of reagents used is critical for reproducible results, we have listed commercial sources whose reagents are acceptable for these procedures. There are many other sources for most of these reagents, but they should be tested for quality to ensure good results. Immunization. A highly purified antigen (optimal total dosage 20-200 pg) is emulsified using two 3-ml Luer-Lock syringes with 18-gaugeneedles connected by Tygon microbore tubing(0.3 X 0.09, Fisher, Pittsburgh, PA, catalog #14-170-15F). The first syringe contains 0.75 ml water or buffer and antigen (solubilized or minced gel pieces), and the second contains an equal volume of Freund's complete adjuvant (FCA) (see photographs of equipment set up in Ref. 5). Be sure to thoroughly vortex the FCA to resuspend the bacteria settled in the bottom of the vial. The immunogen

' H. S. Warren, F. R. Vogel, and L. A. Chedid, Annu. Rev.Immunol. 4,369 (1986).

* J . Freund, K. I. Thompson, H. B. Hough, H. E. Sommer, and T. M. Pisani, J . Immunol. lo

60,383 (1948). B. F. Erlanger, this series, Vol. 70, p. 85. M. Reichlin, this series, Vol. 70, p. 159.

666

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TABLE I1 ANIMALSELECTION FOR ANTIBODY PRODUCTION Advantages

Disadvantages

Rabbits

Easy to maintain Yield large amounts of sera (about 20 mllweek from a large rabbit) Multiple subclasses of IgG bind protein A

Guinea pigs

Easy to maintain Multiple subclasses of IgG bind protein A Generally makes high-titer antibodies

Mice and rats

Easy to maintain

Sheep and goats

Large quantities of antisera can be obtained Immunogen can be shipped offsite for commercial immunization

Quantities of antisera may be limited if very large amounts are required Many rabbits are infected with pasteurellosis, which affects immune response and limits life of animal (pathogen-free animals are available) Only small amounts of antisera can be obtained Blood usually is collected by cardiac puncture, risking loss of animal; can be collected from hind leg with vacuum apparatus Small amounts of antisera obtained Antibody does not bind well to protein A, necessitating use of second antibody for detection Special facilities necessary for housing unless commercial producer employed Only one subclass of IgG binds to protein A, necessitating use of second antibody for detection

Species

and adjuvant mixture is emulsified by passing the mixture back and forth between the syringes until the sample is thick. (Note: Some immunogenadjuvant mixtures will be extremely viscous, while others will not be as thick.) Be sure that the tubing is fitted tightly over the needles and that Luer-Lock syringes are used, or the antigen may be lost if the tubing comes off from the pressure! The emulsified immunogen mixture is injected intradermally (not subcutaneously) into the layers of the skin of a shaved rabbit using a 22-gauge needle. The needle is quickly inserted just under the surface of the skin and enough emulsion is added to form small raised "pockets" in the skin. A total volume of approximately 0.5-1.0 ml should be injected into 10-12 sites along the upper sides (5-6 sitestside) of the rabbit. Be sure that the immunization sites are not too large or too close together to cause serious lesions. The remainder of the emulsion (0.5 ml) is also given subscapularly at this time. Because intradermal injections can be painful, it is preferable to use a light anesthetic prior to immunization

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PREPARATION OF POLYCLONAL ANTIBODIES

(i.e., xylazine at a dosage of 5-10 mglkg). A general schedule for boost immunization is given in Table 111. Bleeding and Processing Serum. We have found that the best rabbit restrainer is one that does not trap the rabbit's neck since rabbits can easily be injured in cages where only the head is exposed. Pull the rabbit's ear through the bars and shave the hair along the outside margin of the ear with a single-edge razor blade. The ear should be rinsed with ethanol and dried. Apply a very small amount of xylene with a cotton swab on the center artery. Never apply on marginal vein. If too much xylene is used, the bleeding will be difficult to stop. Generously apply petroleum jelly on the marginal edge of the ear and on the region just under the ear. Do not apply where the cut is to be made. Once the ear is prepared, arazor blade is used to cut the vein using a firm, flat stroke perpendicular to the vein. Be careful not to cut through the ear! For a few seconds the animal may not bleed; then a steady dripping of blood should start. Blood should be collected in glass tubes because clotting will be more efficient and more serum can be obtained with minimal hemolysis. The animal should stop bleeding immediately or very shortly after the ear is released. Wash ear thoroughly to remove xylene. As soon as the blood clots in the tube, rim the edges of the clot with a wooden applicator or glass pipet. (Note: This is a critical step for obtaining the maximum amount of serum from blood.) Allow the blood

TABLE 111 GENERAL SCHEDULE FOR IMMUNIZATION OF RABBITS FOR PREPARATION OF POLYCLONAL ANTIBODIES -

Day -

-

-

Immunogen dose total

---

Carrier

Site of injection

Freund's complete adjuvant: immunogen ratio 0.5 m1:O.S ml PBS + immunogen Freund's incomplete adjuvant; 0.5 ml: 0.5 ml PBS + immunogen

Multiple sites: intradermal (> 10); 1 site suhscapular

Freund's incomplcte adjuvant

Two to four sites subcutaneous

-

l (preimmune bleeding)

20-200 pg

Two to four sites subcutaneous

31 (test bleed) I f antibody detected:

40

10-100 pg

I f no or very Ion' anfibody titer defected: Freund's complete 10-100 pg adjuvant

Four to six sites intradermal Two sites subscapular

TABLE IV A N D DISADVANTAGES OF ANTIBODY DETECTION METHODS ADVANTAGES Technique

Advantages

"Western" blottinga,

High sensitivity Determines directly which proteins are recognized by the antisera Antibody detection reagents commercially available

ImmunoelectrophoresisC

Yields detailed analysis of antibody specificity Complex antisera and antigen mixtures can be analyzed Usually performed under nondenaturing conditions High sensitivity Detects antibodies to either conformational or sequential determinants Assay can be used to quantify either antibody or antigen May be possible to use crude antigen Large numbers of assays can be easily processed Reagents commercially available High sensitivity Large numbers of assays can be easily processed Detects antibodies to either conformational or sequential determinants Reagents commercially available Detects immunological similarities between antigens Usually performed under nondenaturing conditions Simple to perform and inexpensive

Dot-blot assays

Ouchterlony double diffusione

Disadvantages Generally detects only sequential determinants, thus not definitive proof of specificity Binding properties of different proteins to different papers must be optimized Large quantities of antibody and antigen usually necessary Specialized equipment required May require large quantities of antigen to coat microtiter plates Large number of assays requires specialized equipment Conditions for antigen adherance to microtiter plates must be optimized Yields little or no information on specificity Yields little or no information on specificity Binding properties of different proteins to different papers must be optimized

Salts or detergents can result in false positives Large amounts of antibody and antigen are usually required Not accurate for determining specificity of antibody Antigen-antibody ratio critical for immunoprecipitation; therefore, negative results are meaningless

-

" H. Towbin, T. Staehelin, and J. Gordon, Proc. Natl. Acad. Sci. U.S.A.76,4350 (1979). N . L. Anderson, S. L. Nance, T. W. Pearson, and N. G. Anderson, Electrophoresis 3, 135 (1982). ' B. Weeke, Scand. J. Immunol., Suppl. No. 1, p. 15 (1973). E. Engvall and P. Perlman, Immunochemistry 8,871 (1971). ' 0. Ouchterlony, in "Progress and Allergy" (P. Kallos, ed.), Vol. V, p. 1. Karger, Basel, 1958.

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to clot for 2-4 hr at room temperature, then decant serum and centrifuge at 1000 g for 10 min to remove blood cells. Some additional serum can be but there is usually more obtained by letting the blood clot overnight at 4, hemolysis in this sample. (For photographic details of this procedure, refer to Ref. 5) Fractionation of Zg from Serum. It is often desirable to partially purify antibody from antiserum prior to use. Two commonly used methods are ammonium sulfate fractionation and DEAE chromatography. Ammonium sulfate fractionation is a simple but crude method for antibody partial purification. Slowly add 40 ml saturated ammonium sulfate [77.7% (NH4)*S04,pH 7.2-7.41 to 60 ml of serum while stirring. Continue to stir for 1 hr. Centrifuge at 5000 g for 20-30 min at 22". Resuspend pellet in minimal volume of distilled HzO. Dialyze sample extensively to remove ammonium sulfate. DEAE-Sephacel ion-exchange chromatography yields IgG purified from other immunoglobulin subclasses and most serum proteins. For chromatography dialyze the above sample extensively against phosphate equilibration buffer (made by the addition of 0.02 M NaH2P04.H20to 0.02 M Na2HP04until pH 7.4). Pour the resin as a slurry into the column (1.6 x 20 cm) and let settle. The top of the bed should be at least 2 cm from the top of the column. Equilibrate the bed with three column volumes of equilibration buffer. Apply the sample [approximately 100 mg of (NH4)$304 fraction in 5-20 ml equilibration buffer] to the column and begin collecting 8-ml fractions. The first IgG peak should come off in tubes 8-10. Continue to rinse the column until absorbance returns to background. Switch buffer to 0.02 M phosphate with 0.05 M NaC1. The second IgG peak should come off in another 8 to 10 fractions. IgM is removed with 0.1 M NaCl in TABLE V SUMMARY OF COMMON METHODSUSEDTO CHARACTERIZE ANTIBODIES Antibody characteristics Specificity Quantitation Subclass determination

Cytochemical localization

Methods Western blotting Immunoelectrophoresis Enzyme-linked immunoassay Dot-blot assays Immunoelectrophoresis Enzyme-linked immunoassay Ouchterlony double diffusion Immunocytochemistry Organelle fractionation

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phosphate buffer, and other serum proteins in 0.5-1.0 M NaC1. Regenerate the column by washing with three bed volumes of regeneration buffer (0.2 M phosphate buffer, pH 7.4, made as described for equilibration buffer) and then with equilibration buffer. Fractions can be tested for the presence of IgG by SDS-PAGE. In addition, new products have been designed to isolate IgG by affinity chromatography using Protein A attached to cellulose disks. These may prove to be useful for some applications. It is critical to properly characterize antiserum prior to use. Antibody titer, specificity, subclass, and affinity can greatly affect subsequent experimental protocols. Common methods for antibody detection and characterization are outlined in Tables IV and V and discussed in detail else-

here.^'^"' "

T. M. Timmons and B. S . Dunbar, this volume [51].

[SO] Preparation of Monoclonal Antibodies By BONNIES. DUNBAR and SHERIM. SKINNER

The report by Kohler and ~ i l s t e i n that ' a cell hybrid made by fusing normal spleen cells with malignantly transformed antibody-secreting cells (plasmacytoma or myeloma cell line) can provide a continuous source of antibody of predefined specificity has led to the explosion of the use of "monoclonal antibodies." Because this technology has been so widely used, several excellent texts and reviews have been written which describe this technology in great detai1.2-5The properties of these antibodies as well as the advantages and disadvantages of their use as compared to polyclonal antibodies are summarized in Table I. These guidelines should assist in the choice of determining which type of antibodies are optimal for use in designated experiments. I

G. Kohler and C. Milstein, Nature (London) 256,495 (1975).

* R. H. Kennett, T. J. McKearn, and K. B. Bechtol, "Monoclonal Antibodies."

'

Plenum, New York, 1980. G. Kohler, "Hybridoma Techniques." Cold Spring Harbor Lab., Cold Spring Harbor, New York, 1980. J. W. Goding, "Monoclonal Antibodies: Principles and Practice," 2nd Ed. Academic Press, New York, 1986. H. Zola and D. Brooks, in "Monoclonal Hybridoma Antibodies: Techniques and Applications" (J. Hurrell, ed.), p. I . CRC Press, Boca Raton, Florida, 1981.

METHODS IN ENZYMOLOGY, VOL. 182

Copyright O 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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TABLE I ADVANTAGES AND DISADVANTAGES OF MONOCLONAL ANTIBODIES Major advantages Single homogeneous antibody to a defined antigenic determinant Specific antibody can be used to study functional domain of molecule Large quantities of antibody can be obtained since immortal cell lines can theoretically be developed Antibodies with low-affinity binding can be selected during screening procedures (these antibodies are designed for immunoaffinity chromatography) Major disadvantages Procedure is expensive and time consuming Well-equipped tissue culture facilities are needed Epitope recognized by antibody may be shared among many different antigens not related to antigen of interest Hybridoma cell lines are frequently unstable due to chromosome loss or may be lost because of tissue culture contamination

Antibody Screening Assays

Prior to the generation of monoclonal antibodies it is essential to first establish a sensitive and rapid antibody-screening assay. Because thousands of cell culture samples will usually have to be screened, adequate methods should be developed prior to setting up cell cultures. A variety of methods can be used to determine the presence, as well as the specificity, of an antibody. The method of choice will vary depending on the nature of the antibody to be detected (e.g., polyclonal vs monoclonal) or the nature of antigenic determinants to be recognized by that antibody (sequential, conformational, protein, carbohydrate, cell surface, glycolipid, etc.). These procedures which include immunocytochemical localization methods as well as biochemical methods are summarized in detail e l ~ e w h e r e . ~ - ~ Only the most commonly used assays for screening large numbers of hybndoma cell lines are described here. These include enzyme-linked immunoassays and dot-blot immunoassays. (A variety of commercial kits are now available and methods are established for labeling antibodies for routine assays.) Equipment (e.g., the miniblotter manufactured by Immunetics, Cambridge, MA) has also been developed for the large-scale screening of antibodies by one-dimensional Western blots using monoclonal antibodies. Since the quality of reagents used is critical for reproducible results, we have listed commercial sources whose reagents are acceptable for these procedures. There are many other sources for most of these reagents, but they should be tested for quality to ensure good results. B. S. Dunbar, "Two-Dimensional Electrophoresis and Immunological Techniques." Plenum, New York, 1987.

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Method for Enzyme-Linked Immunoassay (ELZSA)for Screening Monoclonal Antibodies

There are currently large numbers of ELISA procedures for the detection of antigens and antibodies. Of the ELISA methods we have used, the avidin-biotinylated peroxidase complex (ABC) system has been the most successful primarily due to its ~ensitivity.'~~ The reagents and antibodyscreening kits are available from a variety of sources, although we routinely use Vector Laboratories (Burlingame, CA). Antigen Binding to Microtiter Plates. To determine if a microtiter plate will optimally bind the antigen or cells of interest, it may be necessary to test a variety of plastics manufactured for this purpose. The capabilities of the spectrophotometric plate reader must also be considered in choosing a well shape. We routinely use a round-bottomed microtiter plate (Dynatech Immulon #2). It may require significant amounts of antigen to adequately coat the plate, although it may be possible to use a crude antigen preparation. It will be necessary to experiment with a variety of buffers, pH values, and antigen concentrations to find the optimal combination for maximum binding since different antigenic molecules will have distinct binding characteristics. Depending on the purity and nature of the antigen, one may need concentrations from 0.1 to 10 pglml. A basic buffer (pH -9.6) works for most antigens, but this must also be experimentally determined. Some antigens, once bound to plastic wells, are stable enough to allow freezing of the plate containing the antigen-coating solution, or at a later step when the plate contains blocking solution (described below) although since some molecules may lose their antigenicity, this will have to be tested. All incubations should be carried out using a vibrating plate mixer for consistent results and intense color development. We use a microplate mixer (Fisher Scientific, Pittsburgh, PA), which may be placed in a cold room or an oven whenever necessary. Plates should be thoroughly washed between reagent treatments. This can be carried out inexpensively with no special equipment or, if available, a microtiter plate washing apparatus is preferable. To wash, pour solution over the face of the plate until the wells are filled. Shake the plate vigorously on the mixer and pour or pipet out wash solution. (Be careful not to mix samples from one well with another.) Three more such passes result in a very thorough washing, minimizing or eliminating background caused by nonspecific binding of reagents.

' A. Voller, D. Bidwell, and A. Bartlett, in "Manual of Clinical Immunology" (N. R. Rose and H. Friedman, eds.), p. 506. Am. Soc. Microbiol., Washington, D.C., 1976. D. W. Drell, D. M. Wood, D. S. Bundman, and B. S . Dunbar, Biol. Reprod. 30,435 (1984).

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PREPARATION OF MONOCLONAL ANTIBODIES

673

Antibody Detection. Antigen is diluted to a previously established concentration (we use 1 pglml) in antigen-coating buffer (0.1 M Na2C03/ NaHCO, , pH 9.6, for example), and 50 p1 is added to each well. (C-ontrol wells include no antigen, no primary antibody, no secondary antibody.) Plates are incubated for 6 hr at room temperature or overnight at 4"on the microplate mixer. After coating, plates are thoroughly washed using assay buffer (20 rnM PBS, pH 7.3, containing 0.15 M NaCl and 0.05%Tween 20). Blocking of nonspecific binding sites is accomplished using blocking buffer [20 rnM phosphate-buffered saline (PBS), pH 8.0, containing 0. I5 M NaCl and 2% nonfat dried milk] at 100 pllwell, and incubated overnight at 4" with shaking. Plates are again washed thoroughly and the primary antibody is applied. Dilutions of control or sample sera or other antibody-containing solutions are made using diluent (2% nonfat dried milk in assay buffer, pH 7.3). Plates are then incubated overnight at 4". Thorough washing follows and the biotinylated second antibody is applied for 1 hr at room temperature. The dilution, made with the diluent solution described above, must be experimentally determined. For our work, a 1:200 dilution has been sufficient. After thorough washing, the plates are incubated for 30 min at room temperature with a freshly made mixture of 0.3% H202 in methanol (100 pllwell) to remove any endogenous peroxidase activity that might otherwise result in background color with subsequent reagents. After washing as before, the plates are treated with the premixed avidinbiotinylated horseradish peroxidase complex (Vector Laboratories, Burlingame, CA, Vectastin kit) (50 pllwell) for 1 hr at room temperature with shaking. After washing, substrate solution is added (50 pl/well) and incubated for 30 min at room temperature with shaking. Our best results have been achieved with a solution of 0.8 mglml o-phenylenediamine dihydrochloride (OPD) (Bethesda Research Laboratories) in 0.1 M citratelphosphate buffer (pH 5.0) containing 0.3% H202.The OPD substrate can be made at 5x concentration and stored frozen in small aliquots for later use. The citratelphosphate buffer (without H202)can be made ahead of time and stored frozen (as can the diluent and the blocking solution), but Hz02 must be added just prior to solution use. After substrate incubation, the plates are read at 450 nm on a microtiter spectrophotometer. It is important to first scan an untreated plate to blank, and subsequently an uncoated well on the treated plate as the reagent blank. As the reaction cannot be stopped it is important to standardize the time of the substrate reaction for reading consistently between assays.

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Zmmunoblotting. Another type of assay uses immobilization of the antigen on a nitrocellulose membrane. The antigen(s) may be electrophoretically transferred to the membrane from a gel using methods outlined in the chapter on protein blotting in this text,9 allowing probing of the antigen in various states of denaturation, chemical alteration, or purification. Production of Monoclonal Antibodies

Once antibody-screening assays have been established, monoclonal antibody production may be undertaken. Hybridoma antibody-producing cells are made by fusing mouse myeloma cells with mouse lymphocytes. This results in a cell line proliferating indefinitely and secreting an immunologically homogeneous product. By screening the resulting hybridomas, one may select those producing useful antibodies. Zmmunization Procedure

The intradermal immunization procedures normally used for polyclonal antibody production in laboratory animals such as rabbits are difficult to use in mice. Therefore other methods have been developed such as subcutaneous injections with adjuvants, nitrocellulose implants (containing antigen), and intrasplenic injections (refer to reviews in Refs. 2-6 for more detail). Generally, subcutaneous immunizations using antigen emulsified in Freund's complete adjuvant are used as the primary injection. (See protocols for antigen preparation in [49] on polyclonal antibodies.) A variety of immunization strategies can also be used to help in obtaining the type of antibodies desired. For example, a longer immunization time should result in a better chance of obtaining IgG rather than IgM immunoglobulins. If limited antigen is available, an alternative immunization procedure can be followed. Fifty to one hundred micrograms antigen in 0.2 ml Complete Freund's adjuvant is injected into the hind foot pad of the mouse. After 10-12 days, the popliteal lymph node in the mouse hind leg will be swollen, and can easily be dissected from the surrounding fat pad. These cells are fused with myeloma cells using the procedure described for spleen cells (omitting the red blood cell lysis step)." Spleen Cell Preparation

The spleens are removed from immunized mice (24-72 hours after boost) using sterile conditions and are placed into Dulbecco's minimal

' T. Timmons and B. Dunbar, this volume [51]. 'OR. Conitti, G. Rocchetti, P. Gnocci, E. Monandi, and Y. M. Galante, J . Immunol. Methods 99,25 (1987).

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essential media (or other standard MEM) which has been supplemented with 2 rnM glutamine, 100 IUIml penicillin, and 100 pglml streptomycin. The connective tissue splenic capsule is removed and the tissue is minced and cells dispersed. The cell suspension is allowed to settle for 10 min on ice and the supernatant then centrifuged (1600 rpm for 6-7 min). The supernatant is discarded and red blood cells in the pellet are lysed by suspension of the pellet in 5 mllspleen of 0.83% NH4Cl solution. Incubation on ice for 10 rnin completes this step. Equal volumes of medium are then added and the cells washed twice. Viable cells are counted using dye exclusion and a suspension of approximately 100 million cellslspleen is made. Myeloma Cell Preparation

Numerous myeloma cell lines are now commercially available. The optimal cell line should be HGPRT negative, not produce or secrete its own immunoglobulin, and its growth should be stable (preferably in the absence of feeder cells). We have used p3U 1 cell lines with routine success." In carrying out a fusion, the cells are concentrated by centrifugation (1600 g for 6-7 min). It is important to concentrate one-tenth as many myeloma cells as spleen cells. The cells are resuspended in medium containing 15% fetal calf serum, and a viable cell count is made. Fusion Procedure

For cell fusion, the HAT selection method is routinely used.'' Briefly, HAT (hypoxanthine, amonopterin, thymidine) selection utilizes the functional complementation of two different (parental) metabolic defects to produce complete function and therefore survival in the hybrid cells. It is not necessary to select against the growth of unfused lymphocytes since they will not grow in tissue culture. One must select only against the unfused tumor cells. The use of HAT prevents them from growing unless "rescued" by fusion with the lymphocytes which supply the enzyme HGPRT (hypoxanthine-guanine phosphoribosyltransferase), a critical metabolic enzyme which is missing in the cell line. Cell Fusion Protocol. Myeloma and spleen cells are combined at a ratio of 1 :10 in a 50-ml conical centrifuge tube (- 10 ml). They are centrifuged at 1600 rpm for 6-7 min and the supernatant discarded. The cell pellet is gently dislodged and 2 ml of PEG 1000 (pH 8.0) is added. The pellet is resuspended and centrifuged (1600 rpm for 3 min). (Note: The time and efficiency of fusion may depend on the batch of PEG. You may have to try D. W. Yelton, B. A. Diamond, S.-P. Kwan, and M. D. Scharff, Curr. Top. Microbial. Zmrnunol. 81, 1 (1978). " M. L. Gefter, D. H. Margulies, and M. 0 . Scharff, Somat. Cell Genet. 3,231 (1977). "

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several lots for best results.) With PEG still in the sample, 5 ml of serumfree medium is slowly added and the pellet gently resuspended. Centrifugation is then carried out at 1000 rpm for 6 min. The supernatant is discarded and 10-20 ml of serum-containing growth medium is added. The fused cells are resuspended, pipetted into a Petri dish, and incubated for 1-3 hr (5% C 0 2 , 37"). The fused cells are pipetted back into a conical centrifuge tube and centrifuged at 1000 rpm for 10 min. Sufficient HAT medium is added to dilute the cell suspension to lo6 cells/ml (e.g., approximately 100 ml/ spleen). HAT medium stock can be made using 136 pg/ml hypoxanthine, 0.9% pg/ml aminopterin, and 3.88 pg/ml thymidine in the Dulbecco's medium described above. This solution may then be stored in the dark in frozen aliquots. When needed, 1 ml of this stock may then be diluted to 100 ml in the above-mentioned Dulbecco's medium. Cells (2 ml/well) are pipetted into cell culture trays (usually Costar or Linbro 24-well trays are optiomal for initial cloning) and placed in the incubator. The incubator is monitored for 2 weeks at which time one can begin screening the media for secretion of antibodies (one should be able to visualize hybridoma colonies by days 10-14). After 7-10 days, wells can be examined for hybrids and medium can be replaced by HT medium. (HT medium is simply HAT medium without aminopterin.) Subcloning Hybridomas

Hybridomas can be s ~ b c l o n e d ' ,by ~ microscopically selecting and pipetting out individual colonies. Alternatively, they can be subcloned using limiting dilution in which cells are diluted such that, statistically, there should only be one viable cell per well. For example, following subcloning, if more than 37% of wells have no growth, there is a reasonable probability that wells with growth will contain single clone^.^ Multiple subclonings should be carried out to better guarantee the monoclonality of a cell line. At each stage of the subcloning procedure, some cells should be frozen and stored to ensure that a cell line will not be lost. Freezing Hybridoma Cells

We have used the procedure previously described5 to freeze cells. Hybridoma cells are washed once in culture medium and suspended at a concentration of 6 x 106/cells/ml HT medium with 50% fetal calf serum. To this suspension, add dropwise an equal volume of medium containing 30% DMSO. Gently mix while slowly adding medium. Transfer 2-ml aliquots to 2-ml freezing vials and freeze using conventional tissue culture techniques. Cells can be stored in liquid nitrogen at - 190 to - 150".

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Cells are thawed by removing them from the freezer and placing them in a 37" water bath. Immediately after thawing, the contents are diluted by adding dropwise an equal volume of HT medium. After IS min, another 6 ml of medium is added over the next 10 min and the cells are left at room temperature for IS min. The cells are then washed twice in HT medium and placed into the incubator. Characterizing Subclasses of Monoclonal Antibodies

It is generally necessary to determine the subclass of the antibody. For example, if you are not interested in obtaining IgM subclasses, you may be able to eliminate these from the cultures and reduce screening numbers. Also, some subclasses do not bind protein A, e t ~Because . ~ subtyping kits are now available commercially (e.g., Bethesda Research Laboratories) this is easily done using the ELlSA assay. Because some subtyping reagents are not specific for immunoglobulins from different strains of mice, it is helpful to use mice, as spleen donors for antibody production, which are compatible with the antibody-subclassing reagents. In Vitro Immunization and Fusion of Peripheral Lymphocytes

A number of laboratories have updated the use of in vitro immunization method^.'^,'^ These methods were developed because frequently adequate numbers of antigen-specific B lymphocytes are not stimulated during in vivo immunization procedures. As discussed by Reading," this failure may be due to tolerance (antigen-specific nonresponsiveness) or to an antigen hierarchy response (selective responsiveness to one or a few components of the irnmunogen preparation). Effective hybridoma formation may therefore be achieved if in vitro immunization methods are ~ s e d . ' ~Immunization -~* takes place in a matter of days rather than weeks or months. Also, because the normal in vivo regulation of the immune response is not a factor, it may be possible to produce antibodies against molecules normally considered only weakly immunogenic. Although these D. Grarecos, M. Astier, and M. Semeriva, J. Immunol. Methods 103, 169 (1987). S. A. Danielsson, S . A. Muller, and C. A. K . Borrebaeck, Immunology 61,51 (1987). l5 C. L. Reading, this series, Vol. 121, p. 18. l6 B. Sharma and P. I. Terasaki, Cancer Res. 34, 115 (1974). I' R. L. Lundah and D. J. Raidt, Cell. Immunol. 9,60 (1973). M. Schelling, Hybridoma 5, 159 (1986). l9 C. A. K. Borrebaeck and S . A. Moller, J. Immunol. 136,3710 (1986). C. A. K. Borrebaeck, Trends Biotechnol. 4, 147 (1986). *' R. L. Pardue, R. C. Bardy, G . W . Perry, and J. R. Dedman, J. Cell Biol. 96, 1149 (1983). C. L. Reading, J. Immunol. Methods 53,261 (1982). I'

l4

"

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IMMUNOLOGICAL PROCEDURES

[SO1

methods may have some benefits, the drawbacks are the production of predominantly IgM subclasses and the difficulty of standardizing the system. It is generally recommended that the beginner become well versed in methods for conventional preparation of monoclonal antibodies prior to initiating this method. Because human splenic and tonsillar tissue is largely unavailable, it is preferable to use peripheral blood lymphocytes for production of human monoclonal antibodies. This has been successful only recently. Procedures were developed by Danielsson et a!. l 4 using an elegant separation scheme in which lymphocytes were divided into several subpopulations. These were activated and reconstituted to give a population with a specific B:T cell ratio. Careful use of a number of support substances during induction and immunization resulted in several hundred cells/106 B cells which secreted antigen-specific antibodies. The technique is quite intricate, but appears to produce B cells which are amenable to fusion for production of human monoclonal antibodies. Antiidiotypic Antibodies

If a homogeneous antibody (e.g., a myeloma-produced antibody) is used as an antigen, certain portions of the molecule may be recognized as antigenic by the responding immunized host. The portion of an antibody molecule which recognizes its antigenic determinants is a set of unique sites termed "idiotype." These sites are made up of particular amino acid sequences in the hypervariable portion of the variable region of the antibody. The antibodies produced by the host against these sites are therefore termed antiidiotype. Antiidiotypic antibodies have internal images of the original immunogen, and therefore are identified operationally as, antibodies which have ~~-~~ activities which mimic those of the original i m m ~ n o g e n . Antiidiotypic antibodies have been described which mimic such proteins and molecules as insulin23and alprenolol.27

'' K. Sege and P. A. Peterson, Proc. Natl. Acad. Sci. U.S.A. 75,2443 (1978). A. Nisonoff and E. Lamoyi, Clin. Immunol. Immunopathol. 21,391 (1981). B . F. Erlanger, W. L. Cleveland, N . H. Wasserman, B. L. Hill, A. S. Penn, H. H. Ku, and R. Sarangarajan, in "Molecular Basis of Nerve Activity" ( J . P. Changeux, F. Hucho, A. Maelicke, and E. Neumann, eds.), p. 523. de Gruyter, Berlin, 1965. 26 B. F. Erlanger, W. L. Cleveland, N . H. Wasserman, H. H. Ku, B. L. Hill, R. Sarangarajan, R. Rajagopalan, E. Cayanis, I . S. Edelman, and A. S. Penn, Immunol. Rev. 94,23 (1986). " A. B. Schreiber, P. 0. Couraud, C. Ande, B. Vray, and A. D. Strosberg, Proc. Natl. Acad. Sci. U.S.A. 77,7385 (1980). 24

25

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While this technology is just becoming feasible, it has been demonstrated that the antiidiotypic strategy is a potentially powerful approach for the preparation of monoclonal antibodies to receptors which are difficult to isolate in quantities sufficient to be used as antigens.26

r r 7

? i

1

Single-Chain Antibodies via Genetic Engineering

Another method of generating homogeneous antibodies to take advantage of or improve their specificity is the genetic engineering of singlechain a n t i b o d i e ~ . ~These ~ . ' ~ recombinant molecules consist of the two antibody variable regions connected by a linear peptide. While this technology is in its infancy, the potential for low-cost, high-volume production of highly specific antibodies is c ~ n s i d e r a b l e . ~ ~ Acknowledgment We wish to acknowledge the expert clerical help in manuscript preparation given us by Ms. Suzanne Mascola.

7 28

a

S. Cabilly, A. D. Riggs, H. Pande, J. E. Shively, W. E. Holmes, M. Rey, L. J. Pery, R. Wetzel, and H. L. Heynehey, Proc. Natl. Acad. Sci. U.S.A. 81, 3273 (1984). M. A. Boss, J. H. Kenten, C . R. Wood, and J. S. Emtage, Nucleic Acids Res. 12, 3791 (1984).

[5 11 Protein Blotting and Immunodetection By THERESE M. TIMMONS and BONNIE S. DUNBAR

7

Polyacrylamide gel electrophoresis (one- and two-dimensional) has become one of the most widely used techniques for the analysis and characterization of complex protein mixtures.'-5 These gels can be stained directly and proteins visualized by several different method^.^,^ However, because access to proteins within the matrix is limited, the information

' D. Garlin, this volume [33]. B. S. Dunbar, H. Kimura, and T. M. Timmons, this volume [34].

' C. R. Merril, this volume [36].

' B.

a

S. Dunbar, "Two-Dimensional Electrophoresis and Immunological Techniques." Plenum, New York, 1987. B. D. Hames and D. Rickwood, "Gel Electrophoresis of Proteins: A Practical Approach." IRL Press, Washington, D.C., 1988.

METHODS IN ENZYMOLOGY. VOL. 182

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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gathered about individual components is usually restricted to their molec. ~ transfer of proteins separated by ular weight and ~ 1Electrophoretic PAGE onto the surface of an immobilizing membrane makes them more accessible to various reagents and probes and therefore enables further characterization. In addition to direct staining, these "blots" can be probed with lectins, for specific carbohydrate moieties, and with antibodies to identify and characterize antigenic determinants. One powerful application of such protein blots is the identification and characterization of an immobilized antigen by the use of antibody probes, which can be visualized by radiolabeled or enzyme-conjugated second antibodies. (If antibodies are affixed to the membrane, they can be identified and characterized by probing with selected proteins.) A major limitation to this procedure is that the denatured proteins bound to the membrane may no longer contain the same conformational and structural antigenic determinants present in the native protein. Therefore, only antibodies which recognize determinants consisting of a specific amino acid sequence, carbohydrate structure, etc., will be useful in this t e ~ h n i q u e . ~ Protein Electroblotting

A wide variety of transfer methods have been developed.'" However, the conditions for optimal transfer and subsequent binding of a specific protein to a membrane must be determined empirically and may vary for different protein samples. Many parameters affect the efficiency of protein transfer, most of which can be easily manipulated. Some will be discussed below. Since the quality of reagents used is critical for reproducible results, we have listed commercial sources whose reagents are acceptable for these procedures. There are many other sources for most of these reagents, but they should be tested for quality to ensure good results. Selection of Transfer Membrane. Several types of transfer membranes are now available. In addition to standard nitrocellulose, which is the most commonly used up port,'^ several companies now offer nitrocellulose impregnated with a synthetic support, which improves its durability and flexibility without altering its performance. Polyvinylidene difluoride (PVDF) membrane is marketed by Millipore (Bedford, MA), under the trade name Immobilon." Although its protein-binding capacity is slightly

' ' "

L. Anderson, "Two-Dimensional Electrophoresis: Operation of the 1.30-DALT System." Large Scale Biol. Press, Washington, D.C., 1988. H. Towbin, T. Staehelin, and J. Gordon, Proc. Natl. Acad. Sci. U.S.A. 76,4350 (1979). P. Matsudaira, J . Biol. Chem. 262, 10035 (1987). R. Tovey and B. A. Baldo, Electrophoresis 8, 384 (1987). B. Bers and D. Garfin, BioTechniques 3,276 (1985). M. G. Pluskal, M. B. Przekop, M. R. Kavorian, C. Vecoli, and D. A. Hicks, BioTechniques 4,272 (1986).

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68 1

lower than nitrocellulose, it is mechanically stronger and is compatible with many organic solvents. This allows direct protein staining with Coomassie Blue, and direct amino acid composition and sequence analysis of transferred proteins, without interfering with its subsequent use for antibody probing. Transfer Equipment. Several types of transfer units are commercially available. The Bio-Rad Transblot unit (Richmond, CA), the Hoefer unit (San Francisco, CA), and the Electroblot apparatus (E.C. Apparatus Corp., St. Petersburg, FL) each require 4-6 liters of buffer per experiment, and are routinely used for the efficient and reproducible transfer of proteins. Two gels can be transferred simultaneously using these units, but larger units (Pierce, Rockford, IL) are available to transfer 10-20 gels at once. The Bio-Rad Mini Protean I1 system contains a small tank transfer unit for the simultaneous blotting of two minigels in 15-30 min. An alternative transfer apparatus is the semidry electroblotter (Biometra, Bio-Rad, Hoefer, Millipore, and Sartorius, Emeryville, CA) which needs only enough buffer to saturate the filter paper sheets in the gel sandwich. Transfer is complete in 15-30 min. The graphite plate electrodes present in the early models often resulted in incomplete, patchy, and irreproducible transfer. The plates also were extremely susceptible to pitting and corrosion. However, the electrode plates of some newer models are made of more durable platinum (anode) and stainless steel (cathode). We have had excellent results using the TE70 SemiPhor semidry electroblotter available from Hoefer Scientific, which contains these newer electrodes. Either of the buffers described below for use in the tank transfer units can be used with this instrument. After transfer is complete, the gel can be stained for residual proteins and the membrane can be processed as described below. The use of a tank apparatus is currently preferable for protein transfer applications in which antigen is limited, quantitation is important, regulation of temperature during blotting is required, and time is not a critical factor. If large numbers of blots are needed rapidly, antigen is freely available, and qualitative results are sufficient, semidry electroblotters may be more appropriate. Transfer Buffer. The choice of buffer composition depends on the types of gel and membrane selected. The procedure of Towbin7 as modified by Anderson12specifies a Tris-glycine pH 8.3 buffer containing SDS. The recirculating, ice-cooled, high ionic strength buffer used helps prevent the gel from swelling in the absence of methanol during transfer, which can cause poor resolution of the proteins on the membrane. However, 10 rnM

''N. L. Anderson, S. L. Nance, T. W. Pearson, and N. G. Anderson, Electrophoresis 3,135 (1982).

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IMMUNOLOGICAL PROCEDURES

[5 11

3-[cyclohexylamino]- 1-propanesulfonic acid (pH 9.0 or 11.O) plus 10% methanol is suggested by Matsudaira8for transfers from SDS-PAGE minigels to PVDF membrane. Although many variations of electrophoretic transfer of proteins to nitrocellulose have been described, we have found that the procedure that omits alcohol from transfer solutions is generally optimal. Because sodium dodecyl sulfate (SDS) is not rapidly removed from the proteins in the absence of alcohol, the detergent-bound proteins are all initially negatively charged and a more quantitative transfer of proteins is achieved. Furthermore, alcohols or other reagents can alter or modify molecules and may therefore destroy some antigenic determinants. Electroblotting Procedure

The method described originally by Towbin7 as modified by Anderson et a1.12 results in efficient and reproducible protein transfer onto either nitrocellulose (Bio-Rad) and PVDF (Millipore). Electrode buffer: 0.250 M Trizma base 30 g 0.192 M glycine 140 g Final volume 1 liter Prepare as much buffer as needed to fill the chamber of the tank blotter. Note: This buffer is 10 times more concentrated than most methods suggest, but we have found this results in optimal transfer of most proteins and is required for others. Carry out SDS-PAGE separation of proteins (one- or two-dimensional separations, full-size or minigels). It is usually beneficial to include prestained molecular weight markers: their separation during electrophoresis, and the efficiency of their electrophoretic transfer onto a membrane, can be monitored visually. A variety of these standards are now commercially available. Bethesda Research Laboratories (Gaithersburg, MD) offers blue-stained markers, and Amersham (Arlington Heights, IL) offers "rainbow" standards (each marker protein can be identified by its own characteristic color dye). It is important to realize that the "rainbow" dyes detach from their respective proteins if they are allowed to remain in solubilization buffer for any length of time. Therefore the standards must be solubilized in a boiling water bath for no more than 60 sec, and immediately loaded onto the gel and electrophoresed. Transfer membrane, four sheets of filter paper, and two foam pads are cut to the same size as the gel and soaked in electrode buffer. (If the hydrophobic PVDF membrane is used, it must first be rinsed for a few seconds in 100% methanol and then in water before it is placed in electrode

[511

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PROTEIN BLOTTING AND IMMUNODETECTION

buffer. This methanol wetting procedure must be repeated if the membrane is allowed to dry at any point in the transfer and detection process.) The transfer stack is built in the following order: cathode side of unit, foam pad, two sheets of filter paper, gel, membrane, two sheets of filter paper, foam pad, anode side of unit. It is critical to add enough filter paper and foam pads to ensure tight contact of the gel and the membrane in the sandwich unit (for a complete photographic illustration of this procedure, refer to Ref. 4). Close the unit and lower it into the transfer chamber filled with chilled buffer and connect the power supply. Transfer at 1.2A for 24 to 4 hr. (In general, higher acrylamide concentration gels and higher molecular weight proteins will need longer transfer times.) If the buffer warms during the procedure, a recirculating cooling bath may be needed. After transfer is complete, place the membrane into a tray which is slightly larger than the sheet itself to ensure efficient mixing of solutions over the paper. Be sure to place the side of the paper that was next to the gel facing up. The use of prestained markers will help to determine on which side the proteins are immobilized. The transfer is processed as described below. The gel can be fixed and stained to monitor the efficiency of t r a n ~ f e r . ~ . ~ As an alternative, the method described by Matsudaira8 is effective for transfer of proteins from gels onto PVDF membrane, and is less expensive to use. The equipment and procedure described above is used, but the electrode buffer is 10 mM 3-[cyclohexylamino]-1-propanesulfonicacid (pH 11.0)plus 10% methanol. Transfer is accomplished at 0.5 A for 1030 min. The membrane is processed as described below.

-

I ~ m ~ o d _ e t e c t iof o nProteins

The procedure for immunodetection of antigens with antibodies is compatible with either PVDF or nitrocellulose membranes. Supplies and Reagents Tris-buffered saline (TBS)/azide: 10 mM Tris-HC1, pH 7.0 0.9% NaCl 0.02% Sodium azide Primary antibody, second antibody intermediate (if needed) 1 2 5 ~ - ~ a b e lStaphylococcus ed aureus protein A or protein G (Amersham, ICN, Costa Mesa, CA, NEN, Boston, MA, etc.); '25~-labeled IgG directed against species from which primary antibody is obtained Blocking solution: TBSIazide + 3-5% instant nonfat dry milk (or 3% bovine serum albumin)

684

IMMUNOLOGICAL PROCEDURES

1511

Although bovine serum albumin is a more fully characterized and purified reagent than nonfat dry milk, it is expensive to use routinely. A 3-5% solution of nonfat dry milk efficiently blocks most nonspecific bindHowever, the carbohydrates present ing sites for irnmunogl~bulins.~~'~.'~ may interfere with binding of an antibody recognizing a carbohydrate determinant. Other common blocking reagents include nonionic detergents such as PVP-40 (polyvinylpyrrolidone, average M , = 40,000) and Tween 2 0 . ' ~ 3 ' ~ * ' ~ Procedure for Zmmunoblotting. Immediately after protein transfer is completed, place the membrane (protein side up) in a dish and incubate with 100-150 ml blocking solution and shake vigorously at room temperature for 6-24 hr on a rotating or shaking platform that is reliable and can accommodate large numbers of gels. (Note: The best results are achieved when optimal shaking platforms are used. We recommend those available from Pierce Apparatus Branch.) Wash two times with 100-150 ml TBSI azide for 20 min each. Dissolve the primary antibody in blocking solution, in a volume that will just completely cover the membrane. The amount of antibody will depend on the antibody titer and can range from 20 p1 to 10 ml of serum in 60 ml of blocking solution. Add the antibody solution and incubate with vigorous shaking for about 6 hr. (Note: These incubation times may be reduced, depending on the titer and nature of the antibodies. However, to obtain the best initial results, we recommend these conditions for optimal signal with low background.) Wash twice as before, and then wash overnight with vigorous shaking. If a second antibody bridge is required (see below), dissolve it in blocking solution and incubate with vigorous shaking for 6 hr. Wash twice as before, then wash overnight and continue with the protein 12'1 labeling. If no bridge is needed, add '251-labeledsecond antibody or '251-labeledprotein A or G (approximately lo6 cpmltransfer) in blocking solution and incubate with shaking at room temperature for 6 hr. Protein A and protein G are cell wall proteins isolated from specific bacterial strains, and have specific binding sites for certain classes of immunoglobulins. Protein A binds (to varying degrees) most subclasses of IgG, plus IgM, IgA, and 1gD.l' Protein G binds nearly all subclasses of IgG, but not other classes of immunoglobulins. One important property of intact protein G, as isolated from streptococci group G, is the presence of a separate binding site for albumin, which could give ambiguous results "

D. A. Johnson, J. W. Gautxch, J. R. Sportsman. and J. H. Elder, Gene Anal. Technol. 1 , 3

l4

(1984). H . Towbin and J. Gordon, J . Immunol. Methods 72, 313 (1984). J. J. Langone, Adv. Immunol. 32, 157 (1982).

l5

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PROTEIN BLOTTING AND IMMUNODETECTION

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TABLE I PROTEING PROTEINA AND RECOMBINANT RECOGNITION OF IgG FROM VARIOUS ANIMAL SPECIESUSINGDOT IMMUNOBLOT ASSAYS Recognition bya IgG speciesb Mouse Rabbit Guinea pig Pig Human Cat Dog Goat Cow Sheep Horse Rat Chicken "

+ + + , + +, +,

Protein A

Recombinant protein G

+I-

+/-

+++ ++ ++ +++

-

++

++

+I-

+

+I-

+I-

++ ++ ++ ++ +/-

++

++ ++ ++ +/-

Strong to weak recognition;

+ I - , very weak; -, no recognition.

Not all subclasses of IgG are recognized by protein A or recombinant protein G.

in immunodetection experiments using tissue homogenates or serum.163" Recombinant protein G (ICN, Zymed, San Francisco, CA, Bio-Rad, etc.) has been engineered to eliminate this binding site. The species specificity of protein A and recombinant protein G recognition of IgG is summarized in Table If the 125~-labeled protein available does not bind to the IgG of interest, a second antibody bridge can be used: i.e., primary antibody:cat IgG, second antibody:goat anticat IgG, 'Z5~-labeledrecombinant protein G. In the case of monoclonal antibodies raised in mouse, we routinely use a rabbit anti-mouse immunoglobulin second antibody bridge, rather than using '251-labeled protein A or G directly, to produce the cleanest and strongest signal by autoradiography. The transfer membrane can be air dried before processing by autoradiography or it can be exposed to film while damp. To process the wet J. Bjorck and G. Kronvall, J. Immunol. 133,969 (1984).

l6

R. Fahnestock, P. Alexander, J. Nagle. and D. Tilpula, J . Bacteriol. 167, 870 (1986). 8 . Akerstrom, E. Nielsen, and L. Bjorck, J . Biol. Chem. 262, 13388 (1987).

"S . IS

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IMMUNOLOGICAL PROCEDURES

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membrane, drain it and place it on a piece of Whatman filter paper slightly larger than the membrane itself, and wrap in plastic wrap or seal in a plastic bag. After the autoradiogram has been developed, the membrane can be reprobed by exposing it to another primary antibody. If desired, the first primary antibody can be stripped (leaving the immobilized proteins still bound to the membrane) by washing with a low-pH (approximately 2.5) b ~ f f e r , ' ~a, ~ buffered ' solution of 0.5% Tween 20 or other detergent,20or a high concentration of chaotropic agent such as 3 M NH4SCN.20The efficiency of the stripping can be monitored by reexposing the membrane to X-ray film. (This procedure is more effective when the membranes are kept moist during autoradiography .) If the stripped antibodies are neutralized quickly and/or dialyzed exhaustively against TBS, they may be used to probe a second immunoblot. Variations of this technique have been successfully employed to select a specific population of antibodies from a polyclonal antiserum, on the basis of their recognition of a specific antigenic determinant. As an alternative to 12'1, antigens can be visualized directly on the transfer membrane using an enzyme-conjugated second antibody, directed against the IgG of the species from which the primary antibody is obtained. (Protein A and protein G are also available conjugated to the enzymes described below.) The enzymes most commonly used in this procedure, alkaline phosphatase and horseradish peroxidase, are coupled to the formation of a colored product which can be detected by visual inspection of the membrane. The high sensitivity of this type of reagent has both advantages and disadvantages. Results are obtained quickly, but the use of an extremely sensitive detection method can be confusing, especially if the background staining level is high. If the signal-to-noise ratio is too low or the optimal amount of protein is not immobilized on the membrane, and the desired information cannot be obtained, the membrane can not easily be reprobed or stripped. However, if 'Z5~-labeled protein A or G is used, the time of autoradiographic exposure can be varied to obtain the optimal signal. The membrane can be reprobed easily, and with less buildup of background signal than is possible with enzyme-conjugated detection. However, the speed of detection is often an overriding concern, and for a familiar antigen-antibody system, the enzyme-conjugated protocol may be the method of choice. The following procedure can be followed for immunodetection by horseradish peroxidase-conjugated second antibodies (DAKO, Santa Barbara, CA, Miles, Naperville, IL, etc.): (1) Block membrane, wash, and incubate in primary antibody as described above; (2) wash twice quickly

''

l9

J. B. Olmsted, J . Biol. Chem. 256, 11955 (1981). D. E. Smith and P. A. Fisher, J . Cell Biol. 99, 20 (1984).

[5 11

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and then overnight in TBS without azide (azide may interfere with the enzyme activity used for antibody detection); (3) dissolve the appropriate peroxidase-conjugated second antibody in blocking solution without azide, and incubate with vigorous shaking for 1 hr at room temperature. The amount of antibody needed will vary with the titer of the preparation used, and must be determined experimentally; (4) wash the transfer membrane for 30 min in TBSIno azide, with three changes of solution, at room temperature with vigorous shaking; ( 5 )prepare fresh color substrate [20 ml of 100 mM Tris-HC1, pH 7.0, plus 1.0 mg/ml3,3'-diaminobenzidinetetrahydrochloride (Sigma); 20 ml of 0.02% H202 in H20;200 pl of 8.0% NiC12 in H20](6) mix together and pour over transfer; (7) shake at room temperature until color appears; (8) wash with TBSIazide, and air dry. If immobilized proteins are being used to screen a series of monoclonal antibodies or to characterize antisera or antibodies in limited supply, the miniblotter system (Immunetics, Cambridge, MA) is useful. Antigens are transferred from one-dimensional standard-sized or minigels. The rnembrane is blocked, and then is clamped in the Lucite holder. The upper surface has open channels that span the height of the membrane, and require as little as 50 p1 of primary antibody solution each. Detection of bound antibodies is accomplished by any of the methods described above. Immunoblotting Artifacts

Many of the problems encountered with high backgrounds and other artifactual stains on immunoblots can be eliminated. (1) Usually a high background is the result of inadequate blocking of binding sites on the membrane, or inadequate washing. It may be necessary to increase the protein concentration of blocking solution or increase time of incubation with blocking solutions. Be sure that there are sufficient volumes of solutions to cover gels. Even if more dilute solutions are used, better results will be obtained if adequate shaking is used. If an excessive amount of second antibody or labeled probe is used, a high background will frequently be obtained. The system should be optimized to give maximum detection of antigen (signal) without giving high background (noise) (i.e., high signal-to-noise ratio). (2) Uneven background may be due to inadequate washing and can be improved as described above. This can also be a problem if soft plastic dishes are used instead of glass dishes for multiple incubations, or if the membrane is handled improperly. Always use gloves when touching membranes. (3) If little or no antibody binds to the proteins, the antibody may not recognize the denatured form of the antigen, or the antibody titer may be too low. The titer can be increased by affinity purification or concentration of the antibody. (4) Irregular transfer of protein to membranes can be a problem with many tank transfer systems. It is sometimes possible to improve this by adding additional paladium

688

IMMUNOLOGICAL PROCEDURES

[521

wire to the chamber used for electrophoretic transfer. Another common problem is that the paper is not pressed' tightly enough against the gel. This can be solved by adding additional sponges to compress the gel and paper together. The techniques of polyacrylamide gel electrophoresis, protein electroblotting, and immunodetection combine to provide an extremely powerful and sensitive method for the analysis and characterization of complex protein mixtures. Acknowledgment The authors wish to thank Ms. Suzanne Mascola for expert secretarial assistance.

C521 Immunoprecipitation of Proteins By GARYL. FIRESTONE and SANDRA D. WINGUTH

The discovery and use of fixed Staphylococcus aureus (Staph A) as an immunoadsorbent'-3 has been a major advance in routinely using antibodies as sensitive probes for selectively examining the expression of specific protein products from radiolabeled tissue. The Kessler procedure (and its modified versions) exploits the high adsorption capacity of protein A molecules which are found on the cell walls of certain staphylococci strains, for the Fc region of specific IgG and IgM isotypes. The overall strategy of this procedure involves reacting a small amount of radiolabeled antigen with an excess of antibody followed by the addition of enough fixed Staph A containing protein A to bind all appropriate antibodies regardless of whether they contain bound antigens. The advantage of this procedure (or any protocol that employs antibodies affixed to a solid state matrix) is that an immunoprecipitate per se need not be formed to separate immunocomplexes from cellular polypeptides not recognized by the antibodies. Thus, small absolute amounts of radiolabeled antigens can be rapidly and selectively immunoadsorbed to Staph A pellets and quantitatively fractionated away from the bulk polypeptides by simple low-speed centrifugation. Moreover, the Staph A immunoadsorption method is versatile in that it has proved useful for analysis of soluble as well as membraneassociated polypeptides, since the immunoadsorption is particularly efficient in the presence of either nonionic detergents such as Triton X-100

' S. W. Kessler, J . Immunol. 115, 1617 (1975). S. W. Kessler, J . Immunol. 117, 1482 (1976).

' R. D. Ivarie and P. P. Jones, Anal. Biochem. 97,24 (1979), METHODS IN ENZYMOLOGY, VOL. 182

Copyright O 1990 by Academic Press. Inc. All rights of reproduction in any form reserved.

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IMMUNOADSORPTION OF RADIOLABELED PROTEINS

689

and Nonidet P-40 (NP-40) (up to 5-10% detergent) or smaller quantities of certain ionic detergents such as deoxycholate. In fact, as discussed in a later section, the inclusion of detergents helps lower nonspecific binding to the immunoadorbed complexes. Another advantage is that after pelleting the final immunoco,mplex, the subsequent electrophoretic analysis or liquid scintillation counting can then be readily accomplished. Many variations on the original Kessler Staph A procedure have been reported for the immunoadsorption of radiolabeled antigens. Besides employing fixed Staph A, the most common variation is to use a solid-state matrix, such as Sepharose or agarose, that is covalently modified with isolated protein A molecules (or less commonly with appropriate secondary antibodies). Overall, the conditions for the use of either fixed Staph A or a derivatized protein A matrix are similar and these reagents can, for the most part, be interchanged. Both immunoadsorbents are readily available from commercial sources. Fixed Staph A is generally less expensive, tends to have a higher capacity for a given cost, and, if desired, can be easily prepared in large quantities; on the other hand, protein A-derivatized matrices can be reused a second time under certain conditions, generally form a tighter pellet, and have a significantly longer shelf life. The following protocols involve the use of fixed Staph A since we routinely use this matrix for the immunoadsorption of several different classes of soluble and membrane-associated proteins. When first attempting an immunoadsorption, there are several key variables that will differ depending on the type of antigen, antibody preparation (serum vs purified IgG, for example), and the tissue and cell source of the radiolabeled preparation (which can affect the amount of degradation of the given antigen). These key conditions include the duration of incubation, pellet washing conditions, temperature, salt concentration, and buffer pH, as well as the type and concentration of detergents. For each laboratory's antigen and antibody preparations, these conditions need to be empirically optimized for both efficiency of immunoadsorbing a specific antigen and for reduction in nonspecific background. The procedures detailed in this chapter provide a useful starting point for an immunoadsorption using fixed Staph A, as well as a discussion of several useful strategies for reducing the background binding in this assay. We have also outlined in detail an SDS elution-readsorption technique that for many systems essentially eliminates detectable nonspecific binding. Preparation of Radiolabeled Cell Extracts for Immunoadsorption It is important to prepare radiolabeled cell extracts or culture medium in a manner that is compatible with an optimized immunoadsorption. A

690

IMMUNOLOGICAL PROCEDURES

[521

useful starting point is to solubilize radiolabeled cells (or tissues) or secreted proteins in a nonionic detergent in a slightly alkaline buffer with a final salt concentration in the 150-300 mM range. Following a radiolabeling period, the cell culture medium is collected as the secreted fraction, and the cells or tissue are washed in phosphatebuffered saline (PBS). Pellet the cells by centrifugation at 600 g for 5 min (generally the medium speed of a typical table top centrifuge) and solubilize the cell (or tissue) pellet by homogenization in 1% Triton X-100, 5 mM EDTA, 250 mM NaCI, 25 mM Tris-HCl, pH 7.5, at 4" (see preparation of TETN250 buffer). A solubilizing buffer-to-cell ratio (v/v) of approximately 8: 1 should be maintained to allow efficient solubilization of membrane-associated proteins. (Some tissues may require a higher solubilization ratio.) The solubilized cell proteins are then harvested as the supernatant fraction after centrifugation at 20,000 g for 10 min at 4"; discard the nonsolubilized pellet. Secreted fractions or non-membraneassociated protein extracts are prepared for immunoadsorption by the addition of one-tenth volume of a l o x detergent-immunoadsorption buffer (10% w/v Triton X-100,50 mM EDTA, 25 mM Tris-HC1, pH 7.5). The inclusion of detergents helps lower nonspecific background without affecting specific antibody recognition. In certain cases, depending on the experiment, the detergents can be left out or the salt and pH varied. The detergent-solubilized extracts can be stored at -20" or lower for several weeks although the stability of a given antigen of interest will dictate its duration and temperature of storage. The amount of total radiolabeled protein in the solubilized fraction can be determined by precipitation with 10% trichloroacetic acid. One useful protocol is to spot 10-25 p1 of solubilized extract on 4.25-cm Whatman filters (#541), add 10% w/v trichloroacetic acid to the filters, gently swirl for several minutes, discard the solution, and repeat this procedure several times. The washed filters are then dried for 10 min under a heat lamp (place the wet filters on aluminum foil) and the filter-associated radioactivity quantitated by liquid scintillation counting. In the first several washes of the filters, the 10% trichloroacetic acid solution should contain approximately 1 mg/ml unlabeled amino acid corresponding to the employed radiolabel precursor to help compete nonprotein-associated radioactivity from the filters. Stock Solutions and Buffers

All buffers, unless stated otherwise, are stored at room temperature and are stable for several months. Discard if the fluid becomes cloudy. PBS: Dulbecco's phosphate-buffered saline (pH 7.0): 0.20 g KC1

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IMMUNOADSORPTION OF RADIOLABELED PROTEINS

69 1

0.20 g KH2P04 8.00 g NaCl 2.16 g Na2HP04.7H20 Bring to 1 liter with distilled water and mix; the final pH should be 7.0 0.2 M EDTA (pH 7.5) stock buffer: It is important to adjust the pH of the EDTA stock solution since untitrated EDTA will drastically change the pH of the final buffer: 1. To 500 ml of distilled water add 58.46 g of EDTA free acid (ethylenediaminetetraacetic acid; M, 292.3) while mixing with a stir bar 2. Slowly add solid NaOH until close to pH 7.5 3. Finish titration with 1.0 M NaOH and add water up to 1 liter final volume (approximately 20 g of NaOH will be used) 4. If you overshoot the desired pH do not back titrate with HCI since this will increase the overall salt concentration 1.O M Tris-HC1 (pH 7.5) buffer: 1. To approximately 750 ml of distilled water add 121.3 g of Trizma base (reagent grade) while mixing with a stir bar 2. Slowly titrate with concentrated HCI to pH 7.5 3. Add water up to a final volume of 1 liter 4. Do not back titrate with NaOH if you overshoot the desired pH TETN250 immunoadsorption buffer [25 mM Tris-HC1 (pH 7 . 9 , 5 mM EDTA (pH 7 . 9 , 250 mM NaC1, 1% Triton X-1001: 1. Combine 12.5 ml of 1.0 M Tris-HC1 (pH 7 . 9 , 12.5 ml of 0.2 M EDTA (pH 7 . 9 , and 475 ml distilled water 2. Add 7.3 NaCl and stir until dissolved 3. Add 5.0 ml 10% Triton X-100 (vlv) and stir until dissolved (Note: If desired, a HEPES-buffered solution can substitute for the Tris-HC1 buffers) TETNSOO buffer: This solution is made exactly as TETN250 except that 14.61 g of NaCl is added (instead of 7.3 g) Nondetergent Staph A washing buffer (TE): 10 rnM Tris-HC1 (pH 7 . 9 , 5 mM EDTA (pH 7.5): 5 ml of 1.O M Tris-HC1, pH 7.5 12.5 ml of 0.2 M EDTA, pH 7.5 482.5 ml distilled water (Note: this low salt buffer is used as a Staph A washing buffer prior to SDS gel electrophoresis since high salt concentration can affect the electrophoretic fractionation of proteins) 1.0 M SucroseITETN250: Dissolve 34.23 g sucrose in TETN250 buffer to a final volume of 100 ml

692

IMMUNOLOGICAL PROCEDURES

[521

2.0 M Urea/TETNSOO: Dissolve 12.0 g urea in TETNSOO to a final volume of 100 ml 50 mg/ml BSA: Dissolve 50 mg bovine serum albumin (fraction V) per milliliter of TETN250 buffer; 10 to 50 ml are common volumes to make. This solution is stable for several months when stored at 4". The solution will be a little cloudy; discard if it becomes very cloudy. Before use, centrifuge the required volume of BSAlTETN250 for 3 min at approximately 10,000 g in a microcentrifuge at room temperature Solubilized unlabeled cell extract: Solubilize nonradiolabeled cells or tissue in the Triton X-100 containing TETN25O solubilization buffer as described above for the radiolabeled cell extracts. This extract should be stored at -20" and may be thawed and refrozen repeatedly. This extract is used to cover nonspecific sites on the Staph A Antibodies and preimmune sera: The immunoadsorption will work with either serum or isolated antibody preparations. The optimal dilution and/or volume of antibodies will need to be determined for a given cell sample and antibody preparation. If needed, the serum or antibodies should be diluted in PBS and not in a detergent-containing solution. (For immunoadsorption of viral antigens we routinely dilute serum 1: 10 in PBS and then use 5 to 20 pllsample.) The diluted antibodies can be stored at -20". Preimmune serum is used as a control for background in the immunoadsorption; a parallel immunoadsorption containing either pre- or nonimmune serum (or antibodies) is needed to identify the nonspecific proteins that are brought down in the final Staph A pellets

Immunoadsorption of Radiolabeled Antigens: Setting Up The length of this procedure will vary depending on the number of samples, although it can generally be accomplished in several hours. Figure 1 shows the immunoadsorption procedure in a generalized flow chart form and will be referred to throughout this chapter. During the procedure, samples of radiolabeled cell extracts, unlabeled cell extracts, antibodies, preimmune sera, Staph A, and BSA solutions should be kept on ice. This procedure is optimized for microcentrifuge spins at approximately 10,000 g. Label three microfuge tubes per sample or control: 1. The first set of tubes will be used for the preadsorption of the radiolabeled samples.

C-

C521

693

IMMUNOADSORPTION OF RADIOLABELED PROTEINS

Washed Staph A

Radiolabeled e x t r a c t s

Unlabeled c e l l e x t r a c t s

Washed Staph A 10,000

Staph A pellet

discard

g spin

Preadsorbed Radiolabeled extracts

10,000

Preadsorbed Staph A

1

unlabeled cell extracts

f discard

Add t o antibodies I Add P r e a d s o r b e d Staph A

g spin

I

Form Staph A-Antibody-Antigen Complex

Spin immunopellets t h r o u g h sucrose

t Final Staph A p e l l e t washes

7

FIG. 1. Flow chart for the Staph A irnmunoadsorption of radiolabeled proteins.

I

2. The second set of tubes will contain the centrifuged BSA and antibodies; the imrnunoadsorption and Staph A binding reactions will take place in these tubes. Pipette 100 p1 of the 50 mglml BSAITETN250 into each of the second set of tubes. Then add the appropriate volume of antibody (or preirnmune sera). 3. The third set of tubes will contain sucrose cushions for a short step-gradient centrifugation. Pipette 600 p1 of sucroseITETN250 buffer into each of the third set of tubes.

694

IMMUNOLOGICAL PROCEDURES

[521

Preadsorption of Staph A and Radiolabeled Cell Extracts

The Staph A referred to in this procedure is fixed Staphylococcus aureus. There are several commercial sources with specific trade names, for example, Pansorbin from Calbiochem (La Jolla, CA). These preparations generally need to be washed at least once in TETN250 immunoadsorption buffer before using. Wash two equivalent aliquots of.Staph A as follows: 1. Add 10p1 of 10% Staph Alsample plus an additional 30 pl Staph A to each of two microfuge tubes (For example, for 10 samples, prepare two 130-p1 aliquots of 10% Staph A) and fill each tube with TETN250 (-1.5 ml) . 2. Spin for 30 sec in a microcentrifuge at approximately 10,000 g and aspirate off the supernatant fraction. 3. Resuspend one washed Staph A pellet in TETN250 to the original volume and store on ice; this Staph A mixture will be used to preadsorb the radiolabeled samples. The second washed Staph A pellet will be preadsorbed with unlabeled cell extracts (see following procedure). As an initial step to lower nonspecific adsorption of radiolabeled proteins to the Staph A, the radiolabeled cell extracts are first preadsorbed with fixed Staph A in the absence of added antibodies. Simultaneously, the Staph A used for the immunoadsorption is preadsorbed with an appropriate volume of solubilized unlabeled cell extract (see Fig. 1). Preadsorption of Staph A with Unlabeled Cell Extracts 1. Add approximately 250-500 p1 unlabeled cell extract (solubilized in TETN250) to the second washed Staph A pellet and vortex to resuspend the Staph A. 2. Incubate at room temperature for 15 min. 3. Spin down the Staph A in a microcentrifuge for 30 sec, aspirate off the supernates, and add TETN250 to the original volume of the Staph A to reconstitute a 10% solution. 4. Resuspend the Staph A by vortex mixing and store on ice until needed

Preadsorption of Radiolabeled Samples with Washed Staph A

Based on the experimental design, pipette the calculated amount of radiolabeled cell extracts into the appropriately labeled first set of microfuge tubes and equalize the volumes to approximately 300-800 p1 with TETN250. Add 10 p1 of 10% washed Staph A to each tube, mix by vortexing, and

[521

IMMUNOADSORPTION OF RADIOLABELED PROTEINS

695

incubate for 15 min at room temperature. Pellet the Staph A by centrifugation in a microcentrifuge for 3 min. The radiolabeled supernatant fractions represent the preadsorbed radiolabeled extracts and will be used to initiate the antigen-antibody reaction as described below. Formation of Antigen-Antibody-Staph A Complexes and Final Washes Add the preadsorbed radiolabeled cell extracts into the corresponding tubes in the second set (containing BSA and appropriate antibodies) to initiate the antibody-antigen reaction. Dispose of the first set of tubes as radioactive waste. Incubate for 10 min at room temperature with occasional vortex mixing. The short incubation in our procedure helps to reduce nonspecific binding of radiolabeled protein, but allows efficient antibody binding to the antigen. It is important to note that for a given antibody-antigen complex this incubation time will need to be optimized. Any incubations longer than 1 hr should be done at reduced temperatures such as 4". One suggested length of incubation is overnight at 4". Add 10 pl of 10% preadsorbed Staph A to each tube, mix by vortexing, and incubate for 5 min at room temperature. Periodic mixing will help this reaction. A useful approach is to vortex at the 2- and 4-min marks. Gently layer each of the corresponding immunoadsorption reaction mixtures from the second set of tubes over the 600-p1 cushions of 1.0 M sucrose in the appropriately labeled third set of tubes. Centrifuge the Staph A-antibody-antigen complexes through the sucrose by a 3-min spin in a microcentrifuge at room temperature. The nonadsorbed radiolabeled protein will stay in the upper layer. Aspirate off the upper layer down to the sucrose interface. Carefully overlay the sucrose cushion to almost the top of the tube with 2 M ureaITETN5OO in order to wash nonspecifically bound radiolabeled proteins from the walls of each tube. Be careful when layering the urea solution so as not to disturb the Staph A pellets. After a short incubation (1-2 min) aspirate off the urea and sucrose solutions, being careful not to aspirate the Staph A pellets. Wash the Staph A pellets twice; first with TETN250 and then with the nondetergent Staph A washing buffer (10 mM Tris-HC1, pH 7.5, 5 rnM EDTA, pH 7.5). If desired, additional washes can be done at this point in the procedure. In each case, add I ml of washing buffer, resuspend the Staph A by vortex mixing, and pellet the Staph A by a 3-min spin in the microcentrifuge. After each spin, carefully aspirate off the supernatant washing buffer. It is recommended that the vacuum be reduced and/or use a fine tip or needle during the aspiration to minimize the possibility of aspirating away the pellet.

696

IMMUNOLOGICAL PROCEDURES

1521

The final Staph A pellets can either be stored at -20" or they can be solubilized in SDS and readsorbed as described below. If desired, to directly count the pellets, add 100 pl of nondetergent Staph A washing buffer, then add the resuspended Staph A to a scintillation vial, add scintillation fluid, and analyze the samples in a liquid scintillation counter. For electrophoretic analysis, the Staph A pellets are suspended in 25 p1 SDS-gel sample buffer4 and 5 p1 of 0.5 M dithiothreitol (DTT) and then incubated at 100" for 2 min. After a 2-min centrifugation at 10,000 g , the supernatant fractions can be electrophoretically fractionated in SDS-polyacrylamide gels5 Steps that Help Reduce Nonspecific Binding of Radiolabeled Material to Staph A

When immunoadsorbing radiolabeled samples, one of the key problems is the nonspecific binding of proteins to Staph A. Several of the steps mentioned in the above procedure were designed to lower this nonspecific binding; in particular, preclearing the radiolabeled sample with washed Staph A, preadsorbing the Staph A with unlabeled cell extracts, incubating for relatively short times, including a high concentration of BSA in the incubation reactions, and pelleting the staph A through a sucrose cushion. To reduce the nonspecific binding in any given immunoadsorption, certain steps in the procedure can be modified if desired. One particularly useful modification is to increase the stringency of the washing conditions. For example, some laboratories carry out both the antibody and Staph A incubation in Triton X-100-containing solutions with added 0.5% SDS.6 Under these conditions it is important to keep the reactions at approximately 1% Triton X-100 to maintain the antibody-specific binding (see following section). Depending on the quality of the antibody preparation, the stringency of the washes can be increased by adding SDS up to a final concentration of several percent. To immunoadsorb viral proteins, we place 0.5% deoxycholate in all of the incubation and washing buffer^.^,^ Another variation is to wash the Staph A pellets in chaotropic buffers8 such as 2 M urea/l% Triton X-100110 mM Tris-HC1, pH 7.5. One unusual source of nonspecific background is the synthesis of radioactive proteins U. K. Laemrnli, Nature (London) 227,680 (1970). E. J. Platt, K. Karlsen, A. Lopez-Valdivieso, P. W. Cook, and G. L. Firestone, Anal. Biochem. 156, 126 (1986). C. L. Holcomb, W. J. Hansen, T. Etcheverry, and R. Schekman, J. Cell. Biol.

(1988).

' N. J. John and G. L. Firestone, BioTechniques 4,404 (1986).

I. Schauer, S. Emr, C. Grass, and R. Schekman, J. Cell Biol. 100, 1664 (1985).

106, 641

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IMMUNOADSORPTION OF RADIOLABELED PROTEINS

697

from unincorporated radioactive amino acids in the radiolabeled extracts by low levels of bacterial contamination in the antiserum samples. By centrifugation for 3 min in a microcentrifuge, any bacteria in the antiserum samples can be pelleted away. SDS Solubilization and Readsorption Double Immunoadsorption that Virtually Eliminate Nonspecific Background

Depending on the absolute level of the antigen, strength of the antibody-antigen reaction, detergent combination, radioactive precursor used to radiolabel the antigen, and other experimental variables, various levels of nonspecific binding of bulk radiolabeled polypeptides can be observed. This nonspecific background becomes a particularly significant problem when the antigen is expressed at a low level, which necessitates increased X-ray film exposure time or loading of increased starting radioactivity to decrease exposure time. As a result, there are many examples in the literature which display autoradiograms or fluorograms where the desired immunoadsorbed antigen is actually a minor band on an electrophoretic gel dominated by radioactive proteins that nonspecifically adsorb to Staph A pellets; such results are often difficult to analyze. Therefore, to immunoadsorb low-abundance polypeptides we have developed a procedure that effectively eliminates the nonspecific background binding of radiolabeled material to the Staph A immunopellets. As diagrammed in Fig. 2, our technique exploits the solubilization of antigen-antibody complexes and their dissociation from Staph A at concentrations of sodium dodecyl sulfate (SDS) above its critical micellar c~ncentration,~,' and the subsequent rebinding of the immunocomplexes to fresh Staph A after diluting the SDS with a combination of 1% Triton X-100 and 0.5% sodium deoxycholate. This procedure allows the unambiguous fluorographic visualization of low-abundance proteins in the virtual absence of nonspecific background.

SDS Release and Readsorption Resuspend the Staph A pellets in 30 p1 of 1% SDS (wlv in PBS) to release the antigen-antibody complexes from the Staph A immunopellets and incubate at room temperature for 10 min. Vortex to resuspend the pellet; repeat the vortex mixing two or three times during the 10-min incubation. Pellet the Staph A with a 4-min centrifugation in a microcentrifuge (10,000 g) and add each of the supernatant fractions to a corresponding tube containing 100 p1 of 50 mglml BSA, 400 p1 TETN250, and 10 p1 of a 10% antibody solution. This dilutes the SDS in the supernatant fraction by

698

IMMUNOLOGICAL PROCEDURES

Staph A-antibody-antigen

[521

immunocomplexes

D i s r u p t immunocomplexes i n SDS m i c e l l e s

Add excess nonionic d e t e r g e n t s t o b r i n g SDS t o b e l o w i t s CMC

Add antibodies

Add p r e a d s o r b e d Staph A ( r e f o r m s Staph A-antibody-antigen immunocomplexes)

1 Staph A p e l l e t

1

10,000 g spin

Discard supernatants

Final Staph A p e l l e t washes FIG. 2. F l o w chart for the SDS release and readsorption o f Staph A immunopellets.

20-fold, putting the SDS concentration below its critical micellar concentration. Incubate for 15 min at room temperature with periodic mixing, then add 10 pl of the preadsorbed 10% Staph A (see Preadsorption of Staph A) and incubate for an additional 5 min at room temperature. Pellet the Staph A immunocomplexes by a 3-min centrifugation and aspirate off the supernatant fraction. Wash the Staph A immunopellets in TETN250 and then in the nondetergent Staph A washing buffer (10 mM Tris-HC1, pH 7.5,5 mM EDTA) as described earlier. Each time, resuspend the pellets by vortex mixing and spin for 3 min to pellet the Staph A. Carefully aspirate all supernatant solution from the pellet. As mentioned above, it is best to lower the vacuum and/or use a fine tip or needle to aspirate.

[521

IMMUNOADSORPTION OF RADIOLABELED PROTEINS

699

Pellets can be frozen (-20") at this point for subsequent gel electrophoresis or scintillation counting. Comments on SDS Release-Readsorption Reaction

The cellular fractions are more likely to contain radiolabeled protein which nonspecifically associates with the Staph A pellets. For secreted fractions, which contain significantly lower amounts of total nonspecific radiolabeled proteins, the second immunoadsorption step is not as critical. A limitation in the SDS release-readsorption technique is that the final recovery of specifically immunoadsorbed proteins varies depending on several factors such as the antibody preparation, the detergent combinations used, as well as the duration and temperature of the incubations. Under our standard conditions, recoveries for mouse mammary tumor virus polypeptides ranged between 35 and 50% while the final recovery of EGF receptor was approximately 20%. Despite the fact that recovery is not quantitative, the longer exposure times allowed by the virtual absence of background radioactivity actually increase the overall sensitivity for detecting and analyzing specific radiolabeled antigens by fluorographic ~ . ~estimate that our procedure can be and autoradiographic a ~ s a y . We conveniently used for detection of proteins expressed in amounts as low as 0.001% of total cellular proteins. It is also important to point out that additional antibodies must be added to the diluted sample; it appears that SDS solubilization partially inactivates the antibodies added initially. Indeed, our technique has proved useful for the detection of many types of low-abundance proteins and has been used successfully to examine epidermal growth factor,' glucocorticoid receptor^,^ y-glutamyl transpeptidase (y-glutamyltransferose),'o and a'-acid glycoprotein.7 Our technique exploits the ability of SDS to release immunocomplexes from Staph A at concentrations above the critical micellar concentration. The critical micellar concentration of this surfactant will vary depending upon the ionic strength and the temperature of the assay. Potential detergent-induced changes in antigen folding may also significantly influence the final results since the overall procedure demands both a dissociation and a subsequent rebinding to Staph A. In this regard, SDS is known to alter the native conformation of polypeptides. In Kessler's original paper,' several different types of chaotropic reagents were effective in blocking the antibody-protein A interaction. Based on this work,

lo

R. Misefeld, S. Okret, A.-C. Wikstrom, 0 . Wrange, J.-A. Gustafsson, and K. R. Yamamoto, Nature (London) 312,779 (1984). R. Barouki, J. Finidori, M.-N. Chobert, M. Aggerbeck, Y . Laperche, and J. Hanoune, J. Biol. Chem. 259, 7940 (1984).

700

IMMUNOLOGICAL PROCEDURES

[531

we have found that chaotropic agents such as 9 M urea, 3.5 M MgC12,and 0.2 N NaOH were also effective in eluting immunocomplexes from Staph A and reducing nonspecific backgrounds although the final recovery after dilution in Triton X-100-containing buffers is lower than for SDS. However, these agents may be useful for particular proteins, especially those susceptible to unfolding and aggregation following SDS treatment. The virtual elimination of nonspecific background probably reflects a constant proportion of nonspecific adsorption of radioactive proteins during each of the Staph A steps. At the second Staph A immunoadsorption, the starting radioactivity was low enough that the final nonspecific background was effectively eliminated. In agreement with a previous study,3 the background at each Staph A immunoadsorption ranged between 0.1 and 0.3% of input radioactivity. The second immunoadsorption decreases cellular backgrounds by approximately 100-fold from about 2250 parts per million (ppm) to less than 25 ppm. Recovery of specifically bound polypeptides following the second immunoprecipitation step decreases by approximately 50% without qualitatively altering the electrophoretic pattern of immunoadsorbed proteins. Thus, the trade-off in this procedure is a 50% loss in specific recoverable polypeptides for a significant decrease in overall nonspecific background; the net effect being a 30- to 50-fold increase in signal-to-noise ratio, resulting in a more sensitive analysis of the immunoadsorbed proteins. Acknowledgments The authors thank Dave B. Alexander, Caroline P. Edwards, Nancy G. Forger, Cherie L. Holcomb, Emily J. Platt, and Melanie K . Webster for their helpful comments and Christina Cheng for her preparation and typing of this manuscript.

[531Immunoassays By CHARLES W. PARKER

Immunoassays use the binding specificity of an antibody for its specific antigen to measure either the antigen or antibody.' To quantitate the reaction either the antigen or the antibody is labeled. In theory, any label permitting sensitive measurements may be used, but frequently the label is

' C. W. Parker, "Radioimmunoassay of Biologically Active Compounds." Prentice-Hall, Englewood Cliffs, New Jersey, 1976.

METHODS IN ENZYMOLOGY, VOL. 182

Copyright O 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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IMMUNOASSAYS

70 1

a radioactive isotope, and the term radioirnmunoassay then becomes applicable. Alternatively enzymes with high turnover numbers such as horseradish peroxidase, alkaline phosphatase, or /3-galactosidase can be attached to an antibody or antigen and used with fluorogenic or chemiluminogenic substrates for sensitive immunoenzyme measurements. Immunoassays have provided a sensitive, reproducible, convenient, and generally applicable approach to the measurement of molecules of biologic interest. In the most sensitive systems measurements of ferntomole or even atamole quantities of antigen is possible. The potential value of radioimmunoassays for analytical purposes was first pointed out by Berson and Yalow in their studies with i n ~ u l i nThe . ~ radioimmunoassay concept has since been extended to a larger number of other polypeptides and proteins. Irnmunoassays have been shown to be very useful for discriminating between closely related protein species as well as for determining their absolute concentrations. My colleagues and I extended the radioimmunoassay concept to low-molecular-weight drugs (digitalis, opiates) and metabolites (cyclic nucleotides, prostaglandins) which must be chemically coupled to proteins in order to produce antibodies and pointed out the remarkable degree of sensitivity and specificity that was possible even with small chemical determinant^.'^^-'^ In most radioimmunoassays it is the antigen that is labeled, and this type of immunoassay design will be used for illustrative purposes. Quantitation depends on the ability of the unlabeled antigen (Ag) (the unknown) to inhibit binding of the radioactive antigen (Ag*) by antibody (Ab). The process is a simple competition in which Ag occupies a portion of the antibody combining sites, reducing the free Ab available to Ag*: Ag . Ab

* Ag + Ab + Ag* s Ag* . Ab

In performing the assay, fixed concentrations of Ab and Ag* are incubated in the absence and presence of the unknown samples containing Ag. When high sensitivity is needed the assay is carried out in the presence of only S. A. Berson, and R. S. Yalow, Adv. Biol. Med. Phys. 6 , 349 (1958). G. C. Oliver, D. Brasfield, B. M. Parker, and C. W. Parker, J. Lab. Clin. Med. 68, 1002 (1966). G. C. Oliver, B. M. Parker, D. L. Brasfield, and C. W. Parker, J . Clin. Invest. 47, 1035 (1968). G. C. Oliver, B. M. Parker, and C. W. Parker, Am. J. Med. 51, 186 (1971). S. Spector and C. W. Parker, Science 168, 1347 (1970). A. L. Steiner, D. M. Kipnis, R. Utiger, and C. W. Parker, Proc. Natl. Acad. Sci. U . S . A . 64,367 (1969). A. L. Steiner, C. W. Parker, and D. M. Kipnis, J . Biol. Chem. 247, 1106 (1972). B. M. Jaffe, H. R. Behman, and C. W . Parker, J . Clin. Invest. 52, 398 (1973). 'OR. Roberts and C. W. Parker, this series, Vo!. 74, p. 198.

'

702

IMMUNOLOGICAL PROCEDURES

[531

UNLABELED CK (ng)

FIG. 1 . Representative standard inhibition curve with unlabeled MB isozyme of human creatine kinase (CK) taken from Roberts and Parker,Ioshown on the abscissa, and percentage inhibition on the ordinate.

enough Ab to achieve substantial (40-50%) Ag* binding when no unlabeled Ag is present. Sensitivity may also be increased by preincubating the antibody with Ag before adding Ag*. After the Ag* has been added sufficient time is allowed for adequate Ag* binding and then the free and antibody-bound Ag* are separated, and one or the other is measured by radioactive counting. The concentration of Ag in an unknown sample is determined by finding out where the decrease of Ag* binding it produces falls on the standard Ag inhibition curve obtained by adding graded known quantities of Ag to the assay system (Fig. 1). For accurate quantitation the unlabeled Ag standard needs to be the same as the unknown, but the iodinated antigen and unlabeled antigen need not be identical. Antiserum

Conditions for preparing antisera and radioactive markers suitable for immunoassay use are discussed elsewhere in this volume ([49], [50], and [54]), in previous volumes of this series (volumes 70 and 74), and in other sources, but since the quality of these reagents may be quite critical in determining the usefulness of an assay a few comments are necessary here. Obviously each antigen-antibody system has its own special requirements from the point of view of the sensitivity and specificity that are needed. If immunoassay sensitivity or specificity need to be maximized,

[531

703

IMMUNOASSAYS -

-

-

-

--

the immunization of numerous animals under conditions which maximize antibody affinity with screening at multiple points during the immunization process may be needed to find the most suitable antiserum. While many excellent antisera are obtainable commercially, suitable sera for an investigator's individual needs may or may not be available, and the researcher must still validate antibody specificity in any case. Screening under the actual conditions that will eventually be used in the assay (in the presence of serum or tissue) may be helpful in selecting the most appropriate antiserum. Monoclonal antibodies are very useful for developing highly specific immunoassays, but their monospecificity may be a disadvantage from the point of view of immunoassay sensitivity. The sensitivity of an immunoassay depends in part on the spectrum of antigen epitopes that the antiserum recognizes.' The stability of antigen-antibody complexes is increased if the antigen as well as the antibody is operationally multivalent, permitting cross-linking and the formation of lattices. A protein-antiprotein reaction is probably best described by an overall avidity constant (Kav)which is affected in turn by the Ka values of the antibody combining sites for their individual epitopes as well as by the ability of the different antibodies to participate together in cooperative binding.' The effect of antigen valence on complex formation probably is the major explanation for the generally greater sensitivity of immunoassays for proteins and polypeptides than for low-molecular-weight (haptenic) antigens. As a rough rule of thumb, the practical sensitivity of an assay is equal to l/Ka or l/Kav.Ka values for antibody-hapten interactions usually range between lo6 to 101° liters mol-', whereas Kav values for antibody-protein interactions may be as high as 1012to 1OI3liters mole-'. To avoid the loss of multivalency with monoclonal antibodies in proteins which lack repetitive epitopes, mixtures of monoclonal antibodies recognizing different epitopes on the antigen may be very useful. However, if the affinities of the antibodies are not as high as in polyclonal hyperimmune sera, the monoclonal system may still not be optimal. While polyclonal antisera suitable for assay are likely to contain both high- and low-affinity antibodies, at the very dilute serum concentrations used in sensitive immunoassays, only the high-affinity antibodies are likely to be important in antigen binding. In screening antisera, agar gel diffusion and immunoelectrophoresis are of particular value in that they are simple to perform and provide information both on immunological cross-reactivity and on the presence or absence of multiple antigen-antibody systems. However, if a pure radioactive antigen is available, these analyses are just as easily made in the radioimmunoassay itself. Once antisera with intermediate or high titers have been identified, they are evaluated with regard to (1) sensitivity,

704

IMMUNOLOGICAL PROCEDURES

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linearity, and reproducibility of standard antigen inhibition curves; (2) susceptibility to nonspecific inhibition in tissue samples and buffer (see below); (3) reactivity with possible cross-reacting antigens. Usually the minimal quantity of antibody giving the desired level of radioactive antigen binding (generally 40 to 50% of the total radioactive antigen added) is used in the assay. Depending on the antibody and the immune system, this may represent an antiserum dilution of anywhere from 1:100 to 1 :1,000,000. The adequacy of antibody binding must be verified in tissue extracts under the conditions in which the assay will be used. Where there is a generous margin of sensitivity in the system being assayed, reproducibility may be increased by using the same amount of tissue sample with more antibody, setting the sensitivity of the system at a lower working level. However, when assay sensitivity is already a problem, more tissue sample must also be used and problems of nonspecific interference or cross-reactivity may not be improved. As a rule, the antiserum is used without purification, particularly when high dilutions of antibody are employed. In certain situations purified y-globulin fractions are used in order to eliminate serum proteins with undesirable enzymatic or nonspecific binding activity. Almost all the binding activity in hyperimmune sera is in the IgG fraction, so conventional purification procedures for IgG, such as ammonium sulfate precipitation or chromatography on DEAE-cellulose or staphylococcal protein A agarose columns, can be used. Purification may also be desirable for monoclonal antibody preparations obtained from ascites tumors to remove interfering activities. Albumin and most enzymes in serum can be largely eliminated by ammonium sulfate precipitation at 1.6 M ammonium sulfate. The use of adsorption procedures to remove cross-reacting antibodies can be helpful in improving specificity. Adsorption with cross-reacting antigen coupled covalently to Sepharose, polyacrylamide, or agarose is almost always preferable to its use in solution since the equivalence point is likely to be missed and soluble antigen and antigen-antibody complexes may remain in the preparation. IgG antibodies can also be degraded to univalent fragments, but this process generally presents no advantages in terms of specificity and may be undesirable if the antigen is multivalent, since the functional avidity of reaction may be reduced. Antisera ordinarily can be stored at -20' for at least 3 to 4 years with little or no detectible loss in immunologic reactivity. Storage is preferably done in small volumes so that antisera need not be repeatedly thawed and refrozen. Once antisera have been diluted, some degree of instability should be assumed even in the frozen state, and depending on the system fresh dilutions may need to be prepared frequently.

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Antigen (or Antibody) Marker

A factor which places a practical limit on the sensitivity of an assay is the specific activity of the antigen or antibody marker. The lowest amount of antigen that can be measured is approximately equal to the quantity of marker that is needed for accurate detection. In their most sensitive forms radioactive and enzymatic immunoassays are approximately comparable in sensitivity. Regardless of which type of procedure is used, careful attention needs to be given to the conditions of iodination or conjugation to enzyme so that a maximal sensitivity is achieved without unacceptable losses of immune reactivity ([54] in this volume). Many radioimmunoassays involving radioactive antigens are carried out using about 8000-10,000 cprn of radioactive antigen, but larger or occasionally smaller amounts of radioactivity may be used. Generally, bound rather than free radioactivity is determined because the relative change when inhibitor is present is greater. If 8000 cpm of radioactive antigen is added to the sample and the binding is 40% complete, there will be about 3200 cpm of bound radioactivity in uninhibited samples which will have a coefficient of variation of less than 2% if samples are counted for 1 min. Although lower levels of total and bound radioactivity can be used, the gain in sensitivity is usually not that great, and the need for longer counting times or the greater statistical variation in counting if 1-min counts are used is a decided disadvantage. Thus, the quantity of radioactive antigen in the assay is fixed by the practical level of bound radioactivity that is required to discriminate between samples. In immunoassays with antigen-enzyme conjugates the criteria for how much marker antigen to use are much the same as those for iodinated antigens. The goal is to obtain about 40-50% binding in uninhibited samples with enough enzyme activity to detect decreases in binding down to about 5% or less of the original signal. In contrast to radioactive antigen, when antibodies labeled enzymatically or with radioactivity are used in "noncompetitive" radioimmunoassay systems much larger amounts of enzyme activity or lZ51 are used. In this case the quantity of antibody should be capable of binding all of the unknown antigen in the sample. Tissue or Serum Sample

All, or any portion, of the tissue or serum sample may be measured. Depending on the antigen-antibody system and the tissue, cell, or body fluid being studied, measurements may be made either without or with extraction and various degrees of partial purification. In any case, at-

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tempts to minimize proteolysis by the use of protease inhibitors and low temperatures are very frequently desirable. Sample sizes that bring the level of immunoinhibitory activity within the middle range of the standard curve are chosen. When the level of antigen in a tissue is variable, it is desirable to divide tissue extracts into several portions and assay small and large aliquots in order to ensure that the inhibitory activity will fall somewhere in the most sensitive region of the standard curve. Measurements at several sample levels have the added advantage of providing information on whether inhibition curves with endogenous tissue antigens parallel standard antigen inhibition curves in buffer. Such parallelism is a necessary, but not sufficient, condition for establishing that the inhibitory activity in tissue samples is truly antigen specific. Cross-reacting antigens may give parallel or nonparallel inhibition curves. Partially purified tissue samples may also be studied as a further means of validating the assay (see below). If sensitivity is a problem, it is sometimes possible to concentrate antigen in an extract by specific or nonspecific adsorption." Samples can be passed through columns of Sepharose-coupled antibody and then eluted with 6 M guanidine. Up to 500-fold increases in immunoassay sensitivity with apparent recoveries of 85 to 95% were estimated by using this approach. This and similar selective concentration procedures provide a powerful approach to the quantitation of substances too dilute to be measured by routine methods. However, a number of important pitfalls should be kept in mind. They include a failure to obtain quantitative adsorption or elution of antigen, or inadvertent concentration of cross-reacting or nonspecific interfering substances as the antigen is being concentrated. Incubation Conditions

In choosing the assay conditions considerations such as the stability and physiochemical properties of the antigen, the assay sensitivity that is needed, the anticipated time and cost of the assay, and the experience of the investigator may each be important. Assays are often conducted in a final volume of 0.15 to 0.5 ml. However, Ciabattoni recommends using volumes of 2.0-2.5 ml to permit more precise aliquoting and a greater dilution of reagents in the assay . I 2 Dilution has the added advantage that it often helps eliminate nonspecific binding effects in the assay. Obviously, the avidity of the antibody for antigen is an important consideration here. " l2

B. D. Weintraub, Biochem. Biophys. Res. Commun. 39,83 (1970). G . Ciabattoni, in "Radioimmunoassay in Basic and Clinical Pharmacology," p. 181. Springer-Verlag, Berlin, 1987.

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707

IMMUNOASSAYS --

- -

-

-

It therefore seems desirable that the incubation volume be optimized for the antigen-antibody system being studied. Commonly used buffers for dilution include phosphate, borate, or Tris-buffered saline. Usually, the choice of the buffer is not important. Nonetheless, a careful examination of the effect of buffer, pH, ionic strength, and divalent cations should always be made in a new immunoassay system in order to maximize sensitivity and anticipate unexpected sources of interference in the assay. Although assays are usually carried out at neutrality, doing so is not always optimal. ' % I 3 Nonspecific adherence of antigens and haptens (especially hydrophobic haptens) to glass and plastic tubes or pipets may markedly influence measured activity in the immunoassay. With some proteins and polypeptides [adrenocorticotropic hormone (ACTH) and parathormone, for example], nonspecific binding is reduced if plastic tubes are used. The addition of protein to the medium minimizes nonspecific adsorption and also helps avoid denaturation of highly diluted antigens and antibodies. Therefore, assays involving iodinated antigens are generally carried out in proteincontaining buffers. Bovine serum albumin, gelatin, lysozyme, and ovalbumin are commonly used, usually at final concentrations of 1 to 5 mglml. In some systems diluted whole serum or proteins present in the sample itself are just as satisfactory. However, even though added proteins are often beneficial, they should not be used indiscriminately without making an evaluation for possible adverse effects. For example, contaminating enzymes may degrade the marker. Possible additives, apart from buffer and protein, include enzyme inhibitors and chelating agents. In assays lasting longer than 3 days, a bacteriostatic agent, such as sodium azide, 0.1 to 0.2%, may be incorporated into the medium to help avoid microbial growth. Assay conditions that are used in immunoassays vary widely with total incubation times extending from a few minutes to as long as 6 days and should be optimized for the immune system in question. An initial relatively short incubation (5 to 60 min) at 37" or room temperature helps accelerate immune complex formation; however, since almost all assays are completed in the cold, unless the assay must be completed rapidly, it seems preferable to initiate the assay at low temperatures. Cold temperatures should also be maintained during washing. Highly sensitive assays involving complex antigens usually require 24 hr or longer. Too short an incubation time is undesirable because of possible disequilibrium when antibody-bound and free antigen are separated. Depending on whether an equilibrium or nonequilibrium assay is being used, the radioactive antigen ". I. A.

Fischer, U. Binswanger, and F. M. Dietrich, J. Clin.Inuest. 54, 1382 (1974).

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may be added at the same time as the sample or later in the assay. When assay sensitivity needs to be maximized, it is desirable to determine the effect of preincubating nonradioactive antigen with antibody before adding Ag*. The delayed addition of radioactive antigen gives the unlabeled antigen an opportunity to bind to the antibody first. While some assays are not appreciably improved in regard to their sensitivity by the delayed addition of radioactive antigen, others are quite substantially affected.14 Separation Systems

At the completion of the incubation, bound and free Ag* generally must be separated. Methods for separation of free and bound Ag* have included the use of a second antibody, salt or organic solvent precipitation, adsorption onto insolubilized staphylococcal protein A, charcoal or another nonspecific adsorbent, electrophoresis, gel filtration, or two-phase systems in which the antigen or the antibody is attached to a solid phase. With certain exceptions, each of the above procedures is applicable to a large number of different antigen-antibody systems. A variety of factors enter into the selection of a system, including the rapidity and sensitivity required in the immunoassay, possible unusual physiochemical properties of the antigen, the affinity, antibody class, or subclass and specificity of available antisera, the skill of the technical personnel, and the usual number of samples to be processed. Often the separation method that is chosen is based as much on the previous experience of the individual investigator as the peculiarities of the particular antigen-antibody system being studied. Nonetheless, an investigator who is setting up an immunoassay for the first time should carefully review any published results in the same antigen-antibody system, both for possible useful technical details and any concrete evidence that one separation system is preferable to another. Double-antibody immunoprecipitation is probably the most broadly applicable of the separation system^.'^,'^ Obviously enough second antibody should be used to separate all of the first antibody. Each new batch of second antibody must be verified by titration in the assay. The use of a second antibody is not practical when the first antibody has a low titer because large amounts of both antibodies, particularly the second antibody, will then be needed. If a monoclonal IgG antibody is used in a double-antibody system the effectiveness of the second antibody as an immunoprecipitant for the Ig class or IgG subclass of the monoclonal l4 l5 l6

K. Ichihara, T. Yamamoto, M. Azukizawa, and K. Miyai, Clin. Chim. Acta 98,87 (1979). R. D. Utiger, M. L. Parker, and W. H. Daughaday, J. Clin. Invest. 41,254 (1962). A. R. Midgley, Jr. and M. R. Hepburn, this series, Vol. 70, p. 266.

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antibody must be established and the use of an IgG subclass-specific second antibody may be desirable. Relatively prolonged incubation times are sometimes needed in double-antibody systems to maximize the precipitation reaction. However, the use of accelerators of precipitation such as polyethylene glycol may permit the assay to be completed within 5 to 60 min. Single- or double-antibody immunoassays may be performed in solidphase systems. One approach involves antibodies that are noncovalently adsorbed to the walls of a microtiter plate or test tube. Denaturation can be a problem and can decrease the sensitivity of such assays. Nonetheless this type of assay is very frequently used. The nonspecific adsorption step is usually done at pH 9-10. Alternatively, the antibody is attached covalently to disks or beads which are used in suspension. In other variants the antigen is insolubilized instead of the antibody. Some solid-phase immunoassays appear to be particularly sensitive to the exact nature of the complexes formed between antigen and antibody. Although a number of solid-phase immunoassay systems have been shown to give highly satisfactory results, others are less practical due to delayed equilibration times, problems with reproducibility, or high nonspecific binding blanks. Factors affecting the kinetics of antigen-antibody reactions in solid-phase systems have been reviewed.19 The procedure of Nash et al., which utilizes antibody coupled to commercial cross-linked polyacrylamide beads (Immunobeads, Bio-Rad) using a water-soluble carbodiimide, is rapid and r e p r o d u ~ i b l eThe . ~ ~assay is performed in microtiter plates in a volume of approximately 100 p1. At the completion of the incubation the beads are transferred to glass fiber filter strips on a microharvester, washed, and counted. This procedure lends itself readily to the rapid processing of many samples. The beads can be aliquoted and stored frozen at -80' for extended periods prior to use in the assay. "7"

Solid-Phase Zmmunoassay of Human ZgA with Polyacrylamide-Antibody Beads 1. In 96-well microtiter plates, add the following: 0.01 ml sample or standard 0.05 ml '251-labeledhuman IgA (25,000 cpm) in barbital-buffered saA. A. Ansari, L. M. Bahunguna, and H. V . Malling, J . Immunol. Methods 26,203 (1979). W. H. C. Walker, Clin. Chem. 23, 384 (1977). l9 M. Stenberg and H. Nygren, J . Immunol. Methods 113,3 (1988). G. S. Nash, M. V. Seiden, M. G. Beale, and R. P. MacDermott, J. Immunol. Methods 49, 261 (1982). l8

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line (BBS) with 10% fetal calf serum (FCS): The BBS is freshly prepared from 5 X BBS [5,5-diethylbarbituric acid (2875 g), sodium 5,Sdiethylbarbiturate (1 375 g), and NaCl (42.5 g), which are dissolved in 250 ml hot water; CaC12 2H20(0.1 log) and MgC12 - 6H20 (0.508 g) are added and the volume is brought to 1 liter with water] 0.05 ml of a suspension (60-120 pglml) of rabbit anti-human IgA beads (Bio-Rad, Richmond, CA) 2. Incubate overnight at 25" 3. Resuspend beads on a microtiter plate shaker (Bellco, Vineland, NJ). 4. Transfer to glass filter strips on a Microharvester under vacuum (Bellco). 5. Wash 10 times with about 0.2 ml (filling the wells) of 5% FCS in BBS. 6. Remove strips with forceps and count. In general, adsorption assays involving charcoal are based on differences in size or charge of free and bound antigen affecting solid-phase binding. They usually work well with relatively small peptides. They provide rapid measurements but are sensitive to the protein content of the medium and, depending on the number of samples processed, may be unusually subject to intraassay variation. Salt precipitation with ammonium sulfate is a reliable, rapid, and inexpensive method in appropriate systems, but is applicable only to radioiodinated antigens that are soluble in 40 to 50% ammonium sulfate. Polyethylene glycol is also useful for separating antigen-antibody complexes. The charcoal, ammonium sulfate, and polyethylene glycol methods are reviewed in an earlier volume of this ~ e r i e s . ~ ' . ~ ' Once the separation of bound and free antigen has been initiated, nonequilibrium conditions are established and, depending on the conditions of washing and the particular antigen-antibody system, significant amounts of previously complexed antigen may dissociate. This is not necessarily a major disadvantage since relatively low-affinity interactions involving cross-reacting antigens may be particularly subject to reversal. Some investigators have proposed the use of nonequilibrium assays in which the ability of unknown antigen to displace radiolabeled antigen from preformed antigen-antibody complexes is measured.23 While such nonequilibrium assays are potentially useful because of their simplicity and decreased dependence on sample volume, they also may be subject to 22 23

W. D. Odell, this series, Vol. 70, p. 274. T. Chard, this series, Vol. 70, p. 280. F. Cocola, A. Orlandini, G. Barbarulli, P. Tarli, and P. Neri, Anal. Biochem. 99, 121 (1979).

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71 1

seemingly minor variations in assay time and temperature, and special care may be needed to minimize intraassay variation. Washing solutions usually contain the buffer and nonspecific protein inhibitor used in the original incubation mixture. The use of cold washing solutions is almost always desirable. Regardless of the procedure used, separation of free and bound antigens should be performed as rapidly and reproducibly as possible, and for this reason it may be unwise to process too many samples simultaneously. As already noted, solid-phase assays may facilitate the rapid handling of large numbers of samples.

Representative Immunoassays

Immunoassay Using the Charcoal Method The procedure of Walsh and Wong for secretin will be used for illustration: All pipetting procedures are carried out in an ice bath. Samples, standards, antiserum, and labeled secretin are diluted in 0.1 M sodium acetate, pH 4.5, containing 2% serum bovine albumin, 2500 kIU aprotinin (FBA Pharmaceuticals, New York, NY) per milliliter buffer, and 0.02 M EDTA.24Standards are prepared first by diluting the standards to contain 1000, 100, and 10 pglml. Each of these standards is pipetted in amounts of 200, 100, 50, and 20 p1, producing 10 different concentrations with two points of overlap. Standards and serum samples or other unknowns are diluted to contribute a volume of 1 ml to the reaction mixture. For assays of serum specimens, it is desirable to add charcoal-stripped serum to the standard samples to correct for nonspecific interference by serum protein. Aliquots of unknown serum samples (200 and 50 p1) are diluted to 1 ml with the standard buffer to give a final concentration of 1/10 and 1/40 in the reaction mixture. To each assay tube is added 2000 cpm of labeled secretin plus diluted antibody (predetermined to bind 50% of the label) to give a final volume of 2 ml. The nonspecific binding controls contain the diluted label and standard buffer instead of antibody. Tubes are incubated for 24-72 hr at 4". Separation of bound and free labeled peptide is performed with dextran-coated charcoal. The separation mixture contains 20 mg activated charcoal (Mallinckrodt, Paris, KY), 20 mg dextran T-70 (Pharmacia, Piscataway, NJ), and 20 pl 5% bovine serum albumin in a final volume of 0.2 ml. Tubes should be kept on ice during the separation procedure. After thorough mixing, the tubes are centrifuged at 3000 rpm (-2000 g) for 10-15 min and the supernatant solutions are removed by

" J . H. Walsh and H. C. Wong, in "Radioimmunoassay in Basic and Clinical Pharmacology," p. 315. Springer-Verlag, Berlin, 1987.

712

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IMMUNOLOGICAL PROCEDURES

pouring off into a separate tube. Both the pellet containing the free secretin and the supernatant containing the antibody-bound secretin are counted in a gamma scintillation spectrometer for a minimum of 2 min. Double-Antibody Immunoprecipitation Assay The procedure of Roberts et for the rapid determination of creatine kinase (CK) isoenzymes (MM, MB, BB) will be used for illustration. 1. To 0.120-0.165 ml buffer (50 rnM Tris-HC1,20 mM2-mercaptoethanol, 20 mM EDTA, 2 mglml BSA, 0.2% sodium azide final pH 8.5) add 0.01 ml of the first antibody (rabbit anti-MB CK), 1: 10,000, and 0.005-0.170 ml MB standard or the sample 2. Incubate 15 min at 22" 3. Add 0.02 ml 12'1-labeled BB CK (25,000 cpm). 4. Incubate 60 min at 22". 5. Add 0.02 ml of the second antibody [excess anti-rabbit IgG (goat)]. 6. Incubate 15 min at 22". 7. Centrifuge 15 min (3500 g at 4"), remove supernatant by aspiration, and count pellet. Solid-Phase Immunoenzyme Analysis: Enzyme-Linked Immunosorbent Assay (ELISA) The procedure of Katnik et a1.,26 in which a polystyrene microtiter plate is coated with a haptoglobin (Hp) and used to screen for monoclonal Hp antibodies in hybridoma cultures, is representative of a large number of solid-phase immunoenzyme procedures. The wells of polystyrene microtiter plates (Plastomed, Poland) are coated with 200 p1 of human haptoglobin Hp (200 ng) in 0.1 M carbonatelbicarbonate buffer, pH 9.2, at 37" for 1 hr and at 4" overnight. The plates are washed four times with 250 pl of casein buffer (154 mM NaC1,0.5% casein, 10 mM Tris-HC1,0.02% thimerosal, pH 7.6) and unoccupied binding sites are blocked by casein for 2 hr at 37". Fifty microliters of supernatant from mouse hybridoma cultures being screened for anti-Hp antibody cultures and 150 p1 of casein buffer are added and the plates are incubated at 37" for 3 hr with gentle shaking. After rinsing three times at 5-min intervals with casein buffer, the goat antimouse IgG-horseradish peroxidase conjugate (IgG-HRP) (diluted 2000fold with casein buffer) is added (60 nglwell) and the plates incubated for 3 hr at 37". Excess conjugate is then thoroughly removed by washing, and 2S

26

R. Roberts, B. E. Sobel, and C. W. Parker, Science 194,855 (1976). I. Katnik, M. Podgorska, and W. Dobryszycka, J. Zmmunol. Methods

102,279 (1987).

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peroxidase fixed to the wells is detected by the addition of o-phenylenediamine.26The reaction is followed by increases in absorbancy at 492 nm and comparison with a standard peroxidase reaction. The incubation time for the antigen-antibody reaction can be shortened to 30 min if 2% polyethylene glycol ( M , 6000) is present. Assay Controls, Variance, and Repeatability Replicate samples, preferably in triplicate or quadruplicate, should always be analyzed, ideally using at least two different dilutions of the unknown sample. Insofar as possible each sample should be treated identically. The assay must be rigidly standardized in terms of total reaction volume, buffer content, quantity of radioactive antigen, and duration and temperature of incubation. Every assay should include a full antigen standard curve. In an immunoassay of any size, binding controls and antigen standards should be interspersed at the beginning, middle, and end of the assay to detect any systematic variations in the assay related to the number of samples involved. As a part of every assay, several calibration or control measurements must be made, including (1) determination of total antigen radioactivity added to the assay, (2) determination of nonspecific marker binding (counts present when samples containing the antigen marker but no antibody are processed), (3) standard antigen inhibition curves in buffer (and sometimes in tissue extracts as well) (4) extraction or reagent blank controls, especially if new extraction procedures or reagents are being utilized, (5) possibly, incubation controls for damage to the immunoreactivity of the radioactive antigen when tissue extracts are present. The standard curve helps identify day-to-day variations in the assay due to deterioration of the radioactive antigen or an incorrect dilution of antibody. Ideally, incubation mixtures containing standard and unknown antigen should be identical in every respect. Sometimes adsorbants such as charcoal or insolubilized antibody can be used to prepare antigendepleted serum or tissue samples. If completely antigen-free tissue samples are not available (and they often are not), the best approach is to carry out standard curves both in buffer and in tissue extracts. However, this approach is not completely satisfactory, since the standard curve in tissue is superimposed on the background of tissue antigen. The standard curve should cover a broad (at least 1000-fold) range of antigen concentrations, extending from minimal to complete inhibition of radioactive antigen binding. While 2-fold dilutions are normally used in the working part of the standard curve, a narrower dilution span may be desirable in the most important parts of the curve, particularly when standard curves are non-

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linear. Since dilutions of standard antigen are generally very marked, they must be made accurately, since there is no way of directly verifying the antigen concentration once the dilutions have been made. The instability of highly diluted standard solutions can also be a problem and, as noted, the inclusion of protein in the medium is usually desirable to minimize denaturation. Problems in interpretation of course arise when the standard antigen itself is impure or was obtained from a heterologous species. Interassay variation can be a major problem. Large-scale collaborative studies in which immunoassay results have been compared in different laboratories indicate that the major source of immunoassay variation is interassay variation, which may be due to a variety of factors such as fluctuations in the quality of the radioiodinated antigen marker or unstable or improperly prepared antigen standard^.*^^^^ Many different ways have been suggested for treating radioimmunoassay data statistically. Frequently, laboratories use a logit-log transformation of the data to linearize the standard binding curve and permit easy comparisons among assays performed on different day~.'~-~O This method of calculation has been shown to be applicable to a large number of antigen-antibody systems. In general, there appears to be no real advantage to the use of more complex mathematical analyses, but in about 5-10% of assays four-parameter logistic models may have to be used.31 Regardless of what mathematical transformation is used it should be recognized that results are not as precise at the extreme ends of the binding curve. The complete automation of radioirnmunoassays has been described (see, e.g., Ref. 32), although the procedures are relatively complex and expensive, and are practical only in laboratories performing large numbers of assays.32 Validation of Immunoassay

Regardless of the immunoassay system that is used, the operational specificity and sensitivity of the system must be rigorously evaluated under the conditions in which it will be used. It cannot be assumed that immunoassays that give sensitive and reproducible results in buffer free of

'' W. M . , Hunter and I. McKenzie, J . Endocrinol. 79,49P (1978). C. W. Parker, Annu. Rev. Pharmacol. Toxicol. 21, 113 (1981). D. Rodbard, Clin. Chem. 20, 1255 (1974). 30 D. Rodbard, and J. E. Lewald, Acta Endocrinol. (Copenhagen)64,79 (1970). " D. Rodbard, in "Radioimmunoassay in Basic and Clinical Pharmacology," p. 193. Springer-Verlag, Berlin, 1987. 3Z G. Brooker, W. L. Terasaki, and M. G. Price, Science 194,270 (1976). 29

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serum or tissue proteins will necessarily give valid results in biological samples.' To a certain extent each new antigen-antibody system and each new tissue is an individual problem. For example, antiserum which specifically measures thromboxane B2 in serum cannot be used in urine without sample fractionation because of the presence of at least 20 thromboxane BZ metabolite^.^^ Tissue and blood samples may contain interfering rnolecules that degrade the unknown antigen or radiolabeled antigen marker, competitively bind the antigen, decrease antigen binding nonspecifically, exhibit expected or unexpected immunologic cross-reactivity, affect counting efficiency (primarily in assays involving P particle emitters), or interfere with the separation of bound and free Ag*. Antigenic reactivity may be lost either before or during the immunoassay. Careful attention must be given to the adequacy of recovery of the unknown substance from biologic samples. Samples in which the antigen is extensively degraded as it is processed obviously give misleading results. Storage of samples at -70" may be critical for adequate preservation of immune reactivity. In some systems insoluble debris in the tissue sample may be detrimental. Special problems may be observed with partially insoluble antigens as may be the case with recombinant proteins produced in organisms such as Escherichia coli. Falsely high or low immunoassay results may be obtained with these preparations. There may be effects on the assay due to alterations in pH or the presence of salts, organic solvents or detergents which affect antigen-antibody binding. These types of interference may be particularly misleading if samples that have not been processed identically are compared. Because effects such as these are especially common in crude tissue extracts and inhibition of radioactive antigen binding is normally interpreted as a high immunoassay value, during purification the amount of a protein may appear to decrease disproportionately to the actual losses that are occurring. Divalent cations, chelating agents, substrates, coenzymes, anticoagulants, protease inhibitors, antibacterial agents, and reducing agents also may affect immune reactivity depending on the antigen and the particular epitope(s) that is being recognized. While these changes may be minimized by an intelligent choice of reaction conditions, careful controls are needed. Nonspecific interference in the assay is particularly prone to occur when unusually small amounts of antigen are being measured. It is usually unwise to push the sensitivity of the system to its extreme limits. Analysis of the immunoassay system is greatly facilitated if the major forms of the antigen, its metabolites, and possible cross-reacting proteins " C.

Patrono, in "Radioimmunoassay in Basic and Clinical Pharmacology," p. 213. Springer-Verlag, Berlin, 1987.

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are already known, if procedures for their chromatographic separation are already established, if the purified proteins are available in reasonable quantities for labeling and as immunoassay standards, and if the amount of antigenic reactivity in tissue samples is sufficient in quantity and stability to permit its chromatographic behavior to be analyzed in some detail under representative assay conditions. Frequently, part of the purpose of the assay is to distinguish the antigen from structurally related proteins which may be immunologically crossreactive. Initially it is often necessary to physically separate the individual species and determine the contribution of each to overall immunologic reactivity. The antigen itself is often heterogeneous. Many hormones and enzymes are secreted in multiple molecular forms and exhibit immunologic cross-reactivity with structurally related proteins present at comparable or higher concentrations. Moreover, the protein of interest is frequently partially degraded by proteolysis and the fragments may or may not react significantly with the antiserum. Under these circumstances antisera recognizing different epitopes may give considerably different results. Procedures of established value in the validation of immunoassay results include the following: 1. Parallel studies in another assay system using a different principle of measurement (for example, a functional analysis such as measurement of enzymatic activity or a radioreceptor assay) over the full range of concentrations of interest 2. The use of internal standards (the addition of known amounts of purified unlabeled antigen to tissue samples) to see if the expected increase in measured Ag concentration is demonstrated. Superimposition of dilution curves in a linear plot of the data over at least a 100-fold range of concentrations should be seen 3. The use of enzymes or other proteins that can be expected to selectively alter the reactivity of the antigen in the assay8 4. Comparison of the slopes of the Ag dose-inhibition curves in the unknown sample and the standard 5. Demonstration that the immunoreactivity in the tissue samples comigrates with the antigen in question through a series of chromatographic purification steps or is removed under conditions in which the antigen is selectively adsorbed; evaluation of the yield of antigenic reactivity after fractionation both in the expected region and elsewhere in the chromatograph; wherever possible, identification of cross-reacting proteins migrating outside the major antigen region and a direct analysis of their absolute reactivities in the immunoassay if the purified proteins are

[53]

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available. Chromatographic verification is especially strong if several chromatographic systems are used 6. Use of small amounts of radioactive antigen to monitor its recovery during tissue extraction and purification. Instability of the radioactive antigen usually means that the unknown antigen is also unstable 7. Determination of the stability of the radioactive antigen during the immunoassay 8. Evaluation of samples in which the results of the assay are expected to be markedly positive or negative (for example, in animals in which the organ producing the protein has been surgically removed) or tissues subjected to a known pharmacologic stimulator or inhibitor 9. Exchange of samples between different laboratories making the same measurements 10. The use of antisera which may emphasize different epitopes in the same laboratory 11. Serial analyses of stored samples to determine how much decomposition may normally be occurring prior to the assay 12. Control measurements in the presence of pathologically altered tissue or blood samples Obviously the use of a number of these criteria in combination is stronger than any single criterion alone. Strategies to Reduce Immunologic Cross-Reactivity

Although cross-reactivity presents few if any difficulties in some immunoassays, it often places substantial limitations on the interpretation of the data and may be a major source of error. Thus, it is useful at this point to discuss some general types of antigenic cross-reactivity and consider possible practical solutions. For illustrative purposes the different kinds of immunologic cross-reactivity involving proteins fall into three major categories. One type is represented by two proteins, one with determinants A and B and the second with the identical determinant A and an unrelated determinant C. In this case, an antibody directed solely toward B should detect protein AB in the presence of protein AC with no demonstrable cross-reactivity. The problem, then, is to obtain an effective anti-B antiserum, not contaminated by anti-A antibody. The second type of cross-reactivity is illustrated by two proteins, AB and A'C. Here the only determinants that resemble one another are structurally similar, but not identical. In this case the presence of anti-A' antibodies may or may not be detrimental in measurements of AB, depending on the degree of similarity between A and A', although the use of a monospecific anti-B

718

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[531

serum may still be preferable. A third type of cross-reactivity is exhibited by proteins AB, A'B, or AB'. In this case, there is no non-cross-reacting determinant and it may be difficult or impossible to obtain a completely specific antiserum, although the inherent cross-reactivity between A and A' or B and B' may be reduced to a minimum by the proper selection of an antiserum. When a cross-reacting antiserum must be used, chromatographic fractionation of the sample or use of a less cross-reactive radioactive antigen marker may eliminate or minimize the problem. Simultaneous immunoassays in two cross-reacting systems occasionally are useful, but have to be interpreted with caution. Acknowledgment The author would like to thank Mrs. Carolyn Davinroy for her expert secretarial assistance.

Section XI1 Additional Techniques

[541

RADIOLABELING OF PROTEINS

72 1

[541 Radiolabeling of Proteins

The importance of the specific activity of the indicator molecule in developing a highly sensitive immunoassay has been discussed in detail elsewhere, but deserves continuing e m p h a ~ i s . ' Among .~ the commonly available radioactive isotopes for labeling proteins the half-lives range from 8 days (l3lI) and 60 days (12'1) to 12.3 years (3H)and 5730 years (I4C). The high specific activities obtainable for iodine isotopes largely explain the great popularity of radioiodine as an indicator molecule for proteins. As an alternative, enzyme immunoassays which utilize fluorogenic or chemilucinogenic substrates or are coupled to a second enzyme system for amplification can have comparable sensitivities and are increasingly being utilized. Only radioactively labeled proteins will be discussed. Radioactive Indicator Molecules

Based on specific activity considerations alone, 13'1 is theoretically preferable to 12'1. However, when other factors are considered such as potential radiation exposure to laboratory personnel, the rapid loss of 1311 radioactivity, the absence of p-radiation in 12jI, differences in counting efficiency, and relative isotope abundances in commercial radioiodine preparations (I3lI is 8 0 4 5 % 127~), 12'1 is almost always preferable to I3lI. As discussed in [53] of this volume, either the antigen or the antibody may be iodinated. The choice depends on a number of factors, such as the amount of antigen available, its stability and susceptibility to iodination, and possible special requirements of the assay. A variety of procedures are available for the introduction of 12'1 into proteins, involving either the direct iodination of protein amino acid residues or its introduction via a two-stage reaction involving a reactive carrier. Regardless of the iodination procedure, it is important that protein antigens not be altered too extensively. High levels of iodination, although theoretically desirable for assay sensitivity, often denature the antigen either through direct or indirect effects of iodination, decreasing its immune reactivity and stability and making the immunoassay less reliable.'

' C. W. Parker, "Radioimmunoassay of Biologically Active Compounds." Prentice-Hall, Englewood C l i s , New Jersey, 1976. C. W. Parker, Annu. Rev. Pharmacol. Toxicol. 21, 113 (1981).

METHODS IN ENZYMOLOGY, VOL. 182

Copwight O 1990 by Academic Press, Inc. All rights of reproduction in any form resewed.

722

ADDITIONAL TECHNIQUES

[541

As a practical rule of thumb, in the direct iodination methods, it is often optimal to introduce an average of a little less than one atom of iodine per molecule of antigen. This normally will ensure that most of the antigen molecules are iodinated while avoiding overiodination.' Two-stage iodination procedures may be less damaging to proteins, but here, too, overly aggressive iodination is often undesirable. While extensive information is available in the literature on iodination of proteins, each new antigenantibody system must be studied individually and the optimal levels and methods of iodination determined empirically. Regardless of the iodination procedure, precautions should be taken for the possible release of radioactive vapor which is a potential inhalation hazard during the period of time agents such as chloramine-T are active. Iodination reactions should be capped and performed in an efficient fume hood behind lead shielding. Once the desired iodination reaction is completed further iodination is normally inhibited with a reducing agent such as metabisulfite. Wherever possible the initial purification of the iodinated protein is carried out in the fume hood itself. The use of gloves (I2 readily penetrates the skin), a lead apron, and other protective clothing during iodination and initial purification is necessary to reduce exposure of personnel to external radiation. Radioactive iodine waste solutions should be earmarked and disposed of promptly following governmental guidelines. Good organization helps minimize radioactive contamination. All the reagents, pipets, and tubes needed to complete the iodination reaction should be available in an immediately usable form in the iodination hood. If a solution must be freshly prepared the solid reagent is preweighed, placed in a test tube, and dissolved in a previously measured volume of diluent at the time of the iodination. And, of course, a film badge is used to monitor radiation exposure. Direct Iodination Procedures

Direct iodination methods result in most of the radioactivity being incorporated into tyrosyl and histidyl residues. Losses of immune reactivity may be due to the direct steric effects of introduced iodine atoms on amino acids essential for binding, to oxidative damage occurring in the presence of various oxidized iodine species or the iodinating reagent itself, or to changes in protein charge associated with iodine substitution. Known side reactions involving effects of oxidized iodine on protein amino acids include oxidation of the thiol groups of cysteine to disulfide, of the disulfide group of cysteine to sulfonates, of the thioether group of methionine to sulfone, and of the indole group of tryptophan to ~ x i n d o l e In . ~ addition,

' N. M. Alexander, J . Biol. Chem. 249, 1946 (1974).

[541

RADIOLABELING OF PROTEINS

723

chloramine-T reacts directly with methionine residues. Other effects on protein immunoactivity occur through introducing I2'I onto the tyrosyl phenolic ring which reduces the pK, of the OH group from 10 to 8.4 (monoiodotyrosine) or 6.4 (diiodotyrosine). The pK, of iodinated histidylimidazole groups is reduced from 6.0 to 4.2 (monoiodohistidine) or 2.7 (diiodohistidine). Chloramine- T

When the protein to be iodinated is not unduly subject to inactivation by the iodination reagents, the time-honored chloramine-T method first described by Hunter and Greenwood in 1962 is often ~atisfactory.~ Chloramine-T, which releases hypochlorite ion in aqueous solution, is normally used with carrier-free inorganic 12'1- at neutral pH. ChloramineT introduces iodine onto tyrosyl and to a lesser extent histidyl and sulfhydry1 residues, probably through a cationic iodine intermediate. Chlorine is more electronegative than iodine and it converts iodine to a positively charged form that is active in electrophilic substitution reactions.' Related procedures which avoid using aromatic chloramine molecules as oxidizing agents involve the use of Iodogen (see below), hypochlorite, or chlorine gas. The advantages of the chloramine-T method include its reproducibility, rapidity, efficiency of the iodination in the absence of carrier nonradioactive iodine, and avoidance of extremes of pH or organic solvents which might denature the protein. However, some proteins undergo substantial denaturation during iodination with chloramine-T as detected by altered electrophoretic mobility, aggregation, or loss of antigenic reactivity. Although a careful adjustment of the iodination conditions may minimize the iodination damage, it may be preferable to use other methods. Some protein tyrosyl residues are not readily susceptible to iodination by the chloramine-T method due to hydrogen bonding or sequestration in a hydrophobic region. Reactivity may be increased by iodination at a higher or lower pH, in the presence of a water-miscible organic solvent such as dimethylformamide, or with a reversible protein-denaturing agent such as urea present. A representative protocol6 for chloramine-T iodination is given below. 1. Mix the following: 12'1 [I mCi (10 PI)] W. M. Hunter and F. C. Greenwood, Nature (London) 194,495 (1962).

' J. Grassi, J. MacLouf, and P. Pradelles, in "Radioimmunoassay in Basic and Clinical Pharmacology ," p. 91. Springer-Verlag, Berlin, 1987. G. T. Peake, in "Methods of Hormone Radioirnmunoassay," p. 103. Academic Press, New York, 1974.

724

ADDITIONAL TECHNIQUES

[541

0.5 M phosphate buffer, pH 7.6 (100 pl) 20 pg protein (20 p1) 180 pg chloramine-T (50 pl): Chloramine-T and metabisulfite solutions are prepared immediately before use by adding the appropriate volume of buffer to the preweighed solid 2. Stir 20-30 sec at room temperature and add 500 pg sodium metabisulfite (50 pl). 3. Immediately apply to a Sephadex G-50 spin column packed in 0.15 M NaCl-0.01 M phosphate (PBS) as described below. 4. Centrifuge, collect, and pool eluate of iodinated protein. 5. Dilute in PBS containing 10 mglml bovine serum albumin. 6. Store at 4" (short-term use) or freeze. A number of important variables affect the iodination: 1. Chloramine-T concentration: The quantity of chloramine-T must be adequate to neutralize the small amount of reducing agent sometimes I to allow for possible side reactions with protein, present in the N ~ " ~and as well as to oxidize the I-. The concentration of chloramine-T used above is representative of most iodination procedures. Somewhat surprisingly, it often takes more chloramine-T during iodination with carrier-free lZ5Ithan with equivalent quantities of 1 2 7 ~ . 7The level of protein iodination is affected by the concentration and duration of exposure to chloramine-T. Both are kept to a minimum so as to avoid nonspecific chloramine-T damage to the p r o t e h 8 Too high a concentration of chlorinating agent may also promote iodate formation due to overoxidation, decreasing the efficiency of i ~ d i n a t i o n . ~ 2. Protein concentration: Iodination efficiency is markedly affected by protein concentration. Reactions are carried out in a small (30 to 200 p1) volume, which permits the iodination of minute amounts of protein at sufficiently high protein concentrations to achieve an efficient utilization of radioiodine. In the example shown, the quantity of protein (20 pg) and '251 radioactivity (1 mCi) was selected to provide a product of high specific activity. For proteins with a moderate tyrosine content, incorporation of iodine falls from nearly 100% at 1 mg proteinlml to 60 to 70% at 50 pg p r ~ t e i n l m l . 'Major ~ problems arise in attempting to iodinate < I pg be-

' W. M. Hunter, Br. Med. Bull. 30, 18 (1974). R. S. Yalow and S. A. Berson, in "Principles of Competitive Protein-Binding Assays" (W. D. Odell and W. H. Daughaday, eds.), p. 374. Lippincott, Philadelphia, Pennsylvania,

'O

1971. M. R. Redshaw and S. S. Lynch, J . Endocrinol. 60, 527 (1974). W. M. Hunter, in "Handbook of Experimental Immunology" (D. M. Weir, ed.), 2nd Ed., p. 17.1. Blackwell, Oxford, 1973.

[541

RADIOLABELING OF PROTEINS

725

cause of the reduced efficiency of the reaction and difficulties in avoiding overiodination and loss of antigenic reactivity, although some possible guidelines have been ~uggested."~'~ For reasons that are not well understood, iodination in the presence of 2-4 M dimethyl sulfoxide is occasionally helpful in this situation, probably because oxidation of methionine is inhibited.I3 3. ['25~] Iodide concentration: About 1 mCi 1251is typically used for protein iodinations, but this may vary widely depending on the experimental requirements.14 In order to obtain high levels of radioactivity while keeping the reaction volume to a minimum, concentrated solutions of radioiodine (100 mCi/ml or higher) are used, thus permitting the addition of 1 mCi or more in a volume of only 10 p1. Stock 1251 solutions that have been stored for more than 10 days are usually not used for further iodinations. 1251-is supplied as a solution either in dilute NaOH or at neutral pH. Most investigators now use preparations stored without reducing agent. Maintenance at high pH and low temperature partially stabilizes the lZ51.Occasionally, lZ51 preparations iodinate proteins ineffectively, probably because unusually large amounts of iodate are present.5 Since chloride is a halide ion and is susceptible to oxidation some investigators have avoided having chloride ion in the reaction mixture. However, chloramine-T oxidizes I- much more efficiently than CI- at neutral pH and satisfactory iodination is obtained, even at CI- concentrations of 0.1 M or higher. 4. Iodination pH: In the past, concentrated solutions of 1251 obtainable commercially contained significant concentrations of NaOH (up to 0.1 M), and the protein had to be especially well buffered before the radioiodine was added. As already noted, neutralized solutions of ' 2 5 ~are now avail~ the use of 0.25 to 0.5 M phosphate, pH able. Even with basic 1 2 5 solutions 7.2 to 7.8 (or borate, in the pH 8.0-9.0 range), provides adequate buffering capacity while bringing the pH into a range that is appropriate for iodination. The optimal pH for iodination varies, but for most proteins a pH between 7.3 and 7.8 provides effective labeling. 5. Time and temperature: Oxidation of iodine takes place rapidly after the introduction of chloramine-T into the iodination mixture, and rapid mixing is needed to prevent uneven labeling. The reaction is allowed to proceed for anywhere from a few seconds to 10 min at 0 to 24" (usually 1 min or less). 6 . Termination of iodination: While marked dilution of the reaction R. A. Caro, V. A. Ciscato, S. M. V. De Giacomini, and S. Quiroga, Int. J. Appl. Radiat. Isor. 26,527 (1975). l2 J. R. Panuska, Year Immunol. 1985-86 2,97 (1986). l3 E. R. Stanley and L. J. Guilbert, J. Immunol. Methods 42,253 (1981). " F.C. Greenwood and W. M. Hunter, Biochem. J 89, 114 (1963).

I'

726

ADDITIONAL TECHNIQUES

1541

mixture inhibits further iodination, as a rule an excess of a reducing agent, usually sodium metabisulfite, is added to stop the reaction. Reduction at this stage helps minimize release of volatile IZ51 from the reaction mixture. A 1.2- to 1.5-fold molar excess of metabisulfite over chloramine-T is sufficient to reduce all the unreacted chloramine-T and reactive iodine species. 7. Purification: Once the chloramine-T has been reduced, carrier heterologous protein can be added directly to the iodination mixture. Purification is carried out immediately after iodination in order to remove unconjugated iodine oxidation products, free iodide, and reducing agent. The separation can be accomplished by various methods as discussed below. Lactoperoxidase

A very useful approach to protein iodination involves use of the oxidizing enzyme, lactoperoxidase, a protein obtained from unpasteurized bovine milk.'5-'6Iodination is principally on tyrosyl residues, although histidyl and sulfhydryl groups may also participate. The reaction involves three substrates-peroxide, iodide, and the phenolic target that is being i ~ d i n a t e d . ' ~The . ' ~ efficiency of iodination is determined by the absolute concentrations of the target protein, the enzyme, hydrogen peroxide, and iodide. Other peroxidases can also be used for iodinating proteins, but are usually less satisfactory than lactoperoxidase. The ease with which soluble proteins are iodinated with lactoperoxidase varies considerably with the protein, reflecting in large measure the extent of association between the enzyme and the protein. In favorable situations the iodination efficiency approaches 100%. Enzymatic iodination is a less random process than chemical iodination and is favored in regions of the target protein that contain tyrosyl residues that are sterically available to the enzyme. Since the iodinating species is generated in the active site of the enzyme which is in turn bound to the protein being iodinated, oxidative damage to the protein as a whole is minimized. In addition to selecting for accessible tyrosyls, use of a peroxidase also markedly increases the probability of obtaining monoiodo, rather than both mono and diiodotyrosyl, on the protein, as occurs with chloramine-T. Thus, enzymatically labeled proteins will usually be labeled more homogeneously than chemically iodinated proteins. l5 l6

''

J. J. Marchalonis, Biochem. J. 1l3, 299 (1969). M. Momson, G . S. Bayse, and R. G . Webster, Zmmunochemistry 8,289 (1971). M. Momson, this series, Vol. 70, p. 215.

[541

RADIOLABELING OF PROTEINS

727

Comparative studies have indicated that the lactoperoxidase method is as satisfactory as and sometimes preferable to the chloramine-T method for labeling of many proteins.7~'8-20The selectivity of lactoperoxidase iodination may be an important advantage with proteins that are unusually subject to damage during chloramine-T iodination. Enzymatic iodination may also be useful in the iodination of histidyl r e s i d ~ e s . ~Iodination ' of imidazole groups does not occur readily in the chloramine-T method and high levels of alkalinity and oxidant are generally required. For many protein antigens there is little to choose between the two methods. The lactoperoxidase method sometimes fails to provide effective iodination, presumably because the only available tyrosyl residues are inaccessible to the enzyme. A representative iodination using l a ~ t o ~ e r o x i d a sise 'shown ~ below:

1. Mix the following: 1.8 mCi (15 pl) 5 pg protein in 0.05 phosphate buffer, pH 7.5 (25 p1) 4 pg lactoperoxidase in the same buffer (1.5 pl) 0.88 mmo.1 H202 (1 pl). 2. Stir 60 sec at room temperature. 3. Add0.5 p1 of 0.05 M phosphate, pH 7.4. 4. Immediately purify by gel filtration. Important variables in this reaction include the following: 1. Lactoperoxidase preparation: In general, relatively crude commercial preparations of lactoperoxidase are satisfactory, although their use does increase the quantity of contaminating protein in the reaction mixture. Ideally, the lactoperoxidase should be freshly dissolved and diluted, although if concentrated solutions of the enzyme are stored under optimal conditions, most of the iodination activity is preserved for at least several days. There are also potential problems due to variation in commercial lots of lactoperoxidase or changes in activity of the lyophilized protein with age, and it may be desirable to determine the activity of the lactoperoxidase directly. Problems can also occur if the enzyme is contaminated with proteases. The lactoperoxidase itself can be iodinated-in effect, introducing an extraneous iodinated protein into the antigen preparation.19For H. Pinto, A. C. Lerario, I. Torres de Toledo e Souza, B. L. Wajchenberg, E. Mattar, and R. R. Pieroni, Clin. Chim. Acta 76,25 (1977). l9 J. I. Thorell and B. Johansson, Biochim. Biophys. Acta 251, 363 (1971). 20 Y. Miyachi and A. Chrambach, Biuchem. Biophys. Res. Commun. 46, 1213 (1972). K. N. Holohan, R. F. Murphy, R. W. J. Flanagan, K. D. Buchanan, and D. T. Elmore, Biochim. Biophys. Acta 322, 178 (1973).

728

ADDITIONAL TECHNIQUES

[541

this reason insolubilized preparations of lactoperoxidase (coupled to Sepharose by the cyanogen bromide reaction) are sometimes used in order to facilitate the elimination of the enzyme after the iodination reaction is completed.22 Alternatively, the lactoperoxidase may be physically removed by chromatography after iodination. Normally, however, the target protein is labeled so much faster than lactoperoxidase, even at high lactoperoxidase concentrations, that self-iodination of lactoperoxidase is not a problem. With an antigen that labels inefficiently, use of a high antigen concentration, together with a 100-fold lower concentration of lactoperoxidase, may help to minimize the iodination of the enzyme. 2. Hydrogen peroxide: The H202can be added as such or generated in the iodination solution by the reaction of glucose oxidase with D-glucose. If glucose oxidase is used, the glucose solution should be prepared several hours in advance to permit mutarotation of the glucose to the form that is acted upon by the enzyme. As a rule, the quantity of H202added can be used to control the level of iodination, with close to 1 mol of iodine being incorporated per mole of H202.However, some preparations of radioiodine contain small amounts of H202, thus resulting in higher levels of iodination than had been calculated. This is more likely to be a problem with large amounts of 12'1. H202 is unstable in dilute solution and is therefore stored as a concentrated 30% solution at 4" and diluted in water just before use. 3. Protein: Concentrated solutions of protein comparable to those used in the chloramine-T reactions are normally employed. Since catalases are widely distributed in tissue and react rapidly with H202,the possibility that trace amounts are present in the incubation mixture and are competing with lactoperoxidase may need to be considered when impure proteins are being labeled. The antigen solution must be free of reducing agents that interfere with lactoperoxidase activity. Azide, for example, is frequently used as a preservative for protein and is a potent inhibitor of lactoperoxidase. l9 4. Radioactive iodine solution: Iodination with carrier-free ' 2 5 ~is possible, but often rather inefficient, and frequently carrier '"1- at a 2-4 pM final concentration is used to increase the total I- concentration. For reasons that are not entirely clear, difficulties seem to occur in catalytic iodination with certain lots of radioactive iodine. 5. pH and time: In general, efficient iodination is obtained over a pH range from 3.0 to 8.0, which permits the selection of a pH that gives optimal selectivity of labeling, depending on the protein or peptide. The G . S. David, Biochem. Biophys. Res. Commun. 48,464 (1972).

RADIOLABELING OF PROTEINS

liI

729

rate of reaction with tyrosine and most proteins is maximal at pH 4.5 to 5.0, but with certain proteins the optimum may be nearer neutrality.16 Most proteins can be labeled at neutral pH. Under optimal conditions the introduction of iodine into proteins is rapid, even exceeding the rate of iodination with chloramine-T. Lactoperoxidase has been shown to catalyze the incorporation into bovine albumin of up to 6.5 x lo3 mol of iodinelmol proteinlmin (at pH 7.4).'6323Iodination is usually done at room temperature or 37", but can be done at 4". 6. Termination of iodination: The reaction can be stopped or markedly retarded by adding 10 or 20 vol of cold buffer, followed by chromatographic fractionation. If instantaneous inhibition is required, a reducing agent such as cysteine or azide can be included in the diluent. If labeling of the enzyme is a problem and the enzyme cannot be eliminated by gel filtration, its basic isoelectric point usually facilitates its removal.

Iodine Monochloride Iodine monochloride (ICl) is a very effective iodinating agent for proteins. In the originally described ICI technique the I moiety of IC1 was made radioactive by isotope exchange of ' 2 5 ~ - with nonradioactive ICl.24 The exchange is so rapid that efficient protein labeling is obtained when '27~Cl is added to a well-buffered solution of the protein and 12'1- at pH 8.5. Since ICI is stable at low pH this is a rapid and simple method of labeling proteins with 12'1. The major drawback is that the use of lZ7ICl necessitates dilution with corresponding sacrifices in specific activity. However, in the adaptations of Helkamp and his colleagues and of Doran and Spar, 12'1 is generated from I2'I- by oxidation with 103-in an aqueous In excess acid the solution containing HCI and NaCl (as a ~tabilizer).~'-~~ original lz7I-is oxidized quantitatively to ICI as shown in Eq. (1). Na12' IO,

+ 2NaI2'I- + 6HCI -, 3NaCI + 21251C1+ I2'IC1 + 3H20

(1)

AU the radioactivity is thus theoretically available in a reactive form at 66% of the original specific activity. As a rule, however, at least some carrier I2'I- is used, reducing the specific activity. Detailed procedures for iodin-

* M. Monison and G. S . Bayse, in "Oxidases and Related Redox Systems" (T. E. King, H. S. Mason, and M. Momson, eds.), Vol. 1, p. 375. Univ. Park Press, Baltimore, Maryland, 1974. E. Regoeczi, "Iodine Labeled Plasma Protein." CRC Press, Boca Raton, Florida, 1984. 2J R. W. Helkamp, M. A. Contreras, and M. J . Izzo, Int. J . Appl. Radiat. Isot. 13,747 (1967). D. M. b r a n and 1. L. Spar, J . Irnrnunol. Methods 39, 155 (1980). M. A. Contreras, W. F. Bale, and I. L. Spar, this series, Vol. 92, p. 277.

730

ADDITIONAL TECHNIQUES

[541

ation of proteins by exchange with '27~Cl and by oxidation of total lZ51to '25~Cl using 50 mCi or more of 1251 are given in an earlier volume of this series (volume 92). An iodination procedure using Iz5~Clobtained by oxidation of much smaller quantities of ' 2 5 ~is given below. The 125~Cl is generated in the absence of protein and then added with rapid mixing to the buffered protein solution. The efficiency of iodination is considerably lower than in the larger scale oxidative IC1 procedure, but the use of much smaller amounts of 1 2 5 ~ - and relatively little or no 1271is a considerable advantage. 1. Add in the order shown: 1.8 mCi IZ5I-(dried down) 10 p12 mM KI03 10 ~ 1 M3NaCl 5 HCl. 10 ~ 1 0 . 1 M 2. Cap and agitate briefly. 3. Wait at least 10 min. 4. Using a Hamilton syringe, add the above solution over a 1-min period to an efficiently stirred solution of 50 pg of protein in 0.5 mlO.l M borate, pH 9.0. 5. Purify by gel filtration. Important variables are as follow: 1. IC1 solution: The reagents should be added in the order given. If carrier '271- is present, at least a 5-fold molar excess of 103- over total lZ5I-and '271- should be used to ensure effective oxidation of I-. ICl can be generated in larger volumes (-0.5 ml) using the same final concentrations of HC1 and NaCl as in smaller scale reactions. The larger volume facilitates rapid mixing at the time the IC1 solution is added to the protein. Effective iodination is obtained as long as the protein solution has enough buffer to neutralize the substantial amounts of acid that are present. The IC1 that is generated is stable in the presence of NaCl and acid but prolonged storage is undesirable because the lZ51Clmay eventually exchange with lZ71O3-. 2. Reaction with protein: Although IC1 is a workable iodinating agent at acidic pH, most reactions with protein are carried out at pH 8 to 9. Typically, a borate buffer is used. Iodinating activity persists for less than 1 min at this pH. Because the reaction with protein is extremely fast, immediate and thorough mixing is required as the addition is being made. Very rapidly adding a relatively large volume of IC1 solution into a small volume of protein solution facilitates rapid mixing (for example 0.5 ml of IC1 solution may be added to 0.1 ml of protein solution). Volatile radioac-

t

F

;= 'Y 5"r

-

i

T i

-

IT-

--

-7?

-

7 I

1541

RADIOLABELING OF PROTEINS

tivity is considerably less of a problem than in the chloramine-T method and the reaction is usually not quenched with a reducing agent.

lodo-gen Iodo-gen, (1,3,4,6-tetrachloro-3a,6a-diphenylglycoluriI, Pierce Chemical Co.) is a chloramine virtually insoluble in water which can be prepared as a thin film in the iodinating v e ~ s e l . *When ~ * ~ ~an aqueous solution of ' 2 5 ~ - and protein is added, iodination is rapidly initiated. On a weight basis Iodo-gen is about 2.6 times more potent than chloramine-T in terms of iodinating efficiency. However, due to losses of active chlorine during storage of the glycouril, somewhat lower values may be obtained. Since Iodo-gen is immobilized on the wall of the reaction vessel direct exposure of the protein to the oxidant is minimized. The iodination shows the same dependency on protein concentration as seen in soluble chloramine reactions. The reaction vessel can be made of any material that does not significantly adsorb the protein to be iodinated. It preferably has a flat bottom in order that its contents can be stirred magnetically.24 Screwcapped borosilicate vials (Sargent-Welch Co., Skokie, IL) of 12-mm i.d. provide a flat surface area of 113 mm2 and are suitable for most protein iodinations. For vials of this size the Iodo-gen is usually dissolved in 0.1 ml chloroform or dichloromethane. This volume of solvent is optimal for coating. The solvent is allowed to evaporate spontaneously; forcible drying may cause flaking.29It is practical to coat a large number of vials at the same time and store them in a dry and dark place.28Components of the iodination reaction (radioiodide, protein, buffer, and carrier I-, if desired) are mixed in another tube before transfer to the reaction vial. The vial is placed on a magnetic stirrer and the reaction mixture is added. Reaction times of 5-10 min usually are suitable. The reaction is terminated by removing the fluid from the vial and adding a reducing agent. Simply removing the protein solution from the iodination vial does not terminate the reaction since various species of oxidized iodine are still present in solution.

Other Direct Labeling Methods Other direct iodination procedures found to be effective in individual instances, but not widely used, include utilizing an insolubilized chloramine (an N-chlorobenzyl sulfonamide) attached to polystyrene beads

t

LI-

IS 9'

3

-

73 1

P. J . Fraker and J . C. Speck, Biochem. Biophys. Res. Commun. 80,849 (1978). K. H. Sullivan and R. P. Williams, Anal. Biochem. 120,254 (1982).

732

ADDITIONAL TECHNIQUES

[541

(Iodobeads), electrolysis, polyiodide, volatilized IZ, or thallium trichloride. Two-Stage Labeling Methods

In two-stage labeling methods a low-molecular-weight carrier is iodinated in the absence of the protein and then combined with the protein via a reactive group which forms a stable bond with afunctional amino acid. The iodinatable carrier ordinarily contains a phenolic residue as the site for iodination, but an imidazole is also ~uitable.~' The groups reacting with protein normally used in two-stage iodinations are active esters, aldehydes, and imidoesters, all of which interact predominantly with lysyl residues. Most sizable proteins contain a number of lysyl residues which are available for reaction. The most commonly used two-stage reagent is 3-(p-hydroxypheny1)propionic acid-N-hydroxysuccinimide (HPPS), which was introduced as an iodine carrier by Bolton and H ~ n t e r . HPPS ~' has both an easily iodinatable phenolic hydroxyl group and an active ester through which it attaches to lysyl residues. One disadvantage of this method is the loss of positive &-ammoniumcharges as amino groups are substituted. This decrease in positive charges makes the protein more acidic and is accentuated by the negative charges introduced on iodophenolic residues and may lead to substantial decreases in immune reactivity. These effects of charge are superimposed on possible losses in immune reactivity due to substitution of essential lysines or to steric effects of the introduced groups. A two-stage labeling method that avoids losses of positive charges involves using an aliphatic or aromatic aldehyde group for conjugating iodophenolic residues to protein. The aldehyde moiety reacts with protein amino residues to form a reversible (Schiff base) bond which is stabilized by reduction to a secondary amino group with cyanoborohydride or borohydride. The secondary amino group is predominantly in the cationic form at neutral pH, so changes in net protein charge are considerably less marked. Iodinated imidoesters also react with protein Lysyl residues, but preserve the positive charges. Other possibilities include the use of diazotized aniline, iodosulfanilic acid, or a modified Bolton and Hunter reagent with a blocked amino group that is unmasked after conjugation to p r ~ t e i n . ~ ' By comparison with the direct iodination methods, the major drawback S. L. Jeffcoate, Pathol. Biol. 23,903 (1975). A. E. Colton and W. M. Hunter, Biochem. J . 133,529 (1973). R. K. Assoian, P. M. Blix, A. H. Rubenstein, and H. S. Tager, Anal. Biochem. 103, 70 (1980).

RADIOLABELING OF PROTEINS

3

733

to the two-stage procedures is the need for relatively large amounts of radioactive iodine and the somewhat lower specific activities of the final iodinated product usually obtained. On the other hand, there are a substantial number of proteins that can be labeled with the indirect method that The indirect procedure can also be used could not be labeled to iodinate polypeptides, proteins, and low-molecular-weight chemicals which have no tyrosine, but do have amino groups. Active Ester (HPPS) The N-hydroxysuccinimide ester is available commercially either un~ Cilmmol, Amersham, New Englabeled or iodinated with 1 2 5 (1500-4000 land Nuclear). Its preparation, physical properties, and iodination prior to its reaction with protein have been described in detail.31HPPS is stored dry to prevent hydrolysis. It is soluble in water. HPPS is highly reactive with protein amino groups at or near neutral p ~ , 3 5 , 3although 6 other nucleophilic groups may participate to a limited extent. Specific activities as high as 170 pCilpg have been achieved using HPPS.37 A representative procedure for iodination with HPPS is given below.

: t

Reaction with Protein 1 . Cool a vial containing 0.2 pg ['25~]HPPS to O". 2. Add a cold solution of protein (e.g., human growth hormone), 5 pg in 10 pl of 0.1 M borate buffer, pH 8.5. 3. Agitate the vial gently and allow the reaction to proceed for 15 min at 0". 4. Add 0.5 ml of 0.2 M glycine in 0.1 M borate buffer, pH 8.5, and agitate for 5 min. 5. Purify the iodinated protein by gel filtration on Sephadex G-50 (fine),wet packed, eluting with 50 mM phosphate buffer, pH 7.5, containing 0.25% gelatin or on a spin column (see below). 6. In the procedure given the 0.2 pg of ['2SI]HPPSis equivalent to 3.2 mol of ester/mol of protein. The mean incorporation of Iz5Iis about 30%. To iodinate the protein it is added at 4" in a minimal volume of buffer that ensures complete contact between the solution and the dry residue in

r. r

194,855 (1976).

33

R. Roberts, B. E. Sobel, and C. W. Parker, Science

35

G. W. Anderson, J. E. Zimmerman, and F. M. Callahan, J. Am. Chem. Soc. 86, 1839

" R. Roberts and C. W. Parker, this series, Vol. 74, p. 198. (1964).

J. Rudinger and U. Ruegg, Biochem. 1. 133,538 (1973). 37 J. J. Langone, this series, Vol. 70, p. 221. 36

734

ADDITIONAL TECHNIQUES

[541

the vial. Ideally, a protein concentration of 100 pg/ml or higher is used to increase the efficiency of coupling. At this protein concentration the efficiency of the reaction of HPPS with protein may be as high as 50%, but varies depending on the number of protein amino groups and their reactivities. At pH 8.0 the reaction is essentially complete within a few minutes. Since the ester hydrolyzes rapidly at this pH, the use of long reaction times does not appreciably improve reaction efficiency. The reaction is terminated by the addition of an excess amount (typically a 0.1 M final concentration) of a small molecular weight competitor, such as glycine. The reaction products are usually separated by gel filtration. Because of the affinity of iodohydroxyphenylpropionic acid for albumin, gelatin is preferable to albumin as a carrier during purification and in an immunoassay.

Aldehyde (PHAA) Methods using [3H]- or ['4C]formaldehyde for labeling proteins work well when a high specific activity is not needed.38q39Procedures for introducing an iodinated aldehyde into proteins using a phenolic ring attached to an aliphatic or aromatic aldehyde have been used less extensively but appear to have considerable potential. Since aliphatic aldehydes such as p-hydroxyphenylacetaldehyde (PHAA) are more reactive than aromatic aldehydes they permit more efficient protein labeling.40A procedure developed in our laboratory using this reagent is given below: 1. Iodinate 20 nmol HPAA in 10 pl 1 M HEPES, pH 7.4, with I2'~Cl prepared similarly to above, but using 2 mCi I2'I-, 20 nmol 12'1, 3 p l 5 M NaC1, 3 p10. I M K I 0 3 , and 1 pl0.5 M HC1. 2. Purify the [ ' 2 5 ~ ] i o d o - ~ ~on AA Sephadex LH-20 (0.5 x 1 cm) preequilibrated with 0.1 M ammonium acetate, pH 6.0. Wash with 10 p10.1 M ammonium acetate to remove unreacted iodine. Elute [IZ5I]HPAAwith 10 rnM NH40H in 50% methanol, collecting 0.2-ml fractions and pooling on the basis of radioactivity. Using the above iodination conditions up to 0.8 atoms of iodine/molecule of aldehyde is incorporated. 3. React 140 pg protein, 15 p M [ ' 2 5 ] ] ~ ~ Aand A , sodium cyanoborohydride (20 mM in 10 p10.1 M HEPES, pH 7.6) for 16 hr at room temperature. Maintain under nitrogen in rubber-stoppered tube until the reaction is completed 4. Purify by gel filtration. J. Jentoft and D. G . Dearborn, J. Biol. Chem. 254,4359 (1979). R. H. Rice and G . E. Meirns, J. Biol. Chem. 246,831 (1971). 40 J. R. Panuska and C. W. Parker, Anal. Biochem. 160, 192 (1987). 39

[541

RADIOLABELING OF PROTEINS

Removal of Uncoupled 12'

555-

-

7

-

?r

7

4$

--

735

and Storage

Normally, once an iodination reaction has gone for the prescribed time the iodinating agent is inactivated and a carrier protein (gelatin, lysozyme, ovalbumin, or serum albumin) is added to a final concentration of 0.1 to 1 mglrnl. The carrier protein reduces radiation damage during storage by scavenging free radicals and it also decreases nonspecific adsorption to glassware or plasticware. NaI2'I is also often added to a final concentration of 0.01 M or higher to facilitate exchange with IZ51- bound reversibly to proteins in the iodination mixture. The total volume is usually kept below 0.2 ml at this stage to facilitate subsequent purification. The labeled antigen is then rapidly purified to remove unconjugated radioactivity (and in some systems damaged antigen), usually on a short molecular sieve column. Any gel filtration column long enough to separate the iodinated protein from free Iz51- is usually suitable for the separation. A particularly convenient procedure described by Tuszynski et al. involves the rapid centrifugation of the protein iodination mixture through a disposable column containing Sephadex G-25.4' Aside from requiring that the preparation be brought out of the iodination hood, the only disadvantage of this procedure is that no separation of different iodinated protein species from one another is possible, as may be the case for some proteins on ordinary gel filtration columns.42 1. Pack the tip of a 1.5-ml disposable polypropylene microcentrifuge tube (Bio-Rad) with glass wool. These tubes contain a hole in the bottom to allow the eluted buffer to pass through and collect in the centrifuge tube. 2. Add a thick slurry of Sephadex G-25 (1.5 ml packed resin), equilibrated overnight at room temperature in 0.15 M NaC1,0.01 M phosphate, pH 7.4 (PBS) containing 0.1 ml of a 10 rnglrnl solution of the protein used to dilute the iodination mixture. 3. Place in a 12 X 75 mm centrifuge tube (Falcon 2058) and pack by centrifugation at 200 g for 1 min. Discard eluted buffer. 4. Apply iodination mixture (<0.2 ml) and recentrifuge, collecting the eluted protein. 5. Dilute the protein further in PBS containing protein (or another buffer, depending on the storage conditions that are optimal for the protein) and store frozen in small aliquots. G. P. Tuszynski, L. Knight, J. R. Pipemo, and P. N. Walsh, Anal. Biochem. 106, 118 (1980). 42 J. H. Walsh and H. C. Wong, in "Radioimmunoassay in Basic and Clinical Pharrnacology," p. 315. Springer-Verlag. Berlin, 1987.

736

ADDITIONAL TECHNIQUES

[541

Polystyrene-based ion-exchange resins have also been used for purification either in a batch procedure or as a column. Since I- and most of the oxidized iodine species are negatively charged they bind readily to anionexchange resins. However, some iodine species may remain unbound. Ion-exchange resins, such as DEAE- and CM-cellulose, which fractionate proteins on the basis of charge, have been used to separate different species of the same iodinated protein from one another.43 The protein is often diluted further after purification and stored frozen in small aliquots to avoid repeated thawing and refreezing. The possible release of I2'I2 during storage due to breakages of C-I bonds should be considered. Pepper recommends storing p-thromboglobulin iodinated with the Bolton and Hunter reagent in 30% (wlv) sucrose at liquid nitrogen

temperature^.^^ Evaluation of Activity

Proteins may be labeled for use in immunoassays, as substrates for degradative enzymes, in binding experiments with receptors or other binding proteins in tissues, or to determine their distribution and metabolic fate in vivo. Regardless of the purpose, it is important to minimize denaturation and biologic inactivation. Initially, several different iodination methods are compared and the one providing the best combination of sensitivity, specificity and stability is chosen. In evaluating the level of iodination it is useful to keep in mind that 1 mCi of 1 2 5 ~ - theoretically is approximately 0.46 nmol of I. In commercial 1251preparations (-17 Cilmg) 1 nmol lZ5Iis about 0.5 nm01.~3~'When small quantities of protein are being labeled, usually it is assumed that all of the protein is recovered after purification. Since unknown quantities of protein may be lost during purification and significant quantities of weakly bound lZ51may remain, the actual level of protein labeling is usually not known precisely . I 1 The analysis of a labeled protein usually includes a determination of its biologic, biochemical, or immunologic activity after labeling. It is also useful to determine its susceptibility to inactivation during storage or incubation with tissue.' If a binding assay is involved, the specificity and sensitivity of binding under the usual conditions of assay is analyzed. When dealing with a protein being used for the first time in studies of binding, it may be desirable to begin by tagging it lightly with ' 2 5 ~(approximately 1 iodine atom110 or 20 protein molecules) to minimize the possi43

45

S. Linde and B. Hansen, Int. J. Pept. Protein Res. 6 , 157 (1974). D. S. Pepper, in "Radioimrnunoassay in Basic and Clinical Pharmacology," p. 517. Springer-Verlag, Berlin, 1987. R. I. Cam, M. Mansour, D. Sadi, H. James, and J. V. Jones, J . Immunol. Methods 98,201 (1987).

is41

RADIOLABELING O F PROTEINS

737

bility of reduction in binding reactivity during labeling.7 The labeled protein can then be used as an indicator molecule to detect losses in binding activity in other preparations of the protein that are exposed to '271 under more severe iodination conditions. Even though radioactive protein binding is normally maintained in the 40 to 50% range in binding assays in order to maximize sensitivity, it is important at some point to establish that at least 80 to 90% binding is achievable when larger quantities of antibody or binding protein are used. This analysis provides evidence against major losses in protein reactivity as a result of iodination or nonspecific denaturation. Another useful approach to detecting changes in protein binding is to determine if the radioactive and native (uniodinated) proteins have the same affinities for binding. Various dilutions of the binding protein are incubated with several relatively high concentrations of iodinated protein either alone or as a mixture of iodinated and uniodinated protein at the same total protein con~entrations.'~ If the percentage of unbound radioactivity is the same with labeled protein alone as in the labeled-unlabeled protein mixture, it is unlikely that significant iodination damage has occurred. If changes occur, they are more likely to be seen in regions of the binding curve where competition is maximized. Usually, the basis for differences in radiolabeled protein binding is not known, although examples of different iodination methods labeling a protein on different tyrosines are known.46 Even after an iodination method has been selected each new labeled protein preparation must be evaluated both initially and in subsequent assays for its experimental suitability. In some protein systems repurification of labeled protein preparations may be necessary on a weekly or even daily basis. Even with iodinated proteins which are normally stable for many weeks, individual preparations may be obtained which deteriorate much faster. In immunoassay applications antibody selection may be very important. Antibodies to the same antigen may differ markedly in their abilities to recognize the iodinated antigen, particularly if the iodinated antigen is readily denatured. Other analyses are useful in evaluating labeled proteins, particularly in the early stages of study. They include determination of precipitability with trichloroacetic acid and of radiohomogeneity in SDS or isoelectric focusing polyacrylamide gels or on gel or ion-exchange columns. It is not unusual for contaminating proteins to be labeled disproportionately to their content in the iodination mixture. This analysis may also detect marked changes in chromographic or electrophoretic behavior due to aggregation or other forms of denaturation.

* B. Postmann, T. Postmann, E. Nugel, and U. Evers, J . Zmmunol. Methods 79,27 (1985).

738

ADDITIONAL TECHNIQUES

[551

[551 Using Purified Protein to Clone Its Gene

At first, it might seem strange to have a chapter on cloning techniques in a volume on protein purification methods, but cloning can be viewed as the ultimate purification step. Utilizing recombinant DNA methods, it is possible to obtain DNA encoding the entire protein of interest starting with only a limited amino acid sequence. Large amounts of the protein can then be produced by introducing this DNA into an appropriate heterologous expression system. This technique of producing a single polypeptide permits one to definitely ascribe a particular biological activity to a protein. Expression of a protein in a recombinant system is also of obvious utility where the protein to be studied is available in small quantities and/or only after extensive purification procedures. In addition, the complete primary amino acid sequence of a protein can be determined from the recombinant DNA clone. This information, coupled with comparison to previously characterized proteins, can be used to gain tremendous insight into the structural and functional features of the protein. This chapter makes no attempt to review all possible methods for obtaining recombinant DNA clones for a protein. It is my belief that rather than trying a myriad of different methods, it is better to have a few techniques with which one has become familiar, and appreciate the advantages and pitfalls. The focus here is on methods for designing oligonucleotide probes from protein sequence, and the use of these oligonucleotides to isolate the gene or a cDNA. The hybridization and screening methods will be described in detail, along with a few specific examples of where these techniques have been useful. Note that other general approaches to obtaining coding sequence for a protein are possible; for example, if antibodies are available for a protein, methods have been detailed for using immunological reagents as probes. However, these techniques are outside the scope of this chapter, and the reader is referred elsewhere.' Two Types of Oligonucleotides

There are two general types of oligonucleotide probes which can be designed from protein sequence. One is a set of oligonucleotides which are relatively short and contain all possible nucleotide sequences that could

' R. C. Mierendorf, C. Percy, and R. A. Young, this series, Vol. 152, p. 458. METHODS IN ENZYMOLOGY, VOL. 182

Copyright 0 1990 by Academic Press. Inc. All rights of reproduction in any form reserved.

1551

USING PURIFIED PROTEIN TO CLONE ITS GENE

739

code for a stretch of amino acids. Since the genetic code is degenerate, that is, more than one codon codes for a particular amino acid, the length of these probes is limited by the number of different sequences one desires included in the probe pool. The other type of oligonucleotide that may be designed is a relatively long, low degeneracy (or unique) oligonucleotide which reduces the degeneracy (number of different oligonucleotides in a probe) by making guesses as to which codon will be used to code for a particular amino acid. All methods described here are based on the use of two different probes for the same protein. This significantly reduces the number of false signals one obtains when screening a library, and thus the work involved in characterizing incorrect clones, which can be extremely time consuming. In designing a cloning strategy, it must be decided which kind(s) of probes to design, the exact sequence of the oligonucleotide probes to be synthesized, and what kind of recombinant library to screen. The particular approach taken to cloning the coding region for a protein depends greatly on what information you have about the protein. Do you have long stretches of contiguous amino acid sequence? If so, you should be able to design long oligonucleotides as probes. For technical reasons that will become clear later, these are easier to use than other approaches. Do you know a cell type or tissue that makes this protein? The number of different sequences (sequence complexity) in a cDNA library is orders of magnitude lower than in a genomic library, and thus is apt to give fewer positives with any one probe. Is the sequence amino terminal o r internal? As discussed in the example below, depending on the size of the protein and its mRNA, use of probes from the former kind of sequence may make oligo(dT)primed cDNA library screening a problem. If you have multiple fragments of protein sequence, do you know they are from the same protein? Use of probes from two separate amino acid sequences is advantageous, but obviously one must be sure that both are from the same gene product. We have successfully utilized various combinations of two different probelhybridization systems. The first consists of hybridization of a totally degenerate 17-base long oligonucleotide mixture (17-mer) in tetramethylammonium chloride (TMAC). Design of the oligonucleotide probe is relatively easy: the least degenerate 17-mer mixture possible corresponding to the available amino acid sequence is synthesized. Hybridization in TMAC is technically more demanding than in sodium salt-based buffers such as sodium chloride-sodium citrate (SSC), but TMAC salts remove the T , differences between A: T and G :C base pairs.' Consequently conditions may be set more precisely to minimize hybridization to sequences shorter K. A. Jacobs, R. Rudersdorf, S. D. Neill, J . P. Dougherty, E. L. Brown. and E. F. Fritsch, Nucleic Acids Res. 16,4637 (1988).

740

ADDITIONAL TECHNIQUES

[551

than that desired. Having characterized a number of clones obtained using Procedure 2 (below), it seems that the only sequences detected are perfect matches to the 17-mer, or those containing a single mismatch 1 or 2 nucleotides from an end. The other probelhybridization system is a low-degeneracy 32-mer hybridized in SSC. This system adopts the opposite strategy than that above; in other words, one relies on the ability of a relatively long DNA sequence to hybridize even though there are mismatches present. In our experience, an optimal size for a long oligonucleotide is 32 nucleotides, requiring 11 amino acids of contiguous unambiguous sequence. Longer sequences have been but are unnecessary and may in fact give less specificity. Shorter sequences are risky as there is less room for error in the guesses made. Multiple methods have been suggested for making guesses as to which codon are used; all probably make only slight improvement over random guessing. Three elements may be taken into account when deciding what sequence to make: (I) the relative frequencies of each codon for a particular amino acid for a species, (2) the relative infrequency of CpG in mammalian coding sequences, and (3) how destabilizing a wrong guess is. The latter refers to the premise that certain "aberrant" base pairs (other than G:C and A:T) are more stable than others. G:T and A:G mismatches are less destabilizing than A:C and C:T in sodium salts,2 and this is taken into account in designing some of the probes below. The rules of Lathe6 basically address the first two issues and are an easy way to decide on which codons to choose. Example 1: Cloning Using One Long and One Short Oligonucleotide

Consider as an example the cloning of the bone morphogenetic proteins (BMPs). In this case a protein has been purified from bovine bone based on an activity, induction of ectopic bone formation. The active protein is known to electrophorese at 30 kDa on an SDS-polyacrylamide gel under nonreducing conditions, and to contain at least three polypeptide chains. Due to the scarcity of this protein, and the difficulty in its isolation, it is decided not to try to separate the individual polypeptides and obtain sequence on each separately. Instead, the protein was run on an SDS gel, J. J. Toole, J. L. Knopf, J. M., Wozney, L. M. Sultzman. J. L. Buecker, D. D. Pittman, R. J. Kaufman, E. Brown. C. Shoemaker, E. C. Om, G. W. Amphlett, W. B. Foster. M. L. Coe, G. J. Knutson, D. N. Fass. and R. M. Hewick, Nature (London) 312, 342 (1984). A . J. Celeste, V. Rosen, J. L. Buecker, R. Kriz, E. A. Wang, and J. M. Wozney, EMBO J . 5, 1885 (1986). D. Pennica, G . E. Nedwin, J. S. Hayflick, P. H. Seeburg, R. Derynck, M. A. Palladino, W. J. Kohr, B. B. Aggarwal, and D. Goeddel, Narure (London) 312,724 (1984). R. Lathe, J . Mol. Biol. 183, 1 (1985).

1551

74 1

USING PURIFIED PROTEIN TO CLONE ITS GENE

the 30-kDa region cut out, and the protein digested out of the gel by in situ trypsinization. The tryptic fragments were then separated by HPLC, and each individually sequenced. This procedure has the advantages that multiple sequences can be obtained from a single polypeptide, and lack of sequence due to potential blockage of the amino termini is avoided. The following set of tryptic peptide sequences is obtained.

l . ( A ) 2 . ( A ) 3. (N) Y 4. X F 5 . X L

A F L G D I A F Q V Q Q A A Q D M V V E D A Y Y K P S N X A

L D E E D L G D L (G) T

I Q S I V E

where X represents an unknown amino acid residue and uncertain residues are in parentheses. Nothing is known about which tryptic peptides are derived from which of the polypeptides; therefore, probes must be designed to clone each tryptic fragment independently. In addition, it is not known whether the factors are made by cells in bone or made by cells elsewhere in the animal and merely localized to bone as many other growth factors are.' The safest approach is thus to screen a bovine genomic library, where the coding sequence is known to reside. One must be aware, however, that there is always the danger that an intron exists within the coding sequence for the tryptic fragment. Oligonucleotide probes designed across such a site obviously will not hybridize. The only amino acid sequence of the set that is long enough to design a long oligonucleotide is tryptic peptide 1. First, however, one must decide which region of the sequence to make probes from. A good way to start is to backtranslate the amino acid sequence. Many programs are available which will do this. The following uses the UWGCG8 software and the human codon usage table generated from published data.9 Note that bovine codon usage is essentially the same as human. Ala GCC GCT GCA GCG

.44

.29 .19 .09

Phe TTC .60 TTT .40

Leu CTG CTC TTG CTT TTA CTA

.44

.22 .12 .ll .06 .06

G~Y GGC GGA GGG GGT

.39 .23 .22 .16

ASP

Ile

GAC .59 GAT .41

ATC .56 A'M .32 ATA .12

Ala GCC GCT GCA GCG

.44

.29 .19 .09

' P. V. Hauschka, A. E. Mavrakos, M. D. Iafrati, S . E. Doleman, and M. Klagsbrun, J. Biol. Chem. 261, 12665 (1986)

* J. Devereux, P. Haeberli, and 0. Smithies, Nucleic Acids Res. 12, 387 (1984). T. Maruyama, T. Gojobori, S. Aota, and T. Ikemura. Nucleic Acids Res. 14, rlS1 (1986).

742 !,eu CTG CTC TTG CTT TTA CTA

[551

ADDITIONAL TECHNIQUES

.44

.22 .12 .I1 .06 .06

ASP

Glu

Glu

ASP

GAC .59 GAT .41

GAG .61 GAA .39

GAG .61 GAA .39

GAC .59 GAT .41

Leu CTG CTC TTG CTT TTA CTA

G~Y GGC GGA GGG GGT

.44

.22 .12 .I1 .06 .06

.39 .23 .22 .16

Below each amino acid are given the codons which can encode it, and the frequency with which it is used in known human coding sequences. Note that even though Leu has six possible codons, the codon usage is sufficiently skewed so that one can decide on a preferred codon with significant confidence. The other six-codon amino acids, serine and arginine, do not allow this luxury. One is forced essentially to make guesses at two out of the three positions in the codon (Arg) or all three positions (Ser), and therefore these amino acids are avoided if at all possible. Also note that each of the Leu codons is followed by a codon starting with G. If one accepts the fact that intercodon CpGs are infrequent, then elimination of the second most frequently used Leu codon (CTC) makes it even more likely that the most frequently used (CTG) will be correct. In this case there is not much flexibility in deciding on a region for probe design. A 32-mer can be designed using Alal-Glull, using the Lathe rules. Note that this results in several changes relative to just choosing the most prevalent codons. Ala GCC

Phe TTC

Leu CTG

Gly GGC

Asp GAC

lle ATT

Ala GCC

Leu CTG

Asp GAT

Glu GAG

Glu GA

Both of the indicated changes (codons 6 and 9) represent replacements of Cs by Ts to account for the relative infrequency of CpGs. The actual probe we made was the complement of the above sequence, in order to allow for G:T base pairing in the event the choices for the Phez or AspScodons were incorrect. For a second probe, we made a 17-mer mixture spanning the LeusLeul3 codons, this being the least degenerate 17-mer possible from this amino acid sequence (192-fold). This 17-mer is also made on the noncoding strand, as it can be used as a primer for M13 sequencing of a possible clone; the sequence read should correspond to the remainder of the tryptic peptide sequence and can quickly confirm or disprove a clone as being correct. Note that the probe must be synthesized as two separate oligonucleotide pools since it spans an amino acid with six possible codons: 3' 3'

Leu

ASP

Glu

AAY GAN

CTR CTR

CTY CTY

Glu CTY CTY

Asp

Leu

CTR CTR

RA RA

5' 5'

and where Y is C and T, R is A and G, and N is all of the four nucleotides.

[551

743

USING PURIFIED PROTEIN TO CLONE ITS GENE

Now that the two probes have been designed and synthesized, they can be used to screen a bovine genomic library. In this case, 400,000 recombinants of a library were plated and nitrocellulose replicas made and amplified as described in Procedure 1 (below). One set of filters was hybridized to the 17-mer in TMAC (Procedure 2), the others to the 32-mer in SSC (Procedure 3). While it is not necessary to amplify in order to screen with a 32-mer, it is much easier to compare autoradiographs if both sets of filters have either been amplified or not. One clone hybridized to both probes, and was plaque purified. After subcloning a fragment of this clones into M13, the DNA sequence was derived and indeed found to encode not only the desired tryptic fragment but also fragment 2. Comparing the actual sequence of the bovine BMP-1 gene to the 32-mer, it can be seen that four wrong guesses were made, yet the overall homology is still 88%, and clearly the oligonucleotide probe hybridized to the desired sequence. A F L G D l A L D E E D L bBMP-I: PCTCTCTCCAG CTGCCTTCCTTGGGGACATCGCCCTGGACGAGGAGGAC'ITGAGG

1111lI1l l l I l l 1 / l l l I I l I I I I I I

GCClTCCTCGGCGACA'ITGCCCTGGATGAGCA

32-mer:

Also notice that there is undoubtedly an intron just 5' of sequence hybridizing to the oligonucleotide, as there is no basic residue at the appropriate place as demanded by the specificity of trypsin, and a consensus splice acceptor sequence (Y,AG) is present.

Example 2: Cloning Using Two Short Oligonucleotides

Tryptic peptide 3 is not long enough to design a long oligonucleotide, but two 17-mers can be designed which overlap by 14 bases. The oligonucleotides are synthesized to correspond to opposite strands for the reasons mentioned above. Probe 1: Probe 2:

3' 5'

.

Tyr

Gln

Asp

Met

Val

Val

Glu

ATR

GTY CAR

CTR GAY

TAC ATG

CAN GTN

CA GTN

GA

5' 3'

Probe 1 is 32-fold degenerate, while probe 2 is 64-fold degenerate. In this case, a bovine genomic library was screened with probe 1 alone using Procedures 1 and 2. Screening a library in duplicate with a single probe makes detection of clones that hybridize to the probe more obvious. Twenty-five positive clones were picked and replated for secondaries. It is not possible to predict the number of hybridizing sequences one would expect with any given probe. On a random basis, for a 32-fold degenerate 17-mer, if one is screening 400,000 recombinants of a library with 18kilobase (kb) inserts (= 1.4 X 10" b of DNA) the expected number of

744

[551

ADDITIONAL TECHNIQUES

sequences identical to one of the oligonucleotides in the probe pool is 27 (1.4 x 101°/4" x 32). Of course the distribution of sequences in a mammalian genome is nonrandom, so absolute numbers of positive clones vary with the particular probe used. The 25 clones were then screened in TMAC, hybridizing probe 1 to one amplified nitrocellulose replica and probe 2 to the other. Only 1 out of the 25 hybridized strongly to both probes and was plaque purified. Using a plate lysate of this clone, a small amount of bacteriophage DNA was isolated. This DNA was digested with a variety of restriction enzymes, electrophoresed on duplicate agarose gels, the gels dried down, and each hybridized to one of the oligonucleotide probes.'0 In this manner, two items of information may be obtained. (1) a reasonably sized restriction fragment can be determined suitable for subcloning into M13 for DNA sequence analysis and (2) it can be seen that both probes hybridize to the same DNA fragments in all cases, as they should if this clone contains the amino acid sequence desired. Subcloning a 1.2-kb SacI fragment into M13 using both oligos as probes, DNA sequence was derived from both ends of the SacI fragment. The sequence did encode the amino acid sequence sought, with a basic residue appropriately positioned. The DNA sequence also indicates a stop codon in frame immediately 3' of the tryptic fragment. GAGCTCAGCGCCATCTCCATGCTGTACCmGATGAGAATGAGAAGGTGGTAmAAAGAAC E

L

S

A

I

S

M

L

Y

L

D

E

N

E

K

V

V

L

K

N

-

TATCAGGACATGGmGTCGAGGGTTGTGGGTGTCGTFAGCACAGCAAAATAAAATATAAA Y

Q

D

M

V

V

E

G

C

G

C

R

*

Using the larger amount of amino acid sequence derived from this preliminary DNA sequence, a computer search of the protein database indicates that this protein is quite related to the /3 subunits of the inhibins, as well as other growth factors in the transforming growth factor p (TGF-P) family. The bovine genomic fragments obtained in examples 1 and 2 were used as probes to isolate human cDNA clones encoding the entire corresponding proteins. The complete amino acid sequences of the BMP proteins and discussion of their structures is given el~ewhere.".'~ Example 3: Cloning Using Two Long Oligonucleotides

If a long stretch of contiguous amino acid sequence is available for the protein one wishes to clone, the easiest way to proceed is to design two long oligonucleotides. As an example, we wanted to obtain recombinant lo I'

I'

R . B. Wallace and C. G . Miyada, this series, Vol. 152, p. 432. E. A. Wang, V. Rosen, P. Cordes, R. M. Hewick, M. J . Kriz, D. P. Luxenberg, B. S . Sibley, and J. M. Wozney, Proc. Narl. Acad. Sci. U.S.A. 85,9484 (1988). J . M. Wozney, V . ' ~ o s e nA. , J. Celeste, L. M. Mitsock, M. J. Whitters, R. W. Kriz, R. M. Hewick, and E. A. Wang, Science 242, 1528 (1988).

[551

745

USING PURIFIED PROTEIN TO CLONE ITS GENE

clones for cartilage proteoglycan core protein (CPCP), the mRNA for which should be good marker of cartilage formation. A cyanogen bromide cleavage fragment from bovine nasal cartilage proteoglycan core proteinI3 was used to design the probes. Several other fragments of amino acid sequences for this protein had been published, but were all from the amino-terminal region of the protein. Considering that the estimated size of the protein was 225 kDa, it was decided that probes made from these sequences would be too far from the poly(A) of the mRNA to use in screening an oligo(dT) cDNA library. As described above, the sequence is first back-translated: Met ATG 1.0

Ile ATC .57 ATT .31 ATA .I2

T~P TGG 1.0

His CAC .61 CAT .39

Glu GAG .58 GAA .42

L Y ~ AAG .69 AAA .31

G~Y GGT .35 GGC .34 GGA .18 GGG .13

Glu GAG .58 GAA .42

TrIJ TGG 1.0

Asn AAC .69 AAT .31

ASP GAT .50 GAC .50

Val GTG .46 GTC .27 GTT .21 GTA .06

Pro CCC .33 CCA .29 CCT .29 CCG .09

C Y ~ TGT .54 TGC .46

Asn AAC .69 AAT .31

T Y ~ TAC .61 TAT .39

Gln CAG .69 CAA .31

Leu CTG .46 CTC .25 C T I .10 TTG .08 CTA .07 TTA .05

Pro CCC .33 CCA .29 CCT .29 CCG .09

Phe TTC .62 TTT .38

Thr ACC .41 ACT .30 ACA .20 ACG .09

The codon usage frequencies used for this cloning were the ones available at the time this work was done.I4 Using the entire amino acid sequence, two 32-mers can be designed that overlap by only 2 base pairs. The following changes are made relative to designing the probes based on the mostly frequently used codons: C was replaced by T at His4 and A m l l due to the CpG rule; the alternative codons were used at G l u ~Glus, , and Cysl4 to allow possible G:T base pairing (and reduce the risk of A:C mismatches). Both probes were made on the noncoding strand to provide for the latter; for clarity, the probes are shown on the coding strand. Met Ile Trp His Glu Lys Gly Glu Trp Asn Asp Val Pro Probe 1: ATG ATC TGG CAT GAA AAG GGT GAA TGG AAT GA Probe 2: GAT GTC CCC Cys Asn Tyr Gln Leu Pro Phe Thr TGC AAC TAC CAG CTG CCC TTC AC

" J.-P. Perin, F.

Bonnet, J. Jolles, and P. Jolles, FEBS Lett. 176, 37 (1984).

'Q.Strehler and D. North, Mech. Ageing Deu. 18,285 (1982).

-

746

ADDITIONAL TECHNIQUES

[551

For this cloning, there is no reason to start by screening a genomic library. The Swarm rat chondrosarcoma is known to synthesize CPCP, and provides a convenient source of mRNA to make a cDNA library in AgtlO. Fifty thousand recombinants were screened by Procedure 3 with the two probes designed above. Fifty-nine duplicate positives were obtained. One of these, containing a 4.2-kb insert, was restriction mapped and a 350-bp PuuIIIPstI fragment identified which hybridized to both of the probes. The derived amino acid sequence of this region contains a peptide identical to the bovine CPCP at 20 of the 21 residues used to design the probes, thus confirming the identity of the clone. M I W H E R G E W N RatCPCP: CTGCGTAGTGATGATCTGGCATGAGAGGGGTGAATGGAACGA

11111111111111

Probe 1:

1 111111111111 1 1

ATGATCTGGCATGAAAAGGGTGAATGGAATGA

D V P C N Y Q L P F T RatCPCP: GATGTCCCCTGCAATTACCAGCTGCCCTTCACATGTAAAAA Probe 2:

I I I I I I I I I I I I I I1111l11111111111

GATGTGCCCTGCAACTACCAGCTGCCCTTCAC

Comparing the actual sequence to those of the probes, you can see that there are three nucleotide mismatches with the first long oligonucleotide, and only two with the second. One of the three mismatches is due to an amino acid difference relative to the sequence used to design the probes. This is a conservative Lys-to-Arg change, undoubtedly a species difference. Thus by using long oligonucleotides, we have been able to cross species, i.e., use amino acid sequence from bovine to obtain a rat DNA clone. Use of short oligonucleotides in the same situation is extremely risky. Having presented examples of how to design oligonucleotide probes, and how to successfully utilize these probes, what follows is details of the library screening methods we use. Procedure 1: Plating Genomic Libraries with Amplification

Materials Fresh saturated overnight culture of host bacteria Titered bacteriophage genomic library SM (0. I M NaCI, 8 mM MgS04, 50 rnM Tris-CI, pH 7.5, 0.01% gelatin) NZCYM 1.2% agarose plates prewarmed to 37" NZCYM 0.8% top agarose, melted and cooled to 55" Millipore HATF nitrocellulose filters Whatman 3MM paper (46 x 57 cm) Denaturation solution (0.5 M NaOH, 1.5 M NaCI)

[551

USING PURIFIED PROTEIN TO CLONE ITS GENE

747

Neutralization solution (0.5 M Tris-C1, pH 8.0, 1.5 M NaC1) SSC (0.15 M NaCl, 15 mM sodium citrate) 2x SSCPE (0.3 M NaCl, 30 mM sodium citrate, 10 mM sodium phosphate, pH 6.5, 0.1 mM EDTA) ~ 0.5% SDS, 50 mM EDTA) prewarmed Filter washing solution ( 2 SSC, to 50" Plating the Library

1. For each 150-mm plate, mix the following: 0.3 ml bacteria 0.3 ml SM 8000 pfu (plaque-forming units) bacteriophage library 2. Incubate at 37" for 10-20 min. 3. Add 9 ml NZCYM top agarose. 4. Pour onto NZCYM agarose plate and evenly distribute. 5. Let harden at room temperature for 10-20 min in an upright position. 6. Incubate the plates at 37" for 12- 18 hr.

7. Place a nitrocellulose filter on the plate, using forceps or gloved hands. Bend the filter slightly in the middle, lay it on the plate (starting at the middle), and let it lie across the remainder of the plate, making sure no air bubbles are introduced between the filter and plate. 8. Key the filter to the plate with a needle dipped in ink. 9. Let the filter remain on the plate 2 min. 10. Remove the filter and place it bacterial side up on a fresh NZCYM agarose plate. 11. For the second replica, repeat and allow the filter to remain on the plate about 5 min. 12. Allow bacterialbacteriophage to grow at 37" approximately 6 hr, or until plaques are clearly visible and just touching. Processing of Filters

13. Place the filters bacterial side up on Whatman 3MM paper saturated with denaturation solution. 14. Denature 5 min. 15. Transfer the filters to Whatman 3MM paper saturated with neutralization solution. 16. Neutralize 5 min. 17. Transfer the filters to Whatman 3MM papers saturated with 2 x SSCPE; allow to remain 2-5 min.

748

ADDITIONAL TECHNIQUES

[551

18. Transfer the filters to dry Whatman 3MM paper. 19. Allow the filters to remain just until liquid has absorbed into the paper (10-30 min). 20. Place the filters between sheets of Whatman 3MM paper and bake in a vacuum oven, 80°, 2 hr. 21. Lay the filters on filter washing solhtion and allow to wet through. 22. Gently rub the filters with gloved hand to remove all bacterial debris. 23. Transfer to fresh filter washing solution to rinse.

Notes to Method

I. In one screening, we usually plate 50 plates at 8000lplate = 400,000 recombinants. Considering an average unexpanded recombinant library, this is about 2.4 genome equivalents, which gives one a probability of about 91% of having the sequence of choice.15Note that if using expanded libraries, more recombinants must be screened to have the same probability of obtaining the desired clone.I6 2. A third nitrocellulose replica can be made, leaving the nitrocellulose on the plate 10-30 min. 3. We always use nitrocellulose filters, though others have used nylon membranes." In our experience, the bacteria d o not grow well on the nylon. However, one must be careful with nitrocellulose as it becomes very brittle in TMAC, and the filters cannot be subsequently rehybridized. 4. It is convenient to make replicas from five plates at a time (steps 7-1 1). 5. If one is screening a cDNA library using a bacteriophage vector such as AgtlO or AZAP, which yield large plaques, plate 5000 pfulplate and grow approximately 5-6 hr, or until the plaques are just visible and clear. Amplification can then be done overnight (12-18 hr). Procedure 2: Hybridization to 17-Mers in Tetramethylammonium Chloride (TMAC)

Materials TMAC stock solution: Add 300 ml H 2 0 to a 500-g bottle of TMAC (-5.6 M). The actual concentration of TMAC is calculated by taking the refractive index of Maniatis, E. F. Fritsch, and J . Sambrook, "Molecular Cloning: A Laboratory Manual." Cold Spring Harbor Lab., Cold Spring Harbor, New York. 1982. A.-M. Frischauf, this series, Vol. 152, p. 190. K. Jacobs, C. Shoemaker. R. Rudersdorf, S. D. Neill, R. J . Kaufman, A. Mufson, J. Seehra, S. S. Jones, R. Hewick, E. F. Fritsch, M. Kawakita, T. Shimizu, and T. Miyake, Nature (London) 313,806 (1985).

" T.

l6 I'

r

7

1551

i 71

C

USING PURIFIED PROTEIN TO CLONE ITS GENE

749

a 1 :2 dilution in H 2 0 using the equation M = (qD - 1.331)/0.018 ( x 3 for the dilution) TMAC hybridization buffer: 3 M tetramethylammonium chloride (TMAC) 0.1 M sodium phosphate, pH 6.5 1 mM EDTA 5x Denhardt's s o l ~ t i o n ' ~ : 0.6% SDS 100 pglml salmon sperm DNA TMAC wash solution (room temperature and 50"): 3 M TMAC 0.1% SDS Oligonucleotide probe: Phosphorylate to a specific activity of 4 x lo6-1 x 10' cpmlpmol with T4 polynucleotide kinase and [y-32P]ATF"0

Prehybridize the Filters 1. Individually transfer 25 filters to a 150-mm glass crystallizing dish containing 200 ml TMAC hybridization buffer. 2. Incubate the filters with agitation on an orbital shaker (New Brunswick) at 48", 1-2 hr.

?r 3

Hybridize the Filters 3. Transfer the filters to a new crystallizing dish with 200 ml TMAC hybridization buffer at 48". 4. Add radioactive oligonucleotide to give a hybridization buffer of 1-2 x lo6 cpmlml (about 0.1 pmollml). 5. Incubate at 48", 36-60 hr with agitation.

Remove Unhybridized Probe 6. Pour off hybridization buffer. 7. Add TMAC wash solution (room temperature) to cover filters, swirl briefly, and discard solution. 8. Place the filters one by one into another crystallizing dish with 250 ml TMAC wash solution at room temperature. 9. Wash at room temperature for 15 min. 10. Transfer the stack of filters to a crystallizing dish with TMAC wash solution prewarmed to 50°. 11. Incubate at 50°, exactly 1 hr with agitation. 12. Replace TMAC solution with 2 x SSC, 0.1% SDS, at room temperature.

750

ADDITIONAL TECHNIQUES

13. 14. 15. 16.

[551

Agitate at room temperature 10 min. Repeat SSC wash twice. Dry filters on Whatman 3MM paper. Mount between pieces of Saran wrap and autoradiograph.

Notes to Method 1. Others have described similar TMAC hybridization methods2918; this procedure works best for us. 2. It is critical that the washing at 50" be done for only I hr; the wash temperature is close to the T, of the hybrid, and dissociation is time de~endent.~ 3. TMAC hybridization buffer is stable at 4" for up to 2 weeks. 4. TMAC is an irritant and hazardous, and therefore should be used in afume hood. It should be inactivated with chlorine bleach before disposal. Procedure 3: Hybridization in SSC

Materials SSC hybridization buffer: 5 x SSC 0.1% SDS 5 x Denhardt's solution 100 pglml salmon sperm DNA SSC wash solution (5x SSC, 0.1% SDS), 50" Prehybridize the Filters 1. Individually transfer 25 filters to a 150-mm glass crystallizing dish containing 200 ml SSC hybridization buffer. 2. Incubate the filters with agitation on an orbital shaker at 65", 1-2 hr.

Hybridize the Filters 3. Transfer the filters to a new crystallizing dish with 200 ml SSC hybridization buffer at 50" 4. Add radioactive oligonucleotide to give a hybridization buffer of lo5-lo6 cpmlml. 5. Incubate at 50", 12-18 hr with agitation.

'* K. A. Jacobs and A. Celeste, in "Current Protocols in Molecular Biology" (F. M. Ausubel, R. Brent. R . E. Kingson, D. D. Moore, J. A. Smith, J. G. Seidman, and K. Struhl, eds.). Wiley, New York, 1987.

[561

COMPUTERS A N D SEQUENCES

75 1

Remove Unhybridized Probe

6. Pour off hybridization buffer. 7. Add SSC wash solution to cover filters, swirl briefly, and discard solution. 8. Place the filters one by one into another crystallizing dish with 250 ml SSC wash solution at room temperature. 9. Wash at 50" for 15 min. 10. Replace SSC wash solution; invert stack of filters. 11. Wash at 50" for 15 min. 12. Repeat steps 10 and 11. Notes to Method 1. The hybridization conditions that are used are empirically determined. Often a pilot experiment can be performed to adjust the hybridization temperature and/or wash temperature and salt concentration such that a "reasonable" number of positive signals are obtained. 2. Using long oligonucleotides, amplification (Procedure 1) is not necessary. However, it does greatly increase the signal strength when using bacteriophage such as EMBL3, which give small plaques. If amplification is not used, phage such as AgtlO may be plated at a density of 10,00015,00O/plate.

[561 Computer Analysis of Protein Structure

Introduction

This chapter will attempt to help in the analysis of a newly determined protein sequence. The principle theoretical methods to be discussed concern the following: (1) alignment of protein amino acid sequences, (2) prediction of the protein's structural class, (3) prediction of protein secondary, membrane-associated, antigenic, and target sequence structures, (4) delineating protein function and structure by discerning shared sequence patterns or fragments with proteins of well-known function, and (5) use of amino acid characteristic plots in sequence analysis. Achievement of these goals requires a computer and associated programs; very little these days can be performed by hand or with a personal calculator. A list of some databases and computer program packages will Copyright Q 1990 by Academic Press, tnc.

METHODS IN ENZYMOLOGY, VOL. 182

All rights of reproduction in any form reserved.

752

ADDITIONAL TECHNIQUES

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thus be presented. Since the thrust of this chapter will be a facile and readable description of available techniques, references will be kept to a minimum. However, there are many recent reviews and texts filled with ,~ and Mcdetails and references: von ~ e i j n e , ~' a y l o r , *D ~ o l i t t l e Argos C a l d ~ nand , ~ Lewitter and in done.' Aligning Amino Acid Sequences

If a new protein sequence can be aligned with those of others, years of experimental work can be avoided. Homologous proteins will have the same main-chain folding and likely related functions. If a sequence matches one with known three-dimensional structure, a testable model is quickly at hand, bypassing all the human effort in mass purification, crystallization, and X-ray diffraction analysis. If a sequence can be related to that of a biochemically well-characterized protein, then much of that knowledge is transferable. Aligning many distantly related proteins can point to the two or three never-substituted residues essential for function and candidates for site-directed mutagenesis. Domain structure and modes of evolutionary development are also suggested. And imagine, all this gained with, at most, one night's work by a computer. I will consider methods to align sequences in pairs and multiply, ways to find repeats in a sequence and techniques to discover a sequence pattern that has been derived from a template or consensus resulting from several previously known and aligned sequences. In addition, the significance of whole sequence alignments or fragment matches will be examined. Of course, it is always best to compare amino acids and not nucleotides. There are 20 amino acids, and not just 4, and they often substitute according to conservation patterns that can be statistically searched. Using Single Amino Acid Comparisorls

In matching sequences, two obvious rules must be adhered to: ( I ) any amino acid can only be used or aligned once and (2) the alignment must progress along the sequence consecutively from the N- to C-termini. Typically, one sequence is laid along a column and another along a row. In

' G . von Heijne, "Sequence Analysis in Molecular Biology." Academic press, New York, '

1988. W. R. Taylor, Protein Eng. 2,77 (1988). R. F. Doolittle, "Of URFS and ORFS: A Primer on How to Analyse Derived Amino Acid Sequences." Univ. Sci. Books, Mill Valley, California, 1986. P. Argos and P. McCaldon, in "Genetic Engineering, Principles and Methods" ( J . K . Setlow, ed.), Vol. 10, p. 21. Plenum, New York, 1988. F. I. Lewitter and W. P. Rindone, this series, Vol. 155, p. 582.

[561

Dayhoff Mutation Values K K

753

COMPUTERS AND SEQUENCES

1

L 3

A 5

T 7

V 8

6

Needleman-Wunsch Transformed Matrix

W

D

5

8

K

L

A

T

V

W

D

K

73

63

65

57

43

17

8

L 5 1 4

6

9 1 0

6

4

L

54

6_3

55

58

47

18

4

V 6 1 0

8

8 1 2

2

6

V

43

47

45

45

49

-

14

6

W

5

6

2

3

2

2

5

1

W

24

25

18

17

14

37 -

1

D

8

4

8

8

6

1

1

2

D

19

15

19

16

14

9

12 -

T

8

6

9

1

1

8

3

8

T

8

6

9

Alignment:

1

1

8

3

8

K L A T V W D K L - - V W D T

Needleman-Wunsch score: (no GAP penalty)

237

Needleman-Wunsch score: 226 (GAP penalties ( I , E ) = (10,l))

FIG. 1. An example of the Needleman-Wunsch alignment procedure from a search matrix. The leftmost matrix shows the Dayhoff mutation values for all amino acid comparisons. The elements in the path giving the best alignment score from the Needleman-Wunsch (or transformed) matrix are underscored.

matrix fashion, values are placed in each residue cross-comparison position; a zero is used if the amino acids are different and a one if the same (the so-called Unitary Matrix comparison criterion). According t o the method of Needleman and W ~ n s c hthis , ~ matrix is transformed to allow easy recognition of the best alignment path (see Fig. 1 for an illustration; see also Ref. 3 for details). This is done by going to each position in the matrix, starting in the lower right-hand corner, and finding the maximum value of all the possible positions forward (to the lower right) of the one under consideration that could be allowed in a n alignment including insertions and deletions. This value is added to and replaces the one at the position from which all the forward positions are considered. The process is repeated at positions within rows from right to left and then along rows from the bottom to the top of the matrix. The just transformed or replaced values are always used when finding the maximum of the forward positions when moving from one position and row to the next. It is easy to trace the best alignment path through the fully transformed matrix. Start at the upper left corner and try to move to the highest value consistent with alignment rules 1 and 2 discussed previously. Keep a S. B. Needleman and C. D. Wunsch, J . Mol. Biol. 48,443 (1970).

754

ADDITIONAL TECHNIQUES

[561

running sum of the transformed values contained in the trace path. Note that the matrix looks ahead to discern the best path. One could try several (or all) possible paths if necessary; the one with the highest sum at the end of the process is the optimal one. See Fig. 1 for an illustration. The alignment method has one big drawback. All is well if one proceeds along the main diagonal. However, if the best alignment can be achieved by insertion or deletion, a penalty should be exacted as this is a costly evolutionary event in two related proteins. Normally, a penalty is given to initiate a gap, and then a smaller one is added for each additicnal extension of the gap. Penalty (P) = I + EK, where I is the initiating gap penalty value, E is the extending penalty, and the gap is K positions long. P is subtracted from the alignment sum. Different alignments can result from different values of I and E. Which should be selected? The answer is not clear and seems to differ for different sequences (see below). Several criteria, other than the unitary match of amino acids used above, have become available over the years to weight amino acid replacements. They include the minimum base change per codon required to replace one amino acid for another,' some comparative residue characteristics, such as hydrophobicity and size,8 or relative weights with which amino acids substitute in aligned sequence families (Ref. 9, the so-called Dayhoff mutation matrix). This latter symmetric matrix contains the preferences with which amino acids are observed to substitute in aligned sequences of 71 protein families. For example, Val and Leu interchange with a relative preference of 10 while Trp and Asp substitute with a weight of only 1. To search a sequence against an entire database (presently nearly 10,000 sequences) in a reasonable time, a new approach is needed. Pearson and Lipman" developed a list for the search sequence which contains amino acid type and sequence position for each residue. A database sequence is then compared by determining the offset (difference in sequence positions where two sequences have the same residue) for each of its amino acids. All matches having the same offset can be aligned without gaps. Offsets with a high number of matches and over a reasonably long region constitute a quickly determined starting point to test for alignment extendability with the more responsive Dayhoff mutation matrix. The Lipman-Pearson method is fast, but lacks sensitivity due to the initial

' T. H. Jukes and C. R. Cantor, in "Mammalian Protein Metabolism" (H. N . Munro, ed.), 'O

Vol. 3, p. 21. Academic Press, New York, 1969. A. D. McLachlan, J. Mol. Biol. 64,417 (1972). M. 0.Dayhoff, W. C. Barker, and L. T. Hunt, this series, Vol. 91, p. 524. W. R . Pearson and D. J. Lipman, Proc. Natl. Acad. Sci. U.S.A. 85,2444 (1988).

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COMPUTERS A N D SEQUENCES

755

identity and same-offset criteria; however, it works down to about 30% identity between two sequences.

Using Sequence Fragments Alignments considered "standards of truth" can be had by spatial superposition of a-carbon backbone folds known from X-ray diffraction analysis." However, the distribution of residue match and conservation is often not uniform. There may be a sequence of 10 amino acids where half are identical and I is cysteine essential for function, followed by a n insertionldeletion, then the next 30 residues with only 10% identity but good hydrophobicity conservation to preserve a buried 0-sheet, then 12 amino acids with 3 identically conserved glycines essential in turns, and so forth. The variable pattern is a result of ever-changing structural and functional requirements. The single residue approach may well miss long conserved stretches; so, one is forced to compare all sequence segments. First, define a probe or window length (e.g., 25 residues). Then, when aligning every 25-residue span of 1 protein sequence with every such span in the other sequence, select a criterion and perform a summation over each of the 25 values resulting from the matched amino acid pairs. Place this sum in a matrix at the middle sequence positions of the spans; for example, the score is placed at (13, 13) when comparing the N-terminal25 residues of each protein. Repeat this for all possible intercomparisons to fill in the score matrix. Now, transform the score matrix to obtain the optimal alignment. The visual matrix is often called a "dot" matrix where only a dot is placed for values above a certain threshold. Homology is indicated by lines (composed of dots) that are colinear with the main diagonal, but not contiguous where there are deletions and ins~rtions. In aligning two sequences where the identity level is low (15-30%), it is critical to be sensitive. To achieve this, use several criteria and multiple probe lengths or windows. I have tested many amino acid characteristics12 and found the following five to be most sensitive: (1) hydrophobicity, (2) residue bulk o r shape, ( 3 ) preference of a residue to be in the turnstructural conformation, (4) antiparallel strand conformational preference, and (5) amino acid refractivity index, which correlates strong1y with molecular weight. The importance of these parameters seems obvious in conserving protein tertiary folds: hydrophobicity to maintain the interior core, turn preference to ensure the chain returns to form the core, I' I'

M. G . Rossmann and P. Argos, Annu. Rev. Biochem. 50,497 (1981). P . Argos, J . Mol. Biol. 193, 385 (1987).

756

ADDITIONAL TECHNIQUES

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P-preference as strand folds often comprise protein interiors, and sidechain size and shape for close-packed structures. A priori, it is impossible to know which characteristic the protein is required to conserve in a given sequence region. So, an average score can be calculated which is based on the Dayhoff mutation matrix as well as a comparison of the residue characteristics just mentioned (see Ref. 12 for details). The most important regional characteristic will hopefully dominate the average score. After all sequence fragment comparisons at a given window length are completed, the standard deviation of all the scores is determined and the search matrix values are replaced as a number of standard deviations (v)above the matrix mean. This process is repeated for various window lengths (e.g., 5 to 35). A composite search matrix is

080 INTEERASE WINDOW 25 OAYHOFF +5 PARAMETERS

FIG.2. (a) Search matrix, based on residue characteristics,I2 for comparison of integrases from bacteriophages P2 and $80. A constant search window length of 25 residues was used. The symbols indicate the fractional standard deviation (a) range of the search peak heights (S) according to the following scheme: 3.00 5 S < 4.00 (wavy lines); 4.00 5 S 5 4.20 (circles). (b) As (a), except using windows 7 to 25 in steps of 1 with symbol ranges of 3.70 IS 5 4.00 (thin lines): 4.00 5 S < 4.20 (thick lines); 4.21s 5 S 5 4.60 (circles). It is clear that using many windows facilitates delineation of the alignment path by literally "filling it in."

[561

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COMPUTERS AND SEQUENCES

0

46

92

139

185 231 277 080 INTEGRASE WINDOWS 7 TO 25 OAYHOFF +5 PARAMETERS

324

370

416

FIG.2. (continued)

then possible for all the window lengths since standard deviation scores are directly comparable. This approach reduces greatly the noise level and literally "fills in" the homology through the multiple probe lengths. An example is given in Fig. 2. A computer program package written by Rechid, Vingron, and ~ r g o s ' ~ allows the calculation and display of a search matrix on a microVAX or SUN workstation. The user can interact with the search plot and select an alignment. (Write to the author for the program suite.) The search matrix can also be automatically transformed to yield an optimal alignment. SigniJicanceof Alignment

If the residue identity between two sequences is above 30% with uniform distribution over the sequence and there are no long insertions1 deletions, meaningfulness is obvious to the eye and by several mathematical criteria. For most other cases, significance estimation is not straightfor" R.

Rechid, M. Vingron, and P. Argos, CABZOS 5, 107 (1989).

758

ADDITIONAL TECHNIQUES

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ward. Certain regions of the protein sequences may be aligned well, while others may hardly be matched. An alignment may be reasonable over I relatively short stretch (25-50 residues) and yet cannot be extended. Sometimes four or more amino acids show a perfect or near-perfect consecutive match, and yet the remaining regions do not align well. In other cases, the entire sequences can be aligned but with only 15-30% identity. Finally, a choice of different gap penalty values can lead to different alignments. For example, alignments between two tRNA synthetases using only slightly different penalties in the UWGCG package program BESTFIT were quite different, even to the extent of missing seven consecutive residues in some of the alignments (TTRPETL in valyl- and leucyltRNA synthetases). Three approaches have generally been used to assess alignment quality: (1) Monte Carlo methods based on random shuffling of the two sequences and matching by the same alignment procedure, (2) analytical formulas derived for particular alignment algorithms to yield expected match quality in shuffled sequences, and (3) use of real, but unrelated, protein sequences in large reference samples for analysis. In all cases a mean assessment and standard deviation (a)is calculated from the controls. For longer sequence alignments (say, greater than 50 residues), the literature consensus seems to state that greater than 3u is necessary for consideration of the match as "possible." Around 5 or 6 a it becomes "probable" and above 10a, "certain." Barton and Sternberg14have found that scores greater than 6 u yield 75% correct alignments in helices and 6-strands of known tertiary folds. Application of biological knowledge is also important. If the proteins share some function, the alignment is more likely. Of course, no protein would use a sequence of randomly selected letters; careful engineering and bias prevail. Thus, significance estimates relying on real sequences are more trustworthy. Rechid, Vingron, and ArgosI3 have run several unrelated sequence pairs through the residue characteristic technique, produced search matrices and associated alignments, and determined that the average correlation coefficient over five residue properties is +0.282 (not 0.000 as random shuffling would yield). We subtract this from the mean correlation for the alignment to be tested, and divide by the standard deviation of our trial scores which depends on the alignment length. Then in the strict sense, if the answer is greater than 2u, the alignment may well be significant. Alignments with scores somewhat less than 2 a may be considered if the biological relationship is l4

G. J. Barton and M. J . E. Sternberg, Protein Eng. 1,89 (1987).

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compelling. Most studies have been in agreement that the Dayhoff mutation matrix and residue properties are the most sensitive criteria to date. We recently tested two of the most commonly used alignment procedures on five cases taken from tertiary structural superpositions: the Protein Identification Resource ALIGN program and UWGCG's BESTFIT. They both rely on gap penalties, and the results were radically different for various penalty values. For example, in BESTFIT the match for serine protease sequences was 92 and 64% incorrect with [initiating (I), extending ( E ) ] parameters set at (5.0, 0.3) and (3.0, 0.1), respectively. ALIGN under 12 different conditions was never able to find 2 fragments with 7 consecutive identical residues in a pair of aminoacyl-tRNA synthetases. Furthermore, for different sequences the optimal settings were also different. Of course, our trials involved the tougher alignment cases with 15 to 30% identity. In any event, we recommend ( I , E ) = (4.0, 0.2) for BESTFIT and suggest adding (+2) to the Dayhoff matrix elements and then using an overall gap penalty of 20 for ALIGN. The visual interaction with a search matrix based on residue characteristics produced the best results in our trials, while second best was the application of a Needleman-Wunsch transformation procedure to the same search matrix. With any questionable homology it is always important to examine visually the search matrix, especially the more informative ones showing score values calculated over several window sizes. We have encountered cases where the same percentage identity and the same standard deviation significance level have been achieved, yet the search matrices do not prove equally convincing." With mere credibility scores, the distribution of high peaks along the entire path cannot be seen, and the number and size of noise peaks relative to match peaks cannot be assessed; the presence of weak peaks that fill in well a homology path and are unlikely to occur by chance must be visualized. The high credibility score may result from only a small fraction of the entire alignment region, leaving most of the match in doubt. The multiprobe approach and examination of the search matrix can alleviate these pitfalls. Perhaps the best and only significance test is the observance of a good distribution of peaks colinear with the search matrix diagonal. Despite biological and statistical correlations, mistakes can still be made. I compared nucleotide binding domain sequences from alcohol and lactate dehydrogenases. A search matrix resulted with the alignment assessing significant. Both sequence regions were known to bind NAD, yet the alignment (Fig. 3) was nearly two-thirds incorrect relative to the terl5

P. Argos and S. Fuller, EMBO J . 7,819 (1988).

760 LADH LDH(I) LDH(2)

ADDITIONAL TECHNIQUES

"'KIT[

[561

nn 0 0 0

Q G S T C A V F G L G G V G L S V I M G C K A A G - A A R I I G V D I V G V G A V G M A C A I S I L M K D L A D E V A L V D S Y N K I T V V G V G A V G M A C A I S I L M K D L A D E V A L V D V

LADH LDH(1) LDH(2)

I Q E V L T E M S N G G V D F S F E V I G - - - - - - - - - - - - - A G S K ~ V V ~ A ~ A R ~ Q E G ~ - - S - - - - - - - - - - - - - - - - - - - - - - S A ~ S K L V V I T ~ A R Q Q E G E S R L N L V Q

LADH

- - - R L D T M V T A L S C C Q E A Y G V S V I V G V P P D S Q N L S

;:dl; LADH

M - N P M L L L S G R T U K G A I - - - - - - - - - -

LDH(2)

FIG.3. Alignment of the NAD-binding domains from horse liver alcohol dehydregenase (LADH) with that from dogfish lactate dehydrogenase (LDH) as determined from the tertiary '~ The structural superposition [LDH(2)] and from the homology search m a t r i ~ [LDH(I)I. bracketing arrows show the initiation site where the two alignments do not agree. Residues are boxed in LDH(1) and LDH(2) if they are conserved or identical with those in LADH. The computer search yielded 18 and 34 identical and conserved residues, respectively, while the structural superposition ("standard of truth") resulted in only 7 and 16.

tiary structural superposition, which achieved only 12% identity, half that from the computer results. Alignment over short regions (say 10 to 40 residues) presents an assessment problem. Often they contain one or more regions with exactly matching tetramers, pentamers, or even hexamers. Sometimes they simply appear as good but short homologies with a reasonable number of identities and conservations. Beware if these regions involve repeating amino acids such as RRRRR or APAPAP or GSSSGG (R is the one-letter abbreviation for arginine, A for alanine, P for proline, G for glycine, S for serine). Be cautious if the homology cannot be somewhat extended. It has been found that spans up to 11 amino acids long exist in unrelated proteins (e.g., AAAAAAATAAA where T is threonine).16By the 8-mer level, both normal and repeating-type oligopeptides are in evidence; a common tetramer is certainly no guarantee of relationship. Nonetheless, relatively weak patterns can be significant. A Tyr-MetIVallSer-Asp-Asp tetramer flanked by pentamers each containing about three hydrophobic residues is common in many polymerases. In certain nucleotide-binding domains, Glyl6

P. McCaldon and P. Argos, Proteins 4,99 (1988).

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COMPUTERS AND SEQUENCES -

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-

-

-

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-

-

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-

X-Gly-X-X-Gly-X11-20-Lys pattern (X is any residue) occurs often; yet a search of a sequence database produces at least 10 biologically unrelated proteins displaying the pattern such as collagen, histone H I , D-amino-acid oxidase, and ribulose-bisphosphate carboxylase. Another important template consists merely of [Asn,Asp]-X-X-Cys-[Ser,Thr,Glu]Xl-8-[Asp,Glu]-[Asp,Glu,Ser,Thr]-[Asp,Glu] where amino acids in It has been found in large-T brackets are choices at a given po~ition.l'-'~ antigens, ElA proteins, and v-myc and c-myc oncogenes. Recent experimental results show this region to bind to retinoblastoma protein, which probably interacts with DNA and may be involved in cellular immortalization.I9 It is so simple and short and yet s o biologically important; caution may have missed it. Always try to use your biological sixth sense. It is recommended to search the entire sequence database for the short patterns. In the Protein Identification Resource package, the SCAN program works very quickly on exactly specified fragments while MATCH is just a little slower and allows for any-residue (X) positions in the pattern. Intelligenetic's QUEST gives the most flexibility by allowing any number of amino acids at a given pattern position (e.g., Asp or Glu or Gln o r Asn), variable length separators (e.g., an X run that is 11 to 20 residues long), and the like. If it is found that 1 out of every 50 protein sequences in the complete database displays the template, caution is called for. Of course, if the proteins share similar biological function and the patterns are not of the repeating type and contain active-site-like residues (e.g., His o r Asp or Cys and not Phe or Leu or Val), then credibility is further supported. In any event, a few good site-directed mutagenesis or gene fragment shuffling experiments are recommended. Sequence Patterns as Alignment Aid When many sequences have been aligned, certain patterns of residue conservation and identity are discernible over nearly the entire sequences, or more probably, over a short stretch o r two. This information can be of great help to identify sequences of distantly related family members. Many techniques have been proposed to characterize the template. The residue composition or certain amino acid characteristics (small, large, polar, hydrophobic, aromatic, charged, etc.) can be listed for each match position. The compositions can be normalized by that found in proteins " S. Stabel, P. l9

Argos, and L. Philipson, EMBO J. 4,2329 (1985). J. Figgie and J . F. Smith, Nature (London) 334, 109 (1988). P. Whyte, K. J . Buchkovich. J. M. Horowitz, S . H. Friend, M. Raybuck, R. A. Weinberg, and E. Harlow, Nature (London) 334, 124 (1988).

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generally. Gribskov et cleverly couple the exchange weights in the Dayhoff mutation matrix with the allowed residues at each alignment position of the template (called the "profile"). This approach attempts to guess which residues might be substituted given presently known sequences. Exchange weights are even given for gaps and particular residues. The result is a preference value for each amino acid at each alignment position. Suppose you wanted to find in your sequence a 30-residue pattern by ~' take every possible 30-residue segment in the profile m e t h ~ d . Simply your sequence and lay it residue by residue along the template. Sum the preferences of the template amino acid that matches the search sequence amino acid at each template position. After searching all sequence segments and using a typical significance cut-off, a template match may be found. If the template results from an entire sequence, simply lay the sequence along a matrix row and the template (20 preferences for each column position) along a matrix column and place preference scores according to residue matches as described above in the cross-positions. This will result in a typical search matrix, ready for automated alignment procedures. Finally, some pattern searches even allow a specified mixture of secondary structure predictions, flexible lengths, and exact or conserved amino acid matches.22 Care must be taken with these searches. Addition of new sequences (which build up fast these days) can drastically alter the consensus. A cluster of similar sequences from closely related species can unwarrantedly skew the compositional template at an alignment position. Consensus sequences are not always feasible for tertiary structures as the local order of amino acids (that interact well three-dimensionally) is ignored. Finding Repeats within Sequences

Delineating internal sequence repeats is also related to the pattern problem, except that now the pattern is associated with an internal motif. General but similar techniques based on residue characteristics have been A plot is made of some residue property, such as hydrodeveloped .23,24 phobicity, with sequence position. This plot is moved past an identical one by consecutive steps of one sequence position. A correlation coefficient 'O

'' '' '' 24

M. Gribskov, A. D. McLachlan, and D. Eisenberg, Proc. Natl. Acad. Sci. U.S.A.84,4355 (1987). M. Gribskov, M. Homyak, J. Edenfield, and D. Eisenberg, CABIOS 4,61 (1988). R. H. Lathrop, T. A. Webster, and T. F. Smith, Commum. ACM 30,909 (1987). P. Argos, M . Hanei, J. M. Wilson, and W. N. Kelley, J. Biol. Chem. 258, 6450 (1983). Y . Kubota, H. Takahashi, K. Nishikawa, and T. Ooi, J. Theor. Biol. 91,347 (1981).

r-

r:

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between the superimposed curves is calculated at each register site. A plot of this coefficient vs register will show a repeating pattern if there is a conserved characteristic pattern within the sequence. Several amino acid properties can be used to confirm the motif. Smoothing the initial plot helps. The above methods do not take deletions and insertions into account. This problem is eliminated by using self-search matrices with a given probe length and based on Dayhoff mutations and residue properties.'2 A visual indicator of sequence repeats are stacks of high peaks contained in vertical column spans.2sIn this case, a plot of sequence position and the sum of search matrix scores at that position along a vertical column yields a curve of repetitive high peaks at the start site of each motif. In some cases, the method has delineated repeats where in 10 of them, each with about 35 amino acids, only 1 match position contains the same amino acid.26

Multiple Sequence Alignment The automated Needleman-Wunsch technique is easily bogged down by the many paths and scores involved in a large-dimensional matrix needed to align several sequences simultaneously. Then there are all the gap penalty values to try. Even present supercomputers would balk at the problem. So, short cuts have to be found. In my view, two approaches are presently most viable. One uses all sequence pairwise alignments; Barton and Sternberg2' exemplify the approach. They intercompare all sequences and save the alignment for the most related pair and then compare to this pair a third sequence which has the highest possible score in all comparisons with one of the first two sequences. Sequences are similarly added (the sequence most related to any one of the previously added) and compared with the alignment of all previously used sequences. The problems are nontransitivity along with order dependence and gap penalty choice. In fact, once an insertiontdeletion is introduced, it cannot be eliminated despite increasing information introduced from inclusion of further sequences. A second approach recently developed by Vingron and AgrosZ8 relies on profile fitting, previously discussed. The dipeptides contained in each sequence are distinguished and counted. Those sequences that show a high count of the same 2-mers are clustered; they should be closely related. Within each cluster the sequences are aligned so that the largest zs G . M.

Bressan, P. Argos, and K. K . Stanley, Biochemistry 26, 1497 (1987).

26

F.Altruda, V. Poli, G . Kestogno, P. Argos, R. Cortese. and L. Silenga, Nucletc Acids Res.

l7

W, 3841 (1985). G . J. Barton and M. J . E. Sternberg, J . Mol. Blol. 191, 153 (1987). M.Vingron and P. Argos, CABZOS 5, 1 15 (1989).

"

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number of dipeptides can be matched across all the sequences. The sequence regions enclosed by a set of neighboring aligned dimers are then matched from longest to shortest (this minimizes gaps). The longest two are matched by using a transformed matrix based on Dayhoff mutation values. The third is then fitted to the profile of the first two and then the fourth to the profile of the first three and so forth. The procedure is repeated for all dimer-enclosed sequence regions in each of the clusters. The two clusters sharing the greatest number of dipeptides are joined by aligning each of their profiles. A new profile is then determined from their union. The profile of the next sequence cluster, which shares the most dipeptides with either of the previous two clusters, is aligned with the just determined profile. Then a more extensive profile is calculated from the three clusters. The procedure is repeated and can be performed even if a subset contains only one sequence. This method is automatic and fast and reasonably sensitive; it suffers from the required setting of gap penalty values and certain cutoff scores such as those used for clustering. Recommendations

In summary, the following computer tests are recommended for that brand new sequence.

1. Search the entire protein sequence database with a fast algorithm such as that from Pearson and Lipman. 'O Since this method first recognizes homologies through identities in sequence segments and extends the homology with strict gap penalties, it loses sensitivity but will quickly point out obvious homologies (e.g., -30% identity and above). 2. If a good but short homology is found (say 10-40 residues), use a more sensitive method, such as UWGCG's BESTFIT or my approach," to discern if it is extendable. If a homology from functional considerations is probable, definitely test it by a sensitive method. Make sure to compare the sequence and all sequences homologous to it with all known sequences in the family suspected to be related to its primary structure. Try ALL pairwise comparisons. I have seen many cases where only a few sequence pairs produced a convincing search matrix while the others did not fill in the entire path. 3. If nothing looks particularly exciting, use a sensitive technique on the top 20 to 40 Lipman-Pearson finds to be assured that a marginal homology cannot be expanded to a convincing long one. Of course, if other sequences are suspected to match due to functional relationships and Lipman-Pearson does not suggest them, definitely explore for homology by the more sensitive techniques. 4. If any sequence homology, long or short, makes good biological sense, examine it carefully; though it is short, it may be an important

m

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functional span derived from evolution. Check all members of a protein family containing the short stretch and make sure that important functional and structural residues remain the same or are strongly conserved. For short stretches, also check the pattern against the entire database; if too many unrelated proteins display the template, it's a gamble! Structure Prediction

Finding Structural Class The overall or global properties of protein sequences can be used for general predictions of structural class, cellular location, or even function. The methods rely on patterns in n-dimensional space (i.e., n selected properties of each protein sequence). If points cluster together according to the desired classification, then predictions are possible. Proteins can be structurally classified into five groups: all-a (composed almost exclusively of helices and loops), all-6 (@-strandsand connectors), alp (repeating patterns of helix-connection-strand), a + @ (an all-a region followed by an all-@region), and irregular. The properties used for structural class predictions include amino acid composition of the sequence, number of runs of four or more polar and apolar residues, hydrophobic moment (see next section), and a- and @-contentestimated from secondary structure prediction method^.^^-^' Assignment accuracy is around 70%. From the amino acid frequencies alone, Nishikawa et ~ 1could . deter~ ~ mine if a protein was an enzyme with -80% success. Prediction of intra- or extracellular location was also attempted. Klein et ~ 1 even . ~tried~ to classify a protein by function (e.g., globin, muscle protein, inhibitor, toxin). They used a myriad of properties such as net charge, sequence length, residue frequencies, and mean hydrophobicity. Some 75% of the tested sequences were assigned properly. Secondary Structure Prediction Many techniques have been developed since the mid-1970s to predict the location of helices, strands, turns, and coil within a protein's primary structure. The most popular and one of the earliest approaches is a statistij9

H. Nakashima, K. Nishikawa, and T. Ooi, Nucleic Acids Res. W, 5327 (1985).

'OR. P. Sheridan, J . S. Dixon, R. Venkataraghavan, I. D. Kuntz, and K. P. Scott, Biopolymers 24, 1995 (1985). P. Klein and C. DeLisi, Biopolymers 25, 1659 (1986). K. Nishikawa, Y. Kubota, and T. Ooi, J . Biochem. (Tokyo)94,997 (1983). " P. Klein, J. A. Jacquez, and C. DeLisi, Math. Biosci. 81, 177 (1986). "

766

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TABLE I SECONDARY STRUCTURE PROPENSITIES" Amino acid

a-helix

P-strand

Turn

Ala Arg Asn Asp C Y ~ Gln Glu GlY His Ile Leu LYS Met Phe Pro Ser Thr Trp TYr Val -

a

From J. Palau, P. Argos, and P. Puigdomenech, Int. J. Pept. Protein Res. 19, 394 (1982).

cal one developed by Chou and F a ~ m a nThe . ~ ~secondary structure (SS) state of residues within a protein can be determined from the threedimensional structure of the protein (presently about 100 nonhomologous proteins). It is then easy to calculate the preference of a particular amino acid to be in a given SS (i.e., the percentage of a certain amino acid within the particular SS divided by its overall percentage within the database protein sequences). This propensity is thus normalized to 1.00, which indicates neutral preference; less than 1.00 is avoidance of the SS, while greater than 1.00 indicates preference. Table I lists the helix, strand, and turn propensities. The following rules (not given in detail) can be used to elicit a prediction. 1. If four of six residues in a sequence show a helical propensity greater than 1.00, a helix is nucleated. Extension is allowed in both direc34

P. Y. Chou and G. D. Fasrnan, Adu. Enzyrnol. 47,45 (1978).

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tions until helix-breaking tetrapeptides (average propensity less than 1.00) are encountered. Proline can only be in the first three N-terminal positions. The final mean a-propensity must be greater than 1.05 and greater than the mean p-propensity. 2. Three p-formers (propensity greater than 1.00) in a sequence of five residues nucleate a strand structure. Extension and final prediction conditions are similar to those for helices. 3. Overlapping regions predicted to be both in helix and in strand are resolved by the one with greater mean propensity. 4. Turns are predicted when the average turn propensity over four consecutive residues is greater than 1.OO and greater than the mean a- and P-preferences over the same residues. The Garnier-Robson approach35also relies on statistics from known structures but is more formally based on information theory. The technique uses direction and dipeptide interaction by considering the frequency of a central residue and one residue at any position to the left and right of it. By summing the 16 SS statistical preferences of a central residue and 8 on either side for each of 4 conformational states, the largest result provides the SS prediction. The physicochemical approach (exemplified in Ref. 36) capitalizes on the systematic polar and nonpolar properties of residues along a sequence. In an a-helix, successive C, positions occur at rotations of nearly 100°, so their spatial projection down the central helix axis can be drawn on a circle (see Fig. 4). Up to 18 successive C,'s can be uniquely positioned on the projection; the nineteenth will simply fall on the first, the twentieth on the second, and so forth. Note that (18 x 100")/360" =5, the first integer possible given the 360" divisor. Usually, down one side of the helical wheel are found polar residues to face the external aqueous environment and on the other side hydrophobic residues to pack against the protein's internal core (amphipathic helix). This pattern of hydrophobic/hydrophilic residues can be searched to predict a helix. Similarly, a p-strand may show alternating polar/nonpolar residues along the sequence. Eisenberg et ~ 1 . ~use ' these observations to calculate a hydrophobic moment such that, for a helix, vectors with a magnitude corresponding to the residue's hydrophobicity are drawn from the wheel center to the appropriate C, position. For a given sequence region the vectors can be added (hydrophobic moment); if the helix is 198,425(1987). V. I. Lim, J. Mol. Biol. 88, 873 (1974). " D. Eisenberg, R. M. Weiss, and T. C. Terwilliger, Proc. Narl. Acad. Sci. U.S.A. 81, 140 (1984). 35 36

J . F. Gibrat, J. 0. Granier, and B. Robson. J. Mol. Biol.

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FIG.4. An elementary helical wheel projection. The sequence shown is for a putative transmembrane helix in Neurospora crassa proteolipid protein. Note the 100" rotation between successive positions (helix starts at sequence position 56) and the overlapping projections after the first 18 residues. The amphipathy of the helix is indicated by the bold outline and boxed polar residues as well as Gly. which is thought to appear in lipid-associated helices.

amphipathic, the magnitude of the vector will be large and pointing toward the center of the hydrophobic half of the helix. Strands can also be examined except successive vectors point roughly opposite. Selection of a sequence length allows calculations of the moments for all sequence segments and subsequent predictions for results above a given threshold. S ~ e e t ,L ~ 'e ~ i nand , ~ Nishikawa ~ and Ooi40all converged on the notion to utilize consensus secondary structures of oligopeptides in the databank of known tertiary protein structures. A window of 7-12 residues in length is defined. Each such span in the sequence to be predicted is judged for homology to several spans in the known tertiary structures. The consensus structure in the known spans allows a prediction for the unknown span. Moving along the sequence one residue at a time, the process is repeated and the preferred secondary structure at a particular position taken as the prediction. A new wave in secondary structure prediction is utilization of 38

39

"

R. M. Sweet, Biopolymers 25, 1565 (1986). J. M. Levin, B. Robson, and J. 0. Garnier, FEBS Lett. 205,303 (1986). K. Nishikawa and T. Ooi, Biochim. Biophys. Acta 871,45 (1986).

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artificial intelligence programming techniques4' and neural network mode l ~ However, . ~ ~ improvement in prediction accuracy over the earlier methods is not large. Prediction of Other Structures

Kyte and D ~ o l i t t l ehave ~ ~ attempted to predict membrane-spanning regions by assigning hydrophobicities to each of the amino acids and plotting them as a function of sequence position. The curve is smoothed by averaging the hydrophobicities for every segment of 19 residues and placing the mean value at the central position of the segment (e.g., position 10 for 1-19) in the smoothed plot. Regions are predicted as lipid associated if the plot peaks are sufficiently long and the mean segment hydrophobicity is greater than + 1.6 on their scale (see Table 11). MohanaRao and Argos4" calculated Chou-Fasman-like propensity parameters for each of the 20 amino acids (Table 11)from known and strongly suspected membrane-spanning sequence regions in several proteins. A smoothed plot is produced and predictions made based on helix length (at least 12) and plot peak thresholds. Thresholds were assigned such that few lipid-associated helices were predicted in soluble proteins, an important control not always performed by others. In a different approach, Eisenberg et plot hydrophobic moment vs mean hydrophobicity ( < H > ) for 21-residue windows providing is greater than a threshold (Table 11). If the point falls in a particular region of the plot, it can be predicted as membrane spanning. The accuracy of these methods is mostly untested and they do not always agree.' For example, in cytochrome-c oxidase I, the Kyte-Doolittle method predicts only 8 membrane spanners while Eisenberg et al. prefer 12. Predictions of possible antigenic regions in protein sequences are based on residue polarity and antigenic preferences (Table 11). The procedure of Hopp and Woodd6 plots hydrophilicities for each sequence position and smooths using a sliding window average. The highest peaks indicate the regions most likely to be exposed on the protein surface and therefore most likely to be recognized by an antibody. Welling et calculate antigenic 4'

F. E. Cohen, R. M. Abarbanel. I. D. Kuntz, and R. J. Fletterich, Biochemistry 25, 266 (1986).

'IN.

Qian and T. J. Sejnowski, J. Mol. Biol. 202,865 (1988).

"

J. Kyte and R . F. Doolittle, J. Mol. Biol. 157, 105 (1982). J. K. MohanaRao and P. Argos, Biochim. Biophys. Acta 869, 197 (1986).

45

D. Eisenberg, E. Schwarz, M. Komaromy, and R. Wall, J. Mol. Biol. 179, 125 (1984).

47

G. W. Welling, W. J. Weijer, R. van der Zee, and S . Welling-Wester, FEBS Lett. 188,215 (1985).

" T. P. Hopp and K. R. Woods, Proc. Natl. Acad. Sci. U.S.A. 78,3824 (1981).

770

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TABLE I1 SOMEUSEFULINDICES FOR STRUCTURAL PREDICTIONS

Amino acid Ala Arg Asn Asp CYs Gln Glu GlY His Ile Leu LYS Met Phe Pro Ser Thr T~P TYr Val

KyteDoolittle hydrophobicity

Rao-Argos membraneburied helix

Rao- Argos signal sequence preference

Hopp-Woods antigenic preference

Welling antigenic preference

Eisenberg consensus hydrophobicity

1.8 -4.5 -3.5 -3.5 2.5 -3.5 -3.5 -0.4 -3.2 4.5 3.8 -3.9 1.9 2.8 -1.6 -0.8 -0.7 -0.9 -1.3 4.2

preferences in a Chou-Fasman style for each of the 20 amino acids from known antibody sites in sequences and plot the potentials (Table 11). Jemeson and Wolf48use a summation at each sequence position over five different parameters, each weighted for optimal prediction. They include (weights in parentheses) hydrophilicity (0.3), probability of protein surface exposure (0.15), backbone flexibility (0.15). and secondary structure predictions by two methods (0.2, each). Two techniques have been suggested to find possible T cell epitopes. The one of DeLisi and B e r z o f ~ k yis~based ~ on amphipathicity and a-helix periodicity calculations. Rothbard and ~ a y l o r "search for sequence patterns starting with glycine or a charged residue, then two or three hydrophobic residues, and finally a charged or polar residue. MohanaRao and Argo? have listed preferences for residues to be in B. A. Jameson and H. Wolf, CABIOS 4, 181 (1988). C. DeLisi and J. A. Berzofsky, Proc. Narl. Acnd. Sci. U.S.A. 82,7048 (1985). 50 J. B. Rothbard and W. R. Taylor, EMBO J . 7.93 (1988).

49

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signal sequences (Table 11); von ~ e i j n e "calculates the natural log of the ratio of the amino acid composition at a given signal sequence position to that found in proteins generally. Threshold scores are found for predictions. Finally, von HeijneS2has found sequences of certain mitochondrial proteins that are important in targeting proteins to different mitochondrial locations. Secondary Structure Prediction Accuracy and Utility

How accurate are these prediction methods? The answer lies somewhere between 50 and 65%53.54 with the refined predictions' for specialized supersecondary structures reaching as high as 65-75%. The refined predictions are limited in use because they depend on knowledge of a tertiary structure and several alignable sequences and they can predict only specialized folds. The percentage accuracy refers to three-state models (i.e., helix, strand, other) where roughly 33% correctness would be expected from any random selection. It is generally conceded that the better predictions result from averaging over several method^^^.^^ or averaging predictions over several aligned sequences. What is the utility of secondary structure predictions? Since tertiary topology is mostly a collection of secondary structures connected by loop regions, its prediction will probably have to be preceded by knowledge of secondary structures. If a particular sequence span is known to be essential for function, then secondary structure prediction can provide testable methods for experiments; for example, if a helix is predicted and the hydrophobic side faces the functional substrate cavity, then those residues can be modified by site-directed mutagenesis. I have examined the secondary structure of pentapeptide pairs differing by only one amino acid that are present in known tertiary structures and found their SS to be conserved only 20% of the time.57I also recommended residue exchanges that would strengthen or disturb the secondary structure. Predictions can support alignment of distantly related sequences (see a-crystallinss8 or TIM Pbarrels59)or provide support for assignment of the sequence to a supers' G. von Heijne, Nucleic Acids Res. 14,4683 (1986). G. von Heijne, EMBO J . 5, 1335 (1987). '%. Nishikawa, Biochim. Biophys. Acta 748,285 (1983). W. Kabsch and C. Sander, FEBS Lerr. 155, 179 (1983).

'* "

P. Argos, J . Schwarz, and J. Schwarz, Biochim. Biophys. Acta 439, 261 (1976). V. Biou, J. F. Gibrat, J. M. Levin, B. Robson, and J. Garnier, Protein Eng. 2, 185 (1988). P. Argos, J . Mol. Biol. lW,331 (1987). 58 P. Argos and R. J. Siezen, Eur. J. Biochem. 131, 143 (1983). " I. P. Crawford, T. Niermann, and K. Kirschner, Proteins Struct. Funct. Genet. 2, 118

55

56

(1987).

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secondary structural classification. A nonuniform distribution of SS type may point to domain s t r ~ c t u r ePredictions .~ can sometimes explain the conservation patterns of certain residues, such as glycine in a predicted turn region. Likely exposed areas in the sequence can be predicted; these regions may be more available for modification or antibody recognition or use in vaccine de~elopment.~' Hones et ~ 1 . present ~' an example of how all this information can be utilized to provide an experimentally testable structural model of a glucose dehydrogenase. Residue Characteristic Plots

Research reports on new sequences often display plots of sequence position vs some residue property. However, which amino acid characters are the best ones to examine? Nakai et analyzed over 220 properties. They find only five independent (not strongly correlated) sets that center on volume, hydrophobicity, bulkiness (residue shape), strand propensity, turn conformational preference, and helical propensity. These results essentially agree with those I found in attempting to align sequences (see first section on alignment). As previously discussed, these parameters are probably most important to yield and maintain the protein fold. It is suggested that these characteristics (Table 111) or some closely related ones be used for sequence plots. General trends in property plots are not aIways evident without smoothing. Smoothing is usually achieved by averaging the properties over some given window length of odd number and placing the mean value at the central position of the window. For instance, if a probe length of three is used in a sequence containing six residues, then a mean is calculated for amino acids in sequence positions 1 to 3 , 2 to 4 , 3 to 5, and 4 to 6. The respective means are placed in the plot at positions 2, 3, 4, and 5. Positions 1 and 6 can be taken as their actual values or averaged with the nearest neighbor smoothed values. The length of the window should correspond to the structure examined or predicted. For example, in predicting transmembrane helical segments, use a length of about 20 while for antigenic sites, 7 is better. Alternatively, a length of 3 could be used, but smoothing would occur over several cycles (e.g., 10) where the just smoothed curve is smoothed again. P. Argos. S. V. L. Narayana, and N . Nielsen, EMBO J. 4, 1 1 1 1 (1985). J. L. Bittle, R. A. Haughton, H. Alexander, T. M . Shinnick, J. G . Sutcliffe, and R. A. Lerner, Nature (London)298,30 (1982). J . Hones, K. D. Jany, G . P. Fleiderer, and A. F. V. Wagner, FEBS Lett. 212, 193 (1987). 63 K. Nakai, A. Kidera, and M. Kanehisa, Protein Eng. 2,93 (1988). 60

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TABLE I11 RESIDUE PHYSICAL CHARACTERISTICS' USEDIN ARGOSSEQUENCE ALIGNMENT METHOD

Amino acid Ala Arg Asn ASP CYs Gln Glu G~Y His Ile Leu LYS Met Phe Pro Ser Thr

T~P TYr Val

Hydrophobicity

Turn propensity

Residue bulk

Refractivity index

Antiparallel strand propensity

1.01 0.91 0.89 0.84 1.14 0.91 0.92 0.97 0.94 1.22 1.16 0.88 1.12 1.09 0.88 0.87 0.91 1.08 1.04 1.22

0.82 0.93 1.35 1.48 0.84 1.02 1.05 1.73 0.73 0.54 0.62 1.01 0.41 0.61 2.01 1.40 1.08 0.79 1.11 0.49

0.75 0.93 0.83 0.76 0.88 0.94 0.88 0.22 0.89 I .39 1.39 1.02 1.06 1.29 1.13 0.62 1.03 1.41 1.17 1.40

0.23 1.42 0.71 0.64 1.91 0.94 0.92 0.00 1.16 1.02 1.OO 1.13 1.15 1.57 0.58 0.34 0.59 2.27 1.68 0.74

0.90 0.82 0.54 0.54 1.19 0.85 0.85 0.56 1.12 1.54 1.26 0.82 1.19 1.40 0.42 0.87 1.30 1.40 1.68 1.53

" Normalized to 1.00.

Such plots can be utilized to find domains, predict certain structural types, delineate sequence regions with exaggerated properties, and even relate sequences and find subtle repeats within them. The latter goals have been examined by myself and ~ o l l e a ~ u and e s ~Kubota ~ ~ ~ ~et ~ 1 . * ~ . ~TO '' determine if 2 sequences are related, (1) calculate their respective characteristic plots, (2) translate 1 of the plots to the left of the other so that the 25 N-terminal residues of 1 plot overlap the 25 C-terminal residues of the other, (3) calculate a correlation coefficient between the 2 sequence series of 25 overlapping plot characteristics, and (4) move the leftmost curve 1 sequence position to the right and calculate a correlation coefficient again, except now for the characters over the 26 overlapping positions. This 64

Y. Kubota, K. Nishikawa, H. Takahashi, and T. Ooi, Biochim. Biophys. Acta 70, 242 (1982).

774

[561

ADDITIONAL TECHNIQUES

I I

-

-1ob.0

0 .O

100 .O

LAG

200 .O

300 .O

FIG.5. Sum of the correlation coefficients vs the register of the sequence overlap for six amino acid physical characteristic curves. The two sequences compared are the N-terminal portion (residues 1 to 120) of human xanthine-guanine phosphoribosyltransferase and the entire ATP phosphoribosyltransferase sequence. The highest correlation sum near lag 0.0 suggests that both N-terminal regions are structurally homologous and are nucleotide-binding domains.

procedure is repeated for each successive sequence position until the 25 N-terminal positions of the initial leftmost sequence overlap with the 25 C-terminal positions of the initial rightmost sequence. The register or phase of the two plots is called the "lag," which is defined as the difference in sequence position of the first overlapping point in the two plots. Lag vs correlation coefficient is plotted and can be smoothed (Fig. 5). A high correlation coefficient above a control thresholdz3 would indicate a sequence relationship or domain boundary. If the procedures were repeated

-

[561

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COMPUTERS AND SEQUENCES

for a sequence against itself, then periodic correlation peaks would suggest a repeat within the sequence. Correlation plots over coefficients from several characters (e.g., the five recommended) can be averaged for a more sensitive search. Relationships between dehydrogenase nucleotidebinding domains were found23 while other methods failed. The major weakness of the technique is neglect of insertionsldeletions. If they are too long, the points to be correlated are out of phase. The self-search is independent of this problem, but the size of the smoothing window must be chosen according to the length of the smallest repeat unit expected. Databases and Program Packages

Data Bases

The most relevant databases contain amino acid sequences (see Table IV, source I), nucleotide sequences (source 2, if in Europe; source 3, if in the United States), and three-dimensional protein structures (source 4, Table IV). There are many other specialized data collections on frequency of codon usage, polysaccharide sequences, mapped human genomic loci, and so forth. See Ref. 1 for a more extensive listing which is taken from the complete compilation of R. Martinez and C. Burks at the Los Alamos National Laboratory, Theoretical Biology and Biophysics Group (Los Alamos, NM).

TABLE 1V SOURCES OF DATABASES A N D COMPUTER PROGRAMS REFERRED TO

IN

TEXT

1. Protein Identification Resourse (PIR), National Biomedical Research Foundation, Georgetown University Medical Center (3900 Reservoir Road N.W., Washington, D.C. 20007) 2. EMBL Nucleotide Sequence Data Library, European Molecular Biology Laboratory (Postfach 10 22 09, Meyerhofstrasse 1,6900 Heidelberg, FRG) 3. GenBank Genetic Sequence Data Bank (Los Alamos National Laboratories, Los Alamos, NM 87545) 4. Protein Data Bank (c/o T. F . Koetzle, Chemistry Department, Brookhaven National Laboratory, Upton, NY 11973) 5. U. W. Biotechnology Center (1710 University Avenue, Madison, WI 53705) 6. IntelliGenetics (700 East El Camino Real, Mountain View. CA 94040) 7. Applied Genetic Technology (3910 West Valley Drive, Fairview Park, OH 44126) 8. R. Staden, Laboratory of Molecular Biology, MRC Centre (Hills Road, Cambridge CB2 2QH, England) 9. Lipman-Pearson (Department of Biochemistry, Box 440. Jordan Hall, University of Virginia, Charlottesville, VA 22908)

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[561

Program Packages

The selection of an appropriate sequence analysis package depends on your computer and the type of investigations normally pursued. There are many program suites available. One of the most extensive listings is that by Lewitter and Rindone.' However, their information is taken from the largest compilation maintained by the GenBank Software Clearing House (see source 3, Table IV). I will only give a few examples. An excellent overall package of programs that work on VAX computers is acronymed UWGCG (Table IV, source 5); the techniques include prediction, dot plots, pattern look-ups, consensus sequence searches, moment calculations, and much more. For IBM-like PCs, check on the PC GENE package from IntelliGenetics (source 6); besides many of the usual programs, PC GENE has its own amino acid sequence databank (SWISSPROT), a good list of important structure references, and many prediction routines, including those for transmembrane spans. For Apple PCs, check on MAC GENE (source 7). The STADEN package is also a good one for protein and DNA sequence analysis (source 8). The Lipman-Pearson program (written for different computers and called FASTA) works well for quickly searching an amino acid sequence against all those in most any of the important sequence databases; however, sensitivity is usually only good to about 30% identity. Finally, one should always write to authors for any specialized programs.

Section XI11 Concluding Remarks

t571

RETHINKING YOUR PURIFICATION PROCEDURE

779

[57] Rethinking Your Purification Procedure

Every protein purification that you undertake should provide you not only with purified material, but also with considerable information about the protein. Thus, during the course of purification you most likely will learn about the stability of the protein under a variety of conditions, as well as about its size, charge and, perhaps, its affinity properties. You will have learned whether the protein can be concentrated, diluted, dialyzed, o r exposed to a variety of agents. In addition, you may have prepared an antibody against the protein, subjected it to a limited sequence analysis, o r determined whether it has any covalent modifications. All of this information can be of great help in deciding whether you have developed an optimal purification scheme. Obviously, in some cases you may not care. However, if this protein is one you plan on studying in some detail, and you can foresee many purifications ahead, a rapid and efficient (meaning high purity and high yield) purification scheme can save you an enormous amount of work in the long run. By taking advantage of what you have learned about the protein, it generally should be possible to streamline and optimize the procedure a great deal. There is a natural tendency, especially after having spent many months learning to purify a protein, to go with what you know works. Nevertheless, spending some time rethinking your purification procedure will be a worthwhile exercise. A list of things you might want to think about, with some relevant chapters to help you think, are as follows: 1. Am I using the best source of material ([12]-[15])? a. Is the source readily available in large quantities? b. Is the protein associated with a specific subcellular structure that might be a better starting material ([16], [17])? c. Would it be possible to develop a better source by cloning the gene for this protein using either the antibody I have prepared ([49], [50]) o r by synthesizing an oligonucleotide probe based on sequence information I have obtained ([56])? d. Can the protein be overexpressed from the cloned gene ([91-[111)? 2. Are all the steps in the purification scheme necessary and useful? a. Do I extract most of the available activity ([12]-[15])? b. Do any steps lead to unnecessarily large losses or relatively poor purifications, and is there a special reason for including that step? METHODS IN ENZYMOLOGY. VOL. 182

Copyright Q 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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3.

4.

5.

6.

7.

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CONCLUDING REMARKS -

-

-

c. If there are large losses at any step, do I know why, and can it be prevented? d. Are all the steps cost effective? Would a cheaper purification medium or procedure suffice? e. Can any time-consuming procedures be eliminated? Are the various purification steps carried out in the most optimal sequence? a. Are procedures best for larger amounts of material in the beginning of the scheme, and those better for smaller amounts later in the procedure? b. Can I avoid a concentration or dialysis step by changing the order of steps ([7])? Is it necessary to concentrate a sample prior to a column to which the protein binds? c. Are the solution conditions of the last step in the procedure compatible with storage of the protein, or is a solution change necessary? Should I introduce any new steps into the purification procedure? a. Might a new step that takes advantage of the protein's binding properties (i.e., affinity chromatography) be effective ([29], [301)? b. Can the antibody I have prepared be of use for purification? c. Am I taking advantage of all the protein's structural properties in deciding on purification steps? Is the scale of the purification appropriate to the planned uses of the material? a. Can enough material for my needs be obtained in fewer steps by using two-dimensional gel electrophoresis ([4]) or immunoprecipitation ( [52])? Am I using the best assay for my protein considering speed, cost, and accuracy ([5])? Is a high degree of accuracy necessary during the purification? If I have not already done so, can 1learn anything from the literature by examining purification schemes for related proteins (see, for example, Appendix in this volume).

The answers to these questions will give you a good idea whether modification of your purification procedure might be warranted.

Appendix Personal Perspectives on Enzyme Purification

[581

DNA

POLYMERASE

I

783

[581 The Private Life of DNA Polymerase I*

DNA polymerase first came into my view in February 1955 when 50 cpm of [I4C]thymidine out of a million were converted to an acid-insoluble form by a heat-labile component in a crude extract of Escherichia coli. This premature newborn was for a time ignored because I was more impressed by the vigorous conversion of thymidine to phosphorylated forms and the promising purification under way of an E. coli activity which incorporated [ I 4 C ] A ~ Pinto what we presumed to be RNA. With the discovery of polynucleotide phosphorylase announced later that year by Grunberg-Manago and Ochoa,' the enzymatic synthesis of RNA seemed to have been preempted. As a result, my interest in the ATP-incorporation activity waned and my curiosity about the incorporation of thymidine revived. In December, 10 months after I first observed the acid-insoluble thymidine, I repeated the experiment, found the counts to be sensitive to DNase, and set about to purify the miniscule activity responsible for DNA synthesis.

r I

7

DNA: Primer or Template?

The historic papers by Watson and Crick in 1953~ had proposed that the strands of a DNA double helix could serve as templates for replication, and so it is commonly assumed that in my initial search for DNA synthesis in cell-free extracts 2 years later, I included DNA in the reaction mixture to serve that purpose. Not so. I added DNA expecting that it would serve as a primer for growth of a DNA chain because I was influenced by the pioneering work of Carl and Gerty Cori on the growth of a carbohydrate chain by glycogen phosphorylase. In our first report,3 we refer to the added DNA as a source of "active primer." I never thought that I would discover a phenomenon utterly unprecedented in biochemistry: an absolute dependence of an enzyme for instruction by its substrate serving as a template. * This chapter is adapted from "The Early History of DNA Polymerase," Biochim. Biophys. Acra 1000, in press (1989).

' M. Grunberg-Manago and S. Ochoa, J . Am. Chem. Soc. 77,3165 (1955). * J. D. Watson and F. H. C. Crick, Nuture (London) 171,737; 964 (1953). ' A. Kornberg, I. R. Lehman, M. Bessman, and E. Simms, Biochim. Biophys. Acta 21, 197 (1956).

METHODS IN ENZYMOLOGY, VOL. 182

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I had added DNA for another reason. Nuclease action in the extracts was rampant, and I wanted a pool of DNA to surround and protect at least some of the newly incorporated thymidine. Only later did we learn with elation that the added DNA fulfilled two other essential roles. It did serve as a template and was also the source of the missing nucleotides. (The DNA was cleaved by DNases in the extract to nucleotides. These were converted by ATP and five kinases in the extract to the di- and triphosphates of the A, G, C, and T deoxyribonucleosides; the dNTPs were then still unknown.) It was not until 1970 that we realized that DNA polymerase cannot start a chain and requires a primer to do so. Today, when we recognize that the first DNA polymerase, found in E. coli, is prototypical for all DNA polymerases and is widely used in the analysis and engineering of genes and chromosomes, it may be worth recalling crises of doubt and confusion, as well as triumphs, that surrounded this enzyme in its early history. Problems in Purification

Several fractions prepared from the crude extract were needed for incorporation of t h ~ m i d i n e .Of ~ these, two supplied the nucleases and kinases that generated the missing dNTPs and another the "polymerase." From cell extracts prepared by sonic disruption, nucleic acids had to be removed and this was accomplished by a refined precipitation with streptomycin sulfate followed by digestion with ~ N a s eThese . ~ early steps were for a time inconsistent and especially dismaying to Julius Adler at the start of his postdoctoral work in 1957. Despite his superior skills and experience in enzymology, he could not get the procedure to work properly. After he had exhausted our 100-lb supply of cell paste obtained from the Grain Processing Corporation in Muscatine, Iowa, the next batch worked like a charm. In fact, it seemed that every other Muscatine batch proved to be poor and we were inclined to discard the odd ones without even testing. Nucleases in Polymerase

Try as we might, we could not rid our polymerase preparations of nuclease activity. Knowing that DNA polymerase synthesizes a chain in the 5' to 3' direction, it made no sense to me then that the presumably pure enzyme degraded the very 3' end of the chain it would normally be extending. In the absence of the dNTPs needed for synthesis, nucleotide units I. R. Lehman, M. J . Bessman, E. S. Simms, and A. Kornberg, J. Biol. Chem. 233, 163 (1958).

[581

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were cleaved slowly and serially from the 3' end of a DNA chain.' Eventually, we learned that the enzyme removes frayed or mismatched units at the end of a primer, thereby permitting fresh units to be added to the growing chain end only when it is correctly matched to the template chain. We could infer that if the synthesizing enzyme were to make a rare mistake during elongation of a chain, such as inserting a C opposite an A (estimated to happen once in 10,000 times), it would remove the mismatched C before proceeding with extension of the chain. This astonishing proofreading ability of the e n ~ y m ecoupled ,~ with its fine discrimination in the initial choice of correct building blocks during synthesis, reduces errors in the overall process of replication to 1 in 10 million. Having finally accepted a DNA-degrading activity in the very enzyme that makes it, we were unprepared for the observation that nuclease activity in DNA polymerase persists even when digestion from the 3' end is blocked by an analog or Upon examining this activity, we found that a separate domain in the enzyme operates at a nick in a duplex to remove nucleotides from the 5' end of a chain. Then it became clear that polymerase cou!d use this other nuclease function to remove foreign structures in DNA, such as the thymine-dimer lesion of ultraviolet irrad i a t i ~ nand , ~ as we recognized some years later, could perform an essential step in replication by removing the RNA that initiates the start of a DNA chain. Fragments of Enzyme Are Active

I had been puzzled and even dubious about reports of low-molecularweight forms of the p~lymerase.'~." Then we found one. In 1969, the late Maurice Atkinson asked Doug Brutlag for DNA polymerase to use as a size marker for SDS gel electrophoresis. Instead of a band at 109 kDa, there were two, one at 76 and the other at 34 kDa. With a freshly thawed sample in place of the aged but still active enzyme, only a single band was observed at 109 kDa. Because work on Bacillus subtilis was ongoing in the same laboratory, the fresh enzyme was exposed to a B. subtilis extract and separately to trypsin. In each case, proteolytic cleavages yielded the 76-

' A. Kornberg, Science 163, 1410 (1969). D. Brutlag and A. Kornberg, J. Biol. Chem. 247, 241 (1972).

' R. P. Klett, A. Cerami, and E. Reich, Proc. Natl. Acad. Sci. U . S . A .60,943 (1968). ' M. P. Deutscher and A. Kornberg, J. Biol. Chem. 244,3029 (1969).

R. B. Kelly, M. R. Atkinson, J. A . Huberman, and A. Kornberg, Nature (London)224,495 (1 969). 'O A. G . Lezius, S. B. Hennig, C. Menzel, and E. Metz, Eur. J. Biochem. 2, 90 (1967). " L. F. Cavalieri and E. Carroll, Proc. Natl. Acad. Sci. U.S.A. 59, 951 (1968).

786

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[581

and 34-kDa bands"; the large fragment possessed the polymerase and 3'+5' exonuclease activities,I3 the small fragment the 5'+3' exonuclease.14 Thus, two distinct enzyme domains in a single polypeptide are connected by an exposed hinge, a rather novel observation at that time. Klenow and Henningsen independently observed the proteolytic cleavage of DNA polymerase.'s The commercial adoption of their procedure has made the large ("Klenow") fragment (freed of the 5'+3' exonuclease) an available and highly popular reagent in recombinant DNA technology. Synthesis without an Apparent Template

Convinced of the absolute need of the polymerase for a template, we were shocked to discover the massive synthesis of DNA without any added template. After a lag period of hours, the purified enzyme made the alternating copolymers DNA-like polymers of simple c~rnposition~~"': poly(dA . dT) and poly(dG . dC) and the homopolymer pairs poly(dA) . poly(dT) and poly(dG) . poly(dC). These polymers, once made, proved to be superior templates and have been widely used in DNA chemistry and biology. Subsequent studies showed that generation of the polymers de novo could be ascribed to the reiterative replication of short sequences in the immeasurably small amounts of DNA that contaminate a polymerase preparation. '*.I9 Synthesis of Genetically Active DNA

For more than 10 years after the initial r e p ~ r tI, had ~ to find excuses at the end of every seminar to explain why our DNA product had no biologic activity. If the template had been copied accurately, why were we unsuccessful in all our attempts to multiply the transforming factor activity of DNA from Pneurnococcus, Haemophilus, and Bacillus species? Finally, with the discovery of DNA ligase in 1967, a crucial test could be made. We D. Brutlag, M. R. Atkinson, P. Setlow, and A. Kornberg. Biochem. Biophys. Res. Commun.37,982 (1969). " P. Setlow, D. Brutlag, and A. Kornberg, J. Biol. Chem. 247. 224 (1972). l4 P. Setlow and A. Kornberg, J . Biol. Chem. 247,232 (1972). Is H. Klenow and I. Henningsen, Proc. Narl. Acad. Sci. U . S . A . 65, 168 (1970). " H. K. Schachman, J. Adler, C. M. Radding, I. R. Lehman, and A. Kornberg, J. Biol. Chem. 235, 3242 (1960). " C. M. Radding, J. Josse, and A. Kornberg. J . Biol. Chenr. 237, 2869 (1962). C. M. Radding and A. Kornberg, J. Biol. Chem. 237,2877 (1962). l9 A. Kornberg, L. Bertsch, J. F. Jackson, and H. G. Khorana, Proc. Nntl. Acad. Sci. U.S.A. 51,315 (1964). l2

[581

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replicated the singie-stranded circle of phage 4x174 with DNA polymerase and then sealed the complementary product with ligase. The circular product strand was isolated and then replicated to produce a circular copy of the original viral strand, which could now be assayed for infectivity in E. coli." We found the completely synthetic viral strand to be as infectious a s that of the phage DNA we started with!21 After so many years of trying, we could show that DNA polymerase can assemble a 5000-nucleotide DNA chain with the identical form, composition, and genetic activity of DNA from a natural virus. All the enzyme needed was the four dNTPs of A, G, T, and C. At that moment, it seemed there were no major impediments to the synthesis of DNA, genes, and chromosomes. The way was open to create novel DNA and genes by manipulating the building blocks and their templates. DNA Polymerase Cannot Start a Chain

Despite the excitement over the synthesis of a chain of infectious viral DNA, I had felt a certain uneasiness. One of the inferences drawn from the replication of a single-stranded, circular template was that DNA polymerase could start a new chain. Yet we were never able to find direct proof of this. Moreover, we had observed that replication of the circular template was far more efficient if a small amount of boiled E. coli extract was present." Although it seemed unlikely that a random fragment of DNA in the extract would match the viral DNA template accurately enough to serve as a primer, this proved indeed to be the case. DNA polymerase removed the unmatched regions of the partially annealed fragment by proofreading at the 3' end: with generous editing at the 5' end, no trace of the priming fragment remained in the synthetic product. We were left with the question of how a DNA chain is started, how a single-stranded, circular viral DNA is converted to the duplex form upon entering the cell, and how nascent chains are initiated in the replication of virtually all chromosomes. Indeed, Okazaki had shown earlierZZthat chains are started not just once, at the beginning of the chromosome, but repeatedly in staccato fashion during the progress of replication. DNA Polymerase under Indictment

DNA polymerase was called a "red herring" and charged by Nature New Biology in a series of editorials with masquerading as a replication l'

M. Goulian and A. Kornberg, Proc. Natl. Acad. Sci. U.S.A. 58, 1723 (1967). M. Goulian, A. Kornberg, and R. L. Sinsheimer, Proc. Natl. Acad. Sci. U.S.A. 58, 2321

22

K. Sugimoto, T. Okazaki, and R. Okazaki, Proc. Natl. Acad. Sci. U.S.A. 60, 1356 (1968).

lo

(1967).

788

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APPENDIX -

e n ~ y m e . ~The ' replicative role of DNA polymerase was questioned be, ~ ~ appeared to lack the enzyme cause of the Cairns mutant of E. ~ o l iwhich and yet grew and multiplied at a normal rate. In addition to the apparent dispensability of DNA polymerase for cell multiplication and its more estimable qualifications for repair of DNA, the enzyme could not start a chain and genes were being discovered (designated dnaA, dnaB, dnaC, etc.) which strongly implicated many other proteins as essential for a replication process far more complex than we had imagined. With the rising skepticism about the importance of DNA polymerase, there was serious questioning about the validity of the basic mechanism, the building blocks, and the assays of DNA synthesis. At this juncture, a new polymerase was found in E. coli. Named DNA polymerase I1 (pol 11),25926 it was clearly different from the "classic" DNA polymerase (pol I) discovered in the and from still another, DNA polymerase 111 (pol course of purifying pol 11. Pol 111 holoenzyme, a far more elaborate form with many accessory subunits, was to gain recognition as the central enzyme of DNA replication in E. coli. All three polymerases, although differing significantly in structure, proved to be virtually identical in their mechanisms of DNA synthesis, proofreading, and use of the same building The maligned pol I became the standard for all DNA polymerases in plants, animals, and viruses, as well as in E. coli. The gloomy views of Nature New Biology soon disappeared, as did the magazine itself. Editorials, Nature (London),New Biol. 229,65; 230,258; 233,97 (1971). P . De Lucia and J. Cairns, Nature (London) 224, 1164 (1969). 25 T . Kornberg and M. L. Gefter, Biochem. Biophys. Res. Commun. 40, 1348 (1970). 26 R. E . Moses and C. C. Richardson, Biochem. Biophys. Res. Commun. 41, 1557, 1565 (1970). " T. Kornberg and M. L. Gefter, Proc. Natl. Acad. Sci. U.S.A. 68,761 (1971). Kornberg, A. (1980) DNA Replication, W . H. Freeman & Co., San Francisco, pp. 724. 23 24

*'

I391 Purification and Crystallization of Transaldolase from Candida utilis By B. L. HORECKER and 0. TSOLAS In the pentose phosphate pathway for carbohydrate metabolism, the conversion of pentose phosphate to hexose phosphate is accomplished by a series of C2 and C3 group-transfer reactions catalyzed by the enzymes METHODS IN ENZYMOLOGY, VOL. 182

Copyright G 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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TRANSALDOLASE FROM

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transketolase and transaldolase (for a review see Ref. 1). Thiamin pyrophosphate is the coenzyme for t r a n s k e t o l a ~ e ,which ~ ~ ~ transfers the glycolaldehyde group as the covalent dihydroxyethylthiamin pyrophosphate deri~ative.~ For transaldolase, which catalyzes the transfer of a C3fragment corresponding to dihydroxyacetone from donor to acceptor ~ u b s t r a t e no , ~ cofactor could be identified, although the reaction was shown to proceed in two steps via the formation of a relatively stable dihydroxyacetoneenzyme (DHA-TA) complex6.' according to the following equations:

*

Sedoheptulose 7-P + TA D-erythrose 4-P + DHA-TA D-Glyceraldehyde 3-P + DHA-TA TA + D-fructose 6-P

*

The DHA-TA adduct could be isolated from incubation mixtures containing radiolabeled D-fructose 6-phosphate, but lacking the triose or tetrose phosphate acceptors. Following reduction of the complex with sodium b~rohydride,~ the modified amino acid was isolated and identified as N6-0glycerylly~ine.~ This established that a lysine residue at the substratebinding site formed a Schiff base derivative with the DHA In these and later experiments12only one dihydroxyacetone group was found to be incorporated per mole of dimeric protein, suggesting that modification of the active site on one subunit blocked the active site on the second subunit. This evidence for "half-of-the-sites" activity of transaldolase was confirmed by the observation that photoinactivation of the single histidine residue on either subunit resulted in complete loss of catalytic activity.13 Additional evidence for half-of-the-sites activity was obtained in experiments on the exchange of isozyme subunits discussed below. The studies on the structure and mechanism of action of transaldolase

' B. L. Horecker and A. H. Mehler, Annu. Rev. Biochem. 24,207 (1955). ' B. L. Horecker and P. Z. Smym~otis,J. Am. Chem. Soc. 75, 1009 (1953). ' E. Racker, G. de la Haba, and I. G. Leder, J. Am. Chem. Soc. 75, 1010 (1953). '

H. Holzer, R. Kattermann, and D. Busch, Biochem. Biophys. Res. Commun. 7,167(1962). B. L. Horecker and P. 2. Smyrniotis, J. Biol. Chem. 212,811 (1955). R. Venkataraman and E. Racker, J . Biol. Chem. 236, 1883 (1961). S. Pontremoli, B. D. Prandini, A. Bonsignore. and B . L. Horecker, Proc. Natl. Acad. Sci. U.S.A. 47, 1942 (1961). B. L. Horecker, S . Pontremoli, C. Ricci, and T. Cheng, Proc. Nut/. Acad. Sci. U.S.A. 47, 1949 (1961). J. C. Speck, Jr., P. T. Rowley, and B. L. Horecker, J. Am. Chem. Soc. 85, 1012 (1963).

'O

7

"

E. Grazi, P. T. Rowley, T. Cheng, 0.Tchola, and B. L. Horecker, Biochem. Biophys. Res. Commun. 9,38 (1962). B. L. Horecker, P. T. Rowley, E. Grazi, T. Cheng, and 0. Tchola, Biochem Z. 338, 36 (1963).

" 0.Tsolas,

B. L. Horecker, and C. L. Sia, Arch. Biochem. Biophys. 136, 303 (1970).

''K. Brand, 0.Tsolas, and B. L. Horecker, Arch. Biochem. Biophys. WO, 521 (1969).

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described above depended on the availability of large amounts, essentially substrate quantities, of pure enzyme. The earliest preparations, from dried brewer's yeast,'.14 were not adequate for these purposes. The first evidence for a dihydroxyacetone-enzyme intermediate was obtained with the enzyme crystallized from baker's yeast by Venkataraman and ~ a c k e r . ' ~ These experiments were confirmed and extended with crystalline transaldolase from Candida utilis.' From the latter source,16 three forms of transaldolase were separated by chromatography on DEAE-Sephadex17: two primary isozymes, designated isozymes I and 111, and a hybrid species, isozyme 11, formed by exchange of subunits between isozymes I and 111. Only isozyme I11 could be crystallized, and with certain batches of C. utilis in which isozyme I11 was a minor component the yield of crystalline enzyme was poor, despite high specific activity in the yeast extracts. The failure of isozymes I and I1 to crystallize also explains why the mother liquors from the crystalline enzyme preparation invariably contained large quantities of enzyme that could not be induced to crystallize.17 Purification of Transaldolase Isozymes from Candida utilis

The method described here, developed in 1970,17was a modification of the earlier procedure for the isolation of crystalline transaldolase7 and was designed to permit the recovery of isozymes I and I1 from the supernatant solutions after crystallization of isozyme 111. The procedure has been described in detail.17 Briefly, the frozen yeast cakes provided by the commercial supplier were allowed to thaw and autolyze for 1 day at 23". The autolyzed mass was suspended in 2 vol of cold 0.05 M NaHCO, and fractionated with acetone at - 10". The fraction precipitating between 36 and 51% acetone contained the bulk of the transaldolase activity. Crystallization was from 50% saturated ammonium sulfate at pH 5.0. The first precipitate obtained contained both crystals and amorphous material. Because the crystals were found to be relatively insoluble, the amorphous material could be selectively removed by rapid washing of the crystals with small volumes of cold water. Several recrystallizations from cold 55% saturated ammonium sulfate yielded crystals of type 111 transaldolase having a specific activity of 66 unitslmg protein, approximately 160-fold purified with respect to the first extract. B. L. Horecker and P. Z. Smymiotis, this series, Vol. I . p. 381. R . Venkataraman and E. Racker, J . Biol. C h ~ m236, . 1876 (1961). l 6 It was the late Ernest Beuding who first pointed out that commercial preparations of Cundidu utilis, obtained in the processing of paper pulp wastes (Lake States Yeast Corp., Rhinelander, Wisconsin), were exceptionally rich in enzymes of the pentose phosphate pathway. " 0. Tsolas and B . L. Horecker, Arch. Biochem. Bioplzys. 136,287 (1970). l4

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Candida utilis

79 1

Isozyme I and the hybrid isozyme 11, present in the supernatant solution after removal of the first crystals, were recovered after separation by chromatography on DEAE-Sephadex. Elution was with a gradient of KC1 in 0.05 M phosphate buffer, pH 6.5. Isozymes I and I1 eluted at KC1 concentrations of approximately 0.1 and 0.15 M, respectively. A smaller quantity of isozyme 111, probably arising from exchange of subunits between two molecules of isozyme 11 (see below), was also recovered at 0.2 M KC1 and crystallized as described above. At this stage isozyme I was approximately 35% pure. Further purification was accomplished by adsorption on calcium phosphate gel and elution with 10 mM sodium phosphate, pH 7.5, followed by chromatography on a column of DEAESephadex, as described for the first separation of the three isozymic forms. The specific activity of isozyme I was approximately 64 unitslmg. Pure isozyme I1 was prepared by allowing purified preparations of isozymes I and 111 to hybridize, which occurred when mixtures of the two were dialyzed in the cold for 2 days against 50 mM sodium phosphate buffer, pH 6.5, followed by DEAE chromatography. The isozymes after DEAE chromatography were recovered by precipitation with 75% ammonium sulfate. In a method developed for the purification of transaldolase isozyme I from yeast rich in this isozymic form, the acetone and calcium phosphate gel steps were eliminated. I 8 . l 9 The procedure included fractionation on a combined Cellex-DIDEAE-Sephadex column, followed by CM-Sephadex chromatography. Isozyme I was crystallized from 55% saturated ammonium sulfate at pH 4.5. The specific activity was 61 unitslmg, similar to that obtained for the earlier preparation.I7 Rapid Preparation of Crystalline Isozyme III

A more convenient procedure for the isolation of this isozyme, without column chromatography, could be employed with preparations of C . ~ntilis that contain significant quantities of this i ~ o z y m eCandida .~ utilis, dried at low temperature (Lake States Yeast Corporation), was allowed to autolyze in 3 vol (w/v) of 0.1 N sodium bicarbonate for 7.5 hr at 26", and the extract purified by acetone fractionation and adsorption on calcium phosphate gel, as described above. The calcium phosphate gel eluate was precipitated with 80% saturated ammonium sulfate. The pellet was then l8 l9

S. C. Sun, L. Joris, and 0. Tsolas. Arch. Biochem. Biophys. 178,69 (1977). Isozyme I is selectively released if the frozen yeast cake is extracted rapidly without allowing time for autolysis. This procedure does not significantly alter the quantity of isozyme I11 that can subsequently be recovered from the residual yeast pellet. It is therefore unlikely that isozyme 111 is derived by proteolytic modification of native isozyme I. Additional evidence to support this conclusion was provided by a comparison of the amino acid compositions of the two isozymes. See Ref. 12.

792

[591

APPENDIX

successively extracted with 60, 55, and 50% saturated ammonium sulfate.20The ammonium sulfate extracts containing the bulk of the transaldolase activity were allowed to warm to room temperature to initiate crystallization, and then kept in the cold room for several days. The crystals were collected and recrystallized from 55 to 60% saturated ammonium sulfate solution, as described above. Half-o$the-Sites Activity of Candida utilis Transaldolase

Crystalline isozyme 111, with a molecular weight of approximately 65,000, was inactivated when only a single dihydroxyacetone group was incorporated by reduction of the Schiff base intermediate,' although the enzyme was later shown to be a dimeric protein containing two identical subunits.I2 This isozyme, which contains two histidine residues (one per subunit) was also found to be fully inactivated when only one of these histidine residues was destroyed by photo~xidation.'~ Confirmation of this half-of-the-sites activity, and evidence for the location of the active site between the subunits, was derived from hybridization studies carried out with the reduced P-glyceryl forms of transaldolase i~ozymes.'~."Despite the presence of one unmodified subunit, each P-glyceryl derivative was found to be catalytically inactive. Incubation of a mixture containing the modified forms of isozyme I (aa*) and ) ~ ~ to regenerate the active hybrid, presumably isozyme I11 ( ~ p * failed because of steric barriers to the formation of a dimer containing two P-glyceryl groups: aa*

+ @*

kap

+ a*P*

On the other hand, active isozyme 11 could be generated in the expected quantity from a mixture of native isozyme I and modified isozyme 111: aa (active)

+

pp* (inactive)

C

ap (active)

+

a@* (inactive)

Exchange of subunits between isozyme I, with a subunit weight of approximately 38,000, and isozyme 111, with a subunit molecular weight of 20

2' 22

This method for purification of proteins with ammonium sulfate by back extraction follows a procedure recommended by the late Professor Otto Warburg, who found that it provided better separation of proteins. It has the further advantage of requiring only one large-scale centrifugation. Following the first precipitation, only small quantities of extraction solution need to be manipulated and centrifuged. 0. Tsolas and B. L. Horecker, Mol. Cell. Biochem. 1 , 3 (1973). a * and p* refer to subunits carrying the P-glyceryl-lysine residue.

$

'

C

t I

,

1601

DISCOVERY OF GLUTAMINE SYNTHETASE CASCADE

793

approximately 32,000, thus yields a heterodimer that possesses a fully active catalytic center. Evidence from X-ray crystallographic studies will be required to define this active center and corroborate the proposed reaction mechani~m,~' in which residues from both subunits are thought to participate.

[601 Discovery of Glutamine Synthetase Cascade By EARLR. STADTMAN

'

C i

$+

r$

Studies on the regulation of glutamine synthetase (GS) (glutamateammonia ligase) activity were initiated in 1964 when Clifford Woolfolk joined the Laboratory of Biochemistry, National Heart, Lung, and Blood Institute. Woolfolk had just finished his graduate work at the University of Washington under the direction of Helen Whitely and wanted to investigate the regulation of branched metabolic pathways. Specifically, he wanted to continue some studies on the regulation of aspartate metabolism which I had carried out while on sabbatical leave in Georges Cohen's laboratory at the Pasteur Institute several years earlier. Not wanting to compete with Georges Cohen on a problem that was initiated in his own laboratory, I encouraged Woolfolk to select another problem, and suggested that he examine metabolic maps to identify some enzymes which catalyze reactions whose products serve a s substrates in the first step of two or more divergent biosynthetic pathways. In addition, the multifunctional enzyme should be one whose activity could be easily and quickly measured. Woolfolk came up with three suggestions; namely, glutamate dehydrogenase, glutamine synthetase, and phosphoribosylpyrophosphate synthetase. Preliminary studies with glutamate dehydrogenase in extracts of Escherichia coli failed to disclose any unusual regulatory characteristics, so Woolfolk turned his attention to glutamine synthetase. We were delighted to find that the activity of GS in extracts of E. coli was partially inhibited by each of eight different metabolites: histidine, tryptophan, AMP, CTP, carbamyl-P, glucosamine 6-phosphate, alanine, and g1ycine.I-' All these were known to be end products of glutamine metabo-

1 I

'

C . A. Woolfolk and E . R. Stadtman, Biochem. Biophys. Res. Commun. 17,313 (1964). C . A. Woolfolk and E . R. Stadtman, Arch. Biochem. Biophys. 118,736 (1%7). C . A. Woolfolk,B . Shapiro, and E. R. Stadtman,Arch. Biochem. Biophys. 116,177(1966).

METHODS IN ENZYMOLOGY, VOL. 182

1

794

[601

APPENDIX -

-

-

-

-

-

-

-

1is.111.~Woolfolk showed that in addition to feedback inhibition, the level of GS in E. coli was also under rigorous feedback control. The intracellular concentration of the enzyme could be varied more than 20-fold by variations in the availability of nitrogen in the growth m e d i ~ m . ~ Cumulative Feedback Inhibition

After establishing that E. coli GS is subject to multiple feedback inhibition, Woolfolk et obtained homogeneous crystalline preparations of the enzyme. From detailed hydrodynamic5-' and electron microscopic e ~ a m i n a t i o nit, ~was established that the enzyme had a molecular weight of 600K and is composed of 12 identical subunits, arranged in 2 superimposed hexagonal array^.^ On the basis of extensive kinetic measurements, it was demonstrated that at high, nearly saturating concentrations, each one of the eight different feedback inhibitors was by itself able to inhibit only a fraction (10-60%) of the GS activity. However, the inhibition obtained with a combination of any two of the metabolites was greater than with either one alone, and as the number of metabolites present was increased, there was a progressive increase in the extent of inhibition. When all eight metabolites were added together, almost complete inhibition of the enzyme activity was obtained.'.' Although other interpretations were not rigorously excluded, it was deduced from this unusual behavior that each subunit of GS contains a separate binding site for each one of the eight different feedback effectors. This phenomenon was referred to as "cuThe existence of separate binding sites mulative feedback inhibiti~n."'.~ for alanine, glycine, AMP, CTP, histidine, tryptophan, and also for Damino acids was subsequently verified by direct binding studies9 and by the results of ligand-binding measurements utilizing stopped flow fluores~ence,'~-" c a l ~ r i m e t r i c , ' ~and , ' ~ NMR".12 techniques. E. R. Stadtman, in "The Enzymology of Glutamine Metabolism" (S. Prusiner and E. R. Stadtman, eds.), p. 1. Academic Press, New York, 1973. C. A. Woolfolk and E. R. Stadtman, Arch. Biochem. Biophys. U2,174 (1967). B. M. Shapiro and E. R. Stadtman, J . Biol. Chem. 242,5069 (1967). B. M. Shapiro and A. Ginsburg, Biochemistry 7,2153 (1968). R. C. Valentine, B. M. Shapiro, and E. R. Stadtman, Biochemistry 7,2143 (1968). A. Ginsburg, Biochemistry 8, 1726 (1969). lo S. G. Rhee, P. B. Chock, and E. R. Stadtman, in "Frontiers of Biological Energetics" (P. L. Dutton, J. S. Leigh, and A. Scarpa, eds.), Vol. 1, p. 725. Academic Press, New York, 1978. I' S. G. Rhee, J. J. Villafranca, P. B. Chock, and E. R. Stadtman, Biochem. Biophys. Res. Commun. 78,244 (1970). l2 J. J. Villafranca, S. G. Rhee, and P. B. Chock, proc. Natl. Acad. Sci. U.S.A. 75, 1255 (1978). l 3 P. D. Ross and A. Ginsburg, Biochemistry 8,4690 (1969). l4 A. Shrake, R. Park, and A. Ginsburg, Biochemistry 17,658 (1978).

'

u I

r7 I

[601

DISCOVERY OF GLUTAMINE SYNTHETASE CASCADE

795

Adenylylation of GS

The studies on GS regulation took an unexpected turn when the first batch of purified enzyme was nearly exhausted and a second batch was prepared. Much to our consternation, enzyme from the second batch enzyme was not inhibited by either one of the four metabolites, tryptophan, CTP, AMP, or histidine.I5 In other respects, the old and new batches of enzyme appeared to be indistinguishable. They possessed identical hydrodynamic properties, circular dichroism (CD) spectrum, and amino acid composition. Efforts to normalize their responses to feedback inhibitors by a variety of treatments (exposure to heat, sulfhydryl reagents, ionic strength, 0 2 , urea, etc.) were unsuccessful. It was therefore concluded that the difference in feedback inhibition patterns must reflect subtle differences in the properties of the cells from which the enzymes were isolated. Henry Kingdon therefore undertook a systematic investigation of the inhibition patterns of GS in extracts of various batches of E. coli which had been grown under different conditions. His efforts were rewarded by the finding that GS from nitrogen-starved cells was not inhibited by AMP, histidine, tryptophan, or CTP, whereas the enzyme from cells grown on a nitrogen-rich medium was inhibited by all of these metabolites.l5 The discrepancy in the behavior of enzyme preparations from different batches of cells was therefore attributable to variations in E. coli growth conditions. The molecular basis for their differences in feedback inhibition was eventually disclosed by a comparison of the ultraviolet absorption spectrum of highly purified preparations of both forms of enzyme.I6 The spectrum of the enzyme from cells grown on a nitrogen-rich medium exhibited significantly higher absorbance in the region of 260 nm (Fig. 1). Indeed, the difference spectrum obtained when equal amounts of the two preparations were compared directly, one against the other, exhibited a peak at 260 nm (Fig. 1 , inset), suggesting that the preparation from nitrogen-rich medium was associated with a purine derivative. The 260nm-absorbing material was evidently covalently bound to the protein since it was not released from the protein by gel filtration, exhaustive dialysis, treatment with charcoal or with acid ammonium sulfate, or by incubating with 0.3 M HCI or 0.3 M NaOH for 3 hr at 37", o r by precipitation with 15% perchloric acid. Upon treatment with snake venom phosphodiesterase (SVP), AMP was released from the protein and, coincidentally, the enzyme was converted to a form apparently identical to that isolated from nitrogen-starved cells. It was therefore obvious that the enzyme from "

H. S. Kingdon and E. R. Stadtrnan, J. Bacteriol. 94,949 (1967).

''B. M. Shapiro, H. S., Kingdon, and E. R. Stadtrnan,Proc. Natl. Acad. Sci. U.S.A.58,642 (1967).

796

APPENDIX

[601

FIG. 1. Ultraviolet absorption spectra of adenylylated and unadenylylated forms of glutamine synthetase. Dotted line, glutamine synthetase from cells grown in nitrogen-rich medium; solid line, glutamine synthetase from cells grown in nitrogen-limitingmedium;inset, difference spectrum between the two forms. Extinction coefficients are expressed per mole of enzyme of M,600,000.

nitrogen-rich cells contained an adenylic acid residue attached in phosphodiester linkage to some amino acid residue in the enzyme. To identify the site of attachment, the adenylylated enzyme was fragmented by treatment with pepsin and pronase. A decapeptide fragment containing the AMP moiety was isolated from the protease digest by means of charcoal adsorption and paper chromatography." Of particular significance was the finding that the decapeptide contained no serine, threonine, or basic amino acid residues. In fact, a single tyrosine residue was the only residue in the peptide capable of forming a phosphodiester bond with AMP. That the AMP was attached to the tyrosine residue was verified by showing that removal of the AMP from the peptide by treatment with SVP led to exposure of a tyrosine hydroxyl group. This was established by showing that the characteristic increase in absorbance at 293 nm, which is associated with ionization of the tyrosyl hydroxyl group at pH 13, did not occur until after the AMP was released from the peptide by treatment with the diesterase. It was thus established that the catalytic activity and the susceptibility of E. coli GS to feedback regulation is mediated by the ade" B.

M. Shapiro and E. R. Stadtman, J . Biol. Chem. 243, 3769 (1968).

-

-

"

-

[601

DISCOVERY OF GLUTAMINE SYNTHETASE CASCADE

797

nylylation of a unique tyrosyl group in the enzyme. To our knowledge, this was the first example in which the esterification of a tryosine hydroxyl group was found to be implicated in enzyme regulation. Further comparison of the adenylylated and unadenylylated enzymes showed that the adenylylation of GS caused a shift in the pH activity profile and in the divalent cation specificity of the e n ~ y m e . ' ~ . ' ~Thus, ~''-~~ Mg2+was essential for the biosynthetic activity of the unadenylylated ' . pH optienzyme, whereas the adenylylated enzyme required ~ n ~ The mum of the unadenylylated and adenylylated enzymes were 8.0 and 7.0, respectively. In contrast, Mn2+ supported the ability of both enzyme forms to catalyze the nonphysiological y-glutamyl transfer reaction [reaction (I)] Glutamine

+ NH20H

arsenate

ADP

glutamylhydroxamate + NH3

(1)

However, the pH-activity profiles of the two enzymes for catalysis of this reaction were significantly different. Under standardized assay conditions, Mg2+could support transferase activity of the unadenylylated enzyme, but it selectively inhibited the Mn2+-dependenttransferase activity of the adenylylated enzyme. These characteristics were subsequently exploited in the development of a highly sensitive procedure for the estimation of the average state of adenylylation (T)of GS in crude extracts.19 Adenylyltransferase

With the discovery that the activity and feedback control of GS is modulated by the esterification of a tyrosyl residue, it was obvious that E. coli contains an adenylyltransferase capable of catalyzing the adenylylation and deadenylylation of GS. The presence in E. coli extracts of an adenylyltransferase (ATase) that catalyzed transfer of the adenylyl group of ATP to GS was readily verified. When a protein fraction from E. coli extract was incubated with [ 1 4 C ] ~and ~ p the unadenylylated form of GS, [I4C]AMpgroups became attached to the GS and this adenylylation was accompanied by the expected changes in divalent cation specificity, pH-activity profile, and susceptibility to feedback inhibition.'' It was also evident from these studies that up to 12 adenylyl groups (1 per subunit) could be attached to each molecule of GS. The biosynthetic activity of the enzyme under physiological conditions is inversely proportional to the l8

H. S. Kingdon, B. M. Shapiro, and E. R. Stadtman, Proc. Natl. Acad. Sci. U.S.A. 58,1703

(1967).

f l9

E. R. Stadtman, P. Z. Smymiotis, J. N. Davis, and M. Wittenberger, Anal. Biochem. 95, 275 (1979). A. Ginsburg, J. Yeh, S . B . Hennig, and M. D. Denton, Biochemistry 9,633 (1970).

798

APPENDIX

[601

average number (Z) of adenylyl groups bound per molecule. Thus, the adenylylation of a particular subunit in the dodecameric enzyme leads to inactivation of that subunit only. The adenylyltransferase was subsequently purified to homogeneity by Ann Ginsburg in our laboratory2' and also by Ebner et ~ 1in Holzer's . ~ ~ laboratory in Freiburg. It was established that the adenylyltransferase is comprised of a single polypeptide chain ( M , = 130,000). It catalyzes the reversible reaction (2lZ3 GS

+ 12 ATP

GS(AMP),? + 12 PP,

(2)

Relationship between Adenylyltransferase and Holzer's "Inactivation Enzyme"

In 1966, Holzer and associates described an enzyme in E. coli that catalyzed the inactivation of GS.24,25This "inactivase" required the presence of ATP and glutamine. In view of the fact that adenylylation of GS converts it from an ~ ~ ' + - d e ~ e n dform e n t to an Mn2+-dependentform and also the fact that in Holzer's laboratory GS activity was always assayed in the presence of Mg2+,i.e., under conditions where the adenylyled enzyme is inactive, it appeared likely that Holzer's "inactivase" was identical with Kingdon's adenylyltransferase. This was found to be the case. Further studies in Holzer's laboratory as well as our own confirmed that the "inactivase" and adenylyltransferase were one and the same e n ~ y r n e . 'The ~ , ~adenylyltransferase ~ was ultimately purified to homogeneity in both l a b o r a t o r i e ~ . ~Furthermore, '.~~ it was demonstrated in both laboratories that the interconversion of GS between adenylylated and unadenylylated forms occurs in vivo in response to shifts in the nutritional state of E. coli. Thus, as noted earlier, the adenylylated form is favored when E. coli is grown in a nitrogen-rich medium; but when cells are shifted from a nitrogen-rich to a nitrogen-poor medium, the GS was converted from the less active adenylylated form back to the more active unadenylylated form. S. B . Hennig, W. B. Anderson, and A . Ginsburg, Proc. Natl. Acad. Sci. U.S.A. 67, 1761 (1970). E. Ebner, D. Wolf, C. Gancedo, S. Elsasser, and H. Holzer, Eur. J. Biochem. 14, 535 (1970). 23 M. Mantel and H. Holzer, Proc. Natl. Acad. Sci. U . S . A . 65,660 (1970). 24 D. Mecke and H. Holzer, Biochim. Biophys. Acta l22,341 (1966). 25 D. Mecke, K. Wulff, K. Liess, and H. Holzer, Biochem. Biophys. Res. Commun. 24,542 (1966). 26 K. Wulff, D. Mecke, and H. Holzer, Biochem. Biophys. Res. Commun. 28,740 (1967). 21

"

DISCOVERY OF GLUTAMINE SYNTHETASE CASCADE

799

Deadenylylation of Adenylylated GS An activity capable of catalyzing the removal of adenylyl groups from adenylylated GS was detected in crude extracts of E. coli." By means of gel filtration, B. M. Shapiro resolved the extract into two protein fractions, PI and PI1, both of which were required for deadenylylation activity. He established further that the deadenylylation reaction required the presence of UTP and a-ketoglutarate, and was greatly stimulated by orthophosphate or arsenate, and was strongly inhibited by g~utamine.'~ Upon further purification, it became evident that the PI fraction contained a single adenylyltransferase whose ability to catalyze the adenylylation of GS on the one hand and the deadenylylation of adenylylated GS on the other was somehow specified by the PII protein and by the concentrations of ATP, UTP, Pi, a-ketoglutarate, and g l ~ t a m i n e . ' ~ The role of inorganic orthophosphate in this system was clarified by the studies of Anderson and Stadtmar~,~' showing that the deadenylylation of GS does not involve simple reversal of the adenylylation reaction [reaction (I)] but rather involves a phosphorolytic cleavage of the adenylyltyrosine bond of adenylylated GS to form ADP and the unmodified form of GS [reaction (3)]: GS.AMP

+ P i + G S + ADP

(3)

The PI could not be replaced by PPI. However, inorganic arsenate was able to substitute for P,, in which case AMP was the product, presumably obtained by spontaneous hydrolysis of the AMP-As intermediate.

kB

I

4

Tr_ i

Uridylylation of PIIProtein Governs Activity of Adenylyltransferase The role of the PIi protein in the differential regulation of the adenylylation and deadenylylation reactions was further clarified by the studshowing that when the PIIprotein was incubated with ies of Brown et the PI fraction in the presence of ATP, UTP, a-ketoglutarate, and ~ n ~it + , was converted to a form which after reisolation by gel filtration was able, in the absence of UTP, t o stimulate the ability of the PI fraction to catalyze the deadenylylation of adenyly lated GS. Coincidentally, modification of

*'B. M. Shapiro and E. R. Stadtman, Biochem. Biophys. Res. Commun. 30,32 (1968).

B. M. Shapiro, Biochemistry 8,659 (1969). 29 W. B. Anderson, S. B. Hennig, A. Ginsburg, and E. R. Stadtman, Proc. Natl. Acad. Sci. U.S.A.67, 1417 (1970). W. B. Anderson and E. R. Stadtman, Biochem. Biophys. Res. Commun. 41,704 (1970). " M. S. Brown, A. Segal, and E. R. Stadtman, Proc. Natl. Acad. Sci. U.S.A.68,2949(1971). za

i

800

APPENDIX

1601

PI1led to a decrease in its ability to stimulate the adenylylation of GS. The possibility that the PII was covalently modified by a uridine derivative was indicated by the further demonstration that after incubation with [y3'P, I4C]UTP under the above conditions, the reisolated PII contained I4C but no 32P. Moreover, upon treatment with SVD, the modified PII lost its ability to stimulate the deadenylylation reaction. Based on the results of these preliminary experiments, it was suggested that the unmodified form of PII stimulates the ability of adenylyltransferase to catalyze the adenylylation of GS, whereas a uridine derivative of the PII protein is able to stimulate the deadenylylation of adenylylated GS. It was further suggested that a-ketoglutarate and ATP stimulate and that glutamine inhibits the ability of an enzyme in the PI protein fraction to catalyze the modification of PII. It remained to be determined whether the UTP-dependent modification of the PIIprotein was catalyzed by the adenylyltransferase itself or by some other enzyme in the relatively impure PI protein fraction. This question was answered with the demonstration that homogeneous preparations of the adenylyltransferase were unable to catalyze the covalent modification of PI,, and also by the subsequent studies of Mangum et showing that by means of chromatography on DE-52 cellulose, the adenylyltransferase in the PI fraction could be separated from a protein fraction capable of catalyzing both the uridylylation of the PIIprotein and the deuridylylation-of the UMP.PII conjugate. Whether the uridylylation and deuridylylation reactions were catalyzed by separate enzymes or by a single bifunctional uridylyltransferase (UTase) in the PI fraction was not readily solved because instability of the UTase activity eluded its purification. Moreover, the ratio of uridylylation and deuridylylation activities of a given enzyme preparation could be altered by aging or by exposure to mild denaturing conditions. It was not until several years later that Garcia and Rhee33 succeeded in obtaining an apparently homogenous protein preparation of UTase which possessed both uridylylating and deuridylylating activities. Furthermore, by working rapidly they showed that the ratio of both activities remained constant throughout the purification procedure. Final proof that both activities were properties of a single bifunctional enzyme was afforded by the demonstration that a single point mutation in the structural gene led to the loss in expression of both activities. Because the PII preparation used in these studies was still relatively impure, neither its structure nor the stoichiometry could be ascertained. 32

J. H. Mangurn, G . Magni, and E. R. Stadtrnan, Arch. Biochem. Biophys. 158,514 (1973). Garcia and S. G . Rhee, J. Biol. Chem. 258, 2246 (1983).

" E.

[601

DISCOVERY OF GLUTAMINE SYNTHETASE CASCADE

801

Adler et finally succeeded in obtaining homogeneous preparations of the PII protein and demonstrated that it was a protein of about M, 44,000 and was composed of four apparently identical subunits. Furthermore, when this PI1preparation was incubated with UTP, ATP, a-ketoglutarate, Mg2+or Mn2+,and a more highly purified preparation of the UTase, up to 4 mol of UMP (one per subunit) could be covalently bound to each mole of the PIIprotein. From amino acid analysis, it was found that each subunit of the PII protein contained two tyrosine residues. Upon treatment with trypsin, two different tyrosine-containing peptides were generated which were easily separated by two-dimensional electrophoresis on acrylamide gels. Prior to uridylylation of the PII protein, the tyrosine residues in both of the peptides could be iodinated with ' 2 5 ~in the presence of chloramineT. However, after uridylylation, the tyrosine residue in just one of the two peptides could be iodinated. Since a free hydroxyl group is essential for iodination of a tryosyl group, this indicated that the UMP group was attached to the hydroxyl group of just one of the two tyrosine residues in the PII subunit. This conclusion was verified by the further observation that cleavage of the UMP groups from uridylylated PII by treatment with snake venom phosphodiesterase resulted in the exposure of a stoichiometric amount of ionizable tyrosyl hydroxyl groups as disclosed by spectral analysis. It was thus established that, as in the adenylylation of GS, the uridylylation of the PII protein involves the covalent attachment of the mononucleotide to tyrosyl residues.

IG

The Bicyclic Cascade With the above observations, it was evident that the activity of GS is under the fine control of a cascade system composed of two tightly linked interconvertible enzymelprotein cycles, each of which is catalyzed by a bifunctional enzyme (Fig. 2). From detailed analyses of the enzymes in this cascade, it was demonstrated that the activity of GS is subject to regulation by over 40 metabolite^.^^.^^ Some of these reacted with GS directly, whereas others exerted their effects by interactions with one or both of the two bifunctional enzymes. Of these effectors, a-ketoglutarate, glutamate, ATP, UTP, and Pi are of special significance. The latter three compounds serve as cosubstrates in the nucleotidylationldenucleotidylation reactions, whereas glutamine and a-ketoglutarate are allosteric effectors of the bi-

" S. Adler, D. Punch, and E. R . Stadtman, J . Biol. Chem. 250,6264 (1975). '5

E. G . Engleman and S. H. Francis, Arch. Biochem. Biophys. 191,602 (1978). E . R. Stadtrnan and P. B. Chock, Curr. Top. Cell. Regul. 13,53 (1978).

802

[601

APPENDIX

UMP

I I

PPi

J

I I I I

>==< I

UTP

@

ATP

I

GS

ADP

Gin

I

ATa

\

\

- a-

I I

9

ppi

KG GS(AMP),,

Pi

FIG. 2. The bicyclic cascade of glutamine synthetase regulation. Interrelationship between the uridylylation cycle and the adenylylation cycle, and the reciprocal controls of these interconversions by L-glutamine (Gln) and a-ketoglutarate (a-KG) are shown; + indicates stimulation, - indicates inhibition. Abbreviations: GS, glutamine synthetase; PII,regulatory protein; AT, and ATd, the adenylylation and deadenylylation sites, respectively, on the bifunctional adenylyltransferase; UR and UT, the deuridylylation and uridylylation sites, respectively, on the bifunctional uridylyltransferase. In the text UT and UR are referred to as UT, and UTd, respectively (cf. Fig. 4).

functional enzymes. Thus, it was found that glutamine inhibits and a-ketoglutarate stimulates the ability of adenylyltransferase to catalyze the PIIdependent adenylylation of GS at the adenylylation site (AT,) of ATase, whereas each effector exerts an opposite effect on the capacity of ATase to catalyze the deadenylylation of GS at the deadenylylation site (ATd) of the e n ~ y m e . ' ~ *Similarly, ~ ~ . ~ * glutamine was found to inhibit the ability of uridylyltransferase to catalyze the uridylylation of PII at the LIT, site of UTase, but to stimulate its ability to catalyze the deuridylylation of PII.UMP at the UTd site. In contrast, it was established that a-

DISCOVERY OF GLUTAMINE SYNTHETASE CASCADE

f

803

ketoglutarate stimulates the deuridylylation reaction. In addition, it was determined that ATP serves as an essential allosteric activator for the UTase-catalyzed uridylylation of PII, and that nucleoside monophosphates (especially CMP) serve as negative effectors of the deuridylylation

The discovery that GS activity is regulated by the esterification of a tyrosyl hydroxyl group in the enzyme followed by more than two decades the earlier demonstration that covalent modification of an enzyme constitutes a physiological mechanism for the regulation of key metabolic processes. The pioneering studies of Cori, Krebs, Fisher, and Larner showed quite clearly that the phosphorylation of seryl hydroxyl groups on glycogen phosphorylase and glycogen synthetase provided a physiological mechanism whereby the synthesis and degradation of glycogen could be regulated with respect to one another. This gave rise to the popular notion that the covalent modification of an enzyme constituted a physiological "switch" by means of which the activity of the enzyme could be turned "on" or "off" in response to metabolic demand. Despite its attractive simplicity, this concept unrealistically ignored the fact that in order to serve as metabolic switches, activities of the protein kinases and phosphoprotein phosphatases would have to be reciprocally regulated in an "all" or "none" fashion. The alternative possibility that dynamic, cyclic interconversion of an enzyme between covalently modified and unmodified forms provided a means by which an enzyme could be gradually shifted from one level of activity to another was not seriously considered. However, this possibility became evident from studies of Segal et showing that the fraction of GS subunits that could be adenylylated varied in response to changes in the levels of multiple metabolites that govern the activities of the cascade enzymes. When GS was incubated in a mixture containing arbitrary concentrations of ATP, UTP, Pi, a-ketoglutarate, glutamine, Mg2+ and/or ~ n ' +and , the two bifunctional enzymes (ATase and UTase), within a few minutes the level of adenylylation, ii (i.e., the average number of adenylylated subunits per GS molecule), reached a steady-state value. Moreover, a change in the concentration of any one of the five metabolites or Mn2+caused a shift in the steady-state level of adenylylation of GS, either to higher or lower values, depending on which of the metabolite concentrations was altered (Fig. 3). It was established further that after " A . Segal, M. S. Brown, and E. R. Stadtman, Arch. Biochem. Biophys. 161,319 (1974).

804

[601

APPENDIX

C

Effecter Varied

mM

12 0

a-KG

0

None

1 n

7

n

UTP

2

Gln

0.1

MnC12

1.25

n Gln

1

0

0

MINUTES

FIG. 3. Effect of metabolite concentrations on the steady-state level of adenylylated subunits. The heavy line (filled squares) shows the change of n with time when 95 pg of glutamine synthetase was incubated in a mixture containing 20 mM MgC12,20 mM Pi, 1 mM ATP, 1 mM UTP, 15 mM a-ketoglutarate, 0.3 m M glutamine, and partially purified preparations of PI,, ATase (containing also UTase). The other curves illustrate the effect of changing the concentration of only one metabolite in the mixture, as indicated.

assuming a steady-state value of ii, ATP continued to be decomposed. These experiments demonstrated that for a given metabolic condition, a dynamic steady state is established in which the rates of adenylylation and deadenylylation of GS are equal, and that the fractions of GS subunits that are adenylylated in this steady state, and hence the specific catalytic activity of the enzyme, are specified by the relative concentrations of positive and negative effectors (metabolites) that govern the activity of the cascade enzymes. Contrary to the physiological switch concept, it became evident that the metabolic interconversion of enzymes between covalently modified and unmodified forms is a dynamic process which facilitates continuous shifts in the catalytic activities of the enzyme commensurate with metabolic demand. They established further that the decomposition of ATP associated with the cyclic interconversion of enzymes between

7

w

1

I

I

[601

DISCOVERY OF GLUTAMINE SYNTHETASE CASCADE

805

modified and unmodified forms is the price the cell pays to achieve such fine cellular control. Theoretical Analysis of Cyclic Cascade Systems Prompted by the above results, a detailed theoretical analysis of cyclic cascade systems was undertaken in order to define more exactly how such cycles respond to changes in the concentrations of metabolites which aRect the cascade e n ~ y r n e s . To ~ ~ our - ~ ~surprise, this analysis revealed that interconvertible enzyme cascades are endowed with many unique regulatory capacities that had not been previously suspected. Thus it was demonstrated that interconvertible enzyme cascades can respond simultaneously to a very large number of both positive and negative allosteric effectors and thereby generate a multitude of fundamentally different regulatory patterns. These cascades are also capable of signal amplification, i.e., the concentration of a given allosteric effector that is able to provoke a large change in the level of covalently modified interconvertible enzyme can be orders of magnitude below the K,,, for the binding of that effector to the converter enzymes. Cyclic cascades may also serve as rate amplifiers; and therefore can facilitate a change from one steady-state level of covalent modification to another within the millisecond time range. In addition, interconvertible enzyme cascades are capable of generating a cooperative-type (sigmoidal) response to increasing concentrations of a given effector. Verification of Theoretical Predictions

iK

Whereas these studies had focused attention on the dynamics of interconvertible enzyme systems, the kind of detailed in vitro studies needed to verify predictions derived from the theoretical analysis of cyclic cascades was hampered by the instability of UTase, which had precluded the isolation of appreciable quantities of the enzyme. Nevertheless, because the primary function of UTase is to regulate the PI~/P~I.(UMP) ratio, it was evident that the effect of UTase to the overall adenylylation of GS could be mimicked by varying the mole fraction [PII]/[P~~]+[PII-UMP]. Therefore, a massive kinetic study was carried out to determine if the properties of cyclic cascades disclosed by the theoretical analysis could be ~erified.~' " j9

E. R. Stadtman and P. B . Chock, Proc. Natl. Acad. Sci. U.S.A. 74,2761 (1978). P. B. Chock and E. R. Stadtman, Proc. Natl. Acad. Sci. U.S.A. 74,2766 (1977).

806

APPENDIX

[601

To this end, it was necessary to determine experimentally the values of 28 different reaction constants needed to describe the theoretical model and, in addition, to determine how the steady-state level of GS adenylylation varied when GS is incubated with a highly purified preparation of ATase in the presence of various concentrations of glutamine and a-ketoglutarate and at a number of different mole fractions of PII. Results of these studies confirmed that cyclic cascades are in fact endowed with all of the properties which were predicted by the theoretical considerations. Verification of these basic principles was obtained also from studies in which the state of adenylylation of GS was measured in permeabilized E. coli cells following their incubation in buffer containing variable concentrations of ATP, UTP, a-ketoglutarate, glutamine, and Pi.41,42 Application of Molecular Biology Technology

It is perhaps worth noting that the discovery and elucidation of the GS cascade as summarized in the above sections was achieved by the application of classical enzymological approaches and technologies. None of the modem techniques of molecular biology or of biochemical genetics was utilized. Indeed, it is questionable whether the application of these latter techniques would have helped or hindered progress in the elucidation of this remarkable cascade system. Nevertheless, as noted earlier, a quantitative study of this bicyclic cascade system was hampered by the instability of the UTase, which precluded its isolation as a homogeneous protein, and also by the fact that, except for GS, the intracellular concentrations of the cascade enzymes are relatively low, making it difficult to obtain sufficient quantities of homogeneous preparations needed for detailed studies of the bicyclic cascade in vitro. To overcome these problems, we resorted to the use of molecular biology approaches to obtain strains of E. coli which produced 800-, 500-, and 70-fold more UTase, ATase, and PII protein, respectively, than the wild-type Using these strains, Rhee and colleagues obtained a substantial quantity of a homogeneous preparation of each one of the cascade enzymes. In a monumental effort,45they determined the values of 21 interaction constants that govern the proteinlprotein and protein/ U. Mura and E. R. Stadtman, J . Biol. Chem. 256, 13014 (1981). U. Mura, P. B . Chock, and E. R. Stadtman, J . Biol. Chem. 256, 13022 (1981). " S. G. Rhee, S. C. Park, and J. H. Koo, Curr. Top. Cell. Regul. 27,221 (1985). " H. S. Son and S. G. Rhee, J . Biol. Chem. 262,8690 (1987). 45 S. G . Rhee, W. G. Bang, S. C. Park, J. H. Koo, and K. H. Min, in "Dynamics of Soluble and Immobilized Enzymes" (P. B. Chock, L. Tsou, and C. Y. Huang, eds.), p. 128. Springer-Verlag, Amsterdam, 1987. 4'

42

[601

DISCOVERY OF GLUTAMINE SYNTHETASE CASCADE

807

effector interactions which are involved in various steps of the bicyclic cascade. Then, in order to simulate in vitro the characteristics of the GS cascade, a series of experiments was carried out in which highly purified preparations of the cascade enzymes and the PII protein were mixed together in amounts equivalent to those found in crude extracts of E. coli. In this study, the effects of variations in the relative concentrations of glutamine and a-ketoglutarate were examined under three different conditions, namely: (1) in the presence of UTP, Pi, ATP, UTase, and PIIonly (to mimic the operation of the uridylylation/deuridylylation cycle alone), (2) in the presence of ATase, ATP, and varying ratios of PII and PI~.UMP (to mimic operation of the adenylylation/deadenylylationcycle alone), and (3) in the presence of all substrates and cascade enzymes (to mimic the behavior of the coupled bicyclic cascade). Suffice it to say that the results of these studies confirmed in every important detail theoretical predictions of the cascade model, and illustrated directly the remarkable regulatory features of such cascade^.^' It was evident from the results of these studies that interconvertible enzyme cascades serve as metabolic integration systems. By means of allosteric and substrate site interactions, the interconvertible enzymes are programmed to sense fluctuations in the concentrations of a multiplicity of metabolites. This leads to automatic adjustments in the specific activities and kinetic constants of the several cascade enzymes. Through this system the multiple inputs are integrated and registered as a single output, the fractional modification of the target enzyme, and thereby determines its specific catalytic activity. Role of UTase and PIIin Regulation of Glutamine Synthetase Formation

In the initial studies on the regulation of GS activity, Woolfolk noted that the level of GS in E. coli was dependent on the availability of nitrogen in the culture medium. In the meantime, the mechanism that underlies the nitrogen control of GS synthesis was under extensive investigation in the ~.~' In both laboratories, a delaboratory of M a g a ~ a n i k ~and tailed genetic analysis of the GS cascade led eventually to the demonstration that UTase and the PII protein are involved in the nitrogen control of GS levels. Such roles were suggested also by the studies of Rhee and B. Magasanik, Annu. Rev. Genet. 16, 135 (1982). B. Magasanik, TZBS 13,475 (1988). S. Kustu, K. Sei, and J. Keener, in "Regulation of Gene Expression" (I. R. Booth and C. F. Higgins, eds.), p. 139. Cambridge Univ. Press, London, 1986. 49 J. Keener, P. Wong, D. Popham, J. Wallis, and S. Kustu, in "RNA Polymerase and Regulation of Transcription" (W. S. Reznikoff, R. R. Burgess, J. E. Dahlberg, C. A. Gross, M. T. Record, Jr., and M. W. Wickens, eds.), p. 159. Elsevier, New York, 1987. 46

47

"

1 7

808

APPENDIX

8

xPi

ADP I

I I

GS activity

I

I

GS-

GS

GS synthesis

J

ADP

ATP

FIG.4. The cyclic cascade of glutamine synthetase (GS) regulation. Interrelationship between the uridylylation cycle, the adenylylation cycle, and the phosphorylation cycle; the reciprocal controls of these interconversions by L-glutamine (Gln) and a-ketoglutarate (a-KG)are shown; @ indicates stimulation, @ indicates inhibition. Abbreviations: GS, glutamine synthetase; pll, regulatory protein; At, and ATd, adenylylation and deadenylylation sites, respectively, on the bifunctional adenylyltransferase; UT, and UTd, uridylylation and deuridylylation (uridylyl-removing)sites, respectively, on the bifunctional uridylyltransferase. NRI, glnG product also known as NTRC; NRllK and NRlp, glnL product (also known as NTRB) catalyzing phosphorylation and dephosphorylation of NR,, respectively.

colleagues, showing that the repression of GS synthesis which occurs when E. coli is grown in a nitrogen-rich medium is regulated in part by the intracellular concentrations of PII, UTase, and A T ~ sThe ~ .participation ~ ~ of PIIand UTase in the repression of GS synthesis was ultimately clarified by the elegant studies of Ninfa and ~agasanik"and Keener and K ~ s t u . ~ ' They showed that transcription of the structural gene for GS is under the control of several gene products, two of which, glnG (ntrC) and glnL (ntrB), are members of the gln operon. The product of the glnL gene (NRI~)is a protein kinase that catalyzes the phosphorylation of the glnG

" A. J. Ninfa and B. Magasanik, Proc. Natl. Acad. Sci. U.S.A.83,5909 (1986). J. Keener and S. Kustu, Proc. Narl. Acad. Sci. U.S.A. 85,4976 (1988).

ELONGATION FACTORS

809

product (NR,) and thereby converts it to a form, NRI-P, which can activate glnA transcription. However, the cyclic interconversion of the glnG product between phosphorylated (NRI-P) and unphosphorylated (NRI)forms is dependent upon the concentration of the PIIprotein which stimulates the dephosphorylation of NRI-P. Whether the effect of PIIis to convert the NRII from a kinase to a phosphatase or serves only as an effector which together with NRII accelerates the spontaneous dephosphorylation of NRI-P appears unsettled. In any case, it became evident from these studies and those summarized above that the regulation of GS activity on the one hand and the regulation of GS formation on the other are tightly linked via interconversion of PI^ between its unmodified and uridylylated forms (Fig. 4). It is in fact the UTase which via allosteric interactions senses changes in the concentrations of a-ketoglutarate and glutamine and thereby dictates the steady-state levels of PII and PII-UMP;these in turn specify the activities of NRII and ATase, which in turn determine the steady-state levels of NRI-P and adenylylated GS, and hence the rate of GS synthesis and GS activity.

[611 Discovery, Resolution, Purification, and Function of

i

Elongation Factors By KIVIEMOLDAVE

r, E

1

In the early 1950s, most of the research on protein biosynthesis focused on the cellular components required for the incorporation of amino acids into proteins in cell-free extracts in vitro. Phil Siekevitzl found that incubation of rat liver mitochondria, cytosol, and microsomes (fragments of endoplasmic reticulum membranes containing ribosomes, nonribosomal proteins, endogenous mRNA, lipids, etc.) accounted for most of the incorporation of amino acids observed when whole homogenates were used; mitochondrial oxidative phosphorylation was necessary. Zamecnik and Kelle? obtained a more refined system that consisted of microsomes, a nondialyzable heat-labile fraction from the cytosol, and an ATPgenerating system; the energy requirements provided by mitochondrial oxidative phosphorylation were replaced by ATP and an ATP-generating

' P. Siekevitz, J. Biol. Chem. 195, 549 (1952). ' P. C. Zamecnik and E. B . Keller, J. Biol. Chem. 209,337 (1954).

K

METHODS IN ENZYMOLOGY, VOL. 182

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

810

APPENDIX

16 11

system. Subsequently, a specific requirement for GTP, in addition to ATP and an ATP-generating system, was also r e ~ e a l e d . ~ Although this pioneering work in Paul Zamecnik's and others' laboratories contributed much to the development of cell-free systems for studies on in uitro translation, little or no information was available regarding the mechanism of protein biosynthesis or the intermediary biochemical reactions involved. Of course, at this time, there was also no knowledge of aminoacyl-tRNAs, mRNA, the genetic code, translational factors, or initiation, elongation, and termination reactions, just to mention a few. Two highly significant discoveries were made in 1955-1957 that opened the door to the subsequent burst of information in the field of translation and contributed greatly to the development of molecular biology. One of these discoveries was the carboxyl activation of amino acids, by reaction with ATP, catalyzed by a group of enzymes present in the cytosol, which led to the formation of enzyme-bound aminoacyl adenylates.4-sThe other was the discovery of a small ribonucleic acid molecule (tRNA), which accepted the aminoacyl moiety from the enzyme (aminoacyl-tRNA synthetase)-bound aminoacyladenylate intermediate, in the form of a "high-energy" ester A number of indirect experiments suggested that aminoacyl-tRNAs were intermediates in the incorporation of amino acids into protein. For example, ribonuclease inhibited amino acid incorporation in cell-free extracts, and the addition of tRNA- containing fractions enhanced incorporat i ~ n . ' Aminoacylation ~~'~ of tRNA was greater in the absence than in the presence of microsomes in cell-free amino acid-incorporating extracts.1° Kinetic data obtained with intact mouse ascites cell^'^^'^ or with diluted

' E. B . Keller and P. C. Zamecnik, J . Biol. Chem. 221,45 (1956).

' M. B. Hoagland, Biochim. Biophys. Acta 16,288 (1955).

P. Berg, J. Biol. Chem. 222, 1025 (1956). J. A. DeMoss and G. D. Novelli, Biochim. Biophys. Acta 22,49 (1956). ' M. B. Hoagland, E. B. Keller, and P. C. Zamecnik, J. Biol. Chem. 218,345 (1956). * E. W. Davie, V. V. Koningsberger, and F. Lipmann, Arch. Biochem. Biophys. 65, 21 (1956). M. B. Hoagland, P. C. Zamecnik, and M. L. Stephenson, Biochim. Biophys. Acta 24,215 (1957). lo M. B. Hoagland, M. L. Stephenson, J . F. Scott, L. I. Hecht, and P. C. Zamecnik, J. Biol. Chem. 231,241 (1958). " P. Berg and E. V. Ofengand, Proc. Natl. Acad. Sci. U.S.A. 44,78 (1958). l2 R. S. Schweet, F. C. Bovard, E. Allen, and E. Glassman, Proc. Natl. Acad. Sci. U.S.A. 44, 173 (1958). l 3 P. C. Zamecnik, M. L. Stephenson, and L. I. Hecht, Proc. Natl. Acad. Sci. U . S . A .44,73, l4

l5

(1958). S . B. Weiss, G. Acs, and F. Lipmann, Proc. Natl. Acad. Sci. U.S.A.44,305 (1958). P. C. Zamecnik, Harvey Lect. Ser. 54,256 (1958).

1611

ELONGATION FACTORS -

811

-

cell-free extracts from rat liver16 incubated at lower (25-27.5') temperatures than the usual 37", were also consistent with the intermediary formation of tRNA-amino acid complexes in protein synthesis. The most compelling experiment, however, was reported by Hoagland et al.1° in 1958. They found that radioactive leucine was transferred from an isolated tRNA-amino acid preparation to microsomal protein, and that this process required GTP, ATP, an ATP-generating system, and a small amount of the cytosolic fraction precipitated at pH 5, which also contained the aminoacyl-tRNA synthetases. Studies in the author's laboratory at this time were concerned with the role of aminoacyladenylates and aminoacyl-tRNAs as intermediates in protein synthesis. Using aminoacyladenylates prepared by A1 Meister and colleagues by organic synthesis," a graduate student, Keith Wong, prepared a complex consisting of a purified aminoacyl-tRNA synthetase and the corresponding synthetic aminoacyladenylate, by incubation of these two components; this complex was similar to those we prepared enzymatically by incubation of the purified enzyme with its corresponding amino acid and A T P . ' ~ ,On ' ~ addition of tRNA to either of these complexes, the aminoacyl moiety of the enzyme-bound aminoacyladenylate was transferred to the appropriate site on tRNA and on addition of microsomes to the complexes the amino acid was incorporated into protein. However, the finding that a single amino acid was incorporated into protein was not easily interpretable, unless pools of aminoacyladenylates and/or aminoacyl-tRNAs were assumed to be also present in the preparations used. A much more direct and unequivocal way to study these steps in protein synthesis, therefore, appeared to be the examination of the aminoacyltransfer reaction, from isolated aminoacyl-tRNA to microsomal protein, which Lois Grossi, a graduate student, was investigating. Grossi isolated aminoacyl-tRNAs from incubations containing the aminoacyl-tRNA synthetases and tRNA (pH 5-precipitable fraction) from rat liver cytosol, ATP, and a radioactive amino acid as a tracer. When the isolated aminoacyl-tRNAs were incubated with microsomes, transfer of amino acid from tRNA to microsome-associated (endogenous, nascent) polypeptide was obtained in the presence of cytosol, GTP, and a nucleoside triphosphate-generating system or other nucleoside triphosp h a t e ~ . ~However, ~.~' although some of the activity that catalyzed aminoK. Moldave, J. Biol. Chem. 235,2365 (1960). " K. Moldave, P. Castelfranco, and A. Meister, J . Biol. Chem. 234,841 (1959). Is K. K.Wong, A. Meister, and K. Moldave, Biochim. Biophys. Acta 36,531 (1960). l9 K. K. Wong and K. Moldave, J. Biol. Chem. 235,694 (1960). L. G . Grossi and K. Moldave, Biochim. Biophys. Acta 35,275 (1959). 2' L. G . Grossi and K. Moldave, J . Biol. Chem. 235, 2370 (1960). l6

812

APPENDIX

[611

acyl transfer was present in the pH 5-precipitable fraction, most of the activity was recovered in the portion of the cytosol not associated with aminoacyl-tRNA synthetases; that is, the "pH 5 supernatant" fraction. Early studies on the characterization of the cytosolic components were hampered by the marked variability in the stability of the activity, under the conditions used at this time; severe and irreproducible losses in activity were encountered on heating, dialysis, fractionation with ammonium sulfate, chromatography, lyophilization, etc., and activity was not always restored when resolved inactive fractions were combined. Crude preparations tended to be very heat stable while purified ones were labile. The addition of sucrose or glycerol to the purified preparations frequently stabilized the activity to some extent. Also, at relatively low concentrations of the partially purified transferase, activity was proportional to the amount of material used, but higher concentrations usually inhibited incorporation. Eventually, evidence was obtained that one of the soluble components was a protein which was nondialyzable and heat labile, and it was extensively purified by ammonium sulfate fractionation and chromatography on various adsorbents or ion exchanger^.^' One of the steps used was chromatography on charcoal, a procedure not frequently used for protein purification, which removed over 90% of the protein and RNA and all of the nucleotides, but none of the activity. The other essential component was dialyzable and could be replaced by various nucleotides such as GTP. With purified preparations of the aminoacyltransferase factor, the requirement for GTP was absolute and transfer of tRNA-bound amino acids to microsomal protein was achieved with GTP as the only nucleotide. The activity observed with nucleotides other than GTP, in crude preparations of the transferase, reflected their ability to generate GTP. In 1960, Kirsch et ~ 1 described . ~ ~a method for the preparation of purified ribonucleoprotein particles (ribosomes), by extraction of microsomes with deoxycholate, which were active in amino acid incorporation; the ribosomes still contained endogenous mRNA. The use of particles from which many nonribosomal proteins, lipids, etc., were removed represented a significant step in the development of a highly purified system for studies on protein synthesis. June Fessenden, a new graduate student in our laboratory, prepared ribosomes from rat liver microsomes and tested them in the aminoacyl transfer reaction. Much to our surprise, whereas a crude cytosol fraction such as the pH 5 supernatant catalyzed aminoacyl transfer to ribosomal protein, the 500-fold-purified soluble factor that was active with microsomes failed to catalyze the reaction with ribosomes.23 22

23

J. F. Kirsch, P. Siekevitz, and G . E. Palade, J . Biol. Chem. 235, 1419 (1960). J. M. Fessenden and K. Moldave, Biochem. Biophys. Res. Commun. 6 , 2 3 2 (1961).

ELONGATION FACTORS

r:

r: :

4

L

r: 3

E

c !

r: i

i

4

813

After numerous repeats of the experiments and checking and rechecking of the various components and conditions, the only plausible explanation was that, in addition to the soluble factor that we had purified and which was active with microsomes, another activity was also required for aminoacyl transfer to ribosomes. This second activity was present in microsomes, and it could be removed from them with deoxycholate. Indeed, on addition of dialyzed deoxycholate extract to incubations containing GTP, glutathione, and the purified soluble aminoacyltransferase activity, incorporation of amino acids from aminoacyl-tRNA into ribosomes was obtained. Thus, two distinct activities were required for the aminoacyl transfer reaction; one was present in the cytosol (which we referred to then as aminoacyltransferase I) and the other was obtained from microsomes (referred to as aminoacyltransferase 11). One of the more troublesome problems working with the deoxycholate extract from microsomes was the variable level of activity obtained in different extracts. We were able to pinpoint this lack of reproducibility to the variability in the early deoxycholate products purchased. In some cases, the deoxycholate completely inactivated microsomal aminoacyltransferase 11. Therefore, the laboratory routinely purchased small amounts of a large number of batches, from several companies, and tested their ability to extract the activity from microsomes; when active batches were identified, large amounts were ordered and, on occasion, I feared that we would run out of more than just storage space as we threatened to corner the world market in deoxycholate. The finding that crude soluble fractions from rat liver homogenates catalyzed the aminoacyl transfer reaction with ribosomal particles, in the absence of the microsomal arninoacyltransferase 11, suggested that the latter factor was also present in the cytosol. Indeed, resolution of the two activities from the cytosol, after removal of the aminoacyl-tRNA synthetases by precipitation at pH 5, was obtained by fractionation with ammonium sulfate.24 Aminoacyltransferase I was recovered with the protein precipitated at low salt concentrations, while aminoacyltransferase I1 was precipitated from solution only at relatively higher ammonium sulfate concentrations. Neither factor was active by itself, but when incubated together, in the presence of GTP and a sulfhydryl compound, they catalyzed aminoacyl transfer to ribosomes. Years later, at the Fogarty International Conference on Translation: Its Mechanism and Control,25a nomenclature was adopted that designated aminoacyltransferases I and I1 as elongation factors 1 and 2 (EF-1 and EF-2), respectively. Evidence for two

" .IM. . Fessenden and K. Moldave, J . Biol. Chem. 238, 1479 (1963). T. Caskey, P. Leder, K. Moldave, and D. Schlessinger, Science 176, 195 (1972).

8 14

APPENDIX

[611

elongation factors was also reported from Dick Schweet's laboratory, independently, with preparations from r e t i c u l ~ c y t e sand , ~ ~ subsequently from Escherichia coli in Fritz Lipmann's lab~ratory.~' In this mammalian cell-free system, biosynthetically prepared polynucleotides of varying base compositions stimulated the incorporation of individual amino acids from tRNA into polypeptides on ribosome^,^^^^^ and this template-dependent incorporation was consistent with the "universality" of the genetic code proposed from studies with E. coli. Saul Slapikoff, another graduate student, and June Fessenden obtained evidence that the aminoacyl transfer reaction involved the initial transfer of both the aminoacyl and the tRNA moieties to ribosomes, that ribosomeassociated nascent peptides were also esterified to tRNA, and that these two components, ribosome-associated aminoacyl-tRNA and peptidyltRNA, were intermediates in protein s y n t h e ~ i s . ~ ~ ~ ~ ' Because of the lack of reproducibility and significant losses of activity when ammonium sulfate fractionation procedures were used to resolve the factors, alternate procedures were investigated. Eugeniusz Gasior, a visiting investigator from Poland, obtained complete resolution of the two elongation factors using molecular sieve chromatography on columns of Sephadex G-200.32,33Elongation factor 1 was recovered in the void volume of the column, suggesting that the molecular weight of the factor was greater than 300,000; EF-2 was retarded on the column and emerged in an area where proteins with molecular weights near 100,000 were eluted. The molecular weights calculated from sucrose gradient centrifugation analyses were consistent with those estimated from gel filtration. Chromatography on calcium phosphate gel, hydroxylapatite, and DEAE-Sephadex A-50 columns, as well as density gradient centrifugation, have also been used for resolution and purification of the factors. Mike Schneir, a postdoctoral fellow, obtained several forms of EF-1 on gel filtration on Sephadex G-200, using a factor preparation that he had purified by chromatography on h y d r ~ x y l a p a t i t eThe . ~ ~ various forms of EF-1 differed J. 0. Bishop and R. S . Schweet, Biochim. Biophys. Acta 49,235 (1961). D. Nathans, J . E. Allende, T. W. Conway, G . J. Spyrides, and F. Lipmann, "Informational Macromolecules," p. 349. Academic Press, New York, 1963. 28 J. M. Fessenden, J. Cairncross, and K. Moldave, Proc. Natl. Acad. Sci. U.S.A. 49, 82 (1963). J. M. Fessenden and K. Moldave, Nature (London) 199, 1172 (1963). 30 J. M. Fessenden and K. Moldave, Biochim. Biophys. Acta 55,241 (1962). 3' S. Slapikoff, J. M. Fessenden, and K. Moldave, J. Biol. Chem. 238,3670 (1963). 32 E. Gasior and K. Moldave, Biochim. Biophys. Acta 95,679 (1965). 33 E. Gasior and K. Moldave, J. Biol. Chem. 240, 3346 (1965). 34 M. Schneir and K. Moldave, Biochim. Biophys. Acta 166,58 (1968). 26

27

"

[6 11

ELONGATION FACTORS

815

in molecular weight, ranging from below 100,000 to aggregates considerably above 300,000. Purification of EF-2 from rat liver was achieved by Wladyslaw Galasinski, another visiting scientist from Poland. He chromatographed the factor, resolved from EF-1 in pH 5 supernatant with hydroxylapatite, on columns of cellulose phosphate and DEAE-Sephadex A-50, followed by electrofocusing (electrophoresis in a sucrose density-pH gradient app a r a t ~ s )Activity .~~ was assayed by the ability of the EF-2 preparations to catalyze transfer of radioactive leucine from labeled aminoacyl-tRNA to ribosomal protein, in the presence of excess quantities of resolved and partially purified EF-1. Finally, purified ribosomes essentially free of all nonribosomal proteins, were prepared by ultracentrifugation of deoxycholate-extracted ribosomes through discontinuous (0.5-1.0 M ) sucrose gradients, in buffers containing 0.5 M ammonium ~ h l o r i d e . ~ ~ , ~ ' The availability of completely resolved and extensively purified components for peptide chain elongation, and the development of strategies for assaying various intermediates, allowed the examination of the role of elongation factors in the aminoacyl transfer reaction and of the interactions between factors and other essential reactants. Gasior and Fumio Ibuki, a postdoctoral fellow from Japan, found that when EF-1 was incubated with GTP and aminoacyl-tRNA, a complex was formed in which both EF-1 and aminoacyl-tRNA were more stable than either one in the free form.38-40Over 70% of the EF-1 activity was lost within 10 min in the absence of aminoacyl-tRNA, in the presence of nucleoside triphosphates other than GTP, or if deacylated tRNA was used instead of aminoacyltRNA. Hydrolysis of the aminoacyl-tRNA ester bond, which occurs under the usual conditions employed in the aminoacyl transfer reaction, was also prevented. These observations are reminiscent of those frequently obtained with many enzyme-substrate complexes. Additional evidence for a complex containing these components was obtained by molecular sieve chromatography of the reaction products by Prema Rao, a postdoctoral fellow from India4' and, in experiments involving multistep incubations in which some of the later steps included ribosomes, Ibuki showed that the W. Galasinski and K. Moldave, J. Biol. Chem. 244,6527 (1969). L. Skogerson and K . Moldave, Biochem. Biophys. Res. Commun. 27,568 (1967). " L. Skogerson and K. Moldave, Arch. Biochem. Biophys. 125,497 (1968). j8 E. Gasior, F. Ibuki, and K. Moldave, Biochim. Biophys. Acta 114,209 (1966). j9 F. Ibuki, E. Gasior, and K . Moldave, J. Biol. Chem. 241,2188 (1966). K. Moldave, F. Ibuki, P. Rao, M. Schneir, L. Skogerson, and R. P. Sutter, "Regulatory Mechanisms for Protein Synthesis in Mammalian Cells," p. 191. Academic Press, New York, 1968. 4' P. Rao and K. Moldave, Biochem. Biophys. Res. Commun. 28,909 (1967). jJ

l6

"

816

APPENDIX

[611

synthesis of a ternary complex [EF-l.aminoacy1-tRNA.GTP] reflected the formation of an obligatory intermediate between aminoacyl-tRNA and ribosome-bound a m i n o a ~ y l - t R N A ~ "indeed, , ~ ~ ? ~ ~particle-associated ; EF-1 and aminoacyl-tRNA were detected in incubations containing ribosomes and preformed ternary complex. If endogenous nascent peptidyl-tRNA was removed from ribosomes by treatment with puromycin, prior to the addition of ribosomes to the incubations containing the ternary complex, the aminoacyl-tRNA as such was detected on the ribosome; that is, none of the amino acids was recovered in peptide-bonded form.43 Thus, ternary complex formed from EF-1, aminoacyl-tRNA, and GTP became bound to the ribosomal A site, but the tRNA-bound amino acid did not participate in peptide bond formation unless a peptidyl-tRNA was present at the ribosomal P site. Although a number of reports suggested that the binding of aminoacyl-tRNA to ribosomes was the result of a factor-independent, Mg2+-and K+-dependent r e a ~ t i o n , ~the ~ -studies ~~ summarized above and others4' indicated that EF-1 was the binding factor that catalyzed this intermediate step in translation. The observation that puromycin reacted with high salt-washed ribosomes, free of nonribosomal proteins including EF-1 and EF-2, to form a peptide-bonded peptidyl puromycin product, suggested that peptide bond synthesis was catalyzed by an activity (peptidyltransferase) on the ribosomal particle and did not require a soluble factor or GTP.37,40 This suggestion was consistent with the conclusions derived from similar studies with prokaryotic ribosomal particle^.^^-'^ Larry Skogerson, a graduate student in the laboratory, found that 10 to 20% of the rat liver ribosomes in our preparations participated in the formation of peptidyl p ~ r o m y c i n ~ ~ . ~ ~ ; this value corresponded with the number of ribosomes whose sites were available for the enzymatic binding of aminoacyl-tRNA, as assayed in incubations with EF-1 and GTP or its methylene analog GMP-PCP, but in F. Ibuki and K. Moldave, J. Biol. Chem. 243,44 (1968). F. Ibuki and K. Moldave, J. Biol. Chem. 243,791 (1968). T. Nakamoto, T. W. Conway, J. E. Allende, G. J. Spyrides, and F. Lipmann, Cold Spring Harbor Symp. Quant. Biol. 28, 227 (1963). 45 M. Cannon, R. Kmg, and W. Gilbert, J. Mol. Biol. 7 , 360 (1963). G. J. Spyrides, Proc. Natl. Acad. Sci. U.S.A. 51, 1220 (1964). 47 T. W. Conway, Proc. Natl. Acad Sci. U.S.A. 51, 1216 (1964). R. Arlinghaus, J. Shaeffer, andR. Schweet, Proc. Natl. Acad. Sci. U.S.A.51,1291 (1964). 49 R. R. Traut and R. E. Monro, J. Mol. Biol. 10,63 (1964). P. Leder and H. Bursztyn, Biochem. Biophys. Res. Commun. 25,233 (1966). " A. Samir, P. Leder, and D. Elson, Proc. Natl. Acad. Sci. U.S.A. 56, 1794 (1966). 52 M. S. Bretscher and K. A. Marcker, Nature (London) 211,380 (1966). 53 I. Rychlik, Biochinl. Biophys. Acfa 114,425 (1966). 42

43

"

" "

[611

ELONGATION FACTORS

817

the absence of EF-2. Thus, the ribosome preparations used appeared to consist of a mixed population, in which 10 to 20% contained peptidyltRNA in a puromycin-reactive (P site) position and an unoccupied A site; the rest must have contained peptidyl-tRNA at the A site, prior to translocation, and were unreactive to puromycin. These results also explained the incorporation, however limited, of amino acids into protein in incubations without EF-2, in the presence of EF-1, GTP, and ribosomes that had not been trated with puromycin; EF-1 catalyzed the binding of aminoacyltRNA to open A sites in pretranslocated ribosomes, and ribosomal peptidyltransferase then catalyzed the formation of a peptide bond between the endogenous peptidyl moiety on the P site and the incoming amino acid at the A site. Two of the reactions in aminoacyl transfer thus appeared to be defined; the binding of aminoacyl-tRNA was dependent on EF-1 and GTP, and peptidyltransferase on ribosomes did not require an elongation factor or a nucleotide. Evidence for the role of EF-2 in translocation was obtained by Skoger~on.~~ A, couple " ~ , ~ ~of~ ~years ~ earlier, another graduate student, Dick Sutter, had shown that preincubation of ribosomes with EF-2, GTP, and glutathione, prior to the addition of EF-1 and aminoacyl-tRNA, markedly stimulated the initial rate of amino acid incorporations6; his studies not only localized the sulfhydryl requirement directly to EF-2, but suggested the formation of a ribosome.GTP.EF-2 intermediate, a ribosome-mRNA event priming active ribosomes for the addition of the next aminoacyl-tRNA, or both. Skogerson isolated a ribosome. EF-2 complex, containing GTP, by ultracentrifugation of incubations containing , ~ ~ , ~ ~ a ribosomal event these components and g l ~ t a t h i o n e . ~In~ addition, occurred under these conditions that resulted in the priming of ribosomes for the rapid initial acceptance of incoming aminoacyl-tRNAs. Some ribosomes in this mixed population contained peptidyl-tRNA at the A site and, therefore, were unreactive to puromycin; in other ribosomes, and as the result of the ribosomal event catalyzed by EF-2 and GTP, peptidyl-tRNA was translocated to the P site, and was reactive to puromycin; concomitantly, translocation made available additional sites for the enzymatic binding of aminoacyl-tRNA to these ribosomes. Although at one time or another, binding of aminoacyl-tRNA was ~ - ja~direct role for thought to be independent of elongation f a ~ t o r s , ~and one of the elongation factors in peptide bond formation was suggested,48 the studies described above demonstrated that EF-1 and GTP catalyzed L. Skogerson and K . Moldave, J. Biol. Chem. 243,5354 (1968).

" L. Skogerson and K . Moldave, J. Biol.Chem. 243, 5361 (1968). 56

R. P. Sutter and K. Moldave, J. Biol. Chem. 241, 1698 (1966).

818

[611

APPENDIX -

-

the binding of aminoacyl-tRNA to ribosomes, EF-2 and GTP catalyzed translocation of peptidyl-tRNA (and its corresponding codon) from the A to the P site, and peptide bond formation was catalyzed by a ribosomal activity that did not require elongation factor or GTP. Also, the data indicated that two distinct GTP molecules were required for each cycle in chain elongation, in the reactions catalyzed by EF-1 and EF-2, respectively. This latter fact, that two GTPs were required, was hard to swallow by my competitors, colleagues and friends when it was first presented; time, however, has healed the wounds and helped digestion. The importance of purified components in studies on biochemical reaction mechanisms cannot be overemphasized. Somehow, our more recent experiences with purification of factors from a variety of different cells, such as cultured mammalian cells or yeast, seemed to be easier, less painful. True, chromatographic materials that are greater resolving, faster flowing, and gentler, as well as new technologies and sophisticated, automatic fractionation and analytical intrumentation are now available. Still, looking back, I sometimes wonder why it was so difficult, and took so long, to purify some of these proteins in olden days. Acknowledgments I would be remiss if I did not express my gratitude to the many colleagues, my friends, who worked with me on these studies, and to the National Institutes of Health, the National Science Foundation, and the American Cancer Society for their support of my research.

AUTHOR INDEX

8 19

Author Index Numbers in parentheses are footnote reference numbers and indicate that an author's work is referred to although the name is not cited in the text.

A

t

Abarbanel, R. M., 769 Abbott, S. R., 401 Abdel-Meguid, S. S., 649 Abeles, A., 94 Abelson, J., 185, 192(33) Ackers, G . K., 556, 579, 580(24), 587(24) Ackerson, B. J., 551 Acs, G . , 810 Adair, W. L., 533 Adamietz, P., 637 Adamik. R.. 638. 640(69) Adams. C. J., 213 Adams, L. D., 454, 485 Adams, P. C., 277 Adams, R. P., 180 Adams, S. P., 641 Adhya, S., 94, lOl(10) Adler, J., 786 Adler, S., 801 Aebersold, R. H., 495, 604, 613 Agganual, B. B., 740 Aggerbeck, M., 699 Ahmed, M. U., 646 Ahmed, Y., 612 Aitken, A., 640, 642(79) Aiura, M., 401 Akerstrorn, B., 685 Aktories, K., 638, 640(73) Alberts, B., 524 Alexander, A. E., 244,247(2), 248(2), 249(2) Alexander, H., 772 Alexander, N. M., 617, 722 Alexander, P., 684 Allen, E., 810 Men, G., 600, 602 Allen, J. R., 546 Allen, R. C., 425, 428(7), 429(7), 434(7), 435(7), 436(7), 437(7), 438(7), 439(7), 441(7), 460, 462(2), 463(2), 464, 465(2),

466(2), 468(2), 469(2), 470(2), 472(2), 473(2), 475(2), 477(2), 483 AUende, J. E., 814, 816 Alley, V., 484 Allfrey, V. G., 642 Allietta, M. M., 204 Allington, W. B., 82 Alped, A. J., 410 Althaus, F., 640 Altosaar, I . , 3 11 Altmda, F., 763 Alvarez-Gonzales, R., 637, 640 Ames, B., 575, 578(15) Aminabhavi, T. M., 244, 247(7), 248(7) Amit, A. G., 658 Amphlett, G. W., 740 An der Lan B., 460, 462(7), 464(7), 466(7), 474(7) Anacker, E. W., 244, 247(6), 248(6), 249(6) Ande, C., 678 Anderegg, R. G., 416 Andersen. J. P., 241 Anderson, D. L., 265 Anderson, G. W., 733 Anderson, J., 659 Anderson, L., 442, 454(5), 458(5), 680 Anderson, N. G., 442, 454, 668, 681 Anderson, N. L., 454, 668, 681 Anderson, W. B., 798, 799 Anderson, W. F., 659 Andersson, L., 376, 419 Andrews, A. T., 425, 427(5), 428(5), 429(5), 431(5), 434(5), 435(5), 436(5), 437(5), 439(5), 440(5), 441(5), 460, 461(3), 462(3), 463(3), 464(3), 465(3), 466(3), 469(3), 470(3), 472(3), 474(3), 475(3), 477(3), 583 Anford, C., 576 Angal, S., 270, 271(20) Anraku, Y., 504 Ansanger, M., 241

820

AUTHOR INDEX

Ansari, A. A., 709 Aota, S., 741 Aoyagi, Y., 109 Aponte, G. W., 628 Appella, E., 234 Appelt, K., 652 Appleman, J. R., 551 Arakawa, T., 285, 287(7), 292(7), 402, 576, 652 Ardies, C. M., 501 Argos, P., 752, 755, 756(12), 757, 758(13), 759, 760, 762, 763, 767, 769, 770(44), 771, 772, 773(23, 58), 774(23), 775(23) Arlinghaus, R., 816 Armbruster, D. A., 645 Armstrong, D. W., 241, 249 Amold, L., 475 Amott, D. I., 188 Aronson, N. N., Jr., 213 Arraiano, C. M., 102 Arthur, A., 114 Asbell, M. A., 148 Ashwell, G., 78 Askamit, R. R., 633 Askonas, B. A., 463 Assoian, R. K., 732 Astier, M., 677 Aterton, D., 589 Atha, D. H., 301, 303(5), 304(5) Atkinson, M. R., 785, 786 Atkinson, T., 348, 353, 355 Aton, B. R., 514 Attardi, B., 222 Attardi, G., 213, 222 Attri, A. K., 572, 573(12) Attwood, D., 244,247(3,4), 248(3,4), 249(3) Austerberry, C. F., 21 1 Autilio-Gambetti, L., 483, 486(26) Aviv, H., 200 Awdeh, Z. L., 463 Azukizawa, M., 708

B Bachmann, B. J., 140 Backlund, P. S., Jr., 633 Backman, L., 550 Badaracco, G., 160, 168 Bahunguna, L. M., 709

Bajaj, S. P., 635 Baker, D., 159, 161(6) Baker, K., 109, 133, 134, 135(2), 142(3) Baker, P., 272 Baker, T., 106 Balch, W. E., 206 Baldo, B. A., 680, 684(9) Bale, W. F., 729 Balestrieri, C., 399 Ballou, C. E., 164 Ban-Naim, A,, 340 Bang, W. G., 806, 807(45), 808(45) Banno, Y., 501 Barbarulli, G., 710 Bardy, R. C., 677 Barger, B. O . , 478, 481(6) Barik, S., 94 Barker, W. C.. 754 Barkley, R. M., 635 Barmann, M., 638, 640(73) Barnard, E. A., 510, 511(33) Barnardi, G., 329, 333(3), 338(2) Barnes, S. J., 549 Barouki, R., 699 Barr, P. J., 107 Barratt, D. L., 485 Barry, B. W., 246 Barth, H., 401 Bartholome-DeBelder, J., 267, 268(15) Bartles, J. R., 229 Bartlett, A., 672 Barton, G. J., 758, 763 Barzilai, A., 613 Bastia, D., 109, 110 Batteiger, B., 616 Baudhuin, P., 213, 218 Bauer, G. A., 159 Baughan, M., 637 Baumann, H., 402 Bauw, G., 604, 605 Bayley, H., 517 Baynes, J. W., 645, 646 Bayse, G. S., 726, 729 Beale, M. G., 709 Bearden, J. C., Jr., 63 Beattie, G., 51 1, 533 Beaufay, H., 213 Becher, P., 258 Bechtol, K. B., 670 Beckmann, M. P., 501

AUTHOR INDEX

Beckwith, J. R., 110 Beckwith, J., 94, 106, 107(77), 109 Bednar, R. A., 185, 192(34) Beech, W. F., 357 Behrman, H. R., 701 Beier, H., 183 Belasco, J. G., 102 Belch, J., 549 Belew, M.. 419 Belfrage, G., 418 Benacerraf, B., 663 Benedek, K., 399 Benedetto, J.-P., 194 Benedik, A., 267 Benkovic, P. A., 542 Benkovic, S. J., 542, 547 Bennet, A. D., 276 Bennet, T. P., 78 Bennett, H. P. J., 415 Bennett, V., 504 Bennetzen, J . L., 105 Bensadoun, A., 79 Bensdoun, A., 67 Berg, C. T., 514 Berg, J . M., 659 Berg, K., 375 Berg, P., 115, 117, 126, 810 Berg, T. G., 268, 269(19) Berger, W., 645 Bergh, M. L. E., 643 Bergmeyer, H. N., 40 Bergmeyer, H. U., 174 Berkner, K. L., 117 Berkowitz, P., 635 Berman, J., 162 Berman, M. L., 109 Bernardi, G., 333 Bernhard. S. A., 551 Benie, C. P., 508 Bers, B., 680, 684(10) Berson, S. A., 701, 724 Bertsch, L., 786 Berttrand, O., 356 Berzofsky, J. A., 770 Bessman, M., 783, 784, 786(3) Better, M., 109 Betti, A., 415 Bezard, G., 486 Bhaskar, K. R., 534, 535 Bhavandan, V. P., 532

82 1

Bhella, R. S., 31 1 Bidlingmeyer, B. A., 591 Bidwell, D., 672 Bieber, L. L., 59 Biemann, K., 416, 627 Bier, M., 426, 462, 466, 475(12, 13) Bighi, C., 415 Biggs, R., 556, 557 Bikel, I., 118 Binder, A., 191 Bicion, S., 464 Binswanger, U., 707 Biou, V., 771 Birdsall, N. J. M., 508 Birman, S., 505, 507(19) Bisher, M. E., 485 Bishop, J. O., 814 Bisson, L. F., 160, 169(7) Bitter, G. A., 115 Bittle, J. L., 772 Bittner, M., 94, 97(21) Bjorck, J., 684 Bjorck, L., 685 Bjurstrom, E., 152, 153(20) Blacher, R. W., 234 Blacher, R., 80 Black, D. L., 202 Blackshear, P. J., 426,429(9), 431(9), 434(9), 435(9), 43@9), 439(9) Blanchard, J. S., 26, 182, 196 Blasco, F., 504, 505(14), 515(14) Blatt, W. F., 76, 81 Bliss, M., 475 Blix, P. M., 732 Block, S. S., 370 Bloemendal, H., 390 Bloom, J. W., 635 Bloswick, B. P., 501 Blumenfeld, 0. O . , 644, 645 Blundell. T. L., 652 Bock, A., 267 Bock, E., 504 Bock, P., 572, 573(13) Bodner, M., 613 Bohemier, D., 635 Bohlen, P., 213 Bohme, H.-J., 344 Bohni, P. C., 173 Boime, I., 200 Boivin, P., 356

822

AUTHOR INDEX

Bokoch, G. M., 638 Bolivar, F., 271 Bollin, E., Jr., 375 Bollivar, F., 267 Bolton, H., 68, 277 Bonnet, J., 194 Bonsignore, A., 789, 790(7), 791(7), 792(7) Boone, T. C., 268, 269(19) Booth, J. W., 546 Bordier, C., 515, 520 Borrebaeck, C. A. K., 374, 677 Boss, M. A., 679 Bosshard, H. F., 478, 481(7) Botchan, M., 116 Bott, R. R., 648 Boulikas, T., 486 Boulot, G., 658 Bourguignon, J., 549, 550(26) Bouvier, S., 94 Bovard, F. C., 810 Bowman, B. J., 504, 505(14), 515(14) Boyer, H. W., 271 Boyer, P. D., 642 Bradbury, A. F., 628 Bradford, M. M., 1I , 29, 62, 182, 520 Bradley, M. K., 118, 131 Braiman, M. S., 501 Brand, K., 789, 792(13) Branden, C. I., 650 Brandt, P. T., 630 Branton, D., 174, 438,439(31), 441(31) Brasfield, D. L., 701 Brass, K. M., 628 Brauer, M., 642 Braun, D., 591 Bravo, R., 455 Brawerman, G., 101 Brayer, G. D., 650 Bredehorst, R., 637 Breitman, T. R., 642, 643(94) Brems, D. N., 267 Brendel, K., 480 Brennan, R. G., 659 Brenner, S. L., 664 Bressan, G. M., 763 Bretscher, M. S., 816, 817(52) Brevat, A., 199 Brew, S. A., 305 Brewer, S. J., 273

Brick, P., 649, 655 Brightman, A. O . , 503 Brinkley, B. R., 664 Broach, J. R., 161, 173(14) Brodsky, G. L., 635 Brody, T., 634 Brooker, G., 714 Brooks, D., 670, 6710) Brosius, J., 138 Brown, E. L., 739, 740(2), 750(2) Brown, E., 740 Brown, L. R., 485 Brown, M. S., 799, 803 Brown, M., 116 Browne, C. A., 415 Brunden, K. R., 514 Brutlag, D., 785, 786 Bruton, C. J., 348, 353 Buchanan, B. B., 179 Buchanan, K. D., 727 Bucher, T., 289 Buchkovich, K. J., 761 Bucholtz, C., 473 Buecker, J. L., 740 Bujard, H., 94, 100, 101 Bundman, D. S., 672 Bunn, H. F., 645 Burgen, A. S. V., 508 Burgers, P. M. J., 159 Burges, R., 9 Burgess, D. R., 605 Burgess, R. R., 29, 85, 168, 185, 192(32) Burgess, R., 153 Burk, D. J., 548 Burlingame, R. W., 648 Bum, P., 504 Burnett, J. P., 265 Bums, V. W., 542 Burry, C. R., 244, 247(5), 248(5) Bursztyn, H., 816, 817(50) Burton, S. J., 357 Burton, W. G., 415 Burwell, C. B., 454, 455(8) Bury, A. F., 426, 429(11) Busby, T. F., 305 Busch, D., 789 Buss, J. E., 641 Butcher, E. C., 50 Bynum, J. W., 45

AUTHOR INDEX

Cabib, E., 67 Cabilly, S., 679 Cai, G.-Z., 635 Cairncross, J . , 814 Cairns, J., 788 Cairns, L., 534 Caldwell, H. D., 614, 615, 617(8, 151, 625(14, 15) Callahan, F. M., 733 Campbell, D. H., 50 Campbell, W. H., 68 Cann, J. R., 474 Cannistraro, V. J., 101 Cannon, M., 816 Cantor, C. R., 577, 754 Caperelli, C. A., 542 Capucci, L., 160 Caravaggio, T., 474 Cardenas, M. L., 548 Carella, M., 593 Carlquist, M., 628 Carlsson, J., 373, 375(2), 418 Carmichael, G. G., 97 Carneiro, M., 202 Caro, R. A , , 725, 736(11) Caron, M. G., 532 Carr, R. I., 736 Carr, S. A., 601, 627 Carroll, E., 785 Carthew, R. W., 522, 527(6) Cartoni, G. P., 415 Caskey, T., 813 Castelfranco, P. 81 1 Castino, F . , 305 Castrillo, J.-L., 613 Catravas, G . N., 59 Catterall, W. A., 501, 507, 513(24), 515 Cavalieri. L. F., 785 Cawston, T. E., 375 Cayanis, E., 678, 679(26) Cayley, D. S., 29, 85 Celeste, A. J . , 740, 744 Celeste, A., 750 Cepko, C. L . , 116, 119 Cerami, A., 785 Chadha, K. C., 375 Chamberlin, M., 96

823

Chambers, G. K., 353 Chan, H., 272 Chang, A. C. Y . , 97 Chang, C. P., 109 Chang, H. W., 504 Chang, J.-P., 41 1 Chang, J.-Y., 557, 591 Chang, L . M. S., 160, 168 Chang, M., 80 Chang, S., 102 Chao, B. H., 501 Chapman, R. H., 180 Charbonneau, H., 109 Chard, T., 710 Chase, J. W., 649 Chedid, L. A., 665 Chen, H.-C., 638, 640(69) Cheng, S., 234 Cheng, T., 789 Cheng, Y.-F., 591 Chernak, J. M., 103 Chervenka, C. H., 556, 564, 571, 572(9), 573(9), 575(9) Chettur, G., 542 Chicz, R. M., 412, 420 Chillemi, F . , 480, 487(16) Chilson, 0.P., 77 Chin, C. C. Q., 601,627 Chobert, M.-N., 699 Chock, P. B., 794, 801, 805, 806 Chodosh, L. A., 522, 527(6) Chou, P. Y., 766 Chow, F., 272 Chow, T. M., 514 Chrambach, A., 241, 254, 426, 428, 429, 434(8), 435(8), 436(8), 439(8), 440, 441(8), 460, 462(7), 464(7), 466, 474(7), 507, 508, 509(28), 558, 583, 727 Christen, P., 303 Christy, M. R., 635 Chu, B., 584 Chua, N.-H., 175, 177, 186(11) Chuba, P. J . , 485 Chubb, P. J., 454 Chui, D . , 94 Church, A. B., 399 Ciabattoni, G., 706 Ciesiolka, T., 68 Cinti, D. L., 232

826

AUTHOR INDEX

Dragner, R. R., 405 Dravid, A., 76 Draye, J.-P., 218 Drazen, J. M., 539 Drell, D. W., 672 Dreyer, W. J., 495 Drysdale, J. W., 472, 474 Dubray, G.?486 Duhamel, R. C., 480 Duine, J. A , , 644 Dumas, L. B., 161 Dunbar, B. S., 425, 437(2), 442, 450(3), 454(3), 455(3), 458(3), 462, 663, 664, 670, 671, 672, 679, 680(4), 683(4) Dunbar, B., 673, 677(9) Duncan, M. J., 115 Dunn, J. A , , 646 Dunn, J. J., 94, 96 Dunn, P. E., 71 Dunning, H. N., 250 Durrum, E. L., 477, 478 Durschlag, J., 576 Dvororak, P., 41 1 Dwek, R. A , , 629 Dyre, D. R., 644 Dzandu, J. K., 485

Eagon, R. G., 148 Earnest, J . P., 501, 502, 511(4) Eason, R., 564 Easterby, J. S., 549 Eaton, L. C., 267 Eaton, M., 105 Ebel, J. P., 658 Ebert, M. H., 438, 473, 485 Ebner, E., 798 Echols, H., 106 Eckhart, W., 512 Edelhoch, E., 600 Edelman, G. M., 162 Edelman, I. S., 678, 679(26) Edelman, M., 175 Edenfield, J ., 762 Edman, J. C., 234 Efstratiadis, A., 128 Egan, K. M., 115 Egen, N. B., 462, 475

Ehrnstrom, R., 419 Eichberg. J. W., 119 Eichele, G., 303, 658 Einarsson, S., 591 Eipper, B. A., 628 Eisenach, J. C., 503 Eisenberg, D., 762, 767, 769 Eisenberg, S., 522, 524(7), 527(7), 526 Ek, B., 501 Ek, K., 460, 463(9) Ekland, H., 650, 656 Ekman, P., 642 El Rassi, Z., 411, 412, 418 Elder, J. H., 614, 684 Elder, J., 520 Elgersma, O., 380, 382(1), 383(1), 386(1), 389(1) Ellinger, S., 142 Elliot, S. G., 115 Elliott, D. C., 291 Elliott, W. H., 291 Ellis, K. J., 31 Ellis, L. F., 265, 269(4) Ellis, L., 234 Ellis, R. W., 115 Ellisman, M., 613 Elmore, D. T., 727 Elsasser, S., 798 Elson, D., 816, 817(51) Ematage, J. S., 105 Emr, S., 696 Emtage, J . S., 679 Endo, H., 638, 640(70) Engelhorn, S. C., 412 Enger-Valk, B. E., 115 England, S., 568 Engleman, E. G., 801 Engstrom, L., 642 Engvall, E., 668 Enquist, L. W., 109 Epp, O., 499, 647, 648(10) Er-el, Z., 409 Eriksson, K. O., 412 Erlanger, B. F., 665, 678, 679(26) Emst-Fonberg, M. L., 540 Esmon, B., 174 Etchberger, K. J., 128 Etcheverry, T., 696 Eubanks, J. H., 502, 511(4) Eubanks, S. R., 641

AUTHOR INDEX

Evans, W. H., 375, 503, 504(7) Eveleigh, J. V., 357 Evers, U., 737 Everse, J., 77 Eyre, D., 644

F Fahnestock, S. R., 684 Fairbanks, G., 503 Fallon, A,, 415 Fanestil, D. D., 520 Fanning, E., 114 Farr, A. L., 57, 296, 520 Farrar, J. T., 454, 455(8) Fasman, G. D., 767 Fasman, G. R., 566 Fass, D. N., 740 Faulders, B ., 50 1 Fausnaugh, J. L., 341, 410, 414 Fausnaugh, J., 404, 405(21), 412, 413(32) Favorova, 0. O., 194 Fazekas de St. Grogh, S., 478, 479(10), 480(10), 487(10) Feher, G., 651 Feibush, B., 410, 419 Feig, L. A., 113 Felgner, P. L., 71 Felsenfeld, G., 335, 337(11) Fendler, E. J., 250 Fenton, D. M., 268, 269(19) Feracci, H. M., 229 Ferenci, R., 599 Ferguson, B., 548 Ferguson, M. A. J., 629, 630(18) Ferguson, M. A., 631 Ferguson, W. J., 24 Ferreira, P. C. P., 375 Fessenden, J. M., 812, 813, 814 Fiers, W., 105 Figge, J., 116 Figgie, J., 761 Figueroa, A., 419 Finch, J. T., 648 Findlay, J. B. C., 519 Finidori, J., 699 Fink, G. R., Finni, M. D. A., 628

827

Fire, A., 202 Firestone, G. L., 696, 699(5, 7) Fischer, J. A., 707 Fischer, J., 658 Fischer, S. G., 615, 622(10) Fish, W. W., 536, 571, 581(8) Fisher, P. A., 161, 173(14), 686 Flamm, E., 101 Flanagan, R. W. J., 727 Fleiderer, G. P., 772 Fleischer, B., 229, 230 Fleischer, S., 225 Fletterich, R. J., 769 Flexner, C., 117 Florence, A. T., 244, 247(4), 248(4), 251 Fliickiger, R.. 645 Fliigge, U. I., 78 Flugge, U. L., 67 Fontana, A . , 610 Ford, J. C., 399 Foster, D. L., 501, 505, 516(16) Foster, W. B., 740 Fournier, M., 194 Fowler, A. V., 589 Fowler, S., 213 Fox, C. F., 617 Fox, J. L., 659 Fox, R., 196 Francesconi, S. C., 526 Francis, S. H., 801 Frank Jzn, J., 644 Franker, P. J., 731 Frasch, C. E., 486, 615 Fraser, M. J., 118, 121 Frater, R., 479 Frederick, C. A., 659 Freeman, H. C., 485 Frei, B., 638, 640(77) Freifelder, D., 575 French, C. S., 399 Freund, J., 665 Frey, M. D., 462, 475(11) Frey, W. H., 11, 402 Friedman, R. D., 486 Friend, S. H., 761 Fries, E., 68, 254 Frischauf, A.-M., 748 Fritsch, E. F., 124,739,740(2),748,749(15), 750(2) Froehner, S. C., 505

828

AUTHOR INDEX

Fromageot, P., 160 Fromm, H. J., 43 Fryer, H. J . , 68 Fuchs, J. A., 265 Fujikawa, K., 139 Fujimoto, E. K., 60 Fujimoto, E. L., 520 Fujitaki, J. M., 642 Fujiwara, K., 643 Fujiwara, M., 638 Fukuhara, K.-J., 271 Fuller, S., 759 Fullerton, D. S., 501 Furth, A. J., 68, 277, 441

G Gabius, H., 68 Gabius, H.-J., 185, 192(33) Galante, Y. M., 674 Galasinski, W., 815 Gallagher, J. T., 532 Gallegos, C., 115 Gallop, P. M., 634, 644, 645 Gallop, P., 644 Gallwitz, B., 628 Gambetti, P., 483, 486(26) Gancedo, C., 798 Garavito, R. M., 647 Garcia, E., 800, 801(33) Garcia, V., 548 Gardner, K., 504 Garfin, D. E., 462, 473(14) Garfin, D., 386,556,557,558,561(5,6), 569, 582(6), 679, 680, 684(10) Gamier, J. O., 768 Gamier, J., 771 Garrels, J. I., 442, 454, 455(8) Garret, M., 194 Gartner, F. H., 60, 520 Gamey, J. S . , 50 Garvin, A. J., 204 Gasior, E., 814, 815 Gastony, G. J., 180 Gatt, J. A., 645 Gautier, C., 105 Gautxch, H . W., 684 Gawehn, K., 40 GeethaHabib, M., 234

Gefter, M. L., 675, 788 Gegenheimer, P., 184, 185, 192(33) Geller, D. H., 549 Gelsema, W. J . , 402 Gemeiner, P., 354 Geng, X., 405 Gentz, R., 94 Gerard, C., 539 Gerard, N. P., 539 Gerber, G. E., 416 Gerbitz, K. D., 645 Gerlt, J. A., 107 Germain, B. J., 633 Germino, J., 109, 110 Gershoni, J. M., 606 Geynet, P., 510 Ghosh, B., 94, 97(23), 100(14) Ghuysen, J. M., 267, 268(15) Gianazza, E., 387, 460, 463(9), 464(9) Gibbons, R. A., 576 Gibrat, J. F., 767, 771 Gibson, K. D., 415 Giddings, J. C., 465 Giege, R., 647, 658 Giffin, J. C., 115 Gilbert, W., 106, 816 Ginsburg, A., 794, 797, 798, 799 Giordano, T. J., 101 Giovane, A., 399 Glaser, L., 641, 642 Glassman, E., 810 Glatter, O., 567 Glembotsky, C. C., 628 Glockshuber, R., 138, 139(11), 142 Glover, J. S., 617 Gluzman, Y., 117 Gnocci, P., 674 Goding, J. W., 83, 670, 671(4), 675(4) Godson, G. N., 151 Goeddel, D. V., 267 Goeddel, D., 93, 740 Goeke, B. J., 520 Goeke, N. M., 60, 520 Goff, C. G., 637 Goff, S., 105 Gogstad, G. O., 63 Goheen, S. C., 412, 507, 508(22), 514 Gojobori, T., 741 Gold, L., 93, 103 Goldberg, A. L., 265

AUTHOR INDEX - -

- -

-

-

Goldberg, A., 105, 107 Goldberg, S., 152 Goldin, S. M., 515 Goldman, A., 650 Goldman, D. A., 546 Goldman, D., 438, 473, 485, 486, 487, 488(50) Goldring, 0 . L., 241 Goldstein, I. J., 511 Goldwin, T. W., 600 Golgher, R. R., 375 Golinger, J. A., 267 Gomori, G., 33, 36 Gong, Q., 234 Gontero, B., 548 Good, N. E., 24, 26(2) Gooding, D. L., 410 Gooding, K. M., 410, 411,,414 Goodman, C. S., 518 Goodman, H. M., 446, 542 Goodwin, T. W., 175 Gorbunoff, M. J., 329, 333(6), 335, 336 Gordon, J . , 606, 641, 668, 680, 681(7), 682(7), 684 Gorg, A., 438 Gorski, K., 202 Goss, N. H., 643 Goto, T., 161 Gottesman, M. E., 94, lOl(10) Gottesman, M., 94, 101 Gottesman, S., 105, 106 Gough, S. P., 187 Goulian, M., 787 Gouy, M., 105 Grable, J., 659 Graff, D., 50 Graham, F. L., 122 Granier, J. O . , 767 Granstrand, B., 419 Grant, R., 94, 99(11) Grantham, R., 105 Grarecos, D., 677 Grass, C., 696 Grassi, J . , 723, 736(5) Grassman, W., 477 Graves, D. J., 637 Gray, C. P., 416 Gray, J. C., 176, 181(7) Gray, J., 109 Gray, L., 109, 133, 134, 135(2), 136, 142(3)

829

-

Gray, W. R., 634 Grazi, E., 789 Green, A. A., 285, 287(2), 290(2) Greenberg, B. M., 186 Greenberg, J., 207 Greenberg, S. M., 504 Greenwood, F. C., 617, 723, 725

Greesman, G. R., 119 Gregson, R. I., 415 Greutzfeldt, W., 628 Gribskov, M., 762 Griffin, W. C., 245, 248(9) Grisham, L., 336 Grob, P. M., 375 Grodzicker, T., 117 Groenewoud, G., 390 Groner, B., 116 Gronwell, A., 478 Grosjean, H., 105 Gross, C., 106 Gross, E., 272, 610 Grossi, L. G., 811, 812(21) Grossman, A., 106 Groves, W. E., 55, 56(12) Gruissem, W., 177, 186 Grungerg-Manago, M., 783 Guarente, L., 110 Guameros, G., 102 Guilbert, L. J., 725 Gundberg, C. M., 634 Giinther, S . , 438 Gupta, S., 410, 413(32) Gustafsson, J.-A., 699 Gustafsson, P., 94, 97(22) Gygax, P. A., 482

Haas, R., 630 Haas, S. M., 59 Hadcock, J. R., 185, 192(34) Haeberli, P., 741 Haga, T., 508 Haigh, R., 109, 133, 134, 135(2), 142(3) Halasz, I., 416 Hall, B. D., 105 Hallick, R. B., 175, 186 Halliday, J. W., 277 Halonbrenner, R., 303

830

AUTHOR INDEX

Halper, L. A., 550 Halpin, R. A , , 547 Halvorson, H. R., 565, 572, 587(11) Halvorson, H., 572, 573(13) Hamer, D. H., 116 Hames, B. D.. 425, 427(6), 428(6), 429(6), 431(6), 434(6), 435(6), 436(6), 437(6), 439(6), 442. 454(4), 679 Hamlin, R., 648, 649 Hammond, P. M., 353 Hammonds, R. G., 501, 511 Hammons, R. G., Jr., 265 Hampton, J., 614 Hanahan, D., 140 Hanash, S. M., 438 Hancock, K., 615 Hancock, R. J., 486 Hancock, W. S., 414 Hanei, M., 762, 773(23), 774(23), 775(23) Hanley-Bowdoin, L., 177, 186(11) Hannig, K., 477 Hanoune, J., 510, 699 Hansen, B., 187, 736 Hansen, U., 116 Hansen, W. J . , 696 Hao, Y. L., 301 Hara, H., 267, 268(15) Hara, I., 401 Harano, D., 122 Harbrecht, D., 272 Hardy, C.. 116 Harkins, W. D., 245 Harlow, E., 761 Harms, E., 206 Harrington, M. G., 484, 487(27), 490 Hamngton, M., 440, 441(33), 484, 485, 558 Harris, B. K., 201 Harris, C. L., 548 Harrison, C., 29, 85 Harrison, S. C., 659 Hartley , D. L., 264, 265, 266(7) Hartshorne, R. P., 501,507,513(24), 515(24) Hartwell, R. D., 353 Harvey, M. J., 353 Hashimoto, T., 401, 410, 411, 413(40), 419, 509, 513(31) Hasselbacher, C. A., 632 Hatfield, A., 485 Hatfield, C. A., 454 Haufler, C. H., 180

Haughton, R. A., 772 Hauschka, P. V., 634, 741 Havel, H. A , , 267 Hawley, D. K., 94 Hayaishi, O., 637 Hayashi, R., 628 Hayes, C. E., 511 Hayflick, J. S., 740 Hays, W., 272 Hazum, E., 532 Hecht, L. I., 810 Hegeman, G. D., 547 Heideman, W., 501 Heine, J. W., 375, 376(15) Heinemann, F. S., 234, 592, 597 Heintz, N., 201, 202(15) Heldin, C.-H., 501 Helenius, A., 249, 254, 508 Helkamp, R. W., 729 Heller, H. M.. 159 Hellsing, K., 305 Hemdan, E. S., 418 Henderson, L. E., 641 Hennig, S. B., 797, 799, 785 Henning, S . B., 798 Henningsen, I., 786 Henry, J . G., 549 Henson, E., 645 Hentschel, C. C. G., 276 Henzel, W. J., 501, 511 Hepburn, M. R., 708 Herling, W. C., 416 Hermanson, G. T., 60, 520 Hermodson, M. A., 414, 415 Hernandez, T., 102 Heron, I., 375 Hemck, G., 521,524 Herron, G. S., 501, 507 Herskowitz, I., 162 Hess, H. H., 59 Heuckeroth, R. O., 641 Heukeshoven, J . , 485, 514 Hewick, R. M., 495,740, 744 Hewick, R., 748 Hey, Y., 347 Heynehey, H. L., 679 Heyneker, H. L., 267, 271 Hicke, L., 159, 161(6) Hicks, D. A., 605, 681 Hierten, S., 412

-

-

-

-

AUTHOR INDEX

Higgins, P. J., 645 Hilber, D. W., 107 Hill, B. L., 678, 679(26) Hilz, H., 637 Hindle, E. J., 645 Hirose, T., 267, 271 Hirota, Y., 267, 268(15) Hirs, C. W. H., 588, 598(2, 3), 599(3), 647 Hiskey, R. G., 635 Hjelmeland, L. M., 68, 241, 251, 254, 261, 466, 507 Hjerten, S., 329, 409, 516 Ho, Y. S., 94, 99(11) Hoagland, M. B., 810 Hochstrasser, A. C., 490 Hochstrasser, D. F., 487, 489, 490 Hodges, M., 539 Hofman, E., 344, 479 Hofman, K. J., 115 Hogg, P. J., 354 Hoke, R. A., 635 Holcomb, C. L., 696 Holladay, L. A., 265 Holland, B., 138 Holland, I. B., 109, 133, 134, 135(2), 136, 142(3) Holloway, P. W., 506 Holmes, W. E., 679 Holmes, W., 36 Holmgren, A., 650 Holmgren-Konig, M., 119 Holmquist, L., 208 Holohan, K. N., 727 Holtzman, E., 211 Holy, A., 138 Holzer, H., 789, 798 Homans, S. W., 629 Homyak, M., 762 Hones, J., 772 Honig, W., 303 Hood, L. E., 490, 495, 604, 613 Hoogeboom, G., 203 Hooper, J. E., 115 Hopp, T. P., 769 Horecker, B. L., 151, 642, 789, 790, 791(7, 17), 792, 793(21) Horisberger, M., 65 Horowitz, J. M., 761 Horowitz, S. B., 211 Horton, J. R., 535

83 1

Horvath, C., 411, 412, 418 Horwitz, A. R., 109 Hoss, A., 114 Hou, W.-M., 513 Houba-Herin, N., 267, 268(15) Hough, H. B., 665 Howard, B., 117 Howell, K. E., 207 Howell, M. L., 542, 543(7), 544(7), 546(7) Howlett, B., 59, 60(21) Howley, P., 117 Hoyt, M. A., 106 Hsiao, L., 250 Hsu, T. C., 486 Hsu, W. P., 244, 247(7), 248(7) Hu, J.-S., 641, 642(82) Huag, H., 267 Huang, K.-S., 517 Hubbard, A. L., 227, 229 Hubbard, S. C., 213 Hubbell, H. R., 486 Hubbell, I. W., 533 Hubbell, W., 511 Huber, R., 499, 647, 648(10) Huberman, J. A., 785 Hubert, J. J., 512 Hudgin, R. L., 78 Hughes, P., 353, 355 Hughes, W. L., 285, 287(2), 290(2) Hugli, T. E., 600 Hulme, E. C., 508 Humble, E., 642 Humphreys, G. O . , 138 Humphreys, G., 105 Humter, S. M., 65 Hunkapiller, M. W., 490, 495, 604 Hunt, L. T., 754 Hunter, J. B., 65 Hunter, T., 512 Hunter, W. M., 617, 714, 723, 724, 725, 727(7), 732, 733(31), 737(7, 10) Hurwitz, J., 179, 185(18), 192(18) Hutchinson, M. A., 512 Hynes, N. E., 116

Iafrati, M. D., 741 Ibuki, F., 815, 816, 817(40)

832

AUTHOR INDEX

Ichihara, K., 708 Ichikawa, T., 399 Ide, G. J., 171, 172(27) Iesterhelt, D., 647 Ifft, J. B., 559 [kemura, T., 105, 741 Ilazaki, M., 401 Imada, T., 501 Imura, H., 638 Inagami, T., 501 Ingebritsen, T. S., 642 Ingham, K. C., 301, 303(5), 304, 305, 551 Inglis, A. S., 285 Inlow, D., 122 Inman, L., 549 Inouye, H., 276 Inouye, M., 107, 108(81), 149, 267, 268(15) Inouye, S . , 107, 108(81) Inze, D., 605 Irace, G., 399 Irie, S ., 486 Irvin, R. T., 148 Isemura, T., 250 Ishi, Y., 267 Israel, M., 505, 507(19) Israelachvili, J. N., 243, 247(2), 248(2), 249(2) Itakura, A., 267 Itakura, K., 271 Ivarie, R. D., 688, 700(3) Izawa, S., 24, 26(2) Izzo, M. J., 729

Jackson, B. J., 643 Jackson, J. F., 786 Jackson, M. L., 514 Jackson, S. J., 50 Jackson-Machelski, E., 641 Jacobs K. A., 739, 740(2), 750(2) Jacobs, E., 492 Jacobs, K. A., 739, 740(2) Jacobs, K., 748, 750 Jacobs, M. A., 244, 247(7), 248(7) Jacobson, E. L., 637, 640(63) Jacobson, M. K., 637, 640(63) Jacobzone, M., 105 Jacquez, J. A., 765

JafTe, B. M., 701 Jagendorf, A., 59, 60(21), 191 Jahn, G., 142 Jakes, R., 612 James, H., 736 Jameson, B. A., 770 Jarnieson, G. A., 1% Jankovic, D. L., 658 Jansonius, J. N., 303, 647, 658 Jany, K. D., 772 Jazwinski, S. M., 162, 163, 167 Jeang, K.-T., 116, 119 Jeansonne, N. E., 163, 167 Jeffcoate, S. L., 732 Jeffrey, A., 94 Jendrisak, J. J., 153, 168, 185, 192(32) Jenjacobson, L., 659 Jenkins, J., 647 Jentoft, J., 734 Jentsch, S., 613 Jett, M., 196 Jeugebauer, J., 249 Joachirniak, A., 659 Jocelyn, P. C., 376 Johansson, B., 412, 727 Johansson, G., 550 John, M., 332 John, N. J., 696, 699(5) Johnson, A. D., 94, 162 Johnson, B. F., 415 Johnson, D. A., 684 Johnson, E. F., 234 Johnson, E. L., 311 Johnson, J. F., 584 Johnson, K. S., 276 Johnson, L. N., 652 Johnson, M. L., 565, 572, 587(11) Johnson, P., 601 Johnston, M., 156 JollBs, J., 149, 745 Jollts, P., 149, 745 Jonak, Z. L. P., 540 Jonasson, J., 232 Jones, C. G., 63 Jones, C. W., 128 Jones, D. H., 454, 485 Jones, E. R., 244, 247(5), 248(5) Jones, J. V., 736 Jones, K. M., 291 Jones, K., 354

833

AUTHOR INDEX -

Jones, M. D., 115 Jones, N. D., 646 Jones, 0.T., 501, 502, 511(4) Jones, P. P., 688, 700(3) Jones, R. B., 616 Jones, S. S., 748 Jongejan, J. A., 644 Jordan, S. R., 659 Joris, L., 791 Josefsson, B., 591 Josic, D., 402 Josse, J., 786 Joubert, F. J., 301 Jovin, T. M., 426, 434(12) Juarez-Salinas, H., 637, 640(63) Judd, J. C., 615 Judd, R. C., 614, 616(3, 4), 617, 622(3), 625(3, 13, 14, 15) Jukes, T. H., 754 Julich, E., 60 Just, W. W., 464

Kaback, H. R., 501, 505, 516(16) Kabsch, W., 771 Kadam, K. L., 241 Kadonaga, J. T., 521 Kafatos, F. C., 128 Kagawa, Y., 249 Kakkar, V. V., 375 Kam, Z., 651 Kammerer, W., 94 Karnp, R. M., 80, 607, 608(18) Kanai, J., 110 Kanaya, S., 109 Kandel, E. R., 613 Kane, J. F., 264, 265, 266(7) Kanehisa, M., 772 Kannangara, C. G., 187 Kaplan, H. A., 234 Kaplan, N. O . , 77 Karabelnik, D., 303 Karasuyama, H., 116 Karch, K., 416 Karger, B. L., 399, 410, 419 Karin, M., 613 Karlsen, K., 696, 699(5) Karlsson, R., 647, 658

-

-

Karn, J., 290 Karnovsky, M. J., 265 Kassell, B., 181 Kastelein, R. A., 105 Katayama, Y., 106 Katlic, A. W., 532 Katnik, I., 712, 713(26) Kato, Y., 401, 410, 411, 413(40), 419, 509, 513(31) Kattermann, R., 789 Katz, A., 635 Kauffman, E. W., 267 Kaufman, R. J., 740, 748 Kaufman, S., 297 Kavonian, M. B., 605 Kavorian, M. R., 681 Kawakita, M., 748 Kawashima, K., 638, 640(70) Kaye, J. S., 480 Keener, J., 807, 808 Keinan, D., 532 Keinanen, K. P., 539 Keller, E. B., 809, 810 Kellerman, O . , 199 Kelley, W. A., 180 Kelley, W. N., 762,773(23), 774(23), 775(23) Kelly, J. M., 349 Kelly, R. B., 785 Kelly, T. J., 521, 522(4) Kempe, T., 272 Kemper, D. L., 478, 481 Kempf, C., 499, 507(2) Kendall, D. A., 151 Kennedy, L. A., 341, 414 Kennedy, N., 116 Kennedy, R. C., 119 Kennedy, R., 390 Kennedy, T. E., 613 Kennell, D., 101 Kennett, R. H., 670 Kenney, D. M., 534 Kenney, D., 534 Kenny, B., 134, 136, 138, 142(3) Kent, S. B. H., 272, 604 Kenten, J. H., 679 Kenyon, G. L., 547 Kern, H., 206 Kessler, R. J., 520 Kessler, S. W., 688 Khorana, H. G., 416, 501, 517, 786

834

AUTHOR INDEX

Kohler, G., 670,671(3), 675(3,1) Kohr, W. J., 740 Koiw, E., 478 Kolattukudy, P. E., 642 Kolpak, F. J., 658 Kolter, R., 102 Komaromy, M., 769 Komiya, K., 401 Kondo, T., 375 Koningsberger, V. V., 810 Koo, J. H., 806,807(45), 808(45) Koop, D. R., 234 Kopaciewicz, W., 404,405(21), 407 801(18) Kopperschlager, G., 344,479 Kinura, H., 664 Korcakova, J., 411 Kipnis, D. M., 701 Kornberg, A,, 149,783,784,785,786,787 Kirsch, J. F., 812 Kornberg, T., 788 Kirsch, W. M., 635 Kornfeld, R., 643 Kirschenbaum, D. M., 54,55(12) Kornfeld, S., 533,643 Kirschner, K., 771 Korza, G., 234 Kirshner, M. W., 615,622(10) Koshland, D. E., Jr., 632 Kirszbaum, L., 515 Koski, R. A., 115 Kisselev, L. L., 194 Kovacs, K., 599 Kitarnura,T.,410,411,413(40),509,513(31) Kovaleva,G.K., 194 Kivirikko, K. I., 627 Krager, B. L., 399 Kramer, K. J., 71 Klagsbrun, M., 741 Kraszewski, A., 267 Klausner, A., 679 Klavaria, 390 Kratky, O., 567 Kratohvil, J. P., 244,247(6, 7), 248(6, 7), Klee, W.A.,262 Kleid, D. G., 267 249(6) Krauss, H., 176 Klein, P.,765 Krebs, E. G., 642 Klekoski, E. J., 180 Kreibich, G., 229 Klenk, D. C., 60,520 Kresze, G., 50 Klenow, H., 786 Kriegler, M., 116 Klett, R. P., 785 Kriz, M. J., 744 Klevens, H. B., 250 Klosterrneyer, H., 642 Kriz, R., 740 Klug, A., 648 Krohn, R. I., 60,520 Knaus, R., 100 Kronvall, G., 684 Knecht, R., 591 Kroviarski, Y., 356 Knight, D. M., 106 Krug, R., 816 Knight, J., 630 Krutnes, M., 63 Knight, L., 735 Krutzsch, H. C., 641 Knopf, J. L., 740 Krystal, G., 50,55(7) Knox, J. R., 567 Ku, H. H., 678,679(26) Knutson, G. J., 740 Ku, L., 115 Koch, T. H., 635 Kubota, Y., 762,765,773 Koehler, K. A., 635 Kuksis, A,, 630,631(23) Koenen, K., 275 Kukuruzinska, M. A., 643 Koga, S., 277 Kula, M.-R., 303

Khoury, G., 116,119 Khym, J. X., 45 Kidera, A., 772 Kido, H., 642 Kidwell, W. R., 637 Kikuchi, H., 375 Kim, J., 659 Kim, S., 632 Kimmel, A. R., 93 Kimura, H., 425,462,679 King, E. E., 180 King, E. J., 37 Kingson, H. S., 795, 796, 797, 798(16),

AUTHOR INDEX

Kula, R., 153 Kuniak, L., 354 Kuntz, I. D., 765, 769 Kupke, D. W., 567 Kushner, S. R., 101, 102(44) Kushner, S., 102 Kustu, S., 807, 808 Kuter, D. J., 558 Kutny, R., 589 Kuwamura, T., 245, 248(8), 249(8) Kviviriko, K. I., 234 Kwan, S.-P., 675, 678(12) Kyte, J., 769

L L'Italien, J. J., 272 LaBonne, S. G., 161 Labouesse, B., 194 Lad, P. M., 508 Lade, B. N., 94, 100(18) Laemmli, U. K., 425, 429(4), 434(4), 435(4), 519, 605, 614, 615(2), 696 Lagerkvist, S., 591 Lahm, H.-W., 80 Laipis, P., 161 Lamberts, B. L., 478 Lammeli, U. K., 615, 622(10) Lamoui, E., 678 Lampson, G. P., Landford, R., 120 Landis, P. W., 646 Landon, N., 610 Lanford, R. E., 119 Lanford, R., 128, 132 Langley, K. E., 268, 269(19) Langone, J. J., 684, 733 Laperche, Y., 699 LaRochelle, W. J., 505 Larson, D., 202 Larson, E., 59, 60(21) Lartier, J. F., 301 Lasker, J. M., 501 Laskowski, M., Sr., 181 Lasser, G. W., 542, 543(7), 544(7), 546 Lathe, R., 740 Lathrop, R. H., 762 Laue, T. M., 555,556, 557(2), 558(2), 561(2),

Lauer, G., 110 Law, J. H., 71 Law, M. F., 117 Layne, E., 50 Lazar, G., 542 Lazzaroni. J.-C., 109 Leach, B. S., 536 Leavitt, J . , 613 Lebovitz, R. M., 201 Leder, I. G., 789 Leder, P., 200, 813, 816, 817(50, 51) Ledger, R., 354 Lee, C., 438, 439(31), 441(31) Lee, D. P., 415 Lee, J. C., 301, 301(2), 303(2) Lee, L. L., 301, 301(2), 303(2) Lee, T. E., 150 Lees, M. B., 59 Leffert, H. L., 512 Lefkowitz, R. J., 532 Legaz, M. E., 139 LeGendre, N., 605 Legler, G., 60 LeGuern, A., 658 Lehman, I. R., 783, 784, 786 Leighton, F., 213 Leimgruber, W., 213 Leirmo, S., 29, 85 Lemkin, P. F., 454 Lennarz, W. J., 234 Leonard, M. N., 532 Lerario, A. C., 727 Lerner, R. A., 519,614, 772 Lerner, R., 520 Lesbats, B., 505, 507(19) Leskes, A., 229 Leskinen, L., 415 Lesser, D., 659 Levin, A., 438, 439(31), 441(31) Levin, J. M., 768, 771 Levin, O., 329 Levine, A., 94 Levoy Bazari, W., 612 Lewald, J. E., 714 Lewis, J . A., 117 Lewis, M. R., 635 Lewis, R. V., 415 Lewitter, F. I., 752, 776(5) Lezius, A. G., 785 Li, H. P., 50

835

836

AUTHOR INDEX

Li, P., 276 Lian, J. B., 634 Liao, J.-L., 516 Liao, M.-J., 517 Liao, Y. D., 150 Lieber, C. S., 501 Lien, S . , 191 Liess, K., 798 Lieve, L., 148 Light, A., 266 Lilley, R., 105 Lillie, R. D., 31 Lim, V. I., 767 Lin, L. S., 265 Lin, S., 94 Linde, S., 736 Lindman, B., 250 Lindstrom, J. M., 501, 512 Lipkin, L. E., 454 Lipman, D. J., 754 Lipmann, F., 810, 814, 816 Lis, H., 643 Liska, D. J., 635 Liss, A., 255 Litman, B. J., 514 Little, J. R., 51 Little, J. W., 96 Little, S., 105 Littlechild, J. A., 83 Liu, T.-Y., 490, 599, 605 Liu, Y.-C., 354 Livingston, D. M., 116, 118 Lochne, M. A., 416 Loewy, A. G., 636, 642(52) London, E., 517 Long, G., 272 Loomis, W. D., 175, 180(4), 192(4) Loomis, W. F., 519, 520 Lopez-Valdivieso, A., 696, 699(5) Lorber, B., 647 Lorenz, P. B., 250 Lotan, R., 511, 533 Love, W. E., 648 Low, M. G., 630, 631 Lowe, C. R., 353, 354, 355, 357, 361, 511 Lowe, P. A., 270, 271(20), 273, 276 Lowery, R. G., 638 Lowry, 0.H., 50, 57, 296, 520 Lowy, S., 290 Lu, K. C., 401

Lu, S. D., 265 Lubs, H. A., 37 Luckhurst, D. G., 411 Luckow, V. A., 118, 119, 120 Ludden, P. W., 638 Luisi, B. F., 275, 659 Lujan, E., 490, 604 Lukesova, S., 41 1 Lukin, M., 632 Lundah, R. L., 677 Lundblad, R. L., 272 Lunn, C. A., 107 Lunney, J., 558 Luxenberg, D. P., 744 Lynch, S. S., 724

M Ma, A., 227, 229 McAllister, L., 518 MacAlister, T. J., 148 McAllister, W. T., 101 McCaldon, P., 752, 760 McCandless, E. L., 50 McCaslin, D. R., 68, 254, 576 McClure, W. R., 93, 94 McCullough, G. A., 82 MacDermott, R. P., 709 McDonald, P. W., 59 MacDonald, R. C., 151 McDougall, J., 71 McGrath, J. P., 613 McGrath, J., 96 McHenry, C. S., 149 McIlvaine, T. C., 32 McKearn, T. J., 670 McKeekin, T. L., 576 McKenney, K., 102 McKenzie, I. F. C., 515 McKenzie, I., 714 Mackman, N., 109, 133, 134, 135(2), 136, 142(3) McLachlan, A. D., 290, 754, 762 MacLagan, N. F., 11 MacLouf, J., 723, 736(5) McMaster-Kaye, R., 480 McNarnee, M. G., 501, 502, 511(4) McPherson, A., 292, 646, 650, 652

AUTHOR INDEX

g

K

,

McPherson, A., Jr., 303 McPherson, J. D., 646 McPhie, P., 73, 74(6) Macritchie, F., 71 Madden, E. A., 198, 203, 213, 228, 2290) Maddy, A. H., 241 Magasanik, B., 807, 808 Maget-Dana, R., 51 1 Magni, G., 800 Magnusson, S., 375 MahanaRao, J. K., 769, 770(44) Mahoney, W. C., 414, 415 Mahuran, D., 593 Mains, R. E., 628 Maiorella, B., 122, 125 Maizel, J. V., Jr., 427 Maki, S. L., 635 Makino, S., 277 Makkar, H. P. S., 67 Malaba, L., 548 Malamud, D., 474 Malamy, M. H., 151 Mallia, A. K., 60, 520 Malling, M. V., 709 Mamalaki, C., 510, 511(33) Manaranche, R., 505, 507(19) Mandecki, W., 659 Mangum, J. H., 800, 801(32) Maniatis, T., 124, 748, 749(15) Manley, E. R., 551 Manley, J. L., 202 Mann, K. G., 635 Mansour, M., 736 Mantel, M., 798 Manthorpe, M., 68 March, S. C., 512 Marchalonis, J. J., 726 Marcker, K. A., 816, 817(52) Margossian, S. S., 290 Margulies, D. H., 675 Margulies, M. M., 428 Markham, G. D., 48 Marks, D. F., 265 Markwell, M. A. K., 59, 617 Marmorstein, R. N., 659 Marmorstein, R. Q., 659 Marsh, K., 117 Marshall, K., 576 Marshall, R. C., 285 Marshall, T., 436

837

Marston, F. A. O., 264, 265(2), 266(2), 270, 271(20), 273, 276(31) Martensen, T. M., 642, 643(97) Martin, A. J. P., 393 Martin, B., 490 Martin, J. R., 584 Martin, P. L., 201 Martin, R., 575, 578(15) Maruizza, R. A., 658 Maruyama, I. N., 267, 268(15) Maruyama, T., 741 Masiarz, F. R., 162 Masukata, H., 102 Mathews, C. K., 542, 543(7), 544(7), 545(6), 546, 547 Mathewson, N. S., 59 Matson, R. S., 507, 508(22) Matson, S. W., 150 Matsudaira, P., 568, 605, 606(6), 612, 680, 682(8), 683(8) Matthews, B. W., 651, 659 Mattoo, R. L., 182 Mattr, E., 727 Maurer, H. R., 425, 428(7), 429(7), 434(7), 435(7), 436(7), 437(7), 438(7), 439(7), 44 1(7), 460, 462(2), 463(2), 464(2), 465(2), 466(2), 468(2), 469(2), 470(2), 472(2), 473(2), 475(2), 477(2) Maurer, R., 94 Maurey, K. M., 204, 213 Maurizi, M. R., 106 Mavrakos, A. E., 741 May, V., 628 Mayer, R., 645 Mazi6, J. C., 658 Mears, G. E. F., 301 Mecke, D., 798 Meek, J. L., 416 Meezan, E., 480 Mehler, A. H., 789 Meilman, E., 632 Meims, G. E., 734 Meister, A., 811 Melchers, F., 116 Menon, M. P., 312 Mentgas-Hettkamp, M., 60 Menzel, C., 785 Mercer, E. I., 175 Mercier, R., 105 Menil, C. R., 437, 438, 473, 478, 484, 485,

838

AUTHOR INDEX

486,487,488(8,9,29,50), 489,490,582, 679, 683(3) Merz, J. M., 551 Mesecke, S., 50 Messing, J., 94, 140 Messner, P., 567 Metz, E., 785 Metzger, H., 253 Meyer, B. J., 94 Meyer, D. I., 174 Meyer, T. S . , 478 Meyerowitz, E. M., 177 Meyersohn, M., 475 Michaelis, L., 35 Michaels, A. S., 76 Michel, H., 499, 647, 648(10) Michel, M. R., 131 Midgley, A. R., Jr., 708 Miekka, S. I., 304, 305, 551 Mierendorf, R. C., 738 Miki, K., 499, 647, 648(10) Mikulski, A. J., 375 Miller, E. J., 290 Miller, H. I., 101, 106 Miller, J. F., 637 Miller, M. J., 490 Miller, N. T., 410 Miller, P., 605 Miller, S., 312 Millican, A., 105 Mills, D., 180 Milstein, C., 670, 675(1) Min, K. H., 806, 807(45), 808(45) Minton, A. P., 304, 541, 572, 573(12) Mirande, M., 199 Misefeld, R., 699 Mishra, R. K., 503 Mislovicova, D., 354 Misono, K. S., 501 Mitchell, D. E., 82 Mitchell, D. J., 243, 247(2), 248(2), 249(2) Mitsock, L. M., 744 Mitsui, A,, 509, 513(31) Mittal, K. L., 250 Miwa, M., 637, 640(64) Miyachi, Y., 727 Miyada, C. G., 744 Miyai, K., 708 Miyake, T., 748 Moats, W. A., 415

Moen, L. K., 542, 543(7), 544(7), 546(7) Moffatt, B. A., 94, 96, 99(16), 100 Mohr, P., 357 Moir, D. T., 115 Moldave, K., 626, 811, 812, 813, 814, 815, 816, 817 Moldeus, P., 232 Molin, S., 94, 97(22) Moller, J. V., 241 Monandi, E., 674 MONO, R. E., 816, 817(49) Monsigny, M., 51 1 Montanez, C., 102 Montelaro, R. C., 606 Montgomery, D. L., 115 Monthony, J. F.. 464 Moore, J. D., 503 Moore, S., 588, 600 Moos, M., 605 Moos, M., Jr., 490 Mor, J.-R., 157 Moras, D., 647, 658 Morel, N., 505, 507(19) Moreu, P. L., 94 Morgan, E. A., 102 Morgan, S. L., 251 Morii, N., 638 Morino, Y., 643 Moriyama, R., 277 Moms, A,, 532 Moms, C. J. O . , 407 Moms, P., 407 Momson, J. C., 246 Morrison, J . F., 31 Momson, M., 614, 726, 729 Momson, R. D., 650 Morton, D., 202 Morton, R. A., 600 Moses, R. E., 788 Mosher, R. A., 426, 466 Mosmann, T. R., 267 Moss, B., 117 Moss, D. W., 40 Moss, J., 637, 638, 640 Motokawa, Y., 643 Mott, J., 94, 99(11) Moudrianakis, N., 648 Mount, D. W., 96 Mudrij, M., 101 Mueller, S. C., 174

AUTHOR INDEX

c

I

:

LJ It% i

I

Mueller, T. J., 249 Mueller, W. T., 547 Mufson, A., 748 Mukejee, P., 249, 250 Mukunoki, Y., 244, 247(7), 248(7) Muller, R. M., 391 Muller, S. A., 677 Muller-Hill, B., 110, 275 Muller-Platz, C. M., 60 Mullet, J. E., 177, 186(11) Mulligan, R. C., 116, 117, 119 Mumby, S. M., 638 Munakata, K., 109 Mura, U.,806 Murel, A., 382 Murphy, C. I., 118, 119, 120, 125 Murphy, J., 272 Murphy, R. F., 727 Murthy, K., 649 Muth, W. L., 265 Muto, N., 513 Mutt, V., 628 Muzariri, C. C., 548 Myher, J. J., 630, 631(23) Mylin, L. M., 115 Myllyla, R., 627 Mysels, K. J., 244, 247(6), 248(6), 249, 250

N

r

1

Nagahari, K., 109 Nagai, K., 139, 273, 275 Nagashima, F., 643 Nagle, J., 684 Nakagawa, R., 271 Nakagawa, T., 250 Nakagawa, Y., 419 Nakai, K., 772 Nakamoto, T., 816 Nakamura, K., 419, 509, 513(31) Nakao, K., 638 Nakashima, H., 277, 765 Nakata, H., 532 Nance, S. L., 668, 681 Narayana, S. V. L., 772 Narumiya, S., 638 Nash, G. S . , 709 Nash, H. A., 101 Nathans, D., 814

839

Nauta, W. J. H., 482 Navier, M. A., 648 Nawroth, T., 509 Neale, A. D., 349 Nedwin, G. E., 740 Needleman, S. B., 753 Negi, S. S., 67 Neice, R. L., 589 Neidergang, C., 640 Neidhardt, F. C., 106 Neill, S. D., 739, 740(2), 748, 750(2) Nelsestuen, G. L., 634 Nelson, C., 635 Nelson, J. W., 82 Nelson, L., 76 Nemes, P., 473 Neri, P., 710 Neuberger, A., 149 Neuberger, M. R., 599 Neuburger, M., 549, 550(26) Neufeld, E. F., 204 Neuhoff, V., 50 Neville, D. M., Jr., 426, 427(10) Newby, A. C., 249, 508, 509(28) Newhall, W. J., 616 Newman, M. J . , 501, 505, 516(16) Ng, S.-Y., 201 Ngai, K.-L., 648, 650 Ngo, E., 191 Nguyen, N. Y., 440, 490, 605 Nhuyen-Disteche, M., 267, 268(15) Nicaud, J.-M., 109, 133, 134, 135(2), 136 Nice, E. C., 416, 607, 608(20), 613 Nicholson, G. L., 533 Nicholson, S . , 549 Nicolson, G. L., 502, 511 Nicosia, S ., 508 Nielsen, B. L., 485 Nielsen, E., 685 Nielsen, N., 772 Nielsen, T. B., 426 Niermann, T., 771 Nikaido, H., 148 Nilsson, G., 102 Ninfa, A. J., 808 Ninham, B. W., 243, 247(2), 248(2), 249(2) Nishikawa, K., 762, 765, 768, 771, 773 Nishizawa, E. E., 454, 485 Nisonoff, A,, 678 Niwa, M., 267

840

AUTHOR INDEX

Nizushima, S., 109 Nobrega, F. G., 592 Noel, R., 401 Noiva, R., 234 Nordstrom, K., 94, 97(22) North, D., 745 North, T. W., 542, 545(6), 546(6), 547(6) Northcote, D. H., 63 Notvall, L., 119 Novelli, G. D., 810 Nowlan, M. P., 411 Noyes, C. M., 272, 635 Nozawa, Y., 501 Nugel, E., 737 Nugent, K. D., 415 Nusser, E., 480, 487(17), 488(17) Nygren, H., 709

O'Connor, C. M., 632, 633 O'Farrell, P. H., 442, 446, 605 O'Farrell, P. Z., 446 O'Hare, M. J., 416 O'Neil, E. A., 521 Ocho, S., 783 Ochs, D., 438, 487 Odell, W. D., 710 Ogren, W. L., 178 Ogston, A. G., 550 Ohashi, Y., 638 Ohkubo, I., 375 Ohno, Y., 401 Ohsumi, Y., 504 Okamoto, Y ., 645 Okamura-Ikeda, K., 643 Okazaki, M., 401 Okazaki, R., 787 Okazaki, T., 787 Okret, S., 699 Olin, B., 373, 418, 419 Oliver, G. C., 701 Olivera, B. M., 634 Ollis, D. L., 649, 650 Ollis, D., 648, 655 Olmsted, J. B., 686 Olsen, R. W., 510 Olson, B. J., 60, 520 Olson, E. N., 641, 642

Olsson, I., 373, 375(2), 418 Omary, M. B., 512 Ooi, T., 762, 765, 768, 773 Oostra, G. M., 59 Oppenheimer, N. J., 637 Opperman, G., 272 Ordal, E. A., 633 Ordal, G. W., 633 Oren, M., 521 Orkin, S. H., 518 Orlandini, A., 710 Omstein, L., 434 Oroszlan, S., 641 Orozco, E. M., Jr., 177, 186(11) OR, E. C., 740 Orrenius, S., 232 Osbom, M., 427 Osezek, D., 375 Ostman, A., 501 Ostrander, F., 490 Ostrove, S., 390 Ouchterlony, O., 668 Ovengand, E. V., 810 Overath, P., 505, 516(17) Owens, S. M., 475 Ozols, J., 234, 556, 568, 592, 597, 601

Pabo, C. O., 94, 649, 659 Pabst, R., 509 Pace, J. L., 478, 481 Paeratakul, V., 128 Packer, L., 225 Pahlman, S., 409 Paiiliezz, J.-P., 199 Paine, P. L., 211 Palade, G. E., 229, 606, 812 Palau, J., 767 Palladino, M. A., 740 Palmer, J. D., 186 Palusinski, 0. A., 426 Pan, B. T., 113 Pan, Y. C., 80 Pande, H., 679 Panheleux, D., 475 Panuska, J. R., 725, 734 Pao, C. L., 150 Paoletti, E., 117

AUTHOR INDEX

Papahadjopoulos, D., 241 Papasozomenos, S. C. H., 483, 486(26) Papazian, D., 515 Pardee, A. B., 285 Pardue, R. L., 664, 677 Parikh, I., 512 Parish, C. R., 515 Park, R., 794, 805 Park, S. C., 806, 807(45), 808(45) Parker, B. M., 701 Parker, C. S., 202 Parker, C. W., 700, 701, 702(10), 703(1), 707(1), 712, 714, 715(1), 721, 733, 734, 736(1) Parker, H. M., 633 Parker, M. L., 708 Parkison, C., 234 Parkman, R., 534 Parkos, C. A , , 638 Patch, A. M., 415 Patchornik, A., 487, 489, 490(1) Pathak, D., 648 Patrono, C., 715 Paucker, M., 375 Paul, M. H., 478 Paulson, B., 272 Pavlakis, G. N., 116 Payne, D. M., 637, 640(63) Paz, M. A., 644, 645 Peake, G. T., 723 Pearson, J. C., 353, 357 Pearson, J., 414, 415(56) Pearson, T. W., 668, 681 Pearson, W. R., 754 Pedersen, L. G., 635 Peebles, C. L., 185, 192(33) Peltre, G., 475 Pendergast, A. M., 548 Penke, B., 599 Penlava, M. A., 148 Penman, S., 206, 211 Penn, A. S., 678, 679(26) Pennica, D., 740 Pepper, D. S., 736 Percy, C., 738 Perez, C., 116 Perin, J.-P., 745 Perkus, M. E., 117 Perlman, P., 668 Perrin, D. D., 24, 25(4), 28(4), 30(4)

84 1

Perry, G. W., 677 Persson, A. V., 234 Perutz, M. F., 273 Pery, L. J., 679 Peters, K.-R., 585 Peterson, E. A., 375 Peterson, G. L., 50, 53(2), 59(2), 79, 296, 501, 520, 593 Peterson, P. A., 678 Peterson, R. C., 71 Peterson, S. M., 272 Pfannkoch, E., 401, 410, 413(32) F'fitzinger, I., 138, 139(11) Pflieger, G., 60 Philipp, K., 50 Philipson, L., 761 Piccini, A., 117 Pick, L., 179, 185(18), 192(18) Pickett, R. A. 614 Pieroni, R. R., 727 Pihlajaniemi, G. T., 234 Pinto, H., 727 Piotrowski, J. J., 311 Piperno, J. R., 735 Pipes, G. D., 495 Pisani, T. M., 665 Pittrnan, D. D., 740 Pixa, G., 647 Plaisted, S. M., 267 Platt, E. J., 696, 699(5) Platt, T., 94, 99(11) Plevani, P., 160, 168 Pluckthun, A., 138, 139(11), 142 Plumel, M., 34 Plunkett, D., 272 Pluskal, M. G., 605, 681 Podgorska, M., 712, 713(26) Poduslo, J. F., 514 Pohl, T., 80 Polacheck, I., 67 Poli, V., 763 Poljak, R. J., 658 Pollock, J. S., 635 Polson, A., 301 Pommerening, K., 357 Pontremoli, S., 789, 790(7), 791(7), 792(7) Pool, R. R., Jr., 204, 213 Poole, B., 213 Poole, C. F., 310 Popham, D., 807

842

AUTHOR INDEX -

-

Porath, J., 373, 376, 418, 419, 420 Portaliar, R. C., 109 Porter, R. S., 584 Posner, I., 82 Postel, W., 438 Postmann, B., 737 Postmann, T., 737 Potgieter, G. M., 301 Potter, J., 68, 277 Pouwels, P. H., 115 Powell, L. W., 277 Powers, S. D., 416 Powls, R., 549 Poyart, C., 273 Poyton, R. O . , 416 PradeUes, P., 723, 736(5) Prakash, V., 576 Prandini, B. D., 789, 790(7), 79 1(7), 792(7) Pratt, M. E., 484, 485(29), 488(29) Price, B., 336 Price, M. G., 714 Price, P. A., 634, 635 Pricer, W. E., Jr., 78 Priddle, J. D., 68, 277 Princen, L. H., 244, 247(6), 248(6), 249(6) Pringle, J. R., 157, 169 Prives, C., 521 Prouty, W. F., 265 Provenzano, M. D., 60, 520 Pruitt, R. E., 177 Pryde, J. G., 241 Przekop, M B., 681 Ptashne, M., 94, 110, 659 Puett, D., 265 Puigdomenech, P., 767 Pum, D., 567 Pumphrey, J., 106 Punch, D., 801 Purnell, M. R., 637 Pyupe, M., 604

Qian, N., 769 Quadri, F., 353 Quigley, G. J., 658 Quintart, J., 218 Quiocho, R. A., 651 Quiroga, S., 725, 736(11)

Racker, E., 191, 516, 789, 790 Radding, C. M., 786 Rademacher, T. W., 629 Radola, B. J., 460, 462, 464(6), 465(6), 466, 468, 470(25), 475(6, 1l), 479 Rafael, J., 214 Raferty, M. A., 588, 599(4) Raftery, M. A., 503 Ragland, W. L., 478, 481 Ragnarsson, U., 642 Ragone, R., 399 Rahamimoff, H., 515 Rahmsdorf, U., 116 Raidt, D. J., 677 Rajagopalan, R., 678, 679(26) Ramwani, J., 503 Randall, R. J., 57, 296, 520 Ransom, R. W., 510 Rao, M., 81, 94 Rao, P., 815, 816(40), 817(40) Rasmussen, J. N., 187 Rauchsen, P., 536 Raybuck, M., 761 Read, S. M., 63 Reading, C. L., 677 Reading, L., 664 Reber, B. F. X., 515 Rechid, R., 757, 758(13) Record, M. T., 29 Record, M. T., Jr., 85 Reddy, G. P. V., 542, 545(6), 546(6), 547(6) Redinbaugh, M. G., 68 Redman, K. L., 642 Redshaw, M. R., 724 Regan, J. W., 532 Reghetti, P. G., 470, 472, 474 Regnier, F. E., 341, 401, 404, 405, 407, 409, 410,412,413(32), 414,415(56), 416,417, 420 Regnier, F., 391 Regoeczi, E., 729, 731(24) Reich, E., 785 Reichard, P., 547 Reichenberg, D., 312 Reichl, H., 117 Reichlin, M., 665 Reid, L. M., 535 Reinhold, V. N., 629

AUTHOR INDEX

Rcisner, A. H., 473, 479 Rele, M. V., 81 Remold-O'DonneU, E., 534 Restogno, G., 763 Retz, K. C., 68 Reutter, W., 402 Rexach, M., 159, 161(6) Reynolds, E. S., 225 Reynolds, J. A., 241, 426, 576 Reznikoff, W., 93 Rhee, S. G., 794, 800, 801(33), 805, 806, 807(45), 808(45) Rhind, S. K., 273, 276 Rhodes, D. G., 82, 555, 556, 557(2), 558(2), Rhodes, D., 648 Rhodes, R. K., 290 Ricard, J., 548 Rice, R. H., 734 Rich, A., 658 Richards, F. M., 651 Richardson, C. C., 94, 99(15), 788 Richmond, T. J., 648 Richter, C., 638, 640(77) Richter, M. L., 191 Rickwood, D., 442, 454(4), 679 Rieger, F., 51 1 Riggs, A. D., 267, 271 b s , P. D., 276 Righetti, P. G., 387,460,461(1,4), 462(1,4), 463(1, 4, 9), 464(1, 4, 9), 465(1, 4), 466(1, 4), 469(1, 4). 475(1, 4), 477(1, 4), 480, 487(16) Rijken, D. C., 373 Rilbe, H., 466 Rimsay, R. L., 258 Rindone, W. P., 752, 776(5) Rindt, J. P. M., 414, 416(55) Ritschard, W. J., 615 Rivnay, B., 253 Roach, P. J., 642 Roark, D. E., 564 Robbins, P. W., 213 Roberts, B. E., 119 Roberts, F. D., 277 Roberts, J. W., 267 Roberts, R., 701, 702(10), 712, 733 Roberts, T. M., 94, 110, 113, 116, 117

843

Roberts, W. L., 629, 630, 631(23) Robertson, C. A , , 101 Robinson, E. A . , 234 Robinson, J. B., Jr., 355, 549 Robinson, M., 105 Robinson, N. C., 277 Robinson, R. R., 109 Robson, B., 767, 768, 771 Rocchetti, G., 674 Roche, A.-C., 511 Rodbard, D., 428, 429, 558, 583, 714 Rodbell, M., 508 Rodkey, L. S., 464 Rodriguez, H., 501, 51 1 Roeder, R. G., 201, 202(15) Roels, 0.A., 59 Rogers, P., 265 Rogers, S., 94 Roghbard, J. B., 770 Rogozinski, S., 645 Rome, L. H., 204 Ronk, M., 605 Ronnstrand, L., 501 Roozen, A. M. P., 402 Rosebrough, N. J., 57, 296, 520 Rosen, F. S., 534 Rosen, M. J., 250 Rosen, V., 740, 744 Rosenberg, A. H., 94, 100(18) Rosenberg, G. B., 501 Rosenberg, M., 94, 99(7), 102, 104 Rosenberry, T. L., 629, 630, 631(23) Rosenbusch, J. P., 647 Rosenfeld, P. J., 521, 522(4) Rosengren, J., 409 Ross, E., 67 Ross, P. D., 794 Rosset, J., 65 Rossetti, Z. L., 416 Rossmann, M. G., 755 Rossomando, E. F., 38,39(1), 43(1), 48, 316 Rostron, G. M., 645 Roth, R. A., 234 Rothblatt, J. A., 174 Rothblum, L. I., 486 Rothman, J. E., 206 Roumeliotis, P., 401 Rounds, M. A., 404, 405(21) Rowley, P. T., 789 Rubenstein, A. H., 732

AUTHOR INDEX

Rubenstein, M., 416 Rubenstein, P. A., 642 Ruckenstein, E., 411 Rudersdorf, R., 739, 740(2), 748 Rudikoff, S., 106 Rudinger, J., 733 Rudolph, F. B., 43 Ruegg, U., 733 Rushton, B., 648 Russel, G. F. J., 246 Ruther, U., 275 Rutter, W. J., 234 Ruyechan, W. T., 168 Ruygers, C. A., 115 Ryan, C. A., 175 Rychlik, I., 816, 817(53)

Saavedra, R. A., 613 Saballos, H. L., 71 Sabatini, D. D., 229 Sadi, D., 736 Sagai, H., 107, 108(81) Sahlestrom, Y., 305 Saini, R., 635 Saito, S., 249 Saito, Y., 267 Sakiyama, F., 642 Salinovich, O., 606 Sallafranque, M. L., 194 Salsa, M., 148 Salstrom, J., 101 Saltiel, A. R., 630, 63 l(20) Saludjian, A., 658 Sambrook, J., 124, 748, 749(15) Samir, A., 816, 817(51) Sammons, D. W., 454, 485 Samsonov, G. V., 310 Samudzi, C., 659 Samuels, M., 202 Sanchez-Pescador, R., 115 Sandburg, L. L., 71 Sandelius, A. S., 503 Sander, C., 771 Sano, T., 375 Santikam, S., 629 Sara, M.. 567 Sarangarajan, R., 678, 679(26)

Saravis, C. A., 425, 428(7), 429(7), 434(7), 435(7), 436(7), 437(7), 438(7), 439(7), 441(7), 460, 462(2), 463(2), 464(2), 465(2), 466(2), 468(2), 469(2), 470(2), 472(2), 473(2), 475(2), 477(2) Sargent, M. G., 60 Sarver, N., 117 Sasagawa, T., 627 Sasaki, H., 401 Sassenfeld, H. M., 273 Sauer, K., 570 Sauer, R. T., 94 Saul, A., 82 Saunders, C. A., 171, 172(27) Saville, D. A., 426 Sawada, Y ., 401 Scaife, J., 94, 99(7) Scawen, M. D., 353 Schachman, H. K., 786 Schachtele, C. A., 265 Schaffhausen, B. S., 117 Scharff, M. D., 675, 678(12) Scharff, M. O., 675 Schatz, G., 67, 161, 173 Schauer, I., 696 Scheivitz, R. W., 659 Schekman, R., 159, 161(6), 162, 164(17), 174, 696 Schelling, M., 677 Schenk, D. B., 512 Schenkman, J. B., 232 Schering, B., 638, 640(73) Schibler, U., 202 Schimerlik, M. I., 501, 507 Schimmel, P. R., 577 Schleich, T., 285, 287(4), 369 Schleicher, E. D., 645 Schleicher, M., 486 Schlessinger, D., 813 Schleyer, M., 159, 161(6) Schmeissner, U., 102 Schmidt, J., 332 Schmidt, M. C., 100 Schmidt, W. E., 628 Schmuck, M. N., 410, 41 1 Schneider, J. A., 206 Schneir, M., 814, 815, 816(40), 817(40) Schoemaker, J. M., 270, 271(20) Schoenmakers, P. J., 310 Scholla, M. H., 311

AUTHOR INDEX

Schoner, B. E., 265, 269(4) Schoner, R. G., 265, 269(4) Schreiber, A. B., 272, 678 Schuette, S. S., 310 Schultz, L. D., 115 Schumacher, G., 267 Schvartz, I., 532 Schwartz, N. B., 549 Schwarz, E., 769 Schwarz, J., 771 Schweet, R. S., 810, 814 Schweet, R., 816 Schwendener, R. A., 241 Schwimmer, S., 285 Schwyzer, M., 131 Scolnick, D., 117 Scopes, R. K., 9, 55, 56(13), 285, 292(10), 344, 349, 354 Scott, J. F., 810 Scott, J. H., 162, 164(17) Scott, K. P., 765 Scully, M. F., 375 Sebestain, I., 416 Sedgwick, S. G., 94 Sedivy, J., 116 Sedman, A., 438 Sedman, S. A., 473 Seeburg, P. H., 740 Seed, T. M., 196 Seehra, J., 748 Sefion, B. M., 641 Segal, A., 799, 803

l-

1I

Seiden, M. V., 709 Seifter, S., 568, 632, 643, 644, 645 Sejnowski, T. J., 769 Sekine, A., 638 Selander, R. K., 180 Sells, B. H., 55, 56(12) Semeriva, M., 677 Senciall, I. R., 258 Sene, C., 511 Sentenac, A., 160 Sere, P. A,, 548 Servillo, L., 399 Setlow, P., 786 Sezaki, M., 486 Shaeffer, J., 816

845

Shaltiel, S., 409 Shaner, A., 122 Shapiro, A. L., 427 Shapiro, B. M., 793, 794, 795, 796, 797, 798(16), 799, 801(18, 27, 28) Sharma, B., 677 Sharrna, 0.P., 67 Sharon, N., 643 Sharp, P. A., 116, 117, 202, 522, 527(6) Shastry, B. S., 201 Shatzman, A. R., 104 Sheffield, S. B., 50 Sheilagh, R., 258 Shelby, S. L., 93 Shen, S.-H., 271, 272(25) Shenolikar, S., 642 Sheridan, R. P., 765 Sherman, F., 155 Sherwood, R. F., 353, 355 Shifrin, S., 478, 488(9) Shilton, B. H., 646 Shimatake, H., 94 Shimizu, T., 748 Shinnick, T. M., 772 Shinoda, K., 250 Shiraishi, K., 401 Shiveley, J. E., 679 Shively, J. E., 605, 606(9), 607, 608(19) Shoemaker, C., 740, 748 Shore, H. B., 651 Shrake, A., 794 Shuman, H., 109 Shuster, J. R., 115 Shwartz, J. H., 504 Sia, C. L., 789 Sibley, B. S., 744 Siedlecki, J., 190 Siekevitz, P., 229, 809, 812 Siezen, R. J., 771, 773(58) Sigel, E., 510, 511(33) Sigler. P. B., 659 Sihag, R. M., 548 Silberstein, A., 480, 487(17), 488(17) Silenga, L., 763 Silhavy, T. J., 109, 110 Simms, E. S., 784 Simms, E., 783, 784(3), 786(3) Simon, R. H., 335, 337(11) Simons, K., 249, 254, 508 Simpson, D. J., 187

846

AUTHOR INDEX

Simpson, R. J., 599, 607, 608(20), 613 Sims, J. L., 637, 640(63) Singer, S. J., 502 Singh, R. M. M., 24, 26(2) Sinsheimer, R. L., 151, 787 Siu, C. H., 519 Sizmann, D.,267 Sjoberg, B.-M., 547 Skelly, H., 512 Skerra, A., 138, 139(11) Skinner, S. M., 663, 670(3) Skogerson, L., 815, 816(37, 40), 817 Slapikoff, S., 814 Slattery, T. K., 415 Slayman, C. W., 504, 505(14), 515(14) Sletyr, U. B., 567 Slopsema, K., 509 Sluski, R., 635 Sluyterman, L. A. A., 380,382(1), 383(1,2), 386(1, 2). 389(1. 21 Small, D. A. P., 353, 355 Smeal, T., 612 Smith, A. J., 589 Smith, D. B., 276 Smith, D. E., 686 Smith, E. R. B., 478 Smith, G. E., 118, 119, 120, 121, 123, 128 Smith, G. K., 547 Smith, H. O., 103 Smith, I., 211 Smith, J. F., 761 Smith, J. L., 648 Smith, M., 128 Smith, P. K., 60, 520 Smith, R. A., 115, 642 Smith, T. F., 762 Smithies, O., 741 Smulson, M. E., 637 Smymiotis, P. Z., 789, 790, 797 Smyth, D. G., 628 Snajdar, R. M., 501 Snyder, L. R. 415 Snyder, M. A., 632 Sobel, B. E., 712,733 Soby, L. M., 601 Soderberg, L., 391 Sodergerg, B .-O., 650 Soloman, S., 415 Soltis, D. E., 180 Soman, G., 637

Somerlot, L., 438 Somerville, C. R., 178 SornervilIe, L. L., 486 Sommer, H. E., 665 Son, H. S., 806 Sorensen, S. P. L., 31, 34(9) Sottmp-Jensen, T. E., 375 Souter, F., 11 Southern, P. J., 115 Spackman, D. H., 593, 600 Spande, T. F., 600 Spar, I. L., 729 Sparks, J., 271 Sparrman, M., 516 Sparrow, J. T., 414 Speck, J. C.. 731 Speck, J. C., Jr., 789 Spector, S., 701 Spencer, S. A., 501, 511 Spivey, H. O., 551 Sportsman, J. R., 684 Sprague, K. V., 202 Spudich, E., 632 Spudich, J. L., 632 Spurr, A. R., 224 Spyrides, G. J., 814, 816 Srere, P. A., 549, 550 Srinivasan, R., 41 1 Srivastava, D. K.. 551 Stabel, S., 761 Stader, J. A., 109 StadlmWler, J., 138, 139(11) Stadtman, E. R., 793, 794, 795, 796, 797, 798(16), 799, 800, 801, 803, 805, 806 Staehelin, T., 606, 680, 681(7), 682(7) Stange, N., 183 Stanley, E. R., 725 Stanley, K. K., 763 Stanley, S. J.. 638, 640(69) Stark, M. R. J., 134 Stauffer, C. E., 57 Stead, C. V., 355 Steck, T. L., 503 Steele, J. H. C., 576 Steele, W. J., 68 Steiger, B., 229 Stein, S., 80, 213, 415 Stein, W. H., 588 Steiner, A. L., 701 Steiner, J. P., 504

847

AUTHOR INDEX -----

iI .

Steitz, J. A., 202 Steitz, T. A., 649,650,655 Stellwagen, E., 354,355,580 Stempien, M. M., 115 Stenberg, M., 709 Stephens, P., 105 Stephenson, A., 510,5ll(33) Stephenson, M. L., 810 Stem, L. J., 501 Stemberg, J. E., 758 Sternberg, M. .I. E., 763 Sterner, R., 642 Sternglanz, R., 161 Steven, A. C., 485 Stevens, C. F., 517 Stevens, T., 174 Stevenson, R., 311 Stewart, K. K., 74 Stewart, M., 290 Stock, J. B., 632 Stockton, J., 508 Stodola, J. D., 267 Stoll, V. S., 196 Stolzenbach, F. E., 77 Stoops, J. K. 540 Stone, B., 228,229(5) Storm, D. R., 501 Stormo, G., 103 Storrie, B., 198,203,204,213 Stoscheck, C . M., 63 Strahler, J . R., 438 Strang, C. J., 645 Strange, P. G., 532 Strasberg, P. M., 593 Strauber, G.B.,510 Strauch, K., 106,107(77) Strehler, B.,745 Streisinger, G., 149 Strickland, T. W., 268,269(19) Strittmatter, P., 601 Strominger, J. L., 249 Strop, P., 411 Strosberg, A. D., 510,678 Strottmann, J. M., 355 Struhl, K., 154 Struve, W. G., 249 Studier, E., 131 Studier, F. W., 94,96,99(16),100 Subbarao, M. N., 101 Suelter, C. H., 71,176

Sugano, K., 628 Sugden, B., 117 Sugimoto, K., 787 Sugimoto, N., 271 Sugimura, T., 637,640(64) Sugme, R., 273,276(31) Sulkowski, E., 375,419 Sullivan, K. H., 731 Sultzman, L. M., 740 Sumegi, B., 549 Summers, D. F., 494,613 Summers,M. D., 118,119,120,121, 123 Summes, M. D., 128 Sun, S. C., 791 Sundquist, W. I., 272 Sussdorf, D. H., 50 Susskind, M. M., 94 Sussman, M. R., 514 Sutclife, J. G., 772 Sutter, R. P., 815,816(40), 817 Suttie, J. W., 634,635 Swami, K. H. S., 107 Swanson, J., 614 Swartzendruber, J. K., 646 Sweatt, J . D., 613 Sweet, R. M., 768 Switzer, R. C., 478,488(8, 9) Sykes, B. D., 642 Synge, R. L. M., 393 Szewczyk, B., 494,613 Szoka, F., Jr., 241 Szoka, P. R., 272 Szybalski, W., 101

T Tabor, S., 94,99(15) Tachibana, C. Y., 162 Tager, H. S., 732 Tai, H.-H., 513 Tai, Y., 131 Takahara, M., 107,108(81) Takahashi, H., 773 Takahashi, N., 642,643(94) Takahashi, T., 632 Takashashi, H., 762,773(24) Takayanagi, R., 501 Takeda, Y., 659 Tal, M., 480,487(17), 488(17)

848

AUTHOR INDEX

Talbert, L., 277 Talmadge, K., 106 Tamamushi, B., 250 Tamura, M., 501 Tan, L., 513 Tanford, C., 68, 241, 247(1), 248(1), 249(1), 254, 340, 410 Taniguchi, N., 375 Tansanen, K., 234 Tanuma, %-I., 638, 640(70) Tapang, M. A., 411 Tapuhi, Y., 399 Tarli, P., 710 Tarr, G. E., 234, 514 Tarvin, T. L., 591 Taugen, O., 232 Taylor, A., 115 Taylor, B. S., 312 Taylor, J., 454 Taylor, M. W., 128 Taylor, W. D., 547 Taylor, W. R., 752, 770 Tchola, O., 789 Tekamp-Olson, P., 115 Teplow , D. B., 604 Terada, H., 399 Terasaki, P. I., 677 Terasaki, W. L., 714 Terwilliger, T. C., 767 Terzaghi, E., 149 Tevenon, G., 416 Thaller, C., 658 Theill, L. E., 613 Thierry, J. C., 658 Thierry, J.-C., 647 Thoelke. M. S., 633 Thogersen, H. C., 139 Thompson, K. J., 665 Thompson, S. T., 354 Thorell, J. I., 727 Thorgensen, H.-C., 274 Thorrnann, W., 462,466,475(12) Thornburg, R. W., 645 Thorne, C. J. R., 42 Thorner, J., 160, 162, 169(7) Thorpe, S. R., 645, 646 Tiffany, H. L., 428 Tijan, R., 521 Tiller, G. E., 249 Tilpula, D., 684

Timasheff, S. N., 285, 287(7), 292(7), 336, 566, 567, 576, 584, 647, 652 Timmons, T. M., 425, 462, 664, 670, 679 Timmons, T., 673, 677(9) Tinoco, I., 570 Tipton, K. F., 42 Tiselius, A., 329, 329(1) Tolbert, N. E., 59 Tombs, M. P., 11 Tomizawa, J., 102 Tonetti, H., 199 Toole, J. J., 740 Topol, J., 202 Torres de Toledo e Souza, I., 727 Torres, A. R., 375 Toth, I., 542 Touster, O., 213 Tovey, R., 680, 684(9) Towbin, H., 606, 668, 680, 681(7), 682(7), 684 Towery, D. S., 641 Towler, D. A., 641, 642 Townend, R., 567, 584 Traugh, J. A., 548 Traut, R. R., 816, 817(49) Trowbridge, 1. S., 512 Truskolaski, A., 356 Tsagaratos, J., 515 Tsai, C. M., 486, 615 Tsai, S.-C., 638, 640(69) Tsang, V. C. W., 615 Tsolas, O., 789, 790, 791, 792, 793(21) Tsuchiya, T., 249 Tsugita, M., 149 Tsuji, T., 271 Tsunasawa, S., 642 Tuason, D. A., 402 Tuason, V. B., 402 Tucker, I. G., 251 Tudor, G., 474 Tuggle, C. K., 265 Tuhy, P. M., 635 Tulsiani, D. R. P., 213 Turkova, J., 357 Turley, R. B., 68 Tuszynski, G. P., 735 Tweeten, K. A., 415 Tweeten, T. N., 415 Twitty, G. E., 462, 475(12) Tye, B.-K., 162, 522, 524(7), 527(7)

AUTHOR INDEX

Udenfriend, S., 213, 415 Ueda, I., 267 Ueda, K., 637 Uhlin, B. E., 94, 97(22) Umbreit, J. N., 249 Unanue, E. R., 663 Unger, K. K., 401 Unkeless, J. C., 228 Urdea, M. S., 115, 162 Urushizaki, Y ., 643 Utiger, R. D., 708 Utiger, R., 701 Uy, R., 627

Vaara, M., 148 Vagelos, P. R., 540 Valentine, R. C., 794 van Boom, J. H., 658 Van Buskirk, J. J., 635 van Damme, J., 375, 376(15) Van Damme, J., 604 van der Eb, A. J., 122 van der Hoeven, T. A., 232 van der Marel, G., 658 van der Venne, J. L. M., 414, 416(55) van der Zee, R., 513, 514(46), 769 Van Frank, R. M., 265 Van Keuren, M. L., 438, 473, 478, 487, 488(8, 50) Van Montagu, M., 604, 605 van Renswoude, J., 499, 507(2) Vanaman, T., 109 VanBlerkom, J., 455 VanBogelen, R. A., 106 vanBruggen, E. J . F., 585 Vandekerckhove, J., 604, 605, 638, 640(73) Vandlen, R. L., 503 Vanecek, G., 405 Vapnek, D., 94, 97(21) Varon, S., 68 Varshavsky, A., 613 Vartak, H. G., 81 Vasser, M., 94, 97(20), 99(20) Vastola, K., 375 Vauquelin, G., 510

849

Vecoli, C., 681 Vecoli, M. R., 605 Vellekamp, G., 548 Venema, R. C., 548 Venkataraghavan, R., 765 Venkataraman, K., 357 Venkataraman, R., 789, 790 Verbakel, J. M. A., 115 Vesterberg, O . , 460, 461(8), 462(8), 464(8), 465(8) Vidmar, T. J., 454, 485 Vieira, J., 94, 140 Viel, C., 199 Vigny, M., 512 Villafranca, J. J., 794 Villareal, L. P., 126 Vingron, M., 757, 758(13), 763 Vifiuela, E., 427 Viswanathan, S., 199 Vita, A,, 642 Voelkel, K., 161 Vogel, F. R., 665 Volkin, E., 45 Voller, A., 672 von Gabain, A , , 102 von Heijne, G., 752, 771 von Hippel, P. H., 285, 287(4), 369 Vray, B., 678

Waddell, W. J., 11 Wager-Smith, K., 613 Wagner, A. F. V., 772 Wagner, E. M., 645 Wagner, G., 391 Wajchenberg, B. L, 727 Wakil, S. J., 540 Waksman, Y., 532 Walker, I. D., 515 Walker, W. H. C., 709 Walker-Simmons, M., 175 Wall, D. A., 227 Wall, R., 769 Walla, M. D., 646 Wallace, R. B., 744 Wallach, D. F. H., 503 Wallek, M., 612 Waller, J.-P., 199

850

AUTHOR INDEX -

-

-

Wallis, J., 807 Walpole, G. S., 32 Walsh, J. H., 711, 735 Walsh, K. A., 627 Walsh, M. J., 71 Walsh, P. N., 735 Walter, G., 512 Walton, D. J., 646 Wang, A. J.-H., 658 Wang, B. C., 648 Wang, E. A., 740, 744 Wang, J. C.. 161 Wang, J., 138, 570 Wang, K., 486 Warburg, O . , 792 Warren, F., 97, 99(31), 101(31) Warren, H. S., 665 Washabaugh, M. W., 410 Waskell, L., 96 Wasserman, G. F., 547 Wasserman, N. H., 678, 679(26) Watanabe, M., 375 Waters, M. J., 501, 511 Watson, D. H., 354 Watson, J. D., 783 Watterson, D. M., 486 Weaver, L. H., 658 Webb, E. C., 42, 285, 287(3), 410 Weber, K., 427, 558 Webster, R. G., 478, 479(10), 480(10), 487(10), 726, 729(16) Webster, T. A., 551, 762 Weder, H. G., 241 Weeke, B., 668 Wegner, A , , 638 Wehr, C. T., 401 Weigele, M., 213 Weijer, W. J., 769 Weil, P. A., 201 Weinberg, R. A., 761 Weiner, B., 118, 131 Weingraub, B. D., 706 Weinstein, D., 67, 79 Weir, M. P., 271 Weisberg, R. A., 101 Weiss, R. M., 767 Weiss, R., 658 Weiss, S. B., 810 Weissbach, A., 174, 190 Weissbach, H., 174

Weitzman, P. D. J., 549 Weller, P. F., 539 Wellig, G. W., 513, 514(46) Welling, G. W., 509, 769 Welling-Wester, S., 509, 513, 514(46), 769 Welton, A. F., 508 Weser, J., 438 Wessel, D., 67, 78 Westwell, A. E., 244, 247(6), 248(6), 249(6) Wettenhall, R. E. H., 349 Wetzel, R., 679 Wever, P. C., 646 Whalen, W., 94, 100(14) Wheeler, K. P., 513 Whitcombe, T. V., 659 White, F. C., 478, 481(6) White, R. D., 267 White, S. W., 652 White, S., 270, 271(20) Whiting, P. J., 501, 512 Whitters, M. J., 744 Whyte, P., 761 Wible, L., 664 Wick, D. G., 355 Wickerhauser, M., 301 Wickner, W., 149 Wideman, J., 80 Widnell, C. C., 228 Wiebenga, E. H., 585 Wielckens, K., 637 Wiginton, D., 277 Wijdenes, J., 380, 383(2), 386(2), 389(2) Wikstrom, A.-C., 699 Wilchek, M., 358 Wilcox, C., 641, 642(82) Wilf, J.. 304 Williams, A. F., 629, 630(18) Williams, D. C., 265 Williams, E. F., 249 Williams, K. R., 589 Williams, R. P., 731 Williamson, A. R., 460, 461(5), 462(5), 463 Williamson, M. K., 635 Williamson, R. A., 532 Wilson, B. M., 149 Wilson, C. M., 437, 480 Wilson, E. T., 202 Wilson, E., 658 Wilson, J. E., 71 Wilson, J. M., 762, 773(23), 774(23), 775(23)

AUTHOR INDEX

Wilson, K. S., 652 Wilson, S. P., 312 Wilson, T. H., 501, 505, 516(16) Windholz, M., 67 Winget, G. D., 24, 26(2) Winocour, E., 521 Winter, W., 24, 26(2) Winzor, D. J., 354 Wirt, J., 203 Wise, G. E., 485 Witkop, B., 600 Wittenberger, M., 797 Wittmann-Liebold, B., 71 Wold, F., 626, 627 Wolf, D., 798 Wolf, H., 770 Wolska, K., 94 Womack, M. D., 151 Wong, H. C., 102, 711, 735 Wong, J. H., 179 Wong, K. K., 811 Wong, P., 807 Wood, C. R., 679 Wood, D. M., 672 Wood, E. R., 150 Wood, H. G., 643 Wood, T., 548 Wood, W. I., 501, 511 Woodbeny, N. T., 249 Woodford, T. A., 642 Woods, K. R., 769 Woodtli, T., 645 Woolfolk, C. A , , 793, 794 Worsham, L. M. S., 540 Wozney, J. M., 740, 744 Wrange, O . , 699 Wright, C., 116 Wright, E. M., 138 Wright, J. K., 505, 516(17) Wu, C. W., 150 WU, G.-M., 565 Wu, G. J., 200 WU, L.-C. C., 161, 173(14) Wu, S.-L., 399 WU, W. C.-S., 503 Wulff, K., 798 Wychoff, H., 647 Wyckoff, M., 429 Wyers, F., 160 Wypych, J., 268, 269(19)

85 1

Xu, Q.-Y., 605, 606(9)

Yada, Y., 501 Yakis, J., 117 Yalow, R. S., 701, 724 Yamada, M., 117 Yamada, T., 628 Yamaki, M., 501 Yamamoto, K. R., 699 Yamamoto, R., 265 Yamamoto, T., 708 Yamamura, H., 508 Yamasaki, Y., 509. 513(31) Yan, S. B., 402 Yanase, Y., 375 Yancey, S., 102 Yang, D. C. H., 548 Yang, J. T., 567, 584 Yanisch-Peron, C., 94 Yanisch-Pen-on, C., 140 Yanofsky, C., 102 Yansura, D. G., 267 Yao, K., 412 Yarranton, G. T., 138 Yarranton, G., 105 Yeager, R. E., 501 Yee, B. C., 179 Yeh, J., 797 Yelton, D. W., 675, 678(12) Yip, T.-T., 419 Yonehara, S., 375 Yoshihisa, T., 504 Youderian, P., 94 Young, L. S., 202 Young, R. A., 103, 738 Yphantis, D. A., 82, 564, 565, 572, 573(10), 587(11) Yu, H.-A., 551 Yutzey, K. E., 632

Zagorsky, W., 183 Zaidenzaig, Y., 409

852 Zamecnik, P. C., 809, 810 Zapata, G. A . , 635 Zemek, J., 354 Zeppenzauer, E., 656 Zeppenzauer, M . , 656 Zetterqvist, O.,642 Zhang, J., 637 Zhang, Z.-L., 513 Zimmer, H., 50 Zimmerman, J. E., 733 Zimmermann, W., 190

AUTHOR INDEX

Zinn, K., 518 Zipser, D., 105 Zizkovsky, V., 411 Zola, H., 670, 671(5) Zoller, M. J., 128 Zubay, G., 200 Zuong, N. G., 649 Zuong, N.-H., 648 Zurawski, G., 186, 267 Zurawski, S. M., 267

SUBJECT INDEX

853

Subject Index

ACES, pK values at 25",27 N-2-Acetamido-2-hydroxyethanesulfonic acid. See ACES N-2-Acetamidoiminodiacetic acid. See ADA Acetate pK values at 25",27 stock solutions, 32 Acetone, as precipitating agent, 78,289 Acetylcholinesterase, of human erythrocyte, glycoinositol phospholipid membrane anchors, 629-630 analysis of, 630-63 1 Acetylchymotrypsin, interaction with hydroxylated columns, 331 Acetylchymotrypsinogen, interaction with hydroxylated columns, 331 Acetyllysozyme, interaction with hydroxylated columns, 33 1 Acetylribonuclease, interaction with hydroxylated columns, 33 1 Acetyltrypsinogen elution, from hydroxyapatite, 332 interaction with hydroxylated columns, 331 Acid-acetone precipitation, of protein, for amino acid analysis, 592-593 Acids, neutralization, 67 Actin, 195 Activity assay, 10 Acylation, of proteins, analysis of, 640642 ADA, pK values at 25". 27 Adenoviruses, as transient expression vector, 117 Adenylate kinase, heat stability, 298 Adenylyltransferase activity, and uridylylation of P,,protein, 799-801 from Escherichia coli, 797-798 inactivation of glutamate dehydrogenase, 798

Adjuvants, 665 ADP-ribosylation analytical strategies, 639-640 enzymes involved in, 637-638 nonenzymatic, with free ADP-ribose, 638 process of, 637 ADP-ribosyltransferase, assays, 639-640 ADP-ribosyltransferase A, 637 ADP-ribosyltransferase C, 637,638 ADP-ribosyltransferase D, 638 Adsorption, for protein sample concentration, 518-520 Aerosol OT properties of, 248 in protein purification, uses of, 241 AES, pK values at 2s0,28 Affinity chromatography, 83,286-287, 343-344, 376-377. See also Lectin affinity chromatography; Site-specific DNA affinity chromatography advantage of, 417 bifunctional reagents, 377 binding of protein to ligand, 365 blocking unreacted groups, 363-364 buffer conditions, 365 buffer volume to column volume ratio, in scaling up, 379 column, sterilization of, 369-370 column diameter, in scaling up, 378 co~umnregeneration, 368-369 contaminating proteins, 358 coupling of ligand to matrix, 363 coupling or linkage chemistry, 362-363 to detect impurities in protein sample, 556 elution conditions, 367-368 elution profile, measurement of, 368 flow rate, 365-366 in scaling up, 378 general methods, 357-371 isolating cells by, 371-372 matrix, 358-359 mechanical stability of, 379

854

SUBJECT INDEX

of membrane proteins, 499-502 monitoring coupling efficiency, 364-365 nons~ecificinteractions in. 366-367 pretreatment and clean-up steps, 379 principle of, 358 for purification of integral membrane proteins, 510-512 elution buffers for, 510-511 specific ligands for, 5 10-5 1 1 in reconstitution of multienzyme complexes, 550 to remove polybuffer and ampholytes from protein, 390 sample concentration, in scaling up, 378-379 sample loading, 365 sample-to-column volume ratio, in scaling up, 378-379 scale-up, 377-379 solvents, 360 spacer arms in, 360-361 specialized techniques, 371-379 specific vs. nonspecific elution, 367368 Affinity gels covalent coupling gels, 359-360 ligand specificity, 359 preparation, 361-362 sterilization of, 369-370 storage, 370-371 types of, 359 Agarose, 3 10 Agarose gels, for isoelectric focusing, 462463 Aggregation number, 244, 253 Agitation, with abrasiveness, for mechanical lysis, 151-152 Albumins, 286 Alcohol dehydrogenase and lactate dehydrogenase, alignment of NAD-binding domains, 759-760 purification, immobilized dye for, 357 from Z. mobilis, purification of, 349 Aldehydes, aliphatic, a s iodine carriers, 732, 734 Aldimines, 643-645 ALIGN (computer program), 759 Alkaline phosphatase-conjugated second antibodies, immunodetection of proteins with, 686

Alkaline phosphodiesterase, rat liver, 228229 Alkaline phosphodiesterase I assay, 218 organelle specificity, 213 Alkaloids, in plant extracts, 175 Alkyltrimcthylammonium salts, 506 Almadori rearrangement, of aldimines, 645-646 Amide analysis, of proteins, 600-601 Amido Black, 477 dye-dye interactions, 480 staining intensity, 480 Amine buffers, 28 Amino acid analysis accuracy, 601 contamination of sample, 601 cysteine determination, 598-599 equipment, 589-591 HPLC method, 589-591 ion-exchange method, 589-591 of proteins, 587-601 reproducibility, 601 sample hydrolysis, 594-598 sample preparation, 592-594 sensitivity, 601 tryptophan determination, 599-600 Amino acid derivatives, ion-exchange analysis elution positions of, 601 Amino acids affinity to Fe3+ chelate gel, 376 I4C-labeled, intrinsic labeling of peptides with, 617 effects of protein hydrolysis on, 588 hydrophobic, 340 Amino acid sequences aligning Unitary Matrix comparison criterion, 753-754 using multiple sequences, 763-764 using sequence fragments, 755-757 using single amino acid comparisons, 752-755 alignment assessment of, 758-761 significance of, 757-761 analysis, 568, 587-601 protein sample preparation for, 495 computer analysis, recommendations, 764-765

SUBJECT INDEX

databases of, 775 finding repeats within, 762-763 patterns, as alignment aids, 761-762 residue characteristic plots, 772-775 Aminoacyladenylates, 811 Aminoacyltransferase I, 813 Aminoacyltransferase 11, 813 Aminoacyl transfer reaction, 81 1-813 function of elongation factors in, 815818 Aminoacyl-tRNA, 809-8 10 Aminoacyl-tRNA synthetase, 81 1 assay, 41-42 isolation of, 198-200 Aminoacyl-tRNA synthetase multienzyme complex, isolation, 548 p-Aminobenzamidine.2HC1, stock solution, preparation, 181 E-Amino-n-caproicacid, stock solution, preparation, 181 2-Aminoethylsulfonic acid, taurine. See AES Amino groups, in adsorption of proteins to HA, 330-332 2-Amino-2-methyl-1,3-propanediol. See Ammediol 3-Aminopropanesulfonic acid. See APS Ammediol, stock solutions, 36 Ammonia, pK values at 25', 28 Ammonia-formic (or acetic) acid, 30 Ammonium carbonate-ammonia, 30 Ammonium persulfate in isoelectric focusing, 462 pK values at 25", 28 Ammonium sulfate concentration of, determination. 2952% salting out proteins with, 290-296, 410 Ammonium sulfate fractionation, of Ig from serum, 669 Ammonium sulfate precipitation, 69 of ampholytes, from proteins, 390 of inultienzyme complexes, 544-545 purification of proteins with, 792 Ammonyx AO, properties of, 248 Ampholytes caniers, for isoelectric focusing, 464465

separation from proteins, 477 by gel filtration, 390

855

by precipitation with ammonium sulfate, 390 AMS-trypsinogen, elution, from hydroxyapatite, 332 Analogs, in enzyme assay, 48 Analytical centrifuge, 572-573, 575 optical systems for, 572, 578 radius determination with, 572-573 Anion-exchange chromatography, 3 10, 404 Anions influence of, on retention and resolution of selected proteins in IEC, 407 and strength of hydrophobic interactions, 340 Antibodies. See Antiidiotypic antibodies; Monoclonal antibodies; Polyclonal antibodies; Single-chain antibodies Antibody affinity chromatography, for purification of glycoproteins, 534 Antibody domains, expression and secretion of, using hemolysin export pathway, 138-141 Antibody fusion proteins cleavage of, 141 purification of, 141 Antibody screening assays, 671-674 Antigens increasing immune response to, 664-665 purification and preparation, 663-665 Antiidiotypic antibodies, 678-679 Antipain, 181 concentration used, 88 protease class inhibited, 88 Antisera. See also Monoclonal antibodies, production of; Polyclonal antibodies, production of cross-reacting antibodies in, removal of. by adsorption, 704 for immunoassays, 702-704 screening. 703-704 storage, 704 Antitermination, 101 vectors allowing, 101 AOT, properties of, 248 Applied Genetic Technology, address, 775 Aprotinin concentration used, 88 protease class inhibited, 88 as protease inhibitor, 171, 197 stock solution, preparation, 181

856

SUBJECT INDEX

APS. See Ammonium persulfate Arabidopsis thaliana, 177-178 Arginine residues, protein cleavage at, 610 D-Aspartate, L-isoaspartate-methyltransferase, 632 Aspartate (acidic) protease inhibitors, 181 Aspartate residues P-carboxylation of, 635-636 carboxyl methylation of, 632-634 Assays. See also Enzyme assay; Protein assay accuracy, 10 cost, 10 of enzyme activity, development of methods for, 38-39 for estimating protein concentration, criteria, 50 precision, 10 reliability, 10 reproducibility, 10 sensitivity, 10, 50 simplicity, 10 specificity, 10 substrate availability, 10 Atlas (32127, properties of, 247 Atrial natriuretic factor receptor, from bovine adrenocortical cells, purification, 500 Autographica californica nuclear polyhedrosis virus, 117-1 18 wild-type, 123-124 DNA prepared from, 123 Autoradiography, 78 silver stain quenching of, 486-487 Avidin-biotinylated peroxidase complex, for screening monoclonal antibodies, 672-673

Bacillus subtilis, lysis of, 148 Bacteriorhodopsin from Escherichia coli transfected with bacterioopsin gene, purification, 501 renaturation, after purification, 5 16-5 17 Barbital buffer, stock solutions, 35 Bases, neutralization, 67 BCA protein assay, 520

Bead Beater, 161-164 preparation of yeast extracts using, 163164 Bead mills, 151 Beans, growth, 177 Benzamide, stock solution, preparation, 180 Benzamidine concentration used, 88 as protease inhibitor, 88, 170 Benzamidine.HC1, stock solution, preparation, 181 BES, pK values at 2S0, 27 BESTFIT (computer program), 758, 759, 764 Bicinchoninic acid reagent, 182 Bicinchoninic acid (Smith) protein assay, concentration limits of chemicals in, 52-53, 60 Bicine, 29 pK values at 25", 28 Bifunctional agents, 376-377 BigCHAP, 252 properties of, 248 Bile acids, 256 Bile salts, 506 compatibility with divalent cations, 256 spectral properties, 256 Bioaffinity chromatography, 392 Bioaffinity interactions, utilized in HPLC, 417 BioGel, 321, 324 BioGel A, 320 BioGel P, 320 Bio-Rad AGl-X4, 3 11 Bio-Rad Model 422 Electroeluter, 492-495 Biotin, linkage to lipoic acid, 643 Biotin-streptavidin interactions, in sitespecific DNA affinity chromatography, 522 Bisacrylamide, and elution of proteins from gels, 494-495 N,Nf-Bisacrylylcystamine,gel crosslinking with, 441 Bis(2-ethylhexyl)sulfosuccinic acid, sodium salt, properties of, 248 N,N-Bis-3-D-gluconamidopropylcholamide. See BigCHAP N,N-Bis-3-D-gluconamidopropyldeoxycholamide. See Deoxy BigCHAP

SUBJECT INDEX

3-[N-Bis(hydroxyethyI)amino]-2-hydroxypropanesulfonic acid. See DIPS0 N,N-Bis(2-hydroxyethyl)glycine.See Bicine [Bis(2-hydroxyethyl)imino]tris(hydroxymethy1)methane. See Bis-Tris 1,4Bis(4-sulfobutyl)piperazine.See PIBS

N,N'-Bis(3-sulfopropyl)ethylenediamine.

1 5

See EDPS 1,4-Bis(3-sulfopropy1)piperazine. See PIPPS Bis(1,I-triAuoroacetoxy)iodobenzene, treatment of protein, 600-601 Bis-Tris, pK values at 2S0, 27 1,3-Bis[tris(hydroxymethyl)methylamino] propane. See Bis-Tris Bis-Tris propane, pK values at 25", 27 Blenders, 22 BMP proteins, cloning of, 740-743 BNPS-skatole, protein cleavage, 610 Bombyx mori middle silk glands, 199 ovaries, 199 silk, 199 transcription of class I11 genes in, studies of, 200-202 Borate, pK values at 25". 28 Borate buffers, 26 Borax-NaOH buffer, stock solutions, 37 Boric acid-Borax buffer, stock solutions. 36 Boronate affinity gels, for purification of glycoproteins, 534-535 Bovine immunoglobulin G colloidal gold protein assay, standard curve for, 64 Coomassie Blue protein assay, standard curve for, 62 enhanced copper protein assay, standard curve for, 58 ultraviolet spectrum of, 5 1 Bovine serum albumin colloidal gold protein assay, standard curve for, 64 Coomassie Blue protein assav, standard curve for. 62 enhanced copper protein assay, standard curve for, 58 as protein standard, 66 ultraviolet absorbance, 55

857

ultraviolet spectrum of, 51 Bradford protein assay, 29, 520 Braun homogenizer, 161 Brij, spectral properties, 256 Brij 56, properties of, 247 Brij 36T, properties of, 247 Bromphenol Blue, 477 Buffer(s), 11-12, 24-38, 71, 196-197 broad-range, 30-3 1 for chromatofocusing, 382, 384-385 complexation with metals. 26 concentration, 26-28 for electroblotting, 681-682 for immunoadsorption procedures, 690691 neutralization, 67 PK,, 25 preparation of, 29 for protein cleavage, 620 for protein crystallization, 649-650 for protein precipitation, 290 for protein purification, 28-29 in protein solutions, 85 reactions with ninhydrin, 30 for sedimentation equilibrium, 573 selection, 26-29 for solubilization of membrane proteins, 257-258 stock solutions, 28-30 recipes for, 31-38 storage, 54 theory of, 24-26 volatile, 29-30 types of systems for use as, 30 Buffering capacity, 24 expression for, 25 Bulk protein, fractionation of by successive extraction with ammonium sulfate solutions of varying concentration, 294 using solid ammonium sulfate, 294-295 Butanol, for precipitation of proteins, 289

Cacodylate, pK values at 25", 27 Cacodylate buffer, stock solutions, 34 Calf prochymosin, fused to AS-I and AS-11, secretion in E. coli, 136-138 Calmodulin, silver staining, 486

858

SUBJECT INDEX -

Calmodulin-sensitive adenylate cyclase, from bovine cerebral cortex, purification, 501 Candida utilis multienzyme complex, isolation, 548 pentose phosphate pathway enzymes in, 790 transaldolase, 788-793 CAPS, pK values at 2S0, 28 Carbonate, pK values at 25', 27, 28 Carbonate-bicarbonate buffer, stock solutions, 37-38 Carbon-14, half-life of, 72 1 Carboxyl groups, in adsorption of proteins to HA, 330-332 Carboxylic acid buffers, 28 Carboxyl-terminal residues, amidation of, analysis, 627-629 Carboxymethyl. See CM Carboxymethyl cellulose, 81 Carboxypeptidase Y, 633 Carrot root, extract, 185 Cartilage proteoglycan core protein, cloning, 745-746 Catalase assay, 219-220 organelle specificity, 213 Cathodic drift, 466 Cation-exchange chromatography, 3 10, 404 Cations influence of, on retention and resolution of selected proteins in IEC, 408 and strength of hydrophobic interactions, 340 Cauliflower influorescence, extract, 185 cDNA clones, of eukaryotic genes, translation start region for, 103 cDNA library, screening, 738-739 C~EIO 252 , CloElo, 252 C I Z E ~252 . properties of, 247 C12E9, 252 properties of, 247 CIZEIO, 252 properties of, 247 C,,EI,, properties of, 247 Cell aflinity chromatography, 371-373 Cell disruption, effect on isolation of multienzyme complexes, 542

-

-

-

-

Cell fusion, for monoclonal antibody production, 675-676 Cell lysis methods, 195-196 Cells cytoplasmic fraction, preparation, 200202 nuclear extract, preparation, 200-202 Cell suspensions, homogenization, with glycerol, 196 Cellulose, 3 10-3 11 Centricon, 76 Centrifugation, for concentration and desalting of proteins, 83 Centrifuge, 22 Centriprep, 76-77 Cetyltrimethylammonium bromide, critical micelle concentration, 245-246 Chaotropes, 410, 413 added to solubilization buffers, 258 CHAPS, 252, 254, 506 compatibility with divalent cations, 256 effect on lectin affinity, 532 in isoelectric focusing, 466 properties of, 247 in solubilization of aggregating proteins, 258 spectral properties, 256 structure, 255 CHAPS0 compatibility with divalent cations, 256 in isoelectric focusing, 466 spectral properties, 256 Chelating agents, added to buffers, 197 Chelation affinity chromatography, 373376 Chemotaxis, 632 CHES, pK values at 25', 28 Chicken ovalbumin, ultraviolet absorbance, 55 Chinese hamster ovary cells cytoplasmic organelles, isolation of, 206-207 disrupted by low-pressure nitrogen cavitation, 204-206 lysosomes, isolation of, 209-210 mitochondria, isolation of, 208-209 nuclei, isolation of, 210-212 subcellular organelles, isolation of, 203

SUBJECT INDEX

Chloramine-T to label proteins for peptide mapping, 619 protein cleavage, 620 protein iodination method using, 723726 Chloramphenicol acetyltransferase fused to AS-I signal region, secreted from E. coii. 143 and human calcitonin, fusion between, 273 Chloroform/methanoVwater system, used to precipitate proteins, 78 Chloroplast(s), 176 chlorophyll contents, determination of, 188-189 crude, preparation of, 189 detergent lysis, 191 extracts, 186-191 removal of nuclei, 189-190 hypotonic lysis, 190-191 lysate, preparation of, 190-191 purification, 176-177 purified, preparation of, 190 Chloroplast enzymes, reducing agents, 179- 180 3-Cholamidopropyldimethylammonio- 1propane sulfonate. See CHAPS Cholate, 506 Cholic acid compatibility with divalent cations, 256 exchange or removal, 278 sodium salt. See Sodium cholate Chromatin, histone pairs from, purification of, on HA columns, 335, 337 Chromatofocusing, 380-392 application of sample to column, 388389 buffers, 382, 384-385 choice of, 386-387 optimizing, 383 column, 382-383 length of, effect on resolution, 386 packing, 388 optimizing, 383-386 in denaturing agents, 390-391 elution, 389 equipment, 381-383 experiment, 386-391 flow rate, 389

859

effect on resolution, 386 FPLC, 391, 392 gel choice of, 386-387 preparation of, 387-388 high-performance, 391-392 ion exchanger charge on, optimizing, 383 quanitity of, 387 ion-exchange resins, 381-382 mechanism of, 381 monitoring, 389 as one-step separation technique, 390 peristalic pump, 383 pH gradient, slope of, optimizing, 383 pH values, and detergent properties, 257 for purification of integral membrane proteins, 514 reagents, 38 1-382 recorder, 383 reference list, 392 regeneration, 389 resolution of, 391 factors influencing, 383-386 sample preparation, 388 separation of polybuffer or ampholytes from protein, 389-390 UV monitor, 383 Chromatography. See also specific technique to detect impurities in protein sample, 556 for detergent removal, 278, 279 fractionation modes, 392 gels and resins, 23 on immobilized reactive dyes, 343-357 matrices, chemical stability of, 274 stagnant mass-transfer limitations, 393 Chymotrypsin IEC, 407-408 protein cleavage, 612 self-associated, effects on solubility, 304 a-Chymotrypsin, protein cleavage, 620 Chymotrypsinogen, self-associated, effects on solubility, 304 Cibacron dyes, 350-352 Cibacron F3GA, 356 Citrate, pK values at 25", 27 Citrate buffer, stock solutions, 31-32

860

SUBJEC:T INDEX

Citrate-phosphate buffer, stock solutions, 32-33 Click test, 650 Cloning techniques, 738 using one long and one short oligonucleotide, 740-743 using two long oligonucleotides, 744-746 using two short oligonucleotides, 743744 Cloud point, 244-250, 253, 257 CM, 310 CM-BioGel A, 382 CMC, as indicator of strength of detergent binding to proteins, 280 CmRplasmids, 100 Collagen lysine-derived cross-links, 644 methylation of glutamate and aspartate residues, analytical approach to, 633-634 precipitation of, 290 Collagenase, purified by MCAC, 376 Collidine-acetic acid, 30 Colloidal gold protein assay, 63-65 concentration limits of proteins in, 52-53 standard curves for, 64 Colorimetric assays, 57-65 interfering reagent, testing for, 57 microwell plate reader, 68 Color reactions, 10-1 1 Column chromatography accessories to, 23 columns, 22-23 development of, 286-287 Column void volume, 400 Computer analysis, of protein structure, 751-776 Computer programs ALIGN, 759 for aligning amino acid sequences using sequence fragments, 757, 763 BESTFIT, 758, 759, 764 Lipman-Pearson FASTA, 754-755, 776 MAC GENE, 776 MATCH, 761 PC GENE, 776 QUEST, 761 SCAN, 761 sources of, 775 STADEN, 776

UWGCG, 758-759, 776 Concanavalin A, 372, 51 1-512 in glycoprotein purification, 530 Conductivity meter, 21 Coomassie Blue (Bradford) protein assay, 62-63 concentration limits of chemicals in, 5253 cuvettes for, 65 standard curves for, 62 Coomassie Blue G-250, 478-479 Coomassie Blue R-250, 478 protein quanritation using, 487-488 Coomassie Blue staining, 478-479 binding properties, 479-480 dye-dye interactions, 480 general method, 479 for identifying proteins for elution from gels, 491 intensity, 480 and lysine, arginine, and histidine residues, 480 rapid method for isoelectric focusing gels, 479 Coomassie Brilliant Blue R-250, staining in SDS-PAGE, 437-438 Coomassie Violet R-150, 479 Copper staining, in SDS-PAGE, 438-439, 44 1 Covalent chromatography, 376-377 Critical micelle concentration, 244-253 and detergent dialysis, 279-281 of detergents, 506 Critical micelle temperature, 244-245 Crude extract, fractionation of, using solid ammonium sulfate, 294 Crystallography, 646 C12sorbitan Ezo,properties of, 247 CI8:,sorbitan Ezo,properties of, 247 Cuvette(s), 21, 65-66 enzyme assay in, 39-40 Cyanogen bromide, protein cleavage, 610611, 620 Cyclohexylaminoethanesulfonic acid. See CHES 3-(Cyclohexy1)aminopropanesulfonic acid. See CAPS Cysteine determination in proteins, 598-599 performic acid oxidation, 598

SUBJECT INDEX

reduction and alkylation, 598-599 Cysteine (thiol) protease inhibitors, 181 Cystine, performic acid oxidation, 598 Cytochrome P-450, rabbit liver, 234 Cytochrome b5, rabbit liver N-terminal sequence, 234 subunit molecular weight, 234 Cytochrome c heme binding to, 644 IEC, 407-408 Cytochrome oxidase, 229 Cytochrome-c oxidase assay, 214-215 organelle specificity, 2 13 Cytochrome P-450 bound, elution of, 282 eluted from transblots, amino acid analysis, 597 from mouse liver microsomes, solubilization of, 261-262 Cytoplasmic organelles electron microscopy of, 223-225 isolation, preparation of cell homogenates for, 204-206

Databases of amino acid sequences, 775 of nucleotide sequences, 775 of protein structural analyses, 775 of three-dimensional protein structures, 775 Dayhoff mutation matrix, 753-754, 756, 759, 762 DEAE, 310 DEAE-BioGel A, 382 DEAE-cellulose, 311 DEAE-Sephacel, stability of, 274 DEAE-Sephacel ion-exchange chromatography, of Ig from serum, 669-670 DEAE-Toyopearl 650 M, 382 Decanoyl-N-methylglucamide. See MEGA10 3-Decyldimethylammoniopropane I-sulfonate, properties of, 247 n-Decyl-0-D-glucopyranoside,properties of, 247

861

Decylglucoside, 252 Decylmaltoside, 252 Denaturation definition of, 254 during iodination, evaluation of, 736-737 Density gradient centrifugation, for purification of glycoproteins, 535 DeoxyBigCHAP, 252 properties of, 248 Deoxycholate, 506 Deoxycholic acid compatibility with divalent cations, 256 sodium salt. See Sodium deoxycholate Derivative absorption spectroscopy, 399 Desalting. See Protein solution, desalting Detergent(s), 239-253 abbreviations, 247-250 aggregation, principles of, 240-244 anionic, 252, 506 behavior, parameters, 244-250 carboxylic acid-containing, 256 cationic, 252, 506 choosing, 25 1-253 chromatofocusing with, 390 compatibility with divalent cations, 256 concentrations for solubilization, 263 containing stronger acids, and pH, 256 containing weak acids, and pH, 256 effect on functional activity of membrane proteins, 516-517 effect on lectin affinity, 51 1, 531-532 effect on Lowry protein assay, 520 effect on micelle formation, 506 electrical properties, 257 exchange, 277-279 procedures, 279-280 protocol, 281-282 head group, 251-252 hydrogenated, 54 and IEC, 314-316 and immunoadsorption procedures, 688689 ionic, 254, 506 in isoelectric focusing, 466-467 micellar structure adopted by, 241-242 packing parameter, 242-243 monometer, packing parameter, 242-243 nondenaturing properties of, 254-255 nonionic, 252, 254, 257, 506 removal of, 280

862

SUBJECT INDEX

phase separation of membrane proteins with, 515 properties, 246-249, 253 affecting ease of removal, 280-281 temperature effects on, 257 in protein purification, 239 uses of, 241 in protein solutions, 86 removal in analytical procedures, 278-279 chromatographic techniques, 279 by dialysis, 279-280 from membrane proteins, 277-284 by precipitation, 280 in preparative procedures, 277-278 from protein fractions, 506 for reconstitution of integral membrane proteins, 516 typical protocol, 28 1-282 selection, for solubilization of membrane proteins, 254-257 solubility pH dependence of, 256-257 in precipitation medium, 280 for solubilizing integral membrane proteins, 506 spectral properties, 256 stock solutions, preparation of, 263 structure, 239-240, 242-243, 255 cmc and, 246-250 synonyms for, 247-250 tail group, 25 1-252 trade names, 247-250 zwitterionic, 252, 254, 257, 316, 506 Detergent trials, 251 Dextrans, 310 Diacrylylpiperazine and elution of proteins from gels, 490, 494-495 source, 489 N,N1-Diallyltartardiamide,gel cross-linking with, 441 Dialysis, 67-70, 72-75 buffer changes, 72 cross-contamination in, 75 detergent removal by, 279-280 methods, 73-75 molecular weight cutoff, 72-73 optimum protein recovery, 72

for removal of low-molecular-weight solutes, 72 time, 72,74-75 Dialysis halve cells, 74 Dialysis membranes, 73 impurities, removal, 73 pretreatment, 73 semipermeable, 74-75 Dialysis tubing, 73 use of, 73-74 Diazoacetylnorleucine methyl ester, 181 Diethylaminoethyl. See DEAE Digitonin, 252, 506 effect on lectin affinity, 532 precautions with, 532 properties of, 248 solubility, 532 source of, 532 structure, 255 Dihydrophycocyanobilin, binding to proteins, 644 N,N'-Dihydroxyethylenebisacrylamide,gel cross-linking with, 441 Diisopropyl fluorophosphate, as protease inhibitor, 199 Dilute acid, protein cleavage, 610 Dimethylarsinic acid. See Cacodylate Dimethylglutarate, pK values at 25", 27 Dipolar interactions, 287-288 DIPSO, pK values at 25'. 27 Dithiothreitol added to buffers, 197 to prevent oxidation, 504 in protein solutions, 85 as reducing agent for plant enzymes, 179-180 DMSO, chromatofocusing with, 390 DNA genetically active, synthesis of, 786-787 as primer for replication, 783-784 silver staining, 486 synthesis, without template, 786 as template for replication, 783-784 DNA affinity resin, construction, 522-529 DNA-binding proteins, purification, by site-specific DNA affinity chromatography, 521-529 DNA ligase, 786-787 DNA polymerase, 783-788

863

SUBJECT INDEX

fragments, 785-786 activity of, 786 inability to start chain, 787 nuclease activity in preparations of, 784-785 proofreading ability of, 785, 787 purification, problems in, 784 replicative role of, questioning of, 787788 synthesis of DNA, without template, 786 synthesis of genetically active DNA, 786-787 DNA polymerase 11, 788 DNA polymerase 111, 788 I-Dodecanesulfonic acid, sodium salt, properties of, 248 N-Dodecanoylsarcosmate, sodium salt, properties of, 248 Dodecyldimethylamine oxide properties of, 248 in protein purification, uses of, 241 3-Dodecyldimethylammoniopropane 1sulfonate, properties of, 248 N-Dodecyl-N,N-dimethylglycine. See Empigen BB Dodecyl ether, properties of, 247 Dodecylglucoside, 252 Dodecylmaltoside, 252 n-Dodecyl-p-D-rnaltoside, properties of, 248 Dodecyl sulfate, sodium salt. See Sodium dodecyylauryl sulfate Dodecyltrimethylammonium bromide. See DTAB Dolichos bioJorus lectin, purified by MCAC, 376 Dot-blot assays for antibody detection, advantages and disadvantages of, 668 for screening monoclonal antibodies, 67 1 Double-antibody imrnunoassays, 708-709, 712 Double-antibody immunoprecipitation, 708-709, 712 Dro.~ophila,embryos, 195 DTAB, 252 properties of, 248

Dyno-Mill, 151-152 method for preparation of yeast extracts, 160-161

E Eaton press, 160 Edman degradation cycle, 602-603 EDPS, pK values at 25', 27, 28 EDTA added to buffers, 197 concentration used, 88 neutralization, 67 as protease inhibitor, 88, 170, 259, 503 in protein solutions, 85 EGTA, 182 concentration used, 88 as protease inhibitor, 88, 170, 259, 503 Elastin, lysine-derived cross-links, 644 Electroblotting, 680-683 for obtaining N-terminal protein sequence, 604 procedure, 682-683 protein purification by, 604-607 of proteins purified by SDS-PAGE, for peptide mapping, 614-615 selection of transfer membrane, 680-681 transfer buffer, 681-682 transfer equipment, 681 Electroelution protein assay after, 493 of proteins from gels, 488, 492-494 Electrofocusing, detergent in, 241 Electrolysis, protein iodination using, 732 Electron microscopy of organelle preparations, 223-225 of proteins, 585 Electrophoresis, 15, 368, 380. See also Gel electrophoresis; Polyacrylamide gel electrophoresis; SDS-PAGE; Twodimensional gel electrophoresis apparatus, 82 to assess protein purity, 556-562 procedure, 558-561 sample preparation, 558-559 for concentration of proteins, 82-83 detergent in, 241 gels elution of proteins from, 489-490

864

SUBJECT INDEX

gradient to assess protein purity, 559 preparation, 559-560 for molecular weight determination, 581-582 power supply unit, 23 protein purification by, 604-605 protein size analysis, 581-584 analysis of data, 583 method, 582-583 problems and pitfalls, 583-584 for protein subunit determination, 585586 techniques, 69 Electrophoretic elution, of proteins from gel slices, 441, 489-490 Electrophoretic mobility, 460 Electrotransfer, of gel proteins to nitrocellulose or PVDF matrices, 489, 491492 ELISA. See Enzyme-linked immunosorbent assay Elongation factors, 809-818 discovery of, 809-814 function of, 815-818 purification of, 815 resolution of, 814-8 15 Eluents, for chromatofocusing, 382, 384385 Elution of proteins after immunoaffinity chromatography, 512 from gels, 441, 488-495 chemicals for, 489-490 diffusion method, 488 efficiency, 488 electroelution, 488, 492-494 equipment, 489-490, 495 materials, 489-490 yield, 493 from HPLC columns, 608-609 Elutrap, 492-493, 495 EMBL Nucleotide Sequence Data Library, source, 775 Empigen BB, 252 properties of, 248 Empigen OB, properties of, 248 EMTA, pK values at 25', 27 3,6-Endomethylene-l,2,3,6-tetrahydrophthalic acid. See EMTA

Endoproteinase Arg-C, protein cleavage, 612 Asp-N, protein cleavage, 6 12 Lys-C, protein cleavage, 612 Endosomal compartments, labeling of, 216-217 Enhanced alkaline copper (Lowry) protein assay, 29, 57-62, 66, 520 amplification technique, 60 concentration limits of chemicals in, 5253 standard curves, 58 Enzymatic lysis, 147-151 detergent in, 151 of gram-negative bacteria, 148-15 1 of gram-positive bacteria, 147-148 spheroplast formation during, 151 Enzyme(s) contaminants, 2 contributions to cellular economy, 3-4 extraction procedure general considerations, 194-198 optimum, 194 starting material, 194-195 freezing, 151 heat stability, 298 homogeneity, 2 importance to operations of cell, 2 pH optimum, determining, 26 purification buffer components, 196-197 heat and pH denaturative precipitation as adjunctive methods in, 298-299 rationale for, 1-5 specific activity, 42-43 stability evaluation of, 197-198 to perturbations in pH, 298 thermal, 298 effect of various agents on, 197198 stabilization, 196 Enzyme activity components, 38-39 definitions of units, 42-43 latency, 207 measurement of, 38-49 concepts in, 38-43 and rate of reaction, 42 units. 42

SUBJECT INDEX

Enzyme assay amount of enzyme to be added to reaction mixture, 46-47 conditions, optimization, 46-47 continuous, 40, 44-45 coupled, 40, 44-45 design of analysis of primary reaction, 44 analysis of secondary reactions, 44 selection of method of analysis, 44-46 strategy for, 43-49 detection step, 40 discontinuous, 40-42, 44-45 time between samplings and volume of samples, 46-47 initiationlincubation, 40 methods, classification of, 40-42 obtaining initial rate data, 47-48 for previously undescribed activity, developing, 49 quantitative analysis, 43, 48-49 rate of product formation, determination of, 46-47 reaction mixture, 39-40 reduction of data, 40 secondary reactions in, understanding and dealing with, 48 sensitivity, increasing, 47-48 separation step, 40, 41 substrate concentration, 46 range of, 49 termination step, 40 techniques, 45-46 time course of reaction, 46-47 Enzyme cascades, cyclic, theoretical analysis of, 805 Enzyme-conjugated antibody, in immunodetection of proteins, 686-687 Enzyme immunoassay antigen (or antibody) marker, 705, 721 detergent in, 241 Enzyme-linked immunoassay for antibody detection, advantages and disadvantages of, 668 for screening monoclonal antibodies, 67 1-673 Enzyme-linked immunosorbent assay, 712713 Enzyme regulation, steady-state concept of, 803-805

Enzymology classic approach to, 1-2 neoclassical approach, 1-2 Epidermis, precipitation, 298 Epitopes, 663 Epoxide hydrolase eluted from transblots, amino acid analysis, 597 rabbit liver, 234 EPPS, 29 pK values at 25O, 27 Erythrocyte membrane, preparation, 503 Erythrocyte membrane proteins, staining, 485 Escherichia coli adenylyltransferase, 797-798 aminoacyl-tRNA synthetase multienzyme complex, isolation, 548 biased codon usage, 105 clp mutant, 106 culture, 124 degP mutant, 106-107 digestion of cell wall, 149 DNA replication in, DNA polymerases in, 783-788 gene expression in, 93 gentle enzymatic lysis method for, 149150 hemolysin (Hly) secretory process, 132134 secretion of calf prochymosin using AS-I and AS-I1 signal peptides, 136-138 secretion of OmpF-AS-I fusion, 136 use of, 136-141 htpR mutant, 106 initiation of mRNA translation in, 103 liquid shear lysis, 153 Iky mutants, for protein secretion, 109 Ion mutant, 106 lysate, nucleic acid removal from, 153 lysing, in Dyno-Mill Model KDL, starting conditions for, 152 maximizing gene expression in, practice, 111-112 mutants defective in proteases, 105-107 permeabilizing effect of Tris, 148 promoter sequence, 94 protease-defective mutant, method for testing, 107

866

SUBJECT INDEX

protein inclusion bodies in, 265 protein overexpression, 93-1 14, 264-265 recovering proteins from aggregates in, 269-276 secretion, purification, and dimerization of variable domains of antibody McPC603 using hemolysin A signal, 138-141 secretion of cloned gene product, 108109 secretion of fusion proteins, general procedures for, 134-136 secretion of heterologous proteins in, 132-143 future perspectives, 143 optimization of, 141-142 TCA cycle multienzyme complex, isolation. 549 vigorous lysis method for, 150-151 Esterase, rabbit liver N-terminal sequence, 234 subunit molecular weight, 234 Ethanol for precipitation of protein, 78, 289, 296 in protein crytallization, 652 Ethanolamine, pK values at 25", 28 Ethanolamine-HC1, 30 Ethylene glycol, chromatofocusing with, 390 N-Ethylmaleimide, 181 as protease inhibitor, 504 Eukaryotes overexpression of proteins in, 112-132 preparation of extracts from, 194-195 experimental procedures, 198-202 Eukaryotic polypeptides, in inclusion bodies, solvents necessary to solubilize, 266 Evaporation, for protein sample concentration, 518-519 Expression systems bacterial, 264 in eukaryotic cells, development of, 113 Extracts, preparing, 13

Fasciclin 1, 517-5 18 FASTA programs, 754-755, 776 Fast Green, 477 dye-dye interactions, 480

staining intensity, 480 Fast nucleic acid liquid chromatography. See High-performance liquid chromatography Fast protein liquid chromatography, 391392. See also High-performance liquid chromatography Ferguson analysis, of electrophoretic data, 583-584 Fibrinogen human, purified by MCAC, 376 precipitation of, 290, 298 Fibronectin, plasma, and denatured collagen, functional interaction between, 305 Filtration, for protein sample concentration, 518-519 Flat tube microdialyzer, 74 Flavonoids, 175 Fluorescamine, 481 Fluorescent staining effect on protein charge, 478 general method, 481 preelectrophoretic, 480-48 1 sensitivity, 48 1 stains for, 478 Folin-Ciocalteau reagent, 182 Formamide, chromatofocusing with, 390 Formate, pK values at 25". 27 Formic acid, protein cleavage, 620 Fraction collector, 23 Freezing and thawing procedures, 87 French press, 152-153, 160 recovery of protein inclusion bodies from pelleted bacterial cells with, 268 Frictional coefficient, 574 Ns-Fructose-lysine, 645-646 Fructose-lysine residue, formation, 646 Fusion protein, purification strategy, 274276

P-Galactosidase affinity matrix for, 110-1 11 assay, 215-216 organelle specificity, 213 rat liver, assay, 229 j3-Galactosidase fusion proteins, rapid purification of, 110-1 11

SUBJECT INDEX

0-Galactosidase fusion vectors, 109-110 Galactosyl transferase, distribution of, in rat liver homogenate, 231 Gardol, properties of, 248 Gaulin homogenizer. 160 Gelatin standard curve for for colloidal gold protein assay, 64 for Coomassie Blue protein assay, 62 for enhanced copper protein assay, 58 ultraviolet spectrum of, 51 Gel chromatography, for molecular weight determination, 577 Gel electrophoresis. See also Polyacrylamide gel electrophoresis; SDSPAGE; Two-dimensional gel electrophoresis to assess protein purity, 556-562 pitfalls of, 561-562 equipment. 23 gels, 427 denaturing, and assessment of protein purity, 558, 561-562 nondenaturing, and assessment of protein purity, 558, 561-562 high-resolution, gels, elution of protein from, 489 for molecular weight determination, 577 one-dimensional, 425-441 gels, protein elution from, 490-491 principle of, 426-427 slab gels, 427 Gel filtration (chromatography), 15, 69, 70, 79-80, 317-328. See also Size-exclusion chromatography to assess protein purity, 562-563 BioGel supports, 79 column, 3 17 channeling, 58 1 elution volume, 580 void volume, 580 for desalting protein solutions, 79 for detergent removal, 279 matrix, stability of, 274 media, 79 of membrane proteins, 499-502 method, 579-580 for molecular weight determination. 562-563, 579-581

867

principle of, 317-3 18 problems and pitfalls, 581 for purification of integral membrane proteins, 508-510 detergent mixtures for, 509 detergentlprotein ratio for, 509 resolution, 508 yield, 508 to remove polybuffer and ampholytes from protein, 390 Sephadex media, 79 standards, 323, 581 Gel-permeation chromatography. See also Size-exclusion chromatography to assess protein purity, 556, 562-563 for determination of subunit stoichiometry, 587 for diagnosing interacting systems, 587 Gel-staining techniques. 477-488 Gen Bank Genetic Sequence Data Bank, address, 775 Gene amplification, 97, 746-748 Gene expression biased codon usage, potential problem with, 105 in E. coli, 93 maximizing, 93 premature termination and mRNA instability, solutions to potential problems with, 101-102 Gene fusions. to facilitate purification, 275 Gene overexpression, assessing degree of, 98-99 Gene product, cloned localization of, 107 stabilization by specific intracellular localization or secretion, 107-109 Acll Gene product, and P-globin, fusion protein, cleavage of, 273 Genomic library plating, with amplification, 746-748 screening, 746-75 1 Glacial acetic acid, 87 ml, + 25 ml 88% HCOOH in 1 1 liters, 30 Acll-P-Globin fusion protein, solubilization, 274-275 Globulins, 286 Glucoamylase, from A. awamori, purification of, 3 1 1

868

SUBJECT INDEX

Glucose-6-phosphatase, rat liver assay, 229 distribution of, in homogenate, 231 y-Glucosidase, purification of, from yeast extract, 303 P-Glucuronidase, in preparation of yeast extracts, 161-162 Glusulase, in preparation of yeast extracts, 161-162 Glutamate dehydrogenase, 793 bovine liver, precipitated by PEG, 304 Glutamate-ammonia ligase. See Glutamine synthetase Glutamate residues y-carboxylation of, 634-635 carboxyl methylation of, 632-634 Ne-(y-G1utamic)lysinecross-link, 636-637 Glutamine synthetase activity, regulation of, 793-794 adenylylation of, 795-797 and divalent cation specificity, 797 and pH activity profile, 797 deadenylylation of, 799 feedback inhibition, 794-797 formation, role of UTase and PI, in regulation of, 807-809 inactivation of, by adenylyltransferase, 798 regulation, 801-803 steady-state concept of, 803-805 Glutamine synthetase cascade ATase in, 803-804, 806 bicyclic system, 801-803 discovery of, 793-809 molecular biology approach to, 806-807 regulation, 807-809 UTase in, 803-807 verification of theoretical predictions, 805-806 Glycation, nonenzymatic, 645-646 Glycerol added to buffers, 197 cell lysis with, 196 interference with protein crystallization, 649 in protein solutions, 86 in solubilization buffers, 258-259 Glycerol density gradients, for purification of integral membrane proteins, 514515

Glycinamide, pK values at 25", 28 Glycine, pK values at 25", 28 Glycine-HC1 buffer, stock solutions, 31 Glycine max, cv. Williams-82, 177 Glycine-NaOH buffer, stock solutions, 37 Glycohydrolases, 638 Glycoinositol phospholipid membrane anchors, analysis, 626-627, 629-632 Glycoprotein(s) deglycosylation of, 537-539 detection of, 535-539 lectin techniques, 537 lectin affinity chromatography, 530534 expectations, 533-534 lectin selection for, 532-533 scale of experiment, 533 PAS staining of, protocol for, 536 purification, 529-539 by antibody affinity chromatography, 534 on boronate affinity gels, 534-535 density gradient methods, 535 multistep procedure, 533-534 purified, Asn-linked, deglycosylation, 538-539 silver staining, 485 staining, 478 thymol-sulfuric acid staining, 536-537 Glycosylation, 529, 627 analysis of, 643 Glycylglycine, pK values at 2s0, 28 Goats, polyclonal antibody production using, 666 Good buffers, 26, 28, 29, 30 Gradient makers, 23 Gram-negative bacteria digestion of cell wall, 149 enzymatic lysis of, 148-151 addition of freeze-thaw step, 150 outer membrane, permeabilizing, 148149 Gram-positive bacteria, enzymatic lysis of, 147-148 Growth hormone receptor, from rabbit liver, purification, 500 Guanidine, in solubilization of aggregating proteins, 258 Guanidine-HC1, preparation of, 267

869

SUBJECT INDEX -

Guinea pigs, polyclonal antibody production using, 666

Hanging drop method, 653-654, 656 HAT selection, for cell fusion, 675 HCOOH, 25 ml88%, in 1 liter, 30 Heat to precipitate unwanted proteins, 299 termination of enzymatic reactions with, 45-46 Heat denaturation, 288, 299 use of, 299-300 Helical wheel, in secondary structure prediction, 767-768 Hemolysin, secretion from E. coli, 132134 Henderson-Hasselbalch equation, 24-25 HEPES, 29 buffer capacity, 25 pK values at 25", 27 HEPPS, pK values at 2S0, 27 Heptafluorobutyric acid, in RPC, 415 n-Heptyl-P-D-thioglucoside,properties of, 248 Herpesviruses, as transient expression vector, 117 Hexadeiyldimethylammoniopropane Isulfonate, properties of, 248 Hexadecylpyridinium chloride, properties of, 248 Hexadecyltrimethylammonium bromide, properties of, 249 fl-Hexosaminidase assay, 215-216 rat liver, assay, 229 High-performance chromatography, with reactive dyes, 355-356 High-performance liquid chromatography, 14, 15, 69, 309, 391-393 chromatographic modes, 399-421 diode array detector, 398 gradient formation, 396 instrumentation, 394-399 mixers, 395, 398 for obtaining N-terminal protein sequence, 603-604 for protein purificationlpeptide mapping, 625-626

-

pumping pressure, 395 pumping rate, 394 pumping system, 394-396 automated switching valves, 396 pulse dampers, 397-398 selecting, 396 summary of, 397 wettable components of, 394 for purification of proteins and peptides, 607-609 vs. soft-gel systems, 393 solvent delivery, 395 solvent mixing, 395-396 solvents, 396-397 ultraviolet-visible absorbance detectors, 398 High-speed chromatography. See Highperformance liquid chromatography Hoffmeister effect, 410 Homogenizers, 22 cell lysis in, 152-153 Horseradish peroxidase assay, 216-217 interaction with hydroxylated columns, 33 1 organelle specificity, 213 Horseradish peroxidase-conjugated second antibodies, immunodetection of proteins with, 686-687 HPPS, as iodine carrier, 732-734 Hybridoma technology, 675-676 Hybrid protein, cleavage of, in vitro, 271273 Hybrid protein aggregates, solubilization of, 271-273 Hydrophile-lipophile balance number, 244-246, 253 Hydrophobic (interaction) chromatography, 339-343, 392, 409-414 application, 413-414 binding, 342 choice of gel, 341 column regeneration and storage, 343 elution, 342-343 gradient elution times, 413 matrix, stability of, 274 mobile phase, 412 mobile-phase pH, 412-413 principle of, 410 procedures for, 341-343

870

SUBJECT INDEX

to remove polybuffer and ampholytes from protein, 390 vs. reversed-phase chromatography, 340-341 running column, 342-343 selectivity in, 41 1 stationary phase, 411-412 supports, 409-410 mechanism of action, 410 Hydrophobic interactions, 287-288, 339340 Hydrophobicity definition of, 339-340 in protein purification, 343 Hydrophobic media, for removal of detergents, 278 Hydroxyapatite adsorption of proteins to, 329-332 commercial, chromatographic behavior of, 338-339 elution of proteins from, 332-333 Hydroxyapatite chromatography, 329-339 columns elution procedures, 333-339 guidelines for use of, 333-339 loading, 333-334 washing, 334 protein-HA interaction in, 329-332 for purification of integral membrane proteins, 514

capacity for total protein and desired protein in crude extract, 346-347 concentration of, and interaction of retained protein with immobilized dyes, 354 kits, 345-346 matrices for, 353 optimal conditions for elution of desired protein from, 347 regeneration, 349-350 screening, 345-346 sources, 346 storage of, 349-350 Immobilized dye chromatography, 343-357 advantages of, 344-345 alternative procedures to retain additional desired proteins, 354-355 laboratory scale, 347-348 matrix, solid fluorocarbons, 357 principles, 344 representative purification procedures, 348-350 Immobilized dye columns, preparation of, 350-354 Immobilized dye-protein interaction, 355, 357 Immobilized metal affinity chromatography, 373-376, 392, 417-421 advantages of, 418 application, 420-421 N-2-Hydroxyethylpiperazine-N'-ethanechoice of metal, 420 sulfonic acid. See HEPES elution of biomolecules from, 374-375 N-2-Hydroxyethylpiperazine-N'-3-propane- methodology of, 374 sulfonic acid. See EPPS; HEPPS mobile phase, 419-420 Hydroxylamine, protein cleavage, 610 nonspecific electrostatic interactions in, p-Hydroxyphenylacetaldehyde. See PHAA 421 3-(p-Hydroxyphenyl)propionic acid-Nnonspecific hydrophobic interactions in, hydroxysuccinimide. See HPPS 421 pH, 420-421 and pH, 374-375 for separating serum proteins, 375-376 IEC. See Ion-exchange chromatography stationary phase, 418-419 IMAC. See Immobilized metal affinity Immobilon-P, 605,606 chromatography Immune response, enhancement of, 664Imidazole, pK values at 25",27 666 Iminodiacetic acid, for IMAC applications, Immunoadsorption 373-374 of radiolabeled antigens Immobilized dye(s) flow chart for, 693 amount of, determination of, 353 set-up for, 692-693

SUBJECT INDEX

radiolabeled cell extracts for, preparation of, 689-690 Staph A procedure, 688-689 final Staph A pellets electrophoretic analysis, 696 storage, 696 final washes, 695-696 formation of antigen-antibody-Staph A complexes, 695 nonspecific binding of radiolabeled materials to Staph A elimination, by SDS solubilization and readsorption, 697-700 reduction of, 6%-697 preadsorption of radiolabeled samples with washed Staph A, 694-695 preadsorption of Staph A and radiolabeled cell extracts, 694 preadsorption of Staph A with unlabeled cell extracts, 694 stock solutions and buffers for, 690-692 Immunoaffinity chromatography, of membrane proteins, 499-502, 512 elution buffers for, 512 Immunoassays, 700-718 addition of radioactive antigen. delay of, 707-708 additives, 707 antigen (or antibody) marker, 705 antiserum for, 702-704 applications, 701 controls, 713 data analysis, 714 immunologic cross-reactivity in, 715716 reduction of, 7 17-718 incubation conditions, 706-708 incubation time, 707 interference with, 714-715 methods, 700-701 monoclonal antibodies for, 703 nonspecific binding in, 707 polyclonal antibodies for, 703 repeatability, 714 sensitivity of, 701, 703 effect of specificity of indicator molecule, 721 separation systems, 708-7 11 solid-phase, 709

87 1

of human IgA with polyacrylarnideantibody beads, 709-71 1 standard curve, 713 theory of, 700-701 tissue or serum sample for, 705-706 using charcoal method, 709, 711-712 validation of, 714-717 variation, interassay, 714 Immunobeads, immunoassay with, 709711 Immunoblotting, 443 artifacts, 687-688 procedure, 684-687 for screening monoclonal antibodies, 673-674 supplies and reagents, 683-684 Immunodetection, of proteins, 683-687 Immunoelectrophoresis, for antibody detection, advantages and disadvantages of, 668 Immunoenzyme analysis. See Enzymelinked immunosorbent assay Immunogen definition of, 663 purification, 663-665 Immunoglobulin fractionation from serum, 669-670 recognized by protein A and recombinant protein G, 684-685 Immunoglobulin G as protein standard, 66 purification, for immunoassays, 704 ultraviolet absorbance, 55 Immunoprecipitation. See also Doubleantibody immunoprecipitation of proteins, 688-700 Immunostaining, of membrane-bound proteins, 491 Inclusion bodies in baculovirus system, 119 centrifugation, 268 contaminating proteins, 268 in eukaryotic overexpression system, 114 in prokaryotic environment, 114 protein in contaminating nucleic acids, 266-267 purification, 266-267 solubilization of, 265

872

SUBJECT INDEX

washing, 268-269 Initiator codon, position of, preferred environment for, 103 Inorganic buffers, 26 Insect cell, posttranslational modifications of proteins in, 118 Insect cell-baculovirus system, 117-132 baculovirus recombinant plasmid, 120121 confirmation of production of biologically active protein, 131-132 control baculovirus recombinant plasmid, 118-119, 121 details for using, 119-132 equipment, 124- 125 gene product specific antibody to, 124 tests for enzymatic or biological activity of, 124 insect cells for, 12 1-122 plasmids and recombinant DNA technology, gene of interest, 119-120 reagents necessary for construction and use of recombinant baculovirus, 119-121 recombinant virus constructing and recognizing, steps in procedure for, 125-126 detection of protein encoded by, 128132 initial detection methods, 128-130 isolate containing certain sequences, 128 plaque purification of, 127-128 production of full-length peptide, confirmation of, 130- 131 recombinant virus clones isolation of, 126-128 recognition by plaque morphology, 126 selection by genotype, 126-127 selection of sufficient numbers of, 126 specific DNA probe for foreign gene sequence, 124 transfection initial recovery after, 125-126 steps for, 125-126 technology for, 122 transfection efficiency, importance of, 125

virus, 123-124 Insect tissues, labile aminoacyl-tRNA synthetase complex from, 199 IntelliGenetics address, 775 computer program. See QUEST (computer program) Interferons, purified by IMAC, 375-376 Interleukin 2 in E. coli, purified in solubilized, denatured form, 275 in inclusion bodies refolding, 270-27 1 solubilization, 270-27 1 Internal volume, 400 Iodination, of proteins, 617-618, 705, 721722 denaturation during, evaluation of, 736737 direct procedures, 72 1-732 effect on activity, 736-737 effect on immunoactivity, 722-723 level of, evaluation of, 736 procedures, 721-722 removal of uncoupled IZSI,735-736 safety precautions for, 722 storage of iodinated protein, 736 two-stage procedures, 722, 732-734 Iodine monochloride, protein iodination method using, 729-73 1 Iodine-125 half-life of, 721 iodination procedures using, 705, 72 1722 Iodine-13 1 half-life of, 721 iodination procedures using, 721 Iodoacetate, 181 Iodobeads, protein iodination using, 731732 Iodogen labeling of proteins for peptide mapping, 617-619 protein iodination method using, 731 Ion-exchange chromatography, 69, 70, 278, 309-317, 380, 392, 403-409 applications, 407-409 batch vs. open column, 312-313 bed capacity, 3 12 buffer, 315

SUBJEC:T INDEX

column proteins adhering to, 309 size of, 312 column packing, 309 changing displayable counterion, 31 1312 counterions, 310-31 1, 3 15 preparation, 3 11 swelling, 3 11 washing, 31 1 for concentration of proteins, 314 counterions, 310-31 1, 315 to detect impurities in protein sample, 556 displacement salt, 315, 404 elution gradients, 3 15-316 elution in, 404 elution solution, composition of, 314315 graphical presentation of data, 316-317 matrix, stability of, 274 of membrane proteins, 499-502 mobile phase, 404-407 open-column, 309, 313 operation of, 309, 3 13-3 17 packing material, selectivity series, 311312 pH retention maps, 406, 409 pH values, and detergent properties, 257 preparation and loading of sample, 3 13314 principles of, 310 for purification of integral membrane proteins, 5 13 for purification of proteins, 309 retention and resolution of proteins, influence of various anions and cations on, 407-408 salt concentration, monitoring, 315-317 stationary phase, 405 lon-exchange resins, 14-15 Ion exchangers, types of, 310-31 1 Ionic interactions, in protein, 287 Isoelectric focusing, 15, 23, 459-477 additives, 466-467 agarose gels for, 462-463 apparatus, 463 applications of, 460 APS-TEMED system, 462

873

artifacts, 474-475 to assess protein purity, 556 pitfalls, 562 procedure, 561 carrier ampholytes, 464-465 detection of protein bands, 472-473 detergent exchange or removal, 278 detergents for, 257 determining pH gradients, 473-474 by electrophoresis, 380 experimental procedures, 467-475 focusing, 470-472 format of, 461-462 gels casting, 463-464, 468-469 elution of proteins from, 489-490 granulated, 475 preservation of, 473 gradient instability, 466 by ion-exchange chromatography, 380381 materials, 444-445 method, 445-446 microheterogeneity in, 460, 474 nonequilibrium techniques, 446 pH values, and detergent properties, 257 polyacrylamide gels, 462 preparative, 462, 475-477 principle of, 460-461 protein staining, 472-473 protein standards, 474 for purification of integral membrane proteins, 514 quick staining in, 473 rapid Coomassie Blue staining procedure for, 479 resolution, 460, 465-466 Rotofor cell for, 475-477 sample application, 469-470 sample preparation, 469 with SDS-PAGE, 425 silver staining in, 473 stock solutions, 467-468 :soelectric point, 299, 314, 380, 404, 459 determination, with 2D-PAGE, 442-443

Jacalin, in glycoprotein purification, 530

874

SUBJECT INDEX

Keratin, precipitation, 290, 298 Klenow fragment, 649, 786 Kosmotropes, 410, 413 Krafft point, 245

Laboratory chemicals, 20 detection and assay requirements, 20 disposables, 20 equipment and apparatus, 20 fractionation requirements, 22-23 glassware and plasticware, 19-21 setting up, 19-23 small equipment and accessories, 20 supporting materials, 19-21 lac promoter, 95-97 Lactate dehydrogenase assay, 220 organelle specificity, 213 process scale purification of, 356 Lactoferrin, purified by MCAC, 376 P-Lactoglobulin, interaction with HA columns, 330-332 P-Lactoglobulin A, self-associated, effects on solubility, 304 Lactoperoxidase, protein iodination method using, 726-729 Lactose permease, from Escherichia coli, purification, 500 A repressor headpiece, 649 Latency, of organelle-specific enzyme activity, 207 N-Lauryl sarcosinate, 256 compatibility with divalent cations, 256 LDAO, 252 properties of, 248 Lectin affinity chromatography effect of detergent, 531-532 of glycoproteins, 530-534 of membrane proteins. 51 1-512, 531 Lectins in detection of glycoproteins, 537 for glycoprotein purification, 530 protein binding by, 511-512 Lentil lectin, in glycoprotein purification, 530

Leupeptin, 181 concentration used, 88 protease class inhibited, 88 as protease inhibitor, 170-171 Ligands, 11, 15 Lipman-Pearson, address, 775 Lipman-Pearson FASTA computer program, 754-755, 776 Lipopolysaccharides, silver staining, 486 Lipoproteins, staining, 478, 485 Liposome preparation, detergent in, 241 Liquid chromatography, column packing, 309 Liver homogenization, 226 membrane fraction, 225 rate-zonal centrifugation, 227 membranes, preparation of, 225-235 microsomes contaminating membranes of, 227 membrane proteins isolation and solubilization of, for preparative purposes, 231-232 solubilization of, 232-235 yield of, 227 Localization vectors, 107-108 Long terminal repeats, 116 glucocorticoid-regulated, 116 Lowry assay. See Enhanced alkaline copper (Lowry) protein assay LTR. See Long terminal repeats Lubrol, spectral properties, 256 Lubrol PX structure, 255 temperature effects on, 257 Lyophilization, 69, 77-78 Lyophilizer, 77 Lysates, preparation of, 147 Lysine residues, protein cleavage at, 610 Lysis method enzymatic, 147-151 mechanical, 151-153 success of, variables, 147 Lysosomes isolation of, 209-210 purification of, 203 Lysozyme activity of, 148-149 added to sonication mixtures, 153 IEC, 407-408

875

SUBJECT INDEX -

~

p

p

p

p

p

- - -

- -

-

-

Membrane proteins assay, 520 detergent removal from, 277-284 functional activity, 499 functionally active, renaturation of, after purification, 516-517 inactivation, during purification, reactivation after, 513 initial solubilization experiment, protocol for, 263-264 integral, 502 fractionation of, 508-5 15 phase separation, 515 reconstitution of. 516 MAC GENE (computer program), 776 solubilization of, 505-507 a2-Macroglobulin transport specificity fractionation of, as protease inhibitor, 197 515 purified by MCAC, 376 lectin affinity chromatography, 531 Maize. See also Zea mays native growth regimen, 177 determination of solubilized protein, Malate, pK values at 25', 27 264 Maleyllysozyme, interaction with hydroxsolubilization of, 253-264 ylated columns, 33 1 peripheral, 502 Mammalian cells purification of, 504-505 constitutive expression in, 115-1 16 purification, 499-520 subcellular organelles, isolation of, 203 genetic methods, 517-518 a-Mannosidase, organelle specificity, 2 13 immunological methods, 517-518 u-Mannosidase 11, assay, 219 methods, 499-502, 520 Manton-Gaulin press, 152- 153 sample concentration, 518-520 Mass spectrometry. 69 solubilization, 253-264, 502, 504-507, MATCH (computer program), 761 514 MDPF. See 2-Methoxy-2,4-diphenyl-3(2H) buffers glycerol in, 258-259 furanone ionic strength of, 258 MEGA-8, properties of, 249 protease inhibitors in, 259 MEGA-9, 252 urea or chaotropic ions in, 258 properties of. 249 buffer selection, 257-258 MEGA-10. properties of, 247 criteria, 259-260 Memblane(s) detergenttprotein ratio in, 259-260, crude, deglycosylation protocol for, 539 fractionation, 503 262, 506-507 process of, 259-260 large-scale. 226 protein and detergent concentration steps, 225-235 for, 260-263 preparation superfamilies, 5 17 for membrane protein purification, Mercaptoethanol, 67 502-504 for purification of peripheral mem2-Mercaptoethanol brane proteins, 504-505 added to buffers, 197 selective solubilization of, detergent in, in protein solutions, 85 in SDS-PAGE, 426,435-436 241 stock preparation. 263 MES, pK values at 2S0, 27 interaction with hydroxylated columns, 33 1 Lysozyme-AMS, interaction with hydroxylated columns, 33 1 Lyticase, in preparation of yeast extracts, 161-162, 164-166 Lytic baculovirus-insect cell expression system, 117-132 Lytic viruses, mammalian, as expression vectors, 117

876

SUBJEC:T INDEX

Message stability, strategies for enhancing, 101-102 Messenger RNA internal cleavage by RNase III! minimization, 102 turnover and alterations of 5' leader sequence, 101-102 effect of growth rate on, 102 Metal chelate affinity chromatography, 373-376 Metal chelators, 11 Metal ions, in protein crystallization, 649650, 652 Metalloprotease inhibitors, 182 Metallothionein, 116 Methanol for precipitation of proteins, 289 as storage solvent, 322 Methionine residues, protein cleavage at, 610 Methoxatin, 644

2-Methoxy-2,4-diphenyl-3(2H)-furanone, 48 1 2,4-Methylpentanediol, in protein crystallization, 652 Mice liver, 225 monoclonal antibody production using, 674-677 polyclonal antibody production using, 666 Micellar chromatography, detergent in, 241 Micelle molecular weight, and detergent dialysis, 279-281 Micellization concentration-dependent, 244 effect of detergent on, 506 mass-action model, 244 phase-separation model, 244 Michaelis constant, 46 Microcentrifuge, 22 Microdialysis, 656 Microdialyzer, 74 Micromole, 42 Microsomes, removal from extracts, 198 Microwell plate reader, 68 Miniblotter, 671, 687 Minicells, 427

Minifuge, 22 Minigels, 45 1-454 for protein purification, 605 Mini-IEF gels, casting and running, 452453 Mini-SDS-PAGE, casting and running, 453-454 Mini-2D-PAGE, 45 1-454 sample preparation, 452 Mitochondria electron microscopy, 224-225 isolation of, 208-209 purification of, 203 removal from extracts, 198 Molecular sieve chromatography. See Size-exclusion chromatography Molecular weight apparent, sedimentation equilibrium analysis, 564-565 determination with SDS-PAGE, 426-427, 439-440 with 2D-PAGE, 442-443 estimation, with SDS-PAGE, 435 Molecular weight calibration mixtures, 323 Mono(ADP-ribosyl)transferase,637-638 bacterial toxins as, 638 Monoclonal antibodies advantages and disadvantages of, 67067 1 for immunoassays, 703 production of, 670-679 freezing hybridoma cells, 676-677 fusion procedure, 675-676 immunization procedure, 674 myeloma cell preparation, 675 spleen cell preparation, 674-675 subcloning hybridomas, 676 in vitro immunization and fusion of peripheral lymphocytes, 677-678 purification, for immunoassays, 704 subclasses of, characterization of, 677 Mono P, 382 Mono-Q, stability of, 274 Mono-S, stability of, 274 MOPS, pK values at 2S0, 27 MOPSO, pK values at 25', 27 Morpholine, pK values at 25", 28 2-(N-Morpho1ino)ethanesulfonic acid. See MES

877

SUBJECT INDEX -

3-(N-Morpholino)propanesulfonic acid. See MOPS Mouse, liver, microsomes, solubilization of, 261-262 mRNA, of cloned gene, stabilization, 101102 Muconate lactonizing enzyme, crystallization, 650 Multienzyme complex from carrot cell cultures, 542 cofractionation of enzyme activities, 545 criteria for existence of, 545-548 dissociation of, 540-541 dynamic, 540 genetic analysis, 547 intermediates direct transfer from enzyme to enzyme, kinetic test for, 551 incomplete equilibration of pools of, 545-546 steady-state levels of, 546-547 isolation and cell disruption method, 541-542 and dilution of cell contents, 540-541 special problems in, 540-542 kinetic behavior, 545-547 kinetic coupling, assay for, 546-547 in nucleotide biosynthesis, 542-545 protein cross-linking, 547-548 purification, 539-55 1 problems in, 549-550 reconstitution, 550-551 specific, isolation of, 548-550 transient times, 546 from T4 phage-infected E. coli, 542-545 from vertebrate liver, 542 Muscarinic acetylcholine receptor, from pig heart, purification, 500 Myosin, 195 precipitation, 290, 298 N-Myristoylation, of amino-terminal residues, analysis of, 640-642

N NADH-cytochrome-b, reductase, rabbit liver N-terminal sequence, 234 subunit molecular weight, 234

-

-

-

-

--

NADH-cytochrome c reductase, distribution of, in rat liver homogenate, 231 NADPH-cytochrome c reductase, from hamster liver, purification, 501 NADPH-cytochrome-c reductase, rat liver, assay, 229 NADPH-cytochrome-P-450 reductase, rabbit liver, N-terminal sequence, 234 NADPH-reductase, rabbit liver N-terminal sequence, 234 subunit molecular weight, 234 Neurofilament polypeptides, silver staining, 486 NG108 cells, solubilization of opiate receptors from membranes of, by CHAPS, 262 NH,HC03, 24 g, in 1 liter, 30 (NH&C03, 20 g, in 1 liter, 30 Nicotiana tabacum Bright Yellow, 177 cv. SRI, 177 Samsun 6, 177 Nicotinic acetylcholine receptor from chicken brain, purification, 500 purification of, 5 15 from T. californica electric organ, purification, 500 Ninhydrin, buffer reactions with, 30 Nitrocellulose elution of proteins from, 493 transfer of proteins to, 489, 491-492, 680, 682 Nitrogen cavitation, 204-206 Nonanoyl-N-methylglucamide.See MEGA-9 Nonequilibrium pH gradient electrophoresis, in 2D-PAGE, 446 Nonhistone proteins, purified by MCAC, 376 Nonidet P-40 chromatofocusing with, 390 effect on lectin affinity, 531-532 properties of, 247 in protein purification, uses of, 241 N-terminal sequence analysis, 602-613 of N-terminally blocked proteins, 71 cleavage of proteins for, 609-613 requirements for, 603 strategies for obtaining N-terminal protein sequence, 603-604

878

SUBJECT INDEX

Nuclear extract, preparation, 200-202 Nuclei isolation of, 203, 210-212 removal from extracts, 198 Nucleic acids removal from prokaryotic lysates, 153 from yeast extracts, 167-168 ultraviolet absorbance, 55 ultraviolet spectrum of, 51 Nucleolar proteins, silver staining, 486 Nucleoside diphosphatase, purified by MCAC, 376 5'-Nucleotidase distribution of, in rat liver homogenate, 23 1 glycoinositol phospholipid membrane anchor, 632 rat liver, 228-229 Nucleotide biosynthesis, multienzyme complexes in, 542-545 Nucleotide sequences, databases of, 775

0 OBFl purification of, 521-529 titration of, in DNA-binding assay, 527528 Octanoyl-N-methylglucamide, properties of, 249 n-Octyl-@-D-glucopyranoside,properties of, 249 Octylglucoside, 252, 254, 506 in isoelectric focusing, 466 properties of, 249 in protein purification, uses of, 241 structure, 255 Octyl-Sepharose CL-4B, hydrophobic binding properties, 341 Octylthioglucoside, 252 properties of, 249 n-Octyl-@-D-thioglucoside,properties of, 249 OG. See Octylglucoside Oil Red 0 , 478 Oligonucleotide probes

hybridization systems using, 739-740 in SSC, 750-751 one long and one short, hybridization systems using, 740-743 two long, hybridization systems using, 744-746 two short, hybridization systems using, 743-744 types of, 738-740 17-mers, hybridization to, in TMAC, 748-750 Oligosaccharides, analytical deglycosylation, 538-539 OMEGA, properties of, 249 ompA-like leader sequence elements, stabilizing effect of, 102 Open reading frames cloning of, 103-104 of eukaryotic genes, translation start region for, 103 Organellar marker enzyme activities, bookkeeping of, 21 1-213 assay of, 211 Organelle enzymes activity, 213 inhibition of, 213 Organelles intactness, determination of, 207 isolation, 198 subcellular, isolation of, 203-225 Organic solvents as eluents, 316 for precipitation of protein, 296 for protein solubilization, 507 use of, precautions, 297-298 OSG, properties of, 249 Ouchterlony double diffusion, for antibody detection, advantages and disadvantages of, 668 Ovalbumin, IEC, 407-408 Overexpression systems development of, 113-1 14 in E. coli, 93-1 12 in eukaryotes, 112-132 need for, 112 yeast, 114-1 15 Ovomucoid interaction with hydroxylated columns, 331

SUBJECT INDEX

purification of, on HA columns, 335336

Palmitoylation, 642 Papaya lysozyome, commercial 2X recrystallized, purification of, on HA columns, 335-337 Papillomavi~ses,as transient expression vector, 117 Particulate material, removal of, from extract, 198 PAS staining, of glycoproteins, 536 PBE 94, and buffers, for chromatofocusing in different pH ranges, 384-385 PBE 118, and buffers, for chromatofocusing in different pH ranges, 384 (PBE)94, 382 (PBEI118, 382 PC GENE (computer program), 776 Pea. See also Pisum satiua chloroplasts, preparation, 186 genome, 178 growth, 177 growth regimen, 177 leaf mitochondria, multienzyme complex, isolation, 549 Peanut agglutinin, in glycoprotein purification, 530 PEI. See Polyethyleneimine Pentose phosphate pathway, enzymes, in Candida utilis, 790 Pepsin A, protein cleavage, 620 Pepsinogen-AMS, interaction with hydroxylated columns, 33 1 Pepstatin, 181 Pepstatin A concentration used, 88 as protease inhibitor, 88, 170-171, 259 Peptide fragments, in-gel staining, 615, 616 Peptide mapping, 613-626 principle of, 613-614 steps of, 614 Peptides separation, 615, 622-626 visualization, 615 Performic acid oxidation, of cysteine and cystine, 598

879

Peripheral lymphocytes, in vitro immunization and fusion of, for production of monoclonal antibodies, 677-678 Peristalic pump, 23 pH to denature and precipitate unwanted proteins, 299-300 and electric charge on surface of protein molecules, 380-381 isoelectric, 380 of solution of weak acid or base, 24-25 in studying enzymes, 24 PHAA, as iodine carrier, 734 X Phage recombinant (DE3), 96 1,lO-Phenanthroline, 182 Phenolics in plant extracts. 175 and protein determination techniques, 182 Phenylmethylsulfonyl fluoride as protease inhibitor, 170, 197, 259, 503504 stock solution, preparation, 180 Phenyl SPW, stability of, 274 Phenyl-Superose, stability of, 274 pH 5 fraction, 299 pH meter, calibration, 29 Phosphate, pK values at 2S0, 27, 28 Phosphate buffer, 26 stock solutions, 34 Phosphatidylinositol phospholipase C, lysis of glycoinositol phospholipid membrane anchors, 63 1-632 Phosphoamino acids, affinity to Fe3+ chelate gel, 376 6-Phosphogluconate dehydrogenase, purification of, 356 Phosphohydroxylysine, 643 Phospholipase C, from human platelets, purification, 501 Phosphomonoesterases, continuous assay, 40 Phosphopantetheine, binding to proteins, 644 Phosphoproteins, affinity to Fe3+chelate gel, 376 Phosphorylation, 627 analysis of, 642-643 Phototaxis, 632

880

SUBJECT INDEX

PI. See Isoelectric point PIBS, pK values at 2s0, 28

Piperazine-N,N1-bis(2-ethanesulfonic acid). See PIPES

Piperazine-N,N1-bis(2-hydroxypropanesulfonic acid). See POPS0 Piperazine diacrylamide. See also Diacrylylpiperazine source, 495 Pipendine, pK values at 25O, 28 PIPES, pK values at 25", 27 PIPPS, pK values at 25", 28 Pisum sativa, cv. Laxton's Progress No. 9, 177 Plant cells cell walls, 175 chloroplasts, 176 components of, 175-176 mitochondrion, 176 nucleus, 176 organelles, 176 plastid, 176 polyphenols in, 175 vacuole, 175, 176 Plant enzymes, reducing agents, 179- 180 Plant extracts cytoplasmic, of nongreen tissue, 185 from fibrous or phenol-rich leaves, 186 from fleshy beans or seeds, 185 gel filtration, 193 lipid removal from, 192-193 from nonfibrous leaves, 186 nucleic acid removal from, 192- 193 PEI precipitation, 192-193 phenolics in, 175 posttreatment of, 192- 193 preparation of choice of material, 176-178 inhibitor mix for, 184 protocol, 178-192 small-scale, 184 pretreatment, 183 protein determination in, 182 type of, 178-179 whole-cell, from leaves, 185-186 Plants cell breakage, 183- 184 chloroplast activities, purification of, 177 cultivars, 177 genetically characterized lines, 177

homogenization buffer for, 179-182 imbibing, 183 organic flotation, 183 preparation of extracts from, 174-193 proteases in, 175-176 washing, 183 Plasmid with lac operator controlling both T7 and T3 promoters, 100-101 with multicopy inseri in direct head-totail orientation, construction of, 522-524 with nut site downstream of lac promoter, 101 selectable marker, 115 in site-specific DNA affinity chromatography, 521-522 with synthetic pL promoter that contains lac operator sequence in place of oL1, 100 Plasmid pACYC 1771184, 99-100 Plasrnid pACYC 184, 100 Plasmid pACYC177-laciP, 96-97 Plasmid pDL19, 95, 97-98 construction of concatemeric inserts with, 97-98 Plasmid pETI-7, 95 Plasmid pET3, 102 Plasmid pGP1, 96 Plasmid PIN-111-ompA, 107-108 Plasmid pJG201, 110-1 1 1 Plasmid pJL23, 100 Plasrnid pKC30, 95, 104 Plasmid pLG609, 134-136 Plasmid pMLB110718, 109 Plasmid pMOB45, 94, 95 Plasmid pMOB48, 95 Plasmid pRK16F, 95 Plasmid ptacl, 95 Plasmid ptacII, 95 Plasmid pT7 vectors, 95-96 Plasmid pUC18, 96 as translation vector, 104 Plasmid pUC18,19, 95 Plasmid pUC19, 96, 97 as translation vector, 104 Plasmid pV,-lhy, 138-139 Plasmid pV,-lhy, 138-139 Plasmid pWWIO, rho @) independent terminator tR' present in, 102

SUBJECT INDEX

Plasmid pWW12, 95, 100, 102 Plasmid RI drd-19, 97 Plasmid vectors, that provide promoter for transcription of cloned, promoter-less genes, 94-95 Plasminogen activator, purified by MCAC, 376 Platelet-derived growth factor receptor, from pig uterus, purification, 501 PLRP-S, stability of, 274 pL vectors, 94-96, 101 PMSF concentration used, 88 protease class inhibited, 88 POE(8), properties of, 247 POE(9) dodecyl ether, properties of, 247 POE(10) dodecyl ether, properties of, 247 POE(l0) hexadecyl ether, properties of, 247 Polyacrylamide-antibody beads. See Immunobeads Polyacrylamide gel electrophoresis, 679680. See also SDS-PAGE; Twodimensional polyacrylamide gel electrophoresis high-resolution native, 436 protein analysis using, 441-459 Omstein-Davis system, 434, 436 Polyacrylamide gels, 427-428 and background staining with silver stain, 487 granular, 475 for isoelectric focusing, 462 physical characteristics, 428 pore size, 428 Polybuffer, removed from proteins by gel filtration, 390 by precipitation with ammonium sulfate, 390 Polybuffer exchanger groups, 382 Polyclonal antibodies advantages and disadvantages of, 663, 664 animal selection for, 665, 666 characterization, 669-670 definition of, 663 detection methods, advantages and disadvantages of, 668 for immunoassays, 703

88 1

production, 663-670 bleeding and processing serum for, 667-669 fractionation of Ig from serum, 669670 immunization procedure, 665-667 using rabbits, 665-670 Polyethylene glycol, 81 advantages of, 301-302 analytical precipitation curve with, 303304 detergent removal with, 280 mechanism of action, 302 methods for removing, 305-306 precipitation of proteins with, 301-306 in protein crytallization, 652 in reconstitution of multienzyme complexes, 550-551 for removal of detergents, 278 selection of, 302-303 in vapor diffusion experiments, 655 Polyethyleneimine, 310 preparation of, 193 treatment of plant extracts with, 192-193 Polyiodide, protein iodination using, 732 Polyols, added to buffers, 197 Polyoxyethylene derivatives, 506 Polyphenols, inactivation of, 180 Polysaccharides, silver staining, 486 Polysorbate 20, properties of, 247 Polysorbate 80, properties of, 247 Polystyrene, 310-31 1 Polytron homogenizer, 195 Poly(viny1 chloride)-silica sheets, 31 1 Pol ~(vinylidinedifluoride) elution of proteins from, 493 for protein transfer, 489, 491-492, 680682 Ponceau S staining, for identifying proteins for elution from gels, 491-492 POPSO, pK values at 2S0, 27 Pore volume, 400 Postnuclear supernatant, preparation of, 206-207 Potassium chloride, as precipitant and solvent for, 298 Poxviruses, as transient expression vector, 117 Precipitation of membrane proteins, 499-502

882

SUBJECT INDEX

for protein sample concentration, 518519 to remove polybuffer and ampholytes from protein, 390 techniques, 78-79 Precipitation techniques, 285-300 theory, 287-289 limits of, 287-289 Pressure cells, 22 Primary amine buffers, 29 Prochymosin, redox conditions during solubilization and refolding, 270-271 Procion blue H-B, 356 Procion blue MX-R, 355-356 Procion dyes, 350 Procion H-EGN, 356 Procion red HE-3B, 356 Procollagen-lacZ fusion vector, 110111 Prokaryotes liquid shear lysis, 152-153 preparation of extracts from, 147-153 sonication, 153 Promoters, 94 regulatable, 116 Protease A, 169 Protease B, 169 Protease C, 169 Protease inhibitors, 11, 12, 87-88, 169, 180-182, 259, 503-504 added to buffers, 197, 259 precipitated with bulk proteins, 293 in preparation of plant extracts, 176 in protein solutions, 86 in studies with yeast, 170 Proteases in E. coli, 105 and heat denaturation, 300 for limited digestion of proteins, 609 in plants, 175-176 site-specific, protein cleavage with, 611612 in yeast cells, 169 Protein(s). See also Bulk protein acid-acetone precipitation, for amino acid analysis, 592-593 activity assays for, 10 loss of, 88-89

activity-based analysis, to detect impurities, 556, 557 amide content, determination, 600-601 antigenic regions in, prediction of, 769770 binding of, to hydrophobic gels, 342 bulk precipitation, 292-293 precautions, 293 characterization, 557 chemical methods, 567-571 methods, 679-680 scattering methods, 567, 584-585 techniques. 566-568 transport methods, 571-584 charge on, 380, 459 chromatography on hydroxyapatite columns, 329-330 classification of, on basis of solubilities, 286 cleavage buffers for, 620 chemical methods, 610-61 1 reagents, 610 gel slice, 621 lyophilized proteins, 621 NCP strip, 621-622 for N-terminal sequencing of blocked proteins, 609-61 3 reagents, 6 19-620 site-specific chemical methods, 610-61 1 with proteases, 611-612 cofactors, 643-645 colligative properties, 570-571 colorimetric assay, 57-65 composition-based analysis, 568-570 to detect impurities, 556, 557 concentration measurements, 85, 568570 covalent immobilization on glass fiber filters, 495 cyanogen bromide cleavage, 610-61 1 cysteine determination, 598-599 detection postelectrophoretic, 490-495 in 2D-PAGE, 454 determination, in eukaryotic cell extract, 222-223

SUBJEC:T INDEX

electron microscopy, 585 end-group analysis, 568, 602-613 expression of, in recombinant system, 738 fibrous, 287 coiled coil, precipitation of, 290 fractionation with ammonium sulfate, 293-296, 410 apparatus and materials, 22-23 on basis of solubilities, 286 to detect impurities, 555-556 by phase separation, detergent in, 241 globular, 286, 287 heterologous, secretion from E. coli, 132-143 hydrodynamic behavior, characterization of, 574-579 hydrodynamic diameter, 3 18 hydrolysis for amino acid analysis, 594-598 destruction of amino acids in, 588 inactivation, causes of, 84 iodinated activity, evaluation of, 736-737 level of iodination, analysis of, 736 storage, 736 labeling, for peptide mapping, 6 14-61 5, 616-619 labile, stabilization by fusion to P-galactosidase, 109-1 10 mammalian, insect cells and viruses for expression of, 117-132 mass, estimation of, 569-570 membrane-bound, elution of, 489 membrane-spanning regions, prediction of, 769-770 methylated, detection of, 633-634 microgram quantities carboxymethylation, 599 S-pyridylethylation of, 599 microsequencing, 69 minimum molecular weight, determination of, 568-569 mixtures of, salt fractionation of, 289 modifications analysis for, 626-643 in concentration and desalting, 71-72 for crystallization, 648-649 posttranslational, 626-627

883

molecular size, determination, to detect impurities, 555-557 molecular weight, determination, 566587 to detect impurities, 555-557 multifunctional, 539-540 pH optimum, determining, 26 postelectroelution assay, 493 precipitation of, 285-300, 652-653 history, 285-287 organic solvents for, 296-298 with PEG, 301-306 properties, and IEC protocols, 309 prosthetic groups, 643-645 quantitative analysis for, 568 purified by SDS-PAGE, digestion of, 613 solubility, 289 storage, 86-87 purity, determination of, 443, 555-565 quantitation, 50-68 using organic stains, 487 using silver stains, 487-488 in 2D-PAGE, 454-455 radioiodination of, 617-618 radiolabeling of, 721-737 secondary structure and absorbance spectrum of protein, 52 helix, strand, and turn propensities of amino acids, 766 prediction, 765-769 accuracy, 771 utility of, 771-772 secretion. vectors suitable for, 109 separation from ampholytes, 477 without reduction, 436 signal sequences, prediction of, 77077 1 size, determination of, 566-587 solubility, 67, 70 factors affecting, 652-653 in presence of PEG, 302 role of protein-protein interactions, 304 solvent conditions, 85-86 sources, 12-1 3 and IEC protocols, 309

884

SUBJECT INDEX

stability in cell, enhancing, 105-1 10 maintaining, 83-89 during storage, 89 stability and storage study, 12, 86-87 standards, 23, 66-67 for colorimetric assays, 57 storage, 12 structural class, determining, 765 structure, 287 computer analysis of, 75 1-776 prediction, 765-772 three-dimensional, databases of, 775 transitions in, 288 subunits definition of, 566 presence of, determination of, 566587 transblotting, for amino acid analysis, 596-598 tricholoracetic acid/deoxycholate precipitation, for amino acid analysis, 593-594 tryptophan determination, 599-600 ultraviolet absorption maxima, 51-52 ultraviolet spectrum of, 51 viscosity, 584 Protein A immunoadsorption procedure using, 688-689 in immunodetection of proteins, 684-685 Protein aggregates refolding, 270-271 solubilization of, 264-276 cell disruption, 267-268 evaluation of solubilization agents, 269 final protein products, features of, 276 general considerations, 265 reagents, 266, 267, 269 variables, 269 solubilized, purification of, 273-276 Protein assay, 296, 520 accuracy, 780 concentration limits of chemicals in, 5253 cuvettes for, 65-66 general instructions, 65-68 instrumentation, 68 kits, 50 optimal, 780

removal of interfering substances, 67-68 by dialysis and ultrafiltration, 67-68 by neutralization, 67 by precipitation, 67 standards, 66-67 ultraviolet absorption methods, 50-56 absorbance at 205 nm, 55-56 absorbance at 280 nm, 54-55 determination of extinction coefficient for protein of unknown concentration, 56 Protein blots, applications of, 680 Protein crystallization, 646-659 cessation of growth, 651 of complexes, 658-659 conditions for, search for, 653-656 detergent in, 241 low ionic strength, 655-656 nucleation, 65 1-652 postnucleation growth, 65 1 precipitants for, 652-653 procedure, 65 1-658 protein modifications for, 648-649 protein preparation for, 649-650 protein purity required for, 648 salts in, 652-654 seeding, 657-658 stages, 651-652 suitability of protein for, 647-648 temperature in, 652-653 Protein crystals appearance of, 650 cross-linking, 651 large, growth of, 657-658 soaking experiments, 651 solvent content, 651 Protein Data Bar,.., address, 775 Protein elution, from hydroxyapatite, 332333 Protein-DNA crystals, 658-659 Protein-protein crystals, 658 Protein-RNA crystals, 658-659 Protein G, in immunodetection of proteins, 684-685 Protein Identification Resource address, 775 computer program. See ALIGN (computer program); MATCH (computer program); SCAN (computer program)

SUBJEC:T INDEX

Protein kinase, in phosphorylation of proteins, 643 Protein-ligand interactions, effect on protein precipitation with PEG, 304305 Protein Pak, 320 Protein-protein interactions, effect on protein precipitation with PEG, 304305 Protein purification and activity of protein, 9-10, 88-89 additions to protein solvents, 11 by ammonium sulfate precipitation, 792 amount of material required, 9 buffers, 11-12, 28-29 containers, 12 contaminating activities in, 12 for crystallization, 648 by electroblotting, 604-607 by electrophoresis, 604-605 equipment, 21 high-capacity steps, 14-15 by high-performance liquid chromatography, 607-609 intermediate-capacity steps, 15 low-capacity steps, 15 for peptide mapping, 6 14-615 preliminary considerations, 9-12 procedures bulk or batch, 13-14 optimal arrangement of, 70, 779-780 refined, 14-15 rethinking, 779-780 proteolysis in, 87 scale of, 780 scale-up, 13 limitations to, 9-10 by secretion of heterologous proteins from E. coli into culture medium, 132 solutes, 11-12 source of protein, 12-13, 779 steps, arrangement of, 85, 780 strategies and considerations for, 9-15 temperature, 12 volumes, 13 Protein sequencing, 69, 71 Protein solution concentration, 68-69

885

buffer system, 70 chemical destruction of protein during, 71 dry, water-attracting matrices for, 81 by lyophilization, 77-78 and molecular weight of protein, 70 and properties of solutes, 71 and protein volume, 70 strategy to reduce adsorptive loss of protein on surfaces, 71 time required for, 72 by ultrafiltration, 76 desalting, 68-69 buffer system, 70 chemical destruction of protein during, 71 commercially available, prepacked columns, 80 by gel filtration, 79-80 general considerations, 70-72 and molecular weight of protein, 70 and properties of solutes, 71 and protein volume, 70 in RP-HPLC, 80 strategy to reduce adsorptive loss of protein on surfaces, 71 time required for, 72 handling procedures, 84 purified, freezing and thawing, 87 storage conditions, 86-87 trials, 86-87 viscosity, and rate of diffusion, 72 Protein staining for identifying proteins for elution from gels, 491 on Immobilon-P membranes, 606-607 in isoelectric focusing, 472-473 Protein stains, 477-478 and protein contaminants, 561 Proteolysis in crystallization, 649 prevention, 313-314 with membrane proteins, 503-504 in preparation of extracts from yeast, 168-171 during purification, 87, 322 PT7-1,2, 95 PVDF. See Poly(viny1idine difluoride) Pyridine 5 ml

886

SUBJECT INDEX -

-

-

-

+ 50 ml glacial acetic acid in 1 liter, 30

+ 100 ml glacial acetic acid in 1 liter,

30 25 ml, + 25 ml glacial acetic acid in 1 liter, 30 100 ml, + 4 rnl glacial acetic acid in 1 liter, 30 pK values at 25", 27 Pyridine-formic acid, 30 Pyrophosphate, pK values at 25", 27 Pyrophosphohydrolase, reaction catalyzed by, 44 Pyrroloquinoline quinone, 644 Pyruvate kinase, coupled assay, 41

Q QAE-Sephadex A-25, 382 QUEST (computer program), 761

Rabbit bleeding, 667-669 immunization, 665-667 liver, 225 microsomal preparations, SDS-PAGE analysis of, 233 microsomal proteins molecular weight, 234 N-terminal sequence, 234 polyclonal antibody production using, 665-670 reticulocytes, labile aminoacyl-tRNA synthetase complex from, 199 serum, processing, 667-669 serum albumin precursor, from liver, N-terminal sequence, 234 Radioimmunoassays, 368. See also Immunoassays automation of, 714 design of, 701-702 sensitivity of, 701-702 Radioiodination. See Iodination Rat liver, 225 golgi vesicles, isolation of, 229-231 labile aminoacyl-tRNA synthetase complex from, 199

plasma membranes, isolation of, 227229 polyclonal antibody production using, 666 Rat chondrosarcoma, multienzyme complex, isolation, 549 Reaction primary, 44 rate of, 42 secondary, 44, 48 Reactive dye(s), 351, 355 categories, 350-351 Cibacron F series, 356 color index (CI) number, 350-351 commercial samples of, additives, 353 kit, 350 molecular weight, 354 spectral parameters for, 353-354 structures of, 352 synthesis of, 357 Recombinant DNA technology, 271, 738 Redox reagents, for solubilization of protein aggregates, 270-271 Reducing agent(s), 12 in protein solutions, 85 Refractometer, 21 Restriction enzyme inhibitor, negatively charged, crystallization, 650 Reticulocytes, aminoacyl-tRNA synthetase complex from, isolation of, 199-200 Retinoic acid, linkage to proteins, 643 Retinoylation, 642 Reversed-phase chromatography, 70, 340341, 392, 409, 414-417 application, 416-417 matrix, stability of, 274 of membrane proteins, 499-502, 513514 using columns for hydrophobic interaction chromatography, 514 mobile phase, 415-416 peak shape in, 414 as preparative technique, 414 protein denaturation in, 414 stability of, 274 stationary phase, 414-415 Reversed-phase HPLC, 80-81 buffer system, 81 in desalting and concentration of proteins and peptides, 80

SUBJECT INDEX

for obtaining N-terminal protein sequence, 603-604 Reverse micellar bioseparation, detergent in, 241 Rhodopseudomonas uiridis, photoreaction center, purification of, 499 Riboflavin, binding to proteins, 644 Ribosomes aminoacyl transfer to, 812-813 purification of, 8 12-813 Ricinus communis agglutinin I , in glycoprotein purification, 530 RNA. See also Messenger RNA assay, by [2-'4C]uridine incorporation into trichloroacetic acid-precipitable material, 221-222 organelle specificity, 213 RNA polymerase, 93-94 RNase A, crystallization, 650 Rotofor cell, for isoelectric focusing, 475477 Run-away replicon, 95, 97

S100, preparation, 200-202 Saccharomyces, 154 Saccharomyces cereuisiae, strain CYY2 cell harvest, 524 growth, 524 Salivary proteins, silver staining, 486 Salmonella typhimurium leaky mutants, 109 liquid shear lysis, 153 Salting in, 288, 652 Salting out, 410, 652 of proteins, 288-296 Salts in protein crystallization, 652-654 in protein solutions, 85 used to solubilize, precipitate, or fractionate proteins, 290-298 Sand bath, for terminating reactions, 45-46 Sarkosyl NL30, properties of, 248 SB3-12, properties of, 247, 248 SB3-16, properties of, 248 SCAN (computer program), 761 Scenedesmus obliquus, multienzyme complex, isolation, 549

887

Schizosaccharomyces pombe susceptibility to lyticase, 154 susceptibility to zymolase, 154 Scintillation counter, 21 SDS. See Sodium dodecyl/lauryl sulfate SDS-PAGE, 78, 425 applications of, 425-426 catalyst, 431 Coomassie Brilliant Blue R-250 staining in, 437-438 copper staining in, 438-439, 441 detergent exchange or removal, 278 with electroblotting on NCP, for protein purificatiodpeptide mapping, 615, 616, 619 electrode buffer, 43 1 electrophoresis method, 433-435 gels, 427 calibrating, 439 casting, 431-433 detection of proteins in, 437-439 elution of proteins from, 489-490 impurities in, 428 Laemmli system, 429, 434-435 marker proteins, 439 molecular weight determination with, 439-440, 581-582 one-dimensional gels casting and running for second dimension electrophoresis, 446-449 gradient, method for casting, 448449 nongradient, method for casting, 447-448 for protein purificatiodpeptide mapping, 615-616, 622-623 protein in gel slice, 623 solubilized proteins, 622-623 preparative electrophoresis, 440-441 principle of, 429 procedure, 430-436 protein-binding properties, 428 protein purification, for peptide mapping, 614-615 for protein purificatiodpeptide mapping, 605, 615-616, 622 for protein sample concentration, 519520

888

SUBJECT INDEX

proteins purified by, digestion of, 613615 for protein subunit determination, 586 relative mobilities of proteins on (Rf), 440 resolution, 435 resolving gel, 429 formula for, 43 1-432 sample preparation, 433 silver staining in, 438 stacking gel, 429 standards for, 439 stock solutions, 430 two-dimensional, for peptide mapping, 616 variations of method, 435-436 Secretion cloning vectors, 107- 108 Sedimentation, 14 Sedimentation coefficient, 574 interpretation of, 578 Sedimentation equilibrium to assess protein purity, 564-565 buffers for, 573 convection in, 573 data acquisition and analysis, 565 to detect impurities in protein sample, 556, 574 for determination of association constants of mass action-driven macromolecular associations, 587 for determination of protein subunit stoichiometry, 586-587 Sedimentation equilibrium analysis automated, 565 method, 571-573 for molecular weight determination, 571-574, 577 problems and pitfalls, 573-574 Sedimentation velocity to assess protein purity, 556, 563-564 for characterization of hydrodynamic behavior of protein, 574-579 gradient method, 578-579 problems and pitfalls with, 577-579 Sephacryl, 324 Sephacryl HR, 320 Sephadex, 321 Sephadex G, 320 Sephadex G-25, 82

Sephadex G-100, 81 Sepharose, 320, 324 Sepharose CL, 382 stability of, 274 Serine protease inhibitors, 180- 181, 259 Sf9 cells, 117-118 culture, 121 equipment, 124-125 frozen, 121-122 protein expressed in, assay for biological activity of, 131-132 transfection in, 122 Sheep liver, labile aminoacyl-tRNA synthetase complex from, 199 mammary gland, labile aminoacyl-tRNA synthetase complex from, 199 polyclonal antibody production using, 666 spleen, labile aminoacyl-tRNA synthetase complex from, 199 Shine-Dalgarno sequence, 103 Shodex, 320 Shuttle vectors, 116 ~ i a l o ~ h o r i purification, n, multistep procedure, 534 Silkworms, middle silk gland of, homogenization, 195 Silver staining, 78, 482-487 ammoniacal, 482-483 artifacts, 487 background staining, 487 color effects with, 485 diamine, 482-483 for identifying proteins for elution from gels, 491 in isoelectric focusing, 473 nondiamine, 483-484 photodevelopment, 484 protein quantitation using, 487-488 protein-specific, 486 quenching of autoradiography, 486487 reactive groups involved in, 484-485 in SDS-PAGE, 438 sensitivity of, 478 in 2D-PAGE, 443 Single-chain antibodies, genetic engineering of, 679

SUBJECT INDEX

Site-specific DNA affinity chromatography applications, 521 methods, 521-522 procedure, 521, 524-527 for purification of DNA-binding proteins, 521-529 Sitting drop method, 655, 657 Size-exclusion chromatography, 69, 3 18, 392, 399-403 applications, 402-403 to separation problem, 402-403 column cleaning, 402-403 selecting, 401-402 volumetric loading limit, 403 disappearance of desired protein in, 328 elution volume, 318 flow rate, 326-327 high-performance columns, 320-321 repacking, 327 semipreparative and scale preparative, 326-327 matrices, 320-321 mobile phases, 402 packed column matrix parameters, 327 packing materials, 400-401 partition coefficient, 3 18 and molecular weight, 318-319 powdered matrix parameters, 321 practice, 318-328 preliminary screening, 322-324 principle of, 400 purification (-fold) achieved by. 322-323 purification with, enhancement in, 319320 resolution, 319, 326, 401, 403 sample preparation, 322 scaling upward, 326 skewed peaks, 327-328 solvents, 322 suspended matrix parameters, 324-326 total volume, 3 18 trouble shooting, 326-327 using conventional matrix, 324-326 void volume, 3 18 zero elution volume, 318 Skeletal muscle, homogenization, 195 Sodium azide, 11

889

Sodium channel, purification, 515 from rat brain, 500 Sodium chloride, as precipitant and solvent for proteins, 298 Sodium cholate, 252 effect on lectin affinity, 531-532 properties of, 247 in protein purification, uses of, 241 structure, 255 Sodium decane sulfonate, 252 Sodium deoxycholate, 252 effect on lectin affinity, 531-532 properties of, 247 in protein purification, uses of, 241 Sodium dodecane sulfonate, 252 Sodium dodecylhaurate sulfate, in protein purification, uses of, 241 Sodium dodecyl/lauryl sulfate exchange o r removal, 278 properties of, 248 Sodium dodecyl sulfate, 252, 506. See also SDS-PAGE critical micelle concentration, 245-246 effect on functional activity of membrane proteins, 516 effect on lectin affinity, 531-532 Sodium p-hydroxymercuribenzoate, stock solution, preparation, 181 Sodium metabisulfite, as protease inhibitor, 171 Sodium octane sulfonate, 252 Sodium laurocholate, 252 properties of, 249 sodium taurodeoxycholate, 252 properties of, 249 Solute-absorbing matrices, 81-82 Solutes, 71 diffusions of, 72 removal of, 68-69 Solvent-protein interactions, perturbations of, 285, 288-289 Sonication, 153 to recover protein inclusion bodies from pelleted bacterial cells, 267 of yeast, with glass beads, 161 Sonicators, 22 Soybean. See Glycine max Soybean agglutinin, in glycoprotein purification, 530

890

SUBJECT INDEX

Soybean trypsin inhibitor IEC, 407-408 as protease inhibitor, 171, 197 Specific activity, 42-43 Spectrophotometer, 21, 68 Speed Vac concentrator, 77 Spinacea oleracea, hybrid No. 424, 177 Spinach. See also Spinacea oleracea chloroplasts multienzyme complex, isolation. 548 preparation, 186 growth regimens, 177 SP-Sephadex C-25, 382 SSC, oligonucleotide probelhybridization system using, 739-740, 750-751 Staden, R., address, 775 STADEN (computer program), 776 Stagnant mobile-phase mass-transfer problem. 393 Staining. See Gel-staining techniques; Protein staining Stains. See Protein stains; speci$c stain Staphylococcus aureus imrnunoadsorption procedure using, 688-689 lysis of, 147 Staphylococcus typhimurium, permeabilizing effect of Tris, 148 Start codon, 103 Steady-state concept, of enzyme regulation, 803-805 STI-AMS, interaction with hydroxylated columns, 331 Stirred ultrafiltration cells, 76 Stoichiometric displacement model, 405 Subcellular fractionation of membrane proteins, 503 of yeast, 171-174 Subcloning, general method for, 98-99 Substrates, added to buffers, 197 Succinate, pK values at 2S0, 27 Succinate buffer, stock solutions, 33 Succinate-cytochrome-c reductase, distribution of, in rat liver homogenate. 231 Succinylchymotrypsin, interaction with hydroxylated columns, 331 Sucrose density gradients, for purification of integral membrane proteins, 514515 Sulfhydryl agents, 11 Superose, 320

Surfactants, 11-12 SV40, as transient expression vector, 117 SWISSPROT (computer program), 776 Synaptosomal ATP-dependent Ca2+uptake system, purification of, 515 SynChropak, 320 SynChropak AX-300, 382 SynChropak AX-500, 382

T lac promoters, 95-97 Tannins, 175, 180 and protein determination techniques, 182 TAPS, PK values at 25", 28 Taurine derivatives, compatibility with divalent cations, 256 Taurocholic acid, sodium salt, properties of, 249 Taurodeoxycholic acid, sodium salt, properties of, 249 TCA cycle multienzyme complex, isolation, 549 T-cell epitopes, prediction of, 770 T4 dNTP synthetase gel filtration of, 544 genetic analysis, 547 kinetic coupling, assay for, 546-547 purification, 542-545 reactions catalyzed by, 542, 543 TEA, pK values at 25", 27 TEMED, 428, 431-432 in isoelectric focusing, 462 Terminators, at end of cloned gene, and message half-life, 102 Tertiary structure, and absorbance spectrum of protein, 52 TES, pK values at 25', 27 Tetradecyltrimethylammonium bromide, properties of, 249 Tetramethylammonium chloride hybridization to 17-mers in, 748-750 oligonucleotide probelhybridization system using, 739-740 Tetramethylethylenediamine. See TEMED Thallium trichloride, protein iodination using, 732 Thermolysin, protein cleavage, 620

SUBJECT INDEX

Thiols added to buffers, 197 neutralization, 67 Thioredoxin, crystallization, 650 Thylakoid extracts, preparation of, 191192 Thymol-sulfuric acid staining, of glycoproteins, 536-537 Tissue culture cells extract preparation and fractionation, 199-200 as source of enzymes, 195 Tissues, homogenization, 195 T4 lysozyme, 148-149 TMAC. See Tetramethylammonium cNoride Tobacco. See Nicotiana tabucum Torpedo californica, electric organ, preparation, for acetylcholine receptor purification, 502-503 Total permeation volume, 400 Toxic genes potential problem with, 99 repression, 99- 101 Toxins, bacterial, enzymatic activity, 638 T7 polymerase, expression of down regulation of, 100-101 in E. coli, 96 Transaldolase, from Candida utilis, 788793 crystalline, 790 half-of-the-sites activity of, 789, 792-793 isozyme I, purification, 790-791 isozyme 11, purification, 790-791 isozyme 111, purification, 791-792 isozymes, 790 mechanism of action, 789-790 purification of, 790-792 reaction catalyzed by, 789 structure, 789-790 Transblot cell, 491 Transblotting, of protein, for amino acid analysis, 596-598 Transcription, optimizing by genetic fusion t o strong promoters, 93-97 by increased gene dosage, 97-98 Transcriptionally active extracts, 200-202 Transketolase, 788-789 coenzyme, 789 reaction catalyzed by, 789

89 1

Translation of cloned gene, optimizing, 103-105 initiation, sequence determinants for, 103 Translation fusion vectors, 104 Trasylol. See Aprotinin Trichloroacetic acid, as precipitating agent, 78-79 Tricholoracetic acid/deoxycholate precipitation, of protein, for amino acid analysis, 593-594 Tricine, pK values at 25", 28 Triethanolamine. See TEA Triethanolamine-HCl, 30 Triethylamine-CO,, 30 Triethylamine-formic (or acetic) acid, 30 Trifluoroacetic acid, in RPC, 414-417 Trimethylamine-acetic acid, 30 Trimethylamine-C02, 30 Trimethylamine-formic acid, 30 Tris, 26 permeabilization of outer membranes, 148 pK values at 2S0, 28 in protein purification, 29 stock solutions, 35-36 Tris(carboxymethyl)ethylenediamine, for IMAC applications, 373-374 Tris(hydroxymethyl)aminomethane. See Tris 2-~ris(hydroxymethyI)methylamino]ethanesulfonic acid. See TES 3-[[Tris(hydroxymethyl)methyl]amino]propanesulfonic acid. See TAPS N-[Tris(hydroxymethyl)methyl]glycine. See Tricine Triticum aestiuurn, cv. Yamhill, 177 Tritium, half-life of, 721 Triton N-101, spectral properties. 256 Triton X-100, 252, 254 chromatofocusing with, 390 effect on functional activity of membrane proteins, 516 effect on lectin affinity, 531-532 exchange or removal, 278 for CHAPS, 281-282 hydrogenated, 54 properties of, 247 in protein purification, uses of, 241 reduced, 252 properties of, 247

892

SUBJECT INDEX

spectral properties, 256, 316 structure, 255 temperature effects on, 257 Triton X-114, 252 properties of, 247 in protein purification, uses of, 241 tRNA-amino acid complexes, 810-81 1 T7 RNA polymerase, sources for, 96 Tropomyosin, precipitation, 298 Troponin C, silver staining, 486 Trp-tRNA synthetase, purification of, 348-349 Trypanosome variant surface glycoprotein, glycoinositol phospholipid membrane anchors, 629-630 Trypsin heat stability, 298 protein cleavage, 610, 612, 613, 620 Trypsinogen interaction with HA columns, 330-332 isoelectric point, 330 Trypsinogen-AMS, interaction with hydroxylated columns, 33 1 Trypsinogen-COX, elution, from hydroxyapatite, 332 Tryptophan, determination in proteins, 599-600 Tryptophan residues, protein cleavage at, 610 Tryptophanyl-tRNA synthetase, in pancreas, 194 TSK-DEAE SPW, stability of, 274 TSK Phenyl SPW, stability of, 274 TSK-SW, 320 Tubulin, purification of, on HA columns, 336-338 T7 vectors, to simplify translation fusion, 104 Tween 20 properties of, 247 in protein purification, uses of, 241 Tween 80, properties of, 247 Two-dimensional electrophoresis for protein purification, to generate polyclonal antibodies, 664 with thin-layer chromatography, for protein purificationlpeptide mapping, 615-617, 620, 623-625 Two-dimensional gel electrophoresis, gels elution of protein from, 489

elution of proteins from, 489-490 protein elution from, 491-492 Two-dimensional isoelectric focusingSDS-PAGE, for protein purification, 605 Two-dimensional polyacrylamide gel electrophoresis applications of, 442 computer analysis with, 454-455 development of, 442 loading and running individual polyacrylamide gels, 449 minigels, 45 1-454 multiple gradient gels casting, 449-450 loading and running, 450-45 1 materials for, 449 nonequilibrium pH gradient electrophoresis in, 446 problems associated with, 456-457 protein analysis using, 441-459 protein detection methods, 454 in protein purification, 443 protein quantitation in, 454-455 publication format for patterns obtained by, 458-459 reagents for, 442 resolution, strategies for optimizing, 455-457 sample preparation for, 443-444 silver staining in, 443 solubilization procedures, 443-444 standardization of, 442, 457-458 troubleshooting in, 455

U. W. Biotechnology Center, address, 775 Ultracentrifugation, 15, 198 detergent in, 241 Ultracentrifuge, 22 Ultrafiltration, 67-70 applications, 75 concentration polarization, 76 definition, 75-77 membranes for, 75-76 Ultrapore short chain C3, stability of, 274 Ultraviolet absorption, 11 protein assay, 50-56

SUBJECT INDEX

concentration limits of proteins in, 5253 Ultraviolet absorption spectroscopy, 51 Ultraviolet detection, and detergent, 316 Ultraviolet monitor, 23 Ultrogel, 324 Ultrogel A, 320 Ultrogel AcA, 320 Unit, definition of, 42 Urea added to solubilization buffers, 258 chromatofocusing with, 390 in isoelectric focusing, 466-467 preparation of, 267 Urogastrone-polyarginine, expressed in E. coli, purification of, 275 UWGCG ;computer program), 758-759, 776

Vaccinia, as transient expression vector, 117 Vapor diffusion, 652-655 in growth of large crystals, 657 Vectors allowing antitermination, 101 with E. coli lac promoter controlled by lac repressor, 95-97 with phage lambda pL promoter controlled by cI repressor, 94-96 with phage T7 promoter controlled by T7 NA polymerase, 95-96 with run-away replicon, 95,97 with synthetic tac promoter controlled by lac repressor, 95-97 Vertical slab-gel apparatus, 23 Viruses, mammalian, as transient expression vectors, 117 Viscous fingering, 403 Volatilized 12, protein iodination using, 732 V8 protease, protein cleavage, 612, 613, 620

Warburg, Otto, 1 Western blotting, for antibody detection, advantages and disadvantages of, 668

893

Wheat. See also Triticum aestivum growth regimen, 177 Wheat germ cytoplasm extract, 185 extract, small-scale, 184-185 organic flotation, 183 sources, 176-177 storage, 177 Wheat germ agglutinin, 5 11-5 12 in glycoprotein purification, 530 Wheat germ lectin, 372

Yeast. See also Saccharomyces; Schizosaccharomyces pombe ARS DNA-binding protein OBFI, purification of, 521-529 autolysis, 159-160 bacterial contamination, in fermenter runs, 157-158 cell lysis, 159-168 with abrasives, 160-161 choice of buffers, 167 pressure cells for, 160 stabilization of enzymatic activity, 167 cell wall components, 164 enzymatic digestion of, 164-167 coated vesicles from, isolation of, 174 commercial, 154-155 culture media, 155-156 culture vessel, 156-157 disruption of, by agitation with glass beads, 160-164 endogenous inhibitors for proteases A, 8,and C, 169 enzymatic lysis, 161-162 exogenous protease inhibitors for, 170171 extracts lipid removal from, 168 pH, 168 preparation of, 154-174 removal of nucleic acids from, 167168 fractionation of, avoiding proteolysis in, 168-171 genetic analysis, 154

894

SUBJECT INDEX

growth in fermenters, 156-157 monitoring, 157-158 harvesting of cells, cell density for, 158159 as host for overexpression of foreign genes, 114-1 15 mitochondria, preparation of, 173-174 nuclei, preparation, 171-173 protease-deficient strain, 169 proteases present in, 169 storage of cells, 159 strain, choice of, 155 subcellular fractionation, 171- 174 translocation- and glycosylation-competent intracellular membranes from, preparation, 174 vacuole, isolation of, 174

2-314, 316 Zea mays, FR9 x FR37, 177 Zorbax, 320 Zwittergent, 506 Zwittergent 3-08, 252 Zwittergent 3-10, 252 properties of, 247 Zwittergent 3-12, 252 properties of, 248 Zwittergent 3-14, 254 structure, 255 Zwittergent 3-16, properties of, 248 Zymolyase, 161-162 in preparation of yeast extracts, 164-166

ISBN 0-12-213585-7

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