Methods of Biochemical Analysis: v. 30 (Methods of Biochemical Analysis Series: No)

METHODS OF BIOCHEMICAL ANALYSIS Volume 30

Advisory Board

N.G. ANDERSON, Division of Biologkal and Medical Research, Argonne National Laboratories, Argonne, IL 60439 T . H . BUCHER, Institute of Physiologacal Chemistry, and Physical Biochemistry and Cell Biology, University of Munich, 8000 Munich 2, West Germany W.E. COHN, Oak R d g e Nationul Laboratory, T N 37830 P. DOUZOU, Institute of Physico-Chemical Biology, Edmond de Rothschild Foundation, Paris 75005, France S. GATT, Department of Biochemistry,Hebrew University-Hadassah Medical School, Jerusalem, Israel C. JOLICOEUR, Department of ChemlrtT, University of Sherbrooke, Sherbrooke, Que'bec, J l K 2R1, Canada J.H.R. KAGI, Biochemical Institute, University of Zurich, Zurich 8032, Switzerland R.W. LUMRY, Department of Chemistry, University of Minnesota, Minneapolis, M N 55455 B.G. MALMSTROM, Department of Biochemistry and Biophysics, Chalmers University of Technology, S-412 96 Gotebmg, Sweden A. MEISTER, Department of Biochemistry, Cornell Medical College, New York,N Y 10021 R.S. MELVILLE, Bureau of Medical Services, Food and Drug Administration, Retired, 111 12 Kenilworth, Garrett Park, M D 20896 M. OTTESEN, ChemicalDepartment, The Carlsberg Research Center, DK 2500 Copenhagen, Valby, Denmark J .E. SCOTT, Department of Medical Biochemistry, University of Manchester, Manchester M l 3 YPT, England E.C. SLATER, Laboratmy of BiochemistT, B.C.P. Jansen Institute University of Amsterdam, Amsterdam-C., The Netherhnds B.L. VALLEE, Centerfor Biochemical and Biophysical Sciences and Medicine, Harvard University, Boston, M A 02115 P. VENETIANER, Institute of Biochemistry, Hungarian Academy of Sciences, Szeged 6701, Hungary M. WIKSTROM, Department ofhledical Chemistv, University of Helsinki, SF 001 70 Helsinki 17, Finland K. YAGI, Imtitute of Applied Biochemistry, Y a p Memorial Park, Mitab, Gifu 505-01, Japan

METHODS OF BIOCHEMICAL ANALYSIS

Edited by DAVID GLICK Cancer Biology Research Laboratory Stanford University Medical Center Stanford, California

VOLUME 30

An Intersciences Publication JOHN WILEY & SONS NewYork

Chichester

Brisbane

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An Interscience@Publication Copyright 0 1984 by John Wiley & Sons, Inc. All rights reserved. Published simultaneously in Canada. Reproduction or translation of any part of this work beyond that permitted by Section 107 or 108 of the 1976 United States Copyright Act without the permission of the copyright owner is unlawful. Requests for permission or further information should be addressed to the Permissions Department, John Wiley & Sons, Inc. Library of Congress Catalog Card Number: 54-7232 ISBN 0-471-80276-X Printed in the United States of America

10 9 8 7 6 5 4 3 2 1

METHODS OF BIOCHEMICAL ANALYSIS

VOLUME 30

PREFACE

Annual review volumes dealing with many different fields of science have proved their value repeatedly and are now widely used and well established. These reviews have been concerned, not only with the results in the developing fields, but also with the techniques and methods employed, and they have served to keep the ever-expanding scene within the view of the investigator, applier, the teacher, and the student. It is particularly important that review services of this nature should have included the area of methods and techniques, because it is becoming increasingly difficult to keep abreast of the manifold experimental innovations and improvements which constitute the limiting factor in many cases for the growth of the experimental sciences. Concepts and vision of creative scientists far outrun that which can actually be attained in present practice. Therefore, an emphasis on methodology and instrumentation is a fundamental need in order for material achievement to keep in sight of the advance of useful ideas. The volumes in this series are designed to try to meet the need in the field of biochemical analysis. The topics to be included are chemical, physical, microbiological, and if necessary, animal assays, as well as basic techniques and instrumentation for the determination of enzymes, vitamins, hormones, lipids, carboydrates, proteins and their products, minerals, antimetabolites, etc. Certain chapters will deal with well-established methods or techniques which have undergone sufficient improvement to merit recapitulation, reappraisal, and new recommendations. Other chapters will be concerned with essentially new approaches which bear promise of great usefulness. Relatively few subjects can be included in any single volume, but as they accumulate, these volumes should comprise a self-modernizing encyclopedia of methods of biochemical analysis. By judicious selection of topics it is planned that most subjects of current importance will receive treatment in these volumes. The general plan followed in the organization of the individual chapters is a discussion of the background and previous work, a critical evaluaV

vi

PREFACE

tion of the various approaches, and a presentation of the procedural details of the method or methods recommended by the author. T h e presentation of the experimental details is to be given in a manner that will furnish the laboratory worker with the complete information required to carry out the analysis. Within this comprehensive scheme the reader may note that the treatments vary widely with respect to taste, and point of view. It is the Editor’s policy to encourage individual expression in these presentations because it is stifling to originality and justifiably annoying to many authors to submerge themselves in a standard mold. Scientific writing need not be as dull and uniform as it too often is. In certain technical details, a consistent pattern is followed for the sake of convenience, as in the form used for reference citations and indexing. The success of the treatment of any topic will depend primarily on the experience, critical ability, and capacity to communicate of the author. Those invited to prepare the respective chapters are scientists who either have originated the methods they discuss or have had intimate personal experience with them. It is the wish of the Advisory Board and the Editor to make this series of volumes as useful as possible and to this end suggestions will be always welcome.

DAVIDGLICK

METHODS OF BIOCHEMICAL ANALYSIS

VOLUME 30

CONTENTS

The pH Jump: Probing of Macromolecules and Solutions by a Laser-Induced, Ultrashort Proton PulseTheory and Applications in Biochemistry. By Menachem Gutmun .............................................

1

Laser Photolysis in Biochemistry. By Shirley S. Chan and Robert H . Austin .....................................................

105

Density Gradient Electrophoresis of Mammalian Cells. By Abraham Tulp ......................................................... 141 Quantitation of Lipid Transfer Activity. ByJohn R. Wetterau and Donald B. Zalversmit ......................................... 199 Measurement of Oxygen Consumption by the Spectrophotometric Oxyhemoglobin Method. By Octavian Brirzu ...................................................................... 227 Historical Development and Newer Means of Temperature. Measurement in Biochemistry. By Robert L. Berger, Thomas R. Clem, Sr., Victoria A. Harden, and B. W .Mangum ....................................................... 269 Author Index

..................................................................

333

Subject Index

...................................................................

345

Cumulative Author Index, Volumes 1-30 and Supplemental Volume ...................................................... 353 Cumulative Subject Index, Volumes 1-30 and Supplemental Volume ..................................................... 965; vii

Methods of Biochemical Analysis, Volume 30 Edited by David Glick Copyright © 1984 John Wiley & Sons, Inc. METHODS OF BIOCHEMICAL ANALYSIS

VOLUME 30

The pH Jump: Probing of Macromolecules and Solutions by a Laser-Induced, Ultrashort Proton Pulse-Theory and Applications in Biochemistry MENACHEMGUTMAN BiochemGtly, Tel Auiu University, Tel Auiu, Israel

I. Introduction .................................................................................................. 11. Methodology and Instrumentation 1. Dynamics of Proton Dissociation from Excited Molecules ............... 2. Dynamics of Protonation mpounds .................. ............................................. A. Excitation Pulse ............. B. Monitoring Light ................................. ... C. Measuring Equipment ......,............................. D. Geometry of Excit earns ......................... .,,.....,,.,,....................... 111. Kinetics of Proton Dissociation 1. Determination of the Rate of Proton Dissociation .............. 2. The Effect of pK on Rate of Dissociation ......................................... 3. The Effect of the Solvent on the Rate of Proton Dissociation ......... 4. Proton Dissociation in Concentrated Salt Solution ........................... 5. Conclusion ................................................... ....,...,....,.. IV. Detection of the Proton by Its Reaction with the Proton Emitter ....................................... 1. Reactions in a Small, Open, Hydrating Microcavity ......................... ......................................... A. Steady-State Fluorescence B. Time-Resolved Fluorescence ............................... 2. Proton Dissociation in a Poorly Hydrating Site ................................ 3. Proton Dissociation-Recombination in the Inner Space ........................ .............................. ... of a Liposome ........... s ............................................ 4. Discussion and Conch V. The Reaction of the Proton with a Molecular Proton Detector ................. 1. The Detection of Protons by Their Reaction with the Ground... State Anion of the Proton Emitter ......................

1

1 3 4 4 5 5 5 6 6 7 10 10

15 21

22 24 24 27 .33 35 38 43 45

2

MENACHEM GUTMAN

2. The Reaction of Proton with Indicator ............................................. A. Dynamics of Proton Cycle in the Absence of Direct Proton Exchange ...................................................................... B. The Effect of Initial Conditions on the Macroscopic .................... ,............... Parameters ............................... sand Its Effect on the 3. The Direct Proton Exchange between Dynamics of the Proton Cycle ........................................................... 4. Alkalinization Pulse by the Conjugate Base of the Proton Emitter 5. Limitations and Inaccuracies ............................................... A. Reactants Concentration ...................... ................................... B. Accuracy of the Macroscopic Parameters .............................. VI. Kinetics of Protonation of High-Molecular-Weight Structures .................. 1. Protonation of Uncharged Target Adsorbed on Uncharged Carrier 2. T h e Effect of Charge on Rate of Protonation .................................. 3. The Effect of Postprotonation Reaction on the Dynamics ............... A. Simulation of Protonation of Adsorbed Bromo Cresol Green B. Classification of Postprotonation Reactions ........................... VII. Proton Transfer on the Surface of Macromolecular Structure .................. VIII. The Effect of Buffer on the Dynamics of the Proton Cycle ...................... 1. Two-Component Systems: Buffer and Proton Emitter ................... 2. Three-Component System: Emitter, Detector, and Buffer ............. A. Simulative Solution ............................... B. Effect of Initial Conditions .... .,...............,.....,...............

........,...........................

46 47 51

57 62 63 63 65 66 68 69 73 75 78 84

90 91 94 94 96 96 98 99 100 100 101

I. INTRODUCTION The chemiosmotic hypothesis, alias the Mitchell theory, was accepted in biochemistry with a whole glossary of new terms: proton-motive force, proteicity, proton well, local pH, proton-driven reaction, protogenic site, proton symport, etc. All these terms were intended to describe a specific thermodynamic parameter, or chemical mechanism, with the assumption that their meaning is well defined in the Biophysical-BiochemzcacalDictionary. Although these terms are made up of familiar explicit words, the interpretation of some composite terms is only vaguely implicit. Presently, it is generally accepted that the free energy released by proton transfer between phases of different electrochemical proton potential is converted into other forms of chemical energy (ATP synthesis, active transport, redox reaction). Still, the identity of these phases is not agreed upon. The phases are identified with the whole aqueous bulk, a thin nearly

T H E pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

3

unimolecular layer on the surface of a membrane, a single proton trapped in an active site, or even an anhydrous hydrochloric acid in the dry lipid interior of a membrane. The various models describing proton transfer through a transmembranal protein consider an array of hydrophilic semirigid carriers spanning the protein, a water molecule channel, an ice-like microthread, or even approximate the proton channel by Al(0H)s crystal at 300°C. Proton transfer in biochemical systems is measured, in most cases, as an outcome of external force (ATP, redox potential, etc.) mediated by an enzyme. Enzymic turnover is a million to a billion times slower than the basic events of proton transfer. Because of this huge difference in time scale, enzyme-driven proton transfer is blurred by the noncoherent catalysis. By the time the first turnover is completed, the proton had ample time to equilibrate with the whole bulk of the solution. Because of these reasons, my colleagues and I initiated a few years ago a detailed study of proton transfer in an aqueous system, where the event is synchronized by a laser pulse. This technique, using signal averaging, retains the temporal parameters of the event and allows the evaluation of the probabilities of finding a proton in putative environments assigned for it by the different bioenergetic models. During these studies, it became apparent that proton transfer is an extremely sharp instrument for gauging the water in the immediate environment surrounding the site of dissociation. It turned out that the general biological solvent, the water, acquires different properties at the site where biochemical reaction takes place-the surface of the enzyme. These local properties of the water can be measured through the technique of the laser-induced proton pulse, free of perturbation caused by the huge mass of the bulk water. In this chapter, I shall describe the basic methodology of the laserinduced proton pulse. Starting with the initial event of a synchronous proton dissociation, going through the reaction of a proton with other solutes in a true solution, and ending with the complex multiphasic system of protons, macromolecules, and interfaces associated with the real life of biochemical reaction. In each level of complexity, I shall point out the pertinent information available for interpretation and the mode of mathematical and physical analysis. In some cases, I shall also reflect the conclusions on current hypotheses of biochemical proton transfer. 11. METHODOLOGY AND INSTRUMENTATION

The experiments described in this chapter can be carried out in any laser laboratory equipped for monitoring fast photochemical reactions.

4

MENACHEM GUTMAN

1. Dynamics of Proton Dissociation from Excited Molecules This reaction is observed through time-resolved fluorescence measurements. T h e sample is excited by a short laser pulse and the fluorescence intensity at the proper wavelength is followed with time. Thelifetimeofthemeasuredeventsvariesbetween l00psecto -20nsec. The time constant of the measured reaction limits the duration of the excitation pulse. Unless the pulse is shorter than 10%of the lifetime of the measured reaction, the observed signal must be deconvoluted to correct for the time profile of the perturbing event. T h e intensity of the excitation pulse is not critical, yet it is advisable to use low-energy density. High-energy flux enhances the probability of undesired two-photon effects. T h e light source for time-resolved fluorescence can be a nanosecond pulse of Blum-line nitrogen laser, triple harmonics of yttrium-aluminumgarnet (YAG) laser, second harmonics of mode-locked dye, or gas laser. T h e fluorescence decay can be measured with a streak camera, a very fast photomultiplier tube-like Hamamatsu 1294U attached to Tektronix transient digitizer, a box car integrator, or photon counting.

2. Dynamics of Protonation of Ground-State Compounds The reaction is followed through transient absorbance measurements. The sample is excited by intensive laser pulse (0.2-2 MW/cm2) and absorbance changes in the irradiated volume (usually 0.05-0.1 ml) are monitored by a probing light beam at the proper wavelength (see Figure 1).

Figure 1. Optical arrangement and elements needed for transient absorbance measurements. C, observation cell; PL, pulse laser; CWL, C W laser; Mr, mirror; F1, filter; MC, monochromator; PM photo multiplier; Trig, triggering photo diode; T R , transient recorder; AV, signal averager; COM, computer; X-Y, XY recorder.

THE pH JUMP PROBING OF MACROMOLECULES AND SOLUTIONS

5

A. EXCITATION PULSE

The excitation pulse should be less than 10%of the fastest time constant that is measured. A pulse of suitable duration (1- 10 nsec) and energy (0.5- 10 mJ) can be obtained by many commercially available nitrogen or excimer lasers. High-energy input into the solution (more than 10 mJ) can lead to rapid accumulation of undesired photoproducts. Thus, it is better to use a short (1-2 nsec), less intensive pulse (0.5-2 mJ) than massive (5-50 mJ) longer ones (5-20 nsec). The length of the proton cycle is 10-300 pec. Thus, even at high repetition rate (100-400 Hz) available with some gas lasers, the system will relax to its prepulse state before the next pulse. Thus, high repetition rate cannot compensate for low peak power of the laser. I found it practically impossible to use a peak power of less than 50 KW. B. MONITORING LIGHT

The monitoring light should fulfill the following requirement: Its energy-density modulation at the entrance slit of the monochromator should be higher than the energy of the fluorescence emitted from the observation cell. About 1- 10%of the MW excitation pulse is emitted as fluorescence over a wide spectral range. Even if 0.1% of the fluorescence falls at the wavelength of the monitoring beam, it amounts to 10- 100 W of light energy. To prevent it from saturating the photomultiplier, it must be damped below the energy of the oncoming signal. The simplest way to reduce the fluorescent light is to keep the monochromator far from the reaction cell, 1-3 m. The monitoring beam should probe only the irradiated volume, that is, not more than 1- 1.5 mm in diameter, and all of its energy should reach the entrance slit of the monochromator-without the assistance of a lens. A lens will focus both monitoring and fluorescent light and no advantage is gained. The fluorescence spans a wide spectral range, thus the narrower is the wavelength of the probing source, the lesser will be the incremental energy of the fluorescence. Thus the monitoring light should be highly monochromatic collimated intensive beam, that is, the output of a cw laser. C. MEASURING EQUIPMENT

The measuring equipment needed to follow transient absorbance can be as simple as a fast oscilloscope and a Polaroid camera. But a transient recorder coupled to signal averager, q recorder, and computer have a certain advantage.

6

MENACHEM GUTMAN

The measured signals are small-in most cases less than 2-3 mV deviation from the constant voltage of the photomultiplier (-50 mV at 50 R entrance impedance) which corresponds in the above case to AA = log (50 + 2)/50 = 0.017. Many events (50-5000) should be recorded to obtain a clear signal. A few thousand pulses of a megawatt photon flux can bleach even photostable compounds. To avoid this outcome, it is highly recommended to mix the content of the cell. As the irradiated volume is rather small (0.05-0.1 ml), moderate stirring can replenish the irradiated volume with fresh reactants. Under such conditions, 10,000-50,000 events can be recorded without decrease of intensity of signal. Stirring cannot remove transient photoproducts with lifetime of few nanoseconds to -300 Fsec. These photoproducts may appear in varying quantities, depending on the proton emitter, energy density, pH, impurities, etc. Most of these photoproducts, solvated electrons, stable free radicals, and triplets are formed by two-photon reaction and their yield is higher with ground-state ionized emitters (+O-) than with +OH. Thus, it is recommended to keep the pH of the solution below the pK of the proton emitter and to lower the excitation energy density as long as it does not reduce the quality of the measurement. Sometimes these precautions are insufficient. Under these conditions a two-channel signal averager can be very useful for subtracting the imprint of the transient photoproducts from the measured event. D. GEOMETRY OF EXCITATION A N D PROBING BEAMS

The irradiated volume should not be bigger than needed to obtain a good overlap with the probing light. The perpendicular-crossing beams are the geometry of choice (Figure 1), because they allow the use of a constantlength optical path and probing the space very close to the front surface of the cell, where the excitation pulse is at its maximumLThis beam geometry spreads the irradiating beam and lowers the energy density. Lasers with low output should be focused to a small spot that necessitates the colinear alignment of the probing beatn (Figure 1). In such an arrangement, care should be taken that the sample is optically thin. High absorbance of the excitation pulse will render most of the probed space inactive, with concommitant reduction of the measured signal. 111. KINETICS OF PROTON DISSOCIATION

In the classical chemistry of aqueous solutions, proton dissociation is treated as a simple first-order reaction. Actually it is a very complex

THE pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

7

reaction involving more than one step (Huppert et al., 1982).The initial event is the charge separations:

AH,, G A;,

- - - H+

where the dissociating bond of the proton donor stretches to a distance where charge separation takes place and an ion pair is formed. The lifetime of this transient state is very short-comparable with the vibration time, for an OH bond it is about 30 fsec. Compounds with low pK will reach the A- - - . H + state with high frequency whereas those with high pK will have to go through many vibrations until the energy of stretching will overcome the threshold to form the ion pair. The A- - - H+ state is very unstable, being subjected to enormous electrostatic attraction. Thus, no dissociation will take place without the assistance of the solvent. The positively charged proton interacts with the dipole of the surrounding water molecules, and a hydration shell is formed within a time frame of 20-50 fsec (Rao and Berne, 1981), (Warshel, 1982). The stabilized proton can now diffuse away from its conjugated base, but this diffusion, still within the radius of the Coulomb cage, is subjected to electrostatic forces. The rate of the escape of the proton out of the Coulomb cage (where electrostatic force is higher than the thermal energy) is given by Equation (1) (Hauser et al., 1977; Eigen et al., 1964; Eigen, 1964.)

k,

32D

=-

72

6 1 - e-'

where XD is the sum of the diffusion coefficient of the proton and the conju ate base, T is the radius of encounter, and 6 is given by 6 = Z I Z p e lwkT. For r = 5A and residual charge of the conjugate base of - 1, -2, -3, the rate of proton escape out of the Coulomb cage will be 5 x lo", 2 X lo", 0.7 X lo", sec-l, respectively (Eigen et al., 1964). As the rates of proton hydration and escape are comparable for all acids, the big difference between kdiss (or pK) of acids stems from the probability that the dissociating bond will stretch to its A- - - - H+ state. This correlation between the pK and the rate of dissociation is described by the empirical valance bond formalism of Warshel (1982).

B

1. Determination of the Rate of Proton Dissociation

In their first electronic singlet state, hydroxy aromatic compounds are much stronger acids than in their ground state (Weller, 1961; Gutman et al., 1981; Schullman, 1977). The pK shift ApK = pK* - pKo can be

8

MENACHEM CUTMAN

estimated from the wavelength of emission of the neutral and the ionized excited states, using the Forster cycle calculation (Forster, 1950).

where u(+~)-,and u ( + ~are ~ the ) frequency of maximal emission of 40and +OH, respectively,(For compilation of pK* of many compounds, see Ireland and Wyatt, 1976). The rate of proton dissociation can be obtained, either by steady-state or time-resolved measurements. The reaction describing the proton dissociation from the excited molecule is summarized in Scheme I

+OH

k

& $0-*+ H + k- 1

Scheme I The excited molecule can decay, by irradiative ( k ( f ) ) and nonirradiative (Iqn,.))processes into its ground state with a time constant 70 = (k(f) + k(nr))-i. Alternately, it may first dissociate and then decay as an excited anion by the same processes (7'0 = (k'(f) + k'("&*). In a case where dissociation is faster than the decay of +OH*, (Itl>> k ( f ) k,,,) the emission of +OH* will decline very rapidly, due to the depletion of population by the dissociation reaction. The steady-state reflection of this rapid dissociation will be a very weak emission of the +OH* species. Slow dissociation of +OH*, or rapid recombination of the excited anion with the proton (H+-kL1>k1)will enhance the emission of +OH* (observed by steady-state fluorescence)and prolong the decay time of +OH* as observed by time-resolved measurements. The interrelation between the kinetic constants and the steady-state fluorescence of the two species is given by the following equations (Weller, 1961).

+

where is the ratio between the quantum yield measured under the experimental condition (4) and that measured under conditions where

THE pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

9

dissociation is totally supressed (40).This ratio can be approximated by the intensities of the +H* emission measured under experimental conditions and when +OH is dissolved in strong acid or in organic solvent where no dissociation takes place. +'/+A is the ratio of $0-*emission under the experimental conditions (+') and under conditions where no recombination takes place (+A), that is, at pH>>pK*. T~ and TA are the decay lifetime of +OH* and +O-*, respectively. T~ can be approximated by the decay time of +OH* measured at pH << pK* and 7;)is that measured for +O-* at pH>>pK*. By dividing Equations (3) and (4), we obtain

Drawing the ratio of the quantum yields (or the relative intensities) vs. H+ can yield the rate constants, given that the lifetimes of the two states are known. Because of the simplicity of the measurement and the availability of spectrofluorimeters, this method is readily applicable. Still, the lifetime of +OH* and $0-*should be measured and verified for each experimental system. The other method for calculation of the rate of proton dissociation calls for time-resolved measurements. The differential rate equations for OH* and ~$0-*

+

can be integrated to give the time dependence of these two species.

The apparent, macroscopic time constants y1 and y2 are related with the partial rate constant

10

MENACHEM GUTMAN

According to these expressions, the intensity of the +OH*emission will decay as a biexponent, the rapid initial phase y2 represents the reaction as it proceeds until the velocity of dissociation and recombination become equal. The slower phase y1 represents the decay when the two populations (+OH* and +O-*) are in equilibrium with each other. The relative amplitudes of the two phases A r = (a2, - y1)/(y2- yl) and the macroscopic rate constants (y1,y2) allow one to calculate the rate of all partial reactions. The agreement between rate constants calculated by time-resolved measurements and steady-state kinetics is usually'good. In a limiting case, where the rate of recombination is much slower than dissociation pKo > pH >> pK*, the amplitude of the slow phase representing recombination will diminish to zero and the emission of the +OH* state will decay in a single exponent curve with a macroscopic rate constant y2 = k l + k(f,,,) = k l .

2. The Effect of pK on Rate of Dissociation The rate of proton dissociation is controlled by three parameters: the frequency of ion pair formation, the rate of stabilization of the proton by hydration, and the rate of escape out of the Coulomb cage. Measurements carried out in dilute salt solutions, that is, 10- l O O m M , will not be influenced by the two later steps. The activity of the water is invariable whereas the ionic atmosphere will screen the electrostatic attraction. Under such conditions, the rate of dissociation should be a direct function of the probability that the stretching covalent bond will reach the dissociation distance. As demonstrated in Figure 2, this expected correlation is observed over a wide range of pKs. Under these conditions, a reversible dissociation will comply with the relationship Kdiss = k l / k - l . As the recombination reaction for all acids is a diffusion-controlled reaction, we = 10'' * Kdi,,(sec-'). can approximate kl = k-1

-

3. The Effect of the Solvent on the Rate of Proton Dissociation The solvent can affect the rate of dissociation by three independent mechanisms: 1. The rate of proton hydration.

2. The rate of proton diffusion. 3. The dielectric constant of the medium. The first mechanism has already been discussed. The latter two both affect the rate at which the proton escapes out of the Coulomb cage. Diffusion of proton is mediated through rapid exchange of hydrogen bond-covalent bond through the quasistatic continuous network of hy-

THE pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

11

10

Q A

2 8

3

* B

7 6

5

4 3 2 1

1

2

3

4

6

PK

6

7

8

0

Figure 2. Correlation between rate constant of proton dissociation and pK of acids. (M) 8-hydroxypyrene- 1,3,6-trisulfonate, excited state; (U)2-naphthol-3,6-disulfonate,excited state; (A) 2-naphthol-6-sulfonate, excited state; (V) 2-naphthol, excited state; (0)Bromo Cresol Green; (0)Bromo Cresol Purple; (V)Bromo Fymole Blue; (A) 8-Hydroxypyrene 1,3,6 trisulfonate, ground state; (8)2-naphthol 3,6 disulfonate, ground state; ( 0 )2naphthol, ground state.

drogen bonds (Belch et al., 1981). Any rupture of the network will shorten the distance over which the proton can migrate at a rate faster than the relaxation time of the network. Thus, organic solvents, may shorten the fast passage stretch, and slow the proton on its way out of the Coulomb cage. The low dielectric constant of organic solvent will expand the size of the Coulomb cage, which may also lower the probability of successful escape. T o evaluate the relative contribution of each mechanism, Huppert et al., (1981) and Huppert and Kolodney (1981) measured the rate of proton dissociation in organic solvent-water mixtures. The effect of organic solvent on proton dissociation is easily demonstrated through the emission spectrum of HPTS (Figure 3). In water, the rate of dissociation is so fast that 95% of the emission is at the wavelength

12

MENACHEM GUTMAN

450

500

nm

555

Figure 3 . Fluorescence emission spectra of B-hydroxypyrene-l,3,6-trisulfonate in water (---) and in 40% vollvol ethanol water.

of the anion (~$0~"). Increasing the mole fraction of ethanol in the mixture enhances the emission of the neutral species (@OH*)at the expense of that of @O-*,that is, the rate of dissociation is slowed to the extent that the radiative (plus nonradiative) decay of +OH* can successfully compete with the dissociation. T h e kinetic reflection of the enhanced emission of the neutral form is demonstrated in Figure 4. In pure water, @OH*emission decays rapidly (7 = 100 psec) due to dissociation (Figure 4A), but in 50% (vol/vol) of ethanol in water, it is already 25 times longer (Figure 4B) and so is the rise time of 40-* (Figure 4C). T h e dependence of the dissociation rate on the mole fraction of the organic solvent is depicted in Figure 5. As seen in Figure 5 , the rate of dissociation decreases exponentially with the mole fraction of the ethanol. This decrease in rate of dissociation cannot be attributed to the effect of the solvent on the dielectric constant of the solution. At Xethanol= 0.2, the dielectric constant of the mixture is 66.2 (vs. 77.5 of water), but the rate of dissociation is slowed by an order of magnitude. T h e proton conductivity of the water-ethanol mixture decreases with the mole fraction of the solvent, but this decrease is not steep enough to account for the measured effect on the rate of dissociation (see Figure 5). This reasoning

" O S

cn-

8 hydroxy p m n e 2.3,6 T r i sulfonate

Time (psec) (A)

HPS IN E T H A N O L WATER MIXTURE

5 0 VOL.% E T H A N O L O B S E R V E D A T 54.7"

L0

2000 T I M E ( psec) ( B)

Figure 4. Time-resolved fluorescence of 8-hydroxypyrene-1,3,6-trisulfonate in waterethanol mixture. The samples were excited by a 6-psec laser pulse (352 nm) and the emission was recorded by Hammamatsu C939 streak camera combined with optical multichannel analyzer (PAR 1205 D): (A) the emission of the undissociated state, measured in pure water at the spectral range 400-470 nm; (B) the emission of the undissociated state (400-470 nm) measured in 50% voVvol ethanol-water mixture; (C) fluorescence rise time (540 nm) of the dissociated excited form, 45% voVvol ethanol- water mixture.

13

I

HPS IN 'ETHANOl! WATER 'MIXTURi 4 5 VOL.% ETHANOL

fl

ANION FLUORESCENC RISE

J

I

2 3 TIME (nsec)

4

5

(C)

Figure 4. (Continued)

30

0

3. I MOL X E t O H

Figure 5. The dependence of the rate of proton dissociation from excited S-hydroxypyrene- 1,3,6-trisulfonate on the mole fraction of ethanol in water, and the respective proton conductivity of the mixtures. The rate of proton dissociation was measured by time resolved (0)or steady-state (D) fluorescence. The proton conductivity of the solutions (A)is normalized for pure water conductivity. Data taken from Erdey-Grutz and Lengyel (1977).

14

THE pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

15

suggests that the main effect of the organic solvent is to delay the hydration of the proton, increasing the probability of abortive dissociation attempts. Still the present information is insufficient to state whether the slow hydration is the only mechanism that slows dissociation, or whether the expansion of the Coulomb cage and reduced prototropic mobility contribute also to the observed phenomena. 4.

Proton Dissociation in Concentrated Salt Solution

Figure 2 demonstrated the free-energy relationship for proton transfer from various donors to the same acceptor, H20. In the present section, we shall extend these studies by relating the rate to the chemical potential of the acceptor, the water. The water-alcohol mixtures described above are not convenient for measuring the role of water in the proton dissociation reaction. The large Coulomb cage (3081 for 8-hydroxypyrene-1,3,6-trisulfonate (HPTS) (Hauser et al., 1977) and its expansion upon lowering of the dielectric constant, introduces a nontrivial contribution of the ion pair recombination to the observed reaction. Concentrated solutions of strong electrolytes are a much better system. At concentrations above 1M of strong electrolyte, the electrostatic screening shrinks the Coulomb cage to be about the molecular diameter of HPTS. This effective electrostatic screening practically eliminates the role of the Coulomb cage in the recombination. The dissociative step itself will already place the proton out of the range of the electrostatic attraction. Under these conditions, we can investigatethe primary event of proton hydration. What is more, as hydration is a femtoseconds event (Rao and Berne, 1981; Warshel, 1982), only those water molecules that are in the range of the hydration shell can stabilize the dissociating proton. Consequently such studies may be used to gauge the properties of water in microenvironments, such as the active site of an enzyme. The effect of salt on the rate of proton dissociation from excited hydroxypyrene trisulfonate is demonstrated in Figure 6. The effect on the steady-state fluorescence is similar to that shown in Figure 3. The emission of the neutral form is intensified while that of the anion decreases. Figure 7 relates the rates of proton dissociation, as measured by timeresolved fluorescence and steady-state fluorescence with the molar concentrations of LiC104 and MgC12. As in the case of organic solvents (Figure 5),the decreased proton conductivity is insufficient to account for the decrease in the rate of dissociation. The advantage of the strong electrolyte solution, with respect to organic solvent, is their adherence to Rault’s law. Thus the activity coefficient of the water can be easily obtained from the vapor pressure data (Grollman, 1928; Kracek, 1928).

I

I

I

I \ 500

1000

I

I

rF = 180 psec

1500 2000 2500 3 )OO

TIME ( p s e c )

Figure 6. Time-resolved fluorescence of the neutral form of 8-hydroxypyrene- 1,3,6-trisulfonate in concentrated LiC104 solution. Measurements were carried out as in Figure 4. Line (a) 1M LiCIO,; (b) 2.5 M LiCIO,.

Figure 7. T h e variation of the rate of proton dissociation from excited hydroxypyrene trisulfonate on the molar concentration of the salt: (0,0 ) time-resolved fluorescence measurements; (0,U) steady-state fluorescence measurements; (A)proton diffusion coefficient, normalized for pure water (data from Glietenberg et al. 1968). Open symbols, MgCI,; closed symbols, LiCIO,.

16

THE PH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

17

Figure 8 demonstrates the linear log-log correlation between the rate of proton dissociation and the activity coefficient of the water in concentrated solutions of NaCI, LiBr, and MgCI2. The same correlation has been measured for five more compounds whose kdiss values (measured in pure water) differ by five orders of magnithdes (Figure 9). T h e slope of the lines vary with the structure of the compound. Compounds which have a hydrophylic moiety (SO;) or polarizable substituent (Br) in ortho position with the dissociating proton (2-naphthol-3,6-disulfonate and Bromocresol Green) are somewhat less affected by water activity than compounds where a hydrophylic moiety is in a more remote position (2-naphthol-6-sulfonate) or no hydrophylic substitution at all @-naphthol). The realiability of this fluorometric technique for determination of u ( ~ , Ois ) demonstrated in Figures 10 and 1 1. Figure 10 relates the activity of water with the molar concentration of NaC1, as determined by the rate

1

I

u

I

I

46

I

1

0.55

acH,O)

Figure 8. The free-energy relationship between rate of proton dissociation from excited 8-hydroxypyrene-l,3,6-trisulfonate and the activity coefficient of the water. Water activity coefficient was varied by concentrated solution of NaCl (El),LiBr(A), and M g C12 (0,O).

18

MENACHEM GUTMAN

t

1

log

aH20

Figure 9. The dependence of the proton transfer rate on water activity for various excited hydroxy aromatic compounds. (0) 2-naphthol-3,6-disulfonate; (A) 2-naphthol-6,8-disulfonate;(A)8-hydroxypyrene-1,3,6-trisulfonate;(+) 2-naphthol-6-sulfonate;( 0 )2-naphthol; (m)Bromo Cresol Green (ground state). Note the discontinuity of the ordinate.

of proton dissociation from two proton emitters or as measured by the colligative properties of the solution. Figure 1 1 demonstrates the equivalence of u(H?O)as estimated for the same salt solutions by the rate of proton dissociation from two proton emitters. Thus, the kinetic method for determination of u ( H , O ) can be regarded as a reliably accurate technique. The most trivial explanation for the effect of electrolytes on rate of proton dissociation is to consider the effect of salts on the dielectric constant of the solution (see also Equation 1). In concentrated salt solutions, a considerable fraction of the water moiecules are oriented in an hydration shell around the ions; thus, their dielectric constant is smaller than in pure water (Hasted et al., 1948). A decreased dielectric constant will accelerate ion-pair recombination and slow down ion-pair separation.

THE pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

1

2

3

NaCl (M)

4

19

5

Figure 10. The reduction of water activity by high concentration of NaCI. Water activity was measured by the rate of proton dissociation fromexcited hydroxypyrene trisulfonate (A), excited 2-naphthol-6-sulfonate (O),or using the published vapor pressure (0)(Grollrnan, 1928; Kracek, 1928).

The combination of these two effects will lower the probability of proton dissociation in accord with our observation. Yet, this explanation is not applicable for our case. An appreciable decrease of the solution’s dielectric constant occurs above 1 molar of electrolyte. At such concentration (1M) the ionic atmosphere will effectively screen the proton from the electric charge of the conjugated base at a distance of 3 A. Under such effective screening, the contribution of the 10%decrease of the dielectric constant (at 1M NaCl) will have a trivial contribution. Apparently neither electrostatic interactions nor reduced diffusibility of protons is the major cause for the decrease in the proton transfer rate. As these effects are dominating in ion-pair recombination and ion-pair separation, we have to focus our attention to the primary event in proton dissociation: ion-pair formation. In this reaction, the hydrogen of the OH bond of the excited parent molecule forms a hydrogen bond with the nearest H 2 0 molecule, which itself is hydrogen bonded to other water

20

MENACHEM GUTMAN

7

aH20

1

(Pyrene)

Figure 1 1. Correlation between water activity coefficient o f MgC1, and NaC104 solutions as estimated from the rate of proton dissociation from two proton emitters, 2-naphthol6-sulfonate (ordinate) and hydroxypyrene trisulfonate (abcissa). (0)MgCI,; (m)NaC104.

molecules nearby. If the proton moves by 0.5 A, along the lineconnecting it to the nearest H20, the OH bond breaks and H 3 0 + is formed. T h e enthalpy of proton hydration is 270 kcal/mol, whereas the enthalpy of formation of H 3 0 + is estimated to be 170 kcal/mol (Conway, 1964). T h e energy difference of 100 kcal is attributed to further solvation of H 3 0 + by additional water molecules. Within the timeframe of proton dissociation, a stable hydronium ion must be formed: otherwise, the proton will revert to the parent molecule. Molecular dynamic simulations indicate that the formation of a hydration shell around the central ion is completed within 0.05 psec (Rao and Berne, 198 1). Just because the stabilization of proton in the hydration complex is comparable with the OH vibration time (0.03 psec), any perturbation at this step might be crucial for the rest of the reaction to occur. T h e dynamics of proton hydration is too fast to be directly measured, but the thermodynamics of stepwise hydration of proton was measured as clustering of water molecules around free protons in a gas phase (Searcy and Fenn 1974; Kebarle, 1975). Clusters with varying size were observed

T H E p H JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

21

and the respective enthalpy of formation was calculated. The difference in enthalpy of hydration of a proton vs. the cluster number n,designated as -AHo,,,+ shows a remarkable decrease while increasing the cluster number n. The hydration enthalpy difference between a monomer H 3 0 + and a dimer is AHl,z = -32 kcal/mol, while AHo2,3= -22 kcal/mol. These values are comparable with the results obtained by quantum mechanical calculations (Kraemer and Diercksen, 1970;Newton and Ehrenson, 1971). The hydration enthalpy AHo,,,,+l is reaching a limiting value of about - 10 kcal/mol when the cluster number is about 10. Analogous to these results, the enthalpy of proton hydration in solution will also increase with the size of the hydration complex. Yet, in liquid water, one exception should be made: to increase the size of the complex by one water molecule, a water molecule must first be removed from the bulk, with energy investment of 10 kcal/mol (heat of evaporation of water). Therefore, the hydration complex of a proton will not exceed the state where the energy gain of further hydration will be comparable with the heat of evaporation. Using the results of Kebarle (1975) and Searcy and Fenn (1974), we estimate that the hydrating complex in dilute electrolyte solution (u(H,*) = 1) will be of 10 water molecules, or less. In concentrated salt solutions, the vapor pressure is lower than that of pure water, and hence it exhibits reduced water activity. This phenomenon is explained by the fact that a considerable fraction of the water molecules are associated with the hydration of the salt ions. The binding energy of these water molecules (which forms the first and the second hydration shells) to the center ion is larger than 10 kcal/mol; therefore, they are less likely to participate in the hydration of the newly formed proton. To observe successful proton dissociation, the thermodynamic stable complex must be formed within the ion-pair lifetime. The depletion of the solution from water molecules available for this reaction will lower the probability of the successful dissociation. As demonstrated in Figure 9, this function decreases with the activity of the water in the solution. 5. Conclusion

The rate constant of proton dissociation is extremely sensitive to its environment. Except for very strong acids (pK < 0), no dissociation will take place unless water molecules are in the immediate vicinity to act as proton acceptors. What is more, these water molecules must be free to react with the dissociating proton at a time scale comparable with the vibration time (30fsec). During such a short period, the water molecules are practically fixed in space and only those molecules that are at the

22

MENACHEM GUTMAN

distance of the first and second hydration shell will participate in the reaction. The molecular dynamic simulations (Rao and Berne, 1981) demonstrated that the total hydration process of a positive charge ion take place within a shell of less that 4 A. As long as the activity coefficient of the water is measured in homogeneous solution, the kinetics of proton dissociation is just another technique for measuring and probably not the most accurate one. On the other hand, if we can introduce the dissociable proton into a defined microspace, the kinetics of its hydration can be used as a specific method for measuring 0 at the microspace. 2 ) In the next section, we shall describe how the dissociation dynamics are applied for measuring the properties of the space bounded by an active site of a protein.

IV. DETECTION OF THE PROTON BY ITS REACTION WITH THE EXCITED ANION OF THE PROTON EMITTER Immediately after its formation, the hydrated proton reacts in a diffusion controlled reaction with any component in the solution. Reaction of the proton with stable compounds may be followed at any time scale, but the reaction with the excited anion is measurable only during the few nanoseconds of 40-* lifetime. This short observation window through which the reaction is monitored focuses our measurement only to those protons that are at a diffusion distance (within the limited time frame) from +O-*. In a homogenous solution the reaction volume, where $0-* probed for proton, is defined by a radius of r = = ( D L is the diffusion coefficient of H+ and T’ is the lifetime of +O-*). For excited anion with T ’ -6 nsec, r -70 A. If the proton emitter is placed in a microcavity, such as active site of enzyme, the resolution of the observation increases as the probed volume is smaller than the reaction volume. Consequently, the recombination reaction will be faster, reflecting the high formal concentration of protons in the site. If the microcavity is open to the bulk, some of the sites will lose their protons within the +O-* lifetime. A proton that emerges into the bulk is diluted to the bulk pH. In neutral solutions, no proton reentry will take place during the observation window. Thus, those sites that lost their proton will be clearly distinguished from the rest, they are equated with the $0-*molecules that cannot be reprotonated. The detailed analysis of the results furnishes the formalism for probing the interior of a protein (or vesicle) with hydrated protons and concludes about the physical properties of the confined space. It must be stressed that this type of measurements is a close

THE pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

23

approximation to a proton in a box. Each cavity contains a single $0-* and an extra free proton, and within the observation time there is no exchange of matter between the “boxes.” The first reaction taking place in the microscope is the reversible dissociation of the excited state

where the brackets represent events taking place in the cavity. The exchange of the proton between the cavity and the bulk is given by a second reaction {+O-*(aq) + H+(aq))

k23

k32

{+0-*j + H+(aq)

(12)

This rather simple kinetic model is getting obscured by the fact that we cannot measure the protons but the fluorescence of +OH* and +O-*, each decaying to its ground state with a characteristic lifetime (T and T’, respectively). It should also be remembered that the lifetime is affected by radiative and nonradiative transition and the rate of the nonradiative transition may vary with the conditions prevailing in the microspace (Kosower et al., 1975 and 1978; Kosower and Dodiuk, 1978; Dodiuk et al., 1979). The combination of the chemical reactions and the decay of reactants is described in Scheme I1 as a sum of three populations N 1 , N 2 , N 3 corresponding with the three states of the proton and the emitter in the cavity.

24

MENACHEM GUTMAN

and for simplicity we shall define

These three coupled differential equations were solved by the Laplace transform method using the initial conditions Nl(,=o, = 1; N Z ( ~ ==~ ) N3(t=o) = O.* ?'he three populations can be monitored by their emission at the respective wavelength. The emission at hv2 corresponds with N2 and N3 populations and the dynamics are inherently complex. Thus, we shall rather follow the events at hul emission, where only the single population N 1 is observed. The integrated rate equation for N is given in Equation (18). Nl(t) = (k2 - Yl)e-Y't

+ (y2 - k2)e-Y2t

(18)

The function is a sum of t w o terms, one is characterized by a fast (y2)and the other by slow (y1) macroscopic rate constants. The macroscopic rate constants y1 and y2 are the roots of the Laplace polynomial and are given by Equation (19),which relates the macroscopic with the microscopic rate constants Y1.2

=

(kl

+ k 2 ) * V(k1 + k 2 Y 2

- 4 (klk2 - k 1 2 M

(19)

We shall use the expression for analyzing the results of three experimental systems, each representing a typical case of emitter in a microcavity. 1. Reactions in a Small, Open, Hydrating Microcavity A. STEADY-STATE FLUORESCENCE

8-hydroxypyrene- 1,3,6-trisulfonate is a very convenient proton emitter. Its ground state pKo = 7.7 is sufficiently high to work under conditions

'These initial conditions apply only when the ground state of the proton emittor is mostly in its protonated state, i.e., pHGpK,, - 2. If the experiment is carried out under conditions where [+OH] = [+O-],the analyst must excite +OH at a wavelength were 40- has a low extinction coefficient or alternatively modify the initial condition terms during the integration.

THE pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

25

where more than 98% is in the +OH state, and its low pK* (0.5)ensures a very rapid dissociation (Smith et al., 1979; Gutman et al., 1981). The molecule is highly soluble in water, but due to its big hydrophobic surface (Bondi, 1964; Valvani, 1976), it may participate in strong hydrophobic interactions (Melander and Horwath, 1977; Horwath et al., 1976). Thus, in spite of the three sulfono groups, it binds with high affinity to bovine serum albumin (Gutman et al., 1982) and to the heme-binding site of apomyoglobin (Gutman et al., 1982a) (Ka = 7 X 106M-' and 9 x ~ o ~ M -respectively.) ', Figure 12 (line A) depicts the emission spectrum of hydroxypyrene trisulfonate dissolved in diluted buffer (pH 5.0). At this pH, the ground state is fully protonated (pKo = 7.7), but not so the first excited singlet state (pK* = 0.5). The excited molecules dissociate and 95% of the emission is at the wavelength of the excited anion (515 nm). The dissociation can be prevented if the compound is dissolved in acid solution, pH < pK*, such as 2MHCl (line B). Under such conditions, we observe the emission of the neutral form with maximum at 445 nm. Upon ligation to apomyoglobin, the fluorescence of hydroxypyrene trisulfonate consists of two

excitation 400 nm

.-.

450

QOH'HCI

500

nrn

550

Figure 12. Steady-state fluorescence emission of hydroxypyrene trisuIfonate. Fluorescence of 20pM hydroxypyrene trisulfonate. (A) at pH 5.0, (B) in 2M HCI, and (C) in 30@ apomyoglobin pH 5.0. Excitation at 400 nm. Fluorescence measured in arbitrary units at identical instrumental set up.

26

MENACHEM GUTMAN

bands (line C) of equal intensity, typical for the anionic state (515 nm) and the neutral form, slightly shifted to shortened wavelength (435nm). Such shifts are common in organic solvents (Kosower et al., 1975;Kosower, 1968).The total light emission of the two bands of bound hydroxoypyrene trisulfonate is 84% of the emission of the free ligand. The same effects are observed with hydroxypyrene adsorbed to bovine serum albumin (Figure 13).The emission of the neutral form increases to 30% with a distinct blue shift observed as a broad peak between 425 nm and 445 nm. T h e overall emission intensity of bound hydroxypyrene (measured as the area below the emission curves) is 64% of that of the free molecule, probably reflecting the effect of the local dielectric constant. The enhanced emission of the neutral form recorded in these two examples may originate either from rapid recombination of Hf with $0-*(see Scheme 11) or from slow dissociation of +OH* due to reduced activity of water in the cavity (see Figure 9).The contribution of each mechanism to the overall observation cannot be deduced from steadystate fluorescence measurements and necessitate kinetic analysis according to Equation (18).

7-

6S-

Figure 13. Fluorescence emission spectra of free and protein-bound hydroxypyrene trisulfonate. I . I S M hydroxypyrene trisulfonate in water (A) or 1% bovine serum albumin (B) adjusted to pH 6.0. The samples were excited at 400 nm and the emission spectra were measured under identical instrumental set up.

T H E pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

27

B. TIME-RESOLVED FLUORESCENCE

Figure 14 depicts the shape of the laser excitation pulse and the most initial emission of hydroxypyrene bound at the heme-binding site of apomyoglobin. A rapid decay of [+OH*] population is observed, but it slows down after 100 psec (about the middle of the frame). Figure 15 was recorded at a slower streak speed and resolves the phases. The emission of the neutral form decays with rapid kinetics during the first 0.3 nsec (Table I), actompanied by coevolution of the 40-* population (Figure 15). Between 0.3-1.5 nsec, the +OH* population decays while $0-*is in a steady state. Depletion of the +O-* population by radiative plus nonradiative decay is compensated by further dissociation of +OH-*. Only at t > 3 nsec, where the +OH* population is nearly exhausted, does $0-*decay with monotonic first-order kinetics (Figure 16) (7 = 5.9 nsec). The fluorescence dynamics of the same ligand bound to bovine serum albumin is presented in Figure 17. There is a rapid phase of +OH* decay with coevolution of +O-*, followed by a slower +OH* decay ( ~ =1 3.3 nsec). During the initial 1-2 nsec of the second phase the emission intensity of $0-*is practically constant, indicating that +O-* (N2 + N3 population) is replenished by further

i

32ps

c--*

Figure 14. Fluorescence decay time of hydroxypyrene trisulfonate bound to apomyoglobin: (A) 60#4 apomyoglobin, 50#4 hydroxypyrene trisulfonate in lOmM Mes buffer pH 5.0. The emission was measured (in arbitrary units) at a streak speed of 15 mmlnsec at the wavelengths 400-450 nm using BG-3 (3 mm) and GG 400 Schott glass filters. (B) The excitation laser pulse measured under identical conditions as seen by reflection from the front and back surfaces of an empty 0.5-cm cuvette.

...

I

0

1

1

.

1

2

,

3

I 4

B

1

5

6

ins)

Figure 15. Time-resolved fluorescence of neutral and anionic hydroxypyrene trisulfonate bound to apomyoglobin: (A) the emission of the neutral form as measured through BG3, 3 mm, and GG 400 glass filters; (B) the emission of the anionic form measured through a KV 550 glass filter. (A) and (B) were measured at a streak speed of 1.5 mm/nsec with 60@ apomyoglobin, 50@ hydroxypyrene trisulfonate, lOmM Mes buffer (pH 5.0).

I Figure 16. The lifetime of the excited anion of hydroxypyrene trisulfonate bound to apomyoglobin. T h e sample was excited by a nitrogen laser (337 nm, 1 nsec full width at half maximum) and the emission of the anionic form was monitored (in arbitrary units) through a KV 550 filter by a photomultiplier attached to a Tektronix 7912 AD transient digitizer equipped with a 7A19 vertical amplifier.

28

T H E pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

M

‘

29

I

Figure 17. Time-resolved fluorescence of 8-hydroxypyrene-l,3,6-trisulfonatebound to bovine serum albumin. Sample solution: 250p.M hydroxypyrene in 700p.M bovine serum albumin solution pH 5.96. (A) Emission of neutral form measured through BG-3 2-mm filter; (B) emission of anionic form measured through an orange-green 550-nm filter. The time constants of the fast decay of (A) and the rise of (B) are 350 and 290 psec, respectively.

dissociation of +OH* (NI). The decay time of [+O-*] is identical with that measured for the free emitter (Table I). Figure 18A and B are the semilogarithmic plots of [+OH*] decay, as measured for hydroxypyrene trisulfonate at the myoglobin’s heme binding site and the bovine serum albumin site, respectively. Both kinetics follow a biexponential decay: one is very fast with a time constant of less than 1 nsec; the other is slower, yet its time constant is faster than T , indicating that the +OH* population is consumed by more than simple fluorescence decay. The decay of N , is a biexponential reaction, characterized by a fast (y2) and slow (yl) time constant. The amplitude of the slow reaction is represented by the normalized value A,. These three parameters are sufficient for calculating the rate constants of the partial reactions, as described below. The value A, as derived from Equation (18) is A,. = ( k 2 - y1)/(y2 - 71). As both y1 and y2 are measured, k2 can be obtained with no difficulties. The value k2 is the sum of [ k 2 1 * H+], k23, and k’f,n,+,und). The latter is directly measurable. This is the decay constant of +O-* measured at a time where +OH* signal has completely vanished (see Figure 16 and Table I). The rate constant of proton escape from the cavity ( k 2 3 ) can be obtained for y1. This macroscopic rate constant is due to the contribution

02 I 1

I

,

I

2

' I

I

3

I

*ns

0

3

I

4

(B)

Figure 18. Kinetic analysis of the fluorescence decay of neutral hydroxypyrene trisulfonate hound to apomyoglohin (A) or bovine serum albumin (B). The results are taken from experiments carried out as detailed in Figures 15 and 17, using various streak speeds. The values of y,, y2 and AR are listed in Table 11.

30

THE pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

31

TABLE I Macroscopic Rate Constants of 8-hydroxypyrene-1,3,6-trisulfonateFluorescence in Water and in Protein Complexes Decay time (nsec)

Fluorescenting species

Rise time (nsec)

Free +OH, pH = 5 Free +OH, 2M HCl Free $0-,pH = 5 Apomyoglobin4OH Apomyoglobin~OBSA-+OH

-

0.1 1 f 0.01 5.9 ? 0.1

0.11 2 0.01

5.9 2 0.1

BSA-+O-

-

0.52 t 0.2

-

0.29 t 0.02

Y1

0.545 t 0.05 6.1 2 0.1 0.352 t 0.01 0.252 2 0.01 5.76 t 0.1

Y2

-

-

3.2 t 0.1

-

3.3 2 0.1 3.3 -t 0.1

of the two irreversible reactions that consume the bound +OH* population. One reaction is the fluorescence decay and the other is the escape of H+ from the cavity. As long as the proton is in the cavity, +OH* can be reformed by recombination, but once it escapes, the probability of recombination within the lifetime of the excited singlet is practically nil. Insertion of Equations (16) and (17) into Equation (19) leads, in the range pK* < pH C pKo, to a solution y1 = k23 + k,,n,. As the decay times of the free hydroxypyrene in its undissociated state and in its ionized state are the same and equal to that of bound +O-* (see Table I), we can approximate kf,nl-(bund)= k’f,nr(bund). (It is very unlikely that only one species, +OH* or +O-*, will be subjected in the site to selective rapid nonradiative decay.) Using the approximation of k,,, and the calculated values of k23 and k2, the rate of recombination (k21*H+) can be obtained. Finally, using the following expression

y1 + 7 2 = k12 + k21[H+1 + k23

+ kf,m + k’f.7~

(20)

the rate of dissociation in the cavity k I 2 is obtained. The respective values for the dissociation, recombination reaction in the apomyoglobin and the bovine serum albumin are listed in Table 11. There are some similarities between these two sites: the lifetime of +O-*, the rate of proton escape (k23), and even the apparent rate of The implication of these values will be proton recombination (kZ1-[H+]). discussed below. What markedly differentiates the two sites is the rate of proton dissociation (k12). In both sites, the rate of proton dissociation is significantly slower than in water, implying that in these sites the water molecules are at a state that is not suitable for rapid (sub-picosecond) hydration of the discharged proton. The equivalent water activity coefficients, as estimated from the kinetic method described in Section 111. are

-

3 x 10' 3 x 108

"Forster and Volkers (1975).

HPTSr,,, Apomyoglobin Bovine serum albumin

-

1.8 x 109 3.3 x 109

-

0.63 0.31

10'0

6.9 x 10' 2.2 x 109

5.10'" X [H']" 9.3 x 108 9.3 x 108

-

1.38 x 10' 1.3 x 10'

1.66 x 10' 1.6 x 10' 1.7 x 108

The Macroscopic Rate Constant and the Partial Rate Constants of Proton Dissociation, Recombination, and Escape Out of the Apomyoglobin Heme-Binding Site and Hydroxypyrene Binding of Bovine Serum Albumin

TABLE I1

1.oo

0.68 0.8

T H E pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

33

0.68 and 0.8 for the myoglobin and bovine serum albumin, respectively. These values indicate that the water molecules in these two cavities are well below the values of the bulk. The implication of this finding will be discussed below.

2. Proton Dissociation in a Poorly Hydrating Site 2-naphthol-3,6-disulfonate is a very efficient proton emitter (pKo = 9.3; pK* = 0.3).In water, the proton dissociates in less than 100 psec. Binding of 2-naphthol disulfonate to bovine serum albumin increases the emission of +OH* with concommitant decrease of +O-* fluorescence (Figure 19). This effect is concentration dependent (Figure 20), following a typical saturation curve. A Scatchard plot of these results indicate a 1:1 stoichiometry. Each protein molecule carries one binding site ( K , = 2. 106M- ') where the lifetime of the +OH* state is significantly prolonged with respect to that measured in water (Gutman et al., 1982). It should be stressed that in this type of analysis other sites that do not change the emissions intensity ratio with respect to water are not observed. T h e kinetic study of the dissociation of 2-naphthol disulfonate is hampered by some technical limitations. The emission of the +OH* is at a short wavelength, which is already absorbed by the optical components of I

I

I

1

nm

Figure 19. Fluorescence emission spectra of free and protein-bound 2-naphthol-3,6disulfonate. The spectra were recorded at pH 6 with lOOpA4 of the free ligand (a) or in the presence of 140pA4 bovine serum albumin (b). Excitation at 330 nm.

34

MENACHEM GUTMAN

0.5

1.o

Figure 20. Titration of bovine serum albumin by 2-naphthol-3,6-disulfonate. Titration was carried out with 14.7p.M protein (pH = 6.0) and increasing ligand concentration. The fluorescence emission ratio of the dissociatedvs. undissociatedemitter is drawn with respect to the molar ratio of protein to ligand. The emission ratio of free ligand is indicated. Note discontinuity of abcissa and ordinate. (Insert). Sketchard plot of the titration.

our streak camera. Consequently, we miss both the high time resolution and the simple analysis according to Equation (18).Still, the difficulty can be overcome by utilizing another fluorescence property of this proton emitter: the difference in the fluorescence decay time between the neutral and dissociated species. The decay of the neutral state of 2-naphthol3,6-disulfonate is twice as fast as that of the excited anion (Table 111).This difference can be reduced at pH = pK*. Each collision between +O-* and H+ will enable a faster decay pathway for the excited state, through the faster route of +OH*. Indeed, at pH = 1, the lifetime of +O-* is shortened and approaches that of +OH* (compare lines 1,3,and 4, Table 111). As we observe no accelerated decay of 40-* in the protein-emitter complex (lines 6 and 3, Table III), we conclude that the site where $0-*is located cannot confine the free ejected proton for any appreciable time. emission originates must be well exposed to the The site where $0-* bulk, or contain a basic group that can trap the proton and retain it for a time with affinity that will prevent its retransfer to the excited anion. The above conclusion implies that the enhanced +OH* emission represents a reaction in a different site where proton dissociation is suppressed by very low local activity of the water.

THE pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

35

TABLE 111 Fluorescence Lifetime of Free and Bound 2-Naphthol-3,6-Disulfonate

1.

2. 3. 4. 5. 6.

Observed parameter

Experimental condition

Fluorescence lifetime (nsec)

+OH* free +OH* free 40-* free +O-* free +OH*-BSA +O-*-BSA

2M HC1 pH = 7 pH = 7 pH = 1 pH = 5.96 pH = 5.96

13.4 0.08" 22.2 12. 11.1 23.8

"Measured by streak camera as rise time of +O-*.

The slow decay of +OH* would be accompanied by a comparable slow rise of 40-*which is not the case. The rise time of $0-*emission of 2-naphthol disulfonate- bovine serum albumin complex was as fast as measured for the free ligand (trise << 100 psec). This discrepancy implies that the +O-* species that we observe is not formed in the site where +OH* dissociation is slowed by a hundredfold. We have to conclude that the signal we observe is of a mixed population. There are sites where the proton emitter is well exposed to the bulk (or contains an efficient base) and the proton can be rapidly detached from the excited anion. These site(s)are characterized by dynamic properties similar to those measured in water. Besides these sites (not detected by the Scatchard plot), there is one site where the dissociation of the proton is slowed by one hundredfold or more. The inability to measure the dynamics of +OH* prevents more precise information about the properties of such a low u ( H , O ) site.

3. Proton Dissociation-Recombinationin the Inner Space of a Liposome 8-hydroxypyrene- 1,3,6-trisulfonate can be easily trapped in phospholipide vesicles. Once the dye is locked in the vesicle, it leaks out at a rate of 1% per day (Kano and Fendler, 1978).The trapped dye is dissolved in the inner space, as estimated from the low value of its fluorescence polarizability (Rossignole et al., 1982). The leakage of protons out of this space, or their entrance, was measured by Rossignole et al. (1982) and found to be a very slow reaction, with a half-life of a few hours. Thus, if a proton is discharged in such a space by a short laser pulse, the diffusion space of the proton is limited to the inner space and no escape is expected within our short observation time (few nsec).

-

36

MENACHEM GUTMAN

The recombination rate in such a microspace can be estimated according to Goselle et al. (1979). When two reactants, one of which is much smaller and more diffusive (H+) than the other (+O-), are locked in a space with internal diameter R, we can regard the heavier reactant (+O-) as practically immobile target in the center of a reaction sphere. The proton can diffuse through the reaction space, but wherever it penetrates the Coulomb cage of the proton emitter, protonation takes place. The rate constant of the reaction is thus controlled by two radii, the Debye radius (RD), where electrostatic interaction dominates, and the radius of the reaction sphere, out of which the proton cannot escape. The rate constant of this reaction is given by Equation (21)

where ED is the sum of the diffusion coefficient of the proton and the proton emitter. We can approximate ED = D ~ =+9.3 x 1 0 - ~ cm2/sec. R~ = 28 W (Forster and Volker, 1975)and R = 65-80 A, the rate of proton recombination (k2’.H+) is estimated to be 0.4-1.0 X lo9 sec-’, that is, the proton will recombine with its conjugated base within 1 nsec. This time scale is short enough to be followed by the time resolved fluorescence of +OH*. Soybean phospholipid (Asolectine)liposomes were prepared by sonication in the presence of 8-hydroxypyrene- 1,3,6-trisulfonate, and big liposomes were removed by low-speed centrifugation. The free proton emitter was remo-d by ultrafiltration through Amicon filter, or by high-speed sedimentation of the liposomes. The small liposomes were resuspended in water (to give a dye concentration of -5p.M) and the dynamics of +OH* fluorescence was measured using the streak camera or the Tektronix digitizer. A typical time-resolved fluorescence of +OH* is shown in Figure 2 1. The initial rapid decay represents the dissociation of the protons in the liposomes. Each dissociativeevent increases the population of [+O-* + H + ] at the expense of [+OH*] (the square brackets represent an event taking place is a single liposome). Thus, with time the recombination reaction that occurs as the [+O-* + H+] vesicles become more probable, until the velocities of the forward and backward reactions (each taking place in different vesicles) become equal. This corresponds with the appearance of the slow phase, characterized by fluorescence time constant t = 5.3 nsec (measured by Tektronix digitizer). This time constant indicates that there is no other reaction that consumes the [+OH*] population except the normal radiative plus nonradiative decay, that is, there is no proton escape out of the vesicle.

THE pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

37

The rate of proton dissociation was calculated from rapid kinetics measurements (decay time of +OH*, or rise time of +O-*). The rate is comparable with that measured in water, that is, 10" sec-', indicating that the water in the vicinity of the trapped proton emitter is out of the extensive hydration layer of the phospholipid (LeNeveu et al., 1977; Rand et al., 1980; Parsegian et al., 1979; Lis et al., 1982). The rate of proton recombination has been measured either from the time-resolved kinetics or using the steady-state Equation (5). The values calculated by both methods are shown in Figure 22, which relates the rate of the reaction, as calculated by Equation (21) with the radius of the proton-permeable space. The experimental results cluster on the theoretical curve in the range of R = 72 & 7 A, a good approximation with the internal radius of a small liposome (Brauillette et al., 1982). These results can be subjected to more rigorous analysis. The rate constant of protonation of the excited anion of hydroxypyrene trisulfonate is k = 5 X 10'oM-' sec-' (Weller, 1958). Thus, the effective concentration of H + in the reaction sphere (R) is

-

On the other hand, if one proton is discharged in the space defined between the two radii R = 70 A and R D = 35 A, its formal concentration is only 1.2mM. There is an apparent tenfold discrepancy between the

Figure 2 1. Fluorescence decay curve of the neutral form emission of S-hydroxypyrene1,3,6-trisulfonatein water (A) or in the inner space of soybean phospholipids (asolectine) liposomes (B). Excitation by 6-psec pulse, 352 nm. The emission was measured by streak camera, at a streak speed of 7.5 mmlnsec, using KV 375 and BG-3,2-mm filters. (Insert) Semilogarithmic plot of the decay. Note the nonlinearity of the liposomal sample.

38

MENACHEM GUTMAN

k,,xcHf

4x10.

L

I

60

A

1

70

1

80

Figure 22. T h e correlation between the rate of proton recombination with its conjugate base and the internal diameteL of the liposome. Rates of proton recombination were determined either by steady-state fluorescence (W) or time-resolved (0)measurements. The line is drawn according LO Equation (21).

proton concentration deduced from the kinetic data and the one derived from geometric considerations. This discrepancy is eliminated if we consider the special conditions prevailing on the surface of phospholipid membrane. Through a distance of 20-30 8, (depending on the nature of the phospholipids), the activity coefficient of the water, falls in a logarithmic function from the bulk value (a(H20) = 1) down to -0.4 (Parsegian et al., 1979). Thus, there is a layer on the surface of the phospholipid membrane which is an unfavorable solvent for protons. Consequently, the internal volume of the liposome that is available for proton diffusion is less than the actual space as defined by the internal diameter of the liposome. 4. Discussion and Conclusions The dynamics of proton reaction with the excited conjugated base affords a method for measuring the reactions in a space limited by the distance

THE pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

39

the proton can diffuse through the lifetime of the detector ( 7 ’ ) . Thus, even in a free solution, we probe a space with a radius of - 100 A. If we consider the fact that at pH 7 the average distance between protons is -2500A, then it becomesobvious that in dilute solutionsof protonemitters the reaction space has not been penetrated by a “foreign” proton. The proton ejected from the excited molecules is sent on a probing mission, which is monitored by the rate or probability of its recombination with its impatient dispatcher. The first property reported by the ejected proton is the activity coefficient of the water in the probed space. A dissociation reaction taking place on a space comparable in size with the radius of few hydration shells reflects a reduced local activity of the water as slow dissociation. We have encountered this behavior upon following proton dissociation in the heme-binding site of apomyoglobin, the hydroxypyrene trisulfonatebinding sites of bovine serum albumin, and the @-naphtholdisulfonate binding site of the bovine albumin. For each of these sites, a similar 1 was noted. By measuring the rate of proton dissoobservation a(H20)< ciation from protonated indicator adsorbed on micellar surface, we obtained u ( ~ , O = ) 0.7 (see below). Thus we can assume that the activity of water on the interface of a macromolecular structure is reduced with respect to the bulk value. These kinetic measurements corroborate the equilibrium measurements of Parsegian (1979) and his colleagues. Furthermore, the fast kinetic experiments probe a small volume, allowing one to map the activity coefficient’s density of the water in a defined region on a macromolecular surface. This method has been implemented for gauging the soIvent molecule in the heme-binding site of apomyoglobin. The dimensions of the heme binding site are 15 X 15 X 3 A with a 15 X 3 A opening to the bulk (Adams, 1976). Thus all, or most, of the water molecules are in contact with the walls of the cavity and are subjected to strong interactions: hydrogen bonding with the two protonated imidazoles, the charged arginine, and dipole-dipole and dipole-charge interactions with the protein. It is not surprising to find that in such tight environments the activity coefficient of the water is reduced to a level measured in 2.1M MgC12. The reduced activity coefficient in the site is sometimes mixed with the hydrophylic moieties. It is of importance to point out that hydrophylic moieties are those that are responsible for the lowering of in the site. It is the strong interaction with the water molecules that arrests them in a tight hydration shell. In the cases listed above we noted low u ( ~ , O ) values at sites with high association constant ( K A = lo6 - IO’M-’) for highly sulfonated compounds. Thus, it seems to me that hydrophobic interactions might be better discussed in terms of surface tension (Me-

40

MENACHEM GUTMAN

lander and Horwath, 1977; Horwath et al., 1976) and hydrophobic surface area (Valvani et al., 1976) and not be mixed with the presence of charged or hydrophylic groups. The fact that @-naphthol-3-6disulfonate, with a sulfono group at the ortho position with respect to the dissociating hydroxyl, can be tightly bound to a site where the hydration of the proton is at least one hundredfold slower than in water demonstrates that there is no simple correlation between proximity of hydrophylic moiety and of the immediate vicinity. In some cases, we might be able to sense the presence of the hydration layer not by its reluctance to hydrate a proton, but by its reflection of a proton coming from the bulk. That is what was measured in the inner space of a liposome. The proton dissociates at the normal rate, as in bulk water, but the space through which it can diffuse is smaller than the volume defined by the geometry of the liposome. We do observe a major discrepancy between the effective concentration of the proton and the predicted one. This discrepancy is accounted for by the hydration layer of the phospholipids forming the inner shell of the liposome. Any proton approaching this layer will have to perturb its structure, or lose some of its charge dipole stabilization. Thus, the hydration layer acts as reflecting surface that reduces the probability of finding a free proton in this shell. This reflection will probably act mostly as a kinetic barrier in bulk-tosurface proton transfer. Once a proton penetrates it and reacts with a base, its equilibrium parameters will be much less affected by the local activity of the solvent. The reaction of the proton with its emitter-when both are confined in a cavity-should not, a priori, be treated by classical kinetic formalism, but according to stochastic considerations. The short observation time practically isolates the measured site from the bulk, thus the number of the reactants in the observed space is an integer and the dynamics should be treated as a probability function. In a case where the number of reactants (proton emitters) per site is small (most likely one,) the number of identical observed sites is high, and the event is highly synchronized (the perturbation is short with respect to the relaxation time), the difference between the rate constants calculated according to classical formalism or stochastic approach is less than 15% (Vass, 1980).Thus, in most cases the classical formalism can be employed, but its applicability should always be examined. The rate constant of roton recombination, either in a microspace or in aliposome is fast, k 10 sec (Table 11,Figure 22) and measurable with a high degree of accuracy (-20%). In our formalism, this rate is treated as a product of two terms k21[Hf]. This might be misleading, especially when the probed space is very small and u(H,)< 1. It must be stressed that not only dissociation is slowed at low U ( H , ~ )values, but also the rate of

- fs-

THE pH J U M P PROBING OF MACROMOLECULES AND SOLUTIONS

41

recombination. Thus, a straightforward insertion of k12 values measured in bulk, into the (k12-H’) terms is the erroneous estimating of local proton concentration in a microcavity. Only in those cases where k12 is identical with the bulk rate can the bulk value of kP1 be employed for obtaining [H+]. The third rate constant that characterizes the dynamics of proton reaction with +O-* is the rate of proton escape out of the microcavity. This rate is most easily estimated from (-yl) the rate constant of the slow phase of (+OH*) fluorescent decay. During the second phase of decay, the reactants +OH*, +O-*, and H+ in the cavity are in apparent equilibrium (as averaged over the number of sites). Under such conditions, and the provisions that binding does not change the lifetime of the excited ligands, we can assume only two mechanisms that consume the N1population: (1) the irreversible decay of the excited state and (2) the irreversible loss of the proton to the buIk. The rate constant of proton escape is in the range of a few nanoseconds (Table 11), and this has some direct implication in bioenergetics. ~ k23 are established, we Once the orders of magnitude of k ~ 1 . Hand can consider the meaning of the terms “protogenic site” and “local pH” currently used in the bioenergetic literature. A protogenic site, where chemical reaction or conformational change is reversibly converted to a proton-motive force, can produce free protons with a chemical potential 4-5 kcal/mol above the bulk value (Ap.H’ = 150-200 mV). Thus, we are dealing with an event similar to excitation of a proton emitter in the cavity-a pK shift of a dissociable moiety by 3-4 log units. Once these protons are formed in the site, they will diffuse out and equilibrate with the bulk. The net proton flux out of the site will be slowed to zero once the electrochemical potentials of the proton in the bulk is approaching the potential in the site. Once the protogenic event takes place, the proton in the site will be subjected to the same kinetic regulation as we discussed above, it dissociates recombines or escapes into the bulk. Until now, all measurements of proton motive force are limited to the final step: the extrusion of the proton to the bulk. The terms “localized chemiosmosis” or “local pH” were introduced (Westerhoff et al., 1981;DeKouchkovsky and Haraux, 1981)to describe a mechanism where the proton produced at a driving protogenic site will be trapped by a driven protogenic site before it assumes the electrochemical potential of the bulk. This putative scenario has the “energetic advantage” that the energy transferred between the sites is higher (by unmeasurable increments) than the measured potential gradient between the bulk phases separated by the membrane. There are two elements in this mechanism that are presently subjected

42

MENACHEM GUTMAN

to biophysical scrutiny. How long a free proton can be confined in a microenvironment without equilibrating with the bulk and, what is the time-averaged state of a proton in a microenvironment. The lifetime of a free proton in a protogenic site can be estimated from the escape time of the proton as measured above. This few-nanosecond event is lo6 times faster than the millisecond turnover time of protogenic enzymes (ATPase, NADH dehydrogenase, cytochrome b-c complex, cytochrome oxidase, bacterial rhodopsine, etc.). It is very unlikely that any of these enzymes can retain protons in a microenvironment, where the chemical potential is higher than the bulk, for a time period comparable to lo6 escape times. Any proposed mechanism for energy conservation which hinges on trapping and utilizing the proton potential before it equilibrates with the bulk is based on a very low probability event. The time-stable state of the proton in a protogenic site is influenced by the three rate constants that we have considered before: kdiss, (k21.H+) or krec, and he,,. When a protogenic site is functioning under steady-state conditions, then upon averaging over time and population, the fraction of sites retaining a free proton is invariable with time.

And the ratio of sites containing free proton vs. those containing the undissociated AH state is given by the following expression:

The pK of proteinous acids is pK > 4. Thus the fraction of active site retaining a free proton will be or less. Consequently, the true presentation of a proton, even in a protogenic site, is not as a free hydrated proton but as a covalently linked hydrogen atom. Using the same considerations, we can also define the dwelling time of a proton (Tdwejj) as the time a proton is confined in a microspace, either as free proton or bound hydrogen. The dwelling time is given by

, T,, are the time constants for dissociation, recomWhere Tdiss, T , ~ ~and bination, and escape, respectively. We shall now try to apply these time frames to evaluate the temporal requirement from any mechanism that couples the entrapment of the elusive proton with stable energy transformations.

T H E p H JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

43

The stoichiometry of protons per reversible bioenergetic event, like ATP synthesis or reverse electron transport, is higher than two (Gregory and Hammes 1981; Takabe and Hammes, 1981). Thus, the models of coupled reactions must account for the mechanism by which the energy of more than one proton can be stored in the enzyme. The chloroplast ATPase apparently transfers two protons simultaneously, employing a protonable group with pK = 5 for storing these protons (Gregory and Hammes, 1981; Takabe and Hammes, 1981). A group with pK = 5 may retain a proton for a time period of 7 = (10" * Kdiss) = 10 psec. This time frame is very short with respect to the I-msec turnover time of the enzyme. What's more, the enzyme functions very rapidly while pH of the proton donor phase is -7. At such pH, the time constant of protonation of the enzyme 7 = (10" X lo-') = 1 msec is longer than the dwelling time of the proton. Consequently, the formation of the double-protonated enzyme calls for a special mechanism that will prolong the dwelling time of the proton without changing the measured pK. The reduced activity coefficient of the water in a microcavity and the high probability of recombination vs. limited escape rate renders a microcavity, in general, to be efficient storage of protons. Any proton that enters a microcavity-by diffusion mechanisms or generated in the sitewill be delayed in the cavity by a series of recombinations, abortive dissociation attempts (due to reduced water activity),and limited rate of escape. -0.7, may A proton-binding group located in an active site with have a dissociation time 50- to 100-fold longer than in bulk water. A compound with pK = 5 may thus have 7diss (cavity) = 500 psec. This / factor. T ~ ~ In ~ the sites we have value is further affected by the ( T ~ ~ +~ 1) measured, this term may cause another 10-fold delay. Thus, the combination of a proton acceptor plus a microcavity can furnish the proton delay system needed for a stoichiometry of two or more protons for proton motive-coupled reactions. The accumulated protons coming with the bulk potential may be stored in a proton-reactive site, causing a conformational change that affects the equilibrium at the substrate-binding site of the enzyme (Gregory and Hammes, 1981; Takabe and Hammes, 1981).

V. THE REACTION OF THE PROTON WITH A MOLECULAR PROTON DETECTOR In the above section, the proton was employed as a probe of its environment. The kinetic parameters of dissociation, recombination, and escape were the interpretable information. In this section, we shall treat the

44

MENACHEM GUTMAN

proton as a perturbant of chemical equilibria, and the results will be analyzed by relaxation kinetic formalism (Eigen, 1964; Czerlinski, 1966). A proton pulse can be an effective perturbant of most biochemical reactions, but unlike the E jump or Tjump, where the shape of the perturbing function can be determined by external instruments, the time profile of the proton pulse is both measured, modified, and shaped by the perturbed system. The maximal amplitude of the perturbation and the time constant of the decay are functions of the reactants present in the solution. Consequently, instead of assuming a step function perturbation (as in Tjump), or a well-shaped delta function as in E jump, the shape of the proton pulse must be deduced from the response of a proton detector present in the solution. The response of the detector is mediated through its diffusion controlled reaction with the proton. Thus, however fast is this reaction, the detection system is out of phase with the perturbation. The protonation of the indicator is zero at t = 0, when AH+ is maximal, at a time where AH+ is already and reaches its maximal value (AHIn,,) small compared with its initial magnitude. Thus, one of the requirements of the analytical procedure is to yield information about the shape and magnitude of the perturbing function. The method we employ and recommend is a rigorous solution of the nonlinear differential rate equations describing the reacting system. The reaction mixture may contain only one component (+OH) where 40- serves as the proton detector (Section V.l), two components where an indicator (In-) is added to monitor the reaction (Section V.2), or three components where an unobservable proton acceptor (generally called buffer) modulates the observed dynamics by competing, in a directly unmeasurable reaction, with the detector (Section VIII). In all these cases, we shall present a simulative solution that demonstrates the contribution of the various parameters on the observed kinetics. Even in the simple case where only two components are present, many reactions may take place. It is our unhappy experience that taught us that in this realm of fast diffusion-controlled reactions no reaction can a priori be ignored or assumed to be too slow to affect the observed signals. T o avoid such negligence, we employ a computer program that produces a numerical solution to the differential rate equations pertinent to the reaction system. This program reconstructs the time-dependent variable and allows to match the measured values with the computed ones. By systematic varying of the various rate constants of the partial reactions, the computed curve can be shaped to superimpose the experimental results to the satisfaction of the experimentalist (or the referee).

T H E pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

45

1. The Detection of Protons by Their Reaction with the Ground-State Anion of the Proton Emitter The ground-state anion of the proton emitter is the straightforward detector for the ejected protons and represents the simplest system for analysis (Forster and Volker, 1975,Gutman et al., 1981).In theabsenceof other acceptors, the transient increment of H+ concentration (AH+)tis identical with the increment of $0- above its prepulse (equilibrium) concentration (A+O-)t. According to the formalism of chemical relaxation, the concentration of the reactants is given as a sum of the equilibrium concentration plus the incremental deviation from equilibrium, which is the time-dependent variable. For the single-component system

we shall define the incremental dissociaiton induced by the laser pulse as X. The differential rate equation, describing the relaxation of the system from the perturbed state X , = X , to the relaxed state X, = 0 is given below.

k 2 W H - k2X -

k1W- -H+- kIH+X - k 1 W - X

- klX2

W-,

where W H , and ->re h e equilibrium concentrations of these reactants. As k2@H = kI+O- - H + . The equation is reduced to

dx - = -(k2 dt

+ kI(@- + H + ) ) X - klX2

In the classical treatment of chemical relaxation, the nonlinear terms (x2) are neglected with the assumption that the increment X is much smaller than the equilibrium concentration of the reactants. If this assumption is made, the decay of X is characterized by a single exponential decay with time constant: 7-1

=

[k2 + k,(@-

+ H+)]

(28)

Despite this convenience, such approximation is seldom permitted by the initial conditions of the experiment. Suppose that a dilute neutral solution (loo@, pH = 7) of hydroxypyrene trisulfonate (pK = 7.7) is pulsed

46

MENACHEM GUTMAN

with a medium-energy density (0.4MW/cm2) and lOpM of +OH dissociate. In this case, the term X2 = (lo-'') is not negligible with respect to (@- + H + ) * X = (1.6 X lo-"). Thus, the actual decay will not follow a single exponential decay (Pines 1981).Suitable conditions for the single exponential decay call for pH > pK so that &6->> X, or pH<>X. The employment of alkaline initial conditions reduces the fractional increment of the signal (Xlw-) that reduces the signal-tonoise ratio. What is more, it may introduce OH- as a possible proton acceptor in the system. The acidic conditions lead to very rapid recombination, (T < 1psec) which calls for high time-resolution instrumentation. Because of these reasons, it is better not to manipulate the experimental conditions for the sake of simpler kinetic analysis. The numerical analysis is applicable for any initial conditions and allows one to select the conditions according to the biochemical system under investigation. The intrinsic simplicityof the single-component system renders it ideal for measurements of basic reaction in chemistry like the kinetics of recombination in Coulomb cage (Forster and Volkers, 1975), the diffusion coefficient of a proton in water (Gutman et al., 1981) or solids like ice or urea (Pines, 1981). Due to the indirect applicability of these subjects to biochemistry, these subjects will not be further discussed.

2. The Reaction of Proton with Indicator The pH indicators are a large group of compounds with a huge selection of pKs, solubilities, structural properties, and adsorption bands to fit any special demand. As a general rule for monitoring rapid reactions, color changes associated with protonation-dissociation of carbon atoms (like crystal violet or malachite green) should be avoided-as these reactions are not diffusion controlled (Eigen, 1964; Duynstee and Grunwald, 1959a,b).The reaction of amines, azoaraomatic rings, phenols, or carboxylates are fast diffusion-controlled reactions (Eigen, 1964).The time scale of the observations depends on the indicator. In all cases, the protonation is a fast reaction, reaching its maximum in the microsecond time scale. The relaxation of the signal, that is, deprotonation of the indicator, varies with the pK of the indicator. The time constant of the decay can be roughly approximated by T = (5 x 10" - K d i s S ) - l . Thus, indicators with pK = 5 will dissociate in the microsecond time scale whereas those with a pK of 7-8 may linger in their acidic state for approximately 1 msec after the pulse. The reactions that take place in two-component systems, proton emitter, and pH indicator dissolved in water, are given below:

T H E pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

47

plus the following reactions: 1.4 x 10" H+ + O H - . ' H20 +OH + OH- e40- + H 2 0 (hydrolysis) (33) HIn + OH- eIn- + H 2 0 (hydrolysis) (34) The time constant for hydrolysis is approximated by Thydrolysis = K , X 1O5 (sec). Thus, for proton emitters with pKo < 10 this reaction will be too slow to affect of overall dynamics. The reaction of the proton with OH-, should always be considered, due to the high rate constant of this reaction, k = 1.4 X 10"M-' sec-'. In order to avoid efficient competition between In- and OH- for H+, [OH-] in the solution should be at least 100-fold smaller than [In-], that is, better keep the pH of the solution below pH = 8.5. Abiding with the above precautions, we can limit our consideration only to reactions (29) to (31). Reaction (31) is strongly modulated by electrostatic interactions. In a case where both 40- and HIn are negatively charged, the rate of the direct reaction is negligible with respect to reactions (29) and (30). We shall first deal with such a case, and then demonstrate the effect of reaction (31) on the dynamics. A. DYNAMICS OF PROTON CYCLE IN T H E ABSENCE OF DIRECT PROTON EXCHANGE

-

-

The experimental system consists of double charged indicator, Bromo Cresol Green BCG-- + H+ BCGH- (pK 4.7) and triple-charged proton emitter hydroxypyrene trisulfonate: +OH-3 40-4 + H+ (pK = 7.7). Because of the large charge product, Z1Z2 = +4, the contribution of reaction (31)to the relaxing system is rather small, less than 1%, and we can limit our analysis only to reactions (29) and (30). The laser pulse dissociates X molecules of +OH, and the discharged protons react with Y molecules of In-. Thus, the concentration of the reactants at time t is given by

w-

(+O-)t= - + X; (In-)t = In- - Y; (H'), = E++ X - Y

(+OH), = +OH - X (HIn), = m n + Y

48

MENACHEM GUTMAN

At t = 0, X ( 0 ) = XO and Y ( 0 ) = 0. The differential rate equations which describe the time dependence of X and Y are:

d[+O-I dt

--

-

dx

- = (--k'([H+]+ [qz-]) - k*)X dt + k l [ w - ] Y - k l X 2 + klXY

d[In-] dY ----= dt dl

(35)

k3rG-W - (k3([F+]+[ln-]) - k*)Y - k3XY

+ k3Y2

(36)

Three independent parameters control the numerical solution: kl k3, and X,. The other two rate constants (k2 and k4) are already set by the equilibrium constants: K43 = K4/k3; KZI = k 2 / k l . To find the best fit, the two diffusion-controlled rate constants of protonation, k 1 and kJ7 are varied in the range of lo9 to 20 x 10'OM-I sec-' (which is the range of diffusion-controlled reaction between small molecules and H+). The other independent parameter, XO,is estimated from the initial rate of HIn formation. It is assumed that the initial velocity of HIn formation is given by Vinit = k3 [In-][H+]. Usin? the known value of [In-] and an estimation k3 = 5 X 10'oM-l sec- , [H+Io can be estimated with about 30% accuracy. Within this range of estimated parameters, simulations are systematically iterated until a set of parameters (Al, k 3 , X o ) is found which yields the best curve fitted to the experimental one. Figure 23 depicts such a typical simulation. The initial velocity of the reaction led to an estimate forXoof 4-5.5p.M, and within this range ofXo we varied systematically both k l and k 3 . Figure 23A depicts simulations withkl = 1.8 X lO"M-'sec-' andthreevaluesofkg(3,4.2,5X 10'oM-' set-I). In Figure 23B,k3 was kept constant (4.2X 101oM-' sec-') and k l was varied (1.6, 1.8, 2.2 X 10"M-' sec-I). In both figures, Xo = Figure 23. Experimental results and simulated curves of the proton cycle. The experiment was carried out in the presence of 4 0 m Bromo Cresol Green, 1OOphf 8-hydroxypyrene1,3,6-trisuIfonate, pH 5.88, room temperature. Fifty readings of the experimental curve, in the range 0-3.3 psec, at 68-nsec intervals, and 50 readings in the range of 1.0- 17.7 psec at 340-nsec intervals are drawn, together with the simulated curves. (A) The simulated reactions were computed for the following parameters: k l = 18 X lO'"M-' sec-'; X, = 4.25pN; k3 = 3.2 x 10'" (---), 4.2 x 10'" (-), 5.2 x 10'" (--), M-' sec-I. After 5 psec the lines for 4.2 and 5.2 x 10'" M-' sec- practically overlap. (B) T h e simulated reactions were computed for the following parameters: k3 = 4.2 x 10'" M-' sec-'; X, = 4.25pM; k l = 16 X 10'" (---), 18 X 10'" (-), 22 X 10'" (-.-), M - ' sec-'. (C) Simulation of the variation with time of (40-1, (---), (Hln), experimental and simulation (-), and free proton concentration (-.). k l = 18 X 10'" M-' sec-'; X, = 4.25pN; k3 = 4.2 X 10" M - ' sec-'.

'

49

50

MENACHEM GUTMAN

4.0

z

Q

4

I

I

I

-

-

\

-

3.0\,\

';\

-

2.0 j:, \,

-

-

- I

I

I

TABLE I V Association and Dissociation Rate Constants of Protons from the Ground State of Proton Emitters and'hdicators Compound 8-Hydroxypyrene-l,3,6trisulfonate 2-naphthol-3,6disulfonate 2-naphthol-6-sulfonate p-naph tho1 Bromo Cresol Green

has," ( M - ' sec-')

(sec-')

PK

1

18 2 1.5 X 10"

3600

7.7

0.0006

45

9.2

0.004

9.2 9.3 4.95

0.002 0.001 0.0006

7

2

1.5 X 10"

7.6 2 0.4 X 10" 1 2 0.1 x 10'0 4.2 2 0.1 X 10"

hiss

48 5 4 . 7 . 105

"The values given are the average of five to seven independent experiments.

T H E p H J U M P PROBING OF MACROMOLECULES AND SOLUTIONS

5I

et al., 1983) on the experimental one is a very critical evaluation of the accuracy and fit of the simulation, yet because of its unqualitative nature it is inconvenient for comparison between experiments. Thus, we prefer to describe the dynamics of the proton cycle by some measurable parameters-called macroscopic parameters. These are T,,, and Y,,,, which are the time and amplitude coordinates of the maximum of the curve, and two macroscopic rate constants y1 and y2 corresponding with signal build-up and decay. It should be stressed that neither the experimental nor the simulated curves behave as a sum of two exponents. Both the build-up and the decay are nonexponential curves. Thus, we characterize them by approximation. About 60% of the signal is built up and the initial 60% of signal decay are fairly approximated by a mono exponential function. Within these limits, y1 and y2 were calculated and employed as convenient parameters for quantitative characterization of the curve. The usage of this macroscopic parameter for calculation of the partial rate constants is demonstrated in Figure 24. The computer was programmed to produce the value of the macroscopic parameters as a function of k I and k3. Figure 24A depicts the dependence of y l and y2 on ks with a constant value of k l , whereas Figure 24B relates T,, and Y,, with the same parameters. The macroscopic parameters calculated from the experimental curve fit the theoretical curves for k3 = 4 k 0.2 X 101OM-'sec-'. Figure 25, A-D, represents the variation of the macroscopic parameter as a function of kI-for three values of k 3 , 3.2, 4.2, and 6.2 X 101OM-'sec-'. The accurate values of k 1 and k3 are those that will generate simultaneously the correct value of four macroscopic parameters. As seen in Figure 25, that requisite is met by the combination k1 = 1.6 rfr 0.2 X 10"M-'sec-' and k3 = 4.2 x 10'oM-'sec-'. B. T H E EFFECT OF INITIAL CONDITIONS O N T H E MACROSCOPIC PARAMETERS

Figures 23- 25 demonstrate that the simulation technique is suitable for reproducing the outcome of the experiment, thus these calculations may be convenient for understanding the complex relationship between the various parameters appearing in the differential rate equations and the observed macroscopic parameters. The most dominating process during the proton cycle is the continuous competition between +O_Land In- for the protons. In a case of a large perturbation (Xo >> +O-; K-), the initial conditions will have minor effect on the outcome of the proton cycle, but otherwise the dynamics are very much influenced by the prepulse concentration. The

Figure 24. The dependence of the macroscopic parameters on the rate constants of the reaction of the indicator with the proton. The macroscopic parameters were calculated for simulations describing the experimental conditions defined in Figure 23. T h e rate constant k l = 1.8 x 10" M - ' sec-l and X , = 4.25fl were kept constant and k3 was varied from 10'' to 10" M - ' sec-I. The macroscopic parameters with their error bars, which were measured from the experimental results, are indicated. (A) y1 and y2 vs. k,; (B) T,,, and Y,,, vs. k?. T h e vertical lines marked the accuracy of k3 determination.

52

0

I 5

I

I

10

k,

I

15

I

1 20

I

25

10-'0

(A)

0.E

p

+)

0.4

N

P

0.2

0

k,=

10-10

(6 )

Figure 25. The dependence of the macroscopic parameters on the rate constant of proton recombination with the proton emitter anion. The macroscopic parameters were calculated for simulations describing the experimental conditions defined in Figure 23. The frames represent y1 (A), y2 (B), Tmax(C), and Y,, (D) as a function of the rate of protonation of $0-.In each figure, there are three curves calculated for k3 with the values of 3.2 x 10" M-' sec-* (-), 4.2 X 10'' M-' sec-* (-), and 6.2 x 10" M-' sec-' (---). The experimentally determined macroscopic parameters are indicated as parallel horizontal lines. The vertical lines denote the range of k l values that will yield macroscopic parameters compatible with the measured ones.

53

-

a

v)

I

I

I

I

’

I

1

I

0

5

10

k,

a

I I

I 20

15

10“~

(C)

10

-

I

I

I

54

I

I

I

i

I 1

1

I

5

THE pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

55

effect of the prepulse conditions, pH, reactants concentration, and pulse size are represented below. Figure 26 relates the effect of the prepulse pH on the macroscopic parameters ( X , = 4.25pM). Within the range where the experimental results are conveniently measured, the computed macroscopic parameters and the measured ones are essentially identical. It is of interest to point out that at low pH both y1 and y2 increase but at the upper limit y2 does not vary much. This is the range where the concentration increases to such an extent that it competes effectively for any H+ dissociating from HIn, thus y2 approaches the value of It4. The signal size has a clear maximum, determined mostly by the pK values of the proton emitter and the proton detector. At low pH, the depletion of G- limits the formation of HIn, whereas at high pH the absence of undissociated +OH reduces the size of the pulse and increases the competitivity of $0for the protons.

w-

Figure 26. The effect of initial pH on the macroscopic parameters characterizing the Bromo Cresol Green-hydroxypyrene trisulfonate system. The macroscopic parameters were calculated for the experimental conditions described in Figure 23 and the rate constant listed in Table IV. X o = 4.25pW. The experimental values are drawn with their error bars. (A) 71 VS.pH; (B) 72 VS. pH; (C) T,,, VS. pH.

2.

I

I

Figure 26. (Continued)

56

I

THE pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

57

T h e effect of $0- concentration, at a constant pH, on y1 and y2 is given in Figure 27. Increasing the competitiveness of $0- for H + enlarges both y1 and y2. T,,, is shortened and the amount of HIn formed diminishes. It is of interest to note that above certain concentrations of $0-,y2 remains constant. This is the limiting case where $0- competes successfully for any proton dissociating from HIn, consequently the ratelimiting step of the reaction becomes k4. If the proton cycle can be measured under conditions where [$O-] > [In-], then y2 can be accurately equated with kq. This advantage has its drawbacks, for at high excess of $0-,the signal diminishes in size and reduces the accuracy of the measurement. T h e effect of [In-] is given in Figure 28. As expected, there &hardly anyeffect on 439, whereas y1 increases nearly linearly. Once [In-] >> ([$O-] + X), the formation of HIn becomes effectively a pseudo firstorder reaction and y1 will become a linear function of [In-] with a slope of kS. Xo,the size of the proton pulse, depends linearly on the energy density of the excitation beam (Gutman et al., 1981) and the emitter concentrations. As a result, the pulse size may vary between successive experiments. T o estimate the effect of this variance on the macroscopic parameters, we simulated a set of experiments where the pulse size was varied from X o = 1 to 401J.M(Figure 29). During the pulse, the protons are produced together with their conjugate bases and the prepulse pH is regained very rapidly (Figure 23C). Consequently, the effect of the pulse size differs from - the effect on the prepulse concentration of the other reactants, [$O-] o r Y,,, increases steeply with X o , but at higher values the line curves, reflecting the higher probability of an encounter between H + and $0-. T,,, is less sensitive to X o , while the two rate constants, y1 and y2, are nearly independent of the pulse size.

[z-].

3. The Direct Proton Exchange between Reactants and Its Effect on the Dynamics of the Proton Cycle In the above examples, the contribution of a direct proton exchange has been disregarded. Considering the fact that biochemical reactions and physiological events take place in well-buffered solution where the free proton concentration is low (pH > 6) and the buffer concentration is high, the direct proton exchange cannot be neglected. As a matter of fact, the protonation of surface components on a membrane across the unstirred layer is very likely mediated by protonated buffer molecules (McLaughlin and Dilger, 1980; Benz and McLaughlin, 1983). Direct proton exchange can be easily included in our formalism and, under certain experimental conditions, can be measured.

[OO-] (pM1 ( 5)

Figure 27. The dependence of the macroscopic parameters on the $0- concentration. The experimental conditions, except for emitter concentrations, are defined in Figure 23. The rate constants for 8-hydroxypyrene-1,3,6-trisulfonateand Bromo Cresol Green are listed in Table IV. X, = 4.25M. (A) y1 and y2; (B) T,,, and Y,,,.

58

Figure 28. The dependence of the macroscopic parameters on the indicator concentration. See legend to Figure 27.

59

X(o)

(pM) tB)

Figure 29. The dependence of the macroscopic parameters on the pulse size (XJ.The experimental conditions are defined in Figure 23. The rate constants are listed in Table IV. The pulse size, X,, was varied between 1 to 4 0 N . (A) y I and y2; (3)T,,, and Y,,,.

60

T H E pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

61

T h e intensive electrostatic repulsion between the pyrenate trisulfonate anion (2 = -4) and the protonated indicator (BCGH-) lowers the probabiIity of their encounter by a hundredfold (Eigen et al., 1964). Thus, a direct proton exchange between these two reactants is slowed both by slow diffusion (D= 2-3 X cm2 sec-') and electric repulsion. To evaluate whether our experimental system can detect the contribution of a direct reaction, we substituted the highly charged emitter with the neutral one, @-naphthol. T h e protonation cycle of Bromo Cresol Green, in the presence of P-naphthol, is depicted in Figure 30. Calculations that did not account for the contribution of the direct proton exchange (reaction 3 1) gave unsatisfactory simulations. However, including the rate constants of reaction (30) in the differential rate equations dX

(Hf)]+ k 2 + k&H)

= -{k,[(@-) dt

- k(j(h-)}X

+[kl(+O-) - k 5 ( + 0 - ) - k6(+OH )1y -k*X2 + [k* - k5 + KGIXY

dY

- ~,(HI~)W+ (H+)] + k4 + k6(+OH ) + k 5 ( + 5 - ) } Y

- -- [ k 3 ( G 1 - k6(E-) dt

(37)

-{ks[(In-) +k3Y2 + [-k3

_ .

k6 - k5lXY

(38)

led to the simulation given in Figure 30. The rate constant of the spontaneous direction of the proton exchange k5 = 5 x 109M-' sec-' is compatible with the value estimated according to Debye and the viscosity of the solution, but the rate of @-naphtholate protonation ( k , ) also derived from the simulation is only k 1 = 1.5 x 10'oM-'sec-'. This value is only 30% of the value estimated by Weller (1958) for this reaction, or as expected from Debye's equation for diffusion-controlled reaction. The ratio between the measured rate constant of a diffusion-controlled reaction and the value predicted according to Debye's equation is an estimation of the steric factor u. Thus, for protonation of P-naphtholate, u = 0.2-0.3, whereas for proton exchange between protonated indicator and P-naphtholate u 1.0. T h e steric factor u appearing in Debye's equation is a complex function representing not only the geometry of the reacting molecules, that is, the fraction of the reacting surface and axis ratio for nonspheric molecules, but also the rate of rotation of the reactants (Richter and Eigen, 1974; Solc and Stockmayer, 1973; Shoup et al., 1981). A fast rotation of the reactants increases the probability of reaction, but this event must take place before the encounter complex separates. Thus, while fast rotational diffusion increases u, a fast translational diffusion lowers it (Shoup et al., 1981). Consequently, the proton exchange between the rapidly rotating, slowdiffusing molecules P-naphtholate and protonated Bromo-Cresol Green

-

62

MENACHEM GUTMAN 2D

I

I

I

psec

Figure 30. Experimental results and simulated curve for the proton cycle measured with B-naphthol (I&), and Bromo Cresol Green (40 pM) (pH = 7.3). The rate constants for Bromo Cresol Green are taken from Table IV.The simulation curve given in the figure (---) corresponds with k, = 1.0 x 10" M--' sec-I, k5 = 5 x lo9 M - ' sec-I, X o = 2.7p.M.

has a higher steric factor than the reaction between P-naptholate and a free proton. T h e steric factor estimated for the latter reaction (+ -0.3 is comparable with the surface fraction of the hydroxyl on the @-naphthol (Bondi, 1964), indicating that in this reaction the rotational diffusion is not fast enough to affect the outcome of the collision.

4. Alkalinization Pulse by the Conjugate Base of the Proton Emitter In the above sections, we referred only to one product of the photodissociation-the proton. Still, it is produced together with a strong base, the ground-state anion of the proton emitter. T h e reason that the effect of $0-was not observed is due to the experimental conditions that were set to mimimize it. T h e diffusion of the conjugate base is 10 times slower than that of the proton. Thus, the reactions of the proton with other solutes is the dominating event. Furthermore, the electrostatic repulsion between the emitter and the detector reduces the contribution of $0-to the measured dynamics. Once these factors are understood, it is possible to predict and set experimental conditions where the reaction of $0with the indicator will dominate the observed reaction. By setting the experimental pH to be lower than the pK of the proton detector (pH <<

-

T H E pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

63

P K ~ ~[h-] ) , will be very small and further acidification of the indicator will be nil. On the other hand, such initial conditions set [ H G ] concentration to be high, increasing the probability of direct proton exchange with 40- generated by the pulse. A very short time after the pulse, Y can be assumed to be zero. Thus, the time derivative of Y can be either positive or negative, depending on the term of X in Equation (38).

(

$)o

=

[k3(G-) - ke(&-) - ks(HG)]X

(39)

The transition from HIn formation (by reaction with H f ) to HIn consumption (by proton exchange with the increment of +O-) will take place at a certain ratio of [InH]/[In-1:

For each pair of indicator/proton-emitter, there will be a point, pH’, where the response of the indicator will switch its direction. At higher pH values, the indicator will be acidified, whereas at lower pH values alkalinization will dominate. The value of this transition is given by:

k3

K21

k5

K43

Once - >> -,

we can approximate this by:

At the pH below pH’, the reaction with the conjugated base dominates. pH’ differs from the pK by log (k3/k5),the ratio of the rates of Inprotonatiorr(k3) and HIn reaction with a conjugated base (k5). The effect of the initial pH on the direction of HIn response is demonstrated in Figure 31. At pH 7.1, the Bromo Cresol Purple (pK = 6.3) is protonated; at a lower pH (5.12), it gets deprotonated by the reaction with the P-naphtholate formed by the pulse (Gutman et al., 1983).

5. Limitations and Inaccuracies A.

REACTANTS CONCENTRATION

In the present section, I shall point out the sources of inaccuracies and how to minimize them by proper selection of the experimental conditions.

64 A

MENACHEM GUTMAN

I B

Figure 3 1. The effect of pH on the response of Brorno Cresol Purple during the proton pulse. The reaction mixture contained Bromo Cresol Purple (100pM) and P-naphthol (1mM) at pH 7.05 (A) and 5.12 (B). Each tracing is an average of 4096 events. Downward deflection corresponds with protonation of indicator.

We found it convenient to limit the indicator concentration to absorbance range of 0.2-3A. .4t lower concentrations of indicator, the dilution flattens the signal and makes it poorly resolved. T h e rise time becomes shorter, the decay is faster, and the signal diminishes in size (Figure 28). T h e upper limit of indicator concentration is limited by the fluorescence of the proton emitter. It should be remembered that of the 1 MW of excitation energy 10- 100 k W are irradiated as fluorescence. Unless this emission is reduced at the entrance slit of the photomultiplier below the saturation energy of the photomultiplier, nonlinear responses are expected. On the other hand, the transient intensity of the probing beam must be big enough to be recorded. Thus, a combination of intensive monitoring beam and selective damping of the fluorescence are always needed. Once the absorbance of the monitoring beam increases, more light should be introduced to the monochromator with consequent admission of more fluorescence. The concentration of the emitter can vary, in practice, only within a certain range. At low concentration, the perturbation is small whereas at high concentration all of the excitation energy may be absorbed at the first few millimeters of the solution. Under the latter conditions, the size of the perturbation is highly dependent on the distance of the probed space

T H E pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

65

from the front surface of the cuvette. As pointed out in Figure 29, the magnitude of X o has a marked effect on all macroscopic parameters. This may introduce an apparent irreproducibility with every minor change of the monitoring beam’s location. Finally, a possible source of error is the accumulation of photoproducts (Klaning et al., 1973; Lachish et al., 1977). At an energy density of less than 1 MW/cm2, no deviations due to photoproducts were observed (Gutman et al., 1981). Still, whenever many events are accumulated over a long time period, 3-30 ml of irradiated solution and proper mixing are recommended. B. ACCURACY OF THE MACROSCOPIC PARAMETERS

T h e dynamics most accurately measured are the decay of the signal, y2. T h e relaxation is slow enough to be conveniently measured by currently available instruments. The signal relaxation is a complex function; its initial phase is described by a single exponent, but as it proceeds the relaxation slows into a long tail. [Some of the tail might even be attributed to stable photo products (Klaning et al., 1973; Lachish et al., 1977)l. Most of the data, characterized by high signal-to-noise ratio, is clustered in the early exponential decay curve represented by y2. In most cases, y2 can be measured with + l o % error. T h e rate constant of signal rise, yl, is less accurately determined. This signal is a fast event, reaching its maximum in a few microseconds or less. Thus, the ascending section of the curve is limited in resolution; its initial part has an exponential rise but a poor signal-to-noise ratio due to the small signal size. Once the measured values are bigger, the curve begins to deviate from the exponential function and the data become unsuitable for calculating 71. For these reasons, the value for y1 is less accurate than that of y2. In some experiments, the error might be as high as 30%. T,,,, the time when the signal reaches its maximal height, has its own uncertainty due to the flatness of the curve at its maximum and the electronic noise. In the case of a sharply rising, steeply decaying signal, T,,, can be estimated with an error of 270 nsec. On the other hand, slowly decaying signals have undefined maxima and inaccuracy may be as high as 2 1 psec. T h e value of Y,,, for a given experiment can be measured very accurately, with an error of less than 5%. Thus, for the purpose of simulation, the amplitude is a very informative parameter. However, as demonstrated in Figure 29, Y,,, is highly dependent on X o , which is a function of the excitation laser output, pulse-beam dimensions, and observation cell and monitoring-beam geometries. The other parameters, T,,,, yl, and y2 are much less affected by X,. Thus, the amplitude is a

66

MENACHEM GUTMAN

parameter that may vary quite independently between experiments. Therefore, despite the fact that Y,,, can be an informative parameter for a given experiment, it is unsuited for comparison between different experiments. For these reasons, we did not compare the Y,,, values among the experiments. Even though each of the experimental parameters bears its own inaccuracy, the combination of the four leads to unambiguous determination of the reaction rate constants (Figure 25). Table IV lists the means of five to seven independent measurements, carried out under different initial conditions. T h e results are all reproducible within 20%.

VI. KINETICS OF PROTONATION OF HIGH-MOLECULAR-WEIGHT STRUCTURE T h e charge of a macromolecule modifies the ionic atmosphere in its immediate vicinity, as described by the Gouy-Chapman diffused double layer (Adamson, 1960). At the surface of the protein, the concentration of counter ions will exceed that of theothers. As a result, the measured pK of surface groups will be shifted, reflecting the difference between the surface and bulk concentration of the protons (Goldstein, 1972). This surface phenomenon reflects the total charge of the molecule, even of charges located far from the site of protonation. Because of the low dielectric constant of the interior of the protein, the electric field of buried charges propagate to the surface without any attenuation by the ionic atmosphere (Matthew et al., 1979a,b; Matthew and Richards, 1982; Russu et al., 1982). T h e electric charge of the protein (or any other macromolecular structure) affects the rate of protonation of any surface group, as given by the electrostatic terms of the Debye Shmulchowski equation for diffusioncontrolled reactions (Eigen et al., 1964). Besides this charge effect, the probability of protonation of ;I surface group on a protein is modulated by the following factors: 1. T h e rate of encounter between a proton and a specific target on a macromolecular weight structure is a function of the ratio of the target (rt) and macromolecule (r,) radii. 2. T h e surface of a macromolecule forms a two-dimensional diffusion plane. As pointed out by Adam and Delbruck (1968), the probability of encounter between mobile ligand and a target on surface can be higher than encounter in three-dimensional space. 3. T h e matrix through which the proton diffuses, water, changes its properties in the immediate vicinity of the interface. T h e interface is

THE PH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

67

covered by a hydration layer (Le Neveu et al., 1977);(Rand et al., 1980); Parsegian et al., 1979; Lis et al., 1982) characterized by lowered activity coefficient. In this space, the rate of proton transfer will differ from those taking place in the bulk (reactions 16 and 13). What is more, this hydration layer will act as a reflecting surface for an incoming proton. The penetration of a proton into the hydration layer calls for reorientation of the water molecules by the proton (reaction 13).Thus, the hydration layer is a thermodynamically unfavorable space for a free proton, modulating the rate of protonation by a bulk proton. The considerations listed above are concerned with the direct interaction of the proton with its target, yet we also have to consider events that will affect the apparent properties of the measured system. In this category, I include the postprotonation reaction, which can be detected by kinetic analysis but is obscured in equilibrium measurements. The kinetic analysis of protonation (as in reaction 27) is much facilitated by having the equilibrium constants of the reactions. The pK of a surface group depends not only on the total charge of the protein (Tanford, 1955, 1976; Tanford et al., 1955; Tanford and Kirkwood, 1957) but also on the solvent accessibility (Matthew et al., 1979a,b; Matthew and Richards, 1982; Russa et al., 1982). Some of the solvent accessibility abberrations are accounted for by the three mechanisms described above. But solvent accessibility, which reflects the precise configuration of the binding site, points to another factor that must be considered-the selective interactions of the immediate environment with the two forms of the proton acceptor. Highly polarizable microenvironment will favor the charged state of the proton acceptor, whereas local charges will selectively stabilize the counter-charged form of the acceptor. If the acceptor protein can assume two configurations (Tong and Glesmann, 1957; Gutman et al., 1983a) reflecting the selective stabilization energy, we shall observe a postprotonation reaction that will modify the measured equilibrium parameters of the measured system. In such a case, the reaction is not a straightforward reversible-binding-dissociationbut a successive two-step reaction. For such a case, we do not have the advantage of relating the partial rate constant of proton association-dissociation with the observed equilibrium constant, that is, K,# (k4Ik.3). Consequently, the analysis of protonation of macromolecule calls for differentiation between the observed pK (Kobs) and the surface one, the one that reflects the actual step of the fast, reversible protonation. In the following section, we shall define K , = k4/k3. Due to their heterogeneous surface, proteins are too complex to serve as a model system. The one we selected is the indicator-micelle system (Gutman et al., 1981a; Gutman et al., 1983a). Once the formalism is

68

MENACHEM GUTMAN

defined and its applicability is proven, it can be employed in more complex systems like protein or membrane surface. 1. Protonation of Uncharged Target Adsorbed on Uncharged Carrier The simplest model for protonation of a surface group is the reaction between the uncharged indicator Neutral Red (Scheme 111) adsorbed on uncharged micelle of Brij 58. Neutral Red is uncharged in its alkaline state. Its water solubility is extremely low, but it is freely soluble in inorganic solvents. Protonation increases its water solubility. Both the alkaline or acidic state are adsorbed to micelles. T h e distribution ratio between the aqueous and the micellar phases, for the acidic (a)and alkaline (p) states of the indicator, are a = 130 ? 10, p = 1400 100 (Gutman et al., 1983a). T h e apparent pK of the adsorbed indicator is practically independent of ionic strength (Table V), indicating that the surface charge of the micelles is very close to zero (Tanford, 1976). T h e kinetic experiments (Figure 32) were carried out with 501J-M Neutral Red and 40 mg/ml Brij 58 (equivalent to -500@4 of micellar concentration). At this concentration of detergent, 98%of the indicator is adsorbed. T h e initial pH of the experiment (7-7.5) ensured that before perturbation the Neutral Red was mostly deprotonated, whereas the proton emitter (2-naphthol, 3,6-disulfonate, pKo = 9.3) was undissociated. Perturbation of the equilibrium by a laser pulse, dissociates X o molecules of +OH, and the relaxation of the system is described by equations (37) and (38). (Direct proton exchange between +O- and bound indicator can be ignored in this case.) T h e experimental curve and the simulated function are given in Figure 32. T h e rate constants of the reaction are listed in Table V.

*

TABLE V Thermodynamic and Kinetic Parameters for Protonation of Neutral Red Adsorbed on Urij 58 Micelles"

KCI (mM)

p%bs

0 40 100

5.5 ? 0.05 5.55 t 0.05 5.55 t 0.05

-log

k4

k3

5.53 5.60 5.61

kl

kS

(M-' sec-I)

( M - sec- I )

I

7 4 I x 10'' 5.5 4 0.9 X 10" 4.5 4 0.7 x 10"

0.9 2 0.3 x lo'* 1.3 k 0.3 X 10'' 0.9 k 0.3 X 10''

0.006 0.046 0.106

"The experiments were carried out in the presence of 50p.M Neutral Red, 40 mg Brij 58/ml, corresponding with 5OOp.M of micellar concentration), 1 mM of 2-naphthol-3,6disulfonate, and KCI as indicated. T h e k l values appearing in the table were computed simultaneously with k3.

THE pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

69

Scheme I11

;

time (psec) Figure 32. Experimental results and simulation curve for the protonation cycle of Neutral Red adsorbed on Brij 58 micelles. The experiment was carried out in the presence of 1mM 2-naphthol-3,6-disulfonate, 40pM Neutral Red, 40 mg/ml Brij 58 (pH 7.3). The rate constants used for the simulation were k , = 7.0 X lo-'" M - ' sec-'; ks = 0.85 x 10" M-' sec-'; pK, = pK43 = 5.53 X , = 1 O p M . (-) Experimental; (---) simulation.

2. The Effect of Charge on Rate of Protonation The time-independent diffusion-controlled rate constant for reaction between charged reactant is given in Equation (42) (Eigen et al., 1964).

T h e first term, ken, is the rate of encounter between the reactants in absence of electrostatic interaction; the second term represents the contribution of the electrostatic interactions at I = 0, while the last one is a correction for ionic screening at I > 0; x is the radius of the ionic atmosphere [ x = 3.27 x lo7 fi(cm-')I; and rg is the radius of the encounter between the reactants. T h e rate of encounter of small molecules is given by 4?r

ken = -N C D r q a 1000

(43)

70

MENACHEM GUTMAN

where ED is the sum of the diffusion coefficients of the reactants and u is the steric factor (Eigen et al., 1964). The two electrostatic terms are functions of 6,which is the ratio between the Debye radius of the molecule RD = Z1Z2eglEkT (the distance at which the electrostatic force equals to the thermal energy k T ) and rq.

1 At room temperature, we can approximate 6 = 7 * Z l Z p - (given in A

rrj units). The replacement of one reactant by a large molecule bearing a specific site for the small ligand modifies all terms of the equation. The rate of encounter has to account for the fact that the radius of the site (rt)is smaller than the radius of the macromolecule (r,). Furthermore, whereas the slow translational diffusion of the macromolecule can be ignored, the rotational diffusion of the macromolecule becomes crucial. A fast rotation of the macromolecule can compensate for the small radius of the site. In a limiting case of slow rotating macromolecule and a fast translational diffusion of the mobile reactant (a proton in our case) (for details, see Shoup et al., 1981), we can approximate

3 ken = - .ir2NDH+ r(,) 8

-

(44)

The electrostatic interaction terms should also be modified to account for the unequal size of the reactants. The terms appearing in Equation (42) represent the electrostatic potential at the encounter distance rv; if one of the reactants is a macromolecule, we have to calculate the electrostatic potential on its surface and thus r, will replace rq in the two last terms of Equation (42), that is, RD rm

and

~

rmx 1 + r,x

The effect of charge on the rate of protonation of specific site adsorbed on high-molecular-weight structure was measured with the Neutral RedBrij system described above (Equation 42)using the negatively charged detergent, SDS, to modify the micellar charge (Gutman et al., 198la). The charge of these mixed micelles (Fromherz, 1973) was calculated according to Tanford using the pK shift of the bound indicator (Figure 33).The pK of a protonable group carried on the surface of an impenetrable sphere without a radius of exclusion (both solvent and solutes can freely approach the surface of the impenetrable sphere) varies with the charge of the sphere and with the ionic strength of the solution (Tanford, 1976).

T H E pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

71

SDS/rnicelle Figure 33. The dependence of the pK of adsorbed Neutral Red on the average sodium dodecyl sulfate content of mixed micelles.

pWAZ = 0.867W where AZ is the charge increment of the sphere and W, the electrostatic free energy on the sphere surface, given by W=

e3

1

2rmE kT 1 + r,x

(45)

At I + 0, the second term approaches unity. Under such conditions, the value of W for a micelle with r, = 17 A (Tanford, 1973) will be W = 0.205. However, the experimentally measured value (W = 0.0674) is much smaller, indicating that only 33% of the adsorbed SDS molecules are not neutralized by counterions (the extent of neutralization by counterion in pure SDS micelle is 85% (Finstein and Rosano, 1967)). The fractional charge of SDS on micelle and the micellar composition allows calculation of the average charge of the micelles. The micellar charge was also corroborated by the Guoy-Chapman diffused, double-layer model. At equilibrium, the surface charge of the micelle alters the ionic composition of the interface with respect to the bulk concentration. The difference between the actual proton concentration on the interface and the one measured at the bulk by pH electrode is observed as a pK shift of the indicator and is related with the GouyChapman potential (Goldstein, 1972). '4'~ = 60A pK

72

MENACHEM GUTMAN

of which the charge density (or charges per micelle) can be obtained (Adamson, 1960).As seen in Figure 34, the estimates of surface charge by the two methods are compatible. The rate constants of protonation of Neutral Red adsorbed on these mixed micelles was measured, the rates were extrapolated to I = 0. The dependence of the extrapolated values on the micellar charge is given in Figure 35. Similar studies were carried out using photoionization of benzidine derivatives (Narayana et al., 1982),phenothiazine (Alkaitiset al., 1975),or pyrene (Gratzel and Thomas, 1974) dissolved in the hydrophobic core of charged micelles. A laser pulse ejects an electron which is rapidly hydrated and then decays into hydroxy radicals, or reacts with the excited cation. The charge of the micelle has a dramatic effect on the rate of the latter reaction but the presence of the former reaction forbade a precise kinetic analysis of the overall reaction. The adherence of our experimental results to the predicted curve (Figure 35)bears a straightforward implication. Any increment in macro-

SDS yh4

Figure 34. Determination of micellar charge from equilibrium and kinetic measurements. The decrement of micellar charge as a function of sodium dodecyl sulfate added to Brij 58 micelles was calculated from the pK shift according to Gouy-Chapman equation (I = lo&) (A) or from the second-order rate constant of protonation using Debye’s equation (Eigen et al., 1964)for rates measured in the presence of ionic screening (0)at I = 1O M , or from rates extrapolated to I = 0 (0)(Gutman et al., 1981a).

T H E pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

73

15

10

5

Figure 35. The correlation between the second-order diffusion-controlled rateconstant of protonation of adsorbed Neutral Red and the micellar charge. The micellar charge was calculated as described in the text. The continuous line was calculated according to Debye’s equation (Eigen et al., 1964).

molecular charge will be reflected by an immediate pK shift of all protonable group on its surface, and these groups will react with the protons in the solution in a diffusion-controlled reaction; a pH indicator adsorbed on a macromolecule (or membrane) will adjust its state of protonation with the momentary charge of the protein. The use of pH indicator for measurement of rapid changes in charge density of protein has been employed by Carmeli and Gutman (1982). Bromo Cresol Green was adsorbed on bacterial rhodopsin membrane sheets suspended in well-buffered solution ( l O O m M Mes buffer, pH = 5 ) and the photocycle of the bacteriorhodopsin was initiated by 10-nseclaser pulses. The protonation state of the indicator was probed by an He-Ne CW laser and the transient absorption was averaged. In spite of the high buffer capacity of the medium, a transient acidification of the adsorbed indicator was measured-reflecting the charge modulation of the protein and the subsequent redistribution of protons in the Gouy-Chapman diffused double layer.

3. The Effect of Postprotonation Reaction on the Dynamics The pK of a solvent-accessiblegroup on a protein may deviate markedly from the pK of the free species (Russu et al., 1982).This deviation might be a consequence of pK shift by electrostaticinteraction with other charges on the proteih, or result from selective stabilization of one form of the proton acceptor by the immediate vicinity of the protein. For example,

74

MENACHEM GUTMAN

the pK of His $3146of carbomonoxyhemoglobin A, as measured in high ionic salt solution, is pK = 7.85. Such high value cannot be attributed to electrostatic interaction in the solution (Z = 0.lM) but rather to the salt bridge with the nearby carboxyl of Asp p94. Another type of in situ stabilization was noted by Klotz and Fiess (1960). The pK of the dimethylamino naphthalen sulfonyl adduct to bovine serum albumin was measured and found to be lower than that of the free ligand and independent of the ionic strength. The pK shift is attributed to selective stabilization of the uncharged form of the probe by the hydrophobic pocket where it is bound to. To protonate the ligand in the site (or to extract it to a more aqueous phase where the acidic state is stable), an incremental free energy is needed. This free energy is supplied by the 100-fold increase of H + needed to protonate the amine in its hydrophobic pocket. Apparently, the pK shift of a surface group can be qither due to variation of the rate constants of the reaction or reflect some postprotonation reaction. Thus, we must be capable to discriminate in our analysis between the two mechanisms. The model used for this purpose was Bromo Cresol Green (Scheme 1V) adsorbed on Brij 58 micelles (Gutman et al., 1983a). Bromo Cresol Green, due to the solfono group, is water soluble both in the acidic and the alkaline states. This group also prevents it from diffusing through phospholipid bilayers. Due to the large hydrophobic surface of the three aromatic rings, it is well adsorbed to micelles and liposomes both in alkaline and protonated states. In its alkaline state, the phenolate and the quinoid rings are in resonance and the charge is delocalized. This form is fairly soluble in the lipid core of the micelle. A much higher lipophylicity is achieved in the acidic state. The disruption of the resonance by the protonation yields two distinguished nonresonant structures (phenolic and quinoid rings) which are more lipid soluble than the resonant form. Thus, protonation of the indicator on the interface will reorient the molecule in such a way that both phenolic and quinoid rings will have a better contact with the lipid region of the interface. Such reorientation will insert the dissociable proton into an environment where it cannot dissociate (Huppert et al., 1982). Consequently, the dissociation of the

Scheme IV

75

THE pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

proton will be delayed until the phenolic ring reaches the aqueous phase of the interface. Bromo Cresol Green adsorbed on Brij 58 micelles shifts its pK from pK = 4.96 (I+ 0) in water to pK,b, = 6.5 0.05. The distribution ratio of the acidic (a)and alkaline (0) states are a = 30,000 k 1000 and 0 = 1100 2 100. Thus, protonation increases the lipophilicity of the indicator by 30-fold. The pK of the adsorbed indicator is independent of the ionic strength (Table VI), suggesting that the micellar charge is close to zero. T h e simulation of the dynamics of protonation of bound Bromo Cresol Green, using Equations (37) and (38), failed to reproduce the observed dynamics as long as we assumed that the measured pK is a direct function of the forward and backward rates of proton transfer. This inadequacy led to the conclusion that for this case K o b s f k 4 / k 3 and provision must be made for a mechanism where the postprotonation reaction modulates the observed dynamic and equilibrium.

*

A. SIMULATION OF PROTONATION OF ADSORBED BROMO CRESOL GREEN

Protonation of adsorbed Bromo Cresol Green is a fast, diffusion controlled reaction (Gutman et al., 1983a, Gutman et al., 1981a) (see Table VI), implying that the encounter between the proton and the phenolate ring of the alkaline state is the rate-limiting step in HIn formation; that is, the alkaline form of the adsorbed indicator does not insert both resonating rings into an environment where protons cannot diffuse. T h e acidic state of the indicator is 30-fold more lipid soluble than the alkaline state. T h e mechanistic equivalent of this enhanced stability is an insertion of the neutral, proton-bearing hydroxyl into the hydrophobic environment of the micellar surface (Mukerjee and Cardinal, 1978; Dill and Flory, 1980; Narayana et al., 1982). Thus, we have to account for three populations of TABLE VI Thermodynamic and Kinetic Parameters for Protonation of Bromo Cresol Green Adsorbed on Brij 58 Micelles" kin

-RTln -

Salt

(d) pKob, 0 100, KC1 100, KCNS

6.5 2 0.05 '6.5 k 0.05 6.5 k 0.05

PKS 5.35 ? 0.05 5.5 f 0.05 5.3 ? 0.05

k3

kout

(M-' sec-') 0.65 2 0.05 1.00 k 0.05 0.55 ? 0.05

X X X

10'" 10'" 10'"

kou,

(kcal/mol)

(sec-')

-1.56 -1.3 -1.60

150 ? 50 150 50 100 2 50

*

"The experiment was carried out in the presence of 50pW indicator, 40 mg BrlJ 58/ml, and I d of Z-naphthol-3,6-disulfonate.

76

MEkACHEM GUTMAN

the indicator In,,, HIn(,,, and HIn(,) which are the surface-bound form of In-, the surface-bound form of HIn, and the more hydrophobic location of the proton-bearing hydroxyl [HIn(,,]. (The free forms of the indicator are ignored, as they consist of less than 2% of the total indicator.) The equilibrium between the reacting components is given by reactions (46), (47), and (48).

The correlations between the observed equilibrium constant and the various rate constants are k4 k3 K, Kobs =kin I+K,

-

(49)

kOUt

The corresponding differential rate equations are written with three time-dependent variables: X and Y, as defined before, plus a third one, Z, the increment of HIn(,,. HIn,,, is now given by HIn,,) = Y

-

2

The differential rate equations are:

dX

-=

dt dIndt =

[-k,([Rs-] -

+ [Hf]) -k2

dY dt

k3[Gv(

-

[k3([E+] +

]X

+ kl Y - kl X 2 + kl X Y

[In-l)+ k4]Y - k&Y + k3Y2 + k4Z

(51)

(52)

For simulating the reactions summarized by Equations (51-53), we have to negotiate with three independent unknown variables k3, K,, and

T H E pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

77

k,,, (or kin). T o avoid a laborious computer search over a three-dimensional matrix, we tried to estimate the magnitude of k3, the diffusioncontrolled rate constant of protonation of bound Bromo Cresol Green. As discussed by Shoup et al. (198l), the rate constant of a diffusion-controlled reaction of a small ligand with specific site on a macromolecule (ignoring the charge effect) is a function of the site's size. As Neutral Red is comparable in size and adsorbed on the same type of micelle, we can estimate that k3 for Bromo Cresol Green will be similar to that measured for Neutral Red, that is, k3 = 1 x 10'oM-' sec-I. With one variable temporarily determined, we systematically varied the other two. We let the pK, of adsorbed Bromo Cresol Green vary, from the pK measured in water (4.96) up to pKobs, with 0.1 pK unit increments. At each pK value (which sets a certain ratio of kin/kout, Equation 50) we varied k,,, over a wide range (10- lo6) and looked for values that superimposed the simulated curve over the experimental one. Once the simulated curves approximated the experimental one, we varied k3 to ascertain the accuracy of our estimated value; we repeated the search over the pK, and KO,, matrix until superpositioned curves were obtained. T h e result is shown in Figure 36. T h e surface pK for the bound indicator is pK = 5.5, corre-

o 20

40

time

so

80

(psec)

Figure 36. , Experimental results and simulated curve for the protonation cycle of Bromo Cresol Green adsorbed on Brij 58 micelles. The experiment was carried out in the presence of 1mM 2-naphthol-3,6-disulfonate,40 mg/ml Brij 58,57@4 Bromo Cresol Green, l O O m M KCI (pH 7.07). The rate constants used for simulation were k, = 5.0 X 10" M-' sec-'; k3 = 1.0 X M-' sec-'; k,,, = 160 sec-'; X , = 7.75M pK, = 5.51 and pKOb,= 6.5. (-) Experimental; (---) simulation.

78

MENACHEM GUTMAN

sponding to kinkout = 9. The best fit is obtained with k3 = 1 X 10'oA4-' sec-', and k,,, = 150 50 sec-'. The rate constants for this experiment and of those carried out in absence of added salt, or in the presence of lOOmM KCNS are listed in Table VI.

*

B. CLASSIFICATION OF POSTPROTONATION REACTIONS

For the purpose of the coming discussion, we shall assign all systems that undergo a postprotonation reaction into two classes. In class I we include those where the microenvironment stabilizes the nonprotonated configuration, such as the hydrophobic site of dimethyl amino naphthalene in bovine serum albumin (Klotz and Fiess, 1960) or the Neutral Red-Brij micellar system (Gutman et al., 1983a). Class I1 will include those cases where the protonated state is the favored one. In this class, we include the Bromo Cresol Green-Brij micelle, or the 146phistidine of carbomonoxyhemoglobin (Russu et al., 1982). The effect of the stabilization of class-I1 compounds in their protonated state will be analyzed using the Bromo Cresol Green. Protonation of this indicator increases its binding energy from AG = -4.1 kcal/mol, (corresponding with p = 1100) to -6.1 kcal/ mol (a= 30,000). This 2 kcaymol increment is mostly due to the transition of the protonated indicator from the (s) to (c) position (-RT In (kin/ kOuJ = - 1.5kcaymol;Table VI). The remainder ofthe increment (--0.5 kcal/mol) can be attributed to the preferential stabilization of HIn,,, with respect to In-,,). Indeed, the pK, of Bromo Cresol Green is -0.35 pK units (--0.5 kcal/mol) higher than that measured in water (Table Vl). The postprotonation reaction, that is, the translocation of the proton-bearing hydroxyl into a more hydrophobic environment, slows the dissociation of the proton from the adsorbed indicator. We have already demonstrated that proton dissociation necessitates the presence of water molecules which act as proton acceptors (Huppert et al., 1982).Thus, whenever the proton-bearing hydroxyl is inserted into the (c) phase, dissociation is delayed until the indicator resumes its (s)-phase orientation. Consequently, the HIn,,) and HIn,,) have different decay time constants and the ratio of these populations is a function of time and of k;,/k4. Figure 37 demonstrates the effect of the postprotonation event on the following parameters of the observed dynamics: HIn(,), HIn,,), and y2. These parameters were computed for a class-I1compound with the same kinlkOut, pK, and k3 which were measured for Bromo Cresol Green, only that Kin was varied from 10 to lo6 sec-'. In systems were ki,,/k4 << 1, the transient protonation of In-(,) cannot propagate into the (c) phases. At higher values, there is a rapid insertion of HIn,,, into its thermodynamic-favored environment; HIn,,, accumu-

T H E pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

20

79

i

kwt

Figure 37. The dependence of the macroscopic parameters of the protonation cycle of class-I1 compounds on k,,, (or kiJk4). The parameters were calculated for simulation representing the protonation of class-I1 compounds using the rate constants, pK, and pKOb,and concentration listed for the Bromo Cresol Green, Brij 58 system in Figure 36. k,,, (lower abscissa) [or ki,/k4 (upper abscissa)] was varied as indicated. (-) HIn(,,tal) max; (---) HIn(,, max; (-) y2 of HIn(roral) relaxation.

lates even during the brief perturbation and will affect the rate of the relaxation as demonstrated by the dependence of y2 on kin. In contrast to y2, the magnitude of y1 is independent of kin (not shown). Simulated dynamics with varying kin/k4 ratios are given in Figure 38. Figure 38A depicts the dynamics with k;,/k4 = 0.1. In this case, kin is too slow to allow appreciable accumulation of HIn,,,. However, toward the end of the reaction, when the ratio of HIn(,,/HIn(,) increases due to the faster dissociation of HIn,,,, the rate is slowed down. This is observed as a tail in the HIn decay, and in a curvature of the logarithmic plot (Figure 38A, insert). Figure 38B represents a similar stimulation in which kink4 = 1. In this case, the contribution of HIn,,, to the measured dynamics, noted as the biexponential decay, is appreciable much earlier than in Figure 38A. Figure 38C (kink4 = 10) depicts the dynamics when the two populations HIn,,, and HIn(,, are in fast equilibrium, and HIn decays as a single exponential function, characterized by apparent pseudo first-order rate constants. For the specific case where kin >> kq,a direct relationship between the microscopicrate constant and the apparent ones can be obtained. The fast

3

3 I

0

=4 L 3-2

-

1

3

20

0

60

40

%* I /

/

/

/

/

/

0

1

3

a*

5

0

1

1

20

1

1

40

1

1

60

1

1

I

I

I

I

C

1

20

I

1

60 time ( p s e c ) 40

I

80

Figure 38. Simulated dynamics of protonation of class-I1 components at varying rations of kin/k4. The simulations were run for the rate constants pK,, pkb,, pH, and concentration listed for the Bromo Cresol Green, Brij 58 system in Figure 36. (A) ki,/kr = 0.1. (B) ki,lk4 = 1.0. (C) kin/kr = 10. (-) HIn total; (---) HIn,,,. The inserts are the semilogarithmic plots of HIn(cota,trelaxation.

80

T H E pH JUMP: PROBING OF MACROMOLECULES AND S O L U T I O N S

81

exchange of the HIn between its two locations allows us to regard HIn,,) as a steady-state intermediate dHIn,,, - d(Y - 2 ) -=o dt dt This steady-state equation leads to the expressions that relate the apparent pseudo first-order rate constants kS(app) and k4(app) with the kinetic rate constants

and their ratio:

kapp) = kapp)

k4/k3

1 + kin/kout

= Kobs

(see Equation 50)

In the case where both kin and kout are fast, the ratio of the apparent forward and backward rate constants is identical with the dissociation constant determined by equilibrium methods. A different situation prevails in the range where the ratio kink4 is very small. Under such a regime, the brief protonation-dissociation cycle will not propagate into the core, and the dynamics will be controlled only by the rate of the surface reactions. For such specific cases there will be a marked difference between the pK,, derived from the kinetic experiment, and pK,bs obtained by equilibrium measurements. Consequently, it is only the accurate kinetic analysis that can furnish the precise description of the system and yields the infcrmation about the events that follow the protonation. The interrelationship between k&s, K,, and the ratio kin/kOut for class-I compounds is derived by replacing reaction (48)by reaction (56). I"(,)

kin

kou,

,

I"@)

(56)

The replacement leads to Equation (57) which substitutes Equation 50 for class-I compounds.

The numerical analysis of the kinetics of adsorbed Neutral Red (Figure 32) yields a value for K , which is identical with the Kobsmeasured under equilibrium conditions. Thus, for this indicator, adsorbed on Brij 58 micelles (kin/kout) << 1, that is, even the more hydrophobic state of the indicator is preferentially located on the micellar surface rather than in

82

MENACHEM GUTMAN

the core. This observation is in accord with the surface-activity model of Mukerjee and Cardinal (1978). The surface tension of the alkaline indicator is smaller than that of the exposed wettable methylene groups of the micelle (Fromherz, 1981). Thus, in spite of its hydrophobicity, the thermodynamic favored location of Neutral Red is at the interface of the micelle. This location of the proton acceptor accounts for the observed diffusion-controlled protonation. The Neutral Red-Brij 58 system is a simple case of class-I dynamics, yet it is not the general case and the postprotonation reaction may have its effect on the measured dynamics. The effect of kin/kOut on the dynamics of pulse protonation of class-I indicator is depicted in Figure 39. A low ratio implies that the In(=) population is small, and there will be no apparent difference between K, and Kobs (see Equation 57), which is the case for Neutral Red. In such cases, there will be no kinetic abberrations due to the interphase transition. As the ratio kink,,, increases, the contribution of In(=)population will increase, affecting all measured macroscopic parameters. At a very high ratio, the In(,) population is becoming so small that the pulse protonation will decline to nearly unobservable magnitude. This is the most profound distinction between the dynamic properties of two classes. Class-I1 compounds will participate in pulse protonation whatever the kin/kout ratio, as their proton acceptor form is located in the protonpermeable phase. On the other hand, class-I compounds may be totally masked in pulse experiment due to depletion of the basic form from the (s) phase. The effect of the magnitude of k,,, on the observed dynamics is represented in Figure 40. In these calculations the ratio kinlkoUt was ke t constant, but the value of k,,, was varied from 100 sec-' to lo5 sec- . One may expect that the fast equilibration between the (c) and (s) phases may increase the fraction of the indicator being protonated during the pulse, but it is not so. Remember that even for soluble indicator the fraction H I d I n - is a complex function of many variables, thus, the nearly invariance of Y,,, vs. k,, is not in contradiction with theoretical expectations. The major effect of rapid (c) + (s) transition is on the I q C ) population. Fast equilibrium will allow a replenishment of the In(,) population and consequent depletion of In(,) (note the negative sign of Z in Figure 36). Once k,,, 2 lo5 sec-', nearly all of the indicator protonated on the surface is replaced by In migrating from the core-a reaction that slightly increases the total amount of HIn formed. The only parameter of class-I compound that is sensitive to the interphase kinetics is the (c) phase population, for which, presently, we have no specific detection system. A more selective monitoring is needed to differentiate

P

THE pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

83

I -20 I

*

z

0

0 c

-

m

n

P

05-

-10

03

0

km/

kout

Figure 39. The dependence of the macroscopic parameters of the protonation cycle of class-I compounds on hn/kout. The parameters were calculated from simulated dynamics run at the experimental conditions and rate constant of Neutral Red and listed in Figure 32. The values KO,, and pK, were arbitrarily set as 200 sec-’ and 5.5, respectively. (Top) The variation macroscopic rate constants: (-) yI, (---) 7 2 . (Bottom) The variation of maximal amplitude (-) Y , and (---) T,,,.

between the probe located in the (c) and (s) phases. The combination between these methods, the kinetic advantage of the pH jump, and the accuracy of the numerical analysis will permit a more profound probing of hydrophobic microspaces of the proteins.

84

MENACHEM GUTMAN

Figure 40. The dependence of the macroscopic parameters of the protonation cycle of class-I compounds on k,,,. The parameters were calculated for simulation representing the protonationof class-I compounds using the experimentalconditionsand rate constant listed Total change of HIn in Figure 32. pK, = 5.5; kin/k,,, = 9; pulse size low H+. (-) (Ymax); (---) the change in In,,, population (ZmaX).Note the negative sign of Z reflecting the depletion of In(c).

VII. PROTON TRANSFER ON THE SURFACE OF MACROMOLECULAR STRUCTURE Proton transfer between donor and acceptor located on a surface of a protein, or a membrane, is a true representation of many reactions taking place in biochemical catalysis, and is the essence of proton-driven coupled reactions. The ability to measure and analyze the dynamics of protsn flux between two defined sites allows one to probe a specific area of a macromolecule surface. This section will demonstrate how a combination of well adsorbed proton emitter and detector can be used for this purpose. To avoid the complications by the nonhomogeneous surface of a protein, the model system are micelles of the uncharged detergent Brij 58. Studies using phospholipid surfaces and protein are presently under current experimentation. A proton emitter in such experiments should be well adsorbed, yet the dissociating proton must be in contact with the aqueous phase of the interface, otherwise no dissociation will take place. The proton emitter of choice, is P-naphthol. It has an intensive absorption at the wavelength of

T H E pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

85

excitation. T h e water solubility is sufficient for easy manipulation and it is well adsorbed to micelles (in our experiments, the fraction of free Pnaphthol was less than 0.01%) o r to hydrophobic sites. The pK of the ground state (pKo = 9.3) ensures that +OH is the dominant species while pK* = 3 is low enough to make it an efficient proton emitter. The proton detector used is Bromo Cresol Green, for which the mechanism and rate constants are known (see Equation 44). In the absence of micelles, P-naphthol is a very efficient proton emitter, and the protonation of Bromo Cresol Green is easily analyzed by simulation technique (Figure 30). T h e steady-state fluorescence of P-naphthol in water (Figure 4 1) consists of two emission bands, 350 nm (+OH*) and 410 nm (+O-*). Once adsorbed on Brij 58 micelles (4mg/ml), the emission band of $0-*is not observed any more, indicating that the emitter molecules are adsorbed and immerse their hydroxyl in the hydrophobic phase of the micelle. Time-resolved fluorescence reveals that this is not precisely so. T h e fluorescence at h>435 nm has a fast rise time, much faster than that measured for P-naphthol in water, and a decay time characteristic for the

Figure 41. Fluorescence emission spectra of P-naphthol (lo@) in water (A), or adsorbed

to Brij 58 micelles (4 rng/ml) excitation at 300 nm (B).

86

MENACHEMGUTMAN

+O-* species. This fluorescence dynamics indicates that, when adsorbed on the micelle, a small fraction of P-naphthol molecules are oriented in such configuration that their hydroxyl protons are in contact with the aqueous phase. The protonation of adsorbed indicator can proceed by two pathways. The proton may come from the same micelle or from an adjacent one. We shall define the products of each pathway by HInl and HIn2 respectively. The magnitude of HIn, is a function of micelles carrying both emitter and detector HInl=fliigN.nBcG.[Micelle] where iip~=[PN]/[MiCelk] and nBcG=[BCG]/[Micelle).f l is an empirical term reflecting, besides controllable conditions (light intensity, pH, ionic strength), the proton quantum yield and the various rate constants involved in the proton cycle (sections V and VI). The magnitude of HIn2 is related with the concentration of the reactants HIn2 = fyiiPN. [Micelle].iiBc~-[Micelle].where f2 is a proportional factor (measured in M-' units) which reflects the same parameters affecting f , plus those which modulate proton diffusion in the bulk. By themselves f l and f 2 are too complex to be interpreted, but their relative magnitudes characterize the nature of the proton transfer in a given solution. The total product of the two pathways is given by HInT = [BCG] [PN]. (fl [Micellel-' + f2). This relationship is given in Figure 42, where the slope of the line equals (fl [Micellel-' + f2). By

Figure 42. The correlation between the occupancy of a micelle by both emitter and detector, with the maximal amount of protonated detector measured after the excitation pulse. The results are drawn according to equation the HIn, = [BCG] [PN] (fl[MiC]-'+f2). The reactants concentrations in the experiment were 40 mg/ml Brij -58 and p naphthol 100-1000 (500 +M, rnicellar concentration). Bromocresol green 50 a.pH 7.5.

m

THE pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

87

repeating the measurements at different micellar concentrations (not shown) f l and f2 were measured as f l = (2.9 0.02) lo-’ and f2 = 1.58 f 0.05 M-I. As long as the product f 2 - [PN] is smaller than fl the dominating pathway is the proton transfer between reactants adsorbed on the same micelle. Each micelle is an isolated system whereas the macroscopic observation is a sum of many singular stochastic events. To exclude the possibility that the proton transfer takes place in the core of the micelle, it was tested whether a soluble, highly hydrophylic acceptor phosphate anion can compete with the indicator for the proton. As demonstrated in Figure 43, phosphate competes with the indicator whether the emitter and the indicator are adsorbed on the micelle or are free in solution. Thus, the protons are formed on the interface of the micelle and are not confined in any kind of microenvironment that secludes them from the bulk. The analysis of these kinetic measurements should employ a stochastic approach. Each micelle can have a discrete integer number of emitter and detector molecules, as given by the Poisson distribution (Miller, 1978; McQuarri, 1963; Miller et al., 1980). Still, as was argued by Vass (1980), the stochastic dynamics will differ from classical dynamics by not more than 15%.Thus, being aware of this Iimitation, we simulated the experimental results using the classical kinetic equations instead of the stochastic formalism. In these simulations,we accounted for the (S) + (C)transition of the protonated indicator, the K , value and kOu, as derived in Equation (50) (see Table VI) and varied the values of the following parameters: kl, k S , Xo, and [In-] until a satisfactory simulation was obtained (Figure 44). The results of a series of such measurements are listed in Table VII. Because of the discrete nature of the measured events, and the averaged

*

-

a1 a2 c23 0.4 0.5 KPI WM

Figure 43. The competition between phosphate buffer and indicator for the pulse-emitted protons. 40pM Bromo Cresol Green, 200p.M p-naphthol. The reaction was measured in water (0)or in micellar system (250pMmicellarconcentration) (Cl).The resultsare normalized for HInmaxmeasured in the absence of buffer.

TABLE VII Amplitudes and Dynamic Parameters of Proton Transfer between @-Naphtholand Bromo Cresol Green Adsorbed on Brij 58 Micelles"

fi,,

micelle

0.2 0.4 0.5 0.6 0.8 1.0 1.5

2.0 3.0 4.0 6.0

[HInI,,, measured

Effective pulse size

Effective indicator concentration

(W)

k l X lo-'' (M-' sec-I)

k3 x lo-'" ( M - ' sec-')

0.05 0.082 0.100 0.1 12 0.171 0.182 0.267 0.366 0.455 0.513 0.652

0.47 2 0.03 0.60 2 0.03 0.60 2 0.03 0.72 2 0.03 1.00 2 0.05 1.25 2 0.10 1.75 2 0.10 3.00 2 0.10 4.50 2 0.20 6.00 2 0.20 9.00 2 0.20

0.50 2 0.02 0.70 2 0.02 0.75 2 0.02 0.80 5 0.02 0.90 2 0.02 1.05 2 0.02 1.05 2 0.02 1.00 2 0.02 1.00 2 0.02 1.00 2 0.02 1.05 2 0.02

2.50 2 0.25 2.75 2 0.25 1.25 2 0.25 1.60 2 0.15 1.50 2 0.10 1.50 2 0.10 1.50 2 0.10 0.90 5 0.05 0.85 2 0.05 0.62 2 0.03 0.57 0.03

20 2 5 20 5 5 17 ? 5 17 2 5 15 k 5 10 ? 1 9.5 2 0.5 7.0 2 0.5 5.5 5 0.5 5.0 2 0.5 4.5 2 0.5

(W)

(W)

*

"Bromo Cresol Green, 50pM; @-naphthol,0.1 -3.OmM (pH 7.4-7.5); Brij 58, 40 mg/ml (500p.M in micellar concentrations).

Figure 44. Experimental results and simulated dynamics of proton transfer between emitter and detector bound to the same micelle. k l = 2.5 x 10" M-' sec-' and 3.0 x 10" M-' sec-' for upper and lower simulated curves; ks = 2.5 X 10" M-' sec-'; h,,, = 150 pK, = 5.35.40p.M Bromo Cresol Green, 200p.M f3-naphthol, 500 p M Brij 58 (micellar concentration).

T H E pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

89

output of the calculations, the values listed in Table VII call for further anaIysis to derive the physical interpretation of these figures. The amount of protonated indicator detected after the pulse increases linearly with the average content of emitter (Figure 45).The slope of this line corresponds with a yield of 0.003 H+/P-naphthol (mol/mol) which was successfully transferred to the indicator. As the indicator occupies only 10% of the micelles, the dissociation is undetected in 90% of the events. Thus the true yield of protons is 3% of the P-naphthol present. Only 1 of -30 adsorbed P-naphthol molecules is located in such configuration with respect to the micellar surface that the proton can dissociate, in accordance with the negligible emission of adsorbed +O-*. Considering the low efficiency of proton dissociation, the probability of two protons ejected on the surface of the same micelle is very low. Still, the linear correlation between iipN and probability of proton dissociation affects the apparent indicator concentration. A micelle carrying one indicator and few emitters will always record a single proton transfer, but the probability of such an event increases with the number of emitters sharing the same micelle. This increased probability is translated by the classical kinetic formalism as a higher effective concentration of the indicator (Table VII). As long as the physical properties of the micelle are invariable, a linear dependence of the effective indicator concentration on iipN is expected. The deviation from linearity at iipN > 1 may imply that the micelle vary some of its properties. This consideration becomes

1

2

3

-%em

4

5

6

Figure 45. The dependence of the pulse size on the average occupancy number of @-naphthol.5 0 w Bromo Cresol Green; 5 0 0 M micellar concentration and varying pnaphthol. Data taken from Table VII.

90

MENACHEM GUTMAN

evident once we consider the actual distribution of the discrete number of molecules per micelle given by the Poisson distribution

At ii < 0.4, some 80% of the population consists of micelle with only one P-naphthol molecule. At ii = 1.0, 40% of the population contains more than one emitter and at ii = 3 it is -5%. Thus, the higher is the occupation number the less homogeneous is the observed population. As long as we cannot exclude experimentally any secondary interactions between the adsorbed reactants, or modification of the physical properties of the measured system, it is better to limit the interpretation to low occupancy numbers where only one emitter-detector pair share the micelle. The rate constants we measured under these conditions, both for protonation of the indicator and the P-naphtholate are significantly faster than those measured for the free reactants. The rate of protonation of free P-naphtholate is (1 -C 0.1) X 10'oM-' sec-' and free Bromo Cresol Green reacts with the proton at a rate constant of (4.2 0.1) IO'OM-' sec-' (Gutman et al., 1983; Table IV.). The rate of Bromo Cresol Green protonation by a proton produced on the same micelle is 20 times faster than protonation of bound indicator by a proton coming from the bulk 20 X 10" and 1 X 10'oM-' sec-', respectively (Table VII). There may be many reasons for this fast reaction. The most likely one is the nonrandom distribution of distance between the emitter and the detector; it cannot exceed the halfcircumferenceof the micelle (-60 A).Therole of other factors, such as high proton conductivity of the hydration layer, and the two dimensionality of the trajectory (Adam and Delbruk, 1968)is a subject for further research.

*

VIII. THE EFFECT OF BUFFER ON THE DYNAMICS OF THE PROTON CYCLE The experimental systems described above consisted of the minimal number of components: proton emitter and detector. If one wishes to probe a specific site on a protein, he should consider the presence of a third component-the other proton-binding site on the protein. Any species with which the proton may react, referred to here as buffer, will modify the progress of the reaction and perturb the shape of the observed parameter. As the general strategy is to extract the partial rate constant out of the macroscopic ones, we have to retrace the effect of the partial

THE pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

91

reactions of the buffer and evaluate their effect on the macroscopic parameters. T h e reactions which should be considered are (58)-(63). k2

@OH-40kl

HIn

+ H+

k

&In- + H'

(59)

k3

BH B-'

k6 k5

+ H+

plus direct proton exchange between the reactants. k7

H I n + B--BH ks BH

ho + $0- -$OH

+ B-

k12 + $0- /$OH

+ In-

k9

HIn

+ In-

k11

(63)

The corresponding differential rate equations for X,Y , and Z (the increment of buffer protonation) are given in the Appendix. It is obvious that so many variables make a simulative solution a heroic endeavor unless some of them can be obtained by independent experiments. Considering the fact that any application of the proton pulse in an enzymic system must deal with appreciable buffer capacity of the protein or substrates, I shall demonstrate the general procedure of a simulative solution for a buffered solution.

1. Two-Component Systems: Buffer and Proton Emitter T h e three-component system is first dismembered into two-component reaction systems: +OH + indicator (see Section V) and +OH + buffer. Mathematically, these two are identical and can be simulated as detailed above. Yet, practically, there are basic differences between them. T h e monitoring of the indicator-emitter system is through the increment of HIn(Y), while the buffer-emitter pair must be monitored through the transient absorbance of +O-(X) (see Section V.1). In practice this may limit the study to proton emitters that have an absorption band at a wavelength suitable for monitoring.

92

MENACHEM GUTMAN

The second difference concerns the concentration of the reactants. Although indicator concentration can be reduced to the limit of detection, the buffer concentration cannot be easily manipulated. The buffer capacity is generally imposed on the experimental system. Thus, measurements with the buffer-emitter system are carried out in well-buffered solution. Furthermore, while indicator is a noninformative detector at pH < pK, the behavior of the buffer must be considered both above and below its

PK-

The rate constants associated with the buffer system consist of its acid dissociation (k5 and k6) and its direct proton exchange reaction with the emitter (kg and klo). The latter two can be approximated by measuring the effect of the buffer on the dynamics of $0-at pH 7. The proton transfer between $OH and B- can be mediated through proton diffusion (DH+= 9.3 x lop5 cm2/sec) (reactions 58-60) or through the diffusion collision of the reactants (D = 1 X cm2/sec)(reaction 62). In a well-buffered solution near the pK of the reactant ([B- + BH] >> X O ; pH = pK65 = pK21), the increment of Hf after the pulse will be so small that most or all of the relaxation reaction will proceed through the direct proton exchange pathway. Under these specific conditions, the linear approximations of Eigen (1964)are applicable and lead to a simple expression:

-

kobs =

+ BH) kg($GH +B-) + K H ) vs. (WH +K-)/(@+BH)

klo(@-

(64)

A drawing of hobs/(@should yield a straight line with an intercept k l o and slope of k g [Figure 46). It must be stressed that this is an approximative solution that ignores the contribution of the proton diffusion pathway. This omission leads to a systematic error: The equilibrium constant K l o . gas derived by equilib= 5, whereas the value derived rium measurement is K ~ O=.K21/K65 ~ from the rate constant (Figure 46)is kl,,/kg = 23. The approximative values for k g and k l o can be incorporated into a in order to obtain k5 full simulative solution of the relaxation of $0-, (and k6) and refine the values of k l o and kg. The presence of high buffer concentration has a mixed effect on the relaxation of $0-. At pH > pK,j5, B- will compete successfully with $0- for the ejected protons and will slow the relaxation to its prepulse level. At pH < pK, where most of the buffer will be protonated, it will be a poor proton acceptor and efficient proton donor (by direct proton exchange). At this pH range, the buffer will accelerate the decay of $0-to its prepulse level. This effect is demonstrated in Figure 47 which relates the rate of $0-relaxation with the initial pH.

18x10.

10X10*

I

I

I

I

I

lo

1

1

I

-

I 20

C+PhOH BH+PhO'

Figure 46. The effect of buffer on the rate of 40- relaxation after a laser pulse. 100pM hydroxypyrene trisulfonate, 0.1- lOmM of imidazol. The pH of the experiment was varied between 6.5 to 8.5. Intercept = 1.8 X lo9 M-' sec-'. , slope = 7.7 X 10' M-' sec-'. The reaction was monitored at 445 nm using CW HeCd laser as a probing light.

>

6.0

7.0

PH

8.0

Figure 47. The effect of pH on the rate of +O- reprotonation in an emitter-buffer system. loo@ hydroxypyrene trisulfonate, 2mM imidazol buffer. The reaction was followed at 440 nm using CW dye laser. The continuous line is computed for the emitterbuffer system using the rate constants R1 = 1.6 X 10" M-1 sec-'; k5 = 3.5 x 10" M-' sec-'; klo = 2.1 X lo9 M-' sec-I. Measurements in the absence of buffer (0)or in the presence of 2mM imidazol (m).

93

94

MENACHEM GUTMAN

2. Three-Component System: Emitter, Detector, and Buffer A. SIMULATIVE SOLUTION

A simulative solution for the rate equations (detailed in Appendix) describing a three-component system is possible only if the rate constants are previously measured through binary systems (except those given by Equation 61). But once at hand, simulation is feasible as demonstrated in Figure 48. This figure simulates the transient protonation of Bromo Cresol Green by pulse dissociation of 2-naphthol-3,6-disulfonate in presence of 0.25mM imidazol (pK = 7.0). T h e same simulative procedure was used for describing the reaction in a hydroxy pyrene-Bromo Cresol Green - imidazol system and the characteristic rate constants, needed for superimposing of the simulated curve on the measured one, are listed in Table VIII. As evident from the table, the parameters that really govern the dynamics are those that were determined by high degree of accuracy-the reaction of each component ( I t , , k 3 , and It5) with the free proton. These parameters determine the shape of the simulated dynamics and are critical for superimposing the simulation on the experimental curve.

-.2

0

I

I

1

,766

1

I

I

1

2.297 MICRO SECONDS

I

1.532

a

a

1

L

I

3.063 I

1

I

1

3829

Figure 48. Experimental results and simulated curves for a three component reaction. T h e experimental system consisted of 1mM 2-naphthol-3,6-disulfonate, 20pM Bromo Cresol Green, and 0.25mM imidazol (pH 7.0). T h e simulations demonstrate the effect of increasing the buffer concentration; 0.25mM (upper simulation) 0.475; 0.738; 1; 1.3; 1.6 and 2mM (lower curve).

0.25-4

2.0 4.0

1.o

0.5

7.0

5.73 6.1 6.75 5.65 5.9 6.05 5.54

PH

4

0.7 t 0.05 f 0.2

4 t 0.2 4 f 0.2

1.6 t 0.1 1.6 -+ 0.1

f 0.2

f 0.2

4

4

k3 X lo-'' (M-' sec-')

1.6 f 0.1

1.6 f 0.1

(M-' sec-')

k ] x 10-11

2.0

f 0.1

3.25 t 0.25 2.75 f 0.25 2.75 f 0.25 2.75 f 0.25 3.25 t 0.25 2.75 t 0.25 2.75 f 0.5

k5 x lo-'' (M-* sec-')

OIOOpM8-hydroxypyrene-I ,3,6-trisulfonate, 20M Bromo Cresol Green, and imidazole. * I mM 2-naphthol-3,6-disulfonate, 20@ Bromo Cresol Green and imidazole.

Bb

A"

(M)

Imidazol

1

322 3?1

322

322

k7 X lo-' (M-' sec-')

*

f

1

5 2 2

2.5 1 2.5 t 1

2.5

2.5 t 1

k l o x lo-' (M-' sec-')

The Kinetic Parameters Characterizing the Dynamics of a Three-Component System: Emitter, Detector, and Buffer

TABLE VIII

10

<5 <5

<5

<5

k 1 2 x 10' (M-' sec-I)

96

MENACHEM GUTMAN

The direct proton exchange reactions (K7,kg, Klo) are determined only by order of magnitude; within the specified range, they do not change the fitting of the simulation to the experimental results very much. B. EFFECT OF INITIAL CONDITIONS

The effect of buffer concentration on the observed dynamics is demonstrated through the simulated curves in Figure 48 and Figure 49AB. Increasing buffer capacity accelerates the rise and decay of the indicator signal and can shrink it into a small spike (Figure 49B). At substantial buffer capacity, the only reliable parameters are Y,,, and y2. The mechanism by which the buffer affects the proton cycle is the competition between the three acceptors $0-, In-, and B- for the proton. This clearly explains the effect of the initial pH on the proton cycle. At pH 6, where imidazol is mostly protonated, it is a poor competitor for the proton and the indicator signal is big (Figure49A). At pH 8, the buffer is highly deprotonated. The proton emitter (2-naphthol-3,6disulfonate, pKo = 9.3) is not ionized and the buffer-proton reaction dominates the dynamics (Figure 49B). C. DYNAMICS OF T H E BUFFER PROTONATION

The simulation solution to the three-component reaction allows us to visualize the unobserved kinetics, protonation-deprotonation, of the buffer. Figure 50 represents the dynamics of all components participating in the proton cycle. At t = 0,3p,M of$OH were dissociated by the laser pulse forming 3p,M of H + (line A) and $0-(line B). The discharged proton recombines with the indicator to form HIn (line C, observed, and simulated). The ionized proton emitter is reprotonated by diffusion controlled reaction with the free protons and by collision with the imidazole H+ species. (At the pH of the experiment, pH = 5.91, -90% of the imidazole is in its protonated state.) Thus, at the initial state the response of the buffer is in opposite direction to that of the indicator. (line D) (The initial response of the buffer is determined by Equation 39, see Section V-4). The first component that regains its prepulse level is obviously the most reactive one-the free proton. The proton emitter $0-,which is the strongest base in the system, is the next one to relax (line B), whereas the other two components, the indicator and the buffer, are the last where the imidazol, the strongest base of the two, regains the proton robbed by +O-. The three-component system is, chemically and mathematically, a nontrivial system. Any measurement intended to culminate by simulative

- . 2 l ,

1

0

I

I

,766

.

I

I

I

1.532

I

I

I

s

I

I

2.297 MICRO SECONDS

I

I

I

3.063

I

,

I 3.82 9

(A)

c

.6

-.21

0

I

I

I

I .766

I

I

d

1 1.532

3

MICRO

I

I

I 2.297

SECONDS

I

I

I

I

1

3.063

I

I

I

3829

( B)

Figure 49. The effect of pH and buffer concentration on the dynamics of indicator protonation in a three-component (emitter, buffer, detector) system. The simulations are for the experimental system described in Figure 48 at pH < p K w m (A) or pH > pKWfm (B), all at the same ordinate scale. (A) pH = 6.0. Buffer concentrations vary from l.0mM (top) to 1.33, 1.66, and 2.0mM (bottom). (B) pH = 8.0. Buffer concentration varies from 0.25mM (top) to 0.47, 1.0, 1.3, 1.6, and 2mM (bottom).

97

98

MENACHEM GUTMAN

____________-_ ------------

====r --= c-

0

1

,

1

.758

1

1

,

/

,

1.516

1

1

1

,

2 273

MICRO SECONDS

1

1

1

1

3.031

1

1

1

3.789

Figure 50. Visualization of the buffer proton cycle through the simulative solution of the indicator response to a proton pulse. A simulative solution for a three-component system, 8-hydroxypyrene- 1,3,6-trisulfonate (100p.M) Bromo Cresol Green (50p.M) and ImM imidazol (pH 5.91). T h e rate constants of the partial reactions are listed in Table VIII. (a) Free proton concentration. (b) 40-concentration. (c) HIn concentration, measured (-), and simulated (---). (d) BH concentration.

solution necessitates a thorough preparative study with the proper binary systems. The same applies, of course, for studies where a protein marked by a proton detector is the subject under study. Such studies with protein may be facilitated by the selecting of a proton emitter that will have the same charge as the protein-to minimize direct proton exchange between 40- and the protein. Similarly, the initial pH can be adjusted to supress the buffer capacity of the protein. This will simplify the analysis and increase the measured signal. Still each specific case will present its own problems, difficulties, and intuitive improvisations. There is no easy solution for tough problems, but basic ones do merit an effort.

IX. CONCLUDING REMARKS The laser-induced proton pulse is a young, high-resolution method recently introduced to biochemistry. It is a system capable of measuring the diffusion-controlled reaction of a proton with its environment, solvent, and solutes. T h e information derived from these measurements is divided according to the time scale of the event: The primary reaction of proton dissociation, recorded in the nano- and subnanosecond time frames, and slower (microsecond) diffusion-controlled reaction of a proton with other solutes. In the fast reaction of proton dissociation, the observed parameter is the fluorescence decay of the excited proton emitter. This reaction probes the most immediate environment surrounding the dissociating proton.

T H E pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS

99

T h e rates measured reflect directly the physical-chemical properties of the water molecule in the nearby hydration shell. These measurements can gauge the properties of the water in an active site where biochemical catalysis takes place. Insertion of proton emitter in a specific site can be useful to determine the internal geometry of a site in a soluble, functioning protein. The subnanosecond measurement is equivalent to a strobe light that freezes in time a transient conformation of a protein. T h e slower reaction, where the proton reacts with a ground-state molecular proton detector, widens the observation range. It can detect charge modulation of a protein, and the state of the water on an interface. T h e submicrosecond time resolution can discriminate proton exchange between donor - acceptor moieties located on the same structure from reaction with bulk proton. Similarly, it can resolve a complex proton-binding dynamics into initial and postprotonation events and it determines the contribution of each step to the overall measured equilibrium constants. Considering the big variety of proton emitters and detectors, it is not difficult to insert such informative molecules as inhibitors, activators, substrate, o r ligands into specific sites in proteins, membranes, or nucleic acids and study directly the role of water and proton in biochemistry.

APPENDIX The coupled differential equations describing the dynamics of a proton pulse perturbation of a three-component system employ three timedependent variables, X t , Y,, and Zt, which are the incremental dissociation of +OH, and incremental protonation of the indicator and buffer, respectively. The time-dependent concentration of free proton is given by

[H'I1

= Xt -

Yl - Z,.

T h e rate constants appearing in the rate equations are defined in the text as reactions (58-63). T h e differential equations are:

dX dt

-= all

dZ

-= ~ dt

x + a12 Y + a13 2 + bll x2+ b12 XY + bl3 xz

3 X1

+ a32 Y + a33 Z

+ d33 Z 2 + d13 X Z + d23 YZ

100

MENACHEM GUTMAN

Abbreviations

HIn, InBH, B7,7'

D

eo

z

neutral and dissociated forms of a proton emitter in its ground state neutral and dissociated forms of a proton emitter in its first electronically excited singlet state acidic and alkaline forms of indicator acidic and alkaline forms of buffer fluorescent decay time of +OH* and c$O-*, respectively diffusion coefficient eiectronic charge molecular charge

Acknowledgments I am grateful to my good colleagues, D. Huppert who introduced me to the dazzling world of lasers, E,. Nachliel for her invaluable assistance and criticism, and E. Gershon for the long hours he spent in front of the terminals. T h e assistance of B. Wynochansky and J. Jenks in the handling of the manuscript is highly appreciated. T h e study of the laser-induced proton pulse is supported by the American-Israeli Binational Science Foundation (310/82). This manuscript was written during my sabbatical year (1983) in the Department of Chemistry, Massachusetts Institute of Technology.

T H E pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS 101

References Adams, P. A. (1 976), Biochem.J., 159, 37 1- 376. Adam, G. and Delbruck, M. (1968), in Structuru~ChemistlyandMolecuEarBiology,(A. Rich and N. Davidson, Ed.), Freeman, San Francisco, p. 198. Adarnson, A. W. (1960), Physical Chemistly ofsurfuce, Academic Press, New York. Alkaitis, S. A., Beck, G., and Gratzel, M. (1975),J. Am. Chem. Soc., 9 7 , 5723-5729. Belch, A. C., Rice, S. A., and Sceats, M. C. (1981), Chem. Phys. Lett., 7 7 , 455-459. Benz, R. and McLaughlin, S . A. (1983), Biophys.J., 4 1 , 381-388. Bondi, A. (1964),J. Phys. Chem., 68, 441-451. Brouillette, C. G., Segrest, J. P., Ng, T. C. and Jones, J. L. (1982) Biochaktly, 2 1 . 45694575. Carmeli, C. and Gutman, M. (1982), FEBS Lett., 1 4 1 , 88-92. Conway, B. E. (1964), Modern Aspects of Electrochemistry, 3 , 43-70. Czerlinsky, G. M. (1966), Chemical Relaxation, Marcel Dekker, New York. DeKouchkowsky, Y. and Haraux, F. (1981), Bzochzm. Biophys. Res. Commun., 99, 205-212. Dill, K. A. and Flory, P. S. (1980), Proc. Nutl. Acud. Sci. USA, 7 7 , 3115-3119. Dodiuk, H., Kanety, H., and Kosower, E. M. (1979),J. Am. Chem. Soc., 83, 515-521. Duynstee, E. F. J. and Grunwald, F. (1959a),J. Am. Chem. Soc., 81, 4542-4548. Duynstee, E. F. J. and Grunwald, E. (1959b),J. Am. Chem. Soc., 81,4540-4542. Eigen, M. (1964), Angew. Chem. Int. Ed. Engl., 3, 1-19. Eigen, M., Krase, W., Maasse, G., and DeMayer, L. (1964), Prog. Reaction Kinetics, 2 , 286-3 18. Erdy-Grutz, T. and Lengyel, S. (1977), Modern Aspects of Electrochemishy, 12, 1-40, Feinstein, M. E. and Rosano, L. (1967),J. Collotd. Interface Sci., 2 4 , 73-79. Forster, Th. (1950), 2. Electrochem., 5 4 , 42. Forster, Th. and Volkers, A. (1975)’ Chem. Phys. Lett., 3 4 , 1-6. Fromherz, P. (1973), Bzochzm. Biophys. Actu., 365, 270-275. Fromherz, P. (1981), Chem. Phys. Lett., 7 7 , 460-466. Gershon, E. (1982), MSc. dissertation, Tel Aviv University, Tel Aviv. Glietenberg, D., Kutschker, A,, and Stakelberg, M. (1968), Ber. Bunsenges. Phys. C h a . 7 2 , 562-568. Goldstein, L. (1972), Biochemktly, 1 1 , 4072-4084. Goselle, U., Klein, U. K. A., and Hauser, M. (1979), Chem. Phys. Lett., 68, 291-295. Gratzel, M. and Thomas, J. K. (1974),J. Phys. Chem., 7 8 , 2248-2254. Gregory-Dewey, T. and Hammes, G. G. (19Sl),J. Biol. Chem., 256, 8941-8946. Grollman, A. (1928) in I n t e ~ t i o ~ ~ C ~ Tables, t z c a lMcGraw, Hill. New York, vol 111, p. 292. Gutman, M., Huppert, D., and Pines, E. (1981),J. Am. Chem. Soc., 103, 3709-3713. Gutman, M., Huppert, D., Pines, E., and Nachliel, E. (1981a), Biochim. Bzophys. Acfu, 6 4 2 , 15-26. Gutman, M., Huppert, D., and Nachliel, E. (1982), Eur. J . Biochem., 1 2 1 , 637-642. Gutman, M., Nachliel, E., and Huppert, D. (1982a), E u r . J . Biochem., 1 2 5 , 175-181.

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Gutman, M., Nachliel, E., Gershon, E., Giniger, R.,and Pines, E. (1983)J. Am. Chem. SOC., 105,2210-2216. Gutman, M., Nachliel, E., Gershon, E., and Giniger, R. (1983a), Eur. J. Biochem. 134, 63-69. Hasted, J . B., Ritson, M. D., and Collie, C. H. (1948),j. Chem. Phys., 16, 1-3 1. Hauser, M., Haar, M. P., and Klein, U. K. A. (1977),BerBuweng.Physik. Chem.,81,27-30. Horwath, C., Melander, W., and Molnar, I. (1976)J. Chromatog., 125, 129- 156. Huppert, D., and Kolodney, E. (19811,Chem. Phys., 63, 401-405. Huppert, D., Kolodney, E., and Gutman, M. (1978),in PicosecondPhenomenaII,(C. V. Shank, Ed.), Springer Verlag, Berlin, pp. 242-245. Huppert, D., Gutman, M., and Kaufnian, K. J . (l981),Adu. Chem. Phys., 47, 643-679. Huppert, D., Kolodney, E., Gutman, M., and Nachliel, E. (1982),J.Am. Chem. SOC., 104, 6949-6953. Ireland, J. F. and Wyatt, P. A. H. (1976),Adu. Phys. Org. Chem., 12, 131-221. Kano, K . and Fendler, J. H. (1978),Biochim. Biophys. Acta., 504, 289-299. Kebarle, P. (1975) in Thermochemical Informationfrom Gas Phase Ion Equilibria, (P. Au loos, Ed.), Plenum Press, New York. Klaning, W. K., Goldschmidt, R.,Ottolenghi, M., and Stein, G. ( 1 9 7 3 ) , j .Chem. Phys., 59, 1753- 1759. Klotz, I. M. and Fiess, H. A. (1960),Biochim. Biophys. Acta., 38, 57-63. Kosower, E. M. (1968),Introduction to Physical Organic Chemistry,Wiley, New York. Kosower, E. M. and Dodiuk, H. (197E),j. Am. Chem. SOC.,100,4173-4179. Kosower, E. M., Dodiuk, H., and Kanety, H. (1978),j.Am. Chem. Sac., 100, 4179-4188. Kosower, E. M., Dodiuk, H., Tanizawa, K., Ottolenghi, M., and Orbach, N. (1975),j.Am. Chem. Soc., 97, 2167-2178. Kracek, F. C. (1928)in IntentationulCntical Tables. McGraw-Hill, New York, vol 111, p. 351. Kraemer, W. P. and Diercksen, G. H. F. (1970),Chem. Phys. Lett., 5 , 463-465. Lachish, V., Ottolenghi, M., and Stein, G. (1977),Chem. Phys. Lett., 48, 402-406. Le Neveu, D. M., Rand, R. P., Parsegian, V. A., and Gingel, D. (1977),Biophys.j.,18, 209- 230. Lis, L. J., McAlister, M., Fuller, N., Rand, R. P., and Parsegian, V. A. (1982),Biophys.J., 37, 657-666. Matthew, J. B. and Richards, F. M. (1982).Biochemistty,21, 4989-4999. Matthew, J. B., Hanania, G. I. H., and Gurd, F. R. N. (1979a),Biochemistq,18, 1919- 1928. Matthew, J . B., Hanania, G. I. H., and Gurd, F. R. N. (1979b),Btochemktv, 18, 1928- 1936. Melander, W . and Horwath, C . (1977),Biochim. Biophys. Acta., 183, 200-215. McLaughlin, S. A. and Dilger, J. D. (1980),Phys. Rev., 60, 825-863. McQuarrie D. A. (1963),j. Chem. Phy., 38, 433-436. Miller, J . D. (1978),J.Chem. Ed., 55, 776-777. Miller, J . D., Klein, U. K. A., and Houser, M. (1980). Berg. Bunseng. Phys. Chem., 84, 1135- 1140. Mukerjee, P. and Cardinal, C. R. (1978),J. Phys. Chem., 82, 1620- 1627. Narayana, P. A., Li, A. S. W., and Kevan, L. (1982),J.Am. Chem. SOC.,104, 6502-6505.

T H E pH JUMP: PROBING OF MACROMOLECULES AND SOLUTIONS 103 Newton, M. D. and Ehrenson, S. (1971),J. Am. Chem. Soc., 3, 4971-4799. Parsegian, W. A., Fuller, N., and Rand, R. P. (1979), Proc. Natl. Acad. Sci. USA, 76, 27502754. Pines, E. (1981), M.Sc. dissertation, TeI-Aviv University, Tel Aviv. Rao, M. and Berne, J. B. (1981),J. Phys. Chem., 85, 149- 1505. Rand, R. P., Parsegian, U. P., Henry, J. A. C., Lis, L. J., and McAlister, M. (1980), Can.J. Biochem., 58, 959-968. Richter, P. H. and Eigen, M. (1974), Biophys. Chem., 2, 255-263. Rosignol, M., Thomas, P., and Grignon, C. (1982), Biochem. Biophys.Ada., 684, 195-199. Russu, I. M., Ho, N. T., and Ho, C. (1982), Biochemistly,21, 5031-5043. Schullrnan, S. G. (1977), Fluorescence and Phosphorescence Spectroscopy, Pergamon Press, New York. Searcy, J. Q. and Fenn, J. B. (1974),J. Chem. Phys., 6 1 , 5282-5288. Shoup, D., Lipari, G., and Szabo, A. (1981), Biophys.]., 36, 697-714. Smith, K. K., Huppert, D., Gutman, M., and Kaufman, K. J. (1979), Phys. Chem. Lett., 64, 522-525. Solc, R. and Stockmayer, W. H. (1973), Int. J. Chem. Kinet., 5 , 733-752. Tanford, C. (1955),J. Phys. Chem., 59, 788-793. Tan ford, C. (1973), The Hydrophobic Effect: Formation of Micelles and Biologzcal Membranes. John Wiley, New York. Tanford, C. (1976), Physical Chemistry of Macromolecules, John Wiley, New York, p. 457. Tanford, C. and Kirkwood, J. G. (1957),J. Am. Chem. Soc., 79, 5333-5339. Tanford, C., Swanson, S. A., and Shore, W. S. (1955),J. Am. Chem. Soc., 77, 6414-6421. Takabi, T. and Hammes, G. G. (1981), Biochemistry, 20, 6859-6864. Tong, L. K. J. and Glesrnann, M. C. (1957)J. Am. Chem. SOC.,79, 4305-4309. Valvani, S. C., Yalkowsky, H. S., and Amidon, G. L. (1976),J. Phys. Chem., 80, 829-835. Vass, Sz. (1980), Chem. Phys. Lett., 70, 135-137. Warshel, A. (1982),J. Phys. Chem., 86, 2218-2224. Weller, A. (1961), Prog. Reaction Kinetics, I, 189-214. Weller, A. (1958), Z. Phys. Chem. N . F., 17, 224. Westerhoff, H. V., Simonetti, A. L. M., and van Dam, K. (1981),Biochem.J., 200, 193-202.

Methods of Biochemical Analysis, Volume 30 Edited by David Glick Copyright © 1984 John Wiley & Sons, Inc.

METHODS OF BIOCHEMICAL ANALYSIS

VOLUME 30

Laser Photolysis in Biochemistry

s.

SHIRLEY CHAN. Corporate Research Laboratories. Emor, Research and Engineering Co., Annundale. New Jersey. AND ROBERTH . AUSTIN. Department uf Physics. Princeton University. Princeton. New Jersey

1. Introduction

..................................................................................................

2 . Rare-Gas Excimer Laser ..................................................................... 3. Flashlamp-Pumped Organic Dye Laser ............................................. 4 . Nitrogen Laser .................................................................................... 5. Mode-Locked CW Dye Laser ................... .................................. 6 . Shielding ............................................................................................. 111. Signal Acquisition Techniques ..................................................................... 1. Side-On Photomultiplier Tube A. Size and Mechanical Ruggedness ........................................... B. Speed ......................................... ................................... C. Electrical Ruggedness .............................................................. 2. Signal Amplification ........................................................................... 3. High-speed Transient Recorder ....................................................... 4 . Transient Recorder with Signal Averaging and Logarithmic Time Base ........................................................................................... IV . Cryogenic Techniques ............... 1. Helium Cryostat ................................................................................. 2. Sample Preparation ............................................................................ 3. Optical Configuration ... ................. 4 . Temperature Measureme ..... V. Large Perturbation Techniques ................................................................... 1. General Considerations ........... ................................... 2 . Photolysis of Ligated Heme Pr ................................... A. Overall Kinetics ....................................................................... B. Activation Energy Spectrum C . Possible Origin of Activation Energy Spectrum .................... D. Molecular and Spin Tunneling ...

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106 107 107 109

109 110 110 110 111 112 112 112 112 112 113 115 116 116 117 117 118 119 119 120 120 121 123 124

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Vl. The Triplet-State Probes .... 2. Anisotropy Decay

...............................................................................

A. Absorption ............................................................................... B. Phosphorescence ............. ............................................... VII. Intrinsic Photoactivation ............................................................................... 1 . Charge Separation .............................................................................. A. Role of Conform B. Charge-Transfer Band ............................................................ C. Instrumentation ......................... 2. Proton Pumping .......... ................................. VIII. Summary ........... ...................... References .............................

125 125 127 128 128 130 130 132 132 133 134 134 137 137 137

I. INTRODUCTION In this review, we will discuss applications of pulsed lasers as photolysis sources to study kinetics in biological systems. Photolysis is usually interpreted as the breakage of a bond due to the absorption of a photon, but we will stretch the definition a bit and include the creation of long-Iived excited states, such as the triplet state. Chemical kinetics provide a great deal of insight into the mechanism of chemical reactions, as important an aspect as knowledge of the reactants and their products. This is especially true in the biological sciences when enzymes are studied, since in catalysis the important features are the lowering of the potential barrier and the creation of preferential pathways, both of which lead to a change in the kinetics. To obtain as complete a picture as possible of the kinetics, it is important to obtain a maximum time resolution. Many biological reactions occur in a very short time (in fact, down to picoseconds in vision and photosynthesis) so that slower, more traditional techniques such as stopped-flow often lack adequate time resolution. Under fortunate circumstances where the system studied is photosensitive, or where the free-energy change between reactants and products is sufficiently small, then the concentrated energy impulse from a laser is a marvelous tool that can provide a wealth of information concerning the system. The intent of the review is not to offer an exhaustive review of the literature, which would be an overwhelming task. Rather we hope to delineate areas where lasers have played an important role, to discuss the importance of these investigations,and to point to areas where their potential is still untapped.

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Since the authors are experimentalists with physics and biophysics backgrounds, we hope also to provide some practical pointers to those brave enough to develop their own systems. This paper will concentrate on three areas of photolysis where important questions concerning biological systems are addressed. The first will be the well-established but still vital area of the heme proteins that have photolabile ligands. The second area will be with the use of triplet probes on proteins and DNA. The third area covered will be those special proteins that are photochemically active. To avoid making technology take precedence over science, many experimental details will be discussed in the process of our attempt to provide a unifying thread to recent trends in biochemical photolysis. The authors will not attempt to discuss the various aspects of bimolecular kinetics. We will instead stress the aspects of photolysis where the protein is believed to play an important role in the overall kinetic picture.

11. THE PULSED LASERS There are a variety of pulsed lasers suitable for experiments of the type discussed in this paper. There is always a trade-off between cost, convenience, and energy that must be considered. We will consider here only those lasers that have puke durations of one microsecond or less, and the authors’ natural inclination is to those lasers that produce 100 pJ or greater energylpulse. Most pulsed lasers rely either on a very fast and brief excitation source (such as the flash-lamp pumped dye laser), a self-terminating lasing action (the nitrogen laser), or a device in the laser cavity that spoils the gain during the pump cycle and opens quickly (such as in the (2-switched Nd:YAG laser). We give here a brief description of the most important lasers and possible future developments, in order of our opinion of importance. 1. Q-Switched Nd:YAG Laser

This is a solid-state laser that inverts the population of a dilute solution of the rare earth neodynium in a YAG (yttrium-aluminum-garnet) crystal matrix. This laser has its main line at 1063 nanometers (nm), in the near-infrared, a fairly useless wavelength as things go because virtually nothing absorbs there. However, the development of the unstable oscillator configuration in 1972 resulted in a dramatic improvement in beam quality and low divergence that gives incredible harmonic efficiency: frequency doubling to 532 nm in modern Nd:YAG lasers has a 40% conversion efficiency, and similar efficiencies are found for the tripled

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SHIRLEY S. CHAN AND ROBERT H. AUSTIN

355-nm line and quadrupled 266-nm line. Since the crystal matrix is an excellent conductor of heat from the flash-lamp pumped laser rod and because the input threshold energy for the laser is quite low, the Nd:YAG laser can be run easily at a 10-Hz repetition rate and a primary energy/ pulse at 1063 nm of 0.2 joules (J). The duration of the pulse is typically 15-20 nsec, which is adequate for all but the highest-speed studies. Mode-locking of a Nd:YAG laser is the process where ultra-short (30 psec and shorter) pulses are separated by the transit time of a photon in the laser cavity; this has historically been the province of the laser expert, but at least two companies have announced a mode-lock option on their laser which should open the sub-nanosecond pulse world to the scientist whose prime concern is the events in hidher cuvette and not within the laser. As we mention later, it is trivial for a scientist to build a simple dye laser that can be pumped by the doubled or tripled laser to obtain other wavelengths. Another option that is not difficult to build is a stimulated Raman shifter cell containing a gas at 200 psi that with 20-30% conversion efficiency (hydrogen gas) can lase at the Stokes-shifted line, or with less efficiency at the up-shifted anti-Stokes line. Figure 1 shows a general-purpose Nd:YAG laser system that can produce at least 1 mJ/pulse from 260 to 2000 nm. The main expense is the laser itself Nd:YAG lasers are not cheap and a basic oscillator will cost at

Monltorinq Beam: ( 1 ) Tunqsten (2) Arc (o)Hq (b)Xe (3)Loser (a) Ar (b) Kr (c) He Ne (d) He Cd

Nd: YAG Laser (Lonq Pulse, O-Sw1tch.Mode-Lock)

t Microcomputer 11) Average (2)Process (3)Tranrmit

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Sample (I)Cell no I der (2)Cryostat A

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(1)Photodlode r)- (2)Photomultlpller (3)Channel P l a t e

1

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present (1983) at least $45,000. If the experimenter is intrepid, all other options can be built in the lab at minimal expense. An alternative to this essentially steady-state monitoring system has been the pulsed system. Picosecond versions of this have been effectively used by Rentzepis and co-workers (Reynolds et al., 1982),while a slower version has been developed by Eaton and his collaborators (Hofrichter et al., 1983). These two approaches utilize two pulses of light: one to photolyze and the other to generate a continuum monitoring pulse with some delay which is passed through the sample. If the delay can be varied, then a series of pulses can map out an absorption decay profile. We will discuss primarily the nanosecond and slower process in this review, and the reader is urged to look at the above references and the book by Alfano (Alfano, 1982)for information on these exciting developments.

2. Rare-Gas Excimer Laser The rare-gas excimer laser has recently emerged as a powerful source of pulsed UV radiation. The excimer laser utilizes excited-state dimers that form between noble gases, such as argon, and the halogens, such as chlorine and fluorine. Although the output from these lasers is in the UV (308 nm for ArCl, 248 nm for XeCl), these lasers will efficiently drive dye lasers in the visible portion of the spectrum. The outpudpulse is high ( 1 J/pulse is easily achievable in ArCl) and the laser can be run at a high repetition rate for signal averaging. Another advantage of the excimer laser is its ability to also lase N2 (337 nm) and COP (10.6 pm). At present, there is no easy way to achieve mode-locking of these lasers, but the present pulse width of 20 nsec is more than adequate for many experiments. The lack of a strong near-IR line from these lasers also somewhat limits the uses of the excimer laser. The excimer laser, if well designed, has a low noble-gas consumption and can be considerably cheaper ($20,000) than the Nd:YAG laser. 3. Flashlamp-Pumped Organic Dye Laser The flashlamp-pumped organic dye laser is by far the cheapest of the possible lasers and is very simple to operate. Dyes exist from the UV to the near-IR and pulse energies of 1 J/pulse are not uncommon for the better dyes. The main problem with the flash-lamp pumped laser is the difficulty in obtaining high-energy pulses of duration shorter than 100 nsec and the high divergence of the beam (unless a sophisticated oscillatoramplifier scheme is used), which makes for inefficient frequency doubling. However, a very good laser can be obtained for $4000 which makes this laser attractive as a “starter.” Flashlamp technology is evolvingrapidly

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SHIRLEY S. CHAN AND ROBERT H. AUSTIN

so that a careful purchase can give the user a very cost-effective and expandible system.

4. Nitrogen Laser Nitrogen lasers provide an inexpensive ($4000-$20,000) and fast (0.1-2 nsec) source of moderate energy (0.1-2 mJ/pulse) UV light (337 nm) at high repetition rates (to 1 kHz). Because of the necessity of using dye lasers to get visible light, and the rather low energy, the nitrogen laser is of marginal use for photolysis work. However, the recent development of nitrogen lasers of modest cost ($7000) and 100-psec pulse duration (Photochemical Research Associates, 100 Tusla Road, Oak Ridge, Tenn. 37830) affords an interesting alternative to the mode-locked laser. Since the repetition rate of these lasers at present is under 1 kHz singlephoton techniques cannot be used. However, the advent of channel-plate photomultipliers with 300-psec rise times and miniature photomultipliers of 800-psec rise times means that quite excellent time resolution can be obtained by use of a fast box-car amplifier.

5. Mode-Locked CW Dye Laser Mode-locked CW dye lasers are increasingly being used as excitation sources at very low power levels (nJ per pulse) and at very high repetition rates (100 MHz). Because of the picosecond duration of these pulses and the low-energy/pulse most uses have been in fluorescence work. With cavity dumping techniques and clever signal averaging techniques it has been possible to do absorption work, even absorption anisotropy. However, the large argon lasers necessary to drive the dye lasers are very expensive and have short lifetimes of their $20,000 plasma tubes. A recent development that may change this is the continuous wave (CW, or steady emission) Nd:YAG lasers run in mode-lock and frequency doubled by the new, highly efficient frequency doubler crystals. Since the CW Nd:YAG uses simple arc lamps as the excitation source they should be far more reliable to operate and maintain than ion lasers.

6. Shielding Unless the pulsed laser is very well made, it probably will emit electromagnetic interference (EMl) when it is fired, obliterating the desired signal. The book by Ott (1976) has many wise things to say about this subject and is highly recommended. Here are a few of our own fixes: 1. Put the laser in a copper room and bring the beam out. This does work, but is practical only if you have lots of space and patience or if those

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who designed your building were very wise and included shielded rooms in the construction. 2. Put the laser in a copper-screened box. This is effective only if all AC power cords coming in are well choked to the enclosure wall and if all signal cables coming in are either grounded at the enclosure wall or are (better) optically coupled. The lazy way of running an insulated cable into the box creates a wonderful antenna that ruins the shielding. 3. Use double-shielded coaxial cable with BNC connectors (this is a generic term for a coaxial connector used with 50-ohm impedance cable; most high-frequency lab equipment such as oscilliscopeswill have a BNC connector on the front panel) for all low-level lines. Regular BNC cable, type RG-58U, does not have good braid coverage and will pick up radio frequency (RF). Either pass the cable through copper braid (no good for frequencies greater than 50 MHz) or purchase double-shielded cable, such as TRF-58 (Times Fiber Communications Inc., 358 Hall Ave., Wallingford, Conn. 06492). It always is better to amplify small signals at the source before sending down =able, and likewise any small driving signals should be high level coming into the device and then attentuated at the point of application. 4. Many manufacturers put pretty plastic insulating collars on their BNC panel mounts. Disaster! There is no good ground connection at the enclosure wall and the cable shield radiates into the box. There is only one solution: take the panel mounts apart and get rid of the insulating collars. Figure 2 shows where common problems occur.

111. SIGNAL ACQUISITION TECHNIQUES We exclusively consider optical techniques of detection here since that is where the authors’ expertise such as it is resides. In this section, we want to reveal a few of the tricks that we have found helpful in building a high-speed multifunction optical detection apparatus. One of the main problems in fast signal acquisition is development of a suitable active photomultiplier (PM) base. An anode risetime of no more than 20 nsec is desirable and the ability to drive 50-ohm cable with a steady DC voltage of 1.O V is also important. However, the maximum anode DC current that most PMs can handle is about 100 PA. All these facts translate into the need for an active PM base, that is, a base that has a high-speed amplifier driving the cable. In our experiments we have the added requirement of gating the PM “off’’ for sub-microsecond times to do the phosphorescence experiments mentioned briefly in this paper. Can all these features be put into one unit? Yes.

112

SHIRLEY S. CHAN AND ROBERT H. AUSTIN C o pper - S c r e e n

1

Metal Enclosure

Box

,Pi Filter on

Power L in es

Double

(High

Gain

Amplifier a t

Source Att Seams Sealed

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Figure 2. Schematic of common shielding procedures. The source of RF is assumed to be a noisy, pulsed laser. T h e laser should be put in a metal screen box, all power lines coming in should be filtered at the box wall, low-frequency signal lines coming into the box should be choked, while higher frequency lines should be optically coupled if possible. In all cases, the cables coming into the box must be grounded with no breaks to the wall of the box. Sensitive signals should be amplified at the source, signal cables should be double shielded when possible, and power supply lines should be well choked. Again, good grounds at the box wall.

1. SideOn Photomultiplier Tube

First, we would like to point out that side-on PMs, the descendents of the old IP2 1 tubes made by RCA (Princeton, N.3.) are for most applications much better than the more impressive and expensive end-on tubes beloved by high-energy physicists. The side-on has the following benefits:

A. SIZEAND MECHANICAL RUGGEDNESS. The end-on is a large 2-in.diameter PM with a fragile photocathode. The side-on is the size of a old-fashioned vacuum tube with internal cathode. Because of its small size, the side-on is considerably easier to house and support, and is easier to shield from RF sources. B. SPEED.Because of the smaller dimensions of the side-on, anode rise times of 2 nsec are easy 1.0 achieve, while a standard venetian blind end-on tube has 20-nsec rise times. The expensive fast-linear focused end-on tubes can approach the side-on rise time. C. ELECTRICAL RUGGEDNESS.The photocathode of the side-on tube is opaque and plated on a highly conductive metal surface, while the end-on tube is a semitransparent semiconductor forming a thin film. Thus, the cathode of the end-on shows significant fatigue at high current levels due to internal ohmic heating and is easy to damage due to inadvertent exposure to high light levels while at high voltage.

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Unless one really needs the large cathode area of the end-on tubes (and for this problem lenses were created), we find that an excellent tube like the Hamamatsu 928 (Hamamatsu Corp., 420 South Ave., Middlesex, N.J. 08846) to be an extremely versatile performer. This PM has a very broad spectral response from 200 nm to 900 nm, exhibits little if any hysteresis, and can tolerate gross current overloads. A new development has been a miniature PM by Hamamatsu (R1635) that has a risetime of 800 psec. However, since it is an end-on design at present it has limited anode current capability and insufficient red response for many applications.

2. Signal Amplification There are fortunately now on the market extremely fast operational amplifiers (op-amp) with gain-bandwidth products of over 1 Gigahertz [we have in mind the op-amp SN5539 (Signetics Corp., 8 11 Arques Ave., P.O. Box 409, Sunnyvale, Calif. 94087)], which are very stable and have excellent noise figures. (If the jargon here is foreign to you, we recommend the book by Horowitz and Hill, The Art @'Electronics, 1980.) An excellent active base consists of a 20 decibel (dB) gain noninverting (that is, X 1 0 voltage gain) op-amp using the furnished printed circuit depicted by Signetics, with the changes of 50-ohm input and output impedances and the use of 1.O-pFtantalum capacitors on the power supply pins. The resistor divider chain is also put onto a printed circuit for minimum inductance. The top six dynodes are capacitively coupled to an external connection for gating. A negative pulse of -300 V applied to alternate dynodes is an effective gating method. Our base scheme is shown in Figure 3. With this arrangement, we have a multipurpose fast tube that can be used for both absorption, emission, and gated phosphorescence measurements, at very moderate cost. 3. High-speed Transient Recorder

Another problem in high-speed work is the interfacing to a high-speed transient recorder. The two main producers of high-speed transient recorders are Biomation (Gould Inc., Biomation Division, 4600 Ironsides Drive, Santa Clara, Calif. 95050) and LeCroy (LeCroy Research Systems, 700 S. Main St., Spring Valley, N.Y. 10977). The Biomation model 6500 used by us has a 50-ohm input impedance and a maximum sensitivity of 0.25 V full scale and only 6 bits resolution. To see small (sub-millivolt) changes, it is necessary both to amplify the signal and signal average. The amplification is done in two steps. A 5539 amplifier is used to subtract the DC voltage output from the PM, so that the signal appears near ground

100kfi

To Translent Recorder

Figure 3. 100-MHz bandwidth photomultiplier base, capable of 0.5 bsec gated operation. (a) The dynode configuration. The negative high voltage is fed down a 100 kilohm divider chain as shown. Alternate dynodes are pulsed negative with respect to the previous dynode to gate the photomultiplier off. The pulse generator must be capable of driving a 50-ohm load and have less than 100-nsecrise and fall times. In our lab, we use the lamp sync out signal from the laser to gate the PM before the laser pulse. (b) The configuration of the photomultiplier housing. The housing can be removed while all optics are aligned so that the light from a sample can be tracked onto the photomultiplier face to ensure good alignment.

T o - 3 0 0 V Pulrer

- HV

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Electronics’ Figure 4. Schematic diagram of an automatic feedback regulation system for the photomultiplier. The output from the active base (upper left-hand corner) is fed to an amplifier where a DC offset is made. The offset signal (now, close to ground) is compared to a reference voltage and then fed into a summing o p amp (middle amp in bottom left-hand block). The signal from the photomultiplier then in effect acts as an error voltage on the summing amp. The output from the summing amp is fed to a DC-DC voltage converter (Venus Electronics, New York, N.Y.). The negative high-voltage output then drives the photomultiplier. Limiting Zener diodes must be put on the summing amp output so that if the light to the photomultiplier is accidentally blocked the output voltage does not rise to dangerous levels. The time response of the feedback loop must be at least 10 times longer than the characteristic time of any processes of interest, otherwise this arrangement will remove the signal if the feedback gain is sufficiently high!

potential. A subsequent 5539 amplifier provides 30 dB of gain and has sufficient drive for the 50-ohm input impedance of the transient recorder. In our anisotropy measurements we also need to stabilize the gain of the PM relative to the light levels (DC) on the two polarization channels. This is achieved by feeding back the DC PM output to an error amp that controls the PM high-voltage output via a negative feed-back loop. In this way, gain changes due to fluctuations in the monitoring beam intensity are held to less than 0.1%. Figure 4 shows a schematic of the circuits used. In many experiments, the single-shotsignal-to-noiseratio is less than 1, but the sample can be repeatedly excited. Under such situations signal averaging is of enormous use, especially if one is using a high-repetitionrate laser. The basic idea is to use a digital transient recorder to store the signal on a single-shot basis and then average the shots in a computer. For instance, although our Biomation 6500 has only 6-bit (1/64) resolution,

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SHIRLEY S. CHAN AND ROBERT H. AUSTIN

after 1000 averaged sweeps the actual depth of the signal is much deeper, since the presence of noise on the signal leads to a digital averaging between bits (“dithering”).Thus, the 6-bit resolution has posed no problems for us.

4. Transient Recorder with Signal Averaging and Logarithmic Time Base The most efficient use of a transient recorder is, thus, as part of a signal averager. The most cost effective way is to “do it yourself.” Implementation of this plan assumes that the reader has a microcomputer-almost any will do, although we vastly prefer a machine that runs CP/M with the attendant enormous software availability-and a parallel port on the computer. The parallel port is used to control the transient recorder and act as the data input bus. To utilize the 10-Hz repetition rate of a modern Nd:YAG laser, with every laser shot resulting in the storage of 1024 pointskhot, the programming must be done in assembly code for speed. These facts require that the scientist becomes reasonably familiar with hidher microcomputer, but we feel that since a standard microcomputer now costs less than a plain vanilla oscilloscope it is time for all experimentalists to learn basic bit manipulation to fully exploit the computer revolution. The commercial transient recorders have a linear time base. For many of the experiments described here, the signal is of a power-law form and thus has significant components over 3-8 decades in time. Not enough note has been made of the amazing logarithmic transient digitizer developed at University of Illinois (Urbana). In a standard transient recorder the fast analog-digital converter has fast acquisition time that is useful at the maximum sweep rate, but at lower sweep rates the small window of time of the converter means that most of the signal is thrown away. In critical applications, the experimenter must provide an analog smoothing circuit before the input to achieve a reasonable signal-to-noise ratio. However, there is no fundamental reason why the converter cannot run at its full digitization rate and a digital averaging technique be used to automatically provide smoothing at slower times. If this idea is incorporated into a logarithmic sweep to cover the large time scale, then an extremely powerful signal acquisition device is obtained. See Austin et al. (1976) for a complete discussion of the concept of this instrument. Alas, the equipment manufacturers simply have not realized the enormous power and versatility of this autoaveraging time sweeper.

IV. CRYOGENIC TECHNIQUES One innovative aspect of recent work in biochemical photolysis has been the use of cryogenic techniques to study rates and to isolate reaction

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intermediates. The pioneering work of Douzou (1977)and DeVault and Chance (1966) was instrumental in emphasizing the information that could be gained, even though most biological systems work within a narrow temperature range. Indeed, fighting this prejudice has been one trying aspect of those engaged in the field. See the review article by Frauenfelder (1978) for more information.

1. Helium Cryostat The most important piece of equipment is a good helium cryostat. The cryostat must be of the variable temperature variety so that a range of temperatures can be obtained easily, and for optical experiments it is most convenient if the sample is at atmospheric pressure and accessible, as opposed to a vacuum mounted cold-finger. There are two types of cryostats: (1) the flow cryostat that utilizes a continuous flow of liquid helium from a low-loss dewar, or (2) the storage dewar, which holds 2-5 liters of liquid helium and flows the helium down a small capillary tube to the sample chamber. The flow dewar is considerably easier to use than the storage dewar for the nonexpert, but if extended operation of the dewar is contemplated then the storage dewar has a considerably lower consumption rate (0.1 literlhr vs. 2 literslhr). We have found the Janis Dewar Corp. Inc. (22 Spencer Street, P.O. Box 487, Stoneham, Mass. 02180) or Oxford Instruments Inc. (P.O. Box 1829, 222 Severn Ave., Annapolis, Md. 2 1403) to be excellent manufacturers of reliable, efficient dewars. There is no greater agony than a dewar with a cold vacuum leak (well, maybe the jammed fraction collector is worse)!

2. Sample Preparation To do optics experiments at low temperatures often the protein must be put into a glycerollwater mixture that forms a glass at low temperatures. One severe problem with glycerol is the often strong reducing potential of even so-called reagent-grade glycerol. We have found that the Gold-Label glycerol by Aldrich Chemical, Milwaukee, Wisc., to have little if any of this problem. It is also important to keep the salt concentration down to prevent precipitation of the salt at low temperatures. The total concentration of buffer and salt should be kept under 10 mM. Usually, the sample must be thin (0.1 cm or less) because of the loss of optical clarity in a thick glass due to cracking at low temperatures. Because of the required thinness of the sample, high concentrations (greater than 100 pA4 are necessary. One problem with this is the high concentration of reducing agent needed to produce carboxy data. Sodium dithionite is the usual reducing agent, but unfortunately it is difficult to obtain in purified form. At least one group (Reynoldsand Rentzepis, 1982)has reported possible artifacts that can occur if clean dithionite is not used. Anaerobic solutions of

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SHIRLEY S. CHAN AND ROBERT H. AUSTIN

i"lU

Monochromator OrFilters

I

;

\

'\

H e a t Filters

--

F

-

--_

Pulsed

Loref

Mirror

Sample

Figure 5. Optical path for cryostat optics. The arc lamp uses a water filter to remove excess IR before passage of the beam through the bandpass filters. Care must also be taken that the bandpass filters can withstand the excitation laser energy. A glass slide with a hole drilled in the middle and with an aluminum overcoat directs the monitoring beam through a high speed (F/0.75) Fresnel lens for high collection efficiency. The filters afford the highest monitoring intensity, while the monochromator allows scans. Note that with a xenon arc and the feedback loop shown in Figure 4 normalized absorption spectra are obtained during a scan.

dithionite should be used immediately after mixing. Ideally, the protein should be passed down a sizing column to remove the dithionite after reduction, but this can lead to problems with oxygen contamination unless extreme care is taken.

3. Optical Configuration We have found the most flexible optics arrangement for a cryostat to be the one shown in Figure 5. Note the hole in the mirror that allows the excitation beam to impinge on the sample. Back-scattering from the sample is minimized by tilting the dewar windows at an angle to the optics axis of the monitoring beam of light, while narrow-band interference filters block undesired light into the photomultiplier. For many experiments, an intense light is desired to minimize shot noise (photon statistics). We have found that the Photochemical Research Associates arc lamp system, which utilizes a F = 0.75 ellipsoidal reflector, and a mercuryxenon arc light to be a very stable and intense line source.

4. Temperature Measurement and Control

Precise measurement of the sample temperature is also important, and the control of the temperature. Thermocouples can be highly reproduc-

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ible temperature sensors, yet suffer from the low signal voltage generated (millivolts)and the general roll-off of thermoelectric power as the temperature approaches 0 K. Of the common thermocouples, gold-iron has the best low temperature dVldT.A common low-temperature scheme is to use a simple carbon resistor (Allen Bradley, 1201 S. Second Street, Milwaukee, Wisc. 53204 makes the best quality resistors) as a sensor, by measuring the voltage across the resistor when driven by a current source (of about 10 PA). The semiconductor resistors exhibit a very large dRldT as T goes below about 20 K, but are rather insensitive above this temperature. Probably the best sensor is a common silicon signal diode, forwardbiased by a 10 pA current source. The voltage across the diode is about 0.4 V at 300 K and is roughly exponential with decreasing T , rising to about 1.5 V at helium. This is a very convenient voltage and range over which to measure. It is quite straightforward to build a temperature controller using simple op-amps to control the temperature of the sample holder, as long as one is careful to include both offset adjustments to compensate for coolant flow and variable gain in the feed-back look to adjust for differing dVldT in differing sensors, where V is the signal across the sensor. The company Lake Shore Cryogenics, 64 E. Walnut St., Westerville, Ohio 4308 1 is one of the primary suppliers of cryogenic temperature sensors, and the series of articles by Rubin and colleagues (Sample and Rubin, 1977) contain much information on common temperature sensors, and the effect of magnetic fields on their characteristics.

V. LARGE PERTURBATION TECHNIQUES 1. General Considerations A visible photon of wavelength 550 nm carries 2 eV of energy, whereas a photon of 275 nm carries 4 eV. These energies are sufficient to excite directly different electronic states and thus are what we call large perturbations. Visible photons have a unique selective advantage in that band gaps as small as 2-3 eV are rare among normal covalent and ionic materials, hence the lack of color in most proteins. Only in the presence of a highly conjugated system, a charge-transfer band or a transition metal with d-d transitions do we find visible transitions. This affords the experimenter the enormous selective advantage of being able to excite the group that is the reactive center of the molecule because it does have special electronic properties. While our main concern is with photolysis in proteins and nucleic acids, we would be amiss if we did not mention the photolysis of external ligands that can proceed to react with proteins. The most elegant example

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of this is the development of caged ATP (Goldman et al., 1983). This molecule is a chemically inert form of ATP, which can be converted to active form by the absorption of near-UV light to cleave the 2-nitrosoacetophenone group to yield XTP. This photolysis can be made by either a N2 laser pulse o r the doubled ruby laser or tripled Nd:YAG laser. It is the ultimate stopped flow technique! It is unusual to affect directly the action of an enzyme from the absorption of a photon because the lifetime of most singlet excited states (at best, 20 nsec) is too short compared with the turnover time of an enz me. For instance, if the reaction rate of an enzyme is diffusion limited (lO’M-’ sec-’) and the ligand is present at lOP3M concentration then the turnover rate is only lo6 sec-’, so that the added photon energy will only be present during a limited fraction of the cycle.

-

2.

Photolysis of Ligated Heme Proteins

The heme proteins that carry oxygen are intrinsically photosensitive and are one of the easiest systems to apply the technique of photolysis by pulsed lasers. One of the first scientists to use a laser in this system in a pioneering series of experiments on hemoglobin was Quentin Gibson (Gibson, 1959). T h e porphyrin group absorbs visible light strongly (excm-’ and have neartinction coefficients are as high as 200 IT&-’ unity oscillator strength (Antonini and Brunori, 197 1) at several wavelength ranges (400-450,500-600 nm), making it possible to use several different kinds of lasers. After the absorption of a photon into the T* orbitals of the porphyrin ring, the energy is apparently transferred with high efficiency to an antibonding 3dz2orbital in the iron that leads to dissociation of the ligand (Gouterman, 1978). The efficiency of the photolysis is a function of the ligand: those that form linear bonds with the iron (Fez+ - CO, Fe3+ - NO) have near unity quantum yields, while those that form bent arrays (Fez+ - 0 2 , Fez+ - NO) have considerably lower quantum yields. The absorption spectrum of the ligand-bound protein is typically quite different from the ligand-free protein which makes for huge absorption signals that are an experimentalist’s dream. Furthermore, heme proteins in general are available in relatively large quantity and are very soluble in aqueous solution. T h e overall result is that one has an easily photolyzed system with a large transient signal to work with. T h e only drawback might be that the ease of working with this system may make one reluctant to branch out into other areas. However, recent results seem to paint a rather general picture and perhaps Hans Frauenfelder’s characterization of myoglobin as the “hydrogen atom” of biophysics may carry more than a little truth. There are, of course, other proteins that have photosensitive ligands. For instance, hemerthyrin is a nonheme iron-containing protein that also has an easily photolyzed

-

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oxygen- iron bond and exhibits a geminate recombination at lower temperatures (Alberding et al., 1981). A. OVERALL KINETICS

We think that the classic example of what can be done with heme protein kinetics is the series of papers by Frauenfelder et al. (1975, 1976a,b, 1978, 1980)on kinetics of recombination of carbon monoxide (CO) to myoglobin, hemoglobin, and protoheme over the temperature range from 350 to 4 K:

hv

(Protein * CO) (Protein + CO) (Protein) + CO where the left-hand side represents the CO as liganded to the iron before photolysis, the middle expression represents the diffusion of the CO within the protein, and the right-hand side represents the protein and CO as fully dissociated species. Recent work on hemoglobin we feel has somewhat neglected the impact of the original work done by Frauenfelder and his co-workers. We would like to stress that these experiments found three main areas of interest in the recombination kinetics as temperature was lowered:

1. Bimolecular kinetics were replaced by geminate recombination as the solvent viscosity was raised and/or the temperature was lowered (Alberding et al., 1978; Austin et al., 1975; Austin and Chan, 1978; Beece et al., 1980). Woe to those experimenters using long excitation pulse lengths who think a quantum yield is small because the signal is small at sec after the flash: a geminate fast phase can easily give an apparent decrease in the quantum yield due to pulse convolution. 2. The geminate kinetics are of a power law form at low temperatures (that is, of the form [ 1 + tho]-"). This seems to be a universal curve and needs a universal explanation. 3. At temperatures less than about 20 K, the ligand seems to tunnel back to the iron (Alberding et al., 1976b; Alben et al., 1980). B. ACTIVATION ENERGY SPECTRUM

Figure 6 is a brief summary of the kinetics seen. The recombination was found to be not a single-step process with one activation energy barrier, but a process involving possibly multiple barriers in series which are related to the protein structure. In the simple case of a discrete activation barrier the rate constant k is of the form: k = a e-EfksT where a is the infinite temperature rate, kb is Boltzmann's constant, T is the absolute temperature, and E is the internal energy change from the bottom to the saddle point. If nonexponential kinetics are observed, then,

122

SHIRLEY S. CHAN AND ROBERT H. AUSTIN K i n e t i c s of

MbCO i n G l y c e r o - W a t e r

10-6

IO-~

lo2

I

time ( s e c )

I

IO-~ IO-~

I

lo2

time ( s e c )

T=320K

IO-~

IO-~

I

lo2

time (sec)

Figure 6. Kinetics of the recombination of carbon monoxide with myoglobin over the three main temperature regimes. In part (a) only the low-temperaturegeminate, power-law kinetics are seen. In part (b) the lower “blip”shows the beginning of bimolecular kinetics, whereas in (c) the kinetics are totally bimolecular.

in general, the kinetics can be modeled by a Laplace transform of some distribution of activation energies, g(E):

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The idea of g(E)was believed to be related to a distribution of conformational states of a protein. Each conformation had its own activation energy barrier for recombination with its own rate, K(E). In principle, the conformational distribution contains information about the structure of the protein and how it affects the reaction rate of the protein. Thus, it has become clear that the low-temperature kinetics have a great deal to say about protein structure and function, and we feel that this fact is only now becoming realized. C. POSSIBLE ORIGIN OF THE ACTIVATION ENERGY SPECTRUM

Consider the tertiary structure of proteins. The folding of a protein is determined by the interaction of the amino acid groups with themselves and the solvent. There is a range of conformation substrates that is acceptable to nature as near the minimum in free energy and capable of normal function. When the solvent viscosity is low and there is sufficient thermal energy, the protein can “breathe” and change between different possible conformational states. The exchange between states is expected to slow down at lower temperatures and effectively freeze out. At this point, the system is composed of an ensemble of frozen conformers with a Gaussian statistical distribution of density of states, that is, proportional to exp (-ax;) where xo is the equilibrium position of an atom and a is some force constant. While accepting the existence of a distributed energy barrier, Agmon and Hopfield (1983) have recently expanded Frauenfelder’s idea of sequential barriers by proposing a new model. They pointed out that if the above ideas are taken seriously then besides the reaction coordinate there is a second independent variable, namely the protein conformation. The reaction coordinate is taken as the Fe-ligand distance whereas the protein coordinate describes the overall effect of the surroundings on height of the reaction barrier height. As will be discussed later, the spin-change that the iron atom undergoes from spin 0 in ligated state to spin 2 in the five-coordinate state can be used to construct a potential energy surface from the intersecting surfaces for the two spin states. The model demonstrates how the stress of protein or solvent on heme can give rise to a distribution of energy that is responsible for the power law kinetic behavior. Most significantly, they were able to understand qualitatively the power law curve from the simple assumption that the conformational distribution is distributed by a harmonic restoring force that gives rise to a reaction barrier of the form:

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SHIRLEY S. CHAN AND ROBERT H . AUSTIN

where k, is the spring constant for deformation x of the atomic site from the equilibrium position. Using the Arrhenius relation, this then yields a corresponding rate constant:

Since different conformers have different energies, the probability of finding a conformer with energy V at temperature T can be calculated by assuming that the number of molecules with this barrier height is weighted by Gaussian statistics:

1 3 1 -

P ( x ) = [4lTkJ

v(x)]-1’2 exp

-

Moreover, the model tries to account for the temperature kinetics observed in the intermediate range (180-250 K) as being not due to additional barriers in the protein. but a “bound diffusion” where the protein can relax by changing between its conformational states. The relaxation gives an averaging effect of this distribution and the kinetics achieve Arrhenius behavior as observed experimentally. The mean energy has a higher value than the peak of the distribution and therefore the recombination is being slowed down by this higher mean activation energy rather than an additional barrier. The model as it stands still has some deficiencies, but these will be corrected with time. The main message to be gathered from this is the crucial influence that the protein plays upon the binding of the ligand. This has probably always been accepted, but now we see how present day research is clearly bringing out this important fact. D. MOLECULAR AND SPIN TUNNELING

Lastly, we briefly discuss the iow-temperature tunneling. As we mentioned above, one of the three findings of the ligand-hemeprotein recombination work was the tunneling of ligand back to iron at temperatures about and below 20 K (Alben et al., 1980).Tunneling is a quantummechanically possible and predictable transition for two states separated by a finite potential barrier. By the Arrhenius rate equation, as T goes to zero the reaction rate should exponentially fall to zero. When the particle (ligand) no longer has enough kinetic energy (thermal) to jump over the barrier, classically the recombination should stop. However, the process does not stop but becomes temperature independent. This temperature independence is strong evidence of tunneling. Since the particle is either CO or O2 moving to Fe for bond formation, we have a case where the

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molecule tunnels spatially. The spatial molecular tunneling process depends on the width d and the height E of the potential barrier. However, because of the electronic changes that occur during tunneling events, such as spin changes, other terms can enter into the tunneling process and may provide a key insight to ligand reactivity control in biomolecules (Redietal., 1981; Gerstmanetal., 1981;Austinetal., 1982).Muchworkis also needed here to reveal the extent to which the protein can control the tunneling rate, and the possible influence this tunneling process can have on room temperature processes. As recent picosecond results have clearly implied, the role of spin states in these systems may play a major role in the photolysis and recombination (Martin et al., 1983). Photolysis experiments involving tunneling processes require good temperature control below 20 K and low monitoring light levels. The low levels are necessary in the heme proteins because of the slowness of the recombination, and the high quantum yield for photolysis results in substantial photolysis of the sample during the observation period. The tungsten- iodide light makes an excellent source for these studies because of the stability of the light and the relative ease of changing the intensity. If tunneling times greater than about 100 ms are to be measured, then a lock-in arrangement can be used to keep the light levels very low. In this arrangement, the light is chopped mechanically and the lock-in amplifier with high selectivity measures the photomultiplier output at the chopped frequency. In this manner stray light pick-up can be reduced and the lock-in amplifier filtering capabilities can be used to remove unnecessary noise due to too wide a bandwidth. As our kinetic picture matures, the spectroscopy of the intermediate states, whether by optical spectra (Hofrichter et al., 1983), Mossbauer (Marcolin et al., 1979),electron-spin resonance (Yonetaniet al., 1974),or resonance Raman (Spiro, 1981;Ondrias et al., 1983)will allow a complete picture of the simple act of cooperativity in hemoglobin, and perhaps some idea of the role that the heme group plays in truly catalytic process in biology (Sligar and Gunsalus, 1979).

VI. THE TRIPLET-STATE PROBES We have examined the photolysis of heme groups that are incidentally photosensitive. The heme proteins form a special class of proteins yet some rather important results have come from studies from them. Do these results have general validity? We will in this section drift a bit from the subject of photolysis (the rupture of a molecular bond by absorbed photon) and discuss briefly other laser excitation techniques that we believe help us understand the concepts of protein rigidity and the

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SHIRLEY S. CH.4N AND ROBERT H. AUSTIN

diffusion of the protein structure. There are of course other techniques that also shed light (so to speak) on these questions, but we confine ourselves here to laser-based techniques. 1. Importance of Long Lifetimes Any technique that is of interest to us will have a long lifetime so that as wide a range of time as is possible can be examined, and will be sensitive to the conformation and internal dynamics of the biomolecule. We have found that laser excitation into the triplet state is a powerful technique. A few moments of playing with “Silly Putty” will convince the reader of the utility of long lifetime probes. “Silly Putty” is a polymer and acts at short times like an elastic solid because the polymers cannot disentangle themselves. However, at times long compared with the time for a polymer to flow out of the tangle, the substance flows like a liquid. A correct view of the nature of the beast only occurs with as broad a time scan as possible. Perhaps if one measured the elastic constant of “Silly Putty” as a function of time, we would find also a distribution of activation energies! The singlet lifetime is not more than 20 nsec in duration. For many problems in biology this is not enough time to observe events of interest and hence the authors’ preoccupation with the triplet state. Basically, two parameters can be observed in triplet state kinetics: the lifetime of the triplet state and the orientation of the excited dipole moment. If a molecule has a ground state configuration of spin 0, (thus singlet ground state, So = O), the energy level of its singlet excited state (S1 = 0) always lies slightly higher than the triplet state ( T I= 1) so that the deexcitation can in principle go either directly back to the singlet ground state or to the triplet state. However, because there are no spin operators in the electromagnetic field, the transition to the triplet state from the singlet state is forbidden. Most triplet dyes have heavy atoms in order to enhance the singlet- triplet crossing rate due to spin-orbit coupling, which is a relativistic effect and hence goes as Z, the atomic number. Because the triplet state in the absence of spin-operators is forbidden to radiate to the ground state, the triplet lifetime can be quite long, easily 100 Fsec and often longer than 1 msec. Oxygen can quench at the diffusion limit (approximately 10’oM- ’ * sec-’) either excited singlet states of the aromatic amino acids or triplet states. Due to the short lifetime of the singlet state, elevated oxygen pressures were necessary to observe diffusion of oxygen in proteins (Lakowicz and Weber, 1973). However, because of the long triplet state lifetime, such high oxygen pressures are not necessary, and tripletquenching experiments can be done either with modified heme groups or

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with labels. For instance, removal of tbe iron in myoglobin results in the highly fluorescent protoporphyrin group with a high triplet yield. By this means it has been possible to also observe the entry of the oxygen into the porphyrin by the quenching of the triplet lifetime (Austin and Chan, 1978; Alpert and Lindqvist, 1979). In DNA it is possible to use triplet intercalating dyes and monitor the rate at which oxygen can diffuse into the base-pairing region (Poulos et al., 1982).

2. Anisotropy Decay If a polarized laser pulse is used to excite the triplet state then a photoselected excited state population is created, since the excitation probability of a dipole goes as: P(B)= zocos2e where we assume the exciting pulse is linearly polarized along the z axis as seenin Figure 8. The rotational diffusionof the molecule due to Brownian motion is sensitive to the structure of the molecule. For instance, if we define the anisotropy of the excited state population as:

r(t> = (I,,- I J ( I , , + 21,) where I,,is the emission intensity (phosphorescence)or absorption change for light polarization parallel to the excitation polarization and I, is the same quantity perpendicular to the excitation polarization. If the labeled object is a sphere, then the anisotropy decay will be of the form:

2

r(t) = 5 exp

(x) -3kbT t

where c is the radius of the object and q is the viscosity of the medium. Two things to note: the rotational time depends on the volume of the object, and only motion causes depolarization. There is no complicated analysis as in nuclear magnetic resonance (NMR). If the object is nonspherical, then the anisotropy decay is more complex but solvable (see Tao, 1969). The technique really is as straightforward as one could expect. We and other workers have exploited the triplet lifetime to observe the motion of molecules too large to be studied by singlet techniques. Examples are the rotational motion of RNA polymerase (Austin et al., 1983), the rotational diffusion of membrane-bound components (Cherry et al., 1976; Vaz et al., 1979;Jovin et al., 1981)and the motion of the DNA helix

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SHIRLEY S. CIIAN AND ROBERT H. AUSTIN

(Hogan et al., 1982). In all of these studies, the long lifetime of the triplet signal was crucial to study of the biological system. For most proteins, intrinsic triplet lifetimes of aromatics are quite short at room temperatures and thus triplet dye labels must be used. Our own experience has been with the use of triplet probes and triplet anisotropy decay. Since this is somewhat of a new, and we believe under-utilized, field, we will stress it here. Because the triplet yield is usually quite small and either low sensitivity absorption techniques or very low quantum yield phosphorescence measurements must be made, a reasonably highpowered laser is necessary for this kind of experiment. Labeling with reactive dyes can be done either at specific groups (rare) or randomly over the protein surface. The most common reactive group is the isothiocyanate group, (SCN)-, which reacts with primary amines. Since most proteins have a large number of primary amines this is a nonspecific labeling technique. Labels that react with the less-common amino acids afford a chance of specific labeling. Unfortunately, often the more unusual amino acids have critical roles (the sulfur amino acids as bridges between different domains) and thus protein reactivity can be affected. In any case, with any label it is important that reactivity be measured and verified to be unchanged.

3. Modes of Monitoring Triplet Population Triplet state population can be monitored by either absorption or emission. In absorption, there are two modes of operation, both of which are based on the fact that the triplet- triplet excitation spectrum is red-shifted relative to the singlet-singlet-state excitation. Because of this shift, the singlet population is lost due to triplet-state pumping and monitoring wavelengths at major singlet-singlet transitions will show a bleaching. If the monitoring wavelength is set to a maximum of the triplet-triplet band, then an increase in absorption will be observed. The singlet bleaching signal is often larger than the triplet absorption signal, but intense monitoring beams can cause sample bleaching in the singlet technique, but not in the triplet absorption bands since often the dye has virtually no absorbance there. Figure 7 summarizes the possibilities in triplet spectroscopy. A. ABSORPTION

Most of the work has been done using absorption techniques. Although this technique has excellent time resolution, it suffers from sensitivity problems. Photon statistics are the primary source of noise, and it is difficult to overcome this with increasingly stronger laser monitoring

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Xcxcitc

Figure 7. Schematic of various triplet decay options. In part (a) we see the normal singletsinglet transitions. In part (b) is shown the red-shifted triplet absorption (many molecules have a repulsive triplet ground state due to the quantum effect of delocalization). The decrease in singlet absorption due to triplet pumping is known as singlet depletion. In part (c) is shown the emission spectrum of the dye for singlet-singlet transitions (fluorescence). The character represents the Stoke’s shift for this process. Loss of singlet population due to triplet pumping results in a loss of singlet emission intensity. Finally, in part (d) we show the emission due to triplet-singlet emission (phosphorescence).

beams due to destruction of the chromophore. We have found that a practical power limit is about 1.O W/cm2for the beam. A way around this problem is in the triplet emission (phosphorescence).Another problem in triplet absorption work is sample fluorescence and scattered excitation laser light, either of which can cause dead time in the photomultiplier of significant duration. Accordingly, use of a laser as a monitoring beam can be of significant advantage because (1) the narrow spectral width of the laser allows the use of narrow-band laser spike filters and (2)the collimation of the laser beam allows the monitoring beam to be placed a large distance from the sample cuvette, thus the solid angle for scattered light pick-up is correspondingly greatly reduced compared with a system with high-speed lenses. If one is lucky in choice of wavelengths, then a small HeNe laser can be used (632.8 nm). This laser is usually quite stable (we find 0.1% noise from our 5-mW laser made by Aerotech Inc., 101 Zeta

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SHIRLEY S. CHilN AND ROBERT H. AUSTIN

Drive, Pittsburgh, Penna. 15238). The argon lasers are usually much more noisy than counting statistics would indicate due to cavity noise, and are very expensive. Stabilizers are commercially available for the noble gas lasers however and are probably to be preferred over self-made attempts which usually don’t work well due to inequivalent optical paths. If a HeNe laser can be used, be sure to get a cylindricalhousing for ease of mounting, and a polarized beam for stability. B. PHOSPHORESCENCE

In the phosphorescence technique, the triplet emission process is observed instead of triplet absorption. Although phosphorescence yields are quite small, a very large sensitivity enhancement is possible because the only noise is the signal itself. Hence, the signal-to-noise ratio goes as the (signal)”*, and a very large dynamic range can be obtained. Anisotropy decay from lOnM samples is not difficult. Pulsed lasers are important here since the photomultiplier can be gated off during the intense excitation pulse (see instrumentation section) and still obtain sub-microsecond time resolution. 4.

Instrumentation

In the emission mode, the triplet state emission is monitored. Because of the large red shift for emission, use of visible triplet dyes results in near-infrared emission (600- 800 nm). Thus, good red response in the photomultiplier is important. Also important is the proper choice of blocking filter to remove scattered laser light and sample fluorescence, since even if the PM is gated off during the laser pulse, a gross overload of light upon the photocathode will result in serious artifacts and gain reduction after the pulse. A related problem is the serious phosphorescence that many glass filters exhibit. It is quite easy to see this by putting a filter in the doubled 532-nm beam and observe visually with a red filter: bad filters will visibly emit strongly. We have found that a liquid filter can be very low in stray emission, for instance potassium dichromate in a 5-cm x l-cm quartz cuvette is an excellent filter of green light. Our microcomputer is responsible for the anisotropy measurements. To monitor the two orthogonal polarizations, a stepping motor is used to drive a rotating polarizer. In order to average over laser and monitoring beam fluctuations the polarization is rotated every 64 shots on the laser and the signals are stored in separate channels on the computer. An important development for us was the use of a feedback loop from the high-voltage supply to the photomultiplier. If in absorption work the monitoring beam does not have equal intensity in the two channels, an effective gain change occurs that wreaks havoc on the anisotropy. We

=

Pulsed Excltotion

Crystal Po l a r I rer

Tsf

0

PM

Sample

F \

Lens

r e sne I

Liquid F i l t e r

Polar i r e r

v=

Rotating

Filters

/Gated

Monochromator

Figure 8. Optical layout for triplet work. Part (a) shows the absorption set-up, while part (b) shows the emission set-up. In both cases a rotating polarizer is used to sample the intensities for the two orthogonal polarizations. The feedback system is used in part (b) to ensure that the output signal is the same for both polarizations.

Laser

Pulsed

1

Dye Laser

Telescope Lens

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SHIRLEY S. CHAN AND ROBERT H. AUSTIN

utilize a slow feedback network to force the average DC level of the PM output to be constant. In Figure 8, we outline the basic arrangement used in our lab to do precision anisotropy decay studies. One future direction in the ultrafast (10 nsec and less) that will be important is the use of picosecond mode-locked pulses coupled with signal-averaging techniques and new difference techniques to do extraction of small signals out of noise (Waldeck et al., 1981). For example, instead of measuring the change in transmission for the two polarizations as we do in the absorption technique, it is possible to use crossed polarizers with the sample between the polarizers. Then, when an anisotropic population is created, the rotation of the plane of polarization by the sample allows transmission of light. 'This null kind of experiment allows one to circumvent the elaborate streak cameras and with simple optical delays obtain high-quality data with very small pulse energy. We are sure that in the future these new techniques will be increasingly important.

VII. INTRINSIC PHOTOACTIVATION Now we come to those proteins that when photoexcited undergo biologically interesting reactions. Although they are decidedly in the minority in terms of the number of species of proteins that exist, they definitely are overwhelming in terms of their net mass: reaction centers in chloroplasts consist of chlorophyll-protein complexes in which the protein plays a major role. We class the photoactive proteins into three main classes: the chlorophyll - protein complexes, the bacteriorhodopsin protein in purple bacteria, and rhodopsin. The bacteriorhodopsin and rhodopsin molecules are intimately related because they share the same active group rhodopsin whereas the chlorophyll protein has a very similar group to the heme proteins, the porphyrin. If we expect nature to be economical, then we might expect similar mechanisms to exist between the hemes and the chlorophylls, and between the rhodopsins. At first glance, no such connection would seem to exist, since the hemes studied earlier bind ligands while the reaction center separates charges, and the bacteriorhodopsin pumps charge across a membrane while the rhodopsin triggers a nerve impulse. In this speculative section, we will try to convince the reader that some unifying themes might exist.

1. Charge Separation

Let us examine the electron-separation event first. Several theories to explain the transfer of charge in the photosynthetic act exist, but at a perhaps oversimplified level simple time-dependent perturbation theory found in every introductory quantum mechanics textbook (Merzbacher,

LASER PHOTOLYSIS IN BIOCHEMISTRY

133

1970) can provide significant insight. Briefly put, it is assumed that the rate of charge transfer can be calculated from Golden Rule, which states that:

where A is the initial state (unseparated charge) and B is the final state (separated charge), TABis the matrix element connecting the two states as described later, and the delta function (6) expresses the conservation of energy between the energy given up by A and taken up by B. The matrix element T A B is calculated from the perturbing potential that connects the two sites and the initial and final wave functions, "A, and qB: TAB

=J q ~ H ' q ~ d ~ x

A. ROLE OF CONFORMATION DISTRIBUTION

The energy-conserving delta function is actually where the protein seems to play its major role. Because the transfer of charge in this model is sudden, the electron transition is between states of constant atomic position (a nonadiabatic transition). The atoms from which this transfer occurs are bound harmonically to the lattice and when in thermal equilibrium have a Gaussian distribution around the center of the potential: P ( V ) = ( ~ I T ~ ~ T v ) -exp '/~[ - ~ / k b q

where V is the same harmonic potential as we discussed in molecular tunneling. Since the transition is vertical, the new potential surface that the electron ends up on is a function of the original equilibrium position of the atom before the transition. This shift in position is directly tied to the restoring forces that the atom feels. In the integral, the vibrational energy is given by: V(x) = &(x

- xo)2 = p k x - kxxo++kx:

where

A

= +kx:

is the Stoke's shift. Integration of the energy-conserving delta functions over all possible configurations at a temperature T yields the following transfer rate:

134

SHIRLEY S. CHAN AND ROBERT H. AUSTIN

where A A and A B are the Stoke's shifts due to the change in equilibrium position of the atom in the oxidized and reduced states as shown below and E i and E j are the equilibrium redox potentials (Hopfield, 1974). B. CHARGE-TRANSFER BAND

In classical electron transfer studies, excitation of the normal singlet states leads to electron transfer from the excited state. However, because of the dipole moment formed by the separated oxidized and reduced centers, a direct transfer of charge can occur. A similar perturbation theory approach taking into account the harmonic potential sites of the protein yields the prediction that there should be a charge-transfer band with maximum extinction coefficient at photon energy hv,,,:

hv,,, = E i - E i

+ (AA + AB)

Because the Stokes shifts are not expected to be greater than about 1 eV (1 100 nm), these charge-transfer bands lie in the infrared spectrum. To derive maximum information from a charge-transfer experiment (a laser photolysis if you will), it is important that a full time-resolved picture of the subsequent return of the electron to the ground state is obtained. A high-pressure Raman shifter cell with H2 gas at a pressure of 250 lb/in.2 efficiently shifts the 1063-nm main line of the YAG laser by 4000 cm-' to 1900 nm, almost exactly OR the predicted line of charge-transfer bands (Goldstein and Bearden, 1983). Using this efficient wavelength-shifting mechanism, it should be possible to, for the first time, study the timeresolved electron recombination after photo-assisted transfer and carefully check the tunneling theory. The theory of electron transfer and charge transfer are unified and must agree with one another, and, indeed, as we understand the diffusion experiments better, we expect all these experiments to form a consistent picture. C. INSTRUMENTATION

Experiments in this field have often involved extensive signal-averaging of very small signals. In this case, it is imperative to minimize the noise of the system. The brightest lights that offer high beam stability are Hg-Xe arc lamps in a water-cooled elliptical housing. Intensities of 20-50 mW at the Hg lines in a F/1.O optical system are not impossible. Monochromators at this speed are not readily available and do not offer the efficiency that a Hg line optical filter can give. A further problem is the amplification of a very small signal. Modern day op amps such as the chopper-stabilized ICL 7650 by Intersil make it trivial (well, almost) to build a close to Johnson noise-limited amplifier, but the dreaded ground loop has been known to

135

LASER PHOTOLYSIS IN BIOCHEMISTRY

make all efforts for nought. A ground loop arises when two different circuits are at different ground potentials. A current then flows from one circuit to the other one-hopefully through a very small resistance ground strap, but more often down a signal cable. The ohmic drop along this cable can be several millivolts and can be amplified into an intolerable 60-Hz “hum.” As shown in Figure 9, there are three main cures: (1) Plug all instruments into a common outlet strip, with three-prong sockets! (2) Use heavy ground braid to tie all instruments to a common ground point. (3)Use floating inputs with about 10-ohm resistors to true ground and let the diverted ground currents flow in the heavy straps of item 2. The book by Ott (1976) on grounding and shielding techniques is quite valuable here. Figure 10 shows a laser-induced T-jump signal that was used in the paper by Chang and Austin (1982). We see here resolution of voltage changes to one part in lo5, signals that required conquering the ground loop and RF problems. One last warning: make sure your laser manufac-

1 Power Gnd. 4

7r ,/

/

I

/

Single Ground Point

Power Gnd. 2

/ I

-

u Common S i g n a l Source

Figure 9. Schematic of source of ground loops and possible cures. Two discrete amplifiers (1) and (2) are shown. Because of heavy currents that can flow in the power ground paths, and the finite resistances of the ground wires different voltages, V g l and VG, can exist on the chassis boxes of the devices. This difference in voltage can cause currents to flow between the two devices and because of finite resistances in the cable voltages can appear as 60-Hz pick-up ,on the amplifiers. The idea is to force the ground currents to flow in a low impedance path formed by heavy copper braids. The 10-ohm resistors between signal ground and earth ground help in this, as does the single point grounding point, which can be the wall socket or a provided ground wire in the building.

136

SHIRLEY S . CHAN AND ROBERT H. AUSTIN

turer has not synchronized the laser puke rate to the 60-Hz line frequency-if that has been done then the 60 Hz ripple is coherent with the averaging. Disaster! 2. Proton Pumping

A more speculative area lies in the area of the rhodopsin-containing proteins. The remarkable thing about the rhodopsin molecule is the cis-trans isomerization that occurs with unity quantum yield upon absorption of a photon. A great deal of experimental effort has gone into the elucidation of the various steps of the rhodopsin cycle (Applebury et al., 1978)and one of the interesting aspects has been the possibility of the low temperature cis-tram isomerization process. The observation of these transient absorption states can be made using the techniques described in this article. What is not clear is the role that the cis-trans isomerization plays in the hyperpolarization of the rod outer cell. It is curious that a quite similar protein, bacteriorhodopsin, uses the same cis-trans isomerization to pump charge in some way across a membrane. Is there a connection?

c y t =(3pLM

Fez* = 33 rnM Fe31=0.2rnM

2.

3.

4.

TI ME (milliseconds) Figure 10. The T-jump signal for the cytochrome-diron hexacyanidecouple. The vertical scale is approximately V N full scale. Approximately 10’ laser shots where averaged for this trace, with a Hg-Xe lamp as the monitoring light source.

LASER PHOTOLYSIS IN BIOCHEMISTRY

137

VIII. SUMMARY The section on electron tunneling stressed the role that the protein rigidity plays in control of the tunneling rate. The hemeprotein work has somewhat beneath the surface the realization that protein rigidity is playing a role in reaction kinetics, as evidenced by the different distribution functions for activation energies among similar proteins. The DNA work is revealing possible important roles of structural rigidity in gene recognition and expression. The picture seems to be forming that apart from the pure chemical aspects of the placement of amino acids around a site, or the ordering of basepairs, that physical aspects such as structure may be influencing biological behavior. One highly interesting but unproved area is the concept of Davidov nonlinear solitons and topological solitons (Hyman et al., 1981)as a common aspect of energy transduction in biological systems. Since a photon delivers a convenient punch of 1-5 eV of energy to a site, we suspect that photolysis in its many forms will play an essential role in what we hope will be a revolution in bioenergetics. References Agmon, N. and Hopfield, J. J. (1983),j. Chem. Phys., 7 8 , 6947-6959. Alben, J. O., Beece, D., Bowne, S. F., Eisenstein, L., Frauenfelder, H., Good, D., Marden, M. C., Moh, P. P., Reinisch, L., Reynolds, A. H., and Yue, K. T. (1980), Phys. Rev. Lett., 44, 1157-1 160. Alberding, N., Austin, R. H., Chan, S. S., Eisenstein, L., Frauenfelder, H., Gunsalus, I. C., and Nordlund, T. M. (1976a),j. Chem. Phys., 65 4701-4711. Alberding, N., Austin, R. H., Chan, S. S., Eisenstein, L., Frauenfelder, H., and Nordlund, T. M. (1976b), Science, 192, 1009-1004. Alberding, N., Chan, S. S., Eisenstein, L., Frauenfelder, H., Good, D., Gunsalus, I. C., Nordlund, T. M., Perutz, M. F., Reynolds, A. H., and Sorensen, L. B. (1978),Biochemisty, 17, 43-51. Alberding, N., Lavalette, D., and Austin, R. H. (1981)’ Proc. Natl. Acad. Sci. USA, 78, 2307 -2309. Alfano, R. R. (1982), Biological Events Probed by Ultrafast Laser Specroscopy, Academic Press, London. Alpert, B. and Lindqvist, L. (1975), Science, 187, 836-837. Antonini, E. and Brunori, M. (1971), HemoglobinandMyoglobnin TheirReactiomwith Ligands, North-Holland, Amsterdam. Applebury, M., Peters, K., and Rentzepis, P. (1978), Biophys. j.,23, 375-382. Austin, R. H., Beeson, K. W., Eisenstein, L., Frauenfelder, H., and Gunsalus, I. C. (1975), Biochemistry, 14, 5355-5373. Austin, R. H., Beeson, K. W., Chan, S. S., Debrunner, P. G., Downing, R., Eisenstein, L., Frauenfelder, H., and Nordlund, T. M. (1976), Reu. Sci. Instrum., 47, 445-447. Austin, R. H. and Chan, S. S. (1978), Biophys.J., 24, 175-186.

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Austin, R. H. and Hopfield, J. J. (1982), in Ekcfron Transport and Oxygen Utilization (Chien Ho, Ed.), Elsevier North-Holland, New York, pp. 73-80. Austin, R. H., Karohl, J., and Jovin, T. M. (1983), B i m h i s t ? y , 22, 3082-3090. Beece, D., Eisenstein, L., Frauenfelder, H., Good, D., Marden, M. C., Reinisch, L., Reynolds, A. H., Sorensen, L. B., and Yue, K. T. (198O),Biochkby, 19, 5147-5157. Chang, A. M. and Austin, R. H. (1982), J. Chem.Phys., 77, 5272-5283. Cherry, R., Buerkli, A., Busslinger, M., Schneider, G., and Parish, G. (1976), Nature, 263, 389-393. DeVault, D. and Chance, B. (1966). Bzophys.J., 6, 825-847. Douzou, P. (1977), C7yobiochemistty, Academic Press, London. Gerstman, B., Austin, R. H., and Hopfield, J. J. (1981), Phys. Rev. Lett., 47, 1636- 1639. Frauenfelder, H. (1978), Methods Enzymol., 54, 506-532. Gibson, Q. H. (1959), B i o c h . ] . , 71 293-303. Goldman, Y. E., Hibberd, M. G., McCray, J. A., and Trentham, D. R. (1983), Nature, 300, 701-705. Gouterman, M. (1978), in Pmphyinv, vol. 3(A) (D. Dolphin, Ed.), Academic Press, New York, pp. 1-165. Goldstein and Bearden, in preparation. Hiromi, K. (1980), Meth. B b c h . Anal., 26, 137-164. Hogan, M., Wang, J., Austin, R. H., Monitto, C., and Hershkowitz, S. (1982), Proc. Natl. Acad. Sci. USA, 79,3518-3522. Hofrichter, J., Sommer, J., Henry, E., and Eaton, W. (1983), Proc. Natl. Acad. Sci. USA, 80, 2235-2239. Hopfield, J. J. (1974), Proc. Natl. Acad. Sci. USA, 71, 3650-3644. Horowitz, P. and Hill, W. (1980), The Art ofEZectronits, Cambridge University Press, Cambridge. Hyman, J., McLaughlin, D., and Scott, A. (1981), Physica, 3D, 23-44. Jovin, T. M., Bartholdi, M., Vaz, W., and Austin, R. H. (1981), Ann. N.Y. Acad. Sci.,366, 176- 196. Lakowicz, J. R., and Weber, G. (1973), Biochemitq, 12,4154-4161. Martin, J. L., Migus, A., Poyart, C., Lecarpentier, Y., Astier, R., and Antonetti, A. (1983), Proc. Natl. Acad. Sci. USA, 80, 173- 177. Merzbacher, E. (1970), Quantum Mechnnics, John Wiley & Sons, New York, pp. 450-487. Marcolin, H.-E., Reschke, R.,and Trautwein, A. (1979), Eur.J. Biochem., 96, 119- 123. Ondrias, M. R., Friedman, J. M., and Rousseau, D. L. (1983), Science, 220, 615-617. Ott, H. W. (1976), Noise Reduction Techniqws in EZectronic Systems, John Wiley & Sons, New York. Poulos, A. T., Kuzmin, V., and Geacintov, N. (1982),J. Biochem. Btophys. Methods, 6,269281. Redi, M. H., Gerstman, B. S., and Hopfield, J. J. (1981), Biophy.J., 35, 471-484. Reynolds, A. H. and Rentzepis, P. (1982), Biophys. J., 38, 15- 18. Rosenfeld, T., Honig, B., Ottoleng, M., Hurley, J., and Ebrey, T. (1977), Pure Appl. Chem., 49, 341-351. Schurr, M. (1983),J. Chem. Physics (in press).

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Sligar, S. G. and Gunsalus, I. C. (1979), Biochemistry, 18, 2290-2295. Spiro, T. (1981), Israeli J . C b . ,21, 81-86. Tao, T. (1969), Biopolpers, 8, 609-632. Vaz, W., Austin, R. H., and Vogel, H. (1979), Biophys. J., 24, 415-426. Waldeck, D., Cross, A., McDonald, D., and Fleming, G. (1981),J. C b .Phys., 74,33813387. Yapel, A. F. and Lumry, R. (1971), Meth. Biochern. Analy., 20, 169-350. Yonetani, T., Yamarnoto, H., and Iizuka, T. (1974),J . Biol Chem., 249, 2168-2174.

Methods of Biochemical Analysis, Volume 30 Edited by David Glick Copyright © 1984 John Wiley & Sons, Inc.

METHODS OF BIOCHEMICAL ANALYSIS

VOLUME 30

Density Gradient Electrophoresis of Mammalian Cells ABRAHAMTULP. Department of

Biochemistry. Antoni van Leeuwenhoekhuis. The Netherlands Cancer Institute. Amsterdam. The Netherlands

........................................... I . Introduction .................................................. I1 . History ......................................................................... I11. Theory ........................................................................................................... 1. Gravity Effects .................................................................................... 2. Electrophoretic Effects ....................................................................... 3. The Miniaturization Principle ............................................................ IV . Apparatus and Experimental Conditions .................................................... 1. Introduction ........................................................................................ 2. Preparative Electrophoresis Columns ................................................ A. The Buchler Polyprep ............. .................................... B. The Boltz-Todd Device .................................. C Stable-Flow-Free Boundary Electrophoresis (STAFLO) According to Me1 ..................................................................... D. The LKB Column ................................................................... E . Small-Size Focusing Chamber ................................................. F. The ISCO Column .................................................................. G. Quickfit Column ...................................................................... H . Van Oss and Bronson Device ................................................. I . Separation Chamber According to Tulp ............................... a . Device with Movable Electrodes ................................... b. Compact Device ............................................................. 3. Analytical Electrophoresis of Density Gradient ................... A. Transanalyzer ............. ................................................... B. Column with Laser Beam ....................................................... C. Electrophoresis along a Discontinuous Density Interface ..... 4. Experimental Conditions ................................................................... A. Density Solute .......................................................................... B. Ionic Composition ...................................................................

.

14 1

142 143 143 144 147 150 152 153 153 156 158 159 160 160 161 161 161 162 162 166 174 174 175 175 175 175 176

142

ABRAHAM TULP C. Electrodes

............................................

..............................................

....................

............................. G. Thymocytes ............................. 2. Miscellaneous ..................................... 1.

Introduction ........................

..........................

176 176

177

177 177 177 177 178 180 182 182 183 183 184 184 185 186 187 192 192 192

I. INTRODUCTION Differentiation, specialization, maturation, and transformation are generally accompanied by small changes in physical intrinsic properties of cells. Differences in size, density, and surface charge position each cell type in a rather unique part of a size-density-surface charge continuum. It is the purpose of cell separation to transport by some physical means dissimilar, closely neighboring cells from this imaginary space into separate fractions that are obtained after one-dimensional migration of the cells. Cells in aqueous suspension preferably are separated by physical methods under well-defined experimental conditions rather than by chemical modification that influences cellular viability or initiates cellular processes. Moderate physical forces and short separation time greatly contribute to cellular integrity. In this chapter, the application of density gradient electrophoresis to the separation of mammalian cells is described. This process may be defined as the differential zonal movement of cellular particles of anionic character suspended in a stabilized liquid column under the influence of an external electrical field so that collection of differentially moved zones is feasible. Although basically a very simple, low-budget method, it has been

DENSITY GRADIENT ELECTROPHORESIS OF MAMMALIAN CELLS

143

largely superseded by Hannig’s excellent but costly free-flow electrophoresis (FFE) technique (Hannig, 19’71,1972,1978).Application of the FFE technique may be economically prohibitive in small laboratories, whereas density gradient electrophoresis certainly deserves the repeated advocacy of Catsimpoolas and co-workers. The gamut of biological systems that have been resolved in biochemical entities by FFE may then also be attacked by the density gradient electrophoresis technique. 11. HISTORY

Long before electrophoresis emerged as a scientific discipline, Reuss (1809)observed that clay particles in aqueous suspension move under the influence of an electrical field. In 1902 Lillie described the microscopic electrophoretic movement of red blood cells, leukocytes, muscle cells,and isolated nuclei in isotonic sucrose. Application of density gradients for the prevention of gravity-fed disturbances was first put forward by Philpot (1940), stressing the use of short columns, strong gradients, and thin sample layers. Sorof and Ott (1954) used density gradients for the stabilization of moving boundaries in the well-known Tiselius cell. The term “zone electrophoresis” stems from Tiselius (1955) and distinguishes this procedure from moving boundary electrophoresis. Brakke (1953) intro‘ duced the use of density gradients in simple electrophoretic U tubes; he also described the annoying “streaming” phenomenon (Brakke, 1955). Kolin (1955, 1958) was the first to achieve a very rapid separation of an artificial mixture of algae and red blood cells in short columns but with an extremely steep density gradient. Almost unnoticed remained a paper (in French) of Thomas et al. (1969) who separated rabbit alveolar cells by ascending electrophoresis using a very low-ionic-strength buffer in a lactose-glucose isoosmotic density gradient; of the four bands obtained, one contained a pure population of histiocytes. Boltz et al. (1973)made a thorough study of the factors involved in cell density gradient electrophoresis followed shortly after by that of Griffith et al. (1975) using a commercial electrophoresis device. Isoelectricfocusing, essentially an equilibrium method, was applied for the first time to cells by Sherbet et al. (1972). 111. THEORY

Imagine a particle in aqueous solution subjected to gravitational and electrical forces. According to Newton’s second law, the resultant buoy-

144

ABRAHAM TULP

ing, gravitational, frictional, and electrical forces for a spheroidal particle in a viscid medium equal

4 d2x d x 4 - IT pp r3 - = QE - 6nqr - + - I T ( P ~- pM) r3g 3

dt2

dt

3

where pp = density of particle (g/cm3);pm = density of medium (g/cm3); r = radius of particle (cm); q = viscosity of medium (g/cm * sec); x = migration distance (cm); Q = net charge of particle (coulomb); E = electrical field strength (V/cm); and g = gravitational acceleration (cmi sec2). solving Equation (1) yields:

It is easily verified that for a typical mammalian cell (pp = 1.07,r = 5 pm) and at r) = 1.6 CPthe exponential term in Equation (2) IS less than 0.001 within about 2.5 X sec so that Equation (2) reduces to

where M = electrophoretic mobility (cm - sec-'/V * cm-'); the gravity part is recognizableas Stokes' law. Using small programmable calculators, Stokes' law can be easily integrated (Tulp et al., 1981). With respect to density gradient electrophoresis, the two terms in Equation (3) deserve a separate treatment: 1. Gravity Effects

Mammalian cells are large particles so that diffusion effects (Brownian movement) are completely absent, a fact that adds to the electrophoretic resolving power. On the other hand, their large size makes them sediment rather rapidly under the influence of gravity, giving rise to hydrodynamical instabilities. To circumvent gravitational effects, either due to nonideal particle sedimentation or to convection currents, electrophoresis has been performed in a rotating tube according to Hjerten (1970), in an annulus, using electromagnetic propulsion of the liquid content according to Kolin (1967) in a microgravity field in the Apollo-Soyuz project (Van der Hoff and van Oss, 1979;Van der Hoff and Micale, 1979; Van der Hoff et al., 1978),or by vertical arrangement of the FFE chamber

DENSITY GRADIENT ELECTROPHORESIS OF MAMMALIAN CELLS 145

according to Hannig (1971). An alternative for the suppression of hydrodynamic gravity-driven instabilities is the use of density gradients. Stabilization in velocity sedimentation, equilibrium density centrifugation, zonal electrophoresis, and isoelectric focusing, respectively, have as their common denominator a density gradient. Solid supporting media are disadvantageous in that they prevent the migration of large particles like bacteria and cells. The theory that is able to predict the stable banding capacity of zones containing macromolecules in density gradients is firmly established (Svensson et al. 1957; Nason et al. 1969; Sartory, 1969; Meuwissen and Heirwegh, 1970; Winet and Jahn, 1972; Meuwissen, 1973; Plesset et al. 1976; Remenyik et al. 1980). The mechanism that leads to the limited capacity for particles visible by light microscopy is still poorly understood (less than 0.1 % of the amount that can be theoretically layered according to Svensson’s stability criterion) (Svensson et al. 1957) is gravitationally stable. In this process, the interface between cell band and the underlying, denser medium becomes distorted into relatively rapid, descending, thin streamers of less than 1 mm diameter, where collective particle motion occurs. Once formed, the thin streamers are stabilized by diffusion of solute molecules as has been shown by Schumaker (1967). A description that comes close to experience is based upon perturbation analysis. Thus, Mason (1976) has deduced that the maximal particle load, even when the particles are loaded as an inverse gradient into the macromolecular density gradient, equals

where nCrit= concentration of particles that still can be layered without streaming (cells/ml);D = diffusion coefficient of density solute (cm2/sec); dpldx = slope of density gradient (g/cm4);and g = gravity (centrifugal) force (cm/sec2). Mason’s deduction also predicts the growth rate of the instability as well as the diameter of the streaming disturbances. According to Equation (4), low-viscosity-small-diffusion solutes enhance banding capacity. It thus follows that Percoll gradients have a higher banding capacity than sucrose gradients (Tulp et al., 1980b).Gradients have a low capacity for large cells (inversely proportional to cell radius). Figure 1 shows the dependency of critical cell number, before streaming sets in, as a function of the slope of the gradient for a cell of 5-pm radius and for D = 6 x lo-’ cm2/sec,as well as the dependency of cell load upon cell radius in a density gradient

146

ABRAHAM TULP

6 5.10

=O 106

L

I

2

I

4

I

6

-Cell

1

8

I

10

Radius ( f m )

Figure I . Dependency of critical cell load on steepness of density gradient and radius of cellular particle. ncritat unit gravity was calculated from Equation (4).

of gicm’. Table I shows that theoretical and practical cell loads are close. Data taken from a number of unit gravity separation studies indicate that loads seldom exceed 5 X lo6 cellslml and that only small cells (sheep erythrocytes) can be loaded at about 1.5 X lo7 cells/ml. If a large number of cells has to be separated, the low-viscosity density gradient must be strong and either the zonal cell layer must be thick (to the detriment of resolving power) or the effective cross section of the separation column must be large. Many more cells can be layered onto gradients that are close to the density of the cells, but unfortunately viscosity of macromolecules contributing to the density of the gradient is high in the region of p = 1.0’7. Percoll, both for its very low diffusion coefficient and for its low intrinsic viscosity, would be the ideal density gradient solute were it not that the Percoll particles have an electrophoretic mobility of about -0.5 T U (Pertoft et al., 1978). The TU (Tiselius Unit) is defined as cm .

DENSITY GRADIENT ELECTROPHORESIS OF MAMMALIAN CELLS

147

TABLE I Cell Load in Unit Gravity Separation Studies ~~~~~~

~

Cell type" Peripheral blood H Lymphoblasts M Spleen M Erythrocytes S Bone marrow H AKR leukemia M Bone marrow M

Cellular volume (w3) >250 500 -220 31

-

-500 100

~

~

~

~

~~~~~

Cell numberlml (X

References

10-6)

3.2 5.0 3.3 15 5 4

5

Brubaker and Evans (1969) Hayry and Anderson (1976) Miller and Phillips (1969) Peterson and Evans (1967) Rosen et al. (1970) Zeiller and Hansen (1978)

"Abbreviations: H = human, M = mouse, S = sheep.

sec-'/V * cm-' (Catsimpoolas et al., 1976a) and is for anions.

+ for cations and

-

2. Electrophoretic Effects Most large and small molecules and suspended particles have electrophoretic mobilities between 5 and 50 TU. Whereas the gravitational part of Equation (3) has a reliable hydrodynamic theory, the electrophoresis part of Equation (3)expressed in terms of measurable physical properties is still not well understood. From

it follows that the electrophoretic velocity of a cell is proportional to the electrical field strength. In density gradients, both M and E are functions of the migration distance. According to Smoluchovsky, if the product of the Debye-Hiickel constant x times the radius of curvature r is larger than 100 (see Table 11, p. 20, Overbeek and Bijsterbosch, 1979),then the electrophoretic velocity of a cellular particle (a macropolyanion) moving toward the anode equals

where K = specific conductivity (ohm-' - cm-'); E = dielectric constant of the medium (dimensionless); 5 = zeta-potential (volts); i = current (amperes); and q = cross section of separation device (cm2).

148

ABRAHAM TULP

For theoretical limitations and assumptions in general, the reader is referred to Overbeek (1952), Overbeek and Lijklema (1959), Overbeek and Wiersema (1967), and Overbeek and Bijsterbosch (1979), and to Brooks (1973), Brooks and Seaman (1973), Haydon (1961a,b, 1964), Haydon and Seaman (1967), and Sherbet (1978) for cells in particular. At moderate ionic strength (0.6-1 mmho) and for mammalian cell size, electrophoretic mobility is independent of particle size. T w o particles of equal 6 potential but differing widely in size, migrate with equal velocity. It is only at very low conductivity and rather small particle size (1 < XT < 100) that electrophoresis effectuates separation according to size (Wiersema et al., 1966; Hannig et al., 1975; Hannig and Heidrich, 1977). It is doubtful, however, whether cells maintain their structural and functional integrity in buffers of very low ionic strength. Electrophoretic separation of cell size classes seems at present unlikely. Equation ( 5 ) is generally found in electrophoretic studies. Its value for density gradients is limited since 6, E, q,and E are functions ofx. To obtain realistic estimates of the migration velocity as a function of buffer milieu and density gradient composition, one would like to express mobility as a function of the average net surface charge density, which may be assumed to be a constant for a particular cell type. Formulas exist that interconnect the variables in Equation ( 5 ) with more basic natural constants, but it remains to be established whether realistic solutions are thus produced. An extension of Equation (5) in terms of measurable physical constants would permit predictions of the feasibility of density gradient electrophoresis of cells. Even a qualitative, rather than a precise, prediction would contribute to our understanding of the process. Substituting

c=-

4~ud E

where u = net surface charge (C/cm2); d = distance from the charged center to the surface of shear. Whereas d may be expressed as 1 d=-+ai X

(7)

where ai = radius of the counterion forming the ion cloud around the particle, Equation (5) becomes

DENSITY GRADIENT ELECTROPHORESIS OF MAMMALIAN CELLS

149

after neglect of ui. From the Debye-Hiickel theory of the ionic cloud around the particle, it follows, assuming conductivity, viscosity, and dielectric constant to have the same value in the electrical double layer as they have in the bulk liquid, that

‘-J 1OOORTE 8ne2N2p

-_

x

(8)

where p, = ionic strength (gionslliter);R = gas constant (ergldeg * mole); T = absolute temperature; N = Avogadro’s number; and e = elementary charge. Inserting known constants, at 4°C Equation (8) reduces to

k

1

- = 3.33 x 10-9 X yielding

($) -

0

E

x 3.33 x lo-9d:;K -rl

(8b)

(5b)

From Equation (5b) one can conclude that at equal electric field strength, the ratio of electrophoretic velocities at ionic strengths p,1 and p,2 is equal to -

/?=d.

!L v2

K2

since at low ionic strength, specific conductivity is proportional to ionic strength. To speed up electrophoretic separations, it is therefore advisable to lower the conductivity of the buffers (to about 1 mmholcm).There is a limit to the reduction of conductivity because membrane integrity requires a certain electrolyte concentration. For instance Heard and Seaman (1960) showed that starting from an ionic strength of 0.0029, erythrocytesare already electrokineticallymetastable. Problems connected with the removal of Joule heat are also minimized: at equal field strength ~. Equation (5b) is a the ratio of heat production is equal to ( K ~ / K ~ ) ” While good vehicle to demonstrate that electrophoretic mobilities are inversely proportional to the square root of specific conductivity (as is Joule heatkgj, it serves the purpose poorly to connect electrophoretic mobilities, obtained from FFE, analytical laser Doppler spectroscopy (Uzgiris and

150

ABRAHAM TULP

Kaplan, 1974; Kaplan and Uzgiris, 1981)and microelectrophoresis with a cytopherometer (Ambrose, 1965), with measurable physical constants of the gradient. Equation (5b) is applicable in cases where small uncharged molecules (sucrose, Metrizamide, D20) contribute to the density gradient. The situation is radically different when neutral polymers are constituents of the density gradient. Neutral polymers have only a minor positive effect on the dielectric constant of aqueous solutions (Brooks and Seaman, 1973)but electrophoretic mobilities of cells are enhanced considerably in the presence of these polymers (Ponder, 1957; Haydon, 1961a,b, 1964; Brooks and Seaman, 1973). Boltz and Todd (1979) report that fixed chicken erythrocytes have (viscosity corrected) mobilities of -9.8, 17.7, and 26.0 T U in 0 , 5 , and 10% Ficoll solutions, respectively. Brooks (1973) and Brooks and Seaman (1973) proposed that neutral polymers are adsorbed to the cell surface so that the surrounding ionic cloud is changed with marked positive effects on the 5 potential (see also Haydon, 1961a,b). A rather complicated formula was derived by Brooks (1973) that is out of the scope of the present paper. Whatever the causes of the positive effects of neutral polymers on the 5 potential are, it is generally found that cells migrate almost linearly in moderate density gradients of neutral polymers (Catsimpoolas et al., 1975; Boltz et al. 1976, 1978; Catsimpoolas and Griffith, 1977a, 1978; Tulp et al., 1983). Apparently several compensating factors reduce Equation (5)to a constant. For small molecular solutes, the compensating factors are retraceable from Equation (5b). At a constant current supply, i is by definition a constant. Since the specific conductivity K can be expressed at low ionic strength in terms of mobilities (see Kolin, 1967) and since ionic mobilities are inversely proportional to the viscosity, the term ~ ( x ) K ( x )becomes a new constant. Indeed for glucose (Matheka and Geiss, 1965), sucrose (Brakke et al., 1968), D 2 0 (Bronson and Van Oss, 1979), and, over a limited concentration range, Metrizamide (Serwer and Watson, 1981), V K is a constant. For these substances, E varies only slightly over a moderate concentration range (Harned and Owen, 1950). Unfortunately, neither glucose or sucrose are suitable solutes for density gradients due to the osmotic effect upon intact cells but a mixed type gradient of sucrose plus Metrizamide may be quite useful. The term q~ is definitely not a constant for Ficoll400,OOO (Boltz et al., 1973) or Ficoll 70,000 (Tulp et al., 1982a).

3. The Miniaturization Principle For rate-zonal separations, it is general rule that miniaturization of the dimension, in which the physical effective force is acting, brings along a

DENSITY GRADIENT ELECTROPHORESIS OF MAMMALIAN CELLS

151

reduction in separation time. Resolution per unit time is favored by a small initial cell band. The principle is illustrated using Equation (5) but any other velocity equation may be used. Suppose that two types of particles 1 and 2 with 5 potentials 51.0 and 52.0 are layered as a zonal suspension of finite height h (with q o and K) on a liquid denser column with q and K. Assuming that the specific conductivity in sample and column are equal, it follows from Figure 2 that particle 2 migrates at a constant current from the interface I in At seconds a distance 52E i - At

=$rill

QK

A similar particle 2, but originating from the meniscus M, first travelled through h and then migrated into the column in At seconds

Particle 1 migrates from the interface

The cohort of particles 2 is completely separated from all particles 1 if x I J = X z J f l or substituting Equations (5d) and (5e)

and solving for At

Assuming that the neutral polymer present in the liquid column enhances and 52,o by an equal factor a, one obtains both 47~rl0

At=h-

E

(52.0

1

- 51,o)

_ QK - constant h

Figure 2. Resolution per unit time is favored by small initial cell band. See Text. Symbols: = sample height; M = meniscus. I = interface; x = migration distance.

h

152

ABRAHAM TULP

it follows that a n-fold reduction of the sample layer thickness results in a n-fold reduction in separation time. Separation time is independent of the nature of the underlying liquid column, so that Equation (5g) also holds true for density gradients with varying q and K. It also follows that the length of the column that is needed for a given resolution is reduced n-fold upon n-fold reduction of the sample layer thickness. From unit gravity separations, it is known that the minimal sample layering thickness that can be obtained is 0.3 mm (Tulp et al., 1982b,c) by which the limit for separation rapidity is set, unless some way of (electrical) focusing can be used. It is shown in Section IV.2.I.b that the gravity term of Equation (3) gives rise to a focusing effect, provided the density and viscosity of the sample layer differ from the values in the density gradient. T h e principle of miniaturization of the dimension, into which the physical separative force acts, has been applied to other separation techniques as well, that is, to unit gravity separations (Peterson and Evans, 1967; Tulp et al., 1980a, 1982b, c); counter-current thin-layer, two-phase partition (Albertsson, 1965; Albertsson et al., 1982); isoelectric focusing and equilibrium density centrifugation (Beaufay et al., 1974; Tulp et al., 1981). For instance, commercially available isoelectric focusing columns of 27.5-cm length require focusing times of 15-20 hr (Electrofocusing Seminar Notes, LKB part 3 2400-006) or 24-72 h r (Instruction Manual 1-8100-E01),whereas columns of 3.5 cm height require only 2 hr Uonsson et al., 1973) o r 75 min (Frederikson, 1975)o r even down to 10 min (Rilbe, 1973) in a chamber of 1-cm height.

IV. APPARATUS AND EXPERIMENTAL CONDITIONS 1. Introduction

It is only necessary to screen analytical biochemistry to discover that an enormous variety of liquid density gradient electrophoretic columns exists (see also Bloemendal, 1963; Gaal et al., 1980). Several of these devices are entirely suitable o r can be made suitable after minor changes for cell separation purposes. A number of these devices are tabulated in Table I1 together with information on sample layer thickness, effective surface area, wattage, and steepness of the density gradient. T h e electrical field strength is generally a few Vkm; relatively high voltages were used by Boltz et al. (1976) [ 13 Vkm] and by Van Oss and Bronson (1979) [12 Vkm]. T h e effective surface area is small in most devices; large surface areas are found in the device of Tulp et al. (1982a) (50 cm2)and in the Steere and Davis column (122.7 cm2). Specific conductivity in the

DENSITY GRADIENT ELECTROPHORESIS OF MAMMALIAN CELLS

153

majority of devices is about 1 mmhokm. The wattage delivered to each cm3 of density gradient varies widely (by using the conversion factor 1 Watt = 0.24 cal, the adiabatic temperature rise in the gradient can be calculated, assuming that 1 cal heats up 1 cm3of gradient liquid 1°C).It is obvious from Table I1 that several devices can easily absorb much more heat than they actually did thus speeding up separations. Some devices that are not cooled stand a much higher wattage than several externally cooled ones. In the Van Oss and Bronson (1979)device, as much as 0.1 12 W/cm3 is delivered, but this coincides well with the very strong density gkm". Generally, rather weak gradients are gradient of 13.0 X used. Sample layer thickness, the principal determinant of resolving power, is in some cases 6.7 mm. Some of the apparatus tabulated emerged as cell separation columns and these will be described fully.

2. Preparative Electrophoresis Columns Two of the more critical steps in density gradient electrophoresis are undisturbed introduction of the (thin) sample layer into the column and the undisturbed fractionation of the column after electrophoresis. A. T H E BUCHLER POLYPREP

The Biichler Polyprep 100 and 200 (Biichler Instrument, Fort Lee, N.Y.) is an improved version of the device originally designed for polyacrylamide gels by Jovin et al. (1964). For assembly of the apparatus, one may follow the instructions of Catsimpoolas and Griffith ( 1977c), Catsimpoolas et al. (1978), or Platsoucas (1983). Model 200 is depicted in Figure 3. The apparatus consists of a glass column with coolingjacket and a cold finger in the center of the column so that the cross section of the separation compartment is an annulus of 17.6 cm'. To prevent electroosomosis, the annulus is siliconized with 1% solution of Siliclad (Clay Adams, Parsippany, N.Y.) in water. It can be sterilized by 1% formaldehyde. The internal glass cooling tube containing the central elution capillary is positioned 2.5 cm above the rigid porous glass membrane and closed. In the following, the terms lower electrode solution, upper electrode solution, and dense and light gradient solution will be used without explicit reference to the composition of these buffers (see Catsimpoolas and Griffith, 1977c for details). First, via 1 lower electrode solution is introduced up to the glass porous membrane; 80 ml dense Ficoll solution is introduced into the column to the level of the lower surface of the internal cooling finger via port 2, filling the annulus through six ports. Then through 3 a small Teflon tubing is introduced connected with a gradient

P P*

P*

P* P

P*

P*

P* P

P*

P A* A P

P*

A A A

PA(*)

P P P P

-

4 1 3.3 2.7-6.7 4.8

-

6 6.7 6.3 0.5

4.1 1.5 0.8 Not relevant 0.8 2.49 3.53 4 2 4.4-3.2 1 1

-

5.6 5 12 4

-

9.55 1.23 3.5 7.5

-

7.9

1.99 12.8 4-5.4 2.33 4.5-8.36 4.85 4.46 2.44 3.5 1.23

V/cm

7.5 50 12.56 0.75 4.15

0.785 2.01 0.28 2.01 15.8 0.64 0.2 13 41.7 0.75 15.8 4 4.9- 122.7

-

3.68 3.8 3.8

Surface area (cm')

-

-

-

0.8- 1 -

-

-

8.4 1.026

-

1.026

0.99 0.99 0.99 0.835 -1 1.026 0.8 1.026

Conductivity (mmholcm)

200 A0.5%/cm 130 26

-

49 -500 4.8 76 80 220 6.64 18 73

-

6.22 6.64 31.0 6.64

6.67

6.67 6.67

* = not used for cell separation;

-

+

-

-

+

+ + + + + + + + + + + + + +

-

W/cm' Density gradient ( x 10') Cooling (g/cm4)(X lo4)

4 7.25 94 28 15-20 28 33 4 10 25 16 1 5 6 15 1.56 20 24-32 1O(intermit.) 400 68 60 764 60 1.56 20 17.5 20 6.1 15-20 100 10-20 18.5 250 9.5 24 7 112 24 25

Ampere ( X los)

Abbreviations: + = external cooling present; A = analytical electrophoresis; P = preparative electrophoresis; intermit. = current intermitted at regular intervals.

~-~~~~

Svensson et al. (1957) Tulp (1982a, this volume) Van 0 s et al. (1980) Van Oss and Bronson (1979) Van Regenmortel (1 964)

Boltz et al. (1973) Boltz et al. (1976) Boltz and Todd (1979) Borjesson (1981) Brakke et al. (1968) Catsimpoolas and Griffith (1977a) Catsimpoolas et al. (1975) Catsimpoolas and Griffith (1978) Cramer and Svensson (1961) Griffith et al. (1975) Hochstrasser et al. (1967) Lim et al. (1977) LKB column Manson ( 1971) Matheka and Geiss (1965) Platsoucas et al. (1979a) Polson and Russel (1967) Steere and Davis (1968)

Reference

Sample layer thickness (mm)

Values for Sample, Layer Thickness, Surface Area, Wattage, and Density Gradient in Several Types of Stationary Density Gradient Electrophoretic Columns

TABLE I1

DENSITY GRADIENT ELECTROPHORESIS OF MAMMALIAN CELLS 155 dution out

@

m

>>-------coolant

coolant out :.-

upper solutmin

0 uppersolutionout

dcctrode

-1 ): separatm column

codant out upper soivtlon

coolant in bottomsolution in

0

bwer sdutmout

density gradmt

bottomsohrtm

porousglass membrane bwer sdutcon

electmde lower Sokftm in

0

Figure 3. The Buchler-Polyprep200. For a description see Section IV.2.A. [Redrawn from Catsimpoolas and Griffith (19774 with kind permission of the authors and Plenum Press.]

mixer and a linear density gradient of 50 ml dense and 50 ml light Ficoll solution is thus added to the column at a rate of 3 ml/min. Next 125 ml upper electrode solution is layered over the density gradient with increasing speed till it covers the upper platinum electrode. At the interface of density gradient and upper electrode solution, via the Teflon tubing, 5-10 ml sample cell suspension is layered after which the tube is removed. Upper and lower electrode solutions are circulated at 1.2 ml/ min. Electrophoresis is carried out at 4°C at a constant current of 20 mA for about 4.5 hr. Instead of simply draining the column, the following proce-

156

ABRAHAM TULP

dure is advised for superior fractionation. While the current remains on, dense solution is pumped in via 2 at a rate of 1.0 ml/min while simultaneously through the elution capillary within the cooling finger 4 the gradient is pumped out at a rate of 2.9 mllmin. Thus, a sharp interface is formed between the continuously renewed dense solution and the remaining part of the gradient (see also Svendsen, 1972).About 10' cells of small volume (100 pm3) may be processed. B. T H E BOLTZ-TODD DEVICE

One may follow the ontogenesis of this self-made device through the following stages: From the U-tube arrangement (Svensson and Valmet, 1955; Polson and Cramer, 1958; Cramer and Svensson, 1961; Van Regenmortel, 1964; Polson and Russell, 1967; Boltz et al., 1973) via minor changes the apparatus depicted in Figure 4 emerged (Boltz and Todd, 1979). The U-tube arrangements permitted hydrostatic balancing of the electrode reservoirs. The side-arm electrodes in Figure 4 allow easy access to the electrophoretic column. Several variations on this configuration are described by Boltz and Todd (1979), some of which have a geometry that tends to distort cell bands during fractionation. The modular glass- and Lexan-made apparatus depicted in Figure 4A contains a movable oneway cooling tube inserted in the column. The sample input capillary is inside this cooling tube, and permits sample addition to the gradient through four openings half-way down the cooling tube, particularly suited for isoelectric focusing conditions, Figure 4B. The electrode vessels (diameter 5 cm) are connected via a 15%polyacrylamide gel bridge to the column to prevent invasion of electrophoresis products. The platinum wire electrodes are housed in a gas vent while an electrophoresis buffer can be circulated by the electrodes. Otherwise, the lower half of the electrodes are placed in 175 ml saturated NaCl: the electrode reaction is then zero order (less than 0.2%of the ions are converted to H:! or Clp). The upper half of the electrode vessel contains 125 ml 0.01M Pi buffer (pH 7.2). This permits use of the column for several weeks. Briefly, 30 ml of top solution is introduced through the gradient inlet into the column, followed by 25 ml of linear density gradient (light end first, 2%Ficoll plus 6.5%sucrose and 6.4% Ficoll plus 5.7%sucrose). Next, 0.3 ml of a cell suspension in 10% Ficoll plus 5.1% sucrose and 40 ml of dense bottom solution are introduced. A constant field strength of 4-5.4 V/cm at 15-20 mA is applied to the column while electrophoresis is upward. By pumping 15%Ficoll beneath the bottom solutions, fractions are collected from the top.

E 157

F

158

ABRAHAM TULP

C. STABLE-FLOW-FREE BOUNDARY ELECTROPHORESIS (STAFLO) ACCORDING TO MEL

This is basically a continuous method where design is derived from an early invention by Philpot (1940). It has been largely confined to the work of its designer, Mel, who has given the method a firm theoretical basis (Mel, 1964a,b,c) with respect to hydrodynamic feedback principles, migration velocity, laminar flow and density gradient stability. Due to the hydrodynamic properties of this particular separation chamber, an initially flat sample layer turns into arch-shaped (cross section) patterns (laminar defocusing), thus diminishing resolving power on exit from the chamber (Tippetts et al., 1967).Briefly, a Perspex migration chamber (see Figure 5) of 30 X 1.5 X 0.7 cm3 is fed by a multiple channel pump via 12 inlet-outlet combinations. Each inlet delivers a solution of increasing density (from top to bottom). The sample is introduced via the sixth inlet giving a band of 2.5-mm width. The electrode (platinum) compartments are hydrodynamically but not electrically isolated by cellophane membranes from the density gradient. Yeast and bacteria have been electrophoresed in this device (Mel, 1960).The rather limited application of the apparatus also covers the electrophoretic separation of chloroplasts (Packer et al., 1966)and a differential movement of the latter particles in the light and dark (Nobel and Mel, 1966).The electrophoretic mobility of rat erythrocytes was measured (Me1 et al., 1973) in 0.145M NaCl (K = 4 x lo-* mhoicm) at 6.9 Vkm. Clearly, the wattage that can be absorbed by this apparatus is considerable and, in particular, if lower conductivity buffers were used, quite gratifying separations might have been obtained. In view of its merits, it certainly deserved more attention. The chamber can also be used analytically (Catsimpoolas, 1980). &-electrode

I

-a a

c

E

c e

I o p t e e lc t r o d e

Figure 5. STAFLO apparat.us. For description see Section IV.2.C.

DENSITY GRADIENT ELECTROPHORESIS OF MAMMALIAN CELLS

159

D. T H E LKB COLUMN

For isoelectric focusing of mammalian cells, the LKB model 8 101 (110 ml) and model 8102 (440 ml) (Bromma, Sweden) have been widely used. Because the technique is so familiar, the reader is referred to the Instruction Manual, to Sherbet’s books (1978), or to Volume 19 in this series (Haglund, 1971). To circumvent the extraordinary long focusing times needed, Sherbet (1978) devised a side-arm attachment for the LKB equipment, which connects the focusing chamber to the exterior in the middle of the column (Figure 6). This side arm is sealed with a rubber septum. After the pH gradient is established, 2 ml of gradient are with-

e

Figure 6. LKB column. The drawing is taken from the original LKB manual and Sherbet (1978).A = side-arm attachment according to Sherbet. For description see Section IV.2.D.

160

ABRAHAM TULP

drawn from the column via the septum in 4 min, the cells are suspended in this volume, and they are returned to the column (post pH equilibrium loading method) again in 4 min. T h e cells are then focused for 2-3 hr. Sherbet (1978) has mechanized the whole procedure by connecting the focusing column to a p H flow cell and an optical density flow cell for analysis. E. SMALL-SIZE FOCUSING CHAMBER

Sherbet and Lakshmi developed a small-size electrofocusing chamber (Moore and Hibbitt, 1975; Sherbet, 1978) made of Perspex (Figure 7). It consists of two vertical chambers and openings at the top that communicate at the bottom where a needle valve can close the entrance. T h e chamber of 1.4-cm i.d. contains the density gradient (2- 1.5% Ficoll plus 1 % ampholine), while the small-bore chamber (0.5-cm i.d.) contains an

-- I

40mm

@.

c

B

Figure 7. Small-size focusing chamber. For description see Section IV.2.E. [Redrawn from Moore and Hibbit (1975) with kind permission of Dr. H. D. M. Moore and the editor of J. Reprod. Fert.]

DENSITY GRADIENT ELECTROPHORESIS OF MAMMALIAN CELLS

161

electrode. The specimen loading port is sealed by a septum that can be pierced by needle to allow introduction of cellular particles into the column. The column can be drained through a tap via A. The anode solution contained 1M H3P04 and the cathode solution 1M NaOH. The pH gradient was produced by passing a constant current of 1 mA for 16 hr. Then cells are injected through the septum and electrofocused for 2 hr at 2 mA. F. THE ISCO COLUMN

T o our knowledge the ISCO is no longer available (at least in the Netherlands) (Lincoln, Nebr.) Model 630 (150-ml capacity, cross section 4.9 cm2) or Model 212 (30-ml capacity, cross section 0.79 cm2). It was basically developed by Brakke et al. (1968). The electrode chambers are arranged coaxially around the column and separated by two concentric membranes. Near the middle of the electrophoresis column, an optical measuring cell registrates absorbance as the density gradient is lifted upwards. With respect to mammalian cells, the device has been used for the separation of platelets (Carty et al., 1975) and erythrocytes (Gear, 1977). An extensive description of the apparatus limited to isoelectric focusing of proteins was given by Leaback and Wrigley (1976). G. QUICKFIT COLUMN

A very simple electrophoresis column was built from standard Quickfit glassware (Jobling Laboratory Div., U.K.). A Liebig condenser with B24 joints (cat. no. C 1/13) was cooled with water. The electrode chambers were attached to the two ends of the column by flat flange joints (cat. no. FG 15) attached to a cone (cat. no. CNB 24) or a socket (cat. no. SRB 24). Dialysis membranes were placed between the flanges of the two flat flange joints. The Pt electrode chambers were flushed continuously. Electrophoresis time, however, was extremely long: 6- 12 hr (Bflrjesson, 1976). H. VAN OSS AND BRONSON DEVICE

Van Oss and Bronson (1979) devised a rectangular column (2.5 X 0.3 X 15 cm3), the bottom 4-6 cm filled with 2% agarose gel (set in electrophoresis buffer). The column is water jacketed. Wicks consisting of 2% agarose are at the bottom and a filter paper bridge is at the top connecting the anode chamber. The separation chamber is filled first with 0.5 ml D 2 0 (p = 1.124) in low ionic strength buffer (0.013); next cells in 0.2-0.5 ml of equal parts D20/H20 are layered onto the D 2 0 cushion followed by pipetting 1 ml layers of decreasing D 2 0concentration. This procedure yields through rapid diffusion a gradient ranging from 1.072 to 1.02. The

162

ABRAHAM TULP

wicks are connected immediately to an electrical field of 9 to 12 V/cm for 1 hr. After ascending electrophoresis, samples of 0.5 ml are taken from the top by pi etting. The cost of such a D 2 0 gradient is less than $1 .OO. About 1.6 X 10 erythrocytes can be applied.

B

I . SEPARATION CHAMBER ACCORDING TO TULP

With respect to this device, the treatment also involves applications of cell separation (Sections a and b). Applications of the devices A to H are treated separately (Sections V and VII.3). a. Device with Movable Electrodes. Figure 8A shows a photograph of this chamber, Figure 8B shows a drawing with symbols used in the text. The instrument consists of three Perspex circular plates of 24-cm diameter designated T, M, and B. Top (T)and bottom (B) plates, each of 2-cm height are clamped together by six connectors (A) containing strings for the necessary tension. Due to the connectors, bottom and top plates move simultaneously because they are tightly connected to the shaft of the apparatus by screws and fixing pens. A transmission mechanism consisting of a worm-gear is housed in a case (H); it is driven by hand. In the middle plate (M) of 1.5-cm thickness, a hollow separation cylinder of 8-cm diameter and 1.5-cm height is present. The center of this hollow cylinder

Figure 8. T h e electrophoresis chamber. (A) The Perspex apparatus with platinumirridiurn electrodes is described in the text. (B) Abbreviations as used throughout the text. Turning the wheel, W, effects the simultaneous movement of the top and bottom plates whereas the middle plate stays stationary. C , , C p , cocks; f, inflow, outflow of flushing buffer; E, electrode; 0, outlet of separation chamber; i, inlet of separation chamher; T, M, and B, top, middle, and bottom plates, respectively; s, spring.

DENSITY GRADIENT ELECTROPHORESIS OF MAMMALIAN CELLS

163

is at 6.2 cm from the shaft axis. Bottom and top plates are similar in construction housing a cone with flow deflector as well as an electrode compartment (Figure 9) with inflow and outflow tubes (f). In the bottom and top plates, two Teflon cocks (cl and cp) secured with O-rings are present, one for introduction of liquid into the space between the middle plate and a cellophane membrane (m), and one for escape of air. T h e distance between the membrane and middle plate is 0.2 cm. The membrane is held in position by an O-ring (diameter, 8 cm) and a Perspex frame. The membrane (diameter, 9.5 cm) is cut with a scalpel from a wet cellophane sheet. T h e wet membrane is squeezed upon the Perspex frame which is then pressed into the electrode compartment; an O-ring prevents leakage. Thus, the electrode compartments are isolated hydrodynamically but not electrically from the separation chamber by the cellophane membrane. When properly cleaned and stored in position in distilled water, the membrane can be used for at least 3 months. The electrodes (E, Figure 9), diameter 8 cm (type 406029, Drijfhout and Zoons, Amsterdam, the Netherlands) were flat-gauge platinum- iridium (90/10%), 40-mesh, wire diameter 0.016 cm. All parts of the device were demountable. Figure 10 shows, in sequence, the filling procedure. For clarity, the circular plates are represented by blocks in cross section; parts that contain liquid are depicted in shade. First via the cocks, 10 ml 12% Ficoll is introduced into the space f

0

E

H

Figure 9. Schematic diagram of the electrophoresis chamber drawn to scale. Dimensions can be deduced from the top plate, which is 2 cm high. For abbreviations see Figure 8. m, Cellophane membrane; A, connector with internal spring; H, housing for worm gear. A detailed description is given in Section IV.2.1.a. The bottom electrode compartment is identical to the top one. The distance between the cellophane membrane and platinum electrode is 1 cm. Rapid flushing of the electrode compartments proceeds via f.

164

ABRAHAM TULP

+ e--

O / \

n 1

b O /\

____.. *.. I

...............

A

1

I

n

I

................ **f*C*----

Figure 10 Filling of the electrophoresis chamber. For a detailed description see text. Parts that contain liquid are depicted by shading. (a) At right, electrode compartments are filled with electrophoresis buffer. The space between membrane and middle plate is filled as described. At left, the separation chamber with its top and bottom cone is filled with density gradient, cell layer (dotted line), and overlay. (h) The top and bottom plates move to the left but the middle plate is stationary. (c) The electrode compartments are aligned with the hollow separation chamber in the middle plate, flushing of the electrodes is initiated, and electrophoresis started. In (b) and (c), in- and outflow tubes, cocks, and connectors plus springs are omitted for reasons of simplicity. After electrophore3is, top and bottom plates move to the right until the configuration is as in (a) and contents of the chamber are then fractionated via the top cone by pumping in cushion liquid via the bottom cone.

between the middle plate and bottom membrane taking care to remove all air. This procedure was repeated for the top electrode compartment; in that case, the space between the membrane and middle plate was filled with 10 ml electrophoresis buffer. Next, the bottom electrode compartment is filled with electrophoresis buffer from a 2.5-liter reservoir at a constant water pressure of about 10 cm via a polyethylene tube of 50-cm length and 0.4-cm bore. T h e tube efferent from the bottom electrode compartment was 160 cm long and was connected to the top electrode compartment so that the electrophoresis buffer also flushed the top electrode. A 20-cm tube efferent from the top electrode compartment introduced the flushing buffer into a vessel. T h e content of the vessel was reintroduced manually into the reservoir. Prior to electrophoresis, efferent and afferent polyethylene tubes were clamped with forceps. T h e loading chamber is similar to the low-gravity chamber we described in detail previously (Tulp et al., 1980a). In Figure 10a the loading

DENSITY GRADIENT ELECTROPHORESIS OF MAMMALIAN CELLS

165

chamber is'first filled with 12% Ficoll70 in electrophoresis buffer via (i). Next, a linear density gradient of 75 ml ranging from 5 to 11% Ficoll and generated by a gradient mixing device is introduced in 5 rnin via (0)by letting cushion (12%Ficoll) liquid flow out via (i) by gravity. Next, 5 ml of a a cell suspension in 0.5% Ficoll is introduced for 30 sec via (0)followed by 25 ml electrophoresis buffer as an overlay also for 30 sec. The meniscus is lowered for 30 sec until the thin band of cells (0.1 cm), depicted as stippled, is just within the hollow cylindrical part of the chamber. Next, the bottom and top plates are moved in unison for 2 min, manually by the worm-gear to the left (arrows, Figure lob), while the middle plate remains stationary, until (Figure 1Oc) the electrode compartments are above and below the hollow cylindrical part of the separation device. Next, the forceps are removed from the polyethylene tubes and the erectrode compartments are flushed at 100 ml/min. Effluent electrophoresis buffer from the top and bottom plates is collected in a vessel to reduce pH changes in the flushing buffer. Next, electrophoresis is initiated. After electrophoresis, the bottom and top plates are moved to the right for 2 rnin until the top and bottom cones with the flow deflectors are again aligned with the hollow cylindrical separation chamber; 12% Ficoll is introduced via (i)and the contents of the chamber are fractionated via the top cone within 3 rnin.; 25 ml of overlay is collected followed by fractions of 4 ml each.

Example of separation. As prototypic mammalian cells, human erythrocytes treated with neuraminidase and untreated erythrocytes were chosen for their large mobility difference. To lower the migration velocity due to the gravity force, the density gradient ran from 5 to 11% Ficoll. Within 5 min after initiation of electrophoresis, two bands of erythrocytes became visible when the separation chamber was viewed from the side. The distance between these bands increased with time and after 25 rnin the space between the erythrocytes bands was completely transparent and clear. The calculated electric field strength was approximately 2.7 V/cm. Figure 11 shows the separation of treated and untreated erythrocytes after only 25 rnin of electrophoresis at 5°C. Separation was effected within a vertical distance of 0.7 cm. To the eye, the bands were relatively widely spaced, so we judged it appropriate to interpolate the electrophoretic profile (shaded part) to its full depth (fractions of 4 ml each were taken, corresponding with a vertical height of 0.08 cm). Untreated erythrocytes moved 3.77 times more rapidly than the treated erythrocytes. A similar resolving power was obtained at room temperature (not shown). There was no impairment of resolution whether lo' or 10' erythrocytes were applied to the device. Figure 11 also shows that in the absence of

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32[ 30

0

15

10 \D I 0 c

x

-a : 5

0

L

n

s

2

0 Oapth in G r 8 d i e n t ( c m )

Figure 1 1. Separation of treated and untreated human erythrocytes by low-electric-field electrophoresis. Human erythrocytes (5 x 10’) treated with neuraminidase are mixed with 5 x 10’ untreated erythrocytes to a final volume of 5 ml and layered onto a linear gradient of 1.5-cm height. Electrophoresis proceeds for 25 min at 5°C at a constant current of 90 mA. An identical suspension is subjected to velocity sedimentation at unit gravity only. (0)Separation at unit gravity; (0)separation at unit gravity and by electrophoresis. Areas under the migration profile are depicted in shading. Gravity and electric forces act in the same direction (to the right). (Figures 8- 1 1 reproduced with kind permission of the editor of Anal. Biocheni.)

electrophoresis, velocity sedimentation at unit gravity yielded only one band of erythrocytes that sedimented 0.12 cm into the Ficoll gradient (with permission of the editors of Anal. Biochem.).

b. Compact Device. The constructional aspects of the above described separation chamber (Ia) make it rather difficult for wider use. We therefore developed a much simpler compact device under resignation of the excellent hydrodynamic properties of the flow deflectors that guaran-

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teed rapid undisturbed filling and fractionation of the chamber. T h e new device makes a full use of the miniaturization principle (Section 111.3).

Diagram. The diagram (Figure 12A) and a photograph of the complete design (Figure 12B) show that it consists of three compartments clamped together, O-rings preventing leakage. Ionic exchange membranes (diameter 9.5 cm) isolate the separation chamber of small height (1.5 cm) and large cross section (50 cm2) hydrodynamically from the lower and top electrode compartments. T h e lower electrode compartment (I) has an inlet ‘‘2’ and outlet ‘‘0”for rapid electrophoresis buffer flow (140 mI/min) to remove electrolytically developed oxygen formed at the meshes of the Pt electrode (diameter 8 cm, see also Section La). From the bottom outlet, electrophoresis buffer is introduced via a 160-cm long tube of 0.4-cm bore into the top electrode compartment (111)via i; this top compartment contains an excentric cone for the removal of electrolytically generated hydrogen from the top electrode and oxygen (from the bottom electrode compartment). Electrode buffer is fed by gravity from a constant level reservoir at a water pressure of 35 cm, and 2.5 liters of buffer is thus recirculated continuously except during loading of the center part when all tubes are clamped with forceps. Membranes. T h e membranes that isolate the separation compartment from the electrode compartments are ionic exchange membranes fortified with modacrylic fibers. T h e anion-transfer membrane (Type 103QZL-386, Ionics Inc., Watertown, Mass.) is close to the platinum cathode while the cation-transfer membrane (Type 61 AZL 389) is close to the anode. T h e membranes are similar to those used in FFE. Arrangements with two membranes can give rise to the Bethe-Toropoff effect: acid is formed on one side of the membrane and alkali on the other side on passing an electrical current (Svensson, 1948). With two negative membranes like the cellophane membranes described in Ia, the center separation section turns acidic if adequate buffer capacity is not at hand. It is probably due to the Bethe-Toropoff effect that electrophoresis buffer (3B) according to Boltz et al. (1973) did not suffice: 10 min after onset of electrophoresis, a thin whitish layer presumably consisting of insoluble Mg salt developed at the cathode side. The glycine triethanolamine buffer (3B) of Hannig and Zeiller (1969) worked very well. Whereas cellophane membranes are permeable to small uncharged molecules (glycine, sucrose), the ion-exchange membranes used are almost impermeable to such molecules depending on the pore size of the membrane. At 250 mA, in 25 min (typical working conditions) the membranes transport only 0.002 g sucrose and 0.46 ml H 2 0 (Bulletin No. AR 103-3D Ionics Inc.). Attention must be paid to the fact that the membranes are entirely flat, since curved membranes give rise to curved migration bands in the short,

Figure 12. Separation chamber according to Tulp. (A) Diagram of the electrophoresis chamber drawn to scale. For a description see Section IV.2.1.b. Arrows indicate electrophoresis buffer flow. The threecomposingcompartments I, 11, and Ill are shown disassembled. Symbols: a, inlet tube and b, outlet tube separation chamber; m l , cation-transfer and m2, anion-transfer membranes; i, inlet; 0, outlet. The Perspex body is represented by the shaded area. Dark lines represent ion-exchange membranes, dark circles in compartment I1 the O-rings. (B) Photograph of the electrophoresis chamber. The chamber can be rotated along axis P by hand.

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but rather wide, separation column, thus diminishing resolving power. Therefore, the water pressure is equilibrated in electrode compartments and separation chamber by making the level of dense Ficoll'solution (13%)in the gradient mixer equal to the level of electrophoresis buffer in the constant level reservoir (Figure 13). This procedure automatically stretches the membranes. After closing the afferent and efferent tubes of the electrode Compartments, density gradient is introduced into the middle chamber as described below. Preparing the Gradient. First, the chamber is positioned at an inclination of 60" with the vertical (Figure 13). This is made possible because an

P cathode anionic exchange membrane

cationic exchange membrane

Figure 13. Filling procedure of the TULP chamber. For a description see text. T h e circle in 2 denotes the axis around which the separation chamber can be rotated. For simplicitv, in 1,2, and 3 the electrode compartments are omitted. Shaded area represents cell suspension.

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axis is fastened to the outer wall of compartment I1 (Figure 12A). The entire electrophoresis chamber can be made to rotate along this axis at the pivots P in the standard (Figure 12B).The entire electrophoresis chamber can be made to rotate along this axis at the pivots P in the standard (Figure 12B). Dense Ficoll 70 (13%)solution in electrophoresis buffer is introduced via tube (b) of 0.5 cm bore, fixed at an angle of 75" with the cylindrical wall, into the separation chamber until the chamber is completely filled, as well as one limb of the gradient mixer, up to the level permitting flattening of the membranes (see above). Next a linear density gradient of 75 ml ranging from 13 to 3% Ficoll is introduced into the separation compartment within 30 rnin by letting dense Ficoll solution drip from (b),(Figure 138). Next 5 mlcellsuspension (represented by the dotted area in Figure 13 0 )in 1.5% Ficoll is introduced at the top of the inclined chamber via a in 2 min followed by 5 ml electrophoresis buffer as again by letting Ficoll drip from (b). an overlay also in 2 rnin (Figure 13 After stratifying the overlay over the sample layer, both (a) and (b) are clamped with forceps. For this new design, we had to refer to some original ideas developed by Rilbe: turning the chamber slowly back in 4 min to a perfectly horizontal position (Figure 13 @), the sample layer, sandwiched between density gradient and overlay, is squeezed into a layer 1-mm final thickness due to the increase in horizontal cross section (Rilbe and Petterson, 1968; see also McDonald and Miller, 1970; Wells, 1982).The procedure of turning the separation chamber can be automated by a synchronous motor (Rilbe, 1973) but it is done manually in the present set-up. Next the forceps are removed from the tubes that provide for rapid flushing of electrophoresis buffer. After electrophoresis at 250 mA (corresponding with an electrical field strength of about 5.6 Vkm), the circulation of electrophoresis buffer is stopped with forceps and the chamber was carefully and slowly turned upright again after 10 min so as not to disturb the contents of the separation chamber, after which dense Ficoll(l3%)is introduced via tube (b) so that the contents are fractionated via tube (a) at the top in about 10 min. Fractions of 1-2 ml were collected.

a),

Examples of Separation. Figure 14 shows the separation of an artificial mixture of human and rabbit erythrocytes in only 10 min at 4°C and 5.6 Vkm. After 1.5 rnin of electrophoresis, two closely adjacent red cell bands could be observed. There was almost no overlap of the two red cell types and separation was effected within a vertical distance of a few mm. It is evident that longer electrophoresis gives complete separation. Resolving power of the present cassette-like device is apparently very good. Mouse (CBA strain) spleen cells, first freed from erythrocytes on a

DENSITY GRADIENT ELECTROPHORESIS OF MAMMALIAN CELLS

17I

3

.-

9 '0

x *

-In W

0 r

0

L 0)

ni E

3

z

0

Migration D i s t a n c e ( c m 1 Figure 14. Electrophoretic separation of rabbit and human erythrocytes. A mixture of 4 x lo7 rabbit and 5 x lo7 human erythrocytes suspended in 5 ml 1.5% Ficoll 70 are layered to a linear 3-13% Ficoll gradient of 75 ml followed by an overlay of 5 ml, and electrophoresed at 4°C and 250 mA for 10 min. Fractions of 1 ml each were taken. T h e contribution of the two cell types was estimated by electronic sizing. The black bar represents initial cell band-width. (0-0) Rabbit erythrocytes; (0-0) human erythrocytes.

metrizamide cushion, and after electrophoresis for 6 min at 5.6 Vkm, are resolved into two clear-cut visible bands when the separation chamber is viewed from the side. Figure 15 shows the migration profile of 4 x lo' nucleated mouse spleen cells after 15 min only at 250 mA, demonstrating convincingly the presence of the two migrating bands (see also Section V.1.C); the small shoulder in the more rapid migrating fractions (at a migration distance > 1 cm) is composed of erythrocytes predominantly. Separation of nucleated cells took place within 0.5 cm, underlining the excellent hydrodynamic properties of the device. For comparison, the separation of CBA spleen cells with the use of free-flow electrophoresis is given in the inset of Figure 15 (Zeiller and Pascher, 1973). These authors demonstrated that the highest peak in

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0

0.5

1.0

M i g r a t i o n Distance(cm 1 Figure 15. Electrophoresis of murine spleen cells. Murine (CBA strain) spleen cells (4 x 10’) suspended in 5 ml 1.5% Ficoll are layered on a 3-13% Ficoll density gradient followed by an overlay of 5 ml. Electrophoresis proceeds for 15 min at 250 mA and 4°C. Fractions of 1-2 rnl are taken. For comparison, in the inset the separation of CBA mouse spleen cells by free-flow electrophoresis is given. [Redrawn from Zeiller and Pascher (1973) with kind permission of Dr. K. Hannig and Verlag Chemie.]

the migration profile is composed of B cells whereas the more rapidly migrating small peak is composed of T cells. In view of the almost identical migration profiles obtained by FFE and by the present device, w e may conclude that the resolving power is about equal. Because only 15 min is necessary for separation, the method is rapid. Since the density gradient extends from p = 1.02- 1.05, cells are relatively close to their buoying density so that rather large numbers of cells can be layered without a trace of streaming. In Figure 16, 10’ spleen cells from a GR strain mouse are layered onto the density gradient and electrophoresed €or 25 min at 250 mA. Again, two migrating bands can be seen when the chamber is viewed from the side but there is considerable substructure in the migration profile. In-

DENSITY GRADIENT ELECTROPHORESIS OF MAMMALIAN CELLS 173

0

1.o

0.5

Migration Distance ( c m )

Figure 16. Electrophoretic separation of murine (GR strain) lymphoid cells. Unless otherwise stated, cells suspended in 5 ml 1.5% Ficoll were electrophoresed for 25 min at 250 mA at 4°C. Hatched area represents initial cell band width. (*---*) 3.2 x lo' thymocytes; (0-0) 1 x 10' spleen cells are separated by unit gravity sedimentation alone for 25 min; (0-0) 1 x 10' spleen cells are separated by unit gravity sedimentation plus electrophoresis.

deed, different mouse strains show totally different migration profiles Whereas electrophoresis (see Platscoucas and others in Section V. 1C). separates the migrating spleen cells into high-, intermediate-, and lowmobility populations, it is worth mentioning that the cell band under influence of gravity alone remains very thin (Figure 16). At the initial interface of sample layer and density gradient, a jump in viscosity and density occurs using 1.5 to 3% Ficoll70 with the effect that the migrating cell band (by gravity force alone) is compressed by a factor,

rl PP - Po -rlo

PP

- PM

where qo = viscosity of sample layer; q = viscosity of top of the density gradient; po = density of sample medium; and pM = density of top of gradient.

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Whereas the theoretical half-width of the sample layer as loaded is 1 mm, after 25 min sedimentation at unit gravity in the absence of an electrical field it is only 0.36 mm due to the compressing effect of medium viscosity and density (Figure 16). It is this band-width that determines resolving power (Section 111.3). For adequate electrophoretic separations, it is necessary that the unit gravity sedimentation profile be different from that obtained after combined sedimentation and electrophoresis (see our criticism in Section V.1.F). Figure 16 shows the electrophoretic migration of CBA mouse thymocytes. The profile is more or less similar to that obtained by FFE. These T cells have a lower mobility than the B cells from spleen. Low-mobility thymocytes possess the highest 8 antigen content that disappears at the same rate as the negative surface charge increases (Zeiller et al., 1974). These authors suggested the following sequence: thymocytes of low mobility + thymocytes of high mobility + peripheral T cells of high mobility.

3. Analytical Electrophoresis of Cells in Density Gradient Two of the previously described preparative columns can also be used analytically (i.e., Section IV.l.C, Section IV. l.F). A. TRANSANALYZER

An instrument for electrophoresis with continuous optical scanning was developed by Catsimpoolas et al. (1975) and Catsimpoolas and Griffith (1977b). It consists of a cooled electrophoresis cell cassette to accomodate two qLartz columns (0.6-cm i.d., 14.2-cm length). One column carries the sample for analysisand the density gradient. The other is used as reference background. The bottom of the quartz tube is closed by a semipermeable membrane or polyacrylamide gel plug. The quartz tube is coated by methyl cellulose (Dow Chemical Company) to prevent electroosmosis. Buffers are circulated through the upper and lower electrode reservoirs to remove products of electrolysis. The quartz column is moved vertically up and down by a synchronous stepping motor (Catsimpoolas, 1973). Absorbance of light is detected by a photomultiplier as subsequent layers of the column pass the photocell. During electrophoresis, the column is scanned sequentially several times so that a transient electrophoretic analysis of the migrating cells is possible. The device was modified later (Catsimpoolasand Griffith, 1978): the “Transanalyzer” (Bascom-Turner Instruments, Newton, Mass.), contained six removable columns. Five cell samples can be analyzed simultaneously because the columns move side-

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ways while a vertical moving light beam scans the columns. Blueprints of these rather complicated devices may be obtained from Catsimpoolas. B. COLUMN W I T H LASER BEAM

A glass tube, 0.5-cm i.d., 11-cm length, treated with Siliclad, was scanned by a laser beam in the device of Lim et al. (1977)by vertical movement of the density gradient column. The electrical field was repeatedly applied for 60 sec (8 Vkm, 10 mA) and then turned off for 80 sec to allow heat to dissipate. C. ELECTROPHORESIS ALONG A DISCONTINUOUS DENSITY INTERFACE

A discontinuous Ficoll gradient was used by Pradac et al. (1978) to transport a front of erythrocytes horizontally along the Ficollbuffer interface in a cuvette of 2 x 0.2 x 4.5 cm3 at 5.5 V/cm. 4. Experimental Conditions A. DENSITY SOLUTE

The solute contributing to the density increment must be: highly soluble in electrophoresis buffer, of a high density increment per gram, electrically neutral, nontoxic to cells, of a low intrinsic viscosity, not, or only slightly, contributing to osmolarity, and easy to remove after electrophoretic separation. Sucrose has been used in a few cases as a solute for zonal electrophoresis of cells (Carty et al., 1975;Gear, 1977)or for isoelectricfocusingof cells (vide infra),but, it is not desirable due to its osmotic effect. A mixed type isoosmotic gradient of sucrose and Metrizamide (Nyegaard, Oslo, Norway) seems more promising. Ficoll M 400,000 and M 70,000 have been the solutes of choice. Catsimpoolas advised dialysis of Ficoll400,OOO prior to its use since it “dramatical1y”reduced cell clumping. No such clumping effects were found with Ficoll70,OOO. Ficoll gradients are transparent and visual inspection of migrating zones often yield as much information as electronic scanning of the column. Physical properties of Ficoll400,OOO are given by Boltz et al. (1973) and those of Ficoll 70,000 by Tulp et al. (1982a). According to Pretlow et al. (1975), prolonged autoclaving of Ficoll solutions (>16 min) promotes depolymerization and gives rise to osmotic pressure effects. According to Loos and Roos (1974),a 10% Ficoll solution has an osmolarity of about 7.5 mOsm. Apparently due to the autoclaving procedure, it was necessary for Boltz et al. (1973)and Griffith et al. (1975) to vary both sucrose and Ficoll concentrations to obtain an isoosmotic

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density gradient (303 mOsm, from 10% Ficoll plus 5.1% sucrose to 2.5% Ficoll plus 6.35% sucrose in electrophoresis buffer). B. IONIC COMPOSITION

In view of the higher mobilities of cells, buffers of low ionic strength should be used. Buffer capacity should be as high as dilution permits with respect to the stability of cells. Preferentially, ions with low electrophoretic mobilities should be used. Ionic composition as well as the nature of the solute determining osmolarity can be found in a Table of Hannig (1971)depending upon cell type, or in Hannig and Heidrich (1977). Boltz et al. (1973) recommend a buffer of the following composition: 0.2 g KCI, 1.15 g Na2HP04,0.20 g KH2PO4, 0.10 g MgCI2 6H20, 10 g glucose per liter (pH 7.2), specific conductivity 1 mmhokm. This buffer was modified by Griffith et al. (1975) by omitting MgC12 and adding 0.12 g sodium acetate. A buffer, slightly adapted from Hannig and Zeiller ( 1969),containing 18g glycine, 2.24 g triethanolamine, 0.3 g K-acetate, and 7.2 g glucose per liter, pH 7.2 specific conductivity 0.9 mmhokm, is very satisfactory for the separation of lymphoid cells, Tulp (this volume). C. ELECTRODES

The electrodes conventionally used for electrophoresis are noble metal and metal/insoluble metal salt (e.g., Ag/AgCI/Cl-). This electrode requires the installment of barriers to prevent metal ions from entering the separation compartment. Also platinum electrodes, generally used in cell separation studies, must be isolated hydrodynamically from the separation density column to prevent mixing and serious distortion due to electrolytically developed H2 and O2bubbles. In some separation devices (but not yet applied) the use of a less expensive palladium electrode as a cathode might be satisfactory. Palladium dissolves considerable amounts of hydrogen so that no gas is evolved at the cathode and a H2-saturated palladium anode might stop the evolution of oxygen (Neihof and Schuldiner, 1960). D. POWER SUPPLY

Several commercial power supplies for constant current exist. We found a low-cost, regulated power supply (Delta Elektronika BV, Zierikzee, the Netherlands) Type E 060-0.6 for 0-60 V or 0-600 mA entirely satisfactory.

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E. ELECTROENDOOSMOSIS

Due to the surface charge of the separation vessel, waterflow is induced by the electrical force. In the relatively large (cross section) separation columns used for preparative (but not analytical) electrophoresis, electroendoosmosis is very small. Although Rilbe (1977)originally thought that electroosmosis is damped in density gradients, it appeared that at high field strength everywhere at the wall small circulatory liquid movements over very small density intervals occur. These microwhirlpools tend to deteriorate zonal layers and they, rather than heat convection, are the limiting factors for the voltage that can be applied. In cell separation studies, the electrical field strength is only a few V/cm so this kind of disturbance is not operative. Moreover for several types of glass surfaces, coatings have been devised to reduce surface charge and thus electroendoosmosis(Van der Hoff et al., 1977,1978;Van der Hoff and Van Oss, 1979; Van der Hoff and Micale, 1979. Even Perspex (polymethyl methacrylate) may be treated by hydrolysis of the surface in sodium hydroxide followed by neutralization and reaction with Methocel (Van der Hoff et al., 1977).Rilbe (1977)remarks that “even if it were possible to prepare a completely uncharged solid phase, there would be a permanent risk that this complete neutrality could get lost by absorption of solutes with ionogenic groups.”

V. APPLICATIONS

At neutral pH, mammalian cells possess a negative surface charge mainly originating from the carboxylate group of N-acetylneuramic acid residues coupled to glycoproteins. Therefore, cells migrate to the anode.

1. Lymphoid, Blood, and Hemopoietic Cells A. PLATELETS

Platelets of man and rat have been resolved in age groups by Carty et al. (1975). B. ERYTHROCYTES

As prototypic mammalian cells, erythrocytes have been used almost solely to test the resolving power of density gradient electrophoresis columns. To perform a large number of tests, erythrocytes may be fixed by dilute aldehyde solutions (Vassar et al., 1972)to obtain particles that are chemi-

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cally stable and have stable surface charge for several months of storage in physiological saline. In particular, the separation of an artificial mixture of rat, chicken, and rabbit erythrocytes (Boltz et al., 1976; Todd et al., 1979)is a good test of resolving power. Table 111shows the separation of artificial mixtures of various erythrocyte species either on a analytical or preparative scale. The only separation into functional entities has been described by Gear (1977), who obtained a separation of old v. young erythrocytes. Questionable in the experiments is the high ambient osmotic pressure (>889 mOsm) in the density gradient column. C. SPLEEN CELLS

Separation of spleen cells has been studied extensively by Catsimpoolas and co-workers (Catsimpoolas and Griffith, 1975, 1977a,b,c 1978; Catsimpoolas et al., 1980; Griffith et al., 1975, 1976; Platsoucas and Catsimpoolas 1978, 1979, 1980; Platsoucas et al., 1976, 1980). Electrophoretic separation of mouse spleen cells into two visible bands was first reported by Griffith et al. (1975). Athymic nude mouse spleen cells showed only one migration band during electrophoresis (Griffith et al., 1976). Resolution of mouse spleen cells, using analytical quartz and small columns, in at least three distributions after 6 hr migration, was reported by Catsimpoolas and Griffith (1975, 1977a,b, 1978). Evidently resolution TABLE 111 Electrophoretic Separation of Erythrocytes Mixture of erythrocytes

Electrophoresis Analytical Preparative

+ + + Ra,C,R H;R H,Ra(f)lM,Ra H,ghosts R H,C H,H(n) HNf)

+ +

+ + + + + +

Explicitly resolved in

References

R;C R;C;Ra Ra;C;E H;Ra

Boltz and Todd (1979) Boltz et al. (1976) Bronson and Van Oss (1979) Catsimpoolas and Griffith (1975, 1977b) Ra;C;R Gaines et al. (1974) o1d;young eryth. Gear (1977) H ;R/M ;Ra Griffth et al. (1975) Lim et al. (1977) Me1 et al. (1973) Michalik et al. (1980) Tulp et al. (1982a, 1983) Van Oss and Bronson (1979)

Abbreviations: C = chicken; E = equine; H = human; M = mouse; Ra = rabbit; R = rat; (f) = fixed; (n) = neuraminidase treated.

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could have been increased if the sample layers applied to the analytical column were not 2.5 mm high but 1 mm instead. Figure 17 shows that almost complete separation of murine (CBA/ H/TGJ) B and T cells was obtained (Catsimpoolasand Griffith, 1977c)as is evident from the separation of 8-antigen-positive(T)and Ig-positive cells (B), respectively. The high-mobility &positive cells responded in vitro to stimulation by phytohemagglutinin, whereas the low-mobility Ig-positive cells were activated by lipopolysaccharidefrom E. coli. In the intermediate fractions of Figure 17, double negative (for 8 and Ig) was present in a relatively high percentage as can be calculated from Figure 17. These

-s m 0

f

B

60

) .

=

8

40

20

30

40

50

60

70 80 9( fraction number

Figure i7. Electrophoretic separation of murine spleen B and T cells. Spleen cells from a CBA mouse were electrophoresed in Buchler Polyprep apparatus for 4.5 hr at 20 mA and 4°C. [Redrawn from Catsimpoolas and Griffith (1977~)with kind permission of the authors and Plenum Press.]

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results are entirely comparable with those obtained by FFE (Zeiller and Pascher, 1973; Zeiller et al., 1974; Boehmer et al., 1974). Moreover, Platsoucas et al. (1980) found that BALB/c lymphocytes, responding by proliferation to allogenic (CBA/H/TGj)spleen cells in a mixed lymphocyte culture, were exclusively found in the high-mobility fractions. Whereas two peaks with moderate skewness and little substructure are shown, Figure 17, a variable and large number of peaks were detected in the electrophoretic migration profile of spleen cells derived from C57B1/ 6J mice (Platsoucas et al., 1976). These authors ascribe the variability of peak height and peak number to a removal of certain subpopulations in a nonreproducible manner during cell preparation prior to electrophoresis. Their alternate explanation that the variable subpopulations represent dynamic changes of lymphocytes in individual animals due to exposure to different environmental antigens remains open. Not only a putative variability in individual animals was reported but also a straindependency of the electrophoretic profiles was described by Platsoucas and Catsimpoolas (1978, 1979). They suggest that the surface charge of the cells is genetically determined, although its dependency on the H-2 locus is in doubt. For BALB/c mouse spleen cells as well as for other strains (Platsoucas and Catsimpoolas, 1980),an age-related difference in electrophoretic migration profiles was detected. Regardless of age, a separation of T and B cells was always obtained. Thymectomy had profound effects on the age dependency of the electrophoretic profile. Rat spleen cells could also be separated into two populations (B and T cells, Platsoucas and Catsimpoolas, 1979). Platsoucas et al. (1981b) subjected human spleen cells from patients with Hodgkin’s disease to electrophoretic separation. Of the five (pooled) fractions obtained, B lymphocytes were highly enriched in the low-mobility fractions. At high mobility, enrichment of T p cells was measured with minimal contamination of T y cells. In low-mobility fractions, T y cells were significantly enriched, and lymphocytes of intermediate mobility were considerably enriched as a population of rosette-forming cells with Ripley’s serumcoated human red blood cells. In another experiment, these authors separated human splenic T lymphocytes, after rosetting with neuraminidase-treated sheep erythrocytes, into Tp (high mobility) and T y (low mobility) cells, resolution being comparable to that achieved with cells from peripheral blood. D. PERIPHERAL BLOOD CELLS

In 1976 Catsimpoolas et al. suggested that the very clear-cut bimodal electrophoretic migration profile of human blood cells (derived from a

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Lymphoprep interface) was due to the presence of B and T cells, respectively. Depending on the donor, the bimodality was more or less apparent. Lymphocytes, preincubated for 2 hr in autologous plasma, showed a monomodal distribution skewed positively, and ascribed by these authors to “shedding” of charged membrane components. Ault et al. (1976) made a study of the cellular constituents in these electrophoretic profiles. It appeared that monocytes were skewed toward the faster moving fractions; T cells (Ig-negativecells)were distributed fairly uniformly throughout the electrophoretic column. B cells were 65% pure in the slower moving fractions. The overall recovery ranged from 30 to 50% and since most of the cell loss occurred at the time of dilution in electrophoresis buffer, some care is recommended in the use of this low-conductivity buffer (see also criticism of Pretlow and Pretlow, 1979).Bronson and Van Oss (1979) reported the separation of 96% pure human T cells and of about 57% pure cells in the slowest moving fraction. Platsoucas et al. (1979a,b) made a further subdivision and were able to show that Tp (possessing receptors for IgM), Ty (which have receptors for IgG), and T+ (lacking both receptors) could be enriched electrophoretically from T-cell preparations that were first depleted from monocytes and next rosetted with sheep red blood cells. Of the cells applied, that is not counting the loss due to dilution in electrophoresis buffer, 64% were recovered with a viability greater than 95%. The high-mobility fractions contained 85% pure Tp cells, the low-mobility fraction 45% T y cells, and the intermediate mobility fractions were mostly (70%)T+ cells. This particular separation procedure yields cellular preparations that are not modulated with respect to the Fc receptors unlike rosetting techniques with the appropriate rabbit IgM of IgG anti-ox red blood cell antibodies. Platsoucas ( 1983)applied density gradient electrophoresis to the separation of cells from untreated patients with chronic lymphocyticleukemia (B-CLL).The heterogeneity of separated cells could be subdivided with the use of a panel of surface markers. Lymphocytes able to differentiate into plasma cells were highly enriched in the low-mobility fractions and contained the light chain (K-chain) that was already present on the cell surface before stimulation. Using fluorescence-activated cell sorting, it was further demonstrated that immunoglobulin-positivecells had significant increase of cell-surface immunoglobulin expression in the lowmobility fractions. It thus shows that subpopulations of leukemic B cells in different stages of differentiation and maturation can be obtained electrophoretically. Platsoucas and Catsimpoolas (1978) made the observation that peripheral rat blood cells can be separated by electrophoresis in 80% pure B

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(Ig+)cells and in almost 98% pure PHA responsive cells, overlapping of B and T cells in the migration profile was minimal. E. TONSILLAR CELLS

Platsoucas et al. (1980) subjected human faucal tonsil cells to density gradient electrophoresis. Low-mobility fractions were highly enriched with B cells and were contaminated only with 10-3076 T cells. T h e high-mobility fractions were comprised of T cells with minimal contamination of B cells. Intermediate fractions contained approximately equal amounts of T and B lymphocytes. Human tonsillar lymphocytes, preenriched by E rosetting, showed a much more narrow migration profile but purification of Tp, T y , and Ta (IgA receptor) cells was not impressive. if achieved at all. F. BONE MARROW CELLS

Platsoucas et al. (198 la,c) showed that electrophoretic separation of human bone marrow cells resulted in significantly enriched colony (in vitro) forming cells in the high- and intermediate-mobility fractions. T y cells were enriched in the low-mobility fractions and Tk cells in the intermediate ones. These authors explained circumstantially that the contribution of unit gravity sedimentation of cells to the final electrophoretic profile of bone marrow cells is minimal. Although they suggested that unit gravity sedimentation contributed only 15%to the migration profile, we interpret their results quite differently. The sedimentation profile (in the absence of electrophoresis) was nearly similar in shape and band width to the electrophoretic profile (their Figure 3). It would appear, then, that the unit gravity profile is only displaced and that, unless fortituitous combinations of sedimentation velocity and surface charge exist that give rise to an almost similar migration profile, one must conclude that cells all having about the same surface charge were not separated better by the electrical force than they already were by the gravity force. Van Beek et al. (1982) showed that, with unit gravity sedimentation of human bone marrow cells, the relatively fast-sedimenting, large cells are highly enriched myeloid precursor cells, the slowest-sedimenting cells are monocytes (unpublished results), and that cells with intermediate velocities are lymphocytic cells. Also Wells et al. (1977) demonstrated that cells of intermediate and high sedimentation velocity are colony-forming cells. These results are in line with those of Platsoucas et al. (1981a) who found that colony-forming cells are in the intermediate and'high-mobility fractions and that the (large) immature myeloid cells dominated the high-

DENSITY GRADIENT ELECTROPHORESIS OF MAMMALIAN CELLS

183

mobility fraction whereas monocytes are in the low-mobility fractions. Platsoucas et al. (1981~) furthermore showed that in vitro treatment of human bone marrow cells with ubiquitin, but not with thymopoietin, resulted in increased proportions of IgM-bearing cells in the fractions of intermediate electrophoretic mobility. G. THYMOCYTES

Thymocytes from mouse migrated as a single band (Griffith et al., 1975, 1976) and no functional differentiation was recorded.

2. Miscellaneous For demonstration purposes only, Boltz et al. (1973)showed the different migration profile of Chinese hamster cells due to gravity force alone and to the electrical plus gravity force, respectively. Boltz et al. (1976) demonstrated that rat embryonic cells resolved in several electrophoretic subpopulations. The shift in mobility distribution following infection with Herpes simplex virus was ascribed to a depletion of cells from the most rapidly migrating fractions. These authors also showed that human cervical biopsy cells were resolved from erythrocytes and (among others) keratinized squamous cells, a result that can also be obtained by velocity sedimentation at unit gravity. Furcinitti and Hunter (1978) reported on preliminary experiments concerning the electrophoresis of quail femoral medullary cavity cells. Cells from nonestrogenized birds electrophoresed in at least three broad bands, including red blood cells. The highest thymidine incorporation 27 hr after estrogen injection coincided with the peak of large volume cells. BZrjesson et al. (1981) subjected cells from human breast cancer first to velocity sedimentation in a density gradient, and then transferred the whole gradient to an electrophoresis tube. After 6 hr electrophoresis, a correlation between large cancer cells and estradiol uptake was observed. Todd et al. (1981) separated by upward electrophoresis dividing cells of the epithelial cell line T 1;low-mobilitycells were enriched for Gn-phase cells of the cell cycle but this was probably due to the action of the gravity force that pulls larger cells down into the electrophoresis column. Generally, it is more feasible to separate dividing GI-, S- and G2-phase cells by unit gravity alone (e.g., McDonald and Miller, 1970; Tulp et al., 1982~). Human embryonic kidney cells were electrophoresed into a highmobility fraction, possessing the highest level of urokinase and a plasminogen activator, as compared with other fractions (Todd et al., 1981). Todd et al. (1981) deduced from their experiments that a small population of somatropin-secreting cells of low mobility is responsible for the

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production of relatively large quantities of growth hormone in the rat anterior pituitary.

VI. FUTURE PROSPECTS It is hoped that the present review will encourage those active in the field of cell separation to apply low-cost electrophoresis columns to cell-separation problems. In our opinion, much can be expected from the small height and large surface area chamber for rapid electrophoretic separation of cells and cell organelles. Although the device (Section IV.2.1.b) is certainly subject to technical improvements to facilitate operation, the method can open new areas because monoclonal antibodies, reacting with lymphocyte subpopulations under noncapping conditions, may give differential surface charges to various cell classes. These antibody-coated cells may then be separated electrophoretically as in the ASECS- FFE method of Hansen and Hannig (1982). Alternatively, cell surface charge may also be modulated by adhering microspheres coated with specific antibodies (immunospheres) to the cells thus enhancing mobility differences of cells that otherwise have almost equal mobilities (Smolka et al., 1979).

VII. ISOELECTRIC FOCUSING OF CELLS IN DENSITY GRADIENTS 1.

Introduction

In isoelectric focusing (IEF), a “natural” pH gradient is generated by the electrical field increasing from anode to cathode and stabilized by a density gradient (Haglund, 1971).An ampholyte loaded on such a gradient migrates toward a position where its net charge is zero and stays there. The method is essentially an equilibrium method. Whereas ratezonal electrophoresis allows probing of the cell surface to a depth of 1.4 nm beneath the surface of shear, IEF allows probing to a depth of 6-7 nm (Sherbet, 1978). A priori, one may conceive several objections, even leading to scepticism, to the possibility of applying IEF as a method that maintains cellular viability. Just and Werner (1979) and Hannig (1978) in particular have been sceptical. The latter author remarks: “the isoelectric point of cells is not a physical constant as with proteins . . . the importance of cell isoelectric focusing becomes doubtful . . . and cell viability is usually completely

DENSITY GRADIENT ELECTROPHORESIS OF MAMMALIAN CELLS

185

lost.” And even Sherbet (1978) states that “isoelectric focusing does not appear to be suitable for separation of cells, as we had hoped, due to drawbacks inherent in the technique.” These drawbacks can be listed as follows: 1. Under the ambient conditions of IEF (176 ampholine, LKB Produkter, Bromma, Sweden)extremely low specific conductivity is reached: 0.2 mmhokm (Haglund, 1971)or even 0.1 mmhokm (Just and Werner, 1979) corresponding with a very low “ionic strength” of 0.5- 1 mg ion/ liter (Righetti, 1980), affecting the stability of the cellular plasma membrane. 2. Low pH values per se in the separation column must have a deleterious effect on cells. Erythrocytes (Heard and Seaman, 1960; McGuire et al., 1980) and lymphocytes (Mehrishi and Thomson, 1968) are electrokinetically and irreversibly unstable at low pH. Murine lymphoid cells and Chinese hamster fibroblasts show reversible electrokinetic properties after a brief exposure (15 min) to low pH (Greig et al., 1976).Heard and Seaman (1960) showed that the stability of the erythrocyte membrane at low ionic strength and low pH is so impaired that lysis ensues. McGuire et al. (1980) showed that a combination of low pH and low osmolarity favors lysis of cells. These two factors, low “ionic strength” and low pH, weigh more heavily since IEF of cells requires several hours up to 24-48 hr (Leise and Lesane, 1974). Catsimpoolas and Griffith (1977) observed that cells remain focused after about 1 hr but start to defocus after 2 hr, followed by lysis. 3. It is essential that the ampholyte building up the pH gradient does not form complexeswith cellular membrane components, but actually the LKB ampholine does, among others with sulfated and carboxylated polysaccharides (Righetti et al., 1978; Righetti and Gianazza, 1978, 1980; Gianazza and Righetti, 1978).A model for this binding has been proposed by McGuire et al. (1980). The protonated nitrogens, separated by two methylene groups, in the backbone of the carrier ampholyte are thought to bind to sialic acids or sulfate groups attached to the carbohydrate chains of membrane glycopeptides. Moreover, according to Galante et al. (1975), carrier ampholytes form chelates with M2+ metal ions. 4. Insertion of cells at low pH may result in the extraction of cellular proteins. For IEF of HeLa cell metaphase chromosomes, it was found that stripping of basic proteins occurred in the pH gradient (Landel et al., 1972). In this respect, Boltz et al. (1977) and Hammerstedt et al. (1979a) noted that the focusing pH value of sperm cells was related to the pH of sample injection, certainly due to the extraction of basic proteins at low pH. Also sperm became immotile after suspension in electrofocusing

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media (Hammersted et al. 1979a). Despite the fact that PIS of cells are reproducible, they must be considered with caution. 2. The Lack of Theory A reliable theory that predicts IEP of cells based on the pK of charged membrane groups and on their stoichiometry is seriously lacking. When the PI of a cell is not a physical constant, it is even not worthwhile to develop such a theory. Phenomological studies would at least indicate the factors that are involved (Heard and Seaman, 1960).The cell (as a macropolyanion) can be considered as an analog to an amphoteric amino acid. The PI of a cell may be defined as the pH where the number of protonated basic groups is equal to the number of protons dissociated from the acid groups, thus leading to zero surface charge and electrophoretic immobility. Based on very simplified assumptions (Thompson et al., 1978) that the negative surface charge depends on a glycosidic carboxyl group GCH (pK = 2.6) and the positive charge on amino groups NH: (7.6 S pK S 12.5), the following relations hold: GCH NH,f

F=

GC- + H+ NHn-l + H+

in which

The ratio P = total glycosidic carboxyl residues over total N residues can be expressed as (20)

at pH = PI, [GC-] = [NH,f] and solving Equation (20) for [H']

The PI ofE. coli is given as 5.6 (Sherbet, 1978),nearly the same as the PI of an erythrocyte (Just and Werner, 1979). In E. coli the ratio of negative to positive charges is 2 (Sherbet, 1978), whereas in erythrocytes this ratio is as high as 25. Whatever the pKs of membrane groups for the two cell types may be, Equation (20a) will not yield high values of pI for these cell types, as close inspection of Figure 18 shows. Theory and experimental values at present

DENSITY GRADIENT ELECTROPHORESIS OF MAMMALIAN CELLS 4-

I

t

-a

187

I

I 1

I

I

I

2

I

3

’

I

- ’ 1

4

25

P

Figure 18. Theoretical dependency of isoelectric focusing pH (PI) on the ratio of glycosidic carboxyl residuedtotal ‘“”-residues (P). Equation (20a) was applied.

clearly exclude one another. It may well be that at low ionic strength (ambient conditions during IEF) charged groups become “visible” that are not detectable at high ionic strength and low pH. Treatment of the cell-surface charge in the manner of the “smeared site” model for a rigid impenetrable sphere at finite ionic strength (Linderstrflm-Lang and Nielsen, 1959) might bring insight into this problem. Sherbet (1978) derived a formula that connects electrophoretic mobility of a cell at pH 7 with its IP, and for several cell types a good fit was observed. The human erythrocyte however falls completely out of this particular relationship. Righetti et al. (1980) consider this lack of good fit makes the theory suspicious. 3. Applications Being an equilibrium method, cells can be added to the separation column as a zonal layer or may be mixed throughout the gradient at the time the density gradient is introduced into the column. In the latter case, focusing times are extremely long because building up of the pH gradient is a slow process. On banding of the cells at their PI, the banding capacity of the rather weak density gradient can be exceeded according to Mason’s equation (Equation 4 in Section 111.1) and streaming phenomena may occur. Floating of individual cells (at a gradient density > density of the cell) on the other hand is unlikely to occur since this buoying movement at unit gravity is exceedingly slow, as calculation shows.

W

-

3T3 SV-3T3 SV-BHK Py-3T3 3T3+formaldehyd 3T3+ethyleneimine SV-STS+forrnaldehyd SV-3T3+ethyleneimine Butter yellow-induced rat hepatoma

b

Yoshida ascites Ehrlich ascites HeLa y-Globulin-treated Hela Polyoma-transformed B HK Rat liver

a Rabbit: Peripheral lymphocyte Thymocytes Human: Peripheral lyrliphocyte

Cell type

+

Ficoll 400, isoosm.

+

Sucrose, hyperosm. Ficoll 400, isoosm.

+

Sucrose, hyperosm.

+

Dextran 40, hypoosm.

Gradient solute osmolarity

4.6 4.8 4.66 4.78 4.05 8.2 4.1 8.58 4.6

6.4 -5.11 6.5 -5.05

6.35-4.79 5.6 -4.73 6.85-5.32 6.36-4.85

Several peaks

Several peaks Several peaks

Focusing pH (PI)

24

48

Time (hr)

Isoelectric Focusing of Mammalian Cells in Natural pl3 Gradients

TABLE IV

Sherbet (1978)

Sherbet et al. (1972)

Leise and LeSane (1974)

References

CD

oo F

Peripheral lymphocytes (rat) Chicken embryonic: kidney muscle neural retina pigmented retina liver heart brain fibroblast Rat embryonic: kidney liver fibroblast adult rat liver Chicken: bursa thymus Rat embryo Rat ernbryo+HSV Boar sperm(atozoa) Sperm.-seminal vesicles Sperm. t seminal plasma Bull sperm. Rabbit sperm id. off-season Ram sperm:

Meningioma Fetal brain

Human astrocytoma class I1 to IV

Ficoll 400, isoosm.

Ficoll 400, isoosm. t

+

Ficoll400, hypoosm (?)

t

Ficoll 400, isoosm.

Idem

Idem

Sucrose hyperosm., HEPES/MES

+

Ficoll400, isoosm.

+

Ficoll 400, isoosm.

3.18 4.52-4.69 4.47 4.62 6.5 4.5 5.8 5.0 6.8 5.05

4.7 4.8 4.58 4.48

4.35 4.28 4.61 3.89 4.60 4.20 4.38 4.1

4.73 4.38 4.6-3.8 T and B cells

from 4.4 to 5.05

3

2-4 B

3 B 2 E

4.5

25

0.5 E

(Continued)

Hammerstedt et al. (1979a)

Moore and Hibbit (1975)

Thompson et al. (1978)

Rani et al. (1982)

Rao et al. (1979)

Rao (1978)

Hirsch et al. (1977)

Sherbet and Lakshrni (1974)

CD

~

~

citratekitric acid ~~~~~~

Ficoll 400, isoosm.

1

Ficoll 400, isoosm.

t

I

3.5 and 4.7

6.6, 7.5 5.5

4.3 5.2 5.5

4.20

5.2 4.7 4.8 <2.0 6.0 4.98 5.10

Focusing pH (PI)

B

3

B

B

Time (hr)

Boltz et al. (1978)

Boltz et al. (1977)

Hammerstedt et al. (I979b)

References

Abbreviations: HSV = herpes simplex virus; HeLa = human carcinoma of the cervix; PyBHK = polyoma-virus transformed B(aby) H(amster) K(idney) cells; SV = simian virus; 3T3 = mouse fibroblasts; and + = ampholyte forms complexes. Unless otherwise specified (B: Section IV.2.B; E: Section IV.2.E) apparatus in Section IV.2.D was used.

~~

Testicular Cauda epididymal Ejaculated Rabbit ery(glutara1dehyde) Chinese hamster Rat mammary ascites turn. (idem plus prolactin) Rat peritoneal Leukocytes Rat pituitary: chromophobe somatotrophin Bull spermatozoa Human: uterine cervical squam. cervical leukocytes erythrocytes Chicken erythrocytes(f) Rabbit spermatozoa Rat pituitary Rat mammary asc. tumor Chinese hamster

Cell type

Gradient solute osmolarit y

Isoelectric Focusing of Mammalian Cells in Natural p H Gradients

TABLE 1V (Continued)

DENSITY GRADIENT ELECTROPHORESIS OF MAMMALIAN CELLS

191

Sherbet and Lahksmi (1972) have reported IEF of bacterial cells. Kraaipoel and van Duin (1979) separated Chlamydia trachomatis. In Table IVa,b, an extensive survey is given of IEF of mammalian cells. Reproducible PI values are found, but these values cannot be considered as a starting point on which separation of viable mammalian cells can be based. Table IVa gives results obtained under what we consider rather harsh conditions for cell survival: very low or very high osmolarity, long separation time, low pH, and possible complexing action of the ampholine. For instance, we calculate that Leise and Le Sane (1974) performed their separations at about 10 mOsm for 48 hr. It is hard to see how a cell can survive very low osmolarity at low p H for such a long time. Although these authors describe the isolation of cells (80-9076 excluding Trypan Blue), cell survival after 72 hr is very low. Sherbet et al. (1972) have used either sucrose gradient (excessively high osmotic values) or isoosmotic Ficoll gradients. Generally the long focusing time gives rise to two focusing populations, one consisting of dead cells (low PI) and one of living cells. It is noteworthy that Sherbet et al. (1972) report a PI of 6.5 for rat liver cells whereas Rao et al. (1979) give a value of 4.48 and Boltz et al. (1976) of 6.7. Hirsch et al. (1977) isolated almost 80% pure rat B cells by IEF. Thompson et al. (1978) suggested that the PI of rat embryo cells is elevated 0.15 pH units after infection with Herpes simplex virus due to the appearance of basic amino groups of the virions on the cellular surface. Although Rao (1978), Rao et al. (1979), and Rani et al. (1982) used “natural” pH gradients composed of HEPES and MES buffers, the mechanism of stable pH gradient formation still being uncertain in this case, the extremely high osmolarity (due to sucrose) casts doubts on the survival of cells. Manske et al. (1 977) reported viabilities of 80-90% after short IEF giving rise to rather broad focusing peaks, while longer focusing time gives rise to smaller and double peaks accompanied by a dramatic drop in viability to only 20%. Moore and Hibbit (1975) deduced from their experiments that seminal plasma alters the membrane surface charge of boar spermatozoa on ejaculation. Not tabulated, due to the large number of data, are the results obtained by Moore (1979). He showed that for rat, hamster, mouse, and rabbit, spermatozoa released from the caput epidymis had a significantly higher mean isoeleetric point than those from the cauda region. On the other hand, it was not possible to separate a mixture of rat spermatozoa from caput and cauda epididymis by electrofocusing. Boltz et al. (1978) worked out the mathematics of a stable natural pH gradient for a simple citrate buffer system. A stable pH gradient from 3.08 to 7.21 was formed in 0.0015M citrate, that most likely did not form

T

192

ABRAHAM TULP

complexes with membrane macromolecules. We may note that in ampholine natural pH gradients, Chinese hamster cells focused at pH 6.0 (Boltz et al., 1976) but that they focused between p H 3.5 and 4.7 according to Boltz et al. (1978) in a natural citrate pH gradient. Large differences for the PI of rabbit spermatozoa were detected in these two types of pH gradients. Boltz et al. (1978) further showed, although rat mammary tumor cells did not give rise to tumors after IEF they did increase in cell number.

VIII. CONCLUSION Notwithstanding the large number of data, according to Righetti et al. ( 1980) the method is “neither thoroughly explored nor well understood.” It “should be further investigated and exploited.” If isoosmotic, noncomplex-forming natural pH gradients can be devised and if short columns (corresponding with rapid separation time) can be built, a reevaluation of the method of IEF of mammalian cells is certainly worthwhile. Acknowledgments T h e critical comments of Drs. J. A. Aten and H. Spits and Ir. F. Polak are highly appreciated. Thanks are due to Prof. P. Borst for reading the manuscript, Mrs. M. G. Barnhoorn for excellent technical assistance, Mrs. G. G. H. Meijerink for preparing the manuscript, Mr. J. Lomecky for the photographs, and Mr. A. Jans (NKI) for the drawings. Development of the separation devices was done in collaboration with Mr. A. Timmerman. References Albertsson, P. A. (1965), Anal. Biochem., Z I , 121-125. Albertsson, P. A., Andersson, B., Larsson, C., and Akerlund, H. E. (1982), in Methods of Biochemical AnaZysC (D. Glick, Ed.), vol. 28, John Wiley & Sons, New York, pp. 115- 150. Ambrose, E. J. (1965), Cell Electrophoresk, Churchill Ltd., London. Ault, K. A., Griffith, A. L., Platsoucas, C. D., and Catsimpoolas, N. (1976),J. Immunol.,1 1 7 , 1406-1408. Beaufay, H. A., Amar-Costesec, D.,Thines-Sempoux, M., Wibo, M., Robbi, M., and Berthet, J. (1974),J. Cell Biol., 61, 213-231. Bloemendal, H. (1963), Zone Electrophoresis in Blocks and Columns, Elsevier Publishing Co., Amsterdam. Boehmer, H., Shortman, K., and Nossal, G. J . V. (1974),J. Cell Physiol., 83, 231-242. Boltz, R. C. and Todd, P. (1979), in ElectrokznetuSeparationMethods (P. G. Righetti, C. J. Van OSS,and J. W. Van der Hoff, Eds.), Elsevier North-Holland Biomedical Press, Amsterdam, pp. 229-250.

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Boltz, R. C., Todd, P., Streibel, M. J., and Louie, M. K. (1973), Prep. Biochem., 3,383-401. Boltz, R. C., Todd, P., Gaines, R. A., Milito, R. P., Docherty, J. J., Thompson, C. J., Notter, M. F. D., Richardson, L. S., and Mortell, R. (1976),J. Histochem. Cytochem., 24, 16-23. Boltz, R. C., Todd, P., Hammerstedt, R. H., Hymer, W. C., Thompson, C. J., and Docherty, J. (1977), in Cell Separation Methods (H. Bloemendal, Ed.), North-Holland Publishing Co., Amsterdam, pp. 145-155. Boltz, R. C., Miller, T. Y., Todd, P., and Kukulinsky, N. E. (1978), in Electrophoresis 78 (N. Catsimpoolas, ed.), Elsevier North-Holland Inc., Amsterdam, pp. 345-355. BGrjesson, B. W. (1976), Ph. D. thesis, Melbourne, Australia. BGrjesson, B. W. and Sarfaty, G. A. (1981), Cancer, 47, 1828-1833. Brakke, M. K. (1953), Phytopathology, 43, 467. Brakke, M. K. (1955), Arch. Biochem. Biophys., 55, 175-190. Brakke, M. K., Allington, R. W., and Langille, F. A. (1968), Anal. Biochem., 25, 30-39. Bronson, P. M. and Van Oss, C. J. (1979), Prep. Biochem., 9, 61-70. Brooks, D. E. (1973),J. Colloid Interface Science, 43, 687-699. Brooks, D. E. and Seaman, G. V. F. (1973), J. Collozd Interface Science, 43,670-686. Brubaker, L. H. and Evans, W. H. (1969),J. Lab. Clin, Med., 73, 1036-1041. Carty, D. C., Crawford, E. A., and Gear, A. R. L. (1975), Fed. Proc., 34, 289. Catsimpoolas, N. (1973), Ann. N.Y. Acad. Sci., 209, 65-79. Catsimpoolas, N. (1980), Electrophoresis, I , 73-78. Catsimpoolas, N. and Griffith, A. L. (1975), Fed. Proc., 34, 545. Catsimpoolas, N. and Griffith, A. L. (1977a), Anal. Biochem., 80, 555-571. Catsimpoolas, N. and Griffith, A. L. (1977b), in Electrophoresis and Isotachophoresis, (B. J. Radola and D. Graesslin, Eds.), W. de Gruyter & Co., Berlin, pp. 469-479. Catsimpoolas, N. and Griffith, A. L. (1977c), in Methods of Cell Separation (N. Catsimpoolas, ed.), vol. I, Plenum Press, New York, pp. 1-24. Catsimpoolas, N. and Griffith, A. L. (1978), in Electrophoresis 78 (N. Catsimpoolas, Ed.), Elsevier North-Holland Inc., Amsterdam, pp. 59-68. Catsimpoolas, N., Griffith, A. L., and Williams,J. M. (1975), Anal. Biochem., 69,372-384. Catsimpoolas, N., Hjerten, S., Kolin, A., and Porath, J. (1976a), Nature, 259, 264. Catsimpoolas, N., Griffith, A. L., Skrabut, E. M., Platsoucas, C. D., and Valeri, C. R. (1976b), Cellular Immunol., 25, 317-321. Catsimpoolas, N., Griffith, A. L., Gupta, S., Good, R. A., and Platsoucas, C. D. (1980), in Electrophoresis 79 (B. J. Radola, Ed.), W. de Gruyter & Co., Berlin, pp. 607-622. Cramer, R. and Svensson, H. (1961), Experientia, XVII, 49-57. Fredriksson, S. (1975),J. C h r o m t . , I08, 153-167. Furcinitti, P. S. and Hunter, S.J. (1978), in Electrophoresis 78 (N. Catsimpoolas, Ed.), Elsevier North-Holland Inc., Amsterdam, pp. 373-381. Gaal, O., Medgyesi, G. A., and Vereczkey, L. (1980), Electrophoresis in the Separation of Biological Macromolecules, John Wiley & Sons, New York. Gaines, R. A., Boltz, R. C., and Todd, P. (1974), Biophys. J., 15, 245a. Galante, E., Caravaggio, T., and Righetti, P. G. (1975), in Progress in Isoelectric Focusing and Isotachophoresis, North-Holland Publishing Co., Amsterdam, pp. 3- 12. Gear, A. R. L. (1977),J. Lab. Clin. Med., 90, 744-753.

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Methods of Biochemical Analysis, Volume 30 Edited by David Glick Copyright © 1984 John Wiley & Sons, Inc.

METHODS OF BIOCHEMICAL ANALYSIS

VOLUME 30

Quantitation of Lipid Transfer Activity JOHN R. WETTEFUU AND DONALDB. ZILVERSMIT," Division of Nutritional Sciences, Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York

I. Introduction ..................................................................................................... 11. General Considerations for Assaying Transfer and/or Exchange Activities ........................................................................................................... 111. Separation Assays ....................................................... ...................................... 1. Nonexchangeable Markers .................................................................... 2. Cross-Contamination of Isolated Particles ............................................ B. Mitochondria-Small Unilamellar Vesicle C. Microsome-Vesicle .... D. Vesicle-Vesicle

...................... ...........

c. Negatively Charged Vesicles ........................................... E. Unilamellar Vesicle-Erythrocyte F. Small Unilamellar Vesicle-Multi .................... a. Preparation of Multilamell c. Incubations G. Monolayer- Vesicle .............................. ........................... ........... H. Plasma Lipoproteins ...................................................................

.................. IV. Spectroscopic Assays 1. Electron Spin Resonance .......................................................................

200 202 203 203 204 206 206 207 207 208 208 208 209 210 210 '211 211 21 1 21 1 212 213 214 214

*Career Investigator for the American Heart Association. Our experiments reported in this paper were supported in part by NIH grants HL 10940 and 2T32 AM-07158.

199

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JOHN R. WETTERAU AND DONALD B. ZILVERSMIT

2. Fluorescence ........................................................................................... 3. Nuclear Magnetic Resonance ................................................................ 4. Lipid Phase Transition Measurements ................................................. V. Assaying Crude Extracts for Transfer Activities ............................................ VI. Effects of Composition of the Assay Mixture on the Rate of Lipid

............................................................................................... .............................. .......................

ceptor to Donor Particles 2. Composition of Acceptor and Donor

214 216 216 217 218 219 220 220 22 1 222 222 223

I. INTRODUCTION In the late 1960s,Wirtz and Zilversmit (1968)found a soluble factor in rat liver cytosol that accelerated the exchange of phospholipids between biological membranes. Since this discovery, many lipid transfer proteins have been purified. A list of these and their physical properties is found in Table I. Although there has been considerable speculation concerning the physiological role of lipid transfer proteins, their precise function remains in doubt (Wirtz, 1982). Beef brain, heart, and liver have been the most frequently used sources of purified phospholipid transfer proteins. From these tissues, three classes of transfer proteins have been identified: a phosphatidylcholinespecific transfer protein, a phosphatidylinositol-specifictransfer protein, and a nonspecific transfer protein. In addition to the purified proteins shown in Table I, other transfer proteins have been partially purified from a wide variety of tissues and organisms including rat intestine (Lutton and Zilversmit, 1976a), rat lung (Van Golde et al., 1980), sheep lung (Robinson et al., 1978), beef spleen (Metz and Radin, 1982), potato tubers (Kader, 1975),endosperm of germinating castor bean (Douady et al., 1980), Rhodopseudomonas sphaeroides (Cohen et al., 1979), and yeast (Cobon et al., 1976). These proteins originally were called phospholipid exchange proteins. Some proteins are also capable of exchanging one class of phospholipid for another. The terms “homoexchange” and “heteroexchange” were introduced to describe the exchange of lipids within the same class or the exchange of one class of lipids for a second, respectively (Crain and Zilversmit, 1980a). It was found recently that some proteins can facilitate net transfer of phospholipids (Crain and Zilversmit, 1980a; Wirtz et al.,

QUANTITATION OF LIPID TRANSFER ACTIVITY

20 1

TABLE I Purified Lipid Transfer Proteins Mol.

Source Brain, beef Heart, beef Liver, beef Liver, beef Liver, rat Liver, rat Hepatoma, rat Plasma Maize seedlings

I I1 I I1

Wt.

Isoel. Pt.

32,300 32,800 33,500 33,500 28,000

5.3 5.6 5.3 5.6 5.8

I 14,500 I1 14,500

9.55 9.75

Lipid transferred"

References

P1,PC

Helmkamp et al. (1974); Demel et al. (1977)

PI,PC,SPH PC

28,000

8.4

PC,PE,SPH,PA,PG PI,PS,Cholesterol, G M l , CbOse4Cer PC

12,500

8.6-9.0

PE,PC,PI,PS,SPH, Cholesterol

11,200

5.2

SPH,PC,PE,PI+ PS

66,000 58,000

4.8

PL,CE,RE,TG

14,000

8.8

PC,PI

DiCorleto et al. (1979) Kamp et al. (1973); Moonen et al. (1980) Crain and Zilversmit (1980b); Bloj and Zilversmit ( 198 1b) Lutton and Zilversmit (197613); Lumb et al. (1976); Poorthuis et al. (1980) Bloj and Zilversmit (1977); Poorthuis et al. (1981) Dyatlovitskaya et al. (1978) Pattnaik et al. (1978); Morton and Zilversmit (1982) Douady et al. (1982)

"Abbreviations: CE, cholesteryl ester; GbOse4Cer, globotetraglycosylceramide; GM,, II3-a-N-acetylneuraminosyl-gangliotetraglycosy~ceramide; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol, PG, phosphatidylglycerol; PL, phospholipid, PS, phosphatidylserine; RE, retinyl ester; SPH, sphingomyelin; TG, triglyceride.

1980). The general term lipid transfer protein is now used to describe proteins with these transfer activities as well as those proteins that accelerate the transfer of lipids other than phospholipids, for example, cholesteryl ester (Pattnaik et al., 1978) and triglycerides (Rajaram et al., 1980). Lipid transfer proteins have proved to be a useful tool for studying artificial and natural membranes (for a recent review see Bloj and Zilversmit, 198la). With the ability of phospholipid transfer proteins to replace selectively the phospholipid molecules on the exposed surfaces of membranes, information about the asymmetric distribution of phospholipids across a bilayer and the rate of transbilayer movement of phospholipid

202

J O H N R. WETTERAU AND DONALD B. ZILVERSMIT

has been obtained. In addition, lipid transfer proteins have been used to modify the lipid composition of membranes and to study the effects of membrane lipid composition on membrane enzyme activities. Phospholipid derivatives have also been introduced into biological membranes with lipid transfer proteins.

11. GENERAL CONSIDERATIONS FOR ASSAYING TRANSFER AND/OR EXCHANGE ACTIVITIES To quantitate the lipid transfer activity of a protein, one measures the movement of labeled lipids from one membrane, the donor, to a second membrane, the acceptor. Typically, the donor and acceptor membranes are incubated in the presence and absence of transfer protein. After the incubation, the particles are separated and either the loss of radiolabeled lipids from the donor particles or the appearance of radiolabeled lipids in the acceptor particles is quantitated. The rate of lipid transfer in the presence of protein minus the transfer that occurs in the absence of protein is a measure of the lipid transfer activity of the protein. The transfer activity is expressed as a percent of the donor lipid transferred or the number of nmols lipid transferred per unit of time. To determine if the rate of lipid transfer also represents the rate of exchange, it must first be established that lipid exchange occurs between donors and acceptors. Exchange occurs when the rate of lipid transfer from donor to acceptor equals the rate of transfer from acceptor to donor or when the chemical composition of the donor and acceptor membranes does not change during the transfer reaction. A back-transfer of unlabeled and radiolabeled lipids from the acceptor to donor particles must be considered when calculating the lipid transfer rate. When transfer reactions beyond the initial rates are examined, it is necessary to account for the dilution of radioactive lipids in the donor particles by back-transfer of unlabeled lipid from the acceptor particles. Unless this is done, the apparent transfer rate will decrease with time. Equation (1) can be used to determine the transfer rate when backtransfer of unlabeled lipid is significant. B, = Boexp(-kt)

(1)

where Bo is defined as the amount radiolabeled lipid initially present in the exchangeable phospholipid pool of the donor particles; Bt, the amount present at time t ; and K , the fraction of the labeled phospholipid in the exchangeable pool lost per unit time. Equation (2) (McKay, 1938) can be used to correct for back-transfer of unlabeled and labeled lipids from acceptor to donor.

203

QUANTITATION OF LIPID TRANSFER ACTIVITY X

-ln(l - - ) = R t xi where A and B are the total amount of acceptor and donor exchangeable lipid, respectively; x and xi are the amount of exchangeable radiolabeled lipid in the acceptor at time, t , and at infinite time, respectively; and R is the rate of lipid exchange. The pseudo first-order rate constant, k, may be obtained by replacing the rate, R, by kB. Table I1 shows examples of the calculated transfer rates when no corrections are made, when the back-exchange of unlabeled lipid from the acceptor particles is corrected for, and when corrections are made for the back-exchange of both labeled and unlabeled lipid. TABLE I1 Calculated Lipid Transfer Rate With and Without Correction for Back-exchange of Labeled and Unlabeled Lipid" ~

~~

~

~

~~~~

No corrections

Percent of donor label transferred per hour

Column (1) X 50 nrnol",b

5 10 15 20 25 30 35 40 45 50

2.5 5 7.5 10 12.5 15 17.5 20 22.5 25

~

~

Corrected transfer rates Acceptor/donord b,c

2.56" 5.27 8.13 11.16 14.38 17.83 21.54 25.54 29.89 34.66

16,d

56,d

1ob,d

2.63 5.58 8.92 12.77 17.33 22.91 30.10 40.24 57.56

2.58 5.33 8.27 11.43 14.86 18.60 22.70 27.25 32.36 38.18

2.57 5.30 8.20 11.29 14.62 18.20 22.10 26.36 3 1.05 36.30

"Fifty nmol of PL is size of donor pool. til'ransfer rate expressed as nmol transferred/hr. "Corrected for back-exchange of unlabeled lipid. dRatio of acceptor to donor exchangeable lipid pool. The transfers are corrected for back-exchange of labeled and unlabeled lipid.

111. SEPARATION ASSAYS 1. Nonexchangeable Markers Measuring the rate of lipid transfer by an assay that includes the separation of donor and acceptor particles is complicated by the fact that rarely,

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J O H N R. WETTERAU AND DONALD B. ZILVERSMIT

if ever, can two membranes be completely separated with complete recovery of both species. In addition, bulk transfer of the donor membrane to the acceptor may occur by an adhesion or fusion process. These latter mechanisms for lipid transfer must be excluded when measuring the protein-catalyzed transfer of molecular species. To circumvent these problems, nonexchangeable markers are often used. These may be molecules trapped in the aqueous space or incorporated in the membranes of the donor or acceptor particles and remaining with the particles throughout the exchange reaction. It is important that the nonexchangeable marker labels all the particles, either donor or acceptor, to the same extent. Nonexchangeable markers can serve two purposes in an exchange reaction (1) to indicate the recovery of a membrane fraction after the separation procedure or (2) to indicate the contamination of one membrane fraction with another after the separation procedure. When a nonexchangeable marker is used, the decrease in radiolabeled lipids relative to the nonexchangeable marker in the donor, or an increase of radiolabeled lipids relative to the nonexchangeable marker in the acceptor is a measure of the transfer activity. Often membrane proteins serve as nonexchangeable markers in biological membranes. In artificial membranes, a lipid species that is not transferred will serve the purpose. Cholesteryl ester or triglycerides have typically been used when examining phospholipid exchange activities. When artificial membranes are used in an experiment with biological membranes, lipid nonexchangeable markers may be hydrolyzed as a result of hydrolase activities present in the biological membranes (Zilversmit and Hughes, 1976).The resulting free fatty acids, cholesterol, or glycerol moieties may then be lost from the artificial membranes. This is particularly a problem for extended reaction periods.

2. Cross-Contaminationof Isolated Particles If it is not possible to separate the donor and acceptor particles completely in a transfer assay, the accuracy of the assay is dependent upon its design and the corrections made for the contamination of the isolated membranes. T o correct for contamination of substrates, the radioactivity in the contaminating particles should be subtracted from the radioactivity in the recovered particles. This, in effect, can be done by using the relationship in Equation (3)when recovered acceptor particles are contaminated with donor particles.

T

=

T, + D (1

- Tc)

(3)

where T is the fraction of the total radioactivity present in the acceptor and contaminating donor particles, D is the fraction of the donor particles

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205

contaminating the acceptors, and T , is the fraction of the radiolabeled lipid transferred from the donor to acceptor particles. Equation (4)is derived from Equation (3) and is used to calculate the corrected fraction of the donor radiolabeled lipid transferred.

T-D T, = -

1-D

(4)

In equations (3) and (4),the fraction of the labeled lipid in the donor particle after the exchange reaction is subtracted to correct for contamination. In these equations, it is assumed that the acceptor particles are recovered quantitatively. If this is not the case, both the labeled lipid appearing in the acceptor and contaminating donor particles, and the fraction of donor particles contaminating the acceptor particles must be corrected for incomplete recovery of the acceptor particles. To use Equation (4),it is necessary to have a nonexchangeable marker in the donor particles to quantitate contamination of the acceptor. The errors introduced by contamination may be reduced without a correction formula when the apparent background transfer in the absence of transfer protein is subtracted from the total apparent transfer in the presence of protein. If the recovered donor particles are contaminated with acceptor particles, the relationship in Equation ( 5 ) is used to determine the correct percent transfer.

R=l-T,+AT,

(5)

where R is the fraction of the total radioactivity recovered in the contaminated donor particles, A is the fraction of the acceptor particles contaminating the donor particles, and T,is the fraction of the donor radiolabeled lipid transferred. Upon rearrangement, Equation (6) is used to calculate the corrected fraction of the donor radiolabeled lipid transferred.

R-1 T, = A-1 In these formulas, it is assumed that the donor particles are recovered quantitatively. If this is not the case, both R and A must be adjusted for incomplete recovery of the donor particles. The correct choice for the donor and acceptor membrane in a transfer assay can significantly influence the accuracy of the assay and as a result, correction formulas for contaminating particles may not be necessary. This can best be illustrated with an example in which two membranes, A and B, are separated in an assay in which membrane B, the membrane recovered and counted, is contaminated with membrane A. In this case, it

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JOHN R. WETTERAU AND DONALD B. ZILVERSMIT

may be preferable to incorporate the radiolabeled lipids and nonexchangeable marker into membrane B and designate it as the donor membrane in the transfer reaction. Table I11 shows a comparison of the percent error in the calculated transfer rate when membrane B (quantitatively recovered in the assay and contaminated with 1%, 296, or 3% of membrane A) functions as the acceptor or donor particle. As can be seen, the percent error in the calculated transfer rate at low levels of exchange is significantly less when membrane B functions as the donor membrane. TABLE 111 Percent Error in the Calculated Transfer Rate when Particle B Functions as the Acceptor or Donor Particle Percent of particle A contaminating particle B 1%

2%

3%

19.0

9.0 5.7 4.0

38.0 18.0 11.3 8.0

57.0 27.0 17.0 12.0

I .o

2.0

3.0

~

Acceptor

Actual percent exchange 5 10 15 20

Donor

At all percent exchange

3. Specific Assays A. MICROSOME-MITOCHONDRIA

The transfer of phospholipids between mitochondria and microsomes in vitro was first used to measure the activity of lipid transfer proteins (Wirtz and Zilversmit, 1968). In this assay, isolated mitochondria and microsomes are incubated with an appropriate amount of transfer protein. Either particle may be radiolabeled and serve as the donor particle. The exchange reaction is terminated by sedimenting the mitochondria by centrifugation. The change in the radioactivity of either the donor or acceptor particles can be used to calculate the lipid transfer activity. The subcellular fractions of rat tissues may be radiolabeled by injecting [32P]inorganic phosphate or [ 14C]glycerolintraperitoneally. After the radiolabels are incorporated into the phospholipids, the subcellular fractions are then isolated. This procedure simultaneously labels several classes of phospholipids. When this is done, the transfer of an individual

QUANTITATION OF LIPID TRANSFER ACTIVITY

207

class of phospholipids can be determined by isolating that class of lipid before quantitating its radioactivity. Alternatively, individual classes of lipids may be labeled. Techniques to label the polar headgroups of phosphatidylcholine (Wirtz et al., 1972), phosphatidylinositol (Helmkamp et al., 1974), phosphatidylserine (Butler and Thompson, 1975), phosphatidylethanolamine (Lumb et al., 1976), or phosphatidic acid (Possmayer, 1974) are available. Radiolabeled proteins or organelle-specific enzymes may be used as nonexchangeable markers, provided the enzyme activity is stable throughout the transfer reaction. B. MITOCHONDRIA-SMALL UNILAMELLAR VESICLE

In this assay, the transfer of radiolabeled lipids from small unilamellar vesicles to mitochondria is measured Uohnson and Zilversmit, 1975; Crain and Zilversmit, 1980b). Large quantities of mitochondria can be prepared by the procedure of Green et al. (1957) and stored at -20°C. After thawing, they are heated to 80°C for 20 min to destroy mitochondrial lipase which may hydrolyze nonexchangeable markers present in the unilamellar vesicles (Zilversmitand Hughes, 1976). Donor unilamellar vesicles, containing 32P-labeledphospholipids and a trace of [3H]triolein as a nonexchangeable marker, are prepared by sonication. The vesicles, mitochondria, and transfer protein are incubated in buffer containing 1 % fatty acid-poor bovine serum albumin (BSA). The transfer is terminated by sedimenting the mitochondria by centrifugation. An aliquot of the vesicles in the supernatant is then counted. The inclusion of BSA in the incubation increases the vesicle recovery. The extent of transfer is determined from the decrease in the 32P/3Hratio of the unilamellar vesicles. This assay has several advantages: (1) the lipid composition and labeling of the donor particles can be easily controlled; (2) the recovery of vesicles is quite high, typically greater than 90%, although the recovery may depend upon the composition and size of the vesicles; and (3) no labeling of biological membranes is necessary. One disadvantage of this assay is that, for low levels of exchange, the extent of exchange is determined as a relatively small change in an isotope ratio. To avoid this problem, the appearance of label in the mitochondria can be determined; however, some of the convenience of the assay will be lost. C. MICROSOME-VESICLE

The transfer of 14C-labeledphospholipids from microsomes to acceptor vesicles, containing a trace of [3H]cholesteryloleateas a nonexchangeable marker, is measured in this assay (Kamp and Wirtz, 1974; Helmkamp

208

JOHN R. WETTERAU AND DONALD B. ZILVERSMIT

et al., 1974).After incubating the substrates with the transfer protein, the buffer is adjusted to a pH of 5.1 to aggregate the microsomes, which are then quantitatively sedimented by centrifugation. The increase in the 14U3H ratio of the vesicles in the supernatant is used to calculate the lipid transfer. Vesicle recoveries are 60-80%. This assay has been used to measure phosphatidylcholine and phosphatidylinositol transfer and should be adaptable to other lipids by appropriate labeling of the microsoma1 fraction. Provided the microsomes are quantitatively precipitated, this assay, which monitors the appearance of label in the acceptor, is very sensitive. D. VESICLE-VESICLE

Several assays measure the transfer of phospholipids between artificial vesicles. These assays are particularly appealing because they have welldefined acceptor and donor membranes that are easily prepared and, in some cases, can be modified. a. Antigen-Sensitized Vesicles. Ehnholm and Zilversmit ( 1972) incorporated Forssman antigen from sheep red blood cells into vesicles to measure the transfer of lipids between these sensitized vesicles and nonsensitized vesicles. After the substrates were incubated in the presence of exchange protein, the antigen-containing vesicles were aggregated by the addition of anti-Forssman antigen gamma globulin. Following a 3-hr incubation, the sensitized vesicles were sedimented (98%)by centrifugation. The time required to precipitate the sensitized vesicles would make this assay inappropriate for fast exchange reactions.

b. Vesicles Sensitized to Lectins. Sasaki and Sakagami (1978) developed an assay to measure the transfer of phospholipids between concanavalin A(ConA)-reactivevesicles and nonreactive vesicles. The donor membranes contained a-D-mannosyl-(1+3)-a-D-mannosyl-sn- 1,2 diglyceride to make them reactive to concanavalin A. Two different procedures were used to separate the acceptor and donor vesicles after the transfer reaction. In the first procedure, N-ethylmaleimide was added to inhibit the phospholipid exchange proteins and the mixture was then incubated overnight with ConA. The floccules which formed were sedimented by centrifugation and were counted. Due to the long period of time required for the formation of the floccules, the inhibitor of the transfer activity must be effective. In the second separation procedure, ConA coupled to Sepharose 2B was added to the vesicle mixture to preferentially bind the sensitized vesicles. After a 10-min incubation on ice, the mixture was filtered and

QUANTITATION OF LIPID TRANSFER ACTIVITY

209

washed with buffer. The ConA-Sepharose 2B on the filter paper was then counted. About 40% of the reactive vesicles and 6% of the nonreactive vesicles were trapped in the ConA-Sepharose 2B. The recovery of the reactive vesicles could be improved by changing the lipid composition. Kasper and Helmkamp (1981) used Ricinus communis agglutinin to precipitate donor vesicles containing lactosylceramide. The transfer of [3H]phosphatidylcholine from donor to acceptor vesicles, containing cholesteryl ['4C]oleate as a nonexchangeable marker, was measured. Fifteen minutes after the addition of agglutinin, the donor vesicles were sedimented by centrifugation. An aliquot of the supernatant was counted and the transfer was calculated by the increase in the 3H/'4C ratio. Under normal assay conditions, the acceptor vesicle recovery was 92- 98% whereas the supernatant was contaminated with 2-3% of donor vesicles. The transfer did not appear to be affected by glycolipids in the substrate particles. This assay provides several significant advantages: (1) all materials for the assay are readily available; (2) high recovery of acceptor vesicles is possible from a relatively short separation procedure; and (3) the lipid composition of donor and acceptor particles can be manipulated. c. Negatively Charged Vesicles. Hellings et al. (1974) incorporated negatively charged phospholipids into donor vesicles to separate them from acceptor vesicles by ion exchange chromatography. Phosphatidylcholine vesicles containing 7 mol% phosphatidic acid or 9 mol% phosphatidylinositol, were completely retained by a DEAE-cellulose ionexchange column equilibrated with 0.05M KC 1-0.01M glycine-KOH (pH 8.6) buffer. The acceptor particles, containing a nonexchangeable marker and the lipid transferred from the donor, were eluted from the column and counted. The reported recoveries of acceptor vesicles in DEAE ion-exchange assays range from 40%to 70% (Van den Besselaar et al., 1975; Hellings et al., 1974). The low recovery was attributed to nonspecific adsorption of vesicles to the DEAE-cellulose. The recovery was found to depend upon the ratio of vesicle phospholipid to ion exchanger. The composition of the acceptor and donor particles is restricted in this assay. Hellings et al. (1974) found that negatively charged fipids in the donor particles inhibit the phosphatidylcholine transfer catalyzed by the phosphatidylcholine-specifictransfer protein. Therefore, with this assay, it is only possible to measure transfer rates in the presence of a potential inhibitor. McLean and Phillips (1981) used a modification of this assay with a substantially higher recovery of acceptor vesicles. The spontaneous transfer of cholesterol and phosphatidylcholine between vesicles was studied.

210

J O H N R. WETTERAU A N D DONALD B. ZILVERSMIT

Nonlabeled egg yolk phosphatidylcholine vesicles were applied to the DEAE column immediately before the separation step to preequilibrate the column with lipid. The recovery of acceptor vesicles was increased to 80-95%. E. UNILAMELLAR VESICLE-ERYTHROCYTE

The transfer of radiolabeled phospholipids between vesicles and erythrocyte membranes could be used to assay lipid transfer activity. Intact erythrocytes are not an ideal substrate for routine measurements of transfer activity because some transfer proteins do not readily accelerate the transfer of phospholipids from these membranes. Van Meer et al. (1980) found that a very high concentration of the phosphatidylcholinespecific transfer protein was necessary to exchange the phosphatidylcholine of intact red blood cells. Erythrocyte ghosts are a more active substrate for this protein (Bloj and Zilversmit, 1976). However, the nonspecific transfer protein from bovine liver accelerates the exchange of phospholipid between intact erythrocytes and phosphatidylcholine vesicles (Crain and Zilversmit, 1980~). F. SMALL UNILAMELLAR VESICLE-MULTILAMELLAR VESICLE

DiCorleto and Zilversmit (1977)reported that the phospholipid exchange proteins from either beef heart or beef liver were not able to catalyze the exchange of phosphatidylcholine between phosphatidylcholine multilamellar vesicles and small unilamellar vesicles. However, by adding acidic phospholipids to the multilamellar vesicles, protein-enhanced exchange was observed. This assay is versatile and can be easily performed. Both substrates are well-defined particles in which the composition can be readily manipulated. The separation of acceptor and donor membranes by centrifugation is rapid and nearly complete with 98-99% of the multilamellar vesicles being sedimented and with 90% of the small unilamellar vesicles remaining in the supernatant. The exchangeable pool of lipids in the two membranes can be easily determined. When the substrates were prepared by the procedure of DiCorleto and Zilversmit (1977), 70% of the unilamellar vesicle phosphatidylcholine was available for exchange and 7% of the phosphatidylcholine of the phosphatidylcho1ine:phosphatidylethanolamine:cardiolipin (70:25:5, mol%) multilamellar vesicles. Knowledge of the size of the exchangeable pool of lipid is important for predicting the linear range of the assay and determining kinetic parameters. Due to the many potential applications of this assay, a detailed explanation of the procedure follows.

QUANTITATION OF LIPID TRANSFER ACTIVITY

21 1

a. Preparation of Multilamellar Vesicles. The procedure is based on the one by Bangham et al. (1965). Appropriate aliquots of the lipids and antioxidant (butylated hydroxytoluene, 0.1 mol%) are added in CHC13 to a round-bottom flask and mixed thoroughly. The organic solvent is completely removed by evaporation in vacuo and buffer is added to a final concentration of 10 mg lipid/ml buffer (50mM Tris-HC1, 5 m M Na2EDTA, pH 7.4). The flask is swirled by hand until all the lipid is suspended. The dispersion, which appears milky white, is allowed to stand at room temperature for 2 hr. If the vesicles are not used immediately, they can be stored under N2 at 4°C for a few days. Immediately before the exchange reaction, the multilamellar vesicles are centrifuged at 40,OOOg for 15 min. The supernatant is removed, buffer is added, and the vesicles are then resuspended by gentle agitation. b. Preparation of Small Unilamellar Vesicles. Appropriate aliquots of the lipids and antioxidant are added in CHC13 to a screw cap test tube. These should include the 32P-labeledphospholipids and a trace of E3H] triolein which serves as the nonexchangeable marker. The solvent is evaporated in vacuo and 2.0 ml buffer added to a final lipid concentration of 1- 10 mg/ml. The suspension is placed under N2 and vortexed for 10 min. The solution is allowed to stand at room temperature for 30 min and then sonicated in a bath-type sonicator at 10- 15°C (Lab Supplies Co., Hicksville, NY) for 30 min or until the sample is clear. c. Incubations. Unilamellar vesicles (50 nmol phospholipid), multilamellar vesicles (2000 nmol phospholipid), and transfer protein are incubated at 37°C for 30 min in a total of 1 ml buffer. Exchange is terminated by centrifugation at 40,OOOg for 20 min at 4°C. An aliquot of the supernatant containing the unilamellar vesicles is removed and counted. The percent transfer is calculated from the decrease in the 32P/3Hratio of the unilamellar vesicles and the transfer in the absence of transfer protein is then subtracted to determine the protein-stimulated transfer. G. MONOLAYER-VESICLE

In this assay (Demel et al., 1977; Demel et al., 1982), a monolayer, containing I4C-labeled phospholipid, is formed at an air- water interface. Vesicles and exchange protein are injected into the subphase. The loss of radioactivity from the surface is monitored continuously with a gas flow detector. Alternatively, the rate of transfer of radiolabeled lipids is measured by recovering the subphase or monolayer and quantitating the radiolabeled lipids. The difficulty in preparing the monolayers and the

212

IOHN R. WETTERAU A N D DONALD B. ZILVERSMIT

relatively large quantity of transfer protein necessary for the assay would make this assay impractical for routine work. The monolayer assay is ideal for determining the specificity of a protein for transferring a particular class of phospholipids. A knowledge of the size of the exchangeable lipid pool of each class of phospholipids studied is necessary when biological and artificial membranes are used in a specificity study. However, in a donor monolayer all phospholipids are exchangeable; the relative rates of transfer of the different classes of phospholipids will reflect the specificity of the protein for these classes. H. PLASMA LIPOPROTEINS

Although plasma lipoproteins have been used to assay phospholipid transfer, they are more commonly used to study the exchange and transfer of cholesteryl ester and triglycerides by transfer proteins in plasma. The general protocol for the assay is similar to that for the phospholipid experiments previously described, except that nontransferable markers are not commonly used. As a result, the accuracy of the assays depends upon high recoveries of noncontaminated lipoproteins or on the use of a “blank” incubation in the absence of transfer protein. The assay used by Pattnaik et al. (1978) to isolate the cholesteryl ester transfer protein in human plasma measured the transfer of 3H-labeled cholesteryl ester from low density to high density lipoprotein. The transfer reaction was terminated by adding MnCI2 and centrifuging to sediment the low density lipoprotein. A heparin, MnCI2 precipitation step may also be used; however, in the presence of a phosphate buffer, heparin is not necessary for precipitation of low density lipoprotein. Routine assays were found to be reproducible to within 10%. The increase in high density lipoprotein radioactivity is linear with time until about 25% of the initial low density lipoprotein radioactivity is transferred. The lipoproteins may also be separated by ultracentrifugation. When these two methods were compared, the results agreed within 10%. The precipitation technique is much faster (less than 15 min compared with 18 hr) and simpler. Ellsworth et al. (1982) used a small heparin-Sepharose column to separate high density lipoprotein from low density and very low density lipoprotein by adjusting the concentration of NaCl and SDS in the eluting buffer. The results obtained by this method were the same as those gained by ultracentrifugation. Plasma lipoproteins are readily labeled with cholesteryl ester or triglyceride. Fresh plasma is incubated with sonicated dispersions of phosphatidylcholine and radiolabeled apolar lipid in the presence of diethyl

QUANTITATION OF LIPID TRANSFER ACTIVITY

213

p-nitrophenyl phosphate which suppresses 1ecithin:cholesterolacyltransferase activity. The labeled lipoproteins are then isolated by ultracentrifugation. It should be confirmed that the labeled lipids are actually incorporated into the lipoproteins and are not present in the isolated fractions as separate particles. In our studies, low density and high density lipoproteins labeled in this manner demonstrated elution profiles identical to unlabeled lipoproteins on a Bio-Gel A-5m column (Morton and Zilversmit, 1981). Lipoprotein substrates may also be labeled by the addition of radiolabeled cholesterol in ethanol or acetone to fresh plasma which converts the cholesterol to cholesteryl ester by 1ecithin:cholesterolacyltransferase. After the individual lipoprotein classes are isolated, labeled free cholesterol may be removed from high density lipoprotein by incubating with excess, nonlabeled, low density lipoprotein. Similarly, low density or very low density lipoprotein may be incubated with red blood cells for the removal of labeled free cholesterol. Barter et al. (1982) compared the effect of labeling human high-density lipoprotein by a cholesteryl ester transfer method and by the 1ecithin:cholesterol acyltransferase method. They found that 1ecithin:cholesterol acyltransferase labeling of the high density lipoprotein increased the cholesteryl ester content of the high density lipoprotein particles and decreased the fractional rate of cholesteryl ester exchange. Lipoproteins have also been used for studying protein-stimulated phospholipid transfer between lipoproteins (Ihm et al., 1982), and between lipoproteins and vesicles (Damen et al., 1981, 1982).Care must be exercised when high density lipoprotein is used as a substrate in an exchange reaction with vesicles, since vesicles may be disrupted and net transfer of lipid results (Damen et al., 1981).A high density lipoproteinvesicle assay with appropriate precautions has been employed by Damen et al. (1981). I. ADDITIONAL ASSAYS

In addition to the assays described in this section, other assays involving the separation of substrates have been used to quantitate lipid transfer activity. These assays differ primarily in their choice of substrates and the method of separation. Additional biological membranes, which have been used to quantitate transfer activity, include chloroplasts (Tanaka et al., 1980), retinol rod disc membranes (Dudley and Anderson, 1978), intracytoplasmic membrane derived from Rhodopseudomonas sphaeroides (Cohen et al., 1979), myelin (Ruenwongsa et al., 1979), and lamellar bodies (Tsao, 1979).Biological membranes have been isolated by sucrose

2 14

J O H N R. WETTERAU AND DONALD B. ZILVERSMIT

(Patumraj et al., 1982), Percoll (Tanaka et al., 1980),and Metrizamide (Patumraj et al., 1982) gradient centrifugation. In addition, membranespecific gamma globulin fractions have been used to separate biological membranes from artificial membranes (Cohen et al., 1979).

IV. SPECTROSCOPIC ASSAYS Lipid transfer activities are generally determined by assays involving separation of donor and acceptor particles. These techniques have some distinct disadvantages. T h e time-consuming process of separating donors and acceptors is especially troublesome in a kinetic analysis of a transfer reaction when it is necessary to measure the time dependence of lipid transfer reactions. In addition, the separation of donor and acceptor membranes requires that the two particles differ in some respect. Some of the spectroscopic assays used to quantitate lipid transfer provide a means of continuously measuring the transfer without destroying the incubation mixture. In addition, the rate of lipid transfer between two nearly identical membranes can be measured. 1. Electron Spin Resonance Vesicles prepared from spin-labeled phospholipids show a broadening of the electron spin resonance (ESR) spectra due to spin-spin exchange interactions (Maeda and Ohnishi, 1974). The exchange of phospholipid molecules between labeled and unlabeled vesicles is accompanied by the disappearance of this spin- spin exchange broadening. Machida and Ohnishi (1978) used this technique to measure phospholipid transfer catalyzed by the beef liver phosphatidylcholine exchange protein. Donor phospholipid (50 nmol), labeled with nitroxide stearic acid, was mixed with 1 pmol of acceptor vesicle phospholipid and exchange protein. T h e increase with time in the peak height of the low field line of the three-line spectrum was used to determine the transfer rate. Megli et al. (1981) assayed lipid transfer by utilizing the second derivative mode of the spectrometer which exhibited a higher resolution in that mode. Changes in the second derivative spectrum were proportional to changes in the first derivative spectrum.

2. Fluorescence Somerharju et al. (1981) developed an assay for phospholipid exchange activity with the fluorescent phospholipid derivative, 1-acyl-2-parinaroylphosphatidylcholine. When vesicles of this fluorescent phospholipid are

QUANTITATION OF LIPID TRANSFER ACTIVITY

215

prepared, the molecules are self-quenched. When these vesicles are mixed with nonlabeled phosphatidylcholine vesicles and transfer protein, fluorescence increases as the “labeled” phospholipid molecules are inserted into the acceptor phosphatidylcholine vesicles. In a typical experiment, the donor vesicles contained 10 nmol parinaroylphosphatidylcholineand 0.5 nmol phosphatidic acid; the acceptor vesicles contained 200 nmol phospholipid. The continuous increase in fluorescent intensity with time measures the transfer rate in arbitrary units. This assay is very sensitive for determining initial transfer rates. The preparation and handling of the fluorescent phospholipid derivatives is cumbersome, however. Special care has to be taken to prevent degradation of the polyenoic fatty acid. Furthermore, most spectroscopic techniques require calibration to equate the spectral changes with the amount of lipid transfer. It is also important to know whether the rate of transfer of this and other fluorescent and spin-labeled phospholipids is comparable to the transfer rate of more physiological phospholipid molecules. In addition to parinaroyl phospholipids, pyrene fatty acid derivatives may be used. Such phospholipids have a concentration-dependent emission spectrum (Roseman and Thompson, 1980).At low concentrations of the derivative within the bilayer, the fluorescence is maximal at a wavelength below 400 nm. At higher concentrations of the derivative, the excited state monomers can associate with a ground state monomer to form a dimer complex, or eximer, in a diffusion-controlled process. The maximum emission wavelength of the eximer shifts to approximately 470 nm. The ratio of the eximer to monomer fluorescent intensity is proportional to the concentration of the probe molecules within the bilayer. Pownall et al. (1982) incorporated 1 mol% pyrene-labeled sphingomyelin into lipoprotein complexes and measured its rate of protein stimulated transfer to an excess of acceptor particles. Throughout the transfer reaction, monomer emission represents labeled lipid found in both the donor and acceptor complexes, while eximer emission represents labeled lipid in the donor. The eximer fluorescence can therefore be used as a measure of the pyrene-labeled lipid concentration in the donor complex. Roseman and Thompson (1980) used a higher concentration of pyrene-labeled phospholipids (3.8 mol%)in phosphatidylcholine vesicles to measure the rate of spontaneous transfer of these fluorescent phospholipids. An equation was derived that relates the sum of the monomer and eximer intensities in the donor and acceptor vesicles to the extent of lipid transfer. This assay requires a calibration procedure that relates the change in the concentration of the fluorescent derivative in the bilayer to the change in the monomer and eximer fluorescent intensities. Oxygen

216

J O H N R. CVETTERAU A N D DONALD B. ZILVERSMIT

quenching is not a problem in this assay because the ratio of the eximer to monomer fluorescent intensity, which is measured in this assay, is not affected by the presence of oxygen (Correa-Freire et al., 1982). T h e lower concentration of fluorescent lipid required in the pyrene fluorescent assays allows greater flexibility in the lipid composition of the donor particles.

3. Nuclear Magnetic Resonance The induced shift in the ' H and "P nuclear magnetic spectra of phosphatidylcholine unilamellar vesicles by the paramagnetic ions, Pr'+ and Eu", has been used to measure phospholipid exchange (Barsukov et al., 1975). This shift depends on the environment of the phospholipid molecules and is affected by vesicle composition. A linear relationship exists between the paramagnetic induced shift and the concentration of phosphatidylinositol in a phosphatidylcholine vesicle. This relationship was used to quantitate the protein-stimulated exchange of phospholipid between phosphatid ylcholineand phosphatidylinositol vesicles. This method is potentially adaptable to other lipids. A major disadvantage of this technique for quantitating lipid transfer is the large amount of lipid vesicles required for the measurements. Also, 'H nuclear magnetic resonance (NMR) must be done in a D 2 0 solution. This technique has been useful for obtaining information about the mechanism of lipid transfer and the interaction between the transfer protein and the membrane.

4. Lipid Phase Transition Measurements T h e thermotropic phase transition temperature of a vesicle composed of a mixture of dipalmitoyl and dimyristoyl phosphatidylcholine (DPPC and DMPC, respectively) is intermediate between the phase transition temperatures of the single lipid vesicles and reflects the relative concentrations of the two lipids in the vesicle. This can be used to determine the rate of exchange of phosphatidylcholine between two unilamellar vesicles of initially pure phospholipid. This experimental approach has been used previously to study the spontaneous transfer of phospholipids between artificial membranes (Martin and MacDonald, 1976; Duckwitz-Peterlein et al., 1977). Xu et al. ( 1982) used the fluorescence anisotropy of diphenylhexatriene (DPH) in the phosphatidylcholine bilayer to measure the change in the physical state of DPPC and DMPC vesicles upon mixing in the presence of transfer protein. T h e fluorescent measurements were recorded at a temperature intermediate between the phase transition of the two initially pure vesicles. By using flow cytometry, it was possible to measure the fluorescence

QUANTITATION OF LIPID TRANSFER ACTIVITY

217

anisotropy of the DPH in individual vesicles and show that some vesicles are not affected by the transfer protein. These resuIts were confirmed by electron microscopy. It was postulated that a nucleation step in the exchange of phospholipid was necessary. The initial transfer of lipid into a vesicle would in effect be an impurity in the bilayer that could facilitate further transfer. This technique yields important information regarding the transfer process but, like the NMR technique, is not practical for routine quantitation of lipid transfer.

V. ASSAYING CRUDE EXTRACTS FOR TRANSFER ACTIVITIES The most common approach to determine the lipid transfer activities in mammalian tissue is to homogenize the tissue and examine the transfer activities in a postmicrosomal supernatant. Microsomes can be sedimented by a 105,OOOg centrifugation or by adjusting the pH of the tissue homogenate to 5.1 and sedimenting the flocculated microsomes by lowspeed centrifugation. The transfer activities of the supernatant can then be assayed by conventional techniques. To understand the physiological role of the transfer proteins, it is important to use assays for the individual lipid transfer proteins present in the cytosol. There are several approaches that can be used to quantitate specific proteins and activities. Information about the contribution of specific transfer proteins to the total transfer activity of a crude fraction can be obtained by using standard assay techniques. For example, the bovine liver contains the phosphatidylcholine-specific(Kamp et al., 1973),phosphatidylinositol-specific (Helmkamp et al., 1976),and the nonspecific transfer proteins (Crain and Zilversmit, 1980b). By controlling the substrates and the conditions of the assay, it is possible to differentiate between these transfer proteins. The nonspecific transfer protein in bovine liver is the only one of these three transfer proteins capable of transferring phosphatidylethanolamine. Thus, by measuring phosphatidylethanolamine transfer, one is in effect assaying the nonspecific lipid transfer activity. Knowing the relative specificity of this protein for the different phospholipid classes in the assay, it is possible to estimate the contribution of this protein to the sum of phosphatidylcholine transfer activity for the three transfer proteins. The nonspecific transfer protein may be selectively inhibited by increasing the ionic strength of the assay buffer (Crain and Zilversmit, 1980b). Similar approaches may be used to determine the contribution of the phosphatidylinositol-and phosphatidylcholine-specifictransfer proteins to the total phosphatidylinositol and phosphatidylcholine transfer activity. The lipid transfer activity of specific transfer proteins has been shown

218

JOHN R. WETTERAU AND DONALD B. ZILVERSMIT

to be sensitive to the lipid composition of the substrates (Hellings et al., 1974;Wirtz et aI., 1979; Helmkamp, 1980b).Adjusting the lipid composition of the substrates may prove useful for the measurement of individual transfer activities. Antibodies raised against transfer proteins may be used to quantitate transfer activities. The decrease in the transfer activity as measured by a standard assay in a pH 5.1 or 105,OOOg supernatant preincubated with transfer protein-specific antiserum would indicate the contribution of the inhibited protein to the total activity. Helmkamp et al. (1976) used this technique to determine the relative contribution of the phosphatidylcholine-specificand phosphatidylinositol-specific transfer proteins to the total phosphatidylinositol and phosphatidylcholine transfer activities in the bovine brain, heart, and liver. Inhibitors of transfer proteins may be present in the cytosol or plasma. Morton and Zilversmit (198 1) have partially purified a human plasma inhibitor of the triglyceride and cholesteryl ester transfer activities. It is a protein with a molecular weight of approximately 35,000. Crude extracts of potato tuber and the bacteria Rhodopseudomonas sphaeroides appear to be devoid of transfer activity (Kader, 1975; Cohen et al., 1979),but activities could be detected after the initial purification steps. The possible presence of transfer protein inhibitors in cytosol may require the quantitation of transfer proteins by techniques that do not rely on traditional transfer assays. Teerlink et al. (1981) used a doubleantibody radioimmunoassay specific for the phosphatidylcholine transfer protein in rat liver. The assay was effective over a range of 4 to 50 ng of transfer protein. The levels of phosphatidylcholine transfer protein in three lines of Morris hepatomas was found to be in agreement when quantitated by radioimmunoassay (Teerlink et al., 1981) or immunotitration (Poorthuis et al., 1980). In normal liver cells, the amount of phosphatidylcholine transfer protein measured with a transfer activity assay was only about one-third of the same protein measured by radioimmunoassay. Apparently not all of the transfer protein with immunoreactivity possesses transfer activity. Possibly, modulators of transfer activity are present in normal rat liver.

VI. EFFECTS OF COMPOSITION OF THE ASSAY MIXTURE ON THE RATE OF LIPID TRANSFER Many different assays are used to quantitate lipid transfer activity. Some are more suited for routine assays because they are easy to perform and versatile. Other assays more effectively answer questions about the mech-

QUANTITATION OF LIPID TRANSFER ACTIVITY

219

anism and control of the protein-catalyzed transfer. There is no standard assay that compares different proteins in different laboratories. Variations in pH, temperature, ionic strength of the buffer, physical stateof the lipid bilayers, composition of the membranes, and relative concentration of acceptor and donor particles may affect the assay results. Examples will be examined in the following section. 1. Ratio of Acceptor to Donor Particles

A complex relationship exists between the initial rate of lipid transfer and the concentration of acceptor and donor particles (Van den Besselaar et al., 1975; Wirtz et al., 1979).An example of this is illustrated in Figure 1. At a given concentration of acceptor, a low concentration of donor results in a low rate of lipid transfer. When the concentration of donor is increased, so is the transfer rate until a maximum in the transfer rate is reached. A further increase in the donor concentration results in a decline in the transfer rate. In a typical assay, acceptor lipid is present in excess of donor lipid. For example, about four times as much exchangeable acceptor as donor lipid is used in the small unilamellar vesicle-multilamellar vesicle assay (Crain and Zilversmit, 1980b). This minimizes the backtransfer of labeled lipid from the acceptor to the donor particle during the exchange reactions while still maintaining rapid rates of lipid transfer.

O

L

a25 115 .0 a n 1.0 I MNOR CWENTRATIa Figure 1. Effect of donor particle concentration on the initial rate of lipid transfer. The computer-drawn curves are based on the kinetic model by Van den Besselaar et al. (1975) for protein-catalyzed phospholipid exchange. The kinetic constants are for, the transfer of phosphatidylcholine from liposomes containing 12 mol% phosphatidic acid to liposomes containing 2 mol%phosphatidic acid. The three curves represent three different acceptor concentrations indicated above the appropriate curves.

am

220

J O H N R. WETTERAU AND DONALD B. ZILVERSMIT

2. Composition of Acceptor and Donor Particles An alteration in the composition of the acceptor or donor particles may alter the rate at which lipids are transported. The change may be the result of a change in membrane properties, such as fluidity or charge, or a competitive effect. In transfer reactions, lipid molecules compete with each other. In addition, transfer proteins preferentially transfer particular classes of lipids. When the lipid composition of a substrate is modified, this competition between molecules is altered and as a consequence, the lipid transfer rate may change. A. EFFECTS OF FATTY ACYL CHAIN COMPOSITION

Helrnkamp (1980a) studied the effect of the fatty acid composition of the acceptor lipid on the stimulation of phosphatidylinositol transfer from rat liver microsomes to phosphatidylcholine vesicles by bovine brain exchange protein. Acceptor vesicles containing egg phosphatidylcholine or dioleoyl phosphatidylcholine gave approximately the same transfer activity, whereas dielaidoyl phosphatidylcholine or dimyristoyl phosphatidylcholine vesicles produced lower transfer rates. Zborowski and Demel (1982) used the same protein and measured the rate of transfer of phosphatidylinositol from a monolayer to phosphatidylcholine vesicles. Vesicles of egg, dioleoyl, dielaidoyl, and dipalmitoyl phosphatidylcholine, even below its phase transition temperature, all gave equivalent transfer rates. However, a reduced rate was found when dimyristoyl and dilinoleoyl phosphatidylcholine, and other phosphatidylcholines with two polyunsaturated fatty acids, were used. Table IV shows a comparison of the transfer activities measured in the t w o assays. The transfer rates are expressed as a percent of the transfer rate obtained with egg phosphatidylcholine acceptor vesicles. Bozzato and Tinker (1982) found biphasic kinetics of phosphatidylcholine transfer from egg phosphatidylcholine donor vesicles to dipalmitoylphosphatidylcholine:dipalmitoylphosphatidylglycerol (95:5, mol%)multilamellar acceptor vesicles. Biphasic kinetic rates are normally associated with two different pools of phospholipid, a rapidly exchangeable pool and a slowly exchangeable pool, which is due to phospholipid association with membrane proteins or to phospholipid being located in the inner monolayer. In this case, the slow phase of exchange occurred before the labeled lipid of the outer monolayer was exhausted. As the exchange reaction proceeded, the DPPC was incorporated into the outer monolayer of the egg phosphatidylcholine vesicles which resulted in a change in the properties of the vesicles as well as the rate of lipid exchange.

QUANTITATION OF LIPID TRANSFER ACTIVITY

22 1

TABLE IV Transfer Activity of the Bovine Brain Phospholipid Exchange Protein with Acceptor Vesicles of Varying Composition" Donor particle

Acceptor vesicle phosphatidylcholine

Microsomes'

Monolayer'

Egg Dioleoyl Dielaidoyl Dilinoleoyl Dimyristoyl Dipalmitoyl (32°C)

100 99 62 n.d.d 16 n.d.

100 81 97 65 42 99

"Transfer rate expressed relative to that obtained with egg phosphatidylcholine vesicles. 'From Helmkamp (1980). "From Zborowski and Demel (1982). dn.d. = not determined. B. EFFECTS OF PHOSPHOLIPID HEADGROUPS

The effect of different phospholipid head groups on the proteinstimulated transfer by phosphatidylcholine- and phosphatidylinositolspecific proteins has been studied. Contradictory results were obtained for the effect of acidic phospholipids on the transfer of phospholipid by the phosphatidylcholine exchange protein from beef liver. DiCorleto et al. (1977) used small unilamellar vesicle-mitochondria and small unilamellar vesicle-multilamellar vesicles to study the effect of varying amounts of acidic phospholipids incorporated into phosphatidylcholine donor vesicles. Up to 20 mol% phosphatidic acid or phosphatidylinositol in the donor was found to stimulate the transfer of phosphatidylcholine in both assay systems. Wirtz et al. (1979) and Hellings et al. (1974) found different results for the phosphatidylcholine exchange protein with unilamellar and multilamellar vesicles. In these assays, the incorporation of acidic phospholipids (phosphatidic acid or phosphatidylinositol)into the donor particles had an inhibitory effect on the rate of phosphatidylcholine transfer. Helmkamp (1980b) studied the phosphatidylinositol-specificexchange protein from bovine brain. The incorporation of phosphatidic acid, phosphatidylserine, or phosphatidylglycerol into small unilamellar acceptor vesicles had little effect on the transfer of phosphatidylinositol from microsomes. However, when stearylamine was incorporated into the vesicles, the rate of transfer was decreased. A two- to fourfold stimula-

222

J O H N R. WETTERAU AND DONALD B. ZILVERSMIT

tion in the transfer rate was observed with phosphatidylethanolamine added to the vesicles. Interestingly, the ratio of phosphatidylcholine to phosphatidylinositol transferred was affected by the presence of phosphatidylethanolamine, a phospholipid which is not transferred by the protein.

3. Physical State of Substrate Lipid The physical state of the phospholipid in the substrate will affect the rate of lipid transfer. DiCorleto and Zilversmit ( 1977) reported that phosphatidylcholine multilamellar vesicles were not efficient substrates for phospholipid exchange proteins. Unilamellar vesicles of the same composition are excellent substrates. Similar results were found by Machida and Ohnishi (1980) with an ESR assay. They attributed this difference in reactivity to the packing arrangement of the phospholipid molecules in the small unilamellar vesicles with a high degree of bilayer curvature. Dicorleto and Zilversmit (1979) found that ether evaporation vesicles with diameters greater than 1000 A and small unilamellar vesicles, prepared by cholate dialysis, showed similar rates of protein-stimulated phospholipid transfer. These results argue against the concept that the difference in curvature between multilamellar and unilamellar vesicles is responsible for the differences in transfer activity. Kasper and Helmkamp (1981) used egg and dimyristoyl phosphatidylcholine vesicles as acceptor membranes in a transfer assay. By varying the temperature of the exchange reaction and thereby the physical state of the vesicles, they showed that the protein interacts preferentially with vesicles in the liquid-crystalline state. 4. Exchangeable Lipid Pool Knowledge of the size of the exchangeable pool of lipid in a substrate is necessary to determine the rate constants of the exchange process and establish the specificity of a transfer protein for different classes of phospholipids. T h e size of the exchangeable pool of each class of phospholipid must be determined individually because phospholipids are often asymmetrically distributed across biological and artificial membranes (Op den Kamp, 1979). The exchangeable lipid pool in a donor particle can be determined by measuring the labeled lipid remaining in the donor membrane after prolonged incubation of radiolabeled donor membranes with an excess of acceptor particles and transfer protein (Zilversmit and Hughes, 1976). This technique has also been used to determine the asymmetric distribution of phospholipid molecules and their rate of transbilayer movement

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in artificial vesicle (Low and Zilversmit, 1980), microsome (Zilversmit and Hughes, 1977), and erythrocyte (Crain and Zilversmit, 1980c) membranes. The amount of lipid that is subject to rapid exchange appears to vary with the composition of the multilamellar vesicles and their method of preparation. DiCorleto and Zilversmit ( 1977) found that multilamellar vesicles of phosphatidylcho1ine:phosphatidylethanolamine:cardiolipin (70:25:5, mol%) contained 7% of exchangeable phosphatidylcholine. Wirtz et al. (1979) found that 4.5% of the phosphatidylcholine of egg phosphatidylcho1ine:phosphatidicacid (90:10, mol%) multilamellar vesicles was fully exchangeable. Bozzato and Tinker (1982) found that 8.5 5 2% of the phospholipid of annealed dipalmitoylphosphatidylcholine: dipalmitoylphosphatidylglycerol(95:5, mol%) multilamellar vesicles was exchangeable, whereas the phospholipid exchange of nonannealed vesicles was 11 5 3%. References Bangham, A. D., Standish, M. M., and Watkins, J. C. (1965),J. Mol. Biol.,13, 238-252. Barsukov, L. I., Shapiro, Y. E., Viktorov, A. V., Volkova, V. I., Bystrov, V. F., and Bergelson, L. D. (1975), Chem. Phys. Lip&, 14, 211-226. Barter, P. J., Gorjatschko, L., and Hopkins, G. J. (1982), Biochim. Biophys. Acta, 710, 349358. Bloj, B. and Zilversmit, D. B. (1976), Biochemistry, 15, 1277-1283. Bloj, B. and Zilversmit, D. B. (1977),J. B i d . Chem., 252, 1613-1619. Bloj, B. and Zilversmit, D. B. (1981a), Mol. Cell. Biochem., 40, 163-172. Bloj, B. and Zilversmit, D. B. (1981b),J. Biol. Chem., 256, 5988-5991. Bozzato, R. P. and Tinker, D. 0. (1982), Can. J. Biochem., 60, 409-418. Butler, M. M. and Thompson, W. (1975), Biochim. Biqphys. Acta, 388, 52-57. Cobon, G. S., Crowfoot, P. D., Murphy, M., and Linnane, A. W. (1976), Biochim. Biophys. Acta, 441, 255-259. Cohen, L. K., Leuking, D. R. and Kaplan, S. (1979),J. Biol. Chem., 254, 721-728. Correa-Freire, M. C., Barenholz, Y., and Thompson, T . E. (1982), Biochemistry, 21, 12441248. Crain, R. C. and Zilversmit, D. B. (1980a), Biochim. Biophys. Acta, 620, 37-48. Crain, R. C. and Zilversmit, D. B. (1980b), Biochemistry, 19, 1433-1439. Crain, R. C. and Zilversmit, D. B. (1980c), Biochemistry, 19, 1440-1447. Damen, J., Regis, J., and Scherphof, G. (1981), Biochim. Biophys. Acta, 665, 538-545. Damen, J., Regis, J., and Scherphof, G. (1982), Biochim. Biophys. Acta, 712, 444-452. Demel, R. A,, Kalsbeek, R., Wirtz, K. W. A., and Van Deenen, L. L. M. (1977), Bzochzm. Biophys. Acta, 466, 10-22. Demel, R. A., Van Bergen, B. G. M., Van den Eeden, A. L. G., Zborowski, J., and Defize, L. H. K. (1982), Biochim. Biophys. Acta, 710, 264-270.

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DiCorleto, P. E. and Zilversmit, D. B. (1977),Biochemistry, 16, 2145-2150. DiCorleto, P. E. and Zilversmit, D. B. (1979), Biochim. Biophys. Acta, 552, 114- 119. DiCorleto, P. E., Fakharzadeh, F. F., Searles, L. L., and Zilversmit, D. B. (1977), Biochim. Biophys. Actu, 468, 296-304. DiCorleto, P. E., Warach, J. B., and Zilversmit, D. B. ( 1 9 7 9 ) ~Biol. . Chem.,254,7795-7802. Douady, D., Kader, J. C., and Mazliak, P. (1980),Plant Sci. Lett., 17, 295-301. Douady, D., Grosbois, M., Guerbette, F., and Kader, J. C . (1982),Biochim.Biophys. Acta, 710, 143-153. Duckwitz-Peterlein, G., Eilenberg, G., and Overath, P. (1977), Biochim. Bzophys. Acta, 469, 31 1-325. Dudley, P. A. and Anderson, R. E. (1978), FEBS Lett., 95, 57-60. Dyatlovitskaya, E. V., Timofeeva, N. G., and Bergelson, L. D. (1978),J. Biochem., 82, 463 -47 1. Ehnholm, C. and Zilversmit, D. B. (1972), Biochim.Biophys. Actu, 274, 652-657. Ellsworth, J. L., McVittie, L., and Jackson, R. L. (1982),J. Lipid Res., 23, 653-659. Green, D. E., Lester, R. L., and Ziegler, D. M. (1957),Biochim. Biophys. Actu, 23, 516-524. Hellings, J. A., Kamp, H. H., Wirtz, K. W. A,, and Van Deenen, L. L. M. (1974), Eur.J. Biochem., 47, 601-605. Helmkamp, G. M., Jr. (1980a), Biochemistry, 19, 2050-2056. Helmkamp, G. M., Jr. (1980b),Biochim. Biophys. Acta, 595, 222-234. Helmkamp, G. M., Jr., Harvey, M. S., Wirtz, K. W. A,, and Van Deenen, L. L. M. (1974), J. Biol. Chem., 249, 6382-6389. Helmkamp, G. M., Jr., Nelemans, S. A., and Wirtz, K. W. A. (1976),Biochim. Biophys. Acta, 424, 168-182. Ihm, J . , Ellsworth, J . L., Chataing, B., and Harmony, J. A. K. (1982)J. Biol. Chem., 257, 48 18-4827. Johnson, L. W. and Zilversmit, D. B. (1975), Biochim. Biophys. Acta, 375, 165-175. Kader, J . C. (1975), Biochim. Bisphys. Acta, 380, 31-44. Kamp, H. H. and Wirtz, K. W. A. (1974), in Method in Enzymology, Vol. 32, Academic Press, New York, pp. 140- 146. Kamp, H. H., Wirtz, K. W. A., and Van Deenen, L. L. M. (1973),Bzochim.Biophys. Ada, 318, 3 13-325. Kasper, A. M. and Helmkamp, G. M., Jr. (1981), Biochemistty, 20, 146-151. Low, M. G. and Zilversmit, D. B. (1980), Biochim. Biophys. Actu, 596, 223-234. Lumb, R. H., Kloosterman, A. D., Wirtz, K. W. A., and Van Deenen, L. L. M. (1976),Eur.J. Biochem., 69, 15-22. Lutton, C. and Zilversmit, D. B. (1976a), Lip&, 11, 16-20. Lutton, C. and Zilversmit, D. B. (1976b), Biochim. Bzsphys. Acta, 441, 370-379. Machida, K. and Ohnishi, S. (1978), Biochim. Bisphys. Acta, 509, 156-164. Machida, K. and Ohnishi, S. (1980). Biochim. Biophys. Acta, 596, 201-209. Maeda, T. and Ohnishi, S. (1974), Biochem. Biophys. Res. Commun., 60, 1509-1516. Martin, F. J . and MacDonald, R. C. (1976), Biochemistry, 15, 321-327. McKay, H. A. C. (1938), Nature, 142, 997-998.

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McLean, L. R. and Phillips, M. C. (1981), Biochemistry, 20, 2893-2900. Megli, F. M., Landriscina, C., and Quagliariello, E. (1981), Biochim. Biophys. Acta, 6 4 0 , 274-284. Metz, R. J. and Radin, S. (1982),J. Biol. Chem., 2 5 7 , 12901-12907. Moonen, P., Akeroyd, R., Westerman, J., Puijk, W. C., Smits, P., and Wirtz, K. W. A. (1980), Eur. J . Biochem., 1 0 6 , 279-290. Morton, R. E. and Zilversmit, D. B. (1981),J. B i d . Chem., 2 5 6 , 11992-1 1995. Morton, R. E. and Zilversmit, D. B. (1982),J. LipidRes., 2 3 , 1058-1067. O p den Kamp, J. A. F. (1979), Annu. Rev. Biochem., 48, 47-71. Pattnaik, N. M., Montes, A., Hughes, L. B., and Zilversmit, D. B. (1978), Biochim. Biophys. Actu, 530, 428-438. Patumraj, K., Could, A., Subrarnaniam, S., and Slaby, F. (1982),Biochim. Biophys. Actu, 6 9 1 , 37-43. Poorthuis, B. J. H. M., van der Krift, T. P., Teerlink, T., Akeroyd, R., Hostetler, K. Y., and’ Wirtz, K. W. A. (1980), Biochim. Biophys. Actu, 600, 376-386. Poorthuis, B. J. H. M., Glatz, J. F. C., Akeroyd, R., and Wirtz, K. W. A. (1981), Biochim. Biophys. A&, 6 6 5 , 256-261. Possmayer, F. (1974), Bruin Res., 7 4 , 167- 174. Pownall, H. J., Hickson, D., Gotto, A. M., Jr., and Massey, J. B. (1982),Biochim.Biophys.Actu, 7 1 2 , 169-176. Rajaram, 0. V., White, G. H., and Barter, P. (19801,Biochim. Biophys. Actu, 6 1 7 , 383-392. Robinson, M. E., Wu, L. N. Y., Brumley, G. W., and Lurnb, R. H. (1978), FEES Lett., 8 7 , 41-44. Roseman, M. A. and Thompson, T. E. (1980), Biochemistry, 1 9 , 439-444. Ruenwongsa, P., Singh, H., and Jungalwala, F. B. (1979),J. B i d . Chem., 2 5 4 , 9385-9393. Sasaki, T. and Sakagami, T. (1978),Biochim. Biophys. Actu, 5 1 2 , 461-471. Somerharju, P., Brockerhoff, H., and Wirtz, K. W. A. (1981), Biochim. Biophys. Acta, 6 4 9 , 52 1-528. Tanaka, T., Ohnishi, J., and Yamada, M. (1980),Biochem. Biophys. Res. Commun., 9 6 , 394399. Teerlink, T.,Poorthuis, B. J. H. M., Van der Krift, T. P., and Wirtz, K. W. A. (1981),Biochim. Biophys. Acta, 6 6 5 , 74-80. Tsao, F. H. C. (1979), Biochim. Biophys, Acta, 5 7 5 , 234-243. Van den Besselaar, A. M. H. P., Helmkamp, G. M., Jr., and Wirtz, K. W. A. (1975), Biochemistry, 1 4 , 1852- 1858. van Golde, L. M. G., Oldenborg, V., Post, M., and Batenburg, J. J. (1980),J. B i d . Chem.,255, 601 1-6013. Van Meer, G., Poorthuis, B. J. H. M., Wirtz, K. W. A., O p den Karnp, J. A. F., and Van Deenen, L. L. M. (1980), Eur J. Biochem., 1 0 3 , 283-288. Wirtz, K. W. A. (1982), in Molecular Biology of Lipid-Protein Interactions (0.H. Griffth and P. C . Jost, Eds.), Vol. 1, Wiley-Interscience, New York, pp. 151-231. Wirtz, K. W. A. and Zilversmit, D. B. (1968),J. B i d . Chem., 2 4 3 , 3596-3602. Wirtz, K. W. A., Karnp, H. H., and Van Deenen, L. L. M. (1972),Biochim. Bi@hysActu, 2 7 4 , 606-6 17.

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Wirtz, K. W. A., Vriend, G., and Westerman, J , (1979), Eur. J. Biochem., 94, 215-221. Wirtz, K. W. A., De Vaux, P. F., and Bienvenue, A. (1980), B i o c h m i s t ~19, , 3395-3399. Xu, Y. H., Ruppel, D., Ziegler, H., Hartmann, W., and Calla, H. J . (1982),Biochim. Biophys. Acta, 689, 437-443. Zborowski, J. and Demel, R. A. (1982), Biochim. BiophyJ. Acta, 688, 381-387. Zilversmit, D. B. and Hughes, M. E. (1976), Methods Memb. Biol., 7, 211-259. Zilversmit, D. B. and Hughes, M. E. (1977), Biochim. Biophys. Acta, 469, 99- 100.

Edited by David Glick Copyright © 1984 John Wiley & Sons, Inc.

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METHODS OF BIOCHEMICAL ANALYSIS

VOLUME 30

Measurement of Oxygen Consumption by the Spectrophotometric Oxyhemoglobin Method OCTAVIAN BARZU, Unite' de Biochimie des Re'gubtions CeEEuEaires, Dlpartement de Biochimie et Gknktique Mole'culaire, Institut Pasteur, Paris, France

I. 11.

.......................................

228

..................... ..........

230 233 234 234 235 236 242 242 243 244

ween the Rate of HbOp

Deoxygenation and the Oxygen Consumption

......................

111. Purification, Characterization, and Storage of the Oxygen Donors

IV. V. VI.

VII. VIII. IX.

1. Preparation of Hemolysate ........... 2. Chromatographic Purification of HbOp ..................... ..................... ........................... 3. Determination of HbOz Concentration . Instrumentation ............................................................................................ ........................... Technical Procedure .................. 1. Single Measurements of t 2. Repetitive Measurements of the Oxygen Consumption ................. Calculation of Experimental Data ........................... ........................... .......... 1. Comparative Measurements of Oxygen Con Spectrophotometrically Calibrated Substrates .. 2. Measurement of HbOp Deoxygenation at Di ............................................ ,.... Concentrations 3. Polarographic E Consumption in the Presence of the Oxygen Donor .................................................. Simultaneous Determination of Several Photometric Parameters .............. Determination of Hydrogen Peroxide-Generating Oxidases Applications of the Oxyhemoglobin Method for Measurement of ............* ............................. Oxygen Consumption ................................. 1. Determination of Cytochrome Oxid ctivity in Human Liver ...................................................... Homogenates 2. Determination ylalanine Hydroxylase Activity .. 3. Determination of NAD-Linked Dehydrogenases ...........................

227

244 246 246 247 249 250

250 25 1 252

228

OCTAVIAN 4.

BARZU

Measurement of Mitochondria1 Respiration and Oxidative Phoshorylation .....

253 255 256 258 259

9. Determination of R

............................ Abbreviations ................................................................................................ Acknowledgments ............. ................. ........................... References ......

261 262 263 263 264 264

I. INTRODUCTION The measurement of oxygen consumption (uptake) remains an important tool for investigating the oxidative processes in a wide variety of biological materials. “Cellular respiration” results from the activity of a great number of enzymes, either soluble or membrane bound, with various specific activities and different affinities for oxygen. Hence, formidable problems are encountered when attempting to draw conclusions from experiments performed with crude systems. However, the suitable selection of biological material under investigation, or utilization of highly specific inhibitors, could overcome the lack of specificity of oxygen consumption measurements. Since many reactions can be directly or indirectly linked into consumption of oxygen in a well-defined relationship to the oxidized substrate, the design and use of oxygen sensors has had a sharp increase the last two decades. Despite the great diversity of principles and techniques devised, no one can be considered as ideal. Consequently, a new method for measuring oxygen consumption does not necessarily supplant an older method but rather enables new oxidative systems to be studied. Their appropriate use as function of sensitivity, precision, and simplicity enlarges the possibilities of investigating the cellular oxidative processes or assay of different substrates of biological significance. The manometric procedure for measuring oxygen consumption, first introduced by Warburg is still the most accurate method when utilized under appropriately chosen conditions (Dixon, 1943; Umbreit et al., 1964; Slater, 1967).The serious limitations in manometric techniques are the difficulty in following rapid changes in the gas phase and the relatively long time necessary for both equilibration and measurement.

MEASUREMENT OF OXYGEN CONSUMPTION

229

Development of membrane-covered polarographic oxygen sensors gradually replaced the manometric technique by polarography (Chance and Williams, 1956; Clark, 1956; Longmuir, 1957; Kunze and Lubbers, 1973).The use of oxygen electrodes simultaneously with pH, ion-specific, and spectrophotometric measurements clarified many aspects of mitochondrialoxidative phosphorylation (Pressman, 1967; Merola et al., 1971; Starlinger and Lubbers, 1973). The development and design of polarographic oxygen sensors in both closed and open systems are described in many reviews (Davies, 1962; Estabrook, 1967; Degn et al., 1980; Fatt, 1976; Lessler, 1982). Both manometric and oxigraphic procedures have rather low sensitivity as compared with other physical methods such as spectrophotometry, fluorimetry, or radioisotopic analysis. Vaugham and Weber (1970) found that the intensity of pyrenebutyric acid fluorescence is linearly related to the oxygen concentration. Since pyrenebutyric acid was easily taken up by cells (Knoop and Longmuir, 1972; Benson et al., 1980) and by mitochondria (Snow and Jobsis, 1976) without altering the oxidative metabolism, it is one of the most sensitive candidates for indirect detection of the oxygen consumption. Many other analytical applications of this oxygen sensor are expected in the near future. The bacterial luminescence method, despite its great sensitivity, can be applied accurately only in the oxygen concentration range between and 10-8M. Hence, its usefulness is restricted to highly specialized fields like the kinetics of cytochrome oxidase-oxygen reaction (Schindler, 1964; Oshino et al., 1972; Chance et a]., 1978; Lloyd et al., 1981). The 02-sensitive colorimetric indicators occupy an intermediate position on the sensitivity scale between the manometric- polarographic and fluorescent-luminescent methods. This class of measurements contains a large number of micromolecular and macromolecular compounds, many of them being naturally occurring 02-transporting proteins. The privileged position of mammalian hemoglobin (Hb), one of the most completely characterized proteins, is due to several reasons: (1) large amounts of pigment could be easily separated from other erythrocyte proteins; (2) the cooperative binding of oxygen to the four interacting subunits makes hemoglobin an ideal 02-buffering system; and (3) the difference spectra of oxygenated and deoxygenated hemoglobin between 370 nm and 650 nm shows several wavelength ranges suitable to detect variation of oxygen content in the medium, the maximum sensitivity being achieved in the Soret band (Table I). Hill and later Davenport (quoted by Vishniac, 1957) introduced ox muscle myoglobin and Ascaris hemoglobin as a reagent for 0 2 studies on light-dependent evolution of oxygen from cell-free leaf preparations. However, systematic investi-

230

OCTAVIAN BARZU TABLE I Spectral Characteristics of Human Hemoglobin Derivatives"

Wavelength (nm)

HbOB

Hb

MetHb

415 430 436 540 560 570 576 630

125 52 34 14.3 8.5 11.9 15.3 0.2

75 133 99 10.3 12.7 11.0 9.8 1.0

6.10 3.87 3.83 3.88 3.84

-~

"Taken from Antonini and Brunori (1970), van Assendelft and Ziljstra (1975), and Waterman ( 1 978). The numbers correspond to the millimolar extinction coefficients at pH 7, in 0.05-0.1OM phosphate buffer.

gations on the suitability of hemoglobin as an indicator of oxygenconsuming reactions started only in 1967 when mitochondria1respiration was monitored in the presence of human hemoglobin. Since 1967, the use of the HbO2 method was expanded to different biological systems from whole tissues to isolated cells or purified enzymes. The main advantages of Hb02 method over other conventional procedures are the high sensitivity in the most common ranges of volumes (0.2-1.0 ml), the fast response to variation of the oxygen consumption of the samples (due to the prompt dissociation of oxygen from Hb02), and the relative simplicity of taking measurements.

11. PRINCIPLE OF THE HbOo METHOD: RELATIONSHIP BETWEEN THE RATE OF HbO2 DEOXYGENATION AND THE OXYGEN CONSUMPTION The most important characteristic of oxyhemoglobin (Hb02) method is that it operates at low concentrations of molecular oxygen, which are physiological for a variety of cells. The great number of the reactions in which molecular oxygen participates is classified into three major groups (Keevil and Mason, 1978): dioxygenase

+0 2 AH + 0 2 + BH2

A

' A02 monooxwenase

AOH

+ H20 + B

-

-

MEASUREMENT OF OXYGEN CONSUMPTION

AH2 + O2

A

+ H 2 0 2 or AH2 + 1/202

A

23 1

+ H20 (3)

In reaction ( l ) , both atoms of oxygen molecule are transferred to substrate. In reaction (2),one atom of oxygen is reduced to water and the other is transferred to substrate. Microsomal monooxygenases (called also mixed-function oxidases or P-450 enzymes) use as a reductant (BHZ), NADPH. In reaction (3), oxygen is reduced to water or hydrogen peroxide by electron-transferring oxidases. If the substrate (A, AH, or AH2) is in excess of the molecular oxygen concentration, the rate of formation of final product ( H 2 0 , H202,A 0 2 , or AOH) may be described by a . Michaelis- Menten type equation:

In the presence of a finite concentration of oxymyoglobin (MbO2) or HbO2, the total concentration of oxygen (OPt)is the sum of all oxygencontaining species (oxygen dissolved in water, oxygen contained in the pigment molecule, as well the oxygen contained in the reaction product): [02tl=

[02l+

[MbO2l+

[I3201

(5)

As the concentration of soluble oxygen decreases as it is consumed by corresponding enzymes, dissociation of 0 2 from Mb02 occurs according to the equilibrium:

Upon replacing the values for the concentrations of 0 2 by the expression obtained from the equilibrium equation for pigment oxygenation, and taking the necessary derivatives, one obtains the differential relation illustrating that the concentration of Mb02 (or HbO2) varies as a function of time in the presence of an oxygen-consuming system (BArzu and Borza, 1967; BArzu and Jebeleanu, 1971; BArzu et al., 1972).

V

232

OCTAVIAN BARZU

The last equation considers the donor molecule as monomeric (myoglobin or dissociated subunits of hemoglobin) or as tetrameric, but with cooperativity between subunits being lost (HbCPA). A slightly more complicated form of this equation might be obtained if we consider the hemoglobin tetramer, showing the normal cooperativity of subunits in binding the oxygen molecule, as expressed by Hill’sequation or by Adair’s equation in the approximations of Roughton (Biirzu and Jebeleanu, 1971; Biirzu et al., 1972; Biirzu and Satre, 1970). According to Equation (8),three factors influence the rate of pigment deoxygenation, apart from the respiratory rate of the oxygen-consuming system itself: (1) the concentration of the oxygen donor, (2) its affinity for oxygen, and (3) the affinity for oxygen of the oxidase being assayed. In the ideal case, the Thus, oxygen donor concentration is large enough and K,($S w3. the second, third, and fourth terms in the denominator of Equation (8) are much less than 1 , when y is between 0.8 and 0.1, and the deoxygenation rate of pigment is described by a pseudo-zero-order kinetics. Even in those cases where we are far from those ideal conditions ( K S = 1) or where the concentration of Mb02 or H b 0 2 is very low (and the pigment molecule acts as a simple indicator), the interval of linearity of the deoxygenation reaction is long enough to permit precise and reproducible measurements. Hence, by choosing appropriate oxygen donors and the proper region of the dissociation curve of the oxygen donor, the Hb02 method could be used for any oxygen-consuming system from animal, plant, or bacterial cells (Table 11). Since the theoretical estimations using different K: and K values were in good agreement with experimental values, one could select by appropriate simulations the most suitable oxygen donor for a considered oxidase. If in Equation (8) we assume a single y value (0.5), by a simple rearrangement one obtains:

-V- - 1 + 41K

+ 4K:

[Hhl

v0.5

t K2K

(9)

When K: K < which is a very common situation (e.g., when HbOp serves as oxygen donor for mitochondria1 cytochrome oxidase), K: K and 4K; can be neglected and Equation (9) becomes:

V

-=

v0.5

1

4

+-K W t l

V

or - - - l + v0.5

K,Hb

[Hb,] =

(10)

Therefore K t b approximates the e uilibrium constant of the hemoglobin oxygenation reaction. The K&‘ has an important practical use in estimation of the correction factor f , required for the calculation of oxygen consumption by the HbOp method.

MEASUREMENT OF OXYGEN CONSUMPTION

233

TABLE I1 Dependence of VJV Ratio As a Function of the Oxygen Donor Concentration, the Affinity of the Oxygen Donor for the Gas (K) and K: of the Oxidase" VJVb

Y

A

B

C

D

E

0.90 0.80 0.70 0.60 0.50 0.40 0.30 0.20 0.10

0.90 0.96 0.97 0.98 0.98 0.98 0.98 0.98 0.97

0.56 0.75 0.82 0.86 0.88 0.88 0.88 0.88 0.85

0.24 0.43 0.54 0.61 0.65 0.67 0.67 0.66 0.59

0.50 0.79 0.88 0.91 0.91 0.91 0.88 0.82 0.68

0.30 0.39 0.41 0.41 0.40 0.38 0.34 0.30 0.23

"Values of VJV were calculated according to Equation (8) which applies to monomeric pigments. Where hemoglobin was used as oxygen donor, the pigment oxygenation was described by a Hill-type equation (BBrzu et al., 1972)where n indicates the cooperativity between subunits. Two different oxidases were taken as examples, the mitochondria1 cytochrome oxidase (K: = 5 X lO-'M), and Aspergillus niger glucose oxidase (K: = 10-5~). bA = 3mM human hemoglobin (K = 105M-', n = 3), cytochrome oxidase; B = 0.4mM human hemoglobin (K = 105M-', n = 3), cytochrome oxidase; C = 0.lmM human hemoglobin (K = 105M-', n = 3), cytochrome oxidase; D = 0.lmM human myoglobin (K = 106M-'), cytochrome oxidase; and E = 0.4mM human hemoglobin (K = 105M-', n = 3), glucose oxidase.

111. PURIFICATION, CHARACTERIZATION, AND STORAGE OF THE OXYGEN DONORS In principle, the purification of oxyhemoglobin is an easy task since the red cells are separated from plasma by centrifugation, and hemolysis leads immediately to a solution that is 93-95% pure. Usually, the purity of H b 0 2 does not pose a problem and good results can be obtained with nonpurified hemolysates. However, when extending the spectrophotometric method to determinations such as quantitation of ethanol or simultaneous measurement of oxygen consumption and glycolysis of isolated cells, the presence of catalase and glycolytic enzymes in the HbO2 preparations becomes a serious drawback. The selection of a suitable method for Hb02 purification must take into consideration the following criteria: (1) enough hemolysate should be processed to ensure 2000- 5000 determinations of oxygen consump-

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OCTAVIAN BARZU

tion; (2) the procedure must be enough fast to avoid the oxidation of Fe2+; and (3) the purified H b 0 2 must be obtained as concentrated solution so it can be stored for 6- 12 months. One of the best candidates for meeting these requirements is Blue-Sepharose (Cibacron Blue 3G-A Sepharose CL-4B), which acts as a conventional ion-exchanger, combined with a specific affinity for dinucleotide-requiring enzymes (Thompson et al., 1975; Thompson and Stellwagen, 1976).

1. Preparation of Hemolysate Freshly collected o r outdated human blood (the choice of anticoagulant is not important) is centrifuged at 1000- 1500g for 10 min. T h e erythrocytes are washed three times with 0.9% NaCl solution. T h e white cell layer is removed by suction after the final centrifugation. T h e sedimented blood cells are lysed by 30-sec homogenization with an Ultraturrax apparatus (Janke & Kunkel, Breisgau) with 1 vol double-distilled water and 0.1 vol CC14. After 20 min of centrifugation at 2000g in a swinging bucket rotor, the hemolysate (the upper layer) is siphoned into a clean backer and then filtered through glass wool. Since C C 4 and organic solvents in general were shown to bind and even to oxidize or denaturate the HbO2 molecule in several species, the hemolysis could be performed in the absence of any organic solvents. Two vol of double-distilled water added to 1 vol of packed red cells with stirring gives complete hemolysis within 20 min. However, in this case, a high-speed centrifugation (35,00050,000g for 30 min) is required to remove the stroma. If species other than humans are envisaged to serve as an HbOp source, the reader is advised to refer to Riggs (198 1).

2. Chromatographic Purification of HbOn Home-made (Bohme et al., 1972) or commercial Blue-Sepharose in 0.02M K-phosphate buffer (pH 6.9) is packed in chromatographic column. T h e volume of the column is calculated according to the amount of HbO2 to be purified and the binding capacity of the gel. One ml of Blue-Sepharose containing 2.8 pmol dye/ml is suitable for purifying 20 mg Hb02. Hence, a 250-ml column (3 x 35 cm) is suitable for the purification of 5 g Hb02. All chromatography steps are carried out at 2-5"C, at a flow rate of 250-300 mVhr. After equilibrating the column with 400 ml of 0.02M K-phosphate (pH 6.9), 300-400 ml of the hemolysate (diluted to a protein concentration of 12-16 mg/ml with the same buffer) is loaded onto the column. T h e column is washed with 300 ml of the equilibration buffer, at which point the effluent is colorless. HbO2 is eluted with 400 ml of 0.1M triethanolamine-acetate (pH 8.0). Fractions

MEASUREMENT OF OXYGEN CONSUMPTION

235

containing more than 50 mg/ml HbOp are pooled and mixed 1: 1 with glycerol. This Hb02 preparation is practically free of glycolytic enzymes such as lactate dehydrogenase, phosphoglycerate kinase, pyruvate kinase, or glyceraldehyde-3-phosphate dehydrogenase, and contains less than 0.2% of the initial level of catalase (Porumb et al., 1982). After 1 year of storage at +4"C or - 12°C in 50% glycerol, the Hb02 solution is perfectly clear and contains less than 10% methemoglobin (MetHb). The HbOp in glycerol can be used directly for measurement of QOp by dilution with appropriate buffer. When glycerol is undesirable or the amount of MetHb must be minimized, the HbOp is treated as follows. Immediately before use, 3 vol of buffer containing a few crystals of Na dithionite are added to 1 vol of Hb02. The solution is then filtered through a Sephadex G-25 column equilibrated with the desired buffer. Fractions containing HbO2 are pooled, the pigment concentration is determined, and the fractions are kept on ice until use. This preparation is stable for several hours at room temperature and must be used in the same day. Preparation of HbOp derivatives such as HbCPA, HbCPB, P-PMB, and fetal hemoglobin are described in detail in a recent monograph (Antonini et al., 1981).The purity of Hb02 is best checked by measuring the activity of some well-characterized erythrocyte enzymes like catalase, lactate dehydrogenase, glucose-6-phosphatedehydrogenase, or adenylate kinase (Porumb et al., 1982). It should be noted that the most frequently used optical tests for estimatingenzyme activity, with a few exceptions, are not recommended due to the strong absorption of hemoglobin between 330 and 480 nm. To increase the sensitivity in detection of erythrocyte enzymes, fluorimetric assay of pyridine-linked dehydrogenases is recommended in the direction of NADH or NADPH formation. Remember, however, that Hb02 concentrations higher than 0.3 mg/ml quench the fluorescence of reduced pyridine coenzymes. Also, end-point methods followed by colorimetric assay of reaction products are well suited at low enzyme concentrations.

3. Determination of HbOo Concentration The concentration of hemoglobin in freshly prepared hemolysates, containing in principle a single species (HbOp),is easy to estimate by measuring the optical density at 577 nm, where c S 2 is 15.4. A ratio E577/E560 of 1.78 and E577/E542 of 1.06 indicates that practically no MetHb is present in pigment preparations. However, when using aged Hb02 solutions where a mixture of HbOp, MetHb, and in lesser extent Hb coexists, it is better to measure the relative proportion of these species using the three-wavelength determination of Benesch et al. (1973), or the proce-

236

OCTAVIAN BAKZU

dure recommended by the International Committee for Standardization in Hematology, based on the conversion of all hemoglobin derivatives to cyanornethemoglobin. This last compound was shown to be more stable than any other hemoglobin derivative, exhibiting a flat maximum around 540 nm, where is 11.0. Between 630 and 650 nm, Hb02 and Hb absorb weakly, whereas MetHb has E,,,,~ between 3.8 and 4.3 (Benesch et al., 1973); Van Assendelft and Zijlstra, 1975). Therefore the estimation of MetHb in a purified hemoglobin preparation can be performed by a single optical density (O.D.) measurement after previous assay of Hb, as cyanornethemoglobin. 200 mg K3Fe(CN)6,50 mg KCN, and 140 mg KH2P04 are dissolved in 1 liter of double-distilled water. T h e slightly yellow solution having a pH between 7.0 and 7.4 is stable at room temperature for several months if protected from light. T o 0.05 ml hemolysate or purified HbOs, 2.5 ml of reagent are added and after 3 min the O.D. at 540 nm is measured in a l-cm cuvette against either water or reagent. T h e concentration of Hb, in mmol/liter (on heme basis), is Hb, = E540X 51/11, or as mg/ml, Hb, = E j q O x 51 x 16.1/11, where 51 is the dilution factor, 11 is EMof cyanornethemoglobin at 540 nm, and 16.1 is the molecular weight of hemoglobin monomer X For the estimation of MetHb content, the O.D. of the stock solution is measured at 630 nm in 1-cm cuvette after 1/5 dilution with O.1M phosphate buffer, pH 7. MetHb(mmol/liter) = (EGs00.2Hbt) X 5/3.6, where 0.2 is €;&O2 at 630 nm, 5 is the dilution factor, - EEL"'at 630 nm, and at pH 7. For example, if Hb, and 3.6 is EZ"" of stock solution is 2.3mM (or 37 mg/ml), and the measured E630 is 0.22 after 1/5 dilution of stock solution, MetHb (mmoVliter) = (0.22 - 0.2 X 2.315) X Y3.6 = 0.18mM, or 7.7% from the total concentration of hemoglobin. Since the extinction coefficient of MetHb is highly p H dependent (Benesch et al., 1973), it is important to ensure O.D. measurements are performed at pH 7.0 2 0.2.

IV. INSTRUMENTATION Any recording spectrophotometer o r spectralline photometer is suitable for oxygen consumption measurements if meets three important requirements: (1) high monochromator resolution; (2) sensitivity; and (3) stability. Since hemoglobin derivatives have rather narrow absorption bands in the visible spectrum, a perfect calibration of the monochromator is necessary. Spectral-line photometers using low-pressure mercury lamps in conjunction with appropriate interference filters are well suited for utilization of the HbO? method, as there are three different filters for the

ItIEASL’REMENT O F OXYGEN CONSUMPTION

237

region of the Hb02- Hb spectra where there are significant differences (405 nni, 436 nm, and 578 nm). Modern instruments equipped with photomultipliers as light-current transducers allow measurements of‘ small O.D. variations in strongly absorbing mixtures. Commercially available cuvettes having a path of 0.1 - 1.0 cm, and a volume of0.15-3 ml is perfectly suitable. T h e oxygen back-diffusion is eliminated by covering the reaction mixture with paraffin oil (B%rzuand Borza, 1967). Although this procedure proved to be efficient, it requires a thorough cleaning of the cuvette after each measurement. T h e maintenance of optical homogeneity of the reaction mixture containing particles with a pronounced tendency to sediment can be ensured by increasing the viscosity of the reaction mixture (Markert and Frei, 1979), or using a continuous stirring system (Bgrzu, 1978; BSrzu et al., 1980). As practically no “inert” viscous reagents are available, and an increase in viscosity makes it difficult to mix the reaction medium after new additions, the necessity for an efficient stirring system for oxygen consumption measurements using the H b 0 2 method became an imperative. However, it is difficult to achieve efficient stirring in a standard spectrophotometer cuvette. Therefore, a special cuvette for-oxygen consumption measurements was designed (Birzu et al., 1980, Muresan et al., 1980; IXnsoreanu et al., 1981). With an external dimension of 12 X 12 mm2 and a height of 30 n m , the cuvettes are adaptable to any type of commercial spectrophotometer (Figures 1 and 2). T h e internal size, most importantly the distance between the bottom of the cuvette and the place where the light beam crosses the cuvette, depends on the characteristics of particular instruments. In our laboratory we used an Eppendorf spectralline photometer because of its stability

Figure 1 . Glass cuvettes of 0.20-0.24-rnl volume arid 4-mm path, for (A) repetitive or (B) single mrawrements of the oxygen consumption. See text for description.

238

OCTAVIAN BAKZU

Figure 2. Glass cuvettes of (A) 0.5 ml or (B) 0.2 ml for repetitive measurements of the oxygen consumption. See text €or description.

and ease of manipulation. T h e standard cuvette has a volume between 200 and 240 p,l (Figure IB). The interior section of the cuvette is squareshaped (4 X 4 mm2) but the lower part housing the magnetic stirrer is cylinder-shaped with a diameter of 5.5 mm and a height of 3 mm. T h e stopper is made of lucite and extends 12.5- 14 mm from the bottom of the cuvette in such a way that the remaining volume of the cuvette is approximately 200-240 PI. The stopper is fitted with a longitudinal, cylindricalshaped insert which is threaded toward the bottom and allows the injection of various addition via a IO-pI Hamilton microsyringe. T o help the evacuation of air bubbles, the lower surface of the stopper is concave. T h e size of the cuvette may be modified according to the requirements imposed by H b 0 2 concentration, turbidity of the biological material under investigation, or the wavelength chosen for the assay. However, to achieve a good stirring of the reaction medium, it is necessary to maintain an optimal ratio of the internal dimensions of the cuvette. The cuvette is inserted through the top of the changeable cuvette holder made of metal and connected to a separate thermostat (Figure 3). At 8 mm from the bottom of the holder, there are two 8-mm-diameter apertures for the light beam and a side opening for inserting different electrodes when multiparameter measurements are required (Dinsoreanu et al., 1983). The glass cuvette and the thermostated cell holder with the magnetic stirrer underneath are designed to fit exactly onto the Eppendorf photometer (model 1101 M, 61 14 S, o r 61 18) by a simple replacement of its common cuvette holder. The light beam of the photometer crosses the cuvette at a distance between 6 mm and 10 mm from the bottom. This reduces the noise in the O.D. recording due to stirring. T h e estimation of the exact working volume of the cuvette is made individually for each cuvette by determining the dilution of a colored

MEASUREMENT OF OXYGEN CONSUMPTION

239

Figure 3. Mixing attachment (right) and electronic board for power supply (left) for oxygen consumption measurements adaptable to Eppendorf spectralline photometers: (a) changeable cuvette holder thermostatable with separate thermostat; (b) cover of the photomultiplier tube; (c) motor set for magnetic stirrer; (d) switch for power supply; (e) potentiometer for stirring frequency (15- 1800 rpm).

solution (Hb02)of known concentration. The cuvette with the magnetic bean inside is filled with an excess of double-distilled water. T h e plastic stopper is gently pushed into position, while the excess liquid flows through the longitudinal channel. The cuvette is inserted into its holder and the absorption is set to a value between 0 and 0.05 at the desired wavelength. T h e magnetic stirrer and the recorder connected to the photometer are switched on. The recorder should draw a smooth line. Any rapid, low-amplitude oscillations indicate air bubbles are being formed. A few microliters of Hb02 solution of known concentration are injected into the cuvette and the increase of O.D. is monitored with the recorder set at its maximum speed (Figure 5). T h e injected HbO2 is uniformly distributed in the whole cuvette volume in less than 1.5 sec, including the dead time of the recorder. Cuvette volume (pi)= X

e Z o 2 X [HbOz] X cuvette path L S

1.1 HbO, injected

(1 1 )

For example, by injecting 2 ~1 of 1.83mM HbOp solution in 0.4-cm path cuvette, for an extinction increase of 0.11 1 at 577 nm, cuvette volume (1.1) =

15.4 x 1.83 x 0.4 0.1 11

x 2 = 203 pl

(12)

For the purpose of automatic filling and emptying the cuvette, several modifications can be made in the plastic stopper. For example, in Figure

240

OCTAVIAN

BARZU

1A, there is a vertical channel 1 mm in diameter built into one of the side walls of the cuvette, which opens in the lower cylindrical portion of the cuvette. The bottom part of the stopper 4 X 4 mm2 in cross section protrudes into the cuvette. The upper opening of the axial channel of the stopper is covered with a silicone rubber disk, held in position by the top piece (c) which is screwed into the stopper body with 0.8-mm, 0.75-mm thread. Through the channel crossing the top piece, and by piercing the silicone rubber disk, one can insert the needle of a 10-pl Hamilton microsyringe, and reach the inside of the cuvette. For the introduction of the liquid into the cuvette, there is a plastic tube of 1-mm outside diameter and 0.3-mm inside diameter entering the channel built into one of the side walls of the cuvette. This tube is connected to the side branch (a) of the stopper. Evacuation of the cuvette is accomplished by a second side branch (b), which opens into the central channel of the stopper. The cuvettes shown in Figure 2 are used for the same purposes-automatic filling and emptying. However, due to the larger size of their upper chamber (having a cylindrical shape), the access of the solution into the cuvette is via a channel machined into the body of the stopper, instead of the side wall of the cuvette. The major components of the semiautomatic device for the measurement of oxygen consumption shown in Figure 4 are a thermostated glass reservoir of 50-200 ml that contains the reaction mixture and a peristaltic pump to deliver the reaction mixture into the measuring cuvette. The stopper of the glass reservoir is provided with two orifices, one is the entrance of the nitrogen gas required for the partial deoxygenation of HbO2 and the other is the exit. The flow rate is determined by the cuvette

RESERVOIR

PUMP

CUVETTE

Figure 4. Basic design of the device for repetitive measurements of oxygen consumption. S i x text for description.

MEASUREMENT OF OXYGEN CONSUMPTION

24 1

volume, the time interval between two successive measurements, and the need for economizing the reagents. In a single experiment, it is possible to check the cuvette volume, the efficiency of the stirring arrangement, and the optimal pumping rate. The reservoir is filled with buffer or doubledistilled water, and connected through the pump to the measuring cuvette. It is important that the tubes connecting the cuvette and the reservoir are impermeable to oxygen. Preferably, one should use glass tubes of 0.5- 1 mm diameter, joined with natural rubber tubing. The pump is turned on and the liquid is passed from the reservoir into the measuring cuvette, which is filled rapidly, as the "dead" volume is not larger than 1.5 ml. The pump is turned off, and the magnetic stirrer and the recorder are switched on. The speed of the recorder must be at least 10 c d m i n . A few microliters of HbOpof known concentration is injected into the cuvette by piercing the silicone rubber disk. The increase in optical density is registered for about 30 sec, then the pump is turned on. , on The emptying of the cuvette is an exponential process, t 1 ~depending the rate of the pump. As shown in Figure 5, at a rate of 4 mumin, and a 0.5 ml cuvette volume, t l I 2is 4.6 sec, which means that a 99% emptying of the cuvette is achieved in 31 sec. For a greater accuracy of measurements, when a 99.9% emptying is required, 45 sec are necessary for the START PUMP

,

EXTINCTION I NC R E A S €

L

T I M E INCREASE

Hb02

\

Figure 5. Determination of the cuvette volume, the time for mixing the reactants, and the time for emptying the cuvette. The cuvette used in this experiment (Fig. 2A) has a path of 6 mm. By injecting 8 p1 of 2.16mM HbOp solution (first arrow) the extinction increases by 0.672 units,and from this thecalculatedcuvettevolurneis2.16 X 34 X 0.6 X 810.672 = 525 ~ 1The . second arrow indicates the start of the cuvette emptying, at a rate of the pump of 4 ml/min. (Inset) Calculation of the first-order rate constant and t I l 2for the cuvette emptying.

242

OCTAVIAN

BARZU

same rate. With 0.20-0.24m1 cuvettes, generally we used pumping rates of 2 ml/min for no more than 1.5 min. V. TECHNICAL PROCEDURE

In principle, oxygen consumption measurements using the Hb02 method can be performed at any wavelength, provided HbOz-Hb spectra show significant differences in their extinction coefficients. In the Soret band, these differences are maximum at 413 nm (50) and 430 nm (81), as compared with 577 nm (5.5), 560 nm (4.l), and 540 nm (4.0). However, the choice of the working wavelength does not depend only on this single criteria. Other factors, like the pigment concentration, cuvette path length, the absorption due to the biological material itself, the possibility of amplifying the variation of the O.D., and the presence of interfering species in the probe (like cytochromes in concentrated mitochondria1 suspensions) must be considered when selecting the most suitable working wavelength. When single beam instruments are available, with amplification not larger than AE = 0.1 full scale, it is preferable to carry out measurements at 430 nm (or at 436 nm for the photometers supplied with a mercury lamp), to fully benefit on the sensitivity of spectral properties of hemoglobin (Biirzu and Cioara, 1971; Biirzu et al., 1980). It is essential that the final O.D. including the absorption due to biological materials should not be higher than 3.5. At higher values, deviation from the Lamber Beer’s law are to be expected, even when instruments with high-resolution monochromators are used, and most spectrophotometers deviate well before this value. Thus, if a A of 560 nm is selected, where e S o 2 = 8.5 and E~& = 12.7, for a pigment concentration of 0.25mM, the final absorption after complete deoxygenation of HbOz in a 0.6-cm-path cuvette will be 1.9, comfortably below the limit of 3.5 indicated. Thus, oxygen consumption of turbid biological material (E = 1) can be assayed. At 430 nm, where cH& = 131, the maximal concentration of the pigment th’at can be used in 0.4-cm-path cuvettes is O.O65mM, assuming only a weak absorption due to the biological material itself. 1. Single Measurements of the Oxygen Consumption

All reagents, except for the substrate and enzymatic preparation, are placed in the measuring cuvette at a final volume exceeding 10- 15 pl of the measured volume of the cuvette. At a final concentration of Hb02 of 0.07mM, the O.D. after complete deoxygenation of the pigment is 2.8 for

MEASUREMENT OF OXYGEN CONSUMPTION

243

a path of 4 mm at 436 nm. The cuvette is placed into the thermostating unit and the O.D. at 436 nm is adjusted to zero. Nitrogen or argon gas is bubbled until the O.D. reaches a value in the range of 0.6-0.8, corresponding to 30-40% deoxygenation. To ensure a continuous and gentle bubbling of Nz, a device is introduced between the gas bomb and the sample (Bh-zu et al., 1980). After deoxygenation, the plastic stopper illustrated in Figure 1B is pushed gently into position, and the excess liquid fills the longitudinal channel of the stopper. The O.D. is again adjusted between 0 and 0.1, and now both the magnetic stirrer and the recorder are switched on. After temperature and O.D. equilibration, which usually requires 3 min, different additions are injected so that their total volume does not exceed 10 p1(4% maximal dilution of the sample). The O.D. increase due to the oxygen consumption is recorded on full scale in the 0-1, 0-0.5, or 0-0.25 range. All steps involved in spectrophotometric assays are essentially the same as in the oxygraphic method, the only supplementary step being the partial deoxygenation of HbOp. This step requires no more than 0.5- 1 min. To this we add 3 min for temperature and O.D. equilibration as well as the time necessary for the measurement itself, which usually lasts between 1 and 10 min.

2. Repetitive Measurements of the Oxygen Consumption Since the preparation of the sample is often more time-consuming than the determination itself, the need of a semiautomatic, large-scale assay system became evident (Muresan et al., 1980; D h o r e a n u et al., 1981; Dttnsoreanu et al., 1983).The reservoir illustrated in Figure 4 is filled with buffer, substrates, and other reagents, including HbOz (0.07mM final concentration for experiments at 436-nm and 4-mm-path cuvettes).After temperature equilibration, the pump is turned on and the liquid is passed from the reservoir into the measuring cuvette. The recorder will mark a rapid increase of O.D. up to 1.3- 1.4 units, which becomes stabilized after 1-2 min. This value represents the baseline and should be set to the level of 0-0.1 O.D. density units by adjusting the photomultiplier sensitivity. The pump is turned off and nitrogen gas is bubbled gently into the reservoir, taking care to avoid foaming. The deoxygenation of HbOZ in the reservoir requires about 5 min and is accompanied by an obvious change of color toward violet. The degree of deoxygenation can be estimated by turning on the pump and measuring the increase in the O.D. at 436 nm. The pump is once again turned off, the enzyme (or the substrate) solution is injected into the cuvette, and the increase in O.D. due to oxygen consumption is now recorded. To empty the cuvette, the

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OCTAVIAN BARZU

pump is turned on for 1- 1.5 rnin, and the system is ready for the next measurement. After each measurement, samples may be collected for further analytical determinations. O.D. equilibration of the system is the most important factor for the reproducibility of the determinations. It depends upon preventing the diffusion of atmospheric oxygen as well as upon the maintenance of a constant degree of deoxygenation of the hemoglobin in the reservoir. To avoid artifacts caused by the diffusion of atmospheric oxygen into the tubing, the washing of the cuvette should be prolonged by some additional 10- 15sec; thus, the liquid entering the cuvette is of the correct oxygen concentration because it comes directly from the reservoir without having rested in the tubes. By the elimination of preliminary operations, such as pipetting and ensuring adequate nitrogen flow for individual samples, better reproducibility should be attained when working with the semiautomatic system. The standard error of the mean (S.E.M.) from 10 measurements on the same sample is 2 1.1%.

VI. CALCULATION OF EXPERIMENTAL DATA The correspondence between the experimentally measured variation of O.D. and oxygen consumed by different oxidases is described according to the following general formula, keeping in mind that two atoms of oxygen corresponds to 1 mole of deoxygenated Hb02:

QO, (pg-atoms/min.sample) =

2

X

vol

X

AE-

f

X

hElmin

x d

(13)

where vol is the reaction volume (ml), d is the light path length (cm), and AE- is the difference of the millimolar coefficient of extinction of HbOn and Hb at the chosen wavelengths. f represents a correction factor (Equation 10) depending on HbO:! concentration and the affinity for oxygen of both hemoglobin and the respiratory system. The correction factor can be evaluated experimentally in several ways: 1. Comparative Measurements of Oxygen Consumption with Spectrophotometrically Calibrated Substrates When the substrate or the reaction product or both has a characteristic absorption spectra in the visible or ultraviolet ranges, it is possible to calculate the correction factor by measurement of the optical density variations in identical experimental conditions at two selected wavelengths. For example at 334 nm, NAD+ does not show any absorption, whereas

245

MEASUREMENT OF OXYGEN CONSUMPTION

NADH is near its maximum ( E % ~ =~ 6). In a typical experiment for determination of the correction factor, 5.8 kg of bacterial membranes were incubated in a cuvette of 4 mm and 0.203-ml volume with 50mM K-phosphate buffer (pH 7.4) in the absence of HbO2. After injecting 100 nmol NADH, the decrease in O.D. at 334 nm was 0.046Imin. In the presence of 0.066mM HbO2, the O.D. increase at 436 nm due to the oxygen consumed for NADH oxidation to NAD+ and H20 was 0.161/ min. Therefore, NADH oxidized (pmol/min-sample) = -

vol

X

AE334/min

NADH EmM

0.203 x 0.046 6

X

0.4

0.00389 2 x 0.203 x f x 0.161 Q02 (pg-atomslmin-sample) = 65 X 0.4 =

=

0.00251

X

(14)

f

(15)

Since bacterial membranes, like submitochondrial particles, oxidize NADH in a ratio NADH/O equal to 1, it follows that f in this particular case will be 0.00389/0.00251 = 1.55. Table 111 shows the dependence of the correction factor on pH and temperature for respiratory enzymes of rate heart submitochondrial particles and membrane preparations from Citrobacterfreundi, when HbO2 or HbCPA were used as oxygen donors. It TABLE 111 Dependence of the Correction Factor on the Nature of the Oxygen Donor, pH, and Temperature When Bacterial Membranes (Citrobacter freundz) and Rat Heart Submitochondrial Particles Were Used as Oxidases with NADH as Substrate Bacterial membranes

Rat heart particles

Oxygen donor

Temperature

pH 6.2

pH 8.0

pH 6.2

pH 8.0

Hb02 (0.066mM)

15°C 25°C 37°C 15°C 25°C 37°C

1.60 1.61 1.62 1.22 1.23 1.23

1.52 1.52 1.53 1.20 1.20 1.21

1.56 1.58 1.60 1.19 1.19 1.21

1.49 1.50 1.52 1.18 1.18 1.20

HbCPA(0.066mM)

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OCTAVIAN BARZU

can be seen that f varies only slightly with changes in temperature and pH when HbCPA is used as an oxygen donor. When HbO2 is used, dependence on f on temperature is also negligible, however, by increasing pH from 6.2 to 8.0, f decreases by 5.5%.

2.

Measurement of HbOs Deoxygenation at Different Pigment Concentrations

As shown in Equation (lo), when systems having a high affinity for oxygen are used (Kt < 0.4@), the rate of HbOp deoxygenation (V,) extrapolated for infinite concentration of pigment equals the rate of oxygen consumed by the given oxidase: V/V, = 1 KEb/[Hbl]= f . A plot of l/V, or of l/AE436 vs. l/[Hb,] yields the K, for Hb, and from this value one may calculate f for any concentration of pigment (f = ([Hb,] + KEb)/[Hbl]).At temperatures between 25 and 37"C, and at pH values between 7.2 and 7.8, KEb was between 36 and 38@, irrespective of the respiratory system investigated (mitochondria, submitochondrial particles, isolated cells, or purified oxidases). In all these cases, KO, was negligible, and the deoxygenation rate of HbOp at infinite pigment concentration reflects the true rate of oxygen consumption. When HbOp was replaced by HbCPA, the measured KFbCPAvalue was considerably lower (lPp.M)(B~rzu,1978; BPrzu et al., 1980). Thus, for the same pigment concentration (0.07mM),the correction factor is 1.17 (HbCPA) and 1.51 (Hb02),respectively.

+

3. Polarographic Estimation of Oxygen Consumption in the Presence of the Oxygen Donor This method for estimation off is very convenient since only a single assay is required and physical factors such as oxygen solubility, temperature, pH, ionic strength, does not influence the precision of its estimation. As shown by Equation (7), Q02 = V,. + Vo,. When Vy is constant (the interval of linearity of HbOp deoxygenation), V o , remains constant in the same y interval. In addition, at high oxygen concentration, i.e., the initial phase of oxygraphic determination, V, = 0, and Q02 = Vo,. Therefore, when the oxygen consumption of a respiratory system is measured with an oxygen electrode in the presence of Hb02, the oxygraphic curve has two linear segments, and from their slopes the correction factor can be calculated directly (Figure 6). This procedure is applicable to systems having a high affinity for oxygen, providing they are stable during the time required to attain anaerobiosis. To obtain the most accurate values o f f , the measurements should be performed at a concentration of HbOp between 0.07 and 0.30mM. Since the plot o f f as a

MEASUREMENT OF OXYGEN CONSUMPTION

247

mito c hondr ia

t

Figure 6. Oxygraphic measurement of the correction factor using rat liver mitochondria as oxygen-consuming system. The respiratory medium contains in a final volume of 0.5 ml and 24"C, 18OmM sucrose, 50mM KCI, 15mM K-phosphate (pH 7.4), 0.5mM EDTA, 2.5mM MgCI2, 2 m M ADP, 5 m M glutamate, and 0.235mM Hb02 (trace a-b), or 0.242mM HbCPA (trace a-c). The reaction was initiated with 0.88 mg of rat liver mitochondrial protein and recorded until anaerobiosis was attained.

function of l/[Hb,] yields a straight line, the procedure can be extrapolated to HbOp concentrations below 0.07mM.

VII. SIMULTANEOUS DETERMINATION OF SEVERAL PHOTOMETRIC PARAMETERS The simultaneous measurement of several parameters is usually required for determining the stoichiometry and reaction mechanism of a particular oxygen-consuming system. Among the early successful methods for study of mitochondrial oxidative phosphorylation (Chance and Williams, 1956; Merola et al., 1971; Starlinger and Lubbers, 1973)and of hemoglobin dissociation (Imai et al., 1970)was the simultaneous assay of the O.D. and the oxygen concentration with rotatinglvibrating platinum electrode or Clark electrode. By taking advantage of the difference in the spectral properties of the various hemoglobin derivatives,it is possible to measure, in a single experiment, several photometric parameters. In our earliest experiments with the Hb02 method, we showed that by followingchanges at two wavelengths (560 nm and 548 nm), we may follow the mitochon-

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OCTAVIAN BARZU

drial oxygen consumption and the volume changes due to swelling or contraction processes ( B h u et al., 1968).Similarlyby monitoring changes at 433 and 548 nm, it is possible to measure the oxygen consumption as well as oxidation or reduction of cytochrome c (Bk-zu et al., 1972). At 334 nm NAD(P)H is near its maximum absorption, and HbOP-Hb - EZ = 1.3). spectra are in the vicinity of an isosbestic point (.sZo2 Therefore, from measurements at 436 and 334 nm, it is possible to simultaneously determine both the formation or disappearance of NAD(P)H and the oxygen consumption (Figure 7). This procedure permits rapid and accurate estimation of mitochondria1 oxidative phosphorylation, or of hydroxylation reactions that occur in liver microsomes

+lminl-

c

-

0.10

A€

T

I

ADP Succ 1

RLM I

I

Mix

Figure 7. Simultaneous determination of oxygen consumption and NADP+ reduction by phosphorylating rat liver mitochondria. The reaction mixture for 30-40 determinations using repetitive measurement system contained the following reagents in a volume of 100 ml:180mM sucrose, 50mM KCI, 15mM K-phosphate (pH 7.4), 0.5mM EDTA, 2.5mM MgC12, l 0 M glucose, 0.25mM NADP+, and 0.07mM HbO,. After deoxygenation of the reaction mixture in the thermostated reservoir and filling of 0.203-ml and 4-mm path cuvette, 1 ~1 of rat liver mitochondria (31.5 kg of protein) (RLM), 3 ~1 of the mixture containing 10 nmol P',P5-di-(adenosine-S')pentaphosphate,0.5 units of hexokinase, and 0.5 units of glucose-6-phosphate dehydrogenase (Mix), 1 k1 of 40mM ADP were injected. After each addition, the optical density (O.D.) was adjusted to the level displayed before, then 1 FI of 0.2M succinate (Succ.) was injected. The increase of O.D. was followed first at 436 nm, then at 334 nm. From the extinction increases at 436 nm (0.148irnin) and at 334 nm (0.07 limin), using Equations (15) and (16), w e calculated a QO, value of 111 ng-atoms/ minmg of protein, and a rate of NADPH synthesis (which corresponds to ATP synthesis) of 198 n m o l h i n m g of protein. Hence, the ATPiO ratio was 1.79.

249

MEASUREMENT OF OXYGEN CONSUMPTION

(DBnsoreanu et al., 1981).The oxygen consumption is calculated according to the general formula (Equations 13or 15),whereas a modification of Equation (14) is used to calculate the formation or disappearance of N AD(P)H: NAD(P)H (pmol/min.sample) vol

1.3 *x AE43dmin) 65

- -(bE334/min

6xd

1.3 and 65 are the difference of the millimolar extinction coefficients of HbO2 and Hb at 334 and 436 nm, respectively. The sign (k)in Equation (16) depends on whether one follows the formation (+) or the disappear> EZ at 334 nm. ance (-) of NAD(P)H, noting that

&Zoz

VIII. DETERMINATION OF HYDROGEN PEROXIDE-GENERATING OXIDASES When HpO2-generating oxidases are assayed (glucose oxidase, monoamine oxidase, cholesterol oxidase, xanthine oxidase), Hb but not HbOp was shown to be very sensitive to the action of newly formed hydrogen peroxide. The rate of oxidation of Hb to MetHb is dependent on pH (Bh-zu, 1978; Barzu and DBnsoreanu, 1980). In the presence of catalase and a suitable hydrogen donor (methanol or ethanol), H202 is decomposed; therefore, at pH values higher than 7, less than 3% of the deoxygenated pigment is oxidized to MetHb: Glucose + O2 H20p

glucose oxidase

+ RCHpOH-

> gluconolactone

catalase

2H20

+ H202

+ RCHO

In this way the sensitivity of oxygen consumption measurements is increased, that is, 1 mole of oxidized substrate corresponds to 1 mole of oxygen taken up. When catalase is added in absence of a hydrogen donor, 1 mole of the oxidized substrate corresponds to mole of oxygen (Table IV). Enzymes such as glucose oxidase or monoamine oxidase exhibiting ping-pong-type kinetics show dramatic alterations in their kinetic behavior as a function of oxygen concentration (Tipton, 1972;Jain et al., 1973). The glucose oxidase from Aspergillus niger has a K , value for glucose of 15mM when the reaction medium is saturated with air at 25°C. Using the HbO2 method, a proportional decrease of the K , for glucose (I&) and

4

250

OCTAVIAN BARZU TABLE IV

Effect of Methanol, Catalase, and pH on the Rate of HbO2 Deoxygenation, and MetHb Formation during the Oxidation of Glucose by Glucose Oxidase" MetHb formed PH

5.9 6.7 7.6

HbOp deoxygenated Methanol (d) (nmoVminmg protein)

0 100 0 100 0 10 20 30 50 100

190 378 186

373 142 206 242 258 28 1 283

HbOp deoxygenated

(%I

56 17 21

9

12 7.4 5.7 4.6 3.4 3.0

"The reaction medium (final volume 3 ml) contained in 1-cm-path cuvette and at 23°C: 0.1M K-phosphate, 0.02mM HbO,, 1.67mM glucose, 300 units of catalase, and different concentrations of methanol. The reaction was started with 10-pg glucose oxidase (a Sigma preparation), and the rate of H b 0 2 deoxygenation (430 nm) and MetHb formation (420.6 nm) was followed with a Gilford 2400 spectrophotometer.

of the maximal activity (V,) was found. In other words, the term that seems independent of oxygen concentration in oxygraphic and photometric determinations is the ratio V,/K, (Blrzu and Dlnsoreanu, 1980).

IX. APPLICATIONS OF THE OXYHEMOGLOBIN METHOD FOR MEASUREMENT OF OXYGEN CONSUMPTION As discussed in the introduction, any enzymatic activity that is directly (or indirectly) linked to oxygen consumption could be measured by using the HbOp method. However, it is not our purpose to shift all analytical procedures using manometric or polarographic techniques to spectrophotometric assays merely because it is more sensitive. Therefore, in the following sections we will discuss those applications where HbOZ method could offer obvious advantages over other procedures.

1. Determination of Cytochrome Oxidase Activity in Human Liver Homogenates When attempting to assay the oxygen consumption in the small amount of tissues obtained by biopuncture, the spectrophotometric method is in-

MEASUREMENT OF OXYGEN CONSUMPTION

25 1

deed the method of choice, since about 10 mg of tissue is enough to assay some important oxidative enzymes present in whole homogenates. Ten mg of human liver obtained by needle biopsy is homogenized with 0.1 ml of 0.25~4sucrose buffered with IOmMTris-HC1(pH 7.4). Centrifugation at lOOOg for 1 min removes the cell debris. The protein concentration of the supernatant is determined using the sensitive and rapid Coomassie blue binding method (Bradford, 1976).To 10 ~1 of supernatant containing 0.108 mg of protein, 3pl of 2.5% Lubrol WX solution is added (the weight ratio detergendprotein must be situated between 0.5 and 1). The reaction mixture for the assay of human liver cytochrome oxidase contains 0.05M K-phosphate buffer (pH 7.0), 0.02mM human heart cytochrome c (Margoliash and Walasek, 1967),and 0.07mM HbOP, in a volume of 0.24 ml. After partial deoxygenation of HbOn, 1 PI of 0.25M ascorbate (pH 6) is injected in the 4-mm-path cuvette and the increase in absorption (hE43dmin= 0.0102) due to the autooxidation of ascorbate is followed for 2 min at 3’7°C. Thereafter 5 pl of detergenttreated human liver homogenate are injected and the increase in absorption (AE43dmin = 0.126) is recorded again for 2-3 min. The data are analyzed as follows:

QOz (ng-atoms/min.sample)

- 2000 X 0.24 -

X

1.51 X (0.126-0.0102) = 3.22 65 X 0.4

QO, (ng-atomdmin-mgof protein) =

3.22 X 13 = 77.4 0.108 x 5

(19)

(20)

To obtain maximal enzymatic activity, it is essential to follow the conditions describe above. Especially important are the pH and concentration of the phosphate buffer and the presence of human cytochrome c instead of the horse heart cytochrome c (Benga et al., 1972; Benga and Borza, 1975).

2. Determination of Rat Liver Phenylalanine Hydroxylase Activity One g of rat liver is homogenized with 4 ml of 0.25M sucrose buffered with l O m M Tris-HC1(pH 7.4). The homogenate is centrifuged for 1 hr at 105,OOOg and the slightly turbid supernatant (23.5 mg of proteidml) is used as the enzyme source. The reaction medium contains lOOmM Kphosphate buffer (pH 7.4), 0.2mM EDTA, and 0.07 mM HbO2 in a volume of 0.24 ml. After deoxygenation of the reaction mixture, 5 PI of enzyme preparation followed by 2 ~1 of freshly prepared 6,7-dimethyl-

252

OCTAVIAN BARZU

tetrahydropterine are injected into the cuvette. After the optical density is stabilized, 2.5 pl of lOOmM phenylalanine is injected, and the increase in absorption (hE43&nin = 0.0242) is followed for several minutes: QO, (ng-atomshin-mg of protein) -

2000

0.24 X 1.51 X 0.0242 = 5.74 65 X 0.4 x 0.1175 X

(204

The advantage of this sensitive procedure for the assay of phenylalanine hydroxylase activity is that no regenerative system for tetrahydropterine derivative is required (Kaufman, 1971).

3. Determination of NAD-Linked Dehydrogenases Bacterial membranes contain respiratory enzymesresembling those found in inner mitochondria1 membrane (Haddock and Jones, 1977), which oxidize NADH to NAD+ and H 2 0 . C.freundi grown aerobically, possess a particularly active membrane-bound NADH-oxidase activity (1.O- 1.5 pmolhin-rng of protein at 30°C. Since these membranes alone or in the presence of NAD+ do not oxidize substrates such as L-alanine, L-lactate, or ethanol, they act as “indicators” of the activity of lactate dehydrogenase, alanine dehydrogenase, or alcohol dehydrogenase: L-alanine + NAD+ NADH

+ H+ + 3 0 ,

L-alanine dehydrogenase 9

bacterial NADH-oxidase

pyruvate

+ NADH +

NAD+ + H 2 0 (22)

When reaction (22) is 50- 100 times faster than reaction (21), the oxygen consumption is proportional to the activity of L-alanine dehydrogenase (Figure 8). Since NAD+ is continuously regenerated, the enzyme activity is proportional to time, and does not show the typical progressive inhibition due to the accumulation of reduced pyridine coenzyme. This technique allows many dehydrogenases be studied under simple experimental conditions. Moreover, in the presence of excess pyridine-linked dehydrogenase, experiments can be conducted with very low substrate concentrations (1 -5 nmol). Recently Jaworowski et al. (1981) isolated highly purified preparations of NADH dehydrogenase from genetically amplified Escherichzu coli strains. Since the specific activity of NADH dehydrogenase and NADH oxidase of the membranes obtained by genetic manipulations are enough high without requiring further purification, they could probably be used

MEASUREMENT OF OXYGEN CONSUMPTION

253

w

a

pg AlOH

Figure 8. Determination of L-alanine dehydrogenase activity by measuring the oxygen consumption in a “coupled’system involving bacterial NADH-oxidase as indicator enzyme. The oxygen consumption was assayed using a repetitive measurement system with a 4-mm cuvette and 0.203-ml final volume. The cuvette is filled with 0.05M K-phosphate (pH 8), 2 m M NAD+, 0.07mM HbOp, and 40mM L-alanine. The HbOp was deoxygenated by 30-40%, then 5 pl of bacterial membranes from Citrobucterfreundi (BM) correspondihg to 0.18 mg of protein were injected. After addition of bacterial membrane suspension, the O.D. was adjusted to the level displayed before, and varying amounts of purified L-alanine dehydrogenase (Muresan et al., 1983) were injected. The left side of the figure shows a typical recording at 436 nm, by using 0.086 pg of purified L-alanine dehydrogenase (AIDH) fromBaciZZus cereus (AIDH); the right side of the figure shows the dependence of the oxygen consumption on the amount of purified L-alanine dehydrogenase. Citrobucterfreundz (strain 1043) are grown on broth with bactopeptone Difco at pH 7.2 in 1000-ml conical flasks containing 200 ml of culture medium at 37°C up to the late logarithmic phase. The cells are harvested by centrifugation for 45 min at 5000g, then suspended in 50 mi of 0.1M phosphate buffer (pH 7.4) and sonicated at 20,000 Hz and 80 Watts (four pulses of 1 min). The unbroken bacteria and large membrane fragments are removed by centrifugation at 10,OOOgfor 20 min, then the membranes are separated by centrifugation of the supernate at 145,000 g for 1 hr. The membranes are washed with phosphate buffer and centrifuged again at 145,OOOgfor 1 hr, and resuspended in phosphate buffer at a protein concentration of about 40 mg/ml. This preparation is stored at -30” in aliquots of 50 &I, for several months without any significant loss of activity.

as a “coupling” system for measuring a large number of pyridine-linked dehydrogenases and their corresponding substrates. 4.

Measurement of Mitochondrial Respiration and Oxidative Phosphorylation

The most convenient assay of mitochondria1 respiration and ATP synthesis is to measure the changes in oxygen consumption of intact isolated organelles after several additions of ADP with an oxygen electrode (Chance and Williams, 1956; Estabrook, 1967).In exactly the same way, but using much lower amounts of organelles, the HbOp method could

254

OCTAVIAN BARZU

monitor the mitochondria1 respiration and ADP phosphorylation to ATP (BirzuandCioara, 1971; BPrzuetal., 1971; Bengaetal., 1972; Hodirnau et al., 1973; Kezdi et al., 1973; Nessi et al., 1977). Figure 9 shows a typical experiment with rat heart mitochondria. To show the similarities with the familiar polarographic traces, the downward deflection of the photometric recording represents the increase in O.D. at 436 nm. After addition of mitochondria to the assay mixture containing P-hydroxybutyrate as the substrate, the state-4 respiration is recorded. ADP stimulates the mitochondria] respiration four- to seven-fold (state-3 respiration) until all nucleotide is phosphorylated to ATP. T h e state-4 respiration is now restored. To have the maximal rate of mitochondrial respiration, a larger excess of ADP (the third addition of nucleotide in Figure 9) was injected into the cuvette. From the slopes (6) and (a) indicating the state-3 and state-4 respiration of rat heart mitochondria (shown schematically on the right side of the Figure 9), the respiratory control index (RCI = b/a) may 2

7

0.1,E ,

q

11.6 nmoles ADP ~

1

ADP/O=2.71 RCI = 5.53 AOP

Figure 9. Determination of respiratory activity, respiratory control index (RCI) and ADP/O ratio of rat heart mitochondria. Mitochondria were isolated according to Pande and Blanchaer (197 1). The oxygen consumption was determined using the single-measurement system in a 4-mm path cuvette and0.24-ml final volume. The cuvette contained 0.15M KC1, 25mM Tris-HC1 (pH 7.4), 15mM K-phosphate (pH 7.4), 0.5mM EDTA, 5mM P-hydroxybutyrate, and 0.07mM Hb02. At different time interval after partial deoxygenation of Hb02. mitochondria (RHM) and ADP as 1 - ~ 1injections were added to the respiratory medium.

MEASUREMENT OF OXYGEN CONSUMPTION

255

be calculated. Moreover, the amount of oxygen consumed during state-3 respiration can be calculated from the difference E 2 - E = c, and from this the ADPlO ratio. Another procedure to determine oxidative phosphorylation of intact mitochondria or of submitochondrial particles is to measure, in a single photometric experiment, the respiration at 436 nm and the formation of NADPH at 334 nm. NADPH results from the action of hexokinase and glucose-6-phosphate dehydrogenase on ATP (the product of oxidative phosphorylation) and an excess of glucose and NADP+ (Figure 7). Both procedures require only 20-60 pg of mitochondrial protein. Thus, the investigation of respiratory enzymes need not be limited to systems that provide large quantities of mitochondria, Moreover, mitochondria isolated from small amounts of tissue are generally obtained as diluted suspensions, and in some cases (human liver, rat pancreas, or adipose tissue) the high content of lipids combined with an active mitochondrial phospholipase makes these organelles particularly “fragile” (Honjo et al., 1968; Ozawa et al., 1969; Benga et al., 1972; Hodirnau et al., 1973). This fragility is indicated by a rapid loss of the respiratory control, decrease of ADP/O ratio, and the increase in “latent” Mg2+ATPase activity. For greatest stability, the protein concentration of the mitochondrial suspension should be at least 10 mg/ml. Furthermore, the stability of mitochondria from human liver or rat pancreas is enhanced by addition of defatted bovine serum albumin (2 mg/ml),and the addition of 0.4mM nupercaine, which strongly inhibits mitochondrial phospholipase (Scarpa and Lindsay, 1972; Hodh-nau et al., 1973). 5. Measurement of Microsomal Oxidases

Liver microsomes are capable of oxidizing NADPH both in the absence and the presence of xenobiotics such as hexobarbital, which become hydroxylated (Estabrook, 1978; Lu and West, 1978). In the case of “free” oxidation of NADPH (reaction 23), the ratio NADPH/O is 1, whereas with “coupled” oxidation of NADPH (reaction 24), the ratio NADPH/O is 0.5. As under experimental conditions, the two processes run in parallel, and the experimentally obtained NADPH/O ratio will be between 0.5 and 1:

+ H+ + 4 0 2 microsomal oxidases> NADP+ + H20 (23) microsomal oxidases NADP+ + H20 NADPH + H + + 0 2 + RH NADPH

f

-iROH (24)

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OCTAVIAN BARZU

In the presence of EDTA, the lipid peroxidation is completely inhibited (Archakov et al., 1977). Hence, by the simultaneous measurement of the oxygen consumption (noted B) and the oxidation of exogenous NADPH (noted A), it is possible to determine the rate of hydroxylation of hexobarbital (RH). If we denote by x and y the free and coupled NADPH oxidation (the second being equal to the rate of hydroxylation of RH to ROH), we may write x + y = A; x + 2y = B, hence x = 2A - B, and y = B - A. Despite these simplifying assumptions, this method permits rapid and accurate estimations of hexobarbital hydroxylation performed on 20-100 pg of microsomal proteins (Table V). The rate of NADPH oxidation and the rate of oxygen consumption is calculated according to Equations (15) and (16). As indicated in Table V, the calculated rate of hexobarbital hydroxylation is of 5.6 nmoYmin-mgof microsomal protein from control rats, with a threefold increase in animals treated previously with phenobarbital. SKF-525A (diethylaminoethyl-diphenylpropylacetate), a known inhibitor of microsomal oxidases in vitro, inhibits the hexobarbital-induced increase of both oxygen consumption and NADPH oxidation. 6. Oxygen Consumption of Isolated Cells isolated mammalian cells have been frequently used for metabolic studies, cell transformation (mainly neoplastic transformation induced by certain viruses, chemicals, or X-rays), or investigation of specific cellular functions such as synthesis and release of hormones or antibodies, killing of bacteria by oxidases involved in phagocytosis, etc. The large-scale cultivation of mammalian cells is more and more important for producing medically important proteins such as interferon and monoclonal antibodies (Feder and Tolbert, 1983). In all these cases the oxygen consumption measurement still remains an important tool for checking cell viability, membrane intactness, and functional integrity. The HbO2 method for assay of cell oxygen consumption has the advantage of sensitivity, since the measurements can be performed on 3 x lo4 to 3 x lo5 cells. Working with mouse Ehrlich ascite tumor cells respiring with endogenous substrates at 37°C in a medium containing 140mM NaCl, 6 d KCl, and 5- 15 mM K-phosphate buffer (pH 7.4), we found a perfect linearity of the oxygen consumption for a wide range of cells at densities between 2 X lo4 and 5 X lo5. The oxygen consumption begins immediately after the injection of the cell preparation into the reaction medium and it is linear for 7- 10 min in the presence of endogenous substrates. The absence of the lag period found in manometric experiments indicates the fact that in these experiments the achievement

Hexobarbital (1mM) Hexobarbital ( I d ) + SKF-525A (0.2mM) Hexobarbital ( I d ) hexobarbital (1mM) + SKF-525A ( 0 . 2 d )

Additions 1.05 0.82 0.92 1.03 0.75 0.92

20.8 31.4 22.1 27.4 58.4 29.2

28.1 43.9 26.8

~~

NADPH/O

21.8 25.8 20.3

(ng-atoms/min. mg of protein)

O2 consumed

14.5 2.4

5.6 1.8

-

Hexobarbital h ydroxylation rate (nmoVmin-mg of protein)

"Livers from control and phenobarbital-treated rats (a single dose of 0.75 mg/kg 48 hr before sacrifice) are homogenized with 0.25M sucrose buffered with lOmM Tris-HC1 (pH 7.4). The 10% homogenate is centrifuged for 15 min at 12,00Og, to remove cell debris, nuclei, and mitochondria. Th e supernatant is centrifuged at 105,OOOg for 1 hr. The sediment is resuspended in 0.15M KCl containing lOmM Tris-HCl (pH 7.4), and centrifuged again at 105,OOOg for 1 hr. The washed microsomes are used for measurement of oxygen consumption and NADPH oxidation using the repetitive measurement system with a 0.203-ml cuvette having a 4-mm path. 100 ml of the reaction mixture containing 50mM K-phosphate buffer (pH 7.4), 0.5mM EDTA, and 0.075mM HbOp are partially deoxygenated and pumped into the cuvette maintained at 30°C. Microsomes (about 50 pg of protein) are injected. After O.D. equilibration, the reaction was started with 40 nmol NADPH.

Treated with phenobarbital

Control

Rats

NADPH oxidized (nmoVmin.mg of protein)

Oxygen Consumption and NADPH Oxidation by Rat Liver Microsomes"

TABLE V

258

OCTAVIAN BARZU

of a steady-state oxygen consumption is delayed by the slow response of the manometric procedure. The cellular oxygen consumption responds rapidly to the addition of substrates or inhibitors provided they are able to enter the cell. Addition of glucose to intact cells stimulates the oxygen consumption, which lasts for about 20 sec, after which time the inhibition due to the Crabtree effect occurs. 2,4-Dinitrophenol removes the inhibitory effect of glucose. Succinate as well as other intermediates of the citric acid cycle do not activate the oxygen consumption of the intact cells. Dextran-sulfate-treated ascites cells behave differently. The initial activation by glucose is absent, nevertheless the addition of the succinate results in clear activation of the oxygen consumption (BArzu et al., 1980). The values for oxygen consumption on mouse Ehrlich ascites cells obtained at 37°C with endogenous substrates by using the HbO2 method (10 ng-atoms/min*lO6cells, and 32 ng-atoms/min.mg of protein) are significantly higher than those reported by other authors (Suzuki et al., 1968; Gordon and DeHartog, 1968; Cittadini et al., 1973; McCoy et al., 1976). The discrepancies are probably due to the different number of cells per unit assay volume, 20- 50 times lower in spectrophotometric assays than in oxygraphic or manometric ones. Markert and Frei (1978, 1979, 1981) studied the kinetic of oxygen consumption of leukocytes during phagocytosis, in the presence of HbO2 as oxygen donor and indicator of respiration. The respiratory medium in 0.24 ml final volume, 4-mm-path cuvette and at 37°C contained 1lOmM NaCI, 2.2mM KCI, 5.7mM Na2HP04, 1.2mM KH2P04, 0.7mM CaC12, 0.9mM MgCl2, 13.4mM glucose, and 0.06mM Hb02. These authors found oxygen consumption in the resting state of human neutrophils of 0.7 ng-atoms/min.106 cells. After addition of 2 mg of zymozan particles, the maximal rate of oxygen consumption of 10- 12 ng-atoms/min.106 cells was attained after a lag of about 8 min. When preopsonized zymozan was injected, the latency was decreased to less than 1 min. NaF, 2deoxyglucose, and Na-amytal were shown to increase the delay of response to zymozan stimulation as well as to decrease the maximal rate of oxygen consumption. By simultaneous measurement of superoxide anion production and of oxygen consumption of human polymorphonuclear leukocytes stimulated by zymozan particles, Markert et al. (1980) postulated a similar triggering mechanism for these two events.

7. Microspectrophotometric Assay of Oxygen Consumption One of the most important advantages of spectrophotometry in general is the possibility to miniaturize the measuring “cuvette”from 1 pl to 10- 100

MEASUREMENT OF OXYGEN CONSUMPTION

259

pl, without affecting the precision of measurements (Hamberger et al., 1975). Hultborn (1972, 1976), described a technique whereby oxygen consumption rates of lo-’ pg-atomdmin can be measured, thus providing a mean for studying the respiration rates of single cells (Hultborn and Hyden, 1974). Chambers for oxygen consumption measurements are prepared from microscopic slides coated with a thin paraffin film. With a tip of a hypodermic needle held in a micromanipulator, a small round dent of 10- 15 pm is made in the paraffin film, and a drop of 40% HF applied to the dent enables the formation of small circular cavities visible after the removal of paraffin. To increase the measuring capacity, an automatic cuvettechanging device has been developed. The object stage maintained at constant temperature by water circulation from a temperature controlled bath, is moved in six discrete steps by a synchronous motor. Thus, oxygen consumption in six different chambers can be assayed simultaneously. The basic equipment used for microspectrophotometric determinations of oxygen consumption does not differ essentially from the equipment used for other purposes, such as cytophotometry (Hamberger et al., 1975). Using the semimicrotechnique in chambers of about 1 p1, Herlitz and Hultborn (1974)made a comparative study of the classical standard diver technique and the HbOp procedure on samples of 5- 30 pg dry weight of rat corpus luteum tissue. The accuracy of the two methods is equal, but the HbOBmethod proved to be considerably simpler and faster. Using the ultramicrotechnique, the oxygen consumption of single nerve cell from the lateral vestibular nucleus of the rabbits was studied (Hultborn and Hyden, 1974).In the presence of 4mM KCl, the oxygen consumption pllhr-cell, whereas in a medium containing 50mM was of 2.45 x KCl, the oxygen consumption was of 4.64 X pl/hr.cell.

8. Continuous Measurement of Oxygen Consumption of In Vitro-Cultured Embryos Since 1977 a new microtechnique allowing continuous measurement of oxygen consumption in minute regions (0.01 mm2) in the intact chick embryo cultured in vitro under defined metabolic conditions was developed (Raddacz and KuEera, 1977; KuEera et al., 1979; KuEera and Raddacz, 1980; Raddacz and KuEera, 1983). The measuring chamber of about 20 mm in diameter, schematically represented in Figure 10, has a volume varying between 0.3 and 0.6 ml. It is divided in two compartments by a thin and transparent silicone membrane (m). The lower compartment (lc) houses the embryo (e) removed from fertilized eggs at desired

260

OCTAVIAN BARZU

CR

m

\

pump on

+MC

time increase

Figure 10. Schematic representationof the device for microspectrophotometricrecording of chick embryo respiration (Kufera and Raddacz, 1980). See text for description.

stages of development. The viteline membrane, which serves as a natural mechanical support of the embryo, is applied to the silicone membrane separating the two compartments. The lower compartment is perfused by the nutritive medium (NM) containing at pH 7.7, 102.7mM NaC1, 2.012mM KCl, 0.748mM CaC12, 0.787mM MgC12, 0.312mM Na2HP04, 8.92mM NaHC03, and 7.57mM glucose. The upper compartment (uc) is perfused by 1mM HbO2 solution in phosphate buffer containing 0.5mM 2,3-diphosphoglycerate. The perfusion rate is adjustable between 3.5 and 1800 pVmin, and the temperature is maintained at 37°C by warm air. The perfusion chamber is fixed on the microscope stage. This allows a continuous observation of the preparation and local or scanning measurements of absorbance at given stages of the embryonic development. The wavelength of the beam of a high-pressure mercury lamp (L) is selected by an appropriate interference filter (IF) in conjunction with a monochromator (MC).The beam is focused on a defined zone of embryo preparation and transmitted to the photomultiplier tube (PM) connected to a chart recorder (CR)or to a minicomputer. By stopping the perfusion flux, the chamber having the embryo behaves like a closed system and the oxygen consumed by the respiring tissue promotes the dissociation of HbOp which is registered by the increase in optical density at 434.6 nm. After recording the oxygen consumption until anaerobiosis is attained, the perfusion pump is switched on, and the O.D. returns to the initially recorded level, ready for a new measurement. Using this procedure KuEera and Raddacz were able to describe for the first time the spatial variations of the oxygen consumption in the Hensen node, the area pellucida, and area opaca of the chick embryo starting at the stage of definitive primitive streak (stage 4) up to the stage of 10 somites.

26 1

MEASUREMENT OF OXYGEN CONSUMPTION

9. Determination of Rapid Functional Transitions of Respiratory Systems Accurate determinations of the rate of oxygen consumption using oxygen electrodes presents serious technical difficultieswhen dealing with rapid, transient changes in respiratory activity. Capuano et al. (1980) measured the initial rate of oxygen consumption of a suspension of rat liver and beef heart mitochondria, and found that the HbOpmethod gave systematically higher values than the oxygen electrode method (Table VI). The addition of substrates to nonrespiring mitochondria results in an immediate deoxygenation of HbOn, whereas the Clark electrode coated with highsensitivity membrane or standard membrane showed a lag period of 1.5-3 sec. The fast response of the spectrophotometric recordings to variation of the oxygen consumption of the sample is due to the prompt dissociation of the oxyhemoglobin, when the concentration of the oxygen decreases (kl = 34 sec-I). In fact, the dissociation rate constant for different hemoproteins varies widely from 10 sec-' (myoglobin) to 156 sec- (monomeric P-chain of human hemoglobin) (Antonini and Brunori, 1970). Hence, the best candidate for measuring rapid changes of oxygen consumption is the monomeric @chain, and the spectrophotometric method is well suited for following the transient respiratory bursts (Papa et al., 1980a; 1980b).

'

TABLE VI Initial Rates of Oxygen Consumption of Isolated Mitochondria Measured with Clark Electrode or with the H b 0 2 Method" Oxygen consumption (ng-atoms/min.mg of protein)

Mitochondria Beef heart Rat liver Rat liver

Substrate Duroquinol Succinate Succinate + CaCI2

Clark electrode (standard membrane) 95 k 6 53.2 t 2.7 296 t 20

Clark electrode (high-sensitivity membrane) HbOp method 148 k 7 82 t 7 415 k 23

210 212 106 % 4.5 616 f 35

"Taken from Capuano et al. (1980). The respiratory medium contained 1-2.5 mg mitochrondrial protein/ml, 130mM LiCI, 3mM HEPES buffer (pH 7.2), rotenone (0.5 pg/mg of protein), and valinomycin (0.1 pg/mg of protein). When CaCI,-stimulated respiration was determined, valinomycin was omitted, and 2mM K-phosphate (pH 7.2) was added to the medium. HbOf deoxygenation was measured with a dual-wavelength spectrophotometer (A1 = 577 nm; A2 = 568 nm), at 25°C. The final concentrations of duroquinol, succinate, and CaC12 were 19pM, lmM, and 0.69mM, respectively.

262

OCTAVIAN BAKZU

10. Determination of Substrate Concentrations Oxygen sensors are useful not only for oxidative enzyme assays, but also for measuring the concentration of the corresponding substrates. One of the best known examples is the determination of glucose concentration by following with a Clark electrode the oxygen depletion after addition of glucose oxidase to the samples being analyzed (Bergmeyer and Hagen, 1972; Makin et al., 1978; Wolff and Mottola, 1978). T h e same principle can be used for measurement of cholesterol, lactate, and uric acid. Using the HbOp method it is possible to determine glucose in about 1 ~1 of human plasma or serum (Muresan et al., 1980). T h e interval of linearity of extinction increase at 436 nm is between 0.3 and 3 g glucoselliter. Another interesting application of the HbO:, method is the determination of ethanol concentration in the presence of NAD+, alcohol dehydrogenase and bacterial NADH-oxidase as an “indicator” system (Figure 11). Since the standard curve is expressed in terms of the rate of oxygen consumption, the interval of linearity as well as the slope depend on the

0.05

Figure 1 1. Determination of ethanol concentration by measuring the oxygen consumption in the presence of yeast alcohol dehydrogenase, NAD+, and bacterial NADH-oxidase. The oxygen consumption was assayed using the repetitive measurement system with a 4-mm cuvette of 0.203-ml final volume at 30°C. The cuvette is filled with 0.05M pyrophosphate buffer (pH 8.4).2 m M NAD+, and 0.07mM Hb02. After the Hb02 was deoxygenated by 30-40%, 5 PI of bacterial membranes (0.16 mg of protein), and 1 unit of yeast alcohol dehydrogenase (ADH) were injected. After the O.D. was stabilized, varying amounts of ethanol were injected into the cuvette and the extinction increase at 436 nm was followed for another 1-2 min. The left side of the figure shows a typical recording by using 36 nmol of ethanol; the right side of the figure shows the dependence of the oxygen consumption on the amount of ethanol added.

MEASUREMENT OF OXYGEN CONSUMPTION

263

activity of alcohol dehydrogenase and NADH-oxidase. Therefore, the assay of unknown samples must be performed under identical conditions, or even better, by using an “internal” standard. Using this procedure, ethanol concentrations as low as 0.01% in 5 ~1 of human plasma can be easily quantified. The same principle applies for determination of any other substrate that is oxidized by a pyridine-linked dehydrogenase.

X. CONCLUSIONS AND PERSPECTIVES During the last 15 years, the Hb02 method for determining oxygen consumption has found many applications in basic research as well as in clinical chemistry. Assays in the usual range of volumes (0.2-1.0 ml), using general-purpose instruments, allow the estimation of oxygen consumption of whole-tissue homogenates, isolated cells, and mitochondria or microsomes. Though the assays are of higher sensitivity,the results are comparable to those obtained with the oxygraphic or manometric methods. Microspectrophotometric assays using a scanning system revealed regional variations of the oxygen consumption during embryogenesis. As new technologies are developed, the Hb02 method will surely find new applications such as (1) automatic assay of enzymes and metabolites from human serum or tissues obtained by biopuncture and having a diagnostic significance, (2) control of structural and functional integrity of cells or tissues in culture, (3) indicator of cellular transformation induced by viruses or chemicals, in experimental oncogenesis, and (4) simultaneous assay of respiration and glycolysis (using pH microelectrodes), in the physiological range of oxygen concentrations,which would allow a new approach to the study of the Pasteur effect.

Abbreviations HbOz HbOz method Hb Hbt MetHb MbO:! Mb MetMb HbCPA and HbCPB

oxyhemoglobin spectrophotometric assay of oxygen consumption that uses oxyhernoglobin or other hemoproteins as indicator hemoglobin in the deoxygenated form total hemoglobin (HbOp + Hb) methemoglobin oxymyoglobin myoglobin in the deoxygenated form metmyoglobin hemoglobin treated with carboxypeptidase A or carboxypeptidase B, respectively.

264 f3-PMB

Y K: V or Q02

OCTAVIAN BARZU isolated chain of hemoglobin, with p-chloromercurybenzoateblocked-SH groups fractional saturation of the pigment with oxygen (Hb02/Hbt or MbOn/Mb,) Michaelis-Menten constant for oxygen of an oxidase oxygen consumption of biological sample expressed as mol or pmol oxygen consumedhin deoxygenation rate of the oxygen donor for different y values the rate of oxygen consumption during HbOn or Mb02 dissociation as measured with oxygen electrode

Acknowledgments This work was supported by grants from Centre National de la Recherche Scientifique (Laboratoire Associe no. 270),Fondation pour la Recherche Medicale, Institut National de la Sante et de la Recherche Medicale, and Delegation Genkrale a la Recherche Scientifique et Technique. I thank R.J. Hohman for critical comments and helpful discussion, and Florence Durand who kindly printed the photographs. Special thanks are due to Eppendorf Geratebau (Hamburg, GFR) for making possible some new developments described in this work.

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Benson, D. M., Knopp, J. A., and Longmuir, I. S. (1980), Biochim. Biophys. Actu, 5 9 1 , 187-197. Bergmeyer, H. U.and Hagen, A. (1972), Z. Anal. Chem., 2 6 1 , 333-336. Bohme, H. J., Kopperschlanger, G., Schulz,J., and Hoffman, E. (1972),J. Chromutogr., 69, 209-2 14. Bradford, M. (1976), Anal. Biochem., 72, 248-254. Capuano, F., Izzo, G., Altamura, N., and Papa, S. (1980), FEBS Lett., 1 1 1 , 249-254. Chance, B. and Williams, G. R. (1956), Adv. Enzymol., 1 7 , 65-134. Chance, B., Oshino, R., and Oshino, N. (1978), Methods Enzymol., 54, 499-505. Cittadini, A., Scarpa, A., and Chance, B. (1973), Biochim. Biophys. Actu, 2 9 1 , 246-259. Clark, L. C. (1956), Trans. Am. SOC.Artif: Int. Organs, 2 , 41-57. DPnsoreanu, M., Telia, M., Tarmure, C., Oarga, M., Markert, M., Ivanof, A., and Birzu, 0. (1981), Anal. Biochem., I l l , 321-326. DPnsoreanu, M., Markert, M., Lascu, I., Tarmure, C., Frei, J., and BPrzu, 0. (1983), Znt. J . Biochem. 1 5 , 1191 - 1194. Davies, P. (1962), Phys. Tech. Biol. Res., 4 , 137-179. Degn, H., Lundsgaard, J. S., and Petersen, L. C. (1980),Methods Biochem. Anal.,26,47-77. Dixon, M. (1943),Manometric Methods as Applied to the Measurement of Cell Respiration and Other Processes, Cambridge University Press, London and New York. Estabrook, R. W. (1967), Methods Enzymol., 1 0 , 41-47. Estabrook, R. W. (1978), Methods Enzymol., 52, 43-47. Fatt, I. (1976), The Polarogruphic Oxygen Sensor, CRC Press, Cleveland, Ohio, pp. 1-278. Feder, J. and Tolbert, W. R. (1983), Sci. Am., 248, 24-31. Gordon, E. E. and DeHartog, M. (1968), Biochim. Biophys. Actu, 1 6 2 , 220-229. Haddock, B. A. and Jones, C. W. (1977), Bacteriol. Rev., 4 1 , 47-99. Hamberger, L., Herlitz, H., and Hultborn, R. (1975), Methods Enzymol., 39, 403-425. Herlitz, H. and Hultborn, R. (1974), Acta Physiol. Scund., 90, 594-601. HodPrnau, A., Dancea, S., and BPrzu, 0. (1973),J. Cell Biol., 59, 222-227. Honjo, I., Takasan, H., and Ozawa, K. (1968),J. Biochem. (Tokyo) 6 3 , 332-340. Hultborn, R. (1972), Anal. B w c h m . , 47, 442-450. Hultborn, R. and Hyden, H. (1974), Exp. Cell Res., 87, 346-350. Hultborn, R. (1976), in Meas. Oxygen, Proc. Interdiscip. Symp., (H. Degn, I. Balslev, and R. Brook, Eds.), Elsevier, Amsterdam, pp. 89- 102. Imai, K., Morirnoto, H., Kotani, M., Watari, H., Hirata, S., and Kuroda, M. V. (1970), Biochim. Biophys. Actu, 200, 189- 196. Jain, M., Sands, F., and Von Korff, R. W. (1973), Anal. Biochem., 52, 542-554. Jaworowski, A., Campbell, H. D., Poulis, M. I., and Young, I. G. (1981), Biochemistry, 20, 2041-2047. Kaufman, S. (1971), Adv. Enzymol., 35, 245-319. Keevii, T. and Mason, €3. S. (1978), Methods Enzymol., 5 2 , 3-40. Kezdi, M., Mantsch, H. H., Muresan, L., Tarmure, C., and BPrzu, 0.(1973),FEBS Lett., 3 3 , 33-36. Knopp, J. A. and Longmuir, I. S. (1972), Biochim. Biophys. Acta, 2 7 9 , 393-397.

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Kufera, P., Raddacz, E., and deRibaupierre, Y. (1979). Experientiu, 35, 922. Kufera, P. and Raddacz, E. (1980), Respir. Physiol., 39, 199-215. Kunze, K. and Lubbers, D. W. (1973), Adu. Exp. Med. Biol.,37A, 35-43. Lessler, M. A. (1982), Methodc Biocha. Anal., 28, 175-199. Lloyd, D., James, K., Williams, J., and Williams, N. (1981), Anal. Biochem., 116, 17-21. Longmuir, I. S., (1957), Biochem. J.. 65, 378-382. Lu, A. Y. H. and West, S. B. (1978). P h a m . Ther. A . , 2 , 337-358. Makin, H. L. J., Warren, P. J. and Edridge, J. D. (1978), Clin. Chim. Acta, 84, 137-143. Margoliash, E. and Walasek, 0. F. (1967), Methods Enzymol., 10, 339-348. Markert, M. and Frei, J. (1978), Ann. Biol. Clin., 36, 201-204. Markert, M. and Frei, J. (1979), Enzyme, 24, 327-336. Markert, M., Allaz, M. J., and Frei, J. (1980), FEBS Lett., 113, 225-230. Markert, M. and Frei, J. (1981), Adv. Clin. Chem., 22, 125-162. McCoy, G . D., Resch, R. C., and Racker, E. (1976), Cancer Res., 36, 3339-3345. Merola, A. J., Scott, K. M., and Brierley, G. P. (1971), Anal. Bzochem., 41, 455-459. Muresan, L., DPnsoreanu, M., Ana, A., Bara, A,, and BLrzu, 0. (1980),AnaLBiochem., 104, 44-50.

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Pande, S . V. and Blanchaer, M. C. (1971)J. B i d . Chem., 246, 402-411. Papa, S., Guerrieri, F., Lorusso, M., Izzo,G., Boffoli, D., Capuano, F., Capitano, N., and Altamura, N. (1980a), Biochem. J., 192, 203-218. Papa, S., Capuano, F., Markert, M., and Altamura, N. (1980b), FEBS Lett., 1 1 1 , 243-248. Porumb, H., Lascu, I., Matinca, D., Oarga, M., Borza, V., Telia, M., Popescu, O., Jebeleanu, G., and BPrzu, 0. (1982), FEBS Lett., 139, 41-44. Pressman, B. C. (1967), Methods Enzymol., 10, 714-726. Raddacz, E. and Kufera, P. (1977), Experientia, 33, 783. Raddacz, E. and Kufera, P. (1983), Respir. PhYsiOl. 51, 153-166. Riggs, A. (1981), Methods Enzymol. 76, 5-29. Scarpa. A. and Lindsay, J. G. (1972), Eur. J . Biochem., 27, 401-407. Schindler, F. (1964), “Reaction Kinetics of the Cytochrome Oxidase,” Ph.D. Dissertation, University of Pennsylvania. Slater, E. C. (1967), Methodc Enzymol., 10, 19-29. Snow, T . R. and Jobsis, F. F. (1976), Biochim. Biophys. Acta, 440, 36-44. Starlinger, H. and Lubbers, D. W. (1973), Pfliigers Arch., 341, 15-22. Suzuki, R., Sato, K., and Tsuiki, S. (1968), Arch. Biochem. Biophys. 124, 596-603.

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Methods of Biochemical Analysis, Volume 30 Edited by David Glick Copyright © 1984 John Wiley & Sons, Inc.

METHODS OF BIOCHEMICAL ANALYSIS

VOLUME 30

Historical Development and Newer Means of Temperature Measurement in Biochemistry ROBERTL. BERGER,Laboratory ofTechnica1Developnent, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesdu, Maryland, THOMAS R. CLEM, Biomedical Engineering and Instrumentation Branch, Division of Research Seruices, National Institutes of Health, Betksdu, Maryland, VICTORIA A. HARDEN,* Department of History, Emoq University, Atlanta, Georgia, AND B. MANGUM,Centerfor Absolute Physical Quantities, National Bureau of Standarak, Wmhington, D.C.

w.

................. 270 .............. 271

111. Temperature Scal

1. IPTS-68 .............................................................................................. 2. Future Improvements and Extensions of the IPTS ............ 3. Practical Standards for Biochemical and Clinical Laboratories ....... IV. Methods of Measuring- Temperature ............................................................ 1. Liquid-in-Glass Thermometers .......................................................... 2. Dial Thermometers ............................................................................ 3. Bimetallic-Strip Thermometers ......................................................... 4. Gas Thermometers ............................................................................ 5. Resistance Thermometers .................................................................. 6. Thermoelectric Thermometry ........................................................... 7. Radiation Thermometers ................................................................... 8. Noise Thermometers ......................................................................... 9. Resonance Thermometers ...................................

282 286 292 293 294 294 295 295 295 296 298 298

*Fellow, Museum of American History, Smithsonian Institution, Washington, DC (19811982 during which time this writing was done). Certain commercial equipment, instruments, or materials are identified in this paper to specify adequately the experimental procedure. In no case does such identification imply recommendation or endorsement by the National Institutes of Health, the National Bureau of Standards, or the Smithsonian Institution nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.

269

270

R. L. BERGER, T. R. CLEM, V. A. HARDEN, AND B. W. MANGUM 10. Diode Thermometers

.................................... ......................................

1.

VI.

Resistance Measurements

Recent Applications of Modern Te

2. Heat Conduction and Response Time Corrections for Thermal

......................................

Enzyme Reaction Detection .. 4. Standard Temperature Reference Systems for Biochemistry and Clinical Chemistry .................................................................... VII. Conclusions and Forecasts ............ ............................... Acknowledgments ............................................................. References ................................................................

300 30 1 30 1 30 1 302 302 306 306 306 306 307 307 308 308 308 31 1 319 322 327 327 328

I. INTRODUCTION T h e measurement of temperature is one of the most common physical measurements routinely made. It is so common that it is often overlooked as a variable when complex biochemical reactions are being studied. This is unfortunate, because an error in the temperature of a reaction may produce a large error in the results that becomes apparent when the results are compared with those of known standard reactions. For example, if the rate of reaction of an unknown enzyme is being studied at a temperature that is different by 0.1% from the temperature at which the standard reaction was measured, an error as large as 2 - 5 % in the observed rate of reaction can occur. T h e experimental data would not correlate then with the known enzyme reaction rates. Such errors lead to confusion in determining mechanisms and to the large variations that occur even in normal values from one clinical laboratory to another. This article seeks to bring the importance of accurate temperature measurements to the attention of biomedical scientists. We will identify the latest methods of temperature measurement and control as well as new temperature fixed-point standards that are o r will shortly become available.

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27 1

A discussion of the present standards of temperature measurement is preceded by a discussion of the history of thermometry. T h e goal of Sanctorius, the first person to perceive the potential usefulness of the thermometer in medicine, has not changed in principle since the early seventeenth century. He hoped to obtain quantitative measurements rather than subjective appraisals. For us, this should be a caution not to ignore careful temperature measurements as we study complex reactions. Temperature, one of the seven basic physical quantities of the International System (SI) of units, is that property which describes the thermodynamic states of a system and is a measure of that system’s hotness, as expressed in terms of any of several arbitrary scales. It is an indicator of the direction in which energy will flow spontaneously when two bodies are brought into contact, that is, from the hotter body to the colder one. Temperature, unlike mass and volume, is an intensive property, that is, it is independent of the quantity of matter. Any device or system that has one or more physical properties (e.g., electrical resistance, electrical potential, length, pressure at constant volume, or volume at constant pressure) that vary monotonically and reproducibly with temperature may be used to measure temperature. T h e science of the measurement of temperature is called thermometry. I n the past, the measurement of high temperature was known as pyrometry but now that term usually refers to radiation thermometry at any temperature. Although the accuracy of a measurement refers to the difference between the measured value and the true value of the quantity being measured, and the precision of measurement refers to the degree of agreement among repeated measurements of the same quantity, it follows that a set of measurements of the same quantity, it follows that a set of measurements may be very precise but terribly inaccurate. Since in many instances the word “accuracy” is used when inaccuracy is meant and the word “precision” is used when imprecision is meant, perhaps it would be better always to refer to uncertainties of measurement, statistical and systematic, rather than to accuracy and precision.

11. HISTORY OF THERMOMETRY From earliest prehistoric times, people have been interested in the phenomenon of heat. It is estimated that fire was used by Peking man 500,000 years ago. An elevation in body temperature accompanying certain diseases was apparent also to the ancients. Before the fifth century B.C., however, the concepts of heat and temperature remained undefined.

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The philosophers of Greece, whose goal was to understand and describe the nature of the universe, made use of the known properties of heat, but they did not separate the concepts of heat and temperature. They knew, for example, that fire-defined as one of the four elements of the universe-would cause the expansion of air, another primal element. In the second or first century B.C., Heron of Alexandria applied this knowledge to invent a steam turbine, but he considered it only a toy. Philon of Alexandria likewise invented several devices that demonstrated the expansion property of heated air (Cohen and Drabkin, 1948; Sarton, 1959). The Greeks went no further in their investigations, although the manuscripts they left inspired the scientific thinkers of the Renaissance (Taylor, 1941). For approximately 1500 years after these tentative beginnings, the nature of heat and its measurement remained mysterious. Maria, the Jewess of Alexandria, is believed to have invented the first distillation apparatus in the first or second century A.D. No alchemist, however, advanced the understanding of the concepts of heat and temperature beyond what was known by the Greek philosophers. The modern concepts of temperature measurement have their roots in the Scientific Revolution and are linked to two characteristics of that phenomenon: the development of an instrument that extended the ability of the five senses, and the evolution of a quantitative approach to problem solving. The new instrument, of course, was the thermometer. Once the concept of a temperature-measuring instrument was understood, the problems of developing a thermometer that could be standardized and reproduced accurately demanded a quantitative mode of thinking about heat, temperature, and their measurement. Lord Kelvin remarked in the nineteenth century that “when you can measure what you are speaking about and express it in numbers, you know something about it” (Kelvin, 1891- 1894). The history of thermometry is an excellent example of the movement from knowing something vaguely through qualitative evaluation to knowing something definitely through quantitative methods. In the sixteenth century, there was a revival of interest in the mechanical devices of the Greeks. Although it is not known with certainty who at that time first conceived the idea of measuring temperature or “degrees of hotness,” Galileo Galilei is usually credited with the invention of the first thermometer. His writings contain only one reference to the instrument, which he likely did not consider of any importance. His friends and students, however, fortunately recorded a description of the instrument that he invented shortly after 1592.

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The device, which Galileo termed a “thermoscope,” was described by Father Benedetto Castelli, as he recalled an experiment he had seen Galileo conduct in 1603: Galileo took a glass vessel about the size of a hen’segg, fitted to a tube the width of a straw and about two spans long; he heated the glass bulb in his hands and turned the glass upside down so that the tube dipped in water held in another vessel; as soon as the bulb was cooled down, the water rose in the tube to the height of a span above the level in the vessel; this instrument he used to investigate degrees of heat and cold (Bolton, 1900, p. 18; Middleton (1966), pp. 3-26).

The air in Galileo’s device was the thermometric substance, and its expansion or contraction depended upon changes in atmospheric pressure as well as temperature. Because of this, it is more properly called a “barothermoscope,” although its dependence on barometric fluctuations was not recognized until after the invention of the barometer in 1643 (Roller, 1960, pp. 12- 13). Galileo also fitted his barothermoscope with a scale marking off degrees “at pleasure,” but he made no attempt to base the scale on standard temperatures that were reproducible. Galileo apparently did not attempt to measure temperature changes in the human body. One of his colleagues, who was professor of medicine at the University of Padua and who had also experimented with devices to count the pulse, seems to be the first person to attempt to record quantitatively the temperature of the human body. Sancttario Santorio (Sanctorius) recorded the temperatures of many of his patients, believing that Galileo’s instrument would be more accurate than the subjective observations and reports of physician and patient. He developed a scale for the instrument based on two extreme points, the lower being the reading when it was exposed to snow and the upper when it was exposed to the flame of a candle. The barothermoscope, however, could hardly be called a precision instrument. Because no quick solutions were found to the problems of this instrument and others developed soon after, Santorio’s goal of a purely objective measurement of body temperature was not realized for three centuries. Throughout the seventeenth and eighteenth centuries, controversy over the thermometer’s accuracy and value to medicine limited its use. The crude instrument used by Galileo and Santorio, however, represented the first stage-the introduction of the concept-in the development of the modern thermometer. The second stage, in which the original instrument was refined and an acceptable thermometric substance was discovered, began about 163 1 with the development of a liquid-expansion instrument, when Jean Rey, a

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French physician, invented an instrument employing the expansion of water to measure the temperature of his patients. His instrument was a glass bulb and stem similar to Galileo’s except that it was inverted and partly filled with water. The upper end of the stem was left open, so the readings were influenced by evaporation of the water, although not to an appreciable extent by changes in atmospheric pressure (RBller, 1960, p. 13).

Ten years later the Grand Duke Ferdinand I1 of Tuscany, one of the founders of the Florentine Accademia del Cimento (Academy of Experiment), developed a thermoscope using alcohol as the liquid in a glass tube that was hermetically sealed. This advance effectively eliminated the influence of barometric pressure on temperature readings, but it did not solve the problems of calibration and choice of temperature-indicating substance. Using this method, however, the Accademia del Cimento manufactured many temperature-measuring devices. These “marvels of glass blowing” were marked with minute glass beads of different colors attached to the stem. T h e scale was based on a low temperature indicating “the most severe winter cold” and a high temperature representing “the most severe summer heat.” T h e ambiguity in defining such extremes and the difficulty in reproducing them accurately rendered these thermoscopes practically useless. Debate continued about which substance would best register temperature change. There were proponents of alcohol, linseed oil, highly rectified “spirit of wine,” and weakened spirit of wine (Reiser, 19’78).Each of these substances, however, had drawbacks. Spirit of wine boiled at high temperatures and froze at low ones; hence its range of temperature measurement was limited. In addition, different strengths of wine responded differently to temperature changes. Linseed oil adhered to the sides of the thermometer, making difficult an accurate determination of its movement. Mercury proved to be the most acceptable substance. It was easily readable, did not adhere to the tube, conducted heat readily, and had a low freezing and high boiling point. Used first in 1659, quicksilver was later discovered to change its volume more nearly in proportion to changes in temperature than other liquids. One physicist exclaimed in joy over the properties of mercury, “Surely nature has given us this mineral for the making of thermometers” (Cajori, 1929). T h e third stage in the development of the modern thermometer began in 1665 with the development of the first thermometric standard scale. In that year Robert Boyle, Robert Hooke, and Christian Huygens suggested independently that thermometers could be calibrated effectively from a single fixed point. Degrees would represent a standard expansion o r contraction fraction of the volume of the thermometric substance measured at the fixed point. Boyle set the fixed point at the freezing tempera-

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ture of oil of anise seed. Hooke suggested the freezing point of water, while Huygens believed that either the boiling or freezing point of water would be acceptable. A major problem with a single fixed-point scheme was the degree of expansion experienced by different substances as their temperatures changed. Basing a thermometer on the expansion of one liquid would produce degree marks that were not comparable to those based on a different liquid. “We are very much to seek for a standard or certain Measure of Cold,” urged Robert Boyle, “as we have settled Standards for weight, and magnitude, and time, so that when a man mentions an.Acre, or an Ounce, or an Hour; they that hear him, know what he means” (Boyle, 1683). The “certain measure of Cold” that became accepted in the eighteenth century used the concept of two fixed points rather than one. Degrees could be conveniently marked off between the two in equal increments. The first efforts to construct a scale based on two fixed points came in the last third of the seventeenth century. In 1669 the Jesuit Honore Fabri suggested the melting point of snow as the lower temperature and the “greatest summer heat” as the upper (Roller, 1960, p. 15). Joachim Dalence in 1688 refined Fabri’s upper point to a more precisely defined temperature. He adopted the melting point of butter as the higher temperature, defined that temperature as + 10”and the melting point of snow as -lo”, and divided the interval into 20 equal parts. In 1694 Carlo Renaldini, who, like Fabri and Dalence, had been associated with the Accademia del Cimento during its brief existence from 1657 to 1667, proposed the freezing and boiling temperatures of water as the two fixed points. He chose to divide the interval into 12 equal parts. Around 1702, the Danish astronomer Ole Roemer devised a thermometer based on a zero point that represented a mixture of ice and salt and referred to as the “point of artificial freezing” (Cork, 1947). Roemer’s upper point was represented by steam and assigned the value 60”. The use of the sexagesimal system was probably chosen to correspond with that in use in clocks and geometrical figures. Roemer was visited in 1708 by the celebrated maker of meterological instruments, Daniel Fahrenheit. Following this visit, Fahrenheit began to construct thermometers using Roemer’s fixed points, but soon chose to change the denotation of the steam point. He felt that the many fractions involved in common readings-the temperature of the human body was expressed as 22.5”in Roemer’s scale-made the system “inelegant” (Cork, 1947). By 1724 he had completely recalibrated the thermometer. He described his method in Philosophical Transactions: . . . placing the thermometer in a mixture of sal ammoniac or sea salt, ice, and water a point on the scale will be found which is denoted as zero. A second point is obtained if the

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same mixture is used without the salt. Denote this position as 30. A third point, designated as 96, is obtained if the thermometer is placed in the mouth so as to acquire the heat of a healthy man (Fahrenheit, 1724).

Fahrenheit determined the boiling point of water to be 212" using his scale, and subsequently modified the freezing point of water to 32" to represent the interval between these two points by 180", a more rational number than 182". Body temperature was later modified on the scale to 98.6", the "normal" human temperature denoted on clinical thermometers in the United States today. Except for the United States, where thermometers using the Fahrenheit scale are standard for clinical and meteorological reporting, the Fahrenheit scale is no longer in use. The temperature scale accepted in the international scientific community today for measurement in the moderate range is the Celsius scale, originally called the Centigrade scale. Based on the freezing and boiling points of water as the two fixed points, at 0" and loo", respectively, the centigrade scale may have been suggested as early as 1710 by the Swede Elvius. Between 1740 and 1743, it was apparently independently proposed by the Swedish botanist Linnaeus, Christian of Lyons, and, in inverted form, by the Swedish astronomer Anders Celsius. Celsius' scale ("C) made the boiling point of water 0" and the freezing point 100". Because of the association with the "C" for centigrade, however, Celsius is usually credited with the invention of the scale. Other practical temperature scales that bear the name of their originators were proposed by Rene Reaumur in 1730 and W. J. M. Rankine in 1850. Values on the RCaumur ("Re) and Rankine ( O R ) scales are fixed by defining the freezing point of water as 0"Re and 491.67"R and the boiling point of water at one standard atmosphere as 80"Re and 67 1.6'7"R. The Reaumur scale is no longer in use. The Rankine (or absolute Fahrenheit) scale is one of many possible thermodynamic scales and is still used in some aspects of engineering, such as the calculation of the theoretical efficiency of engines. At the same time that mercury-in-glass thermometers were being refined, scientists were examining the behavior of gases. Their conclusions were valuable for the future of temperature measuring devices because gases respond to temperature changes with greater uniformity than liquids. As early as 1660, Robert Boyle published the results of his experiments on the response of gases to changes in pressure. Boyle, however, did not examine changes that occurred when the temperature was varied. The first person to study the effect of temperature change on gases was Guillaume Amontons, who in 1699concluded "that unequal masses of air under equal weights increase equally the force of their spring for equal degrees of heat" (Taylor, 1941). His work began a century of study about the properties of gases that contributed to the development of gas thermometry.

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Amontons stated in general language that the ratio of change in pressure to the original pressure is constant for a given temperature change whether the volume of air measured is large or small. This relationship was found to apply also to other common gases by J, A. C. Charles, a French physician, about 1780. Later, the English scientistJohn Dalton and the French scientists L. J. Gay-Lussac and H. V. Regnault confirmed Charles' conclusion with more accurate experiments. Their work demonstrated that for every degree centigrade rise in temperature, a given volume of gas will increase the pressure by about 1/273. All gases under conditions of low pressures, usually less than two atmospheres, and temperatures above the gas' critical temperature, that is, that point above which the gas cannot be made to liquify under any amount of pressure, obey this law. Then, with the volume at a constant pressure (or conversely the pressure at a constant volume), the following linear relationship holds: v = vo(1

+ at)

(1)

where v is the volume at temperature t "C, vo is the volume at O"C, and a is the coefficient of thermal expansion. A mercury-in-glass thermometer calibrated on the Centigrade scale was generally used for the indication of temperature in those measurements. The coefficient of thermal expansion was determined to be 1/273.15"C-', so that by extrapolation, the lowest possible temperature would be -273.15"C or absolute zero. By introducing the concept of absolute temperature, a new temperature scale based upon the behavior of ideal gases could be set up and thus avoid negative temperatures. Temperatures, 4, on that scale are given by 4 = t("C) + l/a = t("C) + 273.15 "C. Through the use of 4, the general gas law (the law obeyed by an ideal gas) becomes PV- constant

4

(2)

This law is the basis of the gas thermometer, and the scale on which the temperature 4 is defined is known as the ideal-gas or absolute temperature scale. if either the volume or the pressure of a fixed quantity of gas is held constant, then the measurement of the other determines the temperature directly. No gas is ideal, however, so that the gas thermometer is actually based on an approximately-ideal gas and the assumption that departures from ideality can be accurately measured and taken into :&runt. By this means, the thermometer is made to be independent of the properties of real gases and, thus, becomes effectively ideal. The theoretical usefulness of the concept of absolute temperature made possible the construction of gas thermometers that were precise and

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could be compared easily with one another. It also spawned the absolute or Kelvin temperature scale (units, K), named in honor of William Thomson (Lord Kelvin) who suggested the scale and contributed in the nineteenth century to the definition of heat as energy rather than the fluid “caloric”that had been widely accepted in the eighteenth century (Thomson, 1848, 1849). It was not until 1887, however, that a meeting of the Comite International des Poids et Mesures (International Committee on Weights and Measures) (CIPM)in Paris recommended a standard scale of temperature based on the change in pressure of a constant volume of hydrogen under specified conditions. The fixed points of this scale, known as the normal hydrogen or Centrigrade thermodynamic scale, were the temperature of melting ice (0°C)and that of the vapor of boiling distilled water (100°C) under a pressure of one standard atmosphere. In 1889 this standard was adopted formally, based on research conducted at the laboratories of the International Committee in the Breteuil pavilion at Sevres. By 1913 the normal hydrogen scale was established from -40°C to 200°C. In the long debate over the nature of heat, Black, Rumford, Hess, Carnot, Mayer, Joule, Clausius, Kelvin, and Helmholtz established the basic principles of the theory of heat as energy. The first principle, the law of conservation of energy, was widely accepted at the time it was promulgated “because it appeared reasonable and in accord with human intuition” (Lewis and Randall, 1961). The second principle, the law of dissipation of energy-that is, a constant increase in the entropy of the universe-was more slowly accepted. This law, however, ultimately led to the development of the thermodynamic temperature scale discussed below. This scale, which could be made identical to the ideal-gas scale, also considered temperature changes resulting from any type of mechanical or thermal energy. In 1848, William Thomson (Lord Kelvin) proposed such a scale based on the efficiency of an ideal reversible heat engine, which is dependent only on the limits of temperature between which it works (Thomson, 1848,1849). Sadi Carnot had described (Carnot, 1824) the ideal heat engine in 1824; its cycle consists of two isothermal (i.e., compression or expansion of a gas so slowly that there is no change in its temperature) and two adiabatic (i.e., compression or expansion so fast that no heat is lost) paths (Carnot, 1824). Thomson’s scale of thermodynamic temperature T was defined by the equation

where Q1refers to the quantity (joules) of heat extracted from the hot reservoir at a temperature of T I and 42 refers to the quantity of heat

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returned to the cold reservoir at a temperature of T2. T I and T2 are the temperatures on the thermodynamic scale. If an ideal gas is considered as the working substance in a Carnot cycle, then -Q1-

Q2

_ -$1

(4)

$2

Thus, the thermodynamic and the ideal-gas temperature scales become the same if the values are selected to be identical at one finite temperature. Precision thermometry based on the thermodynamic temperature scale had its beginnings with the work of P. Chappuis and of H. L. Callendar during the period from the late 1880s to the early 1900s. Chappuis transferred the hydrogen scale in the range from 0 to lOO”C, provided by his constant-volume hydrogen gas thermometer, to several carefully made mercury thermometers (Chappuis, 1888). These were then used to caIibrate many other mercury thermometers which in turn were to be used in many countries to put temperature measurements on the same scale. The probable uncertainty of those thermometers was stated to be +O.O02”C. An important development in thermometry was the resistance thermometer, first proposed in 1871 by Sir William Siemens, a German engineer living in England. Siemens noted that a change in the electrical resistance of a metallic conductor accompanied a change in temperature and that this property could be used to measure temperature (Siemens, 1871).S. P. Langley invented an even more sensitive resistance thermometer in 1881. It was designed to measure relatively the distribution of energy in the spectrum of the radiation from a hot body and is known as a “bolometer” (Langley, 1881). In the late 188Os, H. L. Callendar produced a workable platinum resistance thermometer (Callendar, 1887). The choice of platinum as a sensor was excellent. It has turned out to be the most stable and accurate thermometer available and is now a standard instrument of the International Practical Temperature Scale (IPTS). Callendar also developed a constant-volume gas thermometer, which he used to calibrate a platinum resistance thermometer up to about 550°C (Callendar, 1887).Another phenomenon noted as early as 1821 was that when two wires of unlike metals were fused together at one end and heated, an electromotive force existed between the other ends of the two wires. T. J. Seebeck’s discovery of these electromotive forces led to the development of thermocouples, thejunctions of metals that were capable of being used to measure temperature (Seebeck, 1826). A variety of metals, the “base” as well as the “noble” metals, have been employed in producing thermocouples. By arranging thermocouples in series to form a “thermopile,” very small changes in temperature can be detected.

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Later developments in temperature measurement employed knowledge about the relationship between temperature and radiation emission, measurable on a spectrometer. Probably first used by H. Le Chatelier in 1892, such a device was termed an “optical pyrometer.” Another temperature-measuring instrument based on radiation from a hot surface was the total radiation pyrometer. This device was proposed by C. Fery in 1902 and modified by others (Fery, 1902). Another development in temperature scales was the statistical mechanical scale. The statistical mechanical temperature of a many-particle system at equilibrium is by definition equal to the thermodynamic temperature. Consequently, any temperature-dependent phenomenon, which can be exactly described by statistical mechanics, can be used to determine the thermodynamic temperature of the system. Four well-known examples of such phenomena are the distribution of radiant energy from a surface (obeying Planck’s radiation law), the magnetic susceptibility of weak paramagnets (nuclear and electronic) at temperatures high compared to their critical (magnetic ordering) temperatures (obeying the Curie Law), nuclear orientation (y-ray anisotropy being the most widely used technique) of magnetically (electronic) ordered systems at ultralow temperatures, and electrical noise in a resistor (obeying the Nyquist relation). Instruments employing each of these phenomena as a temperature-measuring device will be described below. Since thermodynamic temperatures are so difficult to measure accurately by gas thermometry and because of the increasing demand of science and technology for accurate temperature measurements, discussions of an international practical scale covering as wide a temperature range as possible were begun before World War I, discontinued during the war, and then held again in the 1920s.Some desired features of such a scale were that temperatures on the scale should agree as closely as possible with thermodynamic temperatures, that it should be precisely reproducible, and that it should be conveniently and accurately realizable. Such a scale would thereby overcome the practical difficultiesof the direct realization of thermodynamic temperatures by gas thermometry, would unify existing national temperature scales, and would give the users a single, internationally accepted basis for measurements that was in close agreement with the thermodynamic scale. Since most physical laws involving temperature are based on thermodynamic temperatures, it was especially desirable that the values of temperature on any practical scale should be as close as possible to thermodynamic temperatures, or that the differences between the thermodynamic and those of the scale accurately known. Deliberations eventually led to the adoption of the International Temperature Scale of 1927 (ITS-27) by the 7th General Conference on

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Weights and Measures (ComptesRendus, 1927). The scale was developed by the National Bureau of Standards in the United States in cooperation with the Physikalisch-Technische Reichsanstalt of Germany and the United Kingdom’sNational Physical Laboratory (Preston-Thomas, 1972). The scale was based on the freezing and boiling points of water, at 0 and lOO”C,respectively, an interval of 100°Cexactly, and on some fixed points outside this range. In the range of -190 to 660°C of that scale, the platinum resistance thermometer was specified as the interpolation instrument. Slight modifications of ITS-27 were made in 1948. The designation of the unit of temperature, the degree Centigrade, was also replaced by the degree Celsius (Comptes Rendus, 1948). By so doing, all temperature scales were then named after their originators. In 1854,Lord Kelvin recommended that when a single fixed point that was sufficiently stable was developed, it would be preferable to define the scale using only that one point (plus the absolute zero) (Thomson, 1854). Accepting this recommendation, the 10th General Conference in 1954 redefined the Kelvin thermodynamic scale (Comite International, 1955). The redefinition of the scale was accomplished by assigning a value of 273.16 K to the temperature of the triple point of water. The unit, K, was defined as U273.16 of the thermodynamic temperature of the triple point. The zero of the Celsius thermodynamic scale was defined to be 0.01”C below the triple point, that is, a temperature on the scale was to be expressed in terms of its difference from that of the thermal state 0.01 K lower than the triple point of water. The temperature expressed in this way is the Celsius thermodynamic temperature, t , and is defined by t = T - 273.E K

(5)

In order to have a scale that gave temperature as close as possible to thermodynamic temperature, a completely revised and extended version of the IPTS was adopted in 1968 (Comptes Rendus, 1967-1968). This scale with modifications is the current international standard and will be discussed in detail in the following section. One need not conclude that the IPTS-68 is the final temperature scale that will be developed. At the time it was developed, it represented the best present effort to achieve Boyle’s “certain measure of cold” called for over 300 hundred years ago.

111. TEMPERATURE SCALES AND STANDARDS

There are many people making temperature measurements who are not cognizant of what temperature scale is being used, the basis of that scale,

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and the difference between the thermodynamic and practical temperatures, as well as the experimental errors of the measuring device. 1. IPTS-68

The IPTS-68 is the present version of the international temperature scale and it is defined by: (1) assigning values to the temperatures of 11 fixed points, extending from the triple point of hydrogen (13.81 K) to the freezing point of gold (1 337.58 K); (2) specifying interpolation formulae for the specified standard instruments, namely, the platinum resistance thermometer (Metrologia, 1976) between 13.81 K (-259.34"C) and 903.89 K (630.74"C),and the platinum- 10% rhodiudplatinum thermocouple (Cork, 1947)for the range from 903.89 K (630.74"C)to 1337.58 K (1064.43"C);and (3)specifying the Planck law of radiation for temperatures higher than 1337.58 K (1064.43"C),with 1337.58 K as the reference temperature and the value 0.014388 m-K for the second radiation constant C2 (Cork, 1947). The IPTS-68 recognizes the thermodynamic temperature, T, as the basic temperature and defines its unit (the kelvin, K, and not O K ) to be 1/273.16of the thermodynamic temperature of the triple point of water. The IPTS-68 and their assigned temperatures are listed in Table 1. In addition to the defining fixed points of the IPTS-68 given in Table I, there are other, so-called secondary, reference points available. Some of these are listed in Table 11, and some are shown also in Figure 1. The IPTS-68 extended the scales into the region 10 to 90 K and brought the values measured on the scale into agreement with thermodynamic temperatures within the limits of the accuracy of measurement at that time. The scale distinguished between the International Practical Kelvin Temperature, symbol Tss, and the International Practical Celsius Temperature, symbol t68. &5s = T68 - 273.15 K

(6)

The units of t68 and T68 are the same as for t and T, respectively, where T is the kelvin thermodynamic temperature and t is the Celsius thermodynamic temperature (unit is the degree Celsius, "C). The degree Celsius is by definition equal in magnitude to the kelvin. An amended version of the IPTS-68 was adopted in 1975 (Comptes Rendus, 1975). Any measured temperature, T68, was unchanged by that amended version. It differed from the 1968 version only in that an alternative fixed point was introduced (the argon triple point as an alternative to the oxygen boiling point) (see Table I), the specified natural isotopic composition of neon was changed slightly, the reference function for the standard platinum thermometer was given in an improved form,

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TABLE 1 Defining Fixed Points of the IF'TS-68 (amended edition of 1975) Assigned value of international practical temperature" Fixed points Triple point of equilibrium hydrogen* Boiling point of equilibrium hydrogen at a pressure of 33 330.6 Pa (25176 standard Boiling point of equilibrium hydrogenb" Boiling point of neon' Triple point of oxygen Triple point of argon' Condensation point of oxygen"' Triple point of water Boiling point of water' Freezing point of tin" Freezing point of zinc Freezing point of silver Freezing point of gold

t68(Oc)

13.81 17.042 20.28 27.102 54.361 83.798 90.188 273.16 373.15 505.1181 692.73 1235.08 1337.58

-256.34 -256.108 -252.87 -246.048 -2 18.789 - 189.352 - 182.962 0.01 100 23 1.9681 419.58 96 1.93 1064.43

aExcept for the triple points and the equilibrium hydrogen point at 17.042 K, the assigned vaIues of temperature are for equilibrium states at a press of 101 325 Pa (1 standard atmosphere). If differing isotopic abundances could significantly affect the fixed point temperatures, the abundances are specified. bEquilibrium hydrogen means that the hydrogen has its equilibrium wth-pru composition at the relevant temperature. Ortho and para are the designations for the molecular configurations (nuclear spin arrangements) of hydrogen. 'Fractionation of isotopes or impurities dictate the use of boiling points (vanishingly small vapor fractions) for hydrogen and neon and condensation point (vanishingly small liquid fraction) for oxygen. dThe equilibrium state between the solid, liquid, and vapor phase of argon (triple point of argon) at TS8= 83.798 (t68 = 189.352"C)may be used as an alternative to the condensation point of oxygen. T h e equilibrium state between the solid and liquid phases of tin (freezing point of tin) has the assigned value of T68 = 505.1 18 1 K ( t 6 S = 23 1.9681 "C) and may be used as an alternative to the boiling point of water.

the criteria for selection of thermocouples were changed, the values of some of the secondary reference points were changed, a table of estimated uncertainties of the assigned values of the defining fixed points was deleted, and some inconsistencies and deficiencies were removed from and additional information added to the section on supplementary information.

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R. L. BERGER, T. R. CLEM, V. A. HARDEN, AND B. W. MANGUM TABLE I1 Secondary Reference Points International practical temperature

Triple point of normal hydrogen" Boiling point of normal hydrogen" Triple point of neon Triple point of nitrogen Boiling point of nitrogen Boiling point of argon Sublimation point of carbon dioxide Freezing point of mercury Ice pointb Triple point of phenoxybenzene (diphenyl ether) Melting point of gallium Triple point of gallium Triple point of benzoic acid Freezing point of indium Freezing point of bismuth Freezing point of cadmium Freezing point of lead Boiling point of mercury Boiling point of sulfur

13.956 20.397 24.561 63.146 77.344 87.294 194.674 234.314 273.15 300.02 302.922 302.924 395.52 429.784 544.592 594.258 600.652 629.8 1 717.824 ~

-259.194 -252.753 -248.589 -210.004 - 195.806 - 185.856 - 78.476 -38.836 0 25.87 29.772 29.774 122.37 156.634 27 1.442 321.108 327.502 356.66 444.674

~~~

"Normal hydrogen is a mixture of 75% ortho-hydrogen and 25% para-hydrogen. T h e ice point is a very close approximation to the temperature defined as being 0.01 K below the triple point of water.

2. Future Improvements and Extensions of the IPTS In view of the elaborate experimental techniques usually required to make accurate thermodynamic temperature measurements, the need for a practical scale above about 0.5 K that is close to the Kelvin thermodynamic temperature scale remains great. There are several modifications that can be anticipated for a future IPTS. They include assigned values of fixed points that are in closer agreement with thermodynamic temperatures (as determined by recent experiments), extension of the range covered to lower temperature, improved standard instruments for interpolation procedures. It is expected that there will be a scale revision which will encompass the above, and that the new scale will be adopted by about 1987.

POINTS

SECONDARY REFERENCE

4 a

ICamot'r Law) Itm PoVo P+O

(Gas Law1

Vo = Volume at 273.16K

Po = Presiwe JI273.16K

273 16

lm PV

2 p-ro

THERMODYNAMIC TEMPERATURE

=

Conrtantr

= Wawelength

Spec1r.1 Concentratton Of

Black Body Radmnce

c,. c2

h

LA-

Figure 1. The International Practical Temperature Scale and the thermodynamic laws that define it.

5 :

INTERNATIONAL PRACTICAL TEMPERATURE SCALE OF 1968 (AMENDED EDITION OF 1975)

T

T - 2 7 3 16

Fund.ment.1 L a m defining Thermodynamic Tampraturn

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R. L. BERGER, T. R. CLEM, V. A. HARDEN, A N D B. W. MANGUM

3. Practical Standards for Biochemical and Clinical Laboratories Primary standards are those developed and maintained by national standards laboratories such as the National Bureau of Standards. These laboratories develop, maintain, and disseminate standards, such as the International Practical Temperature Scale. The IPTS-68 is disseminated to the users through secondary standards such as calibrated thermometers, fixed point references, and so on (see Table 11). Some of these thermometers are calibrated directly against the defining fixed points and others are calibrated over the range of need by a comparison calibration against a standard interpolating thermometer. This ensures that the basis for temperature measurement, the IPTS-68, is the same everywhere throughout the world. Secondary standards are employed in industrial, academic, and clinical laboratories. Calibrated at appropriate intervals against primary standards at the National Bureau of Standards, secondary standards are used \ to calibrate workrng instruments in the local institution. It is well known, but should nevertheless be emphasized, that there is an accumulation of errors in the progression down the calibration chain. Consequently, for the highest-accuracy work, calibrations should be performed by the primary standards laboratories. Although the specified instruments for realizing the IPTS-68 are the platinum resistance thermometers (PRTs) and the Pt- 1O%Rh/Pt thermocouple, these may not be the most'practical or appropriate choices for use in biochemical applications. In fact, the platinum resistance thermometer almost certainly is not, because of its size, its fragility, and the elaborate ancillary equipment required for making measurements. There are, however, some suitable alternatives for biochemical and biomedical uses. If an inaccuracy of ?O.O3"C, in addition to the thermometers' size and response time, does not prohibit their use, then certain mercury-in-glass thermometers would be suitable. Included in this group would be some total immersion thermometers and the SRM 933 and SRM 934 partialimmersion thermometers (Mangum, 1974; Mangum and Wise, 1974),the latter being available from the National Bureau of Standards. If somewhat better accuracy andor a sensor smaller (with a shorter response time) thanthe mercury-in-glass thermometer is required, then an industrial-grade PRT may be used. Normally, one can expect such PRTs to have an uncertainty of -tO.O2"C, plus whatever statistical uncertainty is present, if the PRTs are calibrated periodically (Mangum and Evans, 1982; Connolly, 1982).Some industrial PRTs are capable of giving better results than this and some much worse (Mangum and Evans, 1982). A few can be stable and reproducible to &O.O05"C, but that represents a small percentage of PRTs and they must be specially selected.

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287

If uncertainties of h0.01"C or less, and/or if very small, fast-response thermometers are required, then thermistors must be used. Of course, they may be used also for less accurate work. Some bead-in-glass probetype thermistors are stable and reproducible (Sappof, 1980),over a l-year period, to the equivalent of +O.OOl"C, when used at temperatures below 100°C. Their response time can be as short as a few milliseconds. There are many different types of thermometers available for use in the biomedical temperature range and they provide a wide choice of stability, sensitivity, and ease of use. Whatever thermometer is used, in either routine or in standardization applications, it must be calibrated periodically against a primary standard, or at least its calibration must be checked periodically at some temperature fixed points (defining fixed points or well-characterized secondary fixed points) in the range of the thermometer. Regular calibrations are particularly important for many of the new electronic thermometers that depend on sensitive resistive elements (e.g., thermistors) as sensors. The low cost, convenience, and high sensitivity of these thermometers make them attractive, but the possibility of significant calibration drift makes it highly desirable to have readily available a means of calibrating them, or for checking their calibration. The quickest, most accurate, and most convenient way of doing the latter is through the use of at least two temperature fixed points available for use. For thermistors, the ice point (0°C) or the triple point (0.01"C) of water and the gallium melting point (29.772"C)are suitable fixed points to be used, and they are readily available to the user. The latter fixed point is available, as SRM 1968, from the National Bureau of Standards (Mangum and Thornton, 1977)and also from commercial sources. The ice point can be prepared easily in the laboratory by using packed distilled-water ice with just enough distilled water added to fill the voids. Either of these fixed points is suitable for use in checking the calibration of industrial PRTs. The ice point is normally used with mercury-in-glass thermometers, although the gallium melting point has been used also for that purpose. Other, accuratelyknown temperature fixed points, conveniently spaced throughout the interval from -40°C to as high as 160"C, are currently being developed and when work on them is completed, they will permit the user to calibrate thermistors, as well as other types of thermometers, in this range. To be most useful to the biomedical community, these temperature reference points should be well-defined, stable, and reproducible to h0.005"C. What are some of the features of high-quality (defining and/or secondary) temperature fixed points that make them suitable for use in calibrating precision thermometers? The melting and freezing behavior of pure and slightly impure mate-

288

R. L. BERGER, T. R. CLEM, V. A. HARDEN, AND B. W. MANGUM

rials is indicated schematically in Figure 2. It is well known that it is virtually impossible to remove completely all impurities from any material, but only those that are soluble in the liquid or the solid, or both, will affect the melting or freezing behavior significantly. A very small quantity of an impurity may simply raise or lower the phase transition temperature, but any significant quantity will also broaden the transition, as indicated in Figure 2c.

ING

Y

a a c

a 0 Y

I+

I

,t

#

COOLING TIME TEMPERATURE REGULATE0 BATH

l,dl

Figure 2. Melting and freezing behavior of pure and slightly impure materials: (a) absolutely pure material with no resistance to thermal conduction and no supercooling; (b) pure material with finite thermal conductivity and supercooling; (c) material with slight impurity content, finite thermal conductivity and supercooling; and (d) method of determining melting and freezing curves.

TEMPERATURE MEASUREMENT IN BIOCHEMISTRY

289

Some of the reasons that the liquid-solid phase transitions of pure metals generally make excellent temperature reference points are ( 1) the latent heat of fusion naturally maintains the sample at the transition temperature until the sample has melted; (2) the high thermal conductivity of most metals assures that temperature gradients in the metal are smail; (3) the metals can usually be highly refined; (4)the degree of supercooling is usually small; and ( 5 ) the pressure dependence of the transition temperature is usually sufficiently small to ignore. The melting behavior of gallium follows quite closely the ideal behavior, as indicated in Figure 2b. Some typical results obtained for about 25 g of gallium in cells similar to those of SRM 1968 (Mangum and Thornton, 1977) are shown in Figures 3 and 4. Figure 3 illustrates the effects of impurities by showing the melting curves of 7Ns, 5Ns, and 3Ns pure gallium; Figure 4 illustrates the effects of different bath temperatures on the melting of a sample of 7Ns pure gallium. In these two figures, the

29.79

fi

0

2

4 6 HEATING TIME IN HOURS

8

Figure 3. Melting curves pure gallium. The solid curve is 7Ns (99.99999%pure), The dashed curve is 5Ns (99.999% pure) and the dot-dashed 3Ns (99.9% pure).

290

R. L. BERGER, T. R. CLEM,V. A. HARDEN, A N D B. W. MANGUM 29.790

cnmiar

-

I a *C

cnA01tll1-a.14 .c

CnADlt*l-a.*-C

29.710

e Y

= E

I-

a a

2

E +

29.770

i s

x

w

RESOLUTION OF OVY

;5

29.760

29.750

tI

0

I

1

I

2

I

3

1 I

1

5

I

6

I

7

I

6

HUT116 TIME (HOURS)

Figure 4. Effect of bath temperature on melting time.

samples were preheated at about 29.70"C. At time 0, the temperature was raised to the value indicated for each curve and regulated at that temperature for the duration of the melt. These curves exhibit the temperature dependence expected for the melting of relatively pure material, In fact, for the curves shown in Figure 4, the temperature variation of the plateau was just that of the instrument resolution (i.e., f 0.0002"C). In contrast to the melting behavior of the 7Ns pure samples, the 5Ns pure gallium took somewhat longer to reach a plateau, and during melting the cell temperature showed a constant rise. Such behavior is to be expected for samples containing small amounts of impurities. Standard platinum resistance thermometers (SPRTs) (Riddle et al., 1972) can be used in fixed-point cells only when the sample sizes are sufficiently great that immersion problems are eliminated. A gallium cell properly constructed for use with SPRTs contains approximately 9001000 g of gallium. Melting curves obtained with SPRTs for two such gallium cells are shown (Mangum and Thornton, 1979) in Figures 5 and 6. Note the flatness of the plateau and the sharp breaks in the curves at the beginning and at the end of the melts, similar to the ideal behavior shown in Figure 2a.

TEMPERATURE MEASUREMENT IN BIOCHEMISTRY 40.

1

I

I

I

I

I

I

I

35.

30. @C

25.

m.

15.

0

I

I

I

1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 Time (minutes)

Figure 5. Gallium melting curves of large cells obtained with standard platinum-resistance thermometers. Scale on left is for the top curve, resistance bridge values ranging from 28581000 to 28585000 are for the middle curve; the remaining scale on the right is for the bottom curve.

Thus, we see that the melting behavior of high-purity gallium is a good example of what to expect of an excellent temperature fixed-point material and that its melting point is at a very convenient temperature for biochemical and biomedical applications. Other temperature fixed points behave in a similar manner, although not all are of the high quality exhibited by gallium. For a more detailed discussion see (Hudson, 1980, Berry, 1979, Guildner and Edinger, 1976).

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R. L. BEKGER, T. R. CLEM, V. A. HARDEN, AND B. W. MANGUM

1

I

I

I

I

20.0 -

-z85w950

15.0

-28581ooo I

1

I

1

I

I

I

IV. METHODS OF MEASURING TEMPERATURE Since temperature measurements are required over such a wide range and diversity of situations, a large number of different types of thermometers with varying levels of accuracy and convenience have been developed over the years. Those most frequently used are based on the expansion of a gas, liquid or solid; on changes in electrical resistance; on the thermoelectric effect; on changes in the thermal radiation of a system; on changes in the thermal (Johnson) noise of electrical resistors: on changes

293

TEMPERATURE MEASUREMENT IN BIOCHEMISTRY

in the resonant frequency of some materials; on spectroscopic changes, on changes in the voltage of semiconductorp-njunction diodes driven at a constant current; and on changes in the magnetic susceptibility of paramagnets. 1. Liquid-in-GlassThermometers

The most familiar thermometer, the mercury-in-glass thermometer, is based on the fact that mercury expands much more rapidly than its glass container. Liquid-in-glass thermometers, containing liquids other than mercury, are used for measurements at temperatures below the freezing point of mercury. Usually a dye is put in the liquid of such thermometers to improve readability. Liquid-in-glass thermometers may be designed for partial-immersion or total-immersion operation. A total-immersion thermometer is one designed to indicate temperatures correctly when the bulb and the entire liquid column are exposed to the temperature being measured. A partial-immersion thermometer is one designed to indicate temperatures correctly when the bulb and a specified portion of the stem are exposed to the temperature being measured. The remaining portion of the stem, referred to as the emergent stem, will be at the ambient temperature, usually different from the temperature being measured. Such thermometers usually have an immersion line to indicate the proper depth of immersion. The principal features of a solid-stem liquid-in-glass thermometer are shown in Figure 7. Not all of the features shown would necessarily be incorporated in any one thermometer. The bulb is the reservoir for the thermometric liquid and contains a volume equivalent to a specific length of the stem capillary; that volume depends upon the coefficients of expansion of the liquid and the bulb glass. For organic thermometric liquids with coefficients of expansion higher than that for mercury, the bulb volumes are correspondingly less. The stem is the glass capillary tube through which the thermometric liquid moves with changes in temperature. The main scale is that scale graduated in degrees or multiples or submultiples of degrees; in many instances the main scale constitutes the rBULB

d

>

AUXILIARY

SCALE

,-STEM

d

f

J

/

d

IMMERSION LINE

\

I

I

?

J

MAIN SCALE

t

1

Figure 7. Features of liquid-in-glass thermometers.

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R. L. BERGER, T. R. CLEM, v. A. HARDEN, AND B.

w. MANGUM

only scale. The auxiliary scale is a short scale, including within its range a reference temperature point such as the ice point, which provides a means for checking the calibration of the thermometer. This scale is added when a suitable reference temperature is not included in the range of the main scale. The expansion chamber is an enlargement at the top end of the capillary bore, having a volume equivalent to not less than the volume of a 20-mm length of the capillary. The expansion chamber is provided to prevent the build-up of excessive pressures in gas-filled thermometers as the thermometric liquid advances toward the top of the scale. The contraction chamber is an enlargement of the capillary bore which reduces a long length of capillary or prevents contraction of the entire liquid column into the bulb. This chamber is introduced below the main scale or between the main scale and an auxiliary scale. The accuracy attainable with a liquid-in-glass thermometer is limited by the characteristics of the thermometer itself. Instability of the thermometric liquid, nonuniformity of capillary bore, and inaccuracies in scale graduation are the important factors. Uncertainties in corrections for the emergent stem may greatly limit the accuracy of partial-immersion thermometers. Generally, partial-immersion thermometers are assigned an uncertainty of +0.3"Cin their calibration, whereas total immersion thermometers may have an uncertainty as small as +O.O3"C.Observer errors add to the uncertainty but with care these can usually be made relatively small. Since a considerable amount of material has been written about the proper calibration and use of liquid-in-glassthermometers (Wise, 19'76), we will not elaborate on all the corrections that must be applied to the indicated temperature values obtained in measurements.

2. Dial Thermometers There are many types of dial thermometers, including liquid, liquidvapor, or gas filled, and their use is very widespread. These thermometers usually consist of a bulb connected via a capillary tube to a Bourdon tube, which is attached through some mechanism to a movable pointer on a scale. The liquid most commonly used in dial thermometers is mercury. Their inaccuracy may range from 2 to 5°C; however, their reproducibility is usually considerably better than that.

3. Bimetallic-Strip Thermometers The principle of the expansion of a solid is employed in bimetallic-strip thermometers, which are comprised of strips of two different metals bonded together, side by side. Since the metals selected for this use have

TEMPERATURE MEASUREMENT I N BIOCHEMISTRY

295

different coefficients of expansion, the bimetallic strip will bend with a change in temperature and this can be used to move an indicator along a scale or to open and close electrical contacts. The latter operation forms a thermostat for use with a furnace or oven. The inaccuracy of bimetallicstrip thermometers may be as much as several degrees Celsius (2-5°C) but their reproducibility can be better by a factor of 10. 4. Gas Thermometers

The most frequently and conveniently used gas thermometer is the constant-volume thermometer, which utilizes the changes in pressure to indicate changes in temperature (Guildner and Thomas, 1982). Another type of gas thermometer is the dielectric-constantgas thermometer (Gugan and Michel, 1980). Measurements in this case depend on the change of the dielectric constant with gas density and, thus, are intensive in their nature, in contrast to the extensive quality of regular gas thermometry. A reference temperature is required for the operation of gas thermometers. Recent gas thermometry results (Guildner, 1980) have uncertainties of about 0.002"C associated with them, but such small uncertainties are very difficult to obtain.

5. Resistance Thermometers There are many different types of resistance thermometers, with each type being most suited for use in a particular temperature region. Included among the resistance thermometers are platinum, copper, nickel, rhodium-iron, and the semiconductor thermometers (arsenic-doped germanium, thermistors, carbon). The quality of each of these types of thermometers varies with the method of its construction. The reproducibility of the highest-quality thermometers of each of the above-named types can be as good as +O. 1 mK in their appropriate temperature range. The industrial-grade-qualityresistance thermometers may be reproducible to only a few tenths of a kelvin.

6. Thermoelectric Thermometry Thermocouples are used for measurements of temperature from a few millikelvins to above 2800°C. The basic thermocouple (Guildner and Burns, 1979)consists of two wires of different materials that, when joined together at one end (the hot junction, say) and connected to a voltage measuring instrument at the other end (the cold junction, say), will produce a voltage that will be a smooth function of the temperature difference between the two junctions. Various pure elements and alloy combinations can be used to form couples that are best suited for particu-

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R. L. BERGER, T. R. CLEM, v. A. HARDEN, AND B. w. MANGUM

lar temperature regions. The most common types of thermocouples are listed in Table 111. Thermocouples are probably the most widely used industrial thermometers because they are inexpensive, rugged, longlasting, and suitable for continuous recording and/or control of temperature. Since thermocouples can be made very small, they can respond rapidly to fast changes in temperature. Because of the nature of thermocouples, an uncertainty of 2O.l"C is about the best that can be attained, even with calibrated thermocouples.

7. Radiation Thermometers Radiation thermometers were developed for measuring temperatures higher than 1064°C; they have the advantage that they are noncontact thermometers. Optical pyrometers measure apparent temperatures of objects by comparing the radiation from the objects over a narrow wavelength band with that of a standard, preferably using a photoelectric detector for the comparison. Corrections for the emissivity of the source must be made to determine the temperature; the preceived temperature may be, and usually is, lower because all of the heat is not radiated. Total-radiation pyrometers measure the whole spectrum of energy radiated by the source. They are less accurate than optical pyrometers but can measure much lower temperatures (of the order of 100°C). This type of pyrometer also requires emissivity corrections. Another technique utilizing radiation from an object as a means of measuring its temperature is that of thermography, that is, the mapping of surface temperature distributions over extended areas. This is fairly widely used in the medical field for the detection of tumors near the surface of the skin and in industrial applications for detection of hot spots, such as defective insulators on power lines, defects in furnace walls, and areas of heat leaks in buildings. Usually, comparison measurements, rather than the actual determinations of temperature values, are made in thermography. Resolutions of 0.05"C to 0.1"C are attainable under the best conditions. Another noncontact technique for measuring high temperatures involves Raman spectroscopy, in particular the nonlinear process known as coherent anti-Stokes Raman spectroscopy (CARS) (Radiation Thermometry, 1982). This technique is finding practical applications in measurements of temperatures of flames (in internal combustion engines, in jet engines) and of hot gases. The imprecision of such temperature measurements is generally a few percent. Recently, a fluoroptic thermometer (Wickersheim and Alves, 1979; Cheng, 1981) has become commercially available. The temperature sensor is comprised of a rare-earth phosphor or mixture of phosphors which,

TABLE 111 Thermocouple Characteristics Thermocouple combinations Type designation"

Temperature range ("C)

B

0 to 1820

E

-270 to 1000

3

-210 to 1200

K

-270 to 1372

R S

T

Materials Platinum-30% rhodium v. platinum-6% rhodium Nickel-chromium alloy v. a coppernickel alloy Iron v. another slightly different copper-nickel alloy Nickel-chromium alloy v. nickelaluminum alloy Platinum-13% rhodium v. platinum. Platinum- 10% rhodium v. platinum. Copper v. a copper-nickel alloy.

-50 to 1767 -50 to 1767 -270to 400

SINGLE-LEG MATERIALS

... N

The negative wire in a combination ...P The positive wire in a combination Platinum-nominal 6 wt% rhodium BN Platinum-nominal 30 wt% rhodium BP EN or TN A copper-nickel alloy, constantan: Cupron? Advance: Thermokanthal JN'; nominally 55% Cu, 45% Ni; often referred to as A d a m Constantan EP or KP A nickel-chromium alloy; Chromel," Tophe1,b T-1: ThermoKanthal KP"; nominally 90% Ni, 10%Cr A copper-nickel alloy similar to, but not always interchangeable with, EN and JN TN; SAMA specification Iron: ThermoKanthal JP," nominally 99.5%Fe JP A nickel-aluminum alloy: Alumel," NiAl,b T-2,d ThermoKanthal KN"; KN nominally 95% Ni, 2% Al, 2% Mn, 1% Si High-purity platinum RS,SN Platinum-13 wt% rhodium RP Platinum-10 wt% rhodium SP Copper, usually Electrolytic Tough Pitch TP The use of trade names does not constitute an endorsement of any manufacturer's products. All materials manufactured in compliance with the established thermoelectric voltage standards are equally acceptable. "The letter type, e.g., Type T, designates the thermoelectric properties, not the precise chemical composition. Thermocouples of a given type may have variations in composition as long as the resultant thermoelectric properties remain within the established limits of error. 'Registered trademark-Wilbur B. Driver Co. "Registered trademark-Kanthal Corp. dRegistered Trademark-Driver-Harris Co. "Registered Trademark-Hoskins Manufacturing Co.

297

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R. L. BERGER, T. R. CLEM, V. A. HARDEN, AND B. W. MANGUM

when excited by the appropriate radiation, exhibit fluorescence and, in addition, have that fluorescent output concentrated into a few sharp emission lines that can be easily separated with narrow-band interference filters. The fluorescence of each of the phosphors has some lines that are a function of temperature and some that are not. The separation, detection, and calibration of the ratio of the temperature-sensitive to the ternperature-insensitive fluorescent lines yield a thermometer over a given temperature range. By taking the ratio, the thermometer becomes essentially independent of the exciting-source fluctuations in output. The exciting (input) and the fluorescent (output) radiation are conducted from and to an optoelectronic device by means of optical fibers. This provides the very important feature of electrical isolation. These thermometers must be calibrated. The manufacturer-quoted imprecision is 20.1"C for a 1-sec measurement time. The probe size is less than l-mm diameter. Although a wide temperature range of operation is possible, the commercial units have a range of from -50 to 200°C.

8. Noise Thermometers Johnson-noise thermometers (Ohte and Iwaoka, 1982) are based on the measurement of the Johnson-noise power or Johnson-noise voltage, and they are now commercially available.Johnson (i.e., thermal) noise voltage is the small fluctuating voltage generated in any electrical conductor by the random motion of the electrons (charged particles). The extent of the motion of the electrons is a function of temperature and, thus, the voltage generated is related to thermodynamic temperature. Noise thermometers may be used from temperatures of a few millikelvins to over lOOO"C, but the commercially available instruments are not suitable for use over this entire range. The inaccuracy of noise thermometers is typically 20.2 to +0.5%, although in specialized cases, they can be considerably better. 9. Resonance Thermometers

Thermometers that are based on the temperature-dependent, resonant frequency of a material are very attractive because the quantity measured is frequency. One such thermometer is the nuclear quadrupole resonance (NQR) thermometer (Ohte and Iwaoka, 1982; Utton, 1967; Ohte et al., 1979; Utton and Vanier, 1976). Nuclear quadrupole resonance is just nuclear magnetic resonance in the absence of magnetic fields. Nuclei with spin I 3 1 posses electric quadrupole moments that, through interactions with electric field gradients produced by valence electrons and by the surrounding ions in the crystalline lattice, cause a splitting of the nuclear energy levels in the absence of a magnetic field. It is the temperature

TEMPERATURE MEASUREMENT IN BIOCHEMISTRY

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variation of these splittings, and hence of the resonance frequency, that gives rise to NQR thermometry. An excellent feature of the NQR thermometer is that the thermometric property involved is a fundamental property of a substance, a unique frequency-temperature relationship that must be established only once and is always thereafter applicable for that substance.Thus, once the frequency -temperature relationship has been determined for a suitable sensor material, such as KC103, that calibration will apply to all other samples of that material provided that the material has been prepared with consistent purity. This, then, eliminates the need to calibrate each thermometer individually as is required for most practical thermometers. Another advantage is that frequency can be easily and accurately measured and the thermometer can be easily made a part of an automated system for temperature monitoring and control. Through the use of standard frequency broadcasts by NBS, the accuracy of the frequency counter used in making measurements can be easily checked. The interaction of the nuclear electric quadrupole moment of 35Cl (1=3/2) of KC103 with the nonhomogeneous field, produced mainly by the valence electrons but with some from the surrounding ions, produces a splitting of the I = 3/2 energy level into two energy levels, each degenerate with respect to the sign of the magnetic quantum number, mz.These energy levels are separated by an energy hv where the frequency v is given by

where e is the electronic charge, (2 the nuclear electric quadrupole moment, q,, the component of the electric field gradient tensor along the principal axis, and h is Planck's constant. The effects of an asymmetric electric field have not been considered here. It has been found that fluctuations in the orientation of the electric field gradient tensor due to torsional vibrations of the C103 group of KC103 account for the temperature dependence of the NQR frequency at low temperatures through changes in the values of the qu. Above 80 K, however, this does not adequately account for the variation since an expansion of the lattice occurs and the molecular vibrations cannot be approximated by harmonic oscillators. As the lattice expands, the distance between the ions increases, causing an additional decrease of qu and an increase in the sensitivity of the thermometer. For KC103,the NQR of 35CIhas been studied from about 10 to 470 K. It was found that the resolution and accuracy of temperature measurements can be about 2 1 mK in the range from 50 to 470 K. Since the width

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R. L. BERGER, T. R. CLEM, V. A. HARDEN, AND B. W. MANGUM

of the 35ClNQR line is approximately 500 Hz, at a resonant frequency of 28 MHz, a determination of the center of the line to only 1% (or 5Hz) gives 1 mK sensitivity. The reproducibility of pure strain-free samples of KC103 is d 2 1 mK near 300 K. Over the entire range of 10-470 K, the inaccuracy of a temperature measurement using this thermometer varies from about 1 to 10 mK, depending on the temperature. A commercial NQR thermometer is available and it is based on the NQR of 35Clin KC103, which has been the substance most studied. Another resonant-frequency thermometer is the quartz crystal resonator (Benjaminson and Rowland, 1972), which, if the crystal is properly cut, is quite linear from about 190 to 525 K. Although this thermometer has excellent resolution, it does exhibit hysteresis and drift. The principle of quartz crystal thermometry is based on the temperature dependence of the piezoelectric resonant frequency of a quartz crystal wafer of a given dimension. The angle of cut of the quartz crystal is selected to give as nearly a linear and yet sensitive correspondence between resonant frequency and temperature as possible. This angle of cut is referred to as an LC (linear coefficient) cut. The temperature sensitivity of the quartz crystal thermometer is about 1000 HzPC. Quartz crystal thermometry is naturally digital; hence, it is independent of lead resistance and does not require four-lead measurements. The resolution of the quartz thermometer depends upon the "counting period" or the integration time; for example, the least significant digit of a l-sec period corresponds to O.OOl"C, of a 10-sec period to O.OOOl"C, and of a 100-sec period to 0.00001"C. The inaccuracy of the quartz crystal thermometer is +O.04O0Cfor the temperature range from -50 to 150°C and +O.O75"C for the range from -80 to 250°C. The short-term stability is 5 X 10-6"C. The long-term stability of a quartz probe is +O.O03"C per month. The major source of error for absolute accuracy measurement is hysteresis of the quartz probe: st0.05"C from -80 to 250"C, *O.O2"C from -50 to 150"C,and ?O.OOl"C over any 10%span from -20 to 120°C. This frequency hysteresis of the probe is produced through mechanical coupling to leads and other effects.

10. Diode Thermometers Semiconductor p-n junction diode thermometers (Swartz and Gaines, 1972; Verster, 1972; Ohte et al., 1982)are becoming widely used throughout the range from liquid helium temperatures (1 K) to about 200°C. The diodes are currently made of germanium, silicon, or gallium arsenide. These thermometers are based on the principle that for forward-biased

TEMPERATURE MEASUREMENT IN BIOCHEMISTRY

30 1

p-n junction diodes, the current varies approximately exponentially with V / T ,where V is the voltage and T is the temperature. At constant current, the junction voltage decreases approximately linearly with increasing temperature. The emitter-base junction diodes of transistors have also been used as thermometers. The long-term reproducibility of diode thermometers is claimed to be kO.0 1°C or better. The instability problems that existed previously are claimed to have been solved. The short-term reproducibility is +O.OOl”C.

11. Liquid-Crystal Thermometers Liquid-crystal thermometers (Rozzell et al., 1974) have come into fairly widespread use in recent years. These thermometers rely on the change in wavelength of reflected light with a change in temperature. The liquid crystals or the liquid-crystal mixtures are selected such that the wavelengths of the reflected light are in the visible region of the spectrum, giving rise to a change in color with a change in temperature. T o serve as thermometers, the liquid crystals must be in their cholesteric phase, and their temperature range of operation is from about 0 to about 70°C. Liquid-crystal thermometers exhibit hysteresis, and care must be exercised in their use to prevent organic solvents from coming in contact with them.

12. Comments Each type of thermometer discussed above has its particular advantages and disadvantages and the selection of a thermometer for a given application must be based on the requirements of that application. Among the considerations that influence the choice of the thermometer are accuracy, sensitivity, reproducibility, size, temperature range, speed of response, durability, and cost. V. MODERN ELECTRONIC METHODS OF SENSOR MEASUREMENT

The great advances that have been made in analog and digital circuitry in the last 10 years have revolutionized our ability to make compact packages that will make measurements on virtually any sensor without “loading” or disturbing the sensor output. The type of instrument needed depends upon the sensor being used. In this section we discuss electrical resistance measurement devices for resistance temperature detectors (RTDs) such as thermistors and platinum resistance thermometers, and voltage mea-

302

R. L. BERGER, T. R. CLEM, V. A. HARDEN, AND B. W. MANGUM

surement devices for thermocouple sensors. There have been many publications that have addressed the classical methods of precisely measuring resistance (Wheatstone bridge) and voltages (potentiometers) (Sturtevant, 1973) and also (Laws, 1918). Consequently,we will not be concerned with those methods; instead, we are describing the types of instruments that a reader may find available commercially. We are mostly concerned with what could be termed “turn-key” systems that require minimal effort on the part of the user to obtain reliable temperature data. 1. Resistance Measurements

Assuming that the potential user has decided which type of RTD is to be used, the requirements for the readout instrument need to be assessed. The simplest and cheapest way to determine the temperature of an RTD is with an ordinary VOM (volt-ohm-miliammeter).This method, while cheap, is not very satisfactory for most applications. A VOM may apply a sufficiently large current to the sensor while its resistance is being measured to produce significant errors due to self-heating of the RTD. Also, this technique yields a value of resistance, and, therefore, a calculation must be made or a conversion table consulted to obtain a value for the RTD temperature. The classical methods of using bridges are cumbersome and, in addition, give only a value of resistance as a result. The accompanying table (Table IV) lists some representative RTD thermometers that are available commercially. This table includes examples of the simplest, cheapest units and also of the more expensive, more sophisticated units. Most instruments available today feature digital display, and since there is very little difference in price between analog and digital meters, there is littlejustification for selecting an analog readout, except for the case in which an instrument functions as a null detector. In addition to these ready-to-use thermometers, there also are available high-resolution, low-excitation power ac resistance bridges that can measure RTD resistances with moderately high accuracy (Berger et al., 1974). Such instruments cost about $2500. Since such instruments measure only a resistance value, the resistance-temperature (R vs. T ) relationships for the RTD must be known accurately to allow the temperature value to be accurately known. An example of this type of instrument is the Model LR-I 10, available from Linear Research, Inc., San Diego, California. A number of handbooks provide a further discussion of more modern bridges. (Handbooks).

2. Voltage Measurements If a decision has been made to use a thermocouple as a sensor, there is a wide range of voltage-measuring devices available from which to choose

Cole-Parmer

United Systems Corporation John Fluke Manufacturing Co.

-200 to +800°C

-190 to +750°C

2180A

K-8129-10

-100 to +600°C

5530

1°C

0.01"C

0.1"C

k 1.25%

+O.l"C

(- 100 to +400"C)

20.3"C

PLATINUM RESISTOR TYPE

2 10

995

860

618

20.15"C

0.05"C

0 to 51°C

46TUC

435

kO.25"C

20 to 43°C

49TA

Yellow Springs Instrument Co. Yellow Springs Instrument Co.

0.01"C

0.01"C 0.01"C

- 10 to + 110°C -10 to + 6 0 T

1006

Hart Scientific

1925

k0.04"C (- 100 to +250"C)

5000

0.01"C

- 183 to +650°C

4200

56-92

784

kO.45"C

~

Special characteristics or comments

~

85

Small, hand-holdable size Battery operation 4-Digit LCD display

142- 168 Analog recorder output 4-Digit LED display 60-600 5-Digit LED display IEEE-488 Bus capability Available, multiprobe accessory available

Recorder output 4-Digit LED display 125- 160 IEEE-488 Capability available 6-Digit LED display 800 IEEE-488 Capability available Recorder output 6-Digit LCD display 25- 100 Portable battery powered 4-Digit LCD display 25- 100 6 Input channels, recorder output, analog meter

Probe

Price U.S. Dollars Basic unit

Inaccuracy

~

k0.05"C k0.03"C

0.01"C

-30 to +lOO°C

5510

United Systems Corporation Instrulab, Inc.

Resolution

Temperature range

Model no.

Manufacturer or distributor

RTD Types, Thermistor Type

TABLE IV

304

R. L. BERGER, T. R. CLEM, V. A. HARDEN, AND B. W. MANGUM

to observe the thermocouple output. As with the RTD, a simple and inexpensive readout system could be obtained using a millivoltmeter or microvoltmeter. This method, however, would be unsatisfactory for most applicationsfor several reasons. Since most thermocouples produce about 20-50 pV/"C, a very sensitive meter would be needed. In addition, the user would have to consult a conversion table to determine the temperature difference, and the value of the temperature being measured could, in the best of circumstances, never be more accurate than that of the reference junction. It is possible to use an ice bath for the reference junction (see previous section). The classical method of using a potentiometer is very cumbersome and the result is a voltage, which presents the same problems as before. Table V lists some representative thermocouple thermometers that use voltage references. They are available commercially, are convenient to use, and will provide respectable resolution and accuracy. The accuracy specifications given in Table V pertain to temperature uncertainties on an absolute basis. For making relative or differential temperature measurements, such as in calorimetric applications, much higher-precision measurements are possible. Using a microvoltmeter or nanovoltmeter with appropriate low-noise and low-drift characteristics, it is possible to measure temperature differences in the microdegree range. Several electronic test instrument manufacturers produce units that are usable in this mode, an excellent example of which is the Keithley Model 181 Nanovoltmeter. Under the best conditions, this instrument can distinguish a change of 10 nV and has an input noise level of 30 nV peak-to-peak. These figures represent millidegree changes for typical thermocouples. Another class of voltage-output thermometer sensors is the silicon integrated-circuit temperature sensor, such as the National Semiconductor LM135. These devices function as Zenor diodes with a breakdown voltage directly proportional to absolute temperature at + 10 mV/K. Their inaccuracy is of the order of 2 1°C over a range of -55 to + 150°C when calibrated at +25"C. The voltage may be measured with an ordinary 3+ 1/2-digit digital voltmeter (DVM); the accuracy of the reading will be limited by the sensor accuracy. A similar thermometer is the Analog Devices AD590 Integrated Circuit Temperature Transducer. This device produces a constant current of 1 pA/K with an absolute accuracy of &0.05"Cover a temperature range of -55 to + 150°C.This current can either be measured directly by a DVM which has a low current-measuring capability, or the current can be used to develop a voltage across a precision resistor of high resistance and then that voltage measured with a 34-digit DVM.

u1

0

B,EJ,K, R,S,T

2 190A

2780-02

John Fluke Manufacturing Co.

United Systems Corporation

J,T,E,K

B,E,J,K, R,S,T

.

2165A

2T

BT

John Fluke Manufacturing Co.

K-8507-20

Cole-Parmer

2K

K-8505-20

870

Keithley

K-

Type

Cole-Parmer

K-8520-40

Model no.

Cole-Parmer

Manufacturer or distributor

0.1"C 1"C

0 to + 140°C Oto+400"C

-180 to +1372"C

-128 to +2471"C overall

0.1OC

0.1"C

1°C

1OC

1"C

-40 to 1999°C

+90 to + 175°C 0 to + l0O0C -100 to +20°C +25 to +45"C -200 to +2328"C overall

0.1"C

1°F

Resolution

-40.0 to 199.9"C

-60 to +2000"F

Temperature range

20.7"C

TYP +0.2 to 0.4%

r0.20"C TYP 2 1 to 22°C

2 1°C

r0.2% of full scale 2 1 digit

+- 1% of reading -+- 1 digit 0 to 1100°F ?0.25% of reading + 1°C

Inaccuracy

Thermocouple-Type Thermometer Systems

TABLE V

520 + 50 min for probe 995 + 50 min for probe 451 + probe

475 + 30- 110 for probe

495 + 30- 110 for probe

155 + 50-200 for probe 199 + 59 for probe

Price U S . dollars

5-Digit display IEEE-488 BUS CAP Multiprobe Acc: avail. 4-Digit LED display Panel mount

4-Digit display IEEE-488 BUS CAP

Battery operation Small size 34 Digit-LCD 2-probe cap Recorder output Small bench top unit LED display 3-Probe Cap Analog model Recorder output 2-probe cap

Battery operation Small size

Special characteristics or comments

306

R. L. BERGER, T . R. CLEM, V. A. HARDEN, AND B. W. MANGUM

3. Digital Recording Methods and Devices Quite often the measurement of temperature during the course of an experiment is a continuous process, and, in addition, it is not an isolated event but is to be related to other events. Consequently, it is desirable to have a method of recording the temperature readings over a period of time without operator attention. Many of the thermometers available accommodate this by providing some type of output that is directly related to temperature. A. ANALOG O U T P U T S

Many of the middle-price-range electronic thermometers available today have a recorder output. Typically, the voltage present there varies from 0 to 0.1 or 1 volt and is directly proportional to the temperature. Generally, this is intended for use with an analog strip-chart recorder to record temperature vs. time or with an x - j recorder to record temperature vs. some other related event. This signal could, however, be converted for use with a digital computer by using some type of analog-to-digital convertor that is connected to the computer. This convertor could be a digital voltmeter with a digital output. B. DIGITAL O U T P U T S

There are three methods in common use by which digital data are communicated between devices. In this section we will discuss these methods. They are; a) Binary Coded Decimal (BCD), b) RS232, and c) IEEE-488 GPIB. With each of these methods, the sending unit and the receiving unit must have the same type of communication port (Handbooks).

a . Binary-Coded Decimal (BCD). BCD output from a digital thermometer comprises four lines of data per digit, weighted in a 1-2-4-8 sequence. There are basically two methods for presenting the BCD data to external devices. One method is to have a set of four lines for each digit, with the total number of output lines equal to four times the number of digits. The other method is to multiplex all digits to the same four data lines and to provide control lines to allow command or determination of which digit is being presented at a particular time. These outputs usually have “TTL compatible” level voltages. TTL, which stands for Transistor-Transistor Logic, is a digital logic family that has been used extensively for the past 10 years. TTL levels are generally 0 +0.8 V for a binary 0 and +3.2 to +5.0 V for a binary 1 (although a 1 can be considered anything above +2.4 V). Each line of the four-digit data lines is called a bit (which is a contraction of binary digit) and the entire group of four is

TEMPERATURE MEASUREMENT IN BIOCHEMISTRY

307

referred to as a byte. When the BCD type of interconnection is referred to as a parallel interface, all four bits of each byte are put out at the same time on parallel lines; the multiplexed method is sometimes referred to as bit-parallel, byte-serial output if the digits (bytes) are output sequentially; or it is referred to as parallel output with hand shaking if the receiving unit tells the sender when to transmit the next byte.

b. RS232 SeriulZntetfuce. EIA RS-232-C is an Electronic Industries Association Standard (EIA, 2001 Eye St., NW, Washington, D.C. 20006) for the “Interface between Data Terminal Equipment and Communication Equipment Employing Serial Binary Data Interchange.” This interface technique is used in numerous applications in the electronics and computer fields for intersystem communications. For thermometers, the temperature data and possibly some identification information, is transmitted one character at a time, sequentially (or serially). Each character is transmitted as a series of, usually, seven ASCII-encoded (U.S.A. Standard Code for Information Interchange, ANSI x3.4-1977, American National Standards Institute, 1430 Broadway, New York, N.Y. 10018)pulses, plus synchronization pulses called start and stop bits. The rate of data transmission and the rate at which the receiver expects to receive it must be matched, and that rate is called the BAUD rate (bits per second). BAUD rates vary, at standardized values, from 110 to 19200 BAUD. Generally, thermometers using this form of data communications operate unidirectionally as far as data are concerned, but the RS232 connection may have some “hand-shaking” characteristics so that the source (the thermometer) will not be sending data when the receiver is not ready to accept it. c. ZEEE-488 General Purpose Interface Bus (GPZB). This is a relatively new technique for communication between electronic devices, and it is an IEEE standard (Institute for Electrical and Electronics Engineers, IEEE 488- 1978, “Digital Interface for Programmable Instrumentation,” IEEE, 345 E. 47th St., New York, N.Y. 10017).Devices with thiscapability generally are controllable via the IEEE-488 in the sense that ranges can be selected, functions can be changed, inputs selected, and so on. Up to 15 instruments, each with a unique address, may be connected together on one GPIB with a controller, thus allowing one computer or data acquisition device to accommodate many measurement devices. The use of GPIB communications requires very little input from the user to get the hardware operational. Most GPIB controllers use a high-level language, such as BASIC, which includes simple inpudoutput commands that simplify using instruments interconnected via the GPIB.

308

R. L. BERGER, T.R. CLEM, V. A. HARDEN, AND B. W. MANGUM

4. Temperature Controllers The control of the temperature of various measurement cuvettes, calorimeters, incubators, etc. has received a great deal of industrial attention in the last 10 years. Because of this, either complete controllers or various integrated circuits can be purchased which allow one to assemble controllers with almost any desired sensitivity or stability. In Table VI, we list a number of'controllers ranging in capability of control from ultrastable to +O.I"C. To use the table intelligently, however, an understanding of the problems associated with temperature control is extremely useful. A detailed discussion of this problem has been given elsewhere (Mudd et al., 1982). Basically, the problem involves operating the controller against at least a 5°C gradient. Thus, if the system being controlled is to be maintained at 25"C, some means must be provided to cool the surface of the device to 17°C since room temperature is often 22 to 25°C. This is most effectively done with a standard low-temperature bath. The controller must then add heat as needed. To do this, and maintain long-term stability, a proportioning system is used that provides a small amount of heating all of the time and adds proportionately larger or smaller amounts as needed. T o ascertain if the system is changing, a differentiating circuit is provided in the controller, and to determine the long-term trend, an integrating circuit is also included. Several such units are listed in Table VI. The controller discussed elsewhere (Mudd et al., 1982) will maintain a calorimeter within +O.OOl"C of a selected value for a number of hours, but it drifts by about t0.01"C over a period of a week. This can be corrected by using a comparison circuit and the digital, linearized thermistor thermometer (Berger et al., 1980).

VI. RECENT APPLICATIONS OF MODERN TEMPERATURE MEASUREMENT METHODS IN BIOCHEMISTRY 1.

Fast Stopped-Flow Thermal Measurements

Measuring the temperature of chemical reactions as a way of determining the extent of reaction has been used for almost 100 years (Callendar, 1902). An early application to the determination of the energy involved in the reaction of hemoglobin with oxygen was made by A. V. Hill (Brown and Hill, 1923). Roughton (Roughton, 1930, 1961) developed a continuous-flow system that was used to measure the reaction of hemoglobin with various ligands. Unfortunately, thermocouple insulation problems were very severe in Roughton's work since the slightest pinhole produced large streaming potentials in the thermocouples. In addition,

Proportional Thermistor or platinum 3002 Proportional Thermistor or platinum PTC 41 Proportional Thermistor or platinum Proportional Thermistor

Leeds and Northrup, Inc. Hart Scientific Company Tronac, Inc.

Commonwealth Technology, Inc.

pp

800 1000 1000 800

00.0 to 99.9"C - 10 to 100°C

-4.0 to 100°C

895

50 to 200°C

0 to 2000°F

0 to 600°C -200 to +20O0C

285

Proportional Platinum or RTD ordoff Proportional Thermocouple

420 1

235

223

185

Controller

620

Athena Controls, Inc.

Yellow Springs Instrument Omega

63-RC on/off

Yellow Springs Instruments Cole-Parmer

Temperature control range

Mercury -59 to + 150°C in glass *Thermistor -70 to + 260°C

Sensor

K2156 Proportional Thermistor -100 to +500"F or platinum RTD 72 Proportional Thermistor 0 to 120°C

K2149 on/off

Cole-Parmer

Type

Model no.

Manufacturer or distributor

50-300

+

.01"C

1°F

1°C 0.1"C

0.005"C

0.05"F

0.05"C

1"C

Stability

Adjustable dead band 0.1 to 30°C Digital readout of temperature, adjustable proportional bandwidth Microprocessor-based unit includes control algorithms and alarm functions

Easy to use, but only good in liquid medium

Special characteristics comments

+0.005"C

+O.O001"C

+O.OOOl"C with Model 408R Bath

+O.O05"C +O.O005"C with Model 5001 Bath

+0.001"

0.2% FS

O.l%FS 0.2% FS

0.5"C

0.05"F

0.05"C

1°C

Precision

50- 150 +0.25"C

50-150

40-100

40-100

40-90

40-90

60-90

55-96

Probe

Approx. price U.S. dollars

Temperature Controllers

TABLE VI

310

R. L. BERGER, T.R. CLEM, V. A. HARDEN, AND B. W. MANGUM

vast amounts of solution were needed. In later work in Roughton's laboratory, Berger started work on a stopped-flow calorimeter utilizing very small thermocouples and new coatings (Berger, 1963; Berger and Stoddart, 1965). At about the same time, Pinsent, Pearson, and Roughton (Pearson et al., 1954) and later Chipperfield (Chipperfield, 1966) with Roughton, greatly improved the continuous-flow calorimeter so that a kinetic experiment could be performed on 100 ml of each reagent. This was in contrast to a volume of only 0.5 ml of each reagent for Berger's system. For continuous-flow measurements, about 2 sec were required for each reading so that the speed of response of the detector was not so important. In the case of the stopped-flow method, however, the time resolution depended upon the sensor response. The need for a better detection system led to the examination of the problem of high noise and low stability of thermistors. As a result, a series of bead thermistors has been produced over the last 20 years that are very fast (circa 5-7 msec), glass encapsulated, have a noise figure that depends only on the Johnson noise, and are very stable. The Johnson-Nyquist Noise (Van Der Ziel, 1954) is given as: e2 =

4kTAfR

where e is the peak-to-peak voltage, k is the Boltzmann constant, T is the temperature in kelvin, Af is the frequency bandpass, and R is the resistance of the thermistor in ohms. Thus, for 1 ohm, 1 Hz, and room temperature e = dGZi-j%= 10-"volts

For a 10-kohm thermistor and a 100-Hz bandpass Thus with 0.3 V across the thermistor, and the usual 4% change in resistance per kelvin for the thermistor, a 1-mK change corresponds to a voltage change of 0.3 x 4 X V = 1.2 x V. Since the peak-topeak voltage is 1.4 times the root mean-square voltage, about 0.01 mK is the limit of detection at this bandpass. It is very difficult to produce the small, fast-response beads at a much lower resistance, but even if it were possible, the best signal-to-noiseratio for most solid-stateamplifiers is for input resistances greater than 1000 ohms. In addition, the inherent llf noise of the best solid-state amplifiers is of the order of 100 to 150 nV for this bandpass. A special ac amplifer was constructed for the work on stopped-flow calorimetry, and it is shown in Figure 8. It is discussed in detail elsewhere (Berger et al., 1974).A commercial version as mentioned earlier from Linear Research, operating at a somewhat lower bandpass,

TEMPERATURE MEASUREMENT IN BIOCHEMISTRY

311

but with excellent results, is available and is used for thermal titration (Watt et al., 1974) where a time resolution of 100 msec is adequate. Using this new detector and bridge, a stopped-flow calorimeter with a 2-msec dead time, a computer-corrected thermistor response of 1 msec, and a sensitivity of 0.1 mK has been constructed. Thus, for a chemical reaction with an enthaphy, Q, of 5 kcal/mole, we can calculate the temperature change, AT, from

Q = MC,AT (1 1) where M is the mass and C , the heat capacity, of the solution. Since both M and C, are approximately 1 for 1 ml of a water solution, the change, AT, will be about 0.125 m"C and thus we can detect about 25 nmol/ml.

2. Heat Conduction and Response Time Corrections for Thermal Detectors

Any heat-detecting device has a finite time response to a change in temperature, and it will, in general, lose some of the heat it has gained to its environment during the time of measurement. A detailed treatment of this problem has been given recently elsewhere (Davidsand Berger, 1964; Berger and Davids, 1965; Davids and Berger, 1969; 1982; Balko et al., 1981) and we will only outline the idea here. CALI BR AT10 W UNBALANCE

L

PMASL ADJUST

SlNL WAVE OSCILLATOR

SENSITIVE

OUTPUT

TIME CONSTANT

BALANCE METER

(A)

Figure 8. Fast, sensitive ac resistance bridge: (a) single-channel stopped-flow, I msec at 0.1 m"C sensitive; (b) differential bridge.

F3

I

w

Figure 8.

I

OSG MODULE

-

J

I

-

MoN'ToR'

+

1

AMPLIFIER MODULE

II

(B)

DlFFERENTIA L FAST THERMISTOR BRIDGE -SIMPLIFIED BLOCK DIAGRAM--

)IFF. A.C. BRIDGE MODULE

(Continued)

L

P

"8" THERMISTO

A" THERMISTOR

I

METER

FtJ

TEMPERATURE MEASUREMENT IN BIOCHEMISTRY

313

To test the response time of any detector, one normally challenges the detector with a step function. For example, consider a flow system (Balko et al., 1981) with a mixer and two driving syringes, an observation tube, and a thermistor as the detector as shown in Figure 9. If acid and base are mixed, a step increase in heat occurs because the reaction is so fast. Thus, 0.02M NaOH and 0.01M HCl mixed in the ratio of one to one will give a temperature rise of about 69 mK since AH for the heat of formation of water at 25°C and in 0.1M KCl is about 13.75 x lo3 cal/mol (Hale et al., 1963).T h e time it takes the temperature of the sensor to rise to lle of its total change is called the sensor's time constant, T. The time to rise half way is t1I2. Since most physical devices respond exponentially, at least to a first approximation, the output E may be expressed as

E = Eo[l - eWat]

(12)

when E = Eole, then a = 117. If we can adequately describe the system physically, we can correct this response time by at least a factor of 10. This is done using the D-B Finite Element Simulation Technique (Berger et al., 1982) or D-B FEST. A detailed analysis is given in (Davids and Berger, 1982; Balko et al., 1981). The basic idea is that if we consider a thermal sensor at temperature T, in contact with a solution whose temperature T , is changing with time due to solution heating during flow or a chemical reaction, then the change in temperature of the sensor in a short time At is

ATs = k(Tc - T,)At

(13)

where k is a constant that depends on geometry, thermal conductivities, densities, and specific heats of both the sensor and the solution. If changes in T , are due to a first-order chemical reaction then

Tc(t)= A (1 -

(14)

One can substitute Equation (14) into Equation (13), and take the limit as ATs and At approach zero. This gives ~

dTs dt

+ kT, = kA [ l - e-"'1

The solution of this is

(

a

Ts=A l + k-a Figure 10 shows the effect of this finite response time of a typical thermistor whose t 1 / 2 values are 10, 20, and 30 msec, respectively.

314

R. L. BERGER, T. R. CLEM, V. A. HARDEN, AND B. W. MANGUM THERMISTOR

STOPPING BLOCK

100 PSI

STOPPING BLOCK

0

100 PSI

I

MIXER

~

THERMISTOR

(6)

Figure 9. Thermal stopped-flow system: (a) conventionalstopped-flow; (b) zero pressure drop stopped-flow.

A problem arises when the reaction is not a simple first-order reaction and heat is lost during the reaction. In this case, we invoke the D-B FEST Method. First, we must divide the system into small cells (qq).In this case, three cells will be adequate: solution, glass coating, and thermistor, all in axial symmetry. The generalization to more cells is straightforward. Gonsider the cells to be numbered 1, 2, and 3 with cell 1 representing the thermistor, cell 2 the glass coating, and cell 3, the solution in the observa-

TEMPERATURE MEASUREMENT I N BIOCHEMISTRY

315

' i i , 40

I

20

0.2

0.4

0.6

0.8

1.0

1.2

1.4 t(sec)

Figure 10. Calculated thermistor response to the temperature change obtained in a firstorder reaction for four different reaction half lives. From top to bottom, the reaction half lives are 10,30,100, and 300 msec, respectively. In each graph, the solid line represents the actual temperature change and the dashed, dotted and dot-dashed curves represent the response of thermistors with t1/2 = 10, 20, 30 msec, respectively.

tion tube. The temperatures of the cells will be denoted by Tq,where i refers to heat transferred in the radial direction a n d j heat transferred in the axial direction; TI^ corresponds to the temperature measured by the thermistor (this is the same as T, of Equation 16). The laws of heat conduction may then be used to calculate the temperature in the reaction chamber, TSj,from the series of measured temperatures, T y assuming that all changes in the temperatures of cells 1, 2, and 3 are due to conduction.

316

R. L. BERGER, T. R. CLEM, V. A. HARDEN, AND B. W. MANGUM

We now assume that each cell q, has a known specific heat Cp,, density

p,, and volume VY. We define B, as the effective conductivity between

cells q, and q2+ S , as the surface area between these cells, and X, as the separation of their centers. The time interval At is chosen to satisfy the stability criteria for the heat conduction calculation. For a tabulation of the temperatures of the cells at different times see Table VII. T11, T12, and TI3 are known from the measurement. By the laws of heat conduction, the amount of heat transferred between cells 1 and 2 is

This transfer of energy is responsible for the observed temperature change in cell 1; therefore,

Solving Equations (17) and (18) for T21,we get

T22can be obtained in a similar manner from T I 2and TI3.To determine TJ1from the changes in temperature of cell 2 during the time interval At, we have to include both the heat exchange between cells 1 and 2 and cells 2 and 3. This gives

This iterative scheme is used to obtain the reaction temperature from the temperature recorded by the thermistor. The reaction temperature T,(t) - T, at time t, is obtained from Equation (20). TABLE VII Temperatures of Cells at Different Times Temperature of cells Time

Cell 1

Cell 2

Cell 3

317

TEMPERATURE MEASUREMENT IN BIOCHEMISTRY

The effectiveness of the response time correction was illustrated by correcting the experimental results obtained with some known reactions by making computer reconstructions. It has been shown also that the technique is internally consistent by generating a simulated calorimeter output and using the correction program to retrieve the input kinetics. The calorimeter simulation program was used to calculate a thermistor output as a result of a fast reaction (step function temperature change in the observation tube. The output of this program was entered into the data reconstruction program, and the results obtained are presented in Figure 1 1. The inset in Figure 11 represents the reconstructed step function from the simulated exponential response of the thermistor. The open circles show the response curve obtained experimentally by the fast-plunge technique. The solid circles represent the reconstructed temperature change as experienced by the thermistor during the plunge. Figure 12 shows data reconstruction of an actual reaction trace obtained by mixing glycylglycine with CO:!in solution. In Figure 12a, the actual oscilloscope trace of the thermistor output (top curve) is shown along with the velocity transducer output (bottom curve). The vertical scale is 20 m"C per division. The thermistor used in this experiment had a response time tIl2 = 7 msed. In Fig. 12b, the computer reconstruction,

0.0 0.0

1

5.0

00

0 5 I

TIME

10.0

(MSEC)

10 I

15.0

I5

2.a

20.0

Figure 1 1. Data reconstruction of an actual thermistor response to a step-function temperature change obtained by plunging the thermistor into a beaker of water: (0)the measured response;(0)thedatacorrected for finite responsetime.(Insert) The reconstructionofa computer-generated response to a step-function temperature change. The reconstructed step function has only a slight distortion close to time zero.

FLOW

tTOPPED

z

(A)

59.8

46.2

35.8 rndegOC

23.8

11 - 9

STOP FLOW

000

000

.052

.lo4

SECONDS (B)

318

.156

* 209

.261

TEMPERATURE MEASUREMENT IN BIOCHEMISTRY

3 19

which has corrected for the thermistor response time, and the experimental data are shown; the curve on the right-hand side is of the observed data. A comparison of the two traces of Fig. 12, a and b indicates that the stopping of the flow and the beginning of the reaction, as revealed by the thermistor trace, is about 8 msec later than the stopping as shown by the velocity transducer. This discrepancy disappears when the data reconstruction procedure is used to obtain the true temperature change in the observation tube. This is shown in Figure 13,where the experimental data and the reconstructed data traces are presented. Here the start of the reaction and the time that flow stopped, as indicated by the flow velocity meter, correspond exactly. Thus, in this example, correcting for the thermistor response time not only shows a faster reaction, as expected, but also resolves an apparent inconsistency between the two traces by shifting the apparent start of the reaction to correspond with the true stopping time of flow. This analysis now makes it possible to make precise fast stopped-flow thermal reaction measurements. An interesting application of these methods has been the study of the isomerization of Cryptrand 21 1 in its reaction with Li and Ca ions by Liesegang (Liesegang, 1981).At the present time, we are using the system in the investigation of the reaction of COPwith hemoglobin (Berger et al., 1978).

3. Thermal Titration and Immobilized Enzyme Reaction Detection The use of modern temperature detection methods in thermal titration has increased considerably in recent years, with several commercial instruments now available. Marini and Martin have recently reviewed this field (Marini and Martin, 1979)extensively so that only a brief discussion will be given here. We have developed a combined pH- thermal differential titration apparatus that is modelled after our earlier single-cellsystem (Berger et al., 1974; Marini et al., 1980).Figure 14 shows the essentials of the instrument. The unique part of this device is that it is under microprocessor control. The computer starts the titration, records the data, and speeds up or slows down the titration automatically if the curve is changing too rapidly. Data-correction programs adjust for response time and Figure 12. Data reconstruction of an actual reaction trace obtained by mixing glyclyglycine with C o p in solution. (a) The actual oscilloscopetrace of the thermistor output (top curve) is shown along with the velocity transducer output (bottom curve). Vertical scale is 20 m"C per division. The thermistor used had a response t l l Z = 7 msec. (b) The computer reconstruction, which corrects for the thermistor response time, along with the raw data are shown. The curve to the right is the observed data.

320

R. L. BERGER, T. R. CLEM, V. A. HARDEN, AND B. W. MANGUM

Figure 13. Reaction of HCI and NaHCOs. (---) The experimental data; (-) puter-corrected curve.

the com-

thermal losses in the system. The sample size is 2-4 ml. Titration times are 120 sec (from pH 2 to pH 10 for a protein such as cytochrome C (Marini et al., 1980, 1980, 1981). In another somewhat similar area, immobilized enzymes have been used in columns with a thermistor as the reaction detector. Several reviews of this work have been published recently (Carr and Bowers, 1980; Klibanov, 1983). Berger and Everse have had success in immobilizing enzymes directly on a glass-coated thermistor bead, thus enabling clinical measurements in vivo of blood glucose from 25 to 1000 mg% glucose. The reaction uses an oxidase that produces hydrogen peroxide, which is then broken down by catalase, yielding an overall heat of 29 kcal/mole. By using a differential thermistor bead system, excellent results were obtained in blood. Unfortunately, the enzymes hydrolyze very quickly, thus greatly limiting the utility of this system. Vurek has used thermistor flakes to build a micro CO2 detection system (Vurek et al., 1975). In that system, differential measurements are used with LiOH on one flake. The reaction with C 0 2 produces about 110 kcallmol, thus making it very sensitive. A commercial version of this instrument is available (W. P. Instruments). One of the uses of new small, sensitive temperature sensors is in

TEMPERATURE MEASUREMENT IN BIOCHEMISTRY

32 1

Figure 14. Differential thermal-pH titration apparatus.

microcalorimetry. One such new sensor is made from n and p type bismuth-telluride grown as single crystals, cut, and fabricated as thermopiles where n type is connected to p type through a copper plate. These thermopiles exhibit a very high Seebeck effect, that is, circa 400 FVPC per junction. Figure 15 shows how they are mounted in a typical calorimeter application. Recent extensive reviews in this series (Spinks and Wadso, 1977; Jolicoeur, 1981) have described the large variety of uses of such thermopiles. We have recently described a new calorimeter specifically designed for biochemical use, which uses this type of thermopile (Mudd et al., 1982).A newly developed unit (Marlow Industries), with hundreds of thermocouples per square inch, has been tested and incorporated into a modified rotor of our calorimeter. The sensitivity is eight times higher than that of our first instruments and three times higher than that of the one described in reference (Mudd et al., 1982). Commercial versions of the NIH-developed fast, thermal stopped-flow, thermal titration, batch,

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R. L. BERGER, T. R. CLEM, V. A. HARDEN, A N D B. W. MANGUM

r2*

Bismuth-Telluride Thermopile, 2 1, mountel in a heat conduction calorimeter. Figure 1 3 the leads; 18, the heater; 20, the copper cell holder; 19, the cell; and 22, the entrance channel.

and flow microcalorimeters are currently available (Commonwealth Technology). Finally, we mention several current applications somewhat outside of biochemistry in the usual sense. Thermography has slowly been coming to the fore. Many of the problems associated with the analysis of thermograms were treated at the Fifth International Symposium on Temperature (Plumb, 1972) in 1972 and new applications were discussed at the Sixth Symposium (Schooley, 1982) in 1982. Of perhaps more current interest is the greatly expanded interest in temperature measurement in hyperthermia and hypothermia. A recent New York Academy of Sciences conference has done an excellentjob of reviewing this (Ann. N.Y. Acad., 1980). Cetas also wrote a general review of thermometry in this field (Cetas, 1968). Perhaps the most exciting new method in thermometry is that of optical fluorescence, which we described earlier. Catheters, wholebody scanners, etc., have been made for use with this method. At this point, 0.01"C is probably the least imprecision that can be obtained with the commercial instrument (Luxtron), with data obtained every 0.1 sec. Improvements are likely, however, as needs are made known to the company. 4.

Standard Temperature Reference System for Biochemistry and Clinical Chemistry

In an earlier section, we discussed the IPTS-68 and mentioned several ways in which that scale can be transferred to the laboratory. While

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323

platinum resistance thermometers, mercury-in-glass thermometers, and thermocouples may be used for that purpose, we believe that bead thermistors, such as the highly stable S-10 and S-15 (Thermornetrics, Inc., Edison, N.J. 08817) thermistors with any of several types of bridges and with several fixed-point reference cells, offer the most practical and convenient method of accomplishing this for the biochemical and clinical laboratories. Let us now examine the accuracy required with regard to absolute temperature for both equilibrium and kinetic measurement to be known to & 1%. [For a general discussion of the variation of reaction rate with temperature, see the article by Bunnett in Techniques in Chemistry,Vol. V I (Lewis, 1974).] Let us discuss the general reaction

kf A+BF=C kb

where kf is the forward rate constant, and Rb the back reaction constant.

where K is the equilibrium constant. I n general, the variation of the equilibrium constant, K, with temperature follows the Van't Hoff Law, i.e., d(ln K) - AH dT RTz

(23)

Upon integration from Ti to T f , we obtain

where Ti and T f are the initial and final temperatures. Assuming AH for the reaction enthalpy to be 12 kcallmol, we can calculate the error for a 0.1"Ctemperature change from, say, 30 to 30.1"Cand from 37 to 37.1"C. Remembering that for thermodynamic calculations we must use the Kelvin scale, the first temperature change is from 273.15 K + 30 = 303.15 K to 303.25 K and the second from 310.15 to 310.25 K. Taking the anti-log of equation (24)

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R. L. BERGER, T. R. CLEM, V. A. HARDEN, A N D B. W. MANGUM

Thus, an error of 0.1"Cat 30°C leads to an error of 0.65%in estimate of concentration for a 12 kcaVmol reaction. At 37"C, the error would be a little less. Consequently, for equilibrium measurements one must know the absolute temperature to 0.03 K if a 1% uncertainty in the data is desired for comparisons between laboratories as recommended by IUB (Expert Panel, 1975). For reaction-rate measurements, the variations of reaction rate with temperature is usually expressed as the Arrhenius equation, which in its integrated form is

*

where k, i s the forward rate constant in Equation (21) (assuming the back reaction can be neglected). E is the activation energy and Zo is the collision factor, assumed to be temperature independent. Taking logarithms In kf

=

In ZO - EIRT

(27)

or log k, = log Zo - E12.303 RT

(28)

If one plots log k f against 1/T for a great many reactions, one obtains a straight line. Since the slope is -E/2.303 R , E is readily evaluated and is called the Arrhenius activation energy. R has the value of 1.987 cal/deg when E is in cal/mol. A great many reactions double their rate for a 10°C rise in temperature. If we take only two temperatures, it is convenient to use the expression

In doubling the rate from 25 to 35°C one has log 2k,, 2 and E = 0.30108 x 4.576 x 308.15 x 298.15110. Thus, E = 12656 cal/mol

- log k f T k = log (30)

For a reaction rate that goes up by a factor of 5, E would be 30,000 callmol, providing other changes, such as the ones Bunnett discusses in some detail, are not occurring. In most clinical chemistry and biochemical reaction studies, the rate is determined by observing the change in concentration. If in Equation (21) a thousandfold excess of A over that of B is used, then the rate of formation of C is independent of A or B and depends only on k f ; we call this a zero-order reaction and

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325

dc

- = kg dt

In this case, the determination of c for a fixed time depends on knowing k f , whose value is temperature dependent. Thus, for E being 12,656 cal/mol, a change of 0.1"C at 30°C will produce a change in the ratio of kfTdkfT1 of

For a first-order reaction: dc

- = kfC dt

(33)

and

c = CO

(1 - eVkft)

(34)

where Co is the value of C at t equals infinity. Many reactions however, must be run in the so-called pseudo first-order mode, that is, A. >> Bo, by say 10 to 100, but neither can now be neglected. Under these circumstances

where A0 and Bo are the initial concentrations of A and B. This neglects the back-reaction. This is usually a reasonable assumption if A. is 100 times larger than Bo and only the first 25-40% of the reaction is used. The integrated form of this equation yields

In both of these cases, to a good approximation, the temperature effect is still mainly due to the variation of the rate constant and, thus, Equation (33) best describes the dependence of C on temperature. Essentially, this gives:

c

CO

[l

- e-kt]

and thus

= 1.007

(37)

R. L. BERGER, T. R. CLEM, V. A. HARDEN, AND B. W. MANGUM

326

Thus, for a change of 0.1"C at 30°C, the absolute temperature should be known to Ifr 0.03 K if 1% data are to be presented for comparisons between laboratories (Expert Panel, 1975). At the present time, to our knowledge, it is doubtful if anyone knows the reaction temperature in a cuvette to better than 0.1"C. Using the IPTS-68 and an S-10 thermistor as the transfer standard, together with the fixed points provided by the gallium melting point and the triple point of water [29.772 and O.Ol"C, respectively], an absolute temperature accurate to & 0.01"Cor better, can be realized in any laboratory in the world for under $5000. A very convenient IPTS-68 transfer standard is provided by the digitally linearized thermistor thermometer, fully described elsewhere (Berger et al., 1980).The latter instrument is a small computer (one card) that holds a constant power (8 pW) across an S- 10 Thermistor. The voltage divided by the current supplied to do this is a direct measure of the resistance of the thermistor on an absolute basis, since the measurement device is directly traceable to NBS standard voltage and current sources. A second input is provided so that a small P20 type probe (Therrnometrics, Inc., Edison, N.J.) as shown in Figure 16,can be used to measure directly the temperature in the reaction vessel. While this probe does not have the high stability of the S-10, it can be readily

1 L * PROG GAIN INST. AMP

CONTROLLED CURRENT SOURCE

HIGH QUALITY OP AMP

I

I

I SYSTEM BUS

PROCESSOR

MEMORY

110

I10

R S 232 OR TTY

I10

CRT DISPLAY

Figure 16. Digital Linearized Thermistor Thermometer traceable to IF'TS-68 to 2 0.002"C absoiute. Absolute "A to D", analog to digital; ''UO', input-output port.

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327

compared with the S-10 and the fixed points, and the PROM reprogrammed, if necessary. A look-up table for every 0.1"C from 0 to 60°C (or 100°C) is placed in a small PROM [Programmable Read Only Memory]. An interpolation algorithm is then used to determine the temperature to the nearest 0.001"C. The equation used to do this (Kilibanov, 1983) is:

RT = R(25"C)exp (Bo +

+

$ + $1

(39)

This equation is fitted to the data on a large computer and the constants Bo, B 1 ,BP,and B3 determined. A look-up table is then generated and put on the PROM. Alternatively, one could have only the constants on the PROM and simply generate the look-up table on the RAM [powered by battery (CMOS RAM) so that it is done only once]. VII. CONCLUSIONS AND FORECASTS

In conclusion, the last 10 years has seen a greatly increased interest in temperature measurement, particularly in the use of thermistors in everything from clinical thermometers, thermal dilution catheters, waterbath regulators, hand-held digital thermometers, and, finally, an "absolute" temperature standard. We can look forward in the next 10 years to bringing temperature standardization to all areas of chemistry, biochemistry and clinical chemistry, as well as to the pasteurization and virus kill point for vaccines. For the present recommendations in biochemistry, see Expert Panel on Enzymes (1975). For the biochemist, the advance of new, sensitivetemperature-measuring devices comesjust at a time when large amounts of very pure proteins, nucleotides, DNA, RNA, lipids, etc., are becoming available. Thus, the ubiquitous nature of heat can be utilized, providing both the thermodynamic and kinetic data needed for any theory of biomolecular interaction. Research now in progress (Schutz et al., 1983) promises several orders of magnitude increase in sensitivity and of measurement speed, so that fast stopped-flow reaction studies on enzyme reactions can be conducted at micromolar enzyme concentrations with a resolving time of 100 psec. Acknowledgments

The authors wish to express their gratitude to Dr. Gearld W. Liesegang, formerly oft e National Heart, Lung, and Blood Institute and presently of Perkin- E mer Corporation, Norwalk, Connecticut, for helpful discussions in the course of preparing this paper; and to Mrs. Pauline Ballew for diligence and patience in typing the manuscript.

5t

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References Ann N.Y. Acad. Sci. (1980), vol. 335. Balko, B., Berger, R. L., and Anderson, K. (1981), Rev. Sci. Instrum., 52, 888-894. Barnes, H. T. (1902), Phil. Trans. R. SOC.(London) A , 199, 149-263. Benjarninson, A. and Rowland, F. (1972), Measurement and Control in Science and Industry, vol. 4, Instrument Society of America, Pittsburgh, pp. 701-708. Berger, R. L. (1963), Temperature, Its Measurement and Control in Science and Industry, vol. 3, part 3, Reinhold Publishing Co., New York, pp. 67-77. Berger, R. L., Balko, B., Borcherdt, W., and Friauf, W. (1968), Rev. Sci. Instrum., 39, 486-493. Berger, R. L., Balko, B., Bowen, P., Paul, R., and Hopkins, H. P., Jr. (1978), Frontiers of Biologtcal Energetics, vol I (L. P. Dutton, J. S. Leigh, and A. Scarpa, Eds.), Academic Press, New York, pp. 698-706. Berger, R. L., Balko, B., Clem, T. R., and Friauf, W. S. (1982), Temperature: Its Measurement and Control in Science and Industry, vol. 5, American Institute of Physics, New York, pp. 897-910. Berger, R. L., Clem, T., Gibson, C., Siwek, W., and Sapoff, M. (1980), Clin. Chem., 26, 1813-1815. Berger, R. L., and Davids, N. (1965), Rev. Sca. Instrum. 36, 88-93. Berger, R. L., Fraiuf, W. S., and Casico, H. E. (1974), Clin. Chem., 20, 1009-1012. Berger, R. L., and Stoddart, L. C. (1965), Rev. Sci. Instrum., 36, 78-84. Berry, K. H. (1979), Metrologk, 15, 89. Bolton, H. C. (1900), Evolution o f t l u Thermometer, 1592-1743, Chemical Publishing Co., Easton, Pennsylvania, p. 18. Boyle, R. (1683), New Experiments and Observations Touching Cold, Richard Davis, London, p. 112. Brown, W. E. L. and Hill, A. V. (1923), Proc. Roy. SOC.(London) B , 94, 297-334. Brown, H. D. (1969), Biochemical Calm'metr), Academic Press, New York. Cajori, F. (1929), A Histoty of Physics, Macmillan, New York, p. 117, as quoted in Taylor, p. 251. Callendar, H. L. (1887), R . SOC.Phzlos. Trans. London, 178, 161. Callendar, H. L. (1902), Phil. Trans. R. SOC.(London) A , 199, 55-148. Carnot, S . (1824), Refictions sur la Puissance Motrice due Feu el sur les Machines Propres a developper cette Puissance, Chez Bachelier, Libraire, Paris. Carr, P. W. and Bowers, L. D. ( 1 9 8 0 Immobilized ~ Enzymes in Analytical and Clinical Chemistry, John Wiley & Sons, New York. Cetas, T . C. (1968), Thmapeutic Heat and Cold, 3rd ed. 0. F. Lehmann, Ed.), Williams and Wilkins, Baltimore, Maryland. Chappuis, P. (1888), Trav. et Mem. Bur. Int. Po& el Mesures, 6, 1. Cheng, A. F. (1981). Measurements and Control 15, 115. Chipperfield, J. R. (1966), Proc. R. SOC.LondonB, 164, 401-410. Cohen, M.E. and Drabkin, I. E. (1948). A Sovrce Book in Greek Science, McGraw-Hill, New York, pp. 326-334.

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Comit6 International des Poids et Mesures (1955),Procb-verbaux des Skances & i‘annke 1954, 24, 81-82, T20-T23, T44-T47; see also Comptes Rendus des Skances de la DixiZme Confkrence Gknkrale des Po& et Mesures, 1954, Gauthier-Villars et Fils, Paris. Commonwealth Technology, Inc., Alexandria, Virginia 22304 Comptes Rendus des Skances de la Septi2me Confkrence Gknh-ale des Po& et Mesures, 1927, Annexe 4, p. 94. Comptes Rendus des Skances de la Neuui2me Confkrence Gknkrale des Poi& et Mesures, 1948, Resolution 3, pp. 55, 63; Resolution 6, p. 64; Annexe 6, p. 89. ComptesRendus de la Treizihe ConfkrenceGtnkrale des Po& et Mesures, 1967 - 1968, Annexe 2, p. Al; Comitk Consultatif de Thermomktrie, 8e session, 1967, Annexe 18; and Metrologm (1969) 5 , 35. Comptes Rendus des Skances de la Quinzihe Confkrence G k n a l e des Po& et Mesures, 1975, Resolution 7, p. 105; Annexe 2, p. Al. Connolly, J. J. (1982), Temperature: Its Measurement and Control in Science and Industry, vol. 5, American Institute of Physics, New York, pp. 815-817. Cork, J. M. (1947), Heat, 2nd ed., John Wiley & Sons, New York, ch. 1. Davids, N. and Berger, R. L. (1964), Comm. ACM, 7, 547-551. Davids, N. and Berger, R. L. (1969), Cum. Mod. Biol., 3 , 169-179. Davids, N. and Berger, R. L. (1982),J. Biochem. Biophys. Meth., 6, 205-217. Expert Panel on Enzymes, Committee on Standards (IFCC) (1975), Clin. Chim. Acta, 61, F11-F24. Fahrenheit, D. G. (1724), Philos. Trans. R . Society (London), 33, 78. Fery, C. (1902), Compt. Rend., 134, 997. Gugan, D., and Michel, G . W. (1980), Metrologia, 16, 149. Guildner, L. A. and Burns, G. W. (1979), “Accurate Thermocouple Thermometry”, in High Temperatures-High Pressures 11, 183 and Manual on the Use of Thermocouples in Temperature Measurement (1981), American Society for Testing and Materials, Baltimore, Maryland. Guildner, L. A., and Edsinger, R. E., (1976), NBS J. of Research 80A, 703. Guildner, L. A. and Thomas, W. (1982), Temperature: Its Measurement and Control in Science and Zdustry, vol. 5, American Institute of Physics, New York, pp. 9- 19. Guildner, L. A. (1980), Accuracy of Realizing Thermodynamic Temperatures by Gas Thermometry, PTB-Mitteilungen 90, 41. Hale, J. D., Izaat, R. M., and Christensen, J. J. (1963),J. Phys. Chem., 67, 2605-2609. Handbooks: (a) Hewlett-Packard application Note No. 290, “Practical Temperature Measurements.” (b) Sheingold, D. H., Transducer Interfacing Handbook, Analog Devices, Inc., Norwood, Mass. 02062. (c) Omega Temperature Measurement Handbook (1982), Omega Engineering, Inc., One Omega Drive, Box 4047, Stamford, Conn. 06907. (d) Transcatalog (1983), Transcat, Box D1, Rochester, N.Y. 14606. (e) Cole-Parmer Instrument Catalog, Cole-Parmer Instrument Company, 7425 North Oak Park Avenue, Chicago, IL 60648. (0 NANMACTemperature Handbook, NANMAC Corporation, 9-1 1 Mayhew Street, Framingham Center, Mass. 01701. Hudson, R. P. (1980), Rev. Sci. Instrum., 51, 871. Jolicoeur, C. (1981), Meth. Biochem. Anal. 27, 171-287. Kelvin, L., (Sir William Thomson) (1891- 1894), Popular Lectures and Addresses, 3 vols., Macmillan, New York, 1, 13.

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Klibanov, A. M. (1983), Sczence, 219, 722-727. Langley, S. P. (1881), A m . J . Sci., 21, 187. Laws, F. B. (1918), Electrical Measuremenh, McGraw-Hill, New York. Lewis, E. S. (Ed.) (1974), Techniques in Chemistry, vol. V I , 3rd ed., Wiley Interscience, New York, pp. 369-488. Lewis, G. N. and Randall, M. (1961), Thermodynamics, 2nd ed., revised by K. S. Pitzer and L. Brewer, McCraw-Hill, New York, p. 75. Liesegang, G. W. (1981),J. Am. Chem. Soc., 103, 953-955. Luxtron, Inc. 1060 Terra Bellas Ave., Mountain View, Calif. 94043. Mangum, B. W. (1974), Clin. Chem., 20, 670. Mangum, B. W. and Evans, G. A., Jr. (i982), Temperature: Its Measurement and Control in Science and Industry, vol. 5, American Institute of Physics, New York, pp. 795-801. Mangum, B. W. and Thornton, D. D., Ed. (1977), Th.e Gallium Melting-Point Standard, NBS Special Publication 48 1, Government Printing Office, Washington, D.C. Mangum, B. W. and Thornton, D. D. (1979), M e t r o l o p . 15, 201. Mangum, B. W. and Wise, J. A. (1974), Description and Use of Precision Thermometers for the ClinicalLaboratory, SRM 933 and SRM 934. NBS Special Publication 260-248, Government Printing Office, Washington, D.C. Marini, M. .A., Marti, G. W., Berger, R. L., and Martin, C. J. (1980), Biopolymers, 19, 885-898. Marini, M. A. and Martin, C. J. (1979), Crit. RPU.Anal. Chem., 8 , 221-285. Marini, M. A,, Martin, C. J.. and Berger, R. L. (1980). Biopolymers, 19, 899-91 1. Marini, M. A., Martin, C. J., Berger, R. L., and Forlani, L. (1981), Eiopolymers, 20, 22532261. Marlow Industries, Inc., 1021 South Juniper Road, Garland, Tex. 75042. M e t r o l o p , 12, 7, (1976). “The 1976 Provisional 0.5 K to 30 K Temperature Scale,” Metrolopa, 15, 65 (1979); also Procb-verbaux des Comitd International des P o d et Me.sures, 4 4 , 1 1 (1976). Middleton, W. E. K. (1966), The History of the Thennometer, Johns Hopkins University Press, Baltimore, Md. Mudd. C., Berger, R. L., Hopkins, H. P., Jr., Friauf, W. S., and Gibson, C. (1982),J. Biochem. Biophys. Methods, 6, 179-203. Ohte, A., Iwaoka, H., Mitsui, K., Sakurai, H., and Inaba, A. (1979), Metrolog-ia, 15, 195. Ohte, A. and Iwaoka, H. (1982), Temperature: Its Measurement and Control in Science and Indust?, vol. 5, American Institute of Physics, New York, pp. 1173- 1180. Ohte, A., Yamagata, M., and Akiyama, K. (1982), Temperature: Its MeasurementandControlin Science and lndustry, vol. 5, American Institute of Physics, New York. pp. 1197-1203. Pearson, L., Pinsent, B. R. W., and Roughton, F. J. W. (1954), Disc. Faruday Soc., 1 7 , 141 - 145. Plumb, H. H. (Ed.) (1972), Temperature, vol. 4, Instrument Society of America, Pittsburgh, Penna. in 3 parts, see Part 111. Preston-Thomas, H. (1972), Temperature, vol. 4, part I Instrument Society of America, Pittsburgh, Penna. pp. 4-7. Radiation Thermometry Session (1982), in Temperature: Its Measurement and Control in Science and Industry, vol. 5, American Institute of Physics, New York, pp. 575-629.

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Methods of Biochemical Analysis, Volume 30 Edited by David Glick Copyright © 1984 John Wiley & Sons, Inc.

Author Index Page numbers in italics indicate pages on which the full reference appears. Austin, R.H., 105, 116, 120, 121,125,

Abrudan, I., 253,266 Adam, G.,66,90,101 Adams, P.A., 39,101 Adamson, A.W., 66,72,101 Agmon, N., 123,137 Akerlund, H.E., 152,192 Akeroyd, R., 201,218,225 Akiyama, K.,330 Alben, J.O., 121, 124,137 Alberding, N.,120, 121,I37 Albertsson, P.A., 152,192 Alfano, R.R., 109,137 Alkaitis, S.A., 72,101 Allaz, M.J.,258,266 Allington, R.W., 150, 154,193 Aloni, Y.,185,195 Alpert, B., 126,137 Altamura, N., 261,265,266 Alten, J.A., 152, 164, 183,197,198 Alves, R.B., 296,331 Amann, R.P., 190,194 Amar-Costesec, D.,152,192 Ambrose, E.J., 150,192 Amidon, G.L., 25,40,I03 Ana, A., 237, 242, 243, 246,258,262,

127, 128, 135,137-138

Ayad, S.R., 185,194

Balko, B., 311,313,319,328 Bangham, A.D., 211,223 Bara, A., 237,242,243,246,258,262,

264,266

Baran, G.J.,154, 175, 178,195 Barenholz, Y.,216,223 Barnes, H.T., 328 Barnhmrn, M.G.,145, 150, 152, 154,

175, 178, 183,198

Barsukov, L.I., 216,223 Barter, P., 201,225 Barter, P.J., 213,223 Bartholdi, M., 127,138 BArzu, O.,231-233, 235,237,238,242,

243,246,248-250,253-255,258, 262, 264-266 Batenburg, J.J., 200,225 Bearden, 134,138 Beaufay, H.A., 152,192 Beck, G.,72,101 Beck, J.D., 182, 183,196 Beece, D.,120, 121, 124,137,138 Beeson, K.W., 116, 120, 121,137 Belch, A.C., 11,101 Bellanti, J.A., 189, 191, 194 Benesch, R., 235,236,264 Benesch, R.E., 235,236,264 Benga, G.,231-233,248,251,254,255, 264 Benjaminson, A., 300,328 Benson, D.M., 229,265 Benz, R., 57,101 Beohar, P.C., 189, 191,196 Bergelson, L.D., 201,216,223,224

264,266

Anderson, K., 311,313,328 Anderson, R.E., 213,224 Andersson, B., 152,192 Ann. N.Y. Acad. Sci., 322,328 Antonetti, A., 125,138 Antonini, E., 120,137, 230,235,261,

264

Applebury, M., 136,137 Archakov, A.H., 256,264 Astier, R., 125,138 Attardi, G.,185,195 Auk, K.A., 181,192

333

334

AUTHOR INDEX

Berger, R.L., 302, 308, 310, 31 1, 313, 319-321,328-331 Bergmeyer, H.U., 262,265 Berne, J.B., 7, 15, 20, 22, 103 Berry, K.H., 291,328 Berthet, J., 152, 192 Bienvenue, A., 200,226 BillesWUe, S., 254, 266 Blanchaer, M.C., 254,266 Bloemendal, H., 152, 192 Bloj, B., 201, 210,223 Bloomfield, V.A., 154, 175, 178,195 Boehmer, H., 180, 192 Boffoli, D., 261, 266 Bohme, H.J., 234,265 Bohn, B., 191, 195 Boltz, R.C.,143, 150, 152, 154, 156, 157, 167, 175, 176, 178, 183, 185, 189-192, 192-1 95, 197 Bondi, A., 25, 62, 101 Borcherdt, B., 328 Bdrjesson, B.W., 154, 161, 183, 193 Borza, V., 231, 237, 251,264 Bowen, P., 319,328 Bowers, L.D., 320,328 Bowne, S.F., 121, 124, 137 Boyle, R.,275, 328 Bozzato, R.P., 220,223 Bradford, M., 251,265 Brakke, M.K., 143, 150, 154, 193 Brierley, G.P., 229, 247,266 Brockerhoff, H.,214,225 Bronson, P.M., 150, 152-154, 161, 178, 181, 193, 198 Brooks, D.E., 148, 150, 177,193, I98 Brossmer, R., 191, 195 Brouillette, C.G., 37, 101 Brown, H.D., 328 Brown, R.P., 185, 197 Brown, W.E.L., 308, 328 Brubaker, L.H., 147, 193 Brumley, G.W., 200,225 Brunori, M., 120, 137, 230, 261,264 Buerkli, A,, 127, 138 Burns, G.W., 295,329 Burns, R.H., 228,267 Busslinger, M., 127, 138 Butler, M.M., 207, 223 Bysterbosch, B.H., 147, 148, 195 Bystrov, V.F., 216,223

Cajori, F., 274,328 Callendar, H.L., 279, 308,328 Campbell, H.D., 252,265 Capitano, N., 261,266 Capuano, F., 26 1,265-266 Caravaggio, T., 185,193 Cardinal, C.R., 75, 82, 102 Carmeli, C., 73, 101 Carnot, S., 278,328 Carr, P.W., 320,328 Cany, D.C., 161, 175, 177, 193 Cascio, H.E., 302, 310, 319,328 Cass, K.H., 234,267 Catsimpoolas, N., 143, 147, 150, 153-156, 158, 175, 176, 178-183,192-194, 196 Cetas, T.C., 322,328 Chan, S.S., 105, 116, 120, 121, 127, 137 Chance, B., 116, 138, 229, 247, 253, 258, 265,266 Chang, A.M., 135, I38 Chappuis, P., 279,328 Chataing, B., 213,224 Cheng, A.F., 296,328 Cherry, R.,127, I38 Chiancone, E., 235,264 Chipperlield, J.R., 310,328 Christensen, J.J., 313,329 Cioara, P.,242, 254,264 Cittadini, A., 258,265 Clark, L.C., 229,265 Clem,T.R., 308, 313,328 Cline, M.J., 182, 198 Cobon, G.S., 200,223 Cohen, L.K.,200, 213, 214, 218,223 Cohen, M.E., 272,328 Cole, J.H., 327,331 Collard, J.G., 152, 164, 183, 197, I98 Collie, C.H., 102 ComitC International des Poids et Mesures, 329 Commonwealth Technology, Inc., 329 Cmptes R d w de la Treidme Conftrence Gkdralt des Paids et Mesures, 1967-1 968, 28 1,329 Cmptes R d w des Stances de la N e u d m e Confhence G h k r a b &s Po& et Mesures,

1948,329

AUTHOR INDEX

C m p e s Rendus des Sparues dc In Quinrikne ConfketlEe G W a l e des Po& et Mesures, 1975, 282,329 Cmnptes Rendus des Sparues a% In Septit?me Confkrme G W a l e des Po& et Mesures, 1927, 281,329 Connolly, J.J., 286, 329 Conway, B.E., 20,101 Cork, J.M., 275,282, 329 Correa-Freire, M.C., 216,223 Corsey, R., 320,331 Crain, R.C., 200, 201, 207, 210, 217, 218, 223,223 Crambach, A., 153, 194 Cramer, R., 154, 156, 193, 196 Crawford, E.A., 161, 175, 177, 193 Cross, A., 132, 139 Crowfoot, P.D., 200,223 Czerlinsky, G.M., 44,101 Darnen, J., 213,223 Dancea, S., 251, 254, 255,264, 265 Dsnsoreanu, M., 237,238,242,243,246, 249,250, 258,262,264-266 Davids, N., 3 11, 3 13,328, 329 Davies, P., 229, 265 Davis, R.E., 154, 197 Debrunner, P.G., 116, 137 Defize, L.H.K., 211, 223 Degn, H., 229,265 De Jonge, H.R.,154,198 DeKouchkowsky, Y., 41,101 Delbruck, M.,66, 90, 101 De Leng, P., 145, 198 Deluca, N., 190, 194 DeMayer, L., 7, 61, 66, 69, 70, 72, 73, 101 Demel, R.A., 201, 211, 220, 221,223,226 httartog, M., 258,265 De Vaux, P.F., 200,226 DeVault, D., 117, 138 Dewanjee, M.K., 178, 183,194 Dhar, S., 189, 191, 196 DiCorleto, P.E., 201, 210, 221-223, 224 Diercksen, G.H.F., 21, 102 Dilger, J.D., 57, 102 Dill, K.A., 75, 101 Dixon, M., 228, 269 Docherty, J.J., 150, 152, 154, 178, 183, 185, 189, 190, 192,193, 195 Dodiuk, H.,23, 26, 101

335

Dombi, G.W., 145,197 Douady, P., 200, 201,224 Douzou, P., 117, 138 Downing, R., 116, 137 Drabkin, I.E., 272,328 Duckwitz-Peterlein. G., 2 16,224 Dudley, P.A., 213, 224 Durney, C.H., 301, 331 Duynstee, E.F.J., 46, 101 Dyatloviukaya, E.V., 201,224 Eaton, W., 109, 138 Ebrey, T., 138 Edridge, J.D.. 262,266 Edsinger, R.E., 291,329 Egbers-Bogaards, M.,182, 198 Ehnholm, C., 208,224 Ehrenson, S., 103 Eigen, M., 7, 44, 46, 61, 66, 69, 70, 72, 73,92, 101, 103 Eilenberg, G., 216, 224 Eisenstein, L., 116, 120, 121, 125, 137 Ek, K., 185, 197 Ellsworth, J.L., 212, 213,224 Erdy-Grutz, T., 14,101 Estabrook, R.W., 229, 253, 255,265 Evans, G.A., Jr., 286,330 Evans, W.H., 147, 152,193, 196 Expert Panel on Enzymes, Committee on Standards (IFCC) (1975), 324, 326,327, 329 Fahrenheit, D.G., 276,329 Fakharzadeh, F.F., 22 1, 224 Fatt, I., 229, 265 Feder, J., 256,265 Feinstein, M.E., 71, 101 Fendler, J.H., 102 Fenn, J.B., 20, 21, 103 Fery, C., 280,329 Fiess, H.A., 74, 78, 102 Fleming, G., 132, 139 Flory, P.S., 75, 101 Forlani, L., 320, 330 Fornerod, M., 254,266 Forster, Th., 8, 32, 36, 45, 46, 101 Frauenfelder, H., 1 17, 120, 121, 124, 137, 138 Fredriksson, S., 152, 193

336

AUTHOR INDEX

Frei, J., 238, 243, 254, 258, 265 Friauf, W.,302,308, 310,313,319, 321, 328,330 Friedman, J.M., 125, 138 Frornherz, P., 70, 82, 101 Fuller, N., 37-39, 67, 102 Fureinitti, P.S., 183, 293 Furukawa, G.T., 290,331 Gaal, O., 152,293 Gaines, R.A., 150, 152, 154, 178, 183, 189, 191-192, 193*195, 197. Gains, J.R., 300, 331 Galante, E., 185, 193 Calla, H.J., 216, 226 Geacintov, N., 127, 138 Gear, A.R.L., 161, 175, 177, 178, 193 Geiss, E., 150, 154, 295 Gershon, E., 50-51, 63, 67, 68, 74. 75, 78, 90,101, 102 Gerstman, B., 125, 238 Gerstman, B.S., 121, 138 Ghysen, W.E.J.M., 154,198 Gianazza, E., 185, 194, 197 Gibson, C., 308, 321,328,330 Gibson, Q.H., 120, I38 Gingel, D., 37, 67, 202 Giniget, R.,50-51, 63, 67, 68, 74, 75, 78, 90,102 Gitanda, V., 183,197 Glaeser, R.M., 158, 178, 195 Glau, J.F.C., 201,225 Glesmann, M.C., 67, 203 Glietenberg, D., 16, 101 Goldman, Y.E., 120, 138 Goldschmidt, R.,65, 102 Goldstein, 134, 138 Goldstein, L.,66, 71, 101 Good, D., 121, 124,137-138 Good, R.A., 154, 178, 180-183,193, 196 Gordon, E.E., 258,265 Gojatschko, L., 213,223 Goselle, U . , 36, 101 Gotto, A.M., Jr., 215, 225 Gould, A., 214,225 Gouterrnan, M., 120,138 Gratzel, M., 72, I02 Gray, I., 189, 191, 194 Green, D., 31 1,332 Green, D.E.,207,224 Gregory-Dewey, T., 43, I01

Greig, R.G., 185, 194 Griffith, A.L., 143, 150, 153-155, 175, 176, 178-181, 183, 192-194, 196 Grignon, C., 103 CroUman, A., 15, 101 Grosbois, M.. 200. 201,224 Gross, E.L., 158, 196 Grover, R., 189, 191, 197 Grunwald, F., 46, 101 Guerbette, F., 200, 201, 224 Guerrieri, F., 261,266 Gugan, D., 295, ?29 Guildner, L A . , 291, 295,329 Guillouzo, A., 143, 297 Gunsalus, I.C., 121, 125, 137, 139 G ~ p t aS., , 154, 178, 180-183,193,196 Curd, F.R.N., 66,67,102 Gutman, M., 7, I 1, 25, 33, 45, 46, 50-51, 57, 63, 65, 67, 68, 73-75, 78, 90, 101-103

Haar, M.P.,7, 15, 102 Hagdahl, L., 145, 154, 197 Hagen, A., 262,265 Hagenberger, B., 177,198 Haglund, H., 159, 184, 185, 194 Hale, J.D., 313,329 Halsall, B., 145,195 HaIsaU, H.B., 145,197 Hamberger. L.,259,265 Hammerstedt, R.H., 150, 152, 154, 138, 183, 185, 186, 189-191,192-294 Hamrnes, G.G., 43,101, 103 Hanania, G.I.H., 66, 67, 102 Hannig. K., 143-145, 148, 167, 174, 176, 180, 184,194, I98 Hansen, E., 147, 184, 194, 198 Haraux, F., 41, I01 Hards, J.M., 177, 198 Harmony, J.A.K., 213,224 Harned, H.S., 150, 194 Hart, A.A.M., 152, 164,197 Hartmann, W., 216,226 Harvey, M.S., 201, 207,224 Hasted, J.B., I02 Hauser, M., 7, 15, 36, 87, 101, I02 Hay, S., 190, 194 Hayson, D.A., 148, 150, I94 Heidrich, H.G., 148, 176, I94 Heirwegh, K.P.M., 145, 195

AUTHOR INDEX Hellings, J.A., 209, 218,221,224 Helmkamp, G.M., Jr., 201,207,209,218, 219,222,224 Henry, E., 109,138 Henry, U.P., 37,67,103 Herlitz, H., 259,265 Hershkowitz, S., 128,138 Hibberd, M.G., 120, I38 Hibbit, K.G., 160, 189, 191,195 Hickson, D., 215,225 Hill, A.V., 308,328 Hill, W., 113, 138 Hiram, S., 247,265 Hirorni, K.,138 Hirsch, R.L., 189, 191,194 Hjerten, S., 144, 147, 180,193,194 Ho, C., 66,67,73,78,103 Ho, N.T., 66,67,73, 78,103 Hochstrasser, H., 154,194 HodArnau, A.,251,254,255, 264,265 Hoffman, E., 234,265 Hofrichter, J., 109,138 Hogan, M.,128,138 Hollande, E., 143,197 Honig, B., I38 Honjo, I., 255,265-266 Hopfield, J.J., 121, 123, 125, 134, 137-138 Hopkins, G.J., 213,223 Hopkins, H.P., Jr., 308,319,321,328, 330 Horowitz, P., 113,138 Horwath, C.,25,40,102 Hostetler, K.Y., 218,225 Hudson, R.P., 291,329 Hughes, L.B.,201,212,225 Hughes, M.E., 204,207,222,223,226 Hultborn, R.,259,265 Hunter, S.J., 183,193 Huppert. D., 7, 11, 25,33,45,46,57,65, 74,78,101-103 Hurley, J., 138 Hyden, H., 259,265 Hyman, J., 137,138 Hymer, W.C., 150, 152, 154, 178, 183, 191, 192,193,197 Ihm, J., 213,224 Iizuka, T.,125,I39 Imai, K., 247,265

337

Inaba, A., 330 Ireland, J.F., 8,102 Ivanof, A., 237,243,265 Iwaoka, H., 298,330 Izaat, R.M., 313,329 IZZO,G., 261,265-266 Jackson, R.L.,212,224 Jahn, T.L.,145,198 Jain, M., 249,265 James, K., 229,266 Jarzynski, J., 327,331 Jaworowski, A., 252,265 Jebeleanu, G., 231,232,235, 264,266 Jobsis, F.F., 229,266 Johnson, C.C., 301,331 Johnson, L.W., 207,224 Jolicoeur, C.,321,329 Jones, J.L., 37,101 Jones, M.N., 185,194 Jonsson, M., 152,194 Jovin, T.,153,194 Jovin, T.M.,127,I38 Jung, S.,235,236,264 Jungalwala, F.B., 213,225 Just, W.W., 184-186,194 Kader, J.C., 200,201,218,224 Kagedal, L.,146,196 Kalsbeek, R., 201,211,223 Kamp, H.H., 201,207,209,217,218, 221,224,225 Kanety, H., 101, I02 Kano, K., I02 Kaplan, J. H., 149, 150,194,198 Kaplan, S., 200,213,214,218,223 Kapoor, N.,182, 183,196 Karohl, J., 127,I38 Karuzina, I.I., 256,264 Kasper, A.M., 209,222,224 Kaufman, K.J., 25,103 Kaufrnan, S., 252,265 Kebarle, P., 20,21,102 Keevil, T.,230,265 Keith, A.D., 185, 186, 189, 190, 194 Kelvin, L., 272,329 Kevan, L., 72,75,102 Kezdi, M.,254,265 Kipp, J.B.A., 198 Kirkwood, J.G., 67,103

338

AUTHOR INDEX

Kitamura, 0.. 255,266 Klaning, W.K., 65, 102 Klein, U.K.A., 7, 15, 36, 87, 101, 102 Klibanov, A.M., 320,330 Kloosterman, A.D., 201,207,224 Klou, I.M., 74, 78, 102 Knopp, J.A., 229,265 Kobdinsky, L., 229,266 Kolin, A., 143, 144, 147, 150, 180, 193-1 95 Kolodney, E., 7, 11, 74, 78, 102 Kooi, M.W., 198 Kopperschlanger, G., 234,265 Korohoda, W., 178,195 Kosower, E.M., 23,26, 101, 102 Kotani, M., 247, 265 Kraaipoel, R.J., 191, 195 Kraan, W., 152, 198 Kracek, F.C., 15,102 Kraemer, W.P., 21, 102 Krase, W., 7, 61, 66, 69, 70, 72, 73, I01 Kntmrine, P.H., 144, 177,198 Kufera, P., 259, 260,266 Kukulinsky, N.E., 190-192, 193 Kunze, K., 229,266 Kunze, M.E., 183, 197 Kuroda, M.V., 247,265 Kutschker, A., 16,101 Kuzmin, V., 127, 139 Laas, T., 146, 196 Labonowski, J.. 178, 195 Lachich, V., 65, 102 Lagakos, N.. 327,331 Lakowicz, J.R., 126, 138 Lakshmi, M.S., 143, 160, 188, 189, 191, 197 Landel, A.M., 144, 150,195 Landriscina, C., 214,225 Langille, F.A., 150, 154, 193 Langley, S.P., 279, 330 Larsson, C., 152, 192 L~SCU, I., 235,238,243,265-266 Laurent, T.C., 146, 196 hvalette, D., 121, Z38 Laws, F.B., 302,330 Leaback, D.H., 161, 195 hcarpentier, Y.,125, 138 Leise, E.M., 185, 188, 191, 195 h Neveu, D.M., 37, 67, I02

Lengyel, S., 14, 101 Lerner, H., 154, 194 Lerner, K., 145, 154, 197 Le Sane, F.,185, 188, 191, 195 Lessler, M.A., 229, 266 Lester, R.L., 207,224 Leuking, D.R., 200, 213, 214, 218,223 Lewis, E.S., 323,330 Lewis, G.N., 278,330 Li, A.S.W., 72, 75, 102 Liebich, H.G., 174, 180, 198 Liesegang, G.W., 319,330 Lijklema,J., 148, 196 Lillie, R.S., I95 Lim, T.K., 154, 175, 178,195 Linderstram-Lang, K., 187, 195 Lindqvist, L., 127, 137 Linnane, A.W., 200,223 Lipari, G., 61, 70,77, 103 Lis, L.J., 37, 67, 102, 103 Lloyd, D., 229,266 Loeb, A.L., 148, 198 Longmuir, I.S., 229, 265-266 h s , J.A., 175, 195 Lords, J.L., 301,331 Lorusso, M., 261,266 Louie, M.K., 143, 150, 154, 167, 175, 176, 183,193 Low, M.G., 223,224 Lu, A.Y.H., 255,266 Lubbers, D.W., 229,247,266 Lurnb, R.H., 200,201,207,224,225 Lumry, R., 139 Lundsgaard, J.S., 229,265 Lutter, R., 152, 183, 198 Lutton, C., 200, 201,224 Luxtrom, Inc., 330 Maasse, G., 7, 61, 66, 69, 70, 72, 73, 101 McAlister, M., 37, 67, 102 McCoy, G.D., 256,266 McCray, J.A., 120, 138 McDonald, D., 132, I39 MacDonald, R.C., 216,224 McGuire, J.K., 185, 195 Machida, K.,214, 222, 224 McKay, H.A.C., 202,224 McLaughlin, D., 137, 138 McLaughlin, S.A., 57, 101, 102 McLean, L.R., 209,225

AUTHOR INDEX McQuame, D.A., 87,192 McVittie, L., 212,224 Maeda, T., 224 Maillard, M., 254,266 Makin, H.L.J.. 262,266 Mangum, B.W., 286,287,289,290,330 Manske, W., 191,195 Manson, W., 154,I95 Mantsch, H.H., 254,265 Marcolin, H.-E., 125,138 Marden, M.C., 121, 124,137 Margel, S., 184,197 Margoliash, E.,25 1,266 Marini, M.A., 311,319,320,330-331 Markert, M., 237,238,243,258,261, 265-266 Marks, G.M., 183,197 Marlow Industries, Inc., 330 Marti, G.W., 320,330 Martin, C.J., 319,320,330 Martin, F.J., 216,224 Martin, J.L., 125,138 Mason, D., 154,195 Mason, H.S., 230,265 Massey, J.B., 215,225 Matheka, H.D., 150, 154,195 Matinca, D., 253,266 Matthew, J.B., 66,67,102 Mazliak, P., 224 Medgyesi, G.A., 152,193 Megli, F.M., 214,225 Mehrishi, J.N., 183, 185,195, 197 Mehta, A., 189, 191,197 Mel, H.C., 158, 178,195-197 Melander, W.,25,40,102 Merola, A.J., 229,247,266 Merzbacher, E., 132-133, 138 Metrologia 12,7 (1976),282,330 Metrologia 15,65 (1979),330 Metz, R.J., 200,225 Meuwissen, J.A.T.P., 145,I95 Meyer, B., 148,194 Micale, F.J., 144, 177,198 Michalik, M., 178,195 Michel, G.W., 295,329 Middleton, W.E.K., 273,330 Migus, A., 125,138 Milito, R.P., 150, 152, 154, 178,183,191, 192,193,197 Miller, J.D., 87, 102

339

Miller, R.G., 147, 195 Miller, T.Y., 185, 190-192, 193,195 Mitsui, K.,330 Moh, P.P., 121, 124,137 Molnar, I., 25,40,102 Monitto, C., 128,138 Montes, A., 201,212,225 Moonen, P., 201,225 Moore. H.D.M., 160, 189, 191,195 Morimoto, H., 247,265 Mortell, R., 150, 152, 154, 178, 183, 191, 192,193 Morton, R.E., 201,213,218,225 Mottola, H.A., 262,267 Mudd, C., 308,321,330 Mukerjee, P., 75,82,102 Munteanu, R., 237,242,243,246,258, 264 Muresan, L., 231-233, 237,243,248,251, 253-255,262,264,266 Murphy, M., 200,223 Nachliel, E., 7,25,33,50-51, 63,67,68, 70,72,74,75,78,90,101, 102 Narayana, P.A., 72,75,102 Nason, P., 145,195 Naughton, M.A., 153,194 Neihof, R., 195 Nelemans, S.A., 217,218,224 Nerren, B.H., 184,197 Nessi, P., 254,266 Newton, M.D., 103 Ng, T.C., 37,I01 Nichols, A.V., 158,197 Nielsen, S.O., 187,195 Nobel, P.S., 158,195, 196 Nordlund, T.M., 116, 120, 121,I37 Nossal, G.J.V., 180,192 Notter, M.F.D., 150, 152, 154, 178, 183, 191, 192,193 Oarga, M., 235,237,243,253,265-266 Ohnishi, J., 213,214,225 Ohnishi, S., 214,222,224 Ohte, A., 298,330 Oldenborg, V., 200,225 Olsen, R.B., 301,331 Ondrias, M.R., 125,138 Op den Kamp, J.A.F., 210,222,225 Opelz, G., 182,198

340

AUTHOR INDEX

Orbach, N., 23,26,102 Oshawa, T., 255,266 Oshino, N.,229,265-266 Oshino, R., 229,265,266 Ott, H.W., 110, 135,138 Ott, M.G., 143,197 Ottolenghi, M., 23, 26,65,102, 138 Overath, P., 216,224 Overbeek, J.T.G., 147, 148,195-197 Owen, B.B., 150,194 Ozawa, K., 255,265-266 Packer, L., 158,I96 Pande, S.V., 254,266 Papa, S.,261,265-266 Parish, G., 127,138 Parsegian, V.A.,3749.67,102, 103 Pascher, G.,171, 172, 174,180,I98 Pattnaik, N.M., 201,212,225 Patumraj, K.,214,225 Paul, R., 319,328 Pearson. L., 3 10,330 Perry, S., 147,197 Pertoft, E.A., 146,196 Perutz, M.F.,120, 121,137 Peters, K.,136,138 Petersen, L.C., 229,265 Peterson, E.A., 147, 152,196 Pettersson, S., 152, 170,194, 197 Phillips, M.C., 209,225 Phillips, R.A., 147,195 Philpot, J.S.L., 143, 158,196 me&, N., 175,196 Pines, E., 7,25,45,46,50-51, 57,63,65, 67, 70,72, 75,90,101, I02 Pinsent, B.R.W., 310,330 Plank, L.D., 183,197 Platsouas, C.D.,153, 154, 178, 180-183, 192,193,196 Plesset, M.S., 145,196 Plumb, H.H., 290, 322,330,331 Polak, F., 152,198 Polson,A., 156,196 Ponder, E., 150,I96 Poorthuis, B.J.H.M., 201,210,218,225 Popescu, O.,235,266 Porath, J., 147, 180,193 Porumb, H., 235,253,266 Possmayer, F., 207,225 Post, M.,200,225 Poulis, M.I., 252,265

Poulos, A.T., 127,138 Pownd, H.J., 215,225 Poyart, C., 125,138 Pradac, J., 175,196 Pradacova, J., 175,196 Resecan, E.,253,266 Pressman, B.C., 229,266 Preston-Thomas, H., 281,300 Pretlow, T.G., 181,196 Pretlow, T.P., 181,I96 Prouzova, O.,175,196 Puijk, W.C., 201,225

Quagliariello, E., 214,225 Racker, E., 256,266 Raddacz, E.,259,260,266 Radiation Thermometry Session (1982), 296,330 Radin, S., 200,225 Raftery, M.A., 185,195 Rajaram, O.V.,201,225 Rand, R.P., 37-39, 67,102,103 Randall, M., 278,330 Rani, S., 189, 191,196 Rao, K.V.,143, 188,189,191,196,197 Rao, M.,7, 15,20,22, 103 Redi, M.H., 121,I38 Regis, J., 213,223 Reinisch, L., 120, 121, 124,137, 138 Reiser, S.J., 274,331 Rembaum, A., 184, 197 Remenyik, C.J., 145,I97 Rentzepis, P., 109, 117,137, 138 Resch, R.C., 256,266 Reschke, R., 125,I38 Reuss, F.F., 197 Reynolds, A.H., 109, 117, 121, 124, 137-138 Rice, S.A., 11, 101 Richardson, L.S., 150, 152, 154, 178, 183, 191, 192,I93 Richter, P.H., 61,103 Riddle, J.L.,290,331 Riggs, A.,234,266 Righetti, P.G., 185, 187, 192,193-195, 197 Rilbe, H.,152, 170,177,194,197 Ritson, M.D., 102 Robbi, M., 152,192 Robbins, S.G., 178, 182,I96

AUTHOR INDEX Robinson, M.E., 200,225 Roller, D., 273-275,331 Roos, D., 175,195 Roozendaal, K.J., 182,198 Rosano, L., 71,I01 Roseman, M.A., 215,225 Rosen, P.J., 147,197 Rosenfeld, T., 138 Rossi-Bernardi, L., 235,264 Roughton, F.J.W., 308,310,330,331 Rousseau, D.L., 125,138 Rowland, F., 300,328 Rozzell, T.C., 301,331 Ruenwongsa, P., 213,225 Ruppel, D., 216,226 Russell, B., 156,I96 Russu, I.M., 66,67,73, 78,I03 Sahgami, T., 208,225 Sakura, H.,330 Sands, F., 249,265 Sapoff, M., 308,328 Sappof, M., 287,331 Sarfaty, G.A., 154,183,193 Sarton, G., 272,331 Sartory, W.K., 145.I97 Sasaki, T., 208,225 Sato, K.,258,266 Satre, M., 232,264 Scarpa, A., 255,258,265-266 Sceats, M.C., 11,101 Schabel, F.M., 147,197 Scherphof, G., 213,223 Schindler, F.,229,266 Schneider, G., 127,138 Schooley,J.F., 322,331 Schuldiner, S., 195 Schullman, S.G., 7,103 Schulz, J., 234,265 Schumaker, V., 145,195 Schumaker, V.N., 145,197 Schurr, M., I38 Schutz, L.S., 327,331 Schwedes, J., 145,195 Scott, A., 137,138 Scott, K.M., 229,247,266 Seaman, G.V.F., 177,193,194,198 Searcy, J.Q., 20,21,103 Searles, L.L., 221,224 Seebeck, T.J., 327,331 segrest, J.P., 37,I01

34 1

Serwer, P., 150,197 Shapiro, Y.E., 216,223 Sherbet, G.V., 143, 148,159,160, 184-188, 191,197 Shore, W.S., 67,103 Shortman, K., 180,192 Shoup, D., 61,70,77,103 Siemens, W.,279,331 Simonetti, A.L.M., 41,103 Singh, H.,213,225 Siwek, W.,308,328 Skeggs, L.T., 154,194 Skrabut, E.M., 180,193 Slaby, F., 214,225 Slater, E.C., 228,266 Sligar, S.G., 139 Sluyser, M., 152,198 Smets, L.A., 182,198 Smith, K.K., 25,I03 Smits, P., 201,225 Smolka, A.J.K., 184,197 Snow, T.R., 229,266 Snyder, R.S., 185,195 Sole, R., 61,I03 Somerharju, P.,214,225 Sommer, J., 109,138 Sorenson, L.B., 120,137,138 Sorof, S., 143,197 Spinks, C.H., 321,331 Spiro, T., 125,139 Stakelberg, M., 16,101 Standish, M.M., 211,223 Starlinger, H., 229,247,266 Stauffer, J.F., 228,267 Steere, R.L., 154,197 Stein, G., 65,102 Stellwagen, E., 234,267 Stockmayer, W.H., 61,103 Stoddart, L.C., 310,328 Stone, A.L., 185,I97 Streibel, M.J., 143, 150, 154, 156, 167, 175,176,183,193 Sturtevant, J.M., 302,331 Subramaniam, S.,214,225 Suzuki, R., 258,266 Svendsen, P.J., 156,I97 Svensson, H., 145,154,156,167,193, 194 Swanson, S.A., 67,103 Swam,J.M., 300,331 Szabo, A., 61,70,77,I03

342

AUTHOR INDEX

Takabi, T., 43,103 Takasan, H., 255,265 Tanaka, T., 213,214,225 Tanford, C., 66-68, 70,71,103 Tanizaw, H., 23,26,102 Tao, T., 127,139 Tarmure, C., 229,237, 238, 243,248, 254,264-266 Taylor, L.W., 272,276,331 Teerlink, T., 218,225 Telia, M., 235,237,243,265,266 Tenforde, T., 158, 178,195 Thines-Sempoux, M., 152,192 Thomas, J.A., 143,197 Thomas, J.K., 72,101 Thomas, P., 103 Thomas, W., 295,329 Thompson, C.J., 150, 152,154,178, 183, 185, 189-192,193,195 Thompson, S.T., 234,267 Thompson, T.E., 215,216,223,225 Thompson, W., 207,223 Thomson, A.E.R., 185,195 Thomson, W., 278,281,331 Thornton, D.D., 287,290,330 Tdinca, R.,254,264 Timmerman, A., 150,178,198 Timofeeva, N.G.,201,224 Tinker, D.O., 220,223,223 Tippets, R.D., 158,I97 Tipps, R.W., 185,195 Tipton, K.F., 249,267 Tiselius, A., 143,197 Todd, P., 143,150, 152,154, 156, 157, 167, 175, 176,178, 183, 185, 186, 189-192,192-195, 197 Tolben, W.R., 256,265 Tong, L.K.J., 67,103 Trautwein, A., 125,138 Trentham, D.R., 120,138 Tsao, F.H.C., 213,225 Tsuiki, S., 258,266 Tulp, A., 145, 150, 152, 154, 164, 175, 178, 182, 183,197,198 Urnbreit, W.W., 228,267 Utton, D.B., 298,331 Uzgiris, E.E., 149,150,194,198 Valeri, C.D., 180, 193 Valmet, E., 156,197

Valvani, S.C., 25, 40,I03 Van Assendelft, O.W., 230,236,267 Van Beck, W.P., 152, 182, 183,198 Vancea, D., 253,266 van Dam, K., 41,103 Van Deenen, L.L.M., 201,207,209-21 1 , 217,218,221,223-225 Van den Besselaar, A.M.H.P., 219,225 Van den Eeden, A.L.G., 21 1,223 Van der Hoff, J.W., 144, 177,198 van der Krift, T.P., 218,225 Van Der Ziel, A., 310,331 Van Duin, A.M., 191,195 vanColde, L.M.G., 200,225 Vanier, J., 291,331 Van Os, C.H., 154,198 Van Oss, C.J., 144, 150, 152-154, 161, 177, 178, 181,193,198 Van Regenmortel, M.H.V., 154, 156,198 Vass, Sz., 87,103 Vassar, P.S., 177,198 Vaughan, W.M., 229,267 Vaz, W., 127,139 Vereczkey, L., 152,193 Verster, T.C., 331 Vilah, C., 143,197 Vishniac, W., 229,267 Vitktorov, A.V., 216,223 Vogel, H., 127,139 Volkers, A., 32,36,45,46,101 Volkova, V.I., 216,223 Von Korff, R.W., 249,265 Vriend, G., 218,219,221,223,226 Vurek, G.G., 320,331 Wadso, I., 321,331 Wagner, G., 174,180,198 Walasek, O.F., 251,266 Waldeck, D., 132,139 Walters, J.A.L.I., 154,198 Wang, J., 128,138 Warach, J.B., 201,224 Warnock, D.G., 320,331 Warren, P.J., 262,266 Warshel, A., 7, 15,103 Watan. H.,247,265 Waterman, M.R., 230,267 Watkins, J.C., 211,223 Watson, R.H., 150,197 Watt, D., 311,331 Weber, G., 126,138,229,267

AUTHOR INDEX

Weir, E.A., 196 Weirsema, P.H., 148, 196, 198 Weller, A., 7, 8, 37, 61, 103 Wells, J.R., 170, 182,198 Werner, G., 184-186,194 West, S.B., 255,266 Westerhoff, H.V., 41, 103 Westerman, J., 201, 218, 219, 221, 223, 225-226 Westra, J.G., 152, 183, I98 Whipple, C.G., 145, 196 White, G.H., 201,225 Wibo, M., 152,192 Wickersheim, K.A., 296,331 Williams, G.R., 229, 247, 253,265 Williams, J., 229, 266 Williams, J.M., 150, 154, 175, I93 Williams, N., 229, 266 Winet, H.,145, 196, 198 Wirth, H., 148,194 Winz, K.W.A., 200, 201, 206, 207, 209-211.214, 217-219, 221, 223, 223-226 Wise, J.A., 286, 289, 294,330-331 Wolff, M. Ch., 262, 267 Wortis, H.H.,143, 175, 176, 178, 183, 194

343

W.P. Instruments, Inc., 331 Wrigley, C.W., 161, I95 Wu, L.N.Y., 200,225 Wyatt, P.A.H., 8, I02 XU, Y.H., 216,226

Yalkowsky, H.S., 25, 40, I03 Yamada, M., 213,214,225 Yamagata, M., 330 Yamamoto, H., 125, 139 Yapel, A.F., 139 Yonetani, T., 125,139 Young, I.G., 252,265 Yue, K.T., 121, 124,137,138 Zborowski, J., 21 1, 220, 221,223,226 Zeiller, K., 147, 148, 167, 171, 172, 174, 176, 180,194, I98 Zettergren, J.G., 196 Zhirnov, G.F., 256, 264 Ziegler, D.M., 207, 224 Ziegler, H.,216, 226 Ziljstra, W.G., 230, 236,267 Zilversmit, D.B., 200, 201, 204, 206-208, 210, 212,213,217,218,221-223, 223-226

Methods of Biochemical Analysis, Volume 30 Edited by David Glick Copyright © 1984 John Wiley & Sons, Inc.

Subject Index Absolute temperature, concept of, 277 Absorption, monitoring triplet state population by, 128-130 Activation energy spectrum, 121-124 possible origin, 123-1 24 Adsorbed Bromo Creson Green, simulation of protonation, 75-78 Alkalinization pulse, proton emitter, 62-63 Amontons, Guillaume, 276 Amplification, signal, 112-1 13 Analog Devices AD590 Integrated Circuit Temperature Transducer, 304 Analog outputs, electronic thermometers, 306 Analytical electrophoresis, cells in density gradient, 174-175 column with laser beam, 175 discontinuous density interface, 175 transanalyzer, 174-1 75 Anisotropy decay, 127-128 Antigen-sensitized vesicles, 208 Assays, to quantitate lipid transfer activity: effects of composition of assay mixture, 218-223 separation, 203-206 specific, 206-2 14 spectroscopic, 2 14-2 17 Barothermoscope, 273 Bimetallic-strip thermometers, 294-295 Binary-coded decimal (BCD), 306-307 Black, Joseph, 278 Bolometers, 279 Boltz-Todd device, 156-157 Bone marrow cells, 182-183 Boyle, Robert, 274-275,276, 281 Biichler Polyprep, 153-156

Calibration, thermometer, 287 Callendar, H.L., 279 Carnot, Sadi, 278-279 Castelli, Father Benedetto, 273 Celsius, Anders, 276 Celsius scale, 276, 281 Chappuis, P.,279 Charge separation, intrinsic photoactivation, 132-135 conformation distribution, 133-1 34 instrumentation, 134-136 transfer band, 134 Charles, J.A.C., 277 Chemiosmotic hypothesis (Mitchell theory), 2 Christian of Lyons, 276 Clausius, Rudolf, 278 Coherent anti-Stokes Raman spectroscopy (CARS), 296 Comitt International des Poids et Mesures (International Committee on Weights and Measures), 278 Concentrated salt solution, proton dissociation, 15-21 Conformation distribution, electronseparation, 133-134 Controllers, temperature, 308, 309 Cryogenic techniques, laser photolysis, 116120 helium cryostat, 116-1 17 optical configuration, 118 sample preparation, 117-1 18 temperature measurement and control, 118-1 19 Cryostat optics, optical path, 118 Curie Law, 280 Dalence, Joachim, 275 Dalton, John, 277

345

346

SUBJECT INDEX

Density gradient electrophoresis, separation of mammalian cells, 141-198 apparatus, 152-175 analytical electrophoresis, 174-1 75 introduction, 152-153 preparative electrophoresis columns, 153-174 applications, 177-184 lymphoid, blood, and hemopoietic cells, 177-183 miscellaneous, 183 experimental conditions, 175-1 77 density solute, 175-1 76 electrodes, 176 electroendoosmosis, 177 ionic composition, 176 power supply, 176 future prospects, 184 history, 143 introduction, 142-143 isoelectric focusing, 184-192 applications, 187- 192 introduction, 184-186 lack of theory, 186-187 theory, 143-152 electrophoretic effects, 147-1 50 gravity effects, 144-147 miniaturization principle, 150-152 Dial thermometers, 294 Digital outputs, electronic thermometer, 306-307 Diode thermometers, 300-301 Discontinuous density interface, electrophoresis and, 175 Dissipation of energy, law of, 278 Electron spin resonance (ESR), 214 Electrophoresis columns, 153-1 74 Boltz-Todd device, 156-1 57 Biichler Polyprep, 153-156 ISCO column, 161 LKB model, 159-160 quickfit column, 161 separation chamber according to TULP, 162-174 compact device, 166-1 74 with movable electrodes, 162-166 small-size focusing chamber, 160-161 STAFLO, 158 Van Oss and Bronson device, 161-162

Electrophoretic effects, mammalian cells, 147-150 Electrophoretic separation of erythrocytes, 178 murine spleen B and T cells, 179 Eivius, 276 Erythrocyte membranes, transfer of radiolabeled phospholipids between vesicles and, 210 Erythrocytes, 177-178 Excited molecules, proton dissociation, 4 Fabri, Honore, 275 Fast stopped-flow thermal measurements, 308-3 11 Ferdinand 11, Grand Duke of Tuscany, 274 Fery, C., 280 Flashlamp-pumped organic dye laser, 109-1 10 Fluorescence, phospholipid exchange activity, 214-216 Focusing chamber, small-size, 160-161 Galileo Galilei, 271-272 Gallium, 287,289-290, 292 Gas thermometers, 295 Gay-Lussac, L.J., 277 Gravity effects, density gradient electrophoresis of mammalian cells, 144-147 Ground-state anion of proton emitter,

4546

Ground-state compounds, dynamics of protonation: excitation pulse, 5 geometry of excitation and probing beams, 6 measuring equipment, 5-6 monitoring light, 5 Helium cryostat, 116-1 17 Helmholtz, Hermann von, 278 Heme proteins, 120-125 activation energy spectrum, 121-124 possible origin, 123-1 24 kinetics, 120-121 molecular and spin tunneling, 124-125 Heron of Alexandria, 272 Hess, Victor, 278

SUBJECT INDEX

High-molecular-weight structure, kinetics of protonation, 66-84 effect of charge on rate, 69-73 postprotonation reaction, 73-84 uncharged target adsorbed on uncharged carrier, 68-69 High-speed transient recorder, 113-1 15 Hooke, Robert, 274-275 Human hemoglobin derivatives, spectral characteristics, 230 Human liver homogenates, cytochrome oxidase activity, 250-251 Huygens, Christian, 274-275 Hydrogen peroxide-generating oxidases, determination of, 249-250 Hydroxypyrene trisulfonate: fluorescence decay time, 27 lifetime of excited anion, 28 macroscopic rate constants, in water and protein complexes, 31 steady-state fluorescence emission, 25 time-resolved fluorescence, 28 IEEE-488 General Purpose Interface Bus (GPIB), 307 Immobilized enzyme reaction detection, thermal titration, 319-322 Incubations, unilamellar vesicles, 21 1 International Practical Temperature Scale (IPTS), 279, 281 International Practical Temperature Scale of 1968 (IPTS-68), 282-285,286 fixed points, 283 future improvements and extensions, 284 secondary reference points, 284 thermodynamic laws, 285 International Temperature Scale of 1927 (ITS-27), 280-28 1 Intrinsic photoactivation, 132-136 charge separation, 132-135 conformation distribution. 133 instrumentation, 134-136 transfer band, 134 proton pumping, 136 ISCO column, 161 Isoelectric focusing (IEF), 184-192 applications, 187-192 introduction, 184-186 lack of theory, 186-187 in natural pH gradients, 188-190

347

Isolated cells, oxygen consumption, 255-258 Johnson-noise thermometers, 298 Joule, James, 278 Kelvin, Lord, 278, 281 Kelvin thermodynamic scale, 281 Langley, S.P., 279 Large perturbation techniques, laser photolysis, 119-125 general considerations, 119-120 heme proteins, 120-1 25 activation energy spectrum, 121-124 kinetics, 121 molecular and spin tunneling, 124-125 Laser-induced proton pulse: effect of buffer on proton cycle, 90-98 three-compound system, 94-98 two-compound systems, 91-93 introduction, 2-3 methodology and instrumentation, 3-6 protonation of ground-state compounds, 4-6 proton dissociation from excited molecules, 4 protonation of high-molecular-weight structure, 66-84 effect of charge on rate, 69-73 postprotonation reaction, 73-84 uncharged target adsorbed on uncharged carrier, 68-69 proton dissociation, 6-22 in classical chemistry, 6-7 in concentrated salt solution, 15-21 effect of pK on rate of, 10 effect of solvent on rate of, 10-15 rate determination, 7-10 proton transfer, surface of macromolecular structure, 84-90 reactions, 22-66 with its emitter, 2 2 4 3 with molecular proton detector, 43-66 Laser photolysis, 105-139 intrinsic photoactivation, 132-136 charge separation, 132-135 proton pumping, 136 introduction, 106- 107

348

SUBJECT INDEX

Laser photolysis (Continued) pulsed lasers. 107-1 11 techniques: cryogenic, 116-1 19 hrge perturbation, 119-125 signal acquisition, 111-1 16 triplet-state probes, 125-132 anisotropy decay, 127-128 importance of long lifetimes, 125-127 instrumentation, 130-132 population, monitoring, 128-1 30 Le Chatelier, H.,280 Leains, vesicles sensitized to, 208-209 Linnaeus, Carolus, 276 Lipid transfer activity, 199-226 assaying crude extracts, 2 17-2 18 effects of composition of assay mixture, 218-223

acceptor and donor particles, 220-222

exchangeable lipid pool, 222-223 physical state of substrate lipid, 222 ratio of acceptor to donor particles, 219

general considerations, 202-203 introduction, 200-202 separation assays, 203-206 cross-contamination of isolated particles, 204-206 nonexchangeable markers, 203-204 specific assays, 206-214 microsomes-mitochondria, 206-208 vesicles, 208-2 13 spectroscopic assays, 2 14-2 17 electron spin resonance, 214 fluorescence, 2 1 4 2 16 nuclear magnetic resonance, 2 16 Liquid-crystal thermometers, 30 1 Liquid-in-glass thermometers, 293-294 LKB column, 159-160 Localized chemiosmosis or “local pH,” 41 Lymphoid, blood, and hemopoietic cells, 177-1 83

bone marrow cells, 182-183 erythrocytes, 177-1 78 peripheral blood cells, 180-182 platelets, 177 spleen cells, 178-1 80 thymocytes, 183 tonsillar cells, 182

Mammalian cells, density gradient electrophoresis, 141-148 apparatus, 152-1 75 analytical electrophoresis, 174-1 75 introduction, 152-1 53 preparative electrophoresis columns, 153-1 74

applications, 177-184 lymphoid, blood, and hemopoietic cells, 177-183 miscellaneous, 183 experimental conditions, 175-177 density solute, 175-176 electrodes, 176 electroendoosmosis, 177 ionic composition, 176 power supply, 176 future prospects, 184 history, 143 introduction, 142-143 isoelectric focusing, 184-192 applications, 187-192 introduction, 184-186 lack of theory, 186-187 theory, 143-152 electrophoretic effects, 147-150 gravity effects, 144-147 miniaturization principle, 150- 152 Maria of Alexandria, 272 Mayer, Alfred, 278 Mercury thermometers, 279 Microsomal oxidases, measurement of, 255-256

Microsomes, 206-208 Miniaturization principle, 150-152 Mitchell theory (chemiosmotic hypothesis), 2 Mitochondrial respiration, 253-255 Mode-locked CW dye laser, 110 Molecular proton detector, reaction of proton, 43-66 alkalinization pulse, proton emitter, 62-63

direct proton exchange, 57-62 ground-state anion of proton emitter, 4546

limitations and inaccuracies, 63-66 macroscopic parameters, 65-66 reactrants concentration, 63-65 pH indicators, 46-57

SUBJECT INDEX

Molecular tunneling process, 124-125 Monolayer vesicle, 211-2 12 Multilamebr vesicle, 210-2 1 1 preparation of, 21 1 Myoglobin. 120, 122 NAD-linked dehydrogenases, 252-253 2-Naphthol-3,5-disulfonate, fluorescence emission spectra, 33 National Bureau of Standards, 286,287 Negatively charged vesicles, 209-2 10 Nitrogen laser, 110 Noise thermometers, 298 Nonexchangeable markers, lipid transfer activity, 203-204 Nuclear magnetic resonance (NMR), 216 Optical pyrometers, 280 Oxidative phosphorylation, 253-255 Oxygen consumption, measurement of,

227-267

applications, HbOn method, 250-263 calculation of experimental data,

244-247

comparative measurements, 244-246 at different pigment concentrations,

246

polarographic estimation, 246-247 hydrogen peroxide-generating oxidases,

249-250

instrumentation, 236-242 introduction, 228-230 purification, characterization, and storage of donors, 233-236 chromatographic purification of HbOp, 234-235 determination of HbOn concentration, 235-236 hemolysate preparation, 234 simultaneous measurement of several parameters, 247-249 technical procedure: repetitive measurements, 243-244 single measurements, 242-243 Oxyhemoglobin (HbOp) method: applications, 250-263 continuous measurement, in vitrocultured embryos, 259-260 cytochrome oxidase activity in human liver homogenates, 250-251

349

determination of substrate concentrations, 262-263 isolated cells, 256-258 microsomal oxidases, 255-256 microspectrophotometric assay of oxygen consumption, 258-259 mitochondrial respiration and oxidative phosphorylation,

253-255

NAD-linked dehydrogenases,

252-253

rapid functional transitions of respiratory systems, 261 rat liver phenylalanine hydroxylase activity, 251-252 consumption measurements, 242-244 groups, 230-233 major characteristic, 230 Peripheral blood cells, 180-182 Philon of Alexandria, 272 pH indicators, reaction of proton with,

46-57

dynamics in absence of direct proton exchange, 47-5 1 effect of initial conditions on macroscopic parameters, 5 1-57 pH jump: effect of buffer on proton cycle, 90-98 introduction, 2-3 methodology and instrumentation, 3-6 protonation of high-molecular-weight structure, 66-84 proton dissociation, 6-22 proton transfer, surface of macromolecular structure, 84-90 reactions, 22-66 with its emitter, 22-43 with molecular proton detector,

43-66

Phosphatidylcholinevesicles, 210 Phosphorescence, monitoring triplet state population by, 130 Planck's radiation law, 280, 282 Plasma lipoproteins, 212-2 I3 Platelets of man and rat, 177 Platinum resistance thermometers, (PRTs), 286, 287 Polarographic estimation, oxygen consumption, 246-247

350

SUBJECT INDEX

Postprotonation reaction, 73-84 adsorbed Bromo Cresol Green, 75-78 classification, 78-84 Proteins. lipid transfer activity, 199-226 assaying crude extracts, 2 17-2 18 effects of composition of assay mixture, 218-223 general considerations, 202-203 introduction, 200-202 separation assays, 203-206 specific assays, 206-214 spectroscopic assays, 214-217 Protonation: ground-state compounds, 4-6 excitation pulse, 5 geometry of excitation and probing beams, 6 measuring equipment, 5 - 6 monitoring light, 5 high-molecular-weight structure, 66-84 effect of charge on rate, 69-73 postprotonation reaction, 73-84 uncharged target adsorbed on uncharged carrier, 6 8 4 9 Proton cycle, effect of buffer on dynamics, 90-98 three-component system (proton emitter, detector, and buffer), 94-98 buffer protonation, 96-98 initial conditions, 96 simulative solution, 94-96 two-component systems (buffer and proton emitter), 91-93 Proton dissociation, 6-22 in classical chemistry, 6-7 in concentrated salt solution, 15-21 effect of pK on rate, 10 effect of solvent on rate, 10-15 excited molecules, 4 poorly hydrating site, 33-38 rate determination, 7-10 scheme I, 8-10 Proton emitter, 22-43 alkalinization pulse, 62-63 dissociation in poorly hydrating site, 33-38 ground-state anion, 45-46 reactions in small, open, hydrating microcavity, 24-33

steady-state fluorescence, 24-26 time-resolved fluorescence, 27-33 Proton pumping, intrinsic photoactivation, 136 Proton reactions, 2 2 4 6 detection with its emitter, 22-43 discussion and conclusions, 3 8 4 3 dissociation in poorly hydrating site, 33-38 small, open, hydrating microcavity, 24-33 molecular proton detector, 43-66 alkalinization pulse, proton emitter, 62-63 direct proton exchange, 57-62 ground-state anion of proton emitter, 45-46 limitations and inaccuracies, 63-66 pH indicators, 46-57 Proton transfer, surface of macromolecular structure, 84-90 Pt-lO%Rh/Ptthermocouple, 286 Pulsed lasers, 107-1 1 1 flashlamp-pumped organic dye, 109-1 10 mode-locked CW dye, 110 nitrogen, 110 Q-switched Nd : YAG, 107-109 rare-gas excimer, 109 shielding, 110-1 11 Purified lipid transfer proteins, 201 Q-switched Nd : YAG laser, 107-109 Quickfit column, 161 Radiation thermometers, 296-298 Rankine, W.J.M., 276 Rankine (or absolute Fahrenheit) scale, 276 Rare-gas excimer laser, 109 Rate constant, proton dissociation, 6-22 in classical chemistry, 6-7 in concentrated salt solution, 15-21 determination, 7-15 effect of pK, 10 effect of solvent, 10-15 Rat liver microsomes, oxygen consumption and NADPH oxidation, 257

SUBJECT INDEX

Rat liver phenylalanine hydroxylase activity, 251-252 Rkaumur, R e d , 276 Rkaumur scale, 276 Reference points, temperature, 287 Regnault, H.V., 277 Renaldini, Carlo, 275 Resistance measurements, thermometer,

302

Resistance temperature detectors (RTDs),

301-304

Resistance thermometers, 295 Resonance thermometers, 298-30 1 Respiratory systems, rapid functional transitions, 26 1 Rey, Jean, 273-274 Riiemer, Ole, 275-276 RS232 serial interface, 307 Rumford, Count, 278 Seebeck, T.J., 279 Sensor measurement, electronic methods,

301-305

Separation assays, 203-2 14 cross-contamination of isolated particles,

204-206

nonexchangeable markers, 203-204 Separation chamber device, 162-1 74 compact, 166-174 diagram, 167 membranes, 167-169 preparing gradient, 169-174 with movable electrodes, 162-166 example of separation, 165-166 Shielding, pulsed lasers, 110-1 1 1 Side-on photomultiplier tube, 112 Siemens, Sir William, 279 Signal acquisition techniques, laser photolysis, 1 1 1-1 16 amplification, 113 side-on photomultiplier tube, 112 transient recorders: high-speed, 113-1 15 with signal averaging and logarithmic time base, 116 Silicon integrated-circuit temperature sensor, 304 Small-size electrofocusing chamber,

160-1 61

351

Small unilammellar vesicle, 210-2 1 1 preparation of, 21 1 Solid-stem liquid-in-glass thermometers,

293

Spectrophotometrically calibrated substrates, oxygen consumption measurements, 244-246 Spectrophotometric oxyhemoglobin method, 227-267 calculation of experimental data,

244-247

comparative measurements, 244-246 at different pigment concentrations,

246

polarographic estimation, 246-247 hydrogen peroxide-generating oxidases,

249-250

instrumentation, 236-242 introduction, 228-230 purification, characterization, and storage of donors, 233-236 chromatographic purification of H b 9 , 234-235 determination of HbOp concentration, 235-236 hemolysate preparation, 234 simultaneous measurement of several parameters, 247-249 technical procedure, 242-244 repetitive measurements, 243-244 single measurements, 242-243 see d o HbOn method Spectroscopic assays, 214-2 17 electron spin resonance, 214 fluorescence, 214-2 1 6 nuclear magnetic resonance, 216 Spleen cells, 178-1 80 Stable-flow-free boundary electrophoresis (STAFLO), 158 Standard platinum resistance thermometers (SPRTs), 290-291 Standard temperature reference system,

322-327

Steady-state fluorescence, 24-26 Temperature measurement, 269-33 1 conclusions and forecasts, 327 digital recording methods and devices,

306-307

352

SUBJECT INDEX

Temperature measurement (Continued) electronic methods, 301-305 resistance measurements, 302 voltage measurements, 302-305 introduction, 270-271 practical standards, 286-292 recent applications, 308-327 fast stopped-flow thermal measurements, 30 1-3 1 1 heat conduction and response time corrections, 311-319 thermal titration and immobilized enzyme reaction detection, 319-322 scales, 281-285 thermometry, 271-281 types of thermometers, 292-30 1 bimetallic-strip, 294-295 dial, 294 diode, 300-301 gas, 295 liquidcrystal, 301 liquid-in-glass, 293-294 noise, 298 radiation, 296-298 resistance, 295 resonance, 298-300 thermoelectric, 295-296 Thermal detectors, heat conduction and response time corrections, 3 11-3 19 Thermal titration, immobilized enzyme reaction detection and, 319-322 Thermoelectric thermometry, 295-296 Thermometry, 271-281 Thermopiles, 279 Thymocytes, 183 Time-resolved fluorescence, 27-33 Tonsillar cells, 182

Transanalyzer, 174-1 75 Transient recorder: high speed, 113-1 16 signal averaging and logarithmic time base, 116 Transition measurements, lipid phase, 216-217 Triplet-state probes. 125-1 32 anisotropy decay, 127-128 importance of long lifetimes, 126-127 instrumentation, 130-132 population, monitoring, 128-130 absorption, 128-130 phosphorescence, 130 Tulp chamber, 162-174 compact device, 166174 with movable electrodes, 162-166 Tunneling processes, ligand-hemeprotein recombination work, 124-125 Unilamellar vesicle, 2 10 incubations, 2 11 Van Oss and Bronson device, 161-162 Vesicles, 208-2 13 antigen-sensitized, 208 monolayer, 211-2 12 negatively charged, 209-210 sensitized to leains, 208-209 unilamellar, 210-211 Voltage measurements, thermometer, 302-305 VOM (volt-ohm-miliammeter), 302 von Helmholtz, Hermann, 278 Yttrium-aluminum-garnet (YAG) laser, 4

Methods of Biochemical Analysis, Volume 30 Edited by David Glick Copyright © 1984 John Wiley & Sons, Inc.

Cumulative Author Index, Volumes 1-30 and Supplemental Volume Acherman, C . J., see Engb, R. W . Alber&on, Per-&, Interaction Between Biomolecules Studied by Phase Partition .............................................. Alb-on, Per-&, Partition Methods for Fractionation of Cell Particles and Macromolecules ................................. Albertsson, P., Anderrcon, B., Larsson, C., and Akerlund, H., Phase Partition-A Method for Purification and Analysis of Cell Organelles and Membrane Vesicles ............................ Alcoch, Nancy W., and MacIntyre, lain, Methods for Estimating Magnesium in Biological Materials ............................ A&, E l k , and W m k , Warren E. C., Enzymatic Methods Used for Diagnosis ................................................ Ames, S t a n k R.,see Embree, Nonis D. Andcrsen, C . A., An Introduction to the Electron Probe Microanalyzer and Its Application to Biochemistry .............. Anderson, N. G.,Preparative Zonal Centrifugation ................. Andrews, P., Estimation of Molecular Size and Molecular Weights of Biological Compounds by Gel Filtration ........................ A d , Mzra, see Grossowicz, Nathan Asboe-Hanren, Gustau, see Blumenkrantz, Nelly Aspen, Anita J., and M&&, Alton, Determination of Transaminase . . Augwtimon, Kh-Bertil, Assay Methods for Cholinesterases ........ Determination of Cholinesterases ............................. Austin, Rob& H., see Chan, Shirk S. Awdch, 2. L., see McLaren, D.S. Baker, S . A., Bourne, E. J., and Whffen, D. H., Use of Infrared Analysis in the Determination of Carbohydrate Structure ........ Balk, M . Earl, Determination of Glutamic and Aspartic Acids and Their Amides ........................................... Barchns, Jack D., see Faull, Kym F. Barks&&, A. D., and Rosenberg, A , , Acquisition and Interpretation of Hydrogen Exchange Data from Peptides, Polymers, and Proteins Bhrzu, Octuvian, Measurement of Oxygen Consumption by the Spectrophotometric Oxyhemoglobin Method ................... B a d , W. S., and Greenway, R. M., Chemical Determination of Estrogens in Human Urine ................................... Bayer, Edward A., and Wilcheh, Meir, The Use of the Avidin-Biotin Complex as a Tool in Molecular Biology .......................

353

VOL.

PAGE

29

1

10

229

28

115

14

1

13

265

15 15

147 27 1

18

1

6 5

SUPP.

131 1 217

3

213

20

103

28

1

30

227

5

337

26

1

354 CUMULATIVE AUTHOR INDEX, VOLUMES 1-30 AND SUPPLEMENT Bell, H e h H., see J q s , Lou& B . Benesch, Reinhold, and Benesch, Ruth E., Determination o f C H Groups in Proteins ........................................... Benesch, Ruth E., see Benesch, Reinhold Benson, E. M., see Storuich, C. A. B e n t b , J . A., Analysis of Plant Hormones ........................ Benzinger, T. H., see Kitzinger, Charlottc Berg, Marie H., see SchwaHz, Samvel Berger, Robert L., Ckm,Thomac R , Sr., Hardm, V u t h A. and Mangum, B.W., Historical Development and Newer Means of Temperature Measurement in Biochemistry.. ................... Bergmonn, Felix, and Dikrtkn, Shabtu?, New Methods for Purification and Separation of Purines .................................... Benon, S o h A,, see Yalow, Rosalyn S. BhaMi, Tarig, see Clump,J. R . B ~ c ~E .I M., , Determination of Carotene ........................ Binnmi3, W . T., Determination of Iodine in Biological Material ..... Bishop. C. T., Separation of Carbohydrate Derivatives by Gas-Liquid Partition Chromatography .................................... Btclckburn, S.,T h e Determination of Amino Acids by High-Voltage Paper Electrophoresis ........................................ Blmu, D. M.,see Holmes, K. C. Blumenkrantz, Nelly, and Asboe-Hansen, Gustav, Methods for Analysis of Connective-Tissue Macromolecules by Determination of Certain Constituents ......................................... Bodanshy, Oscar, see Schwartz, Morton K. Bosscnmain; Irene, see Schwartz, Samuel Bosshard, H a m RudoK Mapping of Contact Areas in Protein-Nucleic Acid and Protein-Protein Complexes by Different Chemical Modification ....................................... B o u h , Alan A., T h e Automated Analysis of Absorbent and Fluorescent Substances Separated on Paper Strips .............. Boulton, A. A., see Majn; J. R. Bourne, E. J., see Baker, S.A. Brantmarh, B. L., see L i d , N . 0. Brauser, BoUO, see Sics, Helmut Bray, H. G.,and Thmpe, W. V., Analysis of Phenolic Compounds of Interest in Metabolism ....................................... Brierby, G. P.,see Lessler, M. A. Broahen, R., and Jacobsen, J., Separation and Determination of Bile Pigments ............................................... Brodie, B m r d B., see Udenfrhd, Sidney B r o o k , Gory, Newer Development in the Determination of Cyclic AMP and Other Cyclic Nucleotides, Adenylate Cyclase, and Phosphodiesterase ....................................... B u d s , Carl A., Tiffany, Thomcrr O., and Scott, Charles D., The Use of a Centrifugal Fast Analyzer for Biochemical and Immunological Analyses ...................................... Bush, I. E., Advances in Direct Scanning of Paper Chromatograms for Quantitative Estimations .................................. Bush, 1. E., Applications of the R MTreatment in Chromatographic Analysis .................................... Erratum ....................................................

10

43

9

75

30

269

6

79

4

22

1 25 1

10

1

13

1

24

39

25

273

16

327

1

27

17

31

22

95

23

189

11

149

IS

357 497

14

CUMULATIVE AUTHOR INDEX, VOLUMES 1-30 AND SUPPLEMENT 355 CaldweU, Karin D., see Giddings, J . Calvin Campbell, I. D., and Dobson, C. M., The Application of High Resolution Nuclear Magnetic Resonance to Biological Systems ... Carstensen, H., Analysis of Adrenal Steroid in Blood by Countercurrent Distribution .................................. Caster, W. O., A Critical Evaluation of the Gas Chromatographic Technique for Identification and Determination of Fatty Acid Esters, with Particular Reference to the Use of Analog and Digital Computer Methods .......................................... Chambers, Robin E., see Clamp J . R. Chan, Shirlq, S., and Amtin, Robert H., Laser Photolysis in Biochemistry .............................................. Chance, Britton, see Maehly, A . C. C h e , Aurin M., The Measurement of Luciferin and Luciferase .... Chinard, Francis P., and H e h , Leslie, Determination of Sulfhydryl Groups in Certain Biological Substrates .............. Christen P., and Gehring, H., Detection of Ligand-Induced and Syncatalytic Conformational Changes of Enzymes by Differential Chemical Modification ....................................... Clamp, John R., and Bhatti, T., and Chambers, R. E., The Determination of Carbohydrate in Biological Materials by GasLiquid Chromatography ...................................... Clark, S t a n k J., see Wotiz,Herbert H . Cleaary, E. G., see Jackson, D. S. Clem, Thomm R., Sr., see Berger, Robert L. Code, Charla F., and McIntyre, Floyd C., Quantitative Determination of Histamine ................................................ Cohn, Waldo E., see Volhin,Elliot Cotlove, Ernest, Determination of Chloride in Biological Materials ... Craig, Lyman C., and King, Te Piao, Dialysis ....................... see also King, Te Pi00 Crane, F. L., and DiuLy, R. A., Determination of Coenzyme Q (Ubiquinone) ................................................ Creech, B. G., see Homing, E . C. Creueling, C. R. and Dab, J . W., Assay of Enzymes of Catechol Amines ............................................ CUT, A. S., The Analysis of Basic Nitrogenous Compounds of Toxicological Importance .................................... Daly, J . W., see Creveling, C. R . Daviakan, Harold M., see Fkhman, William H , Davis, Neil C., and Smith, Emil L., Assay of Proteolytic Enzymes ..... Davic, R.J., see Stohtad, E. L. R. Davis, Robert P.,The Measurement of Carbonic Anhydrase Activity Dean, H. G., see Whitehead,J . K. Degn, H.,L u d g a a r d , J . S., Peterson, L. C., and Omuki, A., Polarographic Measurement of Steady State Kinetics of Oxygen Uptake by Biochemical Samples ............................... Dihstein, Shabtay, see Bergmann, Felix DiuCy, R. A., see Crane, F. L. Dinnnore, Howard, see Schwartz, Samuel Dkche, Za.chak, New Color Reactions for the Determination of Sugars in Polysaccharides ..................................... Dodgmn, K. S., and Spencer, B., Assay of Sulfatases ................

25

1

9

127

17

135

30

105

8

61

1

1

28

151

19

229

3

49

12

277 175

11

279

SUPP.

153

7

39

2

215

11

307

26

47

2

313 211

LO

4

356 CUMULATIVE AUTHOR IXDEX, VOLUMES 1-30 AND SUPPLEMENT Use o f Subzero Temperatures in Biochemistry: Slow Reactions .............................................. Dyer,John R., Use o f Periodate Oxidations in Biochemical Analysis Edwarh, M . A., see Sloruick, C. A. Elving, P.J., O'Rklly, J . E., and Schmakel, C. O., Polarography and Voltammetry of Nudeosides and Nucleotides and Their Parent Bases as an Analytical and Investigative Tool ................... Embree, Nmrir D., Ames, Stanley R., Lehman. Robert W., and. Harris, Philip L., Determination of Vitamin A ......................... Engel, L& L., The Assay o f Urinary Neutral 17-Ketosteroids ..... Engel, R. W., Salmon, W . D., and Ackennan, C.J., Chemical Estimation of Choline ........................................ Engeiman, Karl, see Lovenberg, S. W a k E m & , Lars, see Lindberg, OIa, Everse, Johannes, Ginsburgh, Char& L., and Kaplan, Nathan 0.. Immobilized Enzymes in Biochemical Analysis .................. Faull, Kym F., and B a r c h , Jack D., Negative-Ion Mass Spectrometry, Fused-Silica Capillary Gas Chromatography of Neurotransmitters and Related Compounds ..................................... Felber, J . P., Radioimmunoassay of Polypeptide Hormones and Enzymes ................................................ Fink, Freahick S.,see Kersey, Roger C. Fisher, Susan R., see Giddings, J . Calvin Ftihman, William H., Determination of f3-Glucuronidases ........... Fishman, Willram H., and Davidson, Harold M., Determination o f Serum Acid Phosphatases .................................... Fleck, A.. see Munro, H. N. FwsCn, Sture, and Lindman, B j h , Ion Bonding in Biological Systems Measured by Nuclear Magnetic Resonance Spectroscopy ........ Fraenhel-Conrat, H., Harris,J . Inran, and 4 , A. L., Recent Developments in Techniques for Terminal and Sequence Studies in Peptides and Proteins ...................................... Friedman, Sydnty M., Measurement of Sodium and Potassium by Glass Electrodes ............................................. Frisell, Wilhelm R., and Mackenzi.e, Co.ww G., Determination o f Formaldehyde and Serine in Biological Systems ................ Gale, Ernest F., Determination of Amino Acids by Use of Bacterial Amino Acid Decarboxylases .................................. Gar&& Sven, Determination of Hexosamines ...................... GasheU, Simon J., Analysis o f Steroids by Mass Spectroscopy ........ Giddings,J . Calvin, Myers, Marcus N.,Caldwell, Karin D., and Fisher, Susan R., Analysis of Biological Macromolecules and Particles by Field-Flow Fractionation ...................................... Gofman,John W., see Lalla, Oliver F. de Goklbmg, Nelson D., and OToole, Ann G., Analysis of Cyclic 3',5'-Adenosine Monophosphate and Cyclic 3',5'-Guanosine Monophosphate .............................. Grabar, P k e , Immunoelectrophoretic Analysis ................... Greenway, R. M., see Bauld, W. S. Gross, D.,see Whalky, H.C. S. & Grossman, Shlomo, Oestreicher, G u i h o , and Singer, T h P., Determination of the Activity of Phospholipases A, C, and D ....

Douzou, Pierre, The

22 3

40 1

21

287

4 1

479

1

265

25

135

29

325

22

1

15

77

4

257

27

289

2

359

10

71

6

63

4

6 29

285 289 385

26

79

20 7

1

22

177

111

43

1

CUMULATIVE AUTHOR INDEX, VOLUMES 1-30 AND SUPPLEMENT Grossman, S h k , and Zakut, R i m , Determination of the Activity of Lipoxygenase (Lipoxidase) .................................... Grossowicz, Nathan, and Ariel, Mira, Methods for Determination of Lysozyme Activity ........................................... Guhnun, Menachem, The pH Jump: Probing of Macromolecules and Solutions by a Laser-Induced, Ultrashort Proton Pulse-Theory and Application in Biochemistry .............................. Haegeie, Klaw D., see Thkrot,Jean-Paul G. Haglund, Herman, Isoelectric Focusing in pH Gradien-A Technique for Fractionation and Characterization of Ampholytes Haines, William J., and Karnemaat, John N., Chromatographic Separation of the Steroids of the Adrenal Gland ................ Hanessianr, Stephen, Mass Spectrometry in the Determination of Structure of Certain Natural Products Containing Sugars ....... Harden, Victoria A., see Berger, Robert L. Harris, J . Ieuan, see Fraenhel-Cunrat, H . Harris, Philip L., see Embree, NonG D. Heimegh, K . P . M., Recent Advances in the Separation and Analysis of Diazo-Positive Bile Pigments ............................... Helhman, Leslie, see Chinard, Francis P. Hermans, Jan, Jr., Methods for the Study of Reversible Denaturation of Proteins and Interpretation of Data ......................... Hater, Charles S., see Wikheh, Meir Hiromi, Keitaro, Recent Developments in the Stopped-Flow Method for the Study of Fast Reactions ............................... Hirschbein, L., and G u i l h , N., Characterization, Assay, and Use of Isolated Bacterial Nucleoids .................................. Hjertdn, S., see Porah J. Hjertdn, Stellan, Free Zone Electrophoresis. Theory, Equipment and Applications ............................................ Hjertdn, Stellan, Hydrophobic Interaction Chromatography of Proteins, Nucleic Acids, Viruses, and Cells on Noncharged Amphiphilic Gels ................................ Hoff-Jmgmen, E., Microbiological Assay of Vitamin BIZ ........... Holnon, Ralph T., Measurement of Lipoxidase Activity ............ Measurement of Polyunsaturated Acids ........................ Holmes, K. C., and Blow, D. M . , The Use of X-ray Diffraction in the Study of Protein and Nucleic Acid Structure ................... H m h , Jiri, Polarography of Proteins, Analytical Principles and Applications in Biological and Clinical Chemistry ............... H m i n g , E. C., Vandm Heuvel, W .J . A., and Creech, B. G., Separation and Determination of Steroids by Gas Chromatography ......... Hurvath, C., High-Performance Ion-Exchange Chromatography with Narrow-Bore Columns: Rapid Analysis of Nucleic Acid Constituents at the Subnanomole Level ........................ Huugh, Leslie, Analysis of Mixtures of Sugars by Paper and Cellulose Column Chromatography .................................... Hughes, Graham J . and Wilson, Kenneth, J . , High-Performance Liquid Chromatography: Analytic and Preparative Applications in Protein Structure Determination .............................. Hughes, Thomas R., and Klotz, Irving M., Analysis of Metal-Protein Complexes .....................................

357

25

303

29

435

30

1

19

1

1

171

19

105

22

205

13

81

26

137

28

297

18

55

27 1 2 4

89 81 113 99

13

113

19

435

11

69

21

79

1

205

29

59

3

265

358

CUMULATIVE AUTHOR INDEX, VOLUMES 1-30 AND SUPPLEMENT

Humphrey,J . H., Lung, D. A., and P m y , W. L. M., Biological Standards in Biochemical Analysis ............................. Hutner, S.H., see Stokslad, E. L. R. Jackson, D. S.,and Cleaty, E. G., The Determination o f Collagen and Elastin .................................................. Jocobs, S., The Determination of Nitrogen in Biological Materials ... Jacobs, S., Determination o f Amino Acids by Ion Exchange Chromatography ............................................ Jmobs, Stanley, Ultrafilter Membranes in Biochemistry ............. Jacobsen, C. F.,Ltonk,J., Linderstrm-Lang, K., and Ottesen, M., The pH-Stat and Its Use in Biochemistry ........................ Jacobsen, J.. see B r h s e n , R. James, A. T.,Qualitative and Quantitative Determination of the Fatty Acids by Gas-Liquid Chromatography ......................... Jams, D o u g h R., and Lumty, Rufiu W., Recent Developments in Control of pH and Similar Variables .......................... James, Gordon T., Peptide Mapping of Proteins .................... Jques, Louis B., Determination of Heparin and Related Sulfated Mucopolysaccharides ................................ Japes. Louis B., and Bell, HeZen J., Determination o f Heparin ...... Jar&@, C., and Jar&t&y, O., Biochemical Applications of Magnetic Resonance ....................................... Jardetzhy, O.,see Jarhtzhy, C. Jenden, Donald J., Measurement o f Choline Esters ................. Johnson, George, Gel Sieving Electrophoresis: A Description of Procedures and Analysis of Errors ............................ Joliconcr, Cannel, Thermodynamic Flow Methods in Biochemistry: Calorimetry, Densimetry, and Dilatometry ..................... Jones, Richard T., Automatic Peptide Chromatography ............. JoscfSFon, L. I., and Logcrstedt, S.,Characteristics o f Ribonuclease and Determination of Its Activity .................................. Jukes, ThmMs H., Assay of Compounds with Folk Acid Activity .... Kabara, J . J., Determination and Localization of Cholesterol ....... Kalckar, Hennun M., see Plesnn; P a d Kapcllcr-A&, R., Determination o f Amine Oxidases .............. Kaplan, A., The Determination of Urea, Ammonia, and Urease .... Kamemaat, John N.,see Haines, William J . Kearney, Edna B., see Singer, T h P. Keenan, Robert G., see Saltunan, Bernard E. Kmey, Roger C., and Fink, Freo!mih C., Microbiological Assay of Antibiotics ................................................ King, Te Piao, and Craig, Lyman C., Countercurrent Distribution .... see also Craig, Lyman C. Kitzinger, Charlotte, and Benzinger, T. H., Principle and Method of Heatburst Microcalorimetry and the Determination of Free Energy, Enthalpy, and Entropy Changes ....................... Klotz, INing M., see Hughes, Thumas R. Kobayashi, Yutoka, and Maudrley, David V., Practical Aspects of Liquid-Scintillation Counting ................................. Kolin, Alexandn; Rapid Electrophoresis in Density Gradients Combined with pH and/or Conductivity Gradients ..............

5

65

15 13

25 241

14 22

177 307

4

171

8

1

29 26

137 165

24 7

203 253

9

235

SUPP.

183

29

25

27

18

171 205

9 2 10

39 121 263

SUPP, 17

35 311

1 10

53 201

8

309

17

55

6

259

CUMULATIVE AUTHOR INDEX, VOLUMES 1-30 AND SUPPLEMENT Kopin, Irwin J., Estimation of Magnitudes of Alternative Metabolic Pathways .......................................... K m , Edward D., The Assay of Lipoprotein Lipase in vivo and in vitro .................................................. KuAris, A., New Developments in Determination of Bile Acids and Steroids by Gas Chromatography ............................. Kunkel, Henry G., Zone Electrophoresis ........................... Kurnich, N. B., Assay of Deoxyribonuclease Activity ............... Lugerstedt, S., see Josefsson, L. I. Lulh, Oliver F. de, and Gofman,John W., Ultracentrifugal Analysis of Serum Lipoproteins .......................................... Laursen, Ruhurd A., and M a c U t , Werner, Solid-Phase Methods in Protein Sequence Analysis .................................... Lazarow, Amold, see Patterson, J . W. Leddicotte, George W., Activation Analysis of the Biological Trace Elements .............................................. Lehman, Robert W., Determination of Vitamin E ................... see also Embree, N I ~ YD.I ~ Lekr, Luis F.,see Ponh, Horacio G. Leon&,J., see Jacobsen, C. F. Le Pecq, Jean-Bernard, Use of Ethidium Bromide for Separation and Determination of Nucleic Acids of Various Conformational Forms and Measurement of Their Associated Enzymes ................ Lerner, Aaron B., and Wright, M . Ruth, in vitro Frog Skin Assay for Agents That Darken and Lighten Melanocytes ................. Lessler, M. A., Adaptation of Polarographic Oxygen Sensors for Biochemical Assays .......................................... Lessler, M. A,, and B w l e y , G. P.,Oxygen Electrode Measurements in Biochemical Analysis ...................................... L q ,A. L., see FraetUtel-Conrat, H. L q , H i h B., see Webb,Junius, M . Lindberg, O h , and E m t n ; Lars, Determination of Organic Phosphorus Compounds by Phosphate Analysis ................ Lindcrstrom-Lung, K., see Jacobsen, C. F. L i d , N.O., and Brantmrh, B. L., Preparation and Analysis of Basic Proteins ............................................. Lindman, B j h , see Font%, Sture Lissikhy, Serge, see Roche, Jean Long, D. A.. see Humphrey,J . H. Lovenberg, S. Walter, and Engelman, Karl, Serotonin: The Assay of Hydroxyindole Compounds and Their Biosynthetic Enzymes .................................................... Loveridge, B. A,, and Smales, A. A., Activation Analysis and Its Application in Biochemistry .................................. Lumry, Rujius, see Yapel,AnthonyF.,Jr. Lumry, Rujiw W., see Jams, D o u g h R. Lundquict, Frank, The Determination of Ethyl Alcohol in Blood and Tissues ................................................. Lunhgaard. J. S.,see Degn, H. McCarthy, W.J.. see Winefordner,J . D. Mach&& Werner, see Lawsen, Richard A.

359

11

247

7

145

14 1 9

325 141 1

1

459

26

201

19 2

345 153

20

41

8

295

28

175

17

1

3

1

14

79

SUPP.

1

5

225

7

217

360 CUMULATIVE AUTHOR INDEX, VOLUMES 1-30 AND SUPPLEMENT Mclntire, Ftoyd C., see Code, Charks F. M a d w e , lain, see Alcock, Nancy W . Machmie, Cmmo G., see FriseU, WilhelmR. MacKenzk, S.L., Recent Development in Amino Acid Analysis by Gas-Liquid Chromatography .................................. McKibbin,John M.,The Determinarion of Inositol, Ethanolamine, and Serine in Lipides ........................................ Mchren, D. S.,Read, W . W . C.,A d h , 2.L.,and Tchalian, M., Microdetermination of Vitamin A and Carotenoids in Blood and Tissue .................................................. M c P h o n , Alexander, The Growth and Preliminary Investigation of Protein and Nuclei Acid Crystals for X-Ray Diffraction Analysis Maehly, A. C., and Chance, Britkm, T h e Assay of Catalases and Peroxidases ............................................. Majer, J . R., and BouUa, A. A., Integrated Ion-Current (IIC) Technique of Quantitative Mass Spectrometric Analysis: Chemical and Biological Application .................................... Malmtrimr, Bo C., Determination of Zinc in Biological Materials .... Mangold, Helmut K.. Schmid, H a r d H. 0..and Stahl, Egon, ThinLayer Chromatography (TLC) ................................ Mangum, B.W., see Berger, Rob& L. Margmhcs, Marvin, and V&e, Brrt L., Flame Photometry and Spectrometry: Principles and Applications ..................... Maudsb, David V., see KobaFhi, Y & Mcirter, A h , see As@, Anita J . Muhel, Raymond, see Roche, Jean Micklsen, O w , and Yamamoto,Richard S.. Methods for the Determination of Thiamine ................................... Miller, H& K., Microbiological Assay of Nucleic Acids and Their Derivatives ............................................ Mdner, Kelsty. see Ribi, Edgar Miwa, I., see Oh&, J . Montgomety,Rcx, see Smith, Fred Mullcr, Otto H., Polarographic Analysis of Proteins, Amino Acids, and Other Compounds by Means of the Brditka Reaction ....... Munro, H. N., and Fleck, A., The Determination of Nucleic Acids ... M9en, Marcus N., see G a n g s , J . Calvin NatCLron, Samuel, and W w m d ,William R., Determination of Elements by X-Ray Emission Spectrometry ..................... Neaq, Michael P., see Sktz, W . RudoY Neish, WilliamJ. P., a-Keto Acid Determinations .................. NoveU:, G. David, Methods for Determination of Coenzyme A ...... Oberlem, D d , The Determination of Phytate and Inositol Phosphates .......................................... Oestreicher, GuiUmno, see Grossman, Shlumo O h f a , J., and Miwa, I., Newer Developments in Enzymic Determination of D-Glucose and Its Anomers .................. O&m, K. G., Radiometric Methods for Enzyme Assay ............ Olson,0. E., Palmer, I. S.,and Whitehead,E. I., Determination of Selenium in Biological Materials .............................. O’Rcillg,J . E., see Elving, P. J . onnicrki, A., see Degn, H .

27

1

7

111

15

1

23

249

1

357

21 3

467 327

12

393

3

353

6

191

6

31

11 14

329 113

12

1

5

2

107 189

20

87

21 21

155

21

39

191

CUMULATIVE AUTHOR INDEX, VOLUMES 1-30 AND SUPPLEMENT 361 OToole, Ann G., see Goldberg, Nelson D.

Ottesen, M., see Jacobsm, C. F. Ottesen, Martin, Methods for Measurement of Hydrogen Isotope

Exchange in Globular Proteins ................................ Pdmer, I. S.,see Olson,0. E . Parker, Reno, see Ribi, Edgar Patterson, J. W., and Lazarow, Arnold, Determination of Glutathione Peny, W. L . M., see Humphrey, J . H . Persky, Harold, Chemical Determination of Adrenaline and Noradrenaline in Body Fluids and Tissues ..................... Peterson, L. C., see D e e , H . P l e s w , Paul, and Kalckar, Hermun M., Enzymic Micro Determinations of Uric Acid, Hypoxanthine, Xanthine, Adenine, and Xanthopterine by Ultraviolet Spectrophotometry ........... Po&, Horacio G., and Lebir, Luis F., Measurement of UDPEnzyme Systems ............................................. Porath, J., and Hjerth, S.,Some Recent Developments in Column Electrophoresis in Granular Media ............................ Porter, Curt C., see Silber, Robert H . Pod&, M . D., Gel Electrophoresis in Buffers Containing Urea ..... Pourfarznneh, M., Kamel, R. S.,Landon, J., and Dawes, C. C., Use of Magnetizable Particles in Solid Phase Immunoassay ............. Ranflaub, Jurg, Applications of Metal Buffers and Metal Indicators in Biochemistry .............................................. Radin, Nonnan S., Glycolipide Determination ..................... Ramwell, P. W., see Shaw, Jane E. Read, W ,W. C., see McLaren, D. S. Ribi, Edgar, Parker, Reno, and Milner, Kekey, Microparticulate Gel Chromatography Accelerated by Centrifugal Force and Pressure Robins, Eli, The Measurement of Phenylalanine and Tyrosine in Blood .................................................... Robins, S.P., Analysis of the Crosslinking Components in Collagen and Elastin .................................................. R o c k ,Jean, Lissitzhy, Serge, and Michel, Raymond, Chromatographic Analysis of Radioactive Iodine Compounds from the Thyroid Gland and Body Fluids ....................................... Rock, Jean, Mickl, Raymond, and Licsitrky, Serge, Analysis of Natural Radioactive Iodine Compounds by Chromatographic and Electrophoretic Methods ..................................... Roe, Joseph H., Chemical Determinations of Ascorbic, Dehydroascorbic, and Diketogulonic Acids ..................... Rosmhrantz, Harris, Analysis of Steroids by Infrared Spectrometry .. Infrared Analysis of Vitamins, Hormones, and Coenzymes ...... Roth, Marc, Fluorimetric Assay of Enzymes ....................... Salmon, W. D., see Engel, R. W. Saltrman, Bemard E., and Keenan, Robert G., Microdetermination of Cobalt in Biological Materials ................................. Schayer, Richard W., Determination of Histidine Decarboxylase Activity ....................................... Determination of Histidine Decarboxylase ..................... Schmakel, C. O., see Eluing, P. J. Schmid, Harald H . O.,see Mangold, Helmut K.

20

135

2

259

2

57

3

97

10

107

9

193

14

455

28

267

3 6

30 1 163

22

355

17

287

28

329

1

243

12

143

1 2 5 17

115 1 407 189

5

181

16

273 99

SUPP.

362 CUMULATIVE AUTHOR INDEX, VOLUMES 1-30 AND SUPPLEMENT Schuberi, Jack, Measurement of Complex Ion Stability by the Use of Ion Exchange Resins ......................................... Schuberth,Jan, see Sosbo, S. Bo Schultm, Ham-Rolf, Field Desorption Mass Spectrometry and Its Application in Biochemical Analysis ........................... Schwartz, Morton K., and Boda7uhy, Oscar, Automated Methods for Determination of Enzyme Activity ............................. Schwartz, Morton K., and B&mk>, Oscar, Utilization of Automation for Studies of Enzyme Kinetics ................................ Schwartz, Samuel, Berg, Marie H., Batsenmuier, Irene, and Dimmore, Howard, Determination of Porphyrins in Biological Materials .... Scott, Churlcs D., see Burt& Carl A. Scott, J . E., Aliphatic Ammonium Salts in the Assay of Acidic Polysaccharides from Tissues .............. ............. Seaman, G. R., see Stohtad, E. L. R . Sebaki, Walter, see Werner, Sigurd S e ih , N., Use of the Dansyl Reaction in Biochemical Analysis ...... Seitz, W. Rudolf, and Nearg, Michal P., Recent Advances in .Bioluminescence and Chemiluminescence Assay ................ Shuw, Jane E . , and Ramwell, P. W., Separation, Identification, and Estimation of Prostaglandins .................................. Shibata, Kaz w, Spectrophotometry of Opaque Biological Materials: Reflection Methods .......................................... Spectrophotometry of Translucent Biological Materials: Opal Glass Transmission Method . . .............................. Shore, P . A., Determination of Histamine ......................... Sies, Helmut, and Brawn; B o b , Analysis o f Cellular Electron Transport Systems in Liver and Other Organs by Absorbance and Fluorescence Techniques ..................................... Silber, Robert H.,Fluorimetric Analysis of Corticoids ............... Silber, Rob& H., and Porter,Curt C., Determination of 17,21Dihydroxy-20-Ketosteroidsin Urine and Plasma ................ Singer, T h u u P., see Grossman, Shlomo Singer, Thonrcrc P., Determination of the Activity of Succinate, NADH, Choline, and a-Glycerophosphate Dehydrogenases ...... Singer, Thomas P., and Kearney, Edna B., Determination of Succinic Dehydrogenase Activity ...................................... Sjouall, Jan, Separation and Determination of Bile Acids ........... Sheggs, Helm R., Microbiological Assay of Vitamin BIZ ............. Smales,A. A., see Loveridge, B. A. Smith, Emil L., see DaVrr, Neil C . Smith, Fred, and Muntg-, Rex, End Group Analysis of Polysaccharides ........................................... Smith, Lucilc, Spectrophotometric Assay of Cytochrome c Oxidase . . Smh, S. Bo, and Schuberth, Jan, Measurements of Choline Acetylase ............................................ Spencer, B., see Dodgson, K . S . Spcrty, Warren M ., Lipid Analysis ................................ Spink, Charles H.,and Wadso, Ingemar, Calorimetry as an Analytical Tool in Biochemistry and Biology .............................

3

247

24

313

11

211

16

183

8

22 1

8

145

18

259

23

161

17

325

9

217

7 SUPP.

77 89

26 14

285 63

4

139

22

123

4 12 14

307 97

3 2

153 427

SUPP.

275

2

83

23

1

53

CUMULATIVE AUTHOR INDEX, VOLUMES 1-30 AND SUPPLEMENT Stehl, Egun, see Mangold, Helmut K. St. John, P. A., see Winefordner,J. D. StokFtad, E. L. R., Seaman, G. R., Davis, R. J., and Hutner, S.H.,

Assay o f Thioctic Acid

.......................................

Stmvick, C. A., Benson, E. M., Edwards, M. A., and Woodring,M . J,, Chemical and Microbiological Determination of Vitamin Be ..... Strehler, B m r d L., Bioluminescence Assay: Principles and Practice Strehh, B. L., and Totter,J. R.,Determination of ATP and Related

Compounds: Firefly Luminescence and Other Methods ......... Swartz, Harold M., and Swartz, Sharon M., Biochemical and Biophysical Applications of Spin Resonance .................... Swartz, Sharm M., see Swartz, Harold M. Talalay, Paul, Enzymic Analysis o f Steroid Hormones ............. TcWiun,M., see McLaren, D. S. T h t ,Jean-Paul G., and Haegek, Khw D., Analysis of Morphine and Related Analgesics by Gas Phase Methods .................. Thicrs, Ralph E., Contamination o f Trace Element Analysis and Its Control .............................................. Thorpe, W . V., see Bray, H . G. Tqfany, Thonras O.,see Burtk, Carl A. Tinoco, Zgmuio, Jr., Application o f Optical Rotatory Dispersion and Circular Dichroism to the Study of Biopolymers ................ Tohdm$ SibyuC, The in d r o Determination o f Hyaluronidase ...... T O MJ,. R.,see Strehh, B. L. Treudwell,C. R., see Vahmtny,Gewge V. Tulp, Ahaham, Density Gradient Electrophoresis of Mammalian Cells ............................................ U h f k z d , Sidney, Wekbach, Herbert, and Brodie, Bernard B., Assay of Serotonin and Related Metabolites, Enzymes, and Drugs ........ Ushukov, A. N.,see Vaver, V. A. Vahouny,George V., and Treadwell, C. R., Enzymatic Synthesis and Hydrolysis of Cholesterol Esters ............................... Vallee,Bert L., see Margoshes, Marvin V a h Heuuel, W .J.A., see Homing, E. C. Van PiLrum, John F., Determination o f Creatinine and Related Guanidinium Compounds .................................... Vaver, V. A., and Ushakov,A. N.,High Temperature Gas-Liquid Chromatography in Lipid Analysis ............................ Venkateswarlu,P., Determination of Fluorine in Biological Materials .................................................... Vessey,D. A., see Zakim, D. Vestling,Carl S., Determination o f Dissociation Constants for Two-Substrate Enzyme Systems ............................... Volkin,Elliot, and Cohn, Waldo E., Estimation of Nucleic Acids ...... Volhweider, H. J., Visual Biochemistry: New Insight into Structure and Function of the Genome ........................ Wacher, Warren E. C., see A&, El& Wadro, ingemur, see SpinR, Charks H. Waldemann-Meyer,H., Mobility Determination by Zone Electrophoresis at Constant Current ...........................

363

3

23

12 16

183 99

1

34 1

29

207

8

119

24

1

5

273

18 1

81 425

30

141

6

95

16

219

7

193

26

327

24

93

10 1

137 287

28

20 1

13

47

364 CUMULATIVE AUTHOR INDEX, VOLUMES 1-30 AND SUPPLEMENT Wang, C. H.,Radiorespirometry ................................. Webb,Juniuc M., and Lay,HiuOn B., New Developments in the Chemical Determination of Nudeic Acids ...................... Wed-Malherbe,H., The Estimation of Total (Free + Conjugated) Catecholamines and Some Catecholamine Metabolites in Human Urine ............................................ Determination of Catechol Amines ............................ Weikuin,BmG, Separation and Determination of Amino Acids and Peptides by Gas-Liquid Chromatography .................. W k b a c h , Herbert,see U d e n j d , S k h q Wetterau,John R., and Zilversmit,Donald B., Quanutation of Lipid Transfer Activity ............................................ Werner, Sigurd, and Sebald, Waltcs, Immunological Techniques for Studies on the Biogenesis of Mitochondrial Membrane Proteins . . WhauCjr, H . C. S. a'e, and G r m , D., Determination of Raffinose and Keatose in Plant Products .................................... Whqfen,D. H.,see Barker, S. A. Whitehead,E. I., see Olson,0.E. Whitehead,J. K., and Dean, H . G., The Isotope Derivative Method in Biochemical Analysis ......................................... W h i t e h e , M . W., and ZiMen, F., Isolation and Determination of Neuraminic (Sialic) Acids ..................................... Whaiford, William R., see Ndclson, Samuel Wilchek,Meir, see Bayer, Edward A. Wdchek,Meir, and Hater, Charles S., The Purification of Biologically Active Compounds by Affinity Chromatography ................ Willis,J . B., Analysis of Biological Materials by Atomic Absorption Spectroscopy ..................................... Wilson,Kenneth J., see Hughes, Graham J . Winefordw,J . D.,McCarthy, W .J., and St. John, P.A., Phosphorimetry as an Analytical Approach in Biochemistry ..... Winrlcr, Richard J., Determination o f Serum Glycoproteins ......... W d r i n g , M . J., see Sf&h, C. A. Wotiz,Herbert H., and Clark, Stanlcy J.. Newer Developments in the Analysis of Steroids by Gas-Chromatography ................... Wright, M . Ruth, see Lmrer,Aarm B. Yagi, Kunio. Chemical Determination of Flavins ................... Yapcl,Anthony F.,Jr. and Lumv, Rufw, A Practical Guide to the Temperature-Jump Method for Measuring the Rate of Fast Reactions ............................................... Yalow,Rosalyn S., and Berson, S o h A., Immunoassay of Plasma Insulin .............................................. Yamamdo, Richard S., see Muhehen, Olaf Zakim, D., and Vesscy, D. A., Techniques for the Characterization of UDP-Glucuronyltransfera~,Glucose-6-Phosphatase,and Other Tightly-Bound Microsomal Enzymes .......................... Z U h , F.,see Whitehouse,M. W . Zilvenmit, D d B.,see Wetterau, John R.

15

311

6

1

16 SUPP.

293 119

14

203

30

199

27

109

1

307

16

1

8

199

23

345

11

1

15

369

2

279

18

339

10

319

20

169

12

69

21

1

Methods of Biochemical Analysis, Volume 30 Edited by David Glick Copyright © 1984 John Wiley & Sons, Inc.

Cumulative Subject Index, Volumes 1-30 and Supplemental Volume

Absorbent and Flumescent Substances, The Automated Anal* of, Separated on Paper Strips (Boulton) ............................. Activation Analysis and Its Application in Bhhemisgr (Loveridge and Smales) ...................................... Activahn Analysis of Biological Trace Elemen& (Leddicotte) .......... Adenine, Enzymu Micro Determi&, by Ultraviolet Spectrophotonuhy (Plesner and Kalckar) ......................................... Adrenal Gland, Steroidc of, Chromatographic Separation (Haines and Karnemaat) ..................................... Adrenal Steroids in Blood, Analysis of, by Counttmurrmt Distribdm (Carstensen) ................................................. Adrenaline, Chemical Determinution, in Body Fluids and Tissues (Persky) A f f i @ yChromatography, The Punjication of Biologicallr Active Compounds by Aliphatic AmmoniumSalts in the Assay of Acidic Polysacchdes from Tissues (Scott) .............................. Alterncrtve Metabolic Pathways, Estimutiun of Magnitudes of (Kopin) .... Amine Oxidascs, Delffnrinationof (Kapeller-Adler) .................. AminoAcid Analysis by Gas-Liquid Chrmahgraphy, Recent Developents in, (Mackenrie) ............................ Amino A&, Analysis by Means of Brditka Reaction (Miiller) .......... Amino Acids, Determination by High-Vohge Paper E&ctr@hessis (Blackburn) ................................................. Amino Acids, Determinution by Ion Exchange Chromatography (Jacobs)... Amino A&, Determination by Use of Bactnial Amino Acid Decarboxykues (Gale) ...................................................... AminoA d , Separation and Determination by Gas-Liquid Chromatography (Weinstein) .................................................. AmmoniumSalts, Aliphatic, in the Assay of Acidic Polysacchades from Tissues (Scott) ........................................... Ampholytes,A Techntipfor Fractionation and Charactetization through Grndieptts (Haglund) .................. Isoelechic Focusing in+H Analgesia, Analysis by Gas Phase Metftodr (ThCnot and Haegele) ...... Antibiotics, Microbiological Assay (Kersey and Fink) ................. Application of High R e s o l d m Nuclear Magnetic R e s m n c e to Biological Systems (Campbell and Dobson) ................................ Ascorbic Acid, Chemical Determinution (Roe) ........................ A h u Absorption Spectroscopy, Analysis of Biological Materials by (Willis)

365

VOL.

PAGE

16

327

5 19

225 345

3

97

1

171

9 2

127 57

8

145 247 35

11 SUPP. 27

1

11

329

13 14

177

4

285

14

203

8

145

19 24 1

1 1

153

25 1 11

115 1

1

1

366 CUMULATIVE SUBJECT INDEX. VOLUMES 1-30 AND SUPPLEMENT ATP. Determination of Firejly Luminescence (Strehler and Totter) ..... Avidin.Bwtin. Use of, As Tool in Molecular Biology (Bayer and Wdchek) .................................................... Bacterkd Amino And Decarboxylares in Determination of Amino A d (Gale) ...................................................... Basic Pro&’ns. Prepamtiun and Analysis o f (Lindh and Brantmark) .... Bile A d . Newer Dcvelqpmpnts in the Gm Chromatographic Determination of (Kuksis) ...................................... B 5 A d . Separatiun and Determination of (Sjovall) .................. Bile Pigments. Separation and Determination o f (Brodenen and Jacobsen) .................................... Biochnnical Applicatiollr of Magnetic Resonance (Jardetzky and Jardetzky) .................................... Biochenishy. Historical Developnuw and Nnvtr Means of Temperature Measurement in (Berger. Clem. Harden and Mangum) ........... BiochcniChy. Laser Photolysk in (Chan and Austin) ............... Biological Matmials. Analysis by A t a r u Absmprirm Spectroscopy (Wdlis) . Biological M a h i & . Determindion of Nitrogen in (Jacobs) ............ Biological M a h l s . Determination of Porphyrins in (Schwartz. Berg. Bossenmaier. and Dinsmore) ................................. Bidlogical M a h l s . Determination of Zinc in (Malmstrom) ........... Biological Materials. Methodsfw Estimoring Magnesium in (Alcock and MacIntyre) ...................................... Biological Mnloials. Micr&terminufiun of Cobalf in (Saluman and Keenan) ....................................... Biological Materials. Spectrophotonutty ofi Rt$ection Methods (Shibata) ....... ........................................ Spechophotamet?y of; Opal Gkw M e W (Shibata) ................................... Bwlogical Standards in Biochemical Analysis (Humphrey. Long. and Perry) ................................ Biological Systems. Determination of Sm’ne in (Frisell and Mackenzie) . . Biological Systems Ion Binding in. Memured by Nuclear Magnetic Resonance Spectroscopy (For&n and Lindman) ................... Biological Trace Elementc. ActiuatMi Analysis of (Leddicotte) .......... Bioluminescence Assay: Prin+&s and Practice (Strehler) .............. Bioluminescence and Chiluminescence Assay. Recent Advances in ...... Blood. Analysis of Adrenal Sttroids in. by CountcrGurrenl DishibutMn (Cartensen) ................................................. Blood. Detmnindion of Ethyl Alcohol in (Lindquist) .................. Determination of Adrenaline and Noradrenaline an

.

.

.

............................................

Body Fluidr. Chromatographic Analysis of Radioactive Iodine Cmpuundcf r m (Roche. Lissitzky. and Michel) .................. B q f f c . Containing Urea. Gel Electrophoresis in (Poulik) .............. Calorimehy as a n Analytical Tool in Biochnnistly and Biology ........... Carbohydrate. The Defermination of, in Biological MatniOls by Gas-Liquid Chromatography (Clamp. Bhatti. and Chambers) ................. Carbohydrate Derivatives. Separation of. by Gas-Liquid Partition Chronurtography (Bishop) ..............................

1

341

26

1

4 14

285 79

14 12

325 97

17

31

9

235

30 30 11 13

269 105 1 241

8 3

221 327

14

1

5

181

9

217

7

77

5 6

65 63

27 19 16 23

289 345 99 161

9

7

127 217

2

57

1 14 23

243 455 1

19

229

10

1

CUMULATIVE SUBJECT INDEX. VOLUMES 1-30 AND SUPPLEMENT 367 Carbohydrate Structure. Use o f Infrared Analysis in Determination of (Baker. Bourne. and Whiffen) ................................ Carbonic Anhydrose Activity.Measurements of (Davis) ................. Carotene. Determination of (Bickoff) ............................... (Creveling and Daly) ......................................... Catalases. h a y of (Maehly and Chance) .......................... Catechol Amine Biosynthesis and Metabolism. Assay of Enzymes of Catechohmines and Catecholamine Metabolites. Estimation of Total (Free + Conjugated). in Human Urine (Weil-Malherbe) ............ Catcchol Amines. Determination of (Weil-Malherbe) .................. Cell Parttkles and Macromolecules. Partition Methodr f w Fractimution of (Albertsson) ................................................. Cellular Electron Transport Systems in Liver and Other Organs. Analysis 06bJ Abswbame and Fluorescence Techniques (Sies and Brauser) ........................................... Celluhe Column Chromatography. Analysis of Mixtures of Sugars bJ (Hough) .................................................... C&f'ugal Fast Analyzerfor Biochemical and ImmunologicalAnalyes. The Use of a ................................................. C&@gatiun. Preparative Zonal (Anderson) ....................... Chloride in BWl.gical Materials. Determination of (Cotlove) ............ Cholesterol. Determination and Microsc~@cLocalization of (Kabara) ..... Cholesterol Esters. Enzymatic Synthesis and Hydrolysis of (Vahouny and Treadwell) .................................... Choline. Chemical Estimation of (Engel. Salmon. and Ackerman) ..... Choline Acetyke. MeasurMnents o f (Sorb0 and Schuberth) ........... Choline Estm. Measurement of Uenden) ........................... Cholinesterases. Assay Methods f w (Augustinsson) ................... Cholinesterases. Determination of (Augustinsson) ..................... Chromatographic Analysis. A#licatM?ls of the R M Treahnent in (Bush) ... Chromatographic Analysis. A#lica&ns of &he RMTreatment in. Erratum (Bush) ...................................................... Chromatographic Analysis of Radioactive Iodine Compoundsfrom the Thyroid Ghnd and Body Fluids (Roche. Lissitzky. and Michel) ...... Chromatographic and Electrophoretic Methods. Analysis of Natural Radioactive Iodine Compovnds by (Roche. Michel. and Lissitzky) .... Chromatographic Separation of Steroids of the Adrenal Ghnd (Haines and Karnemaat) ..................................... Chromatography. Gas. in Detennination of Bile Acids and Steroids (Kuksis) ..................................................... Chr&graphy. Gas. Separation and Determinution of Steroids by (Homing. Vanden Heuvel. and Creech) ....................... Chromatography. Gas.Liquid. Determination of &he F q A d by (James) Chromatography. Gas.Liquid. Separati'm and Determination of AminoAcids and Peptides by (Weinstein) .................................... Chromatography. Gas-Liquid Partition. Separation of Carbohydrate Derivatives by (Bishop) ........................................ Chromatography. High-Pevjinmance Llprud: Analytic and Preparative APplUaiiOn in Protein Structure Determination (Hughes and Wilson) ........................................

3 11 4 SUPP. 1

213 307 1 153 357

16 SUPP.

293 119

10

229

26

285

1

205

23 15 12 10

189 271 277 263

16 1 SUPP. SUPP. 5 SUPP. 13

219 265 275 183 1 217 357

14

497

1

243

12

143

1

171

14

325

11 8

69

14

203

10

1

29

59

I

368 CUMULATIVE SUBJECT INDEX. VOLUMES 1-30 AND SUPPLEMENT Chromatography. High Temperature Gas.L+. in L i e Analysis (Vaver and Ushakov) ........................................ Chromatography. Ion Exchange. Determinatkm o f Amino A& by (Jacobs) ..................................................... Chromatografihy. Paper and Cellulace Column. Analysis of Mixtures of Sugars by (Hough) ........................................... Chromatography. of Proteins. N w L k A&. V i w e s . and CeUs on Noncharged Amphiphilu Gels. Hydrophobic Interaction. (Hjerth) ..... Chromatography. Thin-layer (TLC) (Mangold. Schmid. and Stahl) .... Cobalf. Mirrodctrmrination of, in Bwrogical Matowls (Saltzman and Keenan) .................................................... CoenzymeA. M e w fm DetmninotiOn of (Novelli) .................. CoenzymeQ, DeknniMhbn of (Crane and Dilley) ................... Coenzymes.Infrared Analysis of (Rosenkrantz) ...................... Collagen and Elastin. Analysis of the Crosslinking Cmponenfs in (Robins) CoUugen and Elastin. The Detmnination of (Jackson and Cleary) ...... Color Reacfions. New. for Detenninatiun of Sugars in Polysaccharides (Dische) ........................................ ..I.. ........ Column Electrophoresis in Granular Media. Some R e c d Devekpneni3 (Porath and Hjerten) ......................................... Complexes, Metal Protein. Analysts of (Hughes and Klotz) ............ CmplexIon Solubcluy. Measurement Use of Ion Exchange Resins (Schubert) .................................................. Connective-TissueMacromolecules. Analysis by Detnnrifiation of C m i n Cm(Blumenkrantz and Ash-Hansen) ................. ConiaminaciOn in Trace Ekment Analjsis and Its Control (Thiers) ...... C&&. Fluorimehic Analysis of (Silber) .......................... Countercurrent Dirhibution (King and Craig) ....................... Countercurrent Dishibutia. Analysis of Adrenal Stctoidc in Blood by (Carstensen) ................................................. Creatinine and Rehated Gunnidinium C m p n u k . DetenniMEion of (Van Pilsum) ................................................ Current. Canstant. Mobiky Det?nni& by Zone Electrophoresis at (Waldmann-Meyer) .......................................... Cyclic 3'.5 '.Admasine M o m p h p b and Cyclic 3'J '-Gunmine M o n o p h a p h . Analysis of (Goldberg and OToole) .............. C ~ l i AMP c and Other Cyclic Nucleotdes. Adenykate Cyckase. and Phosphodiesterase. Newer Developmnrts in the DetmninOtimt o f (Brooker) ................................................... Cyckxhromec Oxidare. Spectrophoktric Assay of (Smith) ............ Dansyl Reaction. Use of the. in Biochemual Analysis (Seider) ........... Dehydroascorbic Acid. Chemical DctmninotMn of (Roe) ................ Dehydrogenases. Detmninatiun of the Actiwly of Succinate. NADH. Choline a-Clycerophphaie (Singer) .................................... Denaiuration. Reversible. of Proleins. Methodr of Study and Intngrccalion ofData fw (Hermans.Jr.) ..................................... Dmcilp Gradients. R a e Electrophoresis in (Kolin) ................... Deoxyribonuclease Actiwly. Assay of (Kurnick) ....................... Diagn~~ciC. Enzycrtrc Metha& o f (Amador and Wacker) .............. Ddysis (Craig and King) .......................................

26

327

14

177

1

205

27 12

393

5 2 11 5 28 15

181 189 279 407 329 25

2

313

9 3

193 265

3

247

24 5 14 10

39 273 63 201

9

127

7

193

13

47

20

1

22 2 18 1

95 427 259 115

22

123

13 6 9 13 10

81 259 1 265 175

89

CUMULATIVE SUBJECT INDEX. VOLUMES 1-30 AND SUPPLEMENT 369

Diaw-Positive Bile Pigncents. Recent Advances in the Separahn and Analysis of (Heinvegh) ........................................ Dffraction. X.ray. in the Study of Protein and Nuchic Acid Structure (Holmcs and Blow) .......................................... Z.O K .rD .oZ se.t7.i Detmninativn in Urine and Plusma (Silber and Porter) ........................................... DikeiogulrmicAcid. Chemical Determination of (Roe) .................. Dissociation Conrtants. Determination 05for Two-SubstrateEnzyme Systnnr (Vestling) ................................................... Electron Probe Microanalyzer. A n Introduction to. and Its Applicahbn to Biochemistq (Andersen) ....................................... Electron Spin Resonance. Biochemical and Biophysical Applicdon o f (Swartz and Swartz) .......................................... Electrophoresis. Free Zone. T h e q . Equipment. and ApPlUatiuns (Hjerten) ................................................... Electrophoresis. Gel. in Buflers Containing Urea (Poulik) .............. Electrophoresis. Gel Sieving; A DesrriptMn of Procedures and Anulysk o f E r r a uohnson) ............................................. Electrophoresis. Paper. Detmninatbn of Amino A& at High-Voltage by (Blackburn) ................................................. Electrophoresis. Rapul. in Dencity Gradimts Combined with pH o d o r Conducrivaty Gradients (Kolin) .................................. Electrophoresis. Zone (Kunkel) .................................... Electrophoresis. Zone. Constant Cuwent Mobility Detmnination by (Waldmann-Meyer) .......................................... Electrophoresis in Granular Media. Column. Some Recent Deve@nmts (Porath and Hjerten) ......................................... Electrophoretic Methodr. Analysis of Natural Radioactive Iodine Comfiounds by (Roche. Michel. and Lissitzky) .............................. Elements. Determinution 05by X-Ray Evnission Spectrometry (Natelson and Whitford) ..................................... EnthaIpy and Enlropr Changes. Detnminahbn by Heatburst Microcuiurimetry (Kitzinger and Benzinger) ...................... EnzymaticM e w . in Diagnosis (Amador and Wacker) ............. Enzyme Actim$y.Rutonrated M e t h d for Detffninntion o f (Schwartz and Bodansky) .................................................. EnzymeAssay. R a d k t r i c Methods o f (Oldham) .................... EnzymeKinetics. Utilization of Autonuationfor Studies of (Schwartz and Bodansky) ..................................... Enzymes. Assay of in Catcclrol Amine Bimynthesisand Me&lism (Creveling and Daly) ......................................... Enzymes. Detection o f Lend-Induced and Syncatrrlytic C o n f m h d Changes of by D i f l e r d l Chemical Mod$cahbn (Christen and Gehring) ...................................... Enzymes. FZuorhetric Assay of (Roth) .............................. Enzymes. Immobilized. in Biochemical Analysis (Everse. Ginsburgh. and Kaplan) .............................. Enzymes. Proteolytic Assay of (Davis and Smith) ..................... Enzymes. Relaud to Serotonin. Assay of (Udenfriend. Weissbach. and Brodie) ..........................

22

205

13

113

9 1

139 115

10

137

15

147

29

207

18 14

55 455

29

25

13

1

6 1

259 141

13

47

9

193

12

143

12

1

8 13

309 265

11 21

211 191

16

183

SUPP.

153

28 17

151 189

25 2

135 215

6

95

370

CUMULATIVE SUBJECT INDEX. VOLUMES 1-30 AND SUPPLEMENT

Enzyme Systems. Two Substrate. DetmniMtion of Dissm-ktion Conrtantsfor (Vestling) ................................................... EnzymicAnalysis of Steroid Honnoncs (Talalay) ...................... EnzymicDetenniMtMn of D-Glucose and Its Anomen. New Dtoelopmntts in (Okuda and Miwa) ........................................ Estrogens. Chemical Determination of, in Human Urine (Bauld and Greenway) ....................................... Ethanolamine. Determinolion oJ in Lipids (McKibbin) ................ F & y Acid Esters. A Critical Evaluation of the Gas Chromatagrophic Techniquefor Identij5cation and Detmnination 4 with PariinJar Refmeme to the Use of Analog and Digttal Computer Methods (Caster) FuUy A d . DelmniMtion by Gas-Liquid Chromatography (James) ...... Field Desorption M a s Spectrometly: Application in Biochemical Analysis (Schulten) ................................................... Field-Flow Fractionation. Analysis of Bwbgical Matromoleds and Particles By (Giddings. Myers. Caldwell. and Fisher) ............. Firejty Luminescence. DetnrniMtion of A T P by (Strehler and Totter) ... Flame Photometry. Principles and Applicalions (Margoshes and Vallee) Flavim. Chemical Determination of (Yagi) ........................... F1uid.s. Body. Chemical Determination of Adrenuline and Noradrenuline in (Persky) ..................................................... Fluids. Body. Chraurtographu Analysis of Radioactive Iodine Compounds from (Roche. Lissitzky. and Michel) ............................ Flumimetric Analysis of Cwticoidr (Silber) ...... Fluorine. Determinnfiun in Biological Uaierials (Venkateswarlu) ....... Folu Acid Actimty.Assay of Compounclr Wth uukes) .................. FmIdehyde. Determination ofi in Biological Systems (Frisell and Mackenzie) ....................................... F r a c W i u n of Cell Particles and Motromolecules. Partition Melhodr for (Albertsson) ................................................. Free EWB Changes. DetenniMtion by Heatburst Microcahimetly (Kitzinger and Benzinger) .................................... Frog Skin Assayfor Agents that Darken and Lightm Melanocyfa (Lerner and Wright) ......................................... Gas-Llguul Chromatography. The Determination in Carbohydrates and Biological MatPrialr (Clamp. Bhatti. and Chambers) .............. Gel Electrophesis in Buffers Containing Urea (Poulik) ............... f3-Glucuronidoses. Determination of (Fishman) ....................... UDP.GlucuranyLtramfmase. Glucose.6.Phosphatae. and Other Tightly-Bound Microsoma1 Enzymes. Techniquesfor the C h a r a c ~ i z a t i aof (Zakin and Vessey) . . . . . . Gluiumic and Aspartic A d and Their A d s . DetmniMcion of (Balk) .. Glulathionc. Determination of (Patterson and Lazarow) .............. Glycolipid DetnrniMtirm (Radin) .................................. Glycoprotcinr. Serum. Determinatiun of (Winder) ..................... Gradfents. Densily. Rapid Electrophesk in (Kolin) ................... Heatburst Microcalmimetly. Primiple ond Methads of. and DetermiMtion of Free Energy. Enthalpy. and Entropy Changes (Kitzinger and Benzinger) .................................... Heparin. Determination of Uaques and Bell) ........................

.

10 8

137 119

21

155

5 7

337 111

17 8

135 1

24

313

26 1 3 10

79 34 1 353 319

2

57

1 14 24 2

243 63 93 121

6

63

10

229

8

309

8

295

19 14 15

229 455 71

21 20 2 6 2 6

1 103 259 163 279 259

8

309 253

7

CUMULATIVE SUBJECT INDEX. VOLUMES 1-30 AND SUPPLEMENT 371 Heparin. Deternination of (Jaques) ................................ HexosamincS. Determimation of (Gardell) ............................ High-Performance Ion-Exchange Chromatografihy with Narrow-Bore Columns: R a g Analysis of Nucleic Acid Constituents at the Subnanomole Level (Horvath) .................................. Histamine. Determinuha of (Shore) ............................... Histamine. Quantitative Determination of (Code and McIntire) ........ Histidine Decarbqlase. Determination of (Schayer) .................. Histidine Decarbqyhe Activiv. DetermiMh'on of (Schayer) ............ Hormones. Infrared Analysis of (Rosenkrantz) ....................... Hormones. Plant. Analysis of (Bentley) ............................. Hormones, Steroid. Enzymic Analysis of (Talalay) ..................... Hyalurmiduse. in vitro Determination (Tolksdorf) ................... Hydrogen Exchange Data. Acquisition and Interpretation of. from Peptidcs. Polymtrs. and Proteins (Barksdale and Rosenberg) ................ Hydrogen Isotope Exchange in Globular Proteins. M e wfor Mearurement (Ottesen) .................................................... Hydrophobic Inh-action Chromatography o f Prothns. Nucleic Acidr. and Celk on Noncharged Amphiphilic Gels. (Hjertkn) ................... Hypoxanthine. Enzymic M k o Determination. by UltraGolet Spectrophotmneby (Plesner and Kalckar) ......................... Immunoarsay of Plasma Insulin (Yalow and Berson) ................. 1rnmunoclcch.ophmetit Analysis (Garbar) ............................ Immunological Techniquesf m Studies 071 the Biogenesis of Mitochondd Membrane Proh'ns (Werner and Sebald) ........................ Infrared Analysis. Use of. in the Determination of Carbohydrate Stmcture (Baker. Bourne. and Whiffen) ................................ Infrared Analysis o f Vitamins. Hormones, and Cmzymes (Rosenkrantz) Infrared Spectromeq. Analysis of Steroids by (Rosenkrantz) ........... Inositol. Determination of, in L i w s (McKibbin) ..................... Iodine. in Biological Matnial. Determination of (Binnerts) ............. Iodine Compoud. Natural Radioactive. Analysis by Chromatographic and Electrophoretic Me& (Roche. Michel. and Lissitzky) ............ Iodine Compound. Radioactive. from Thyroid Gland and Body Fluids. Chromatographic Analysis (Roche. Lissitzky. and Michel) ........... Ion Binding in Biological Systems Measured by Nuclear Magnetic Resonance Spectrascopr (Forsen and Lindman) ................... Ion Exchange Resins. Measurement of Complex Ion Stability by Use of (Schubert) .................................................. Isolated Bacterial Nuclewids. Charactekatim. Assay. and Use of (Hirschbein and Guillen) ..................................... Isotope Derivative Method in Biochemical Analysis. The (Whitehead and Dean) ....................................... Kestose. Determination. in Plant Products (de Whalley and Gross) ..... a-Keto Acid Determinations (Neish) ................................ 17.Ketosteroids. Urinary Neutral. Assay of (Engel) .................... Lipme. Lipoprotein. Assay of, in vivo and in vitro (Korn) ............. Lip& Analysis (Sperry) ......................................... L i M s . Determination of Inosdol. Ethanolamine. and Serine in (McKibbin) ..................................................

24 6

203 289

21

16 5 9 8 1

79 89 49 99 273 407 75 119 425

28

1

20

135

27

89

SUPP*

3

SUPP*

3 12

7

97

69 1

27

109

3 5 2 7 22

213 407 1 111 251

12

143

1

243

27

289

3

247

28

297

16 1 5 1 7 2

1 307 107 459 145 83

7

111

372

CUMULATIVE SUBJECT INDEX. VOLUMES 1-30 AND SUPPLEMENT

L i e Transfer Activity, Quaniitaiim of (Wetterau and Zilversmit) ..... Lip.p.0t.i. L i p e . Assay 05in vivo and in vitro (Korn) .............. Lipoprotkns. Serum. Ultraentrifugal Analysis (de Lalla and Gofman) . . Lipaxidare Actively, M e a s u r m of (Holman) ....................... Lipoqgenase (Lipoxidate). Detffminationof the Actidy of (Grossman and Zakut) ....................................... ~ -Counting. Practical ~ Aspecis o f ~ ' (Kobayashi and Maudsley) .................................... Lucrferin and Luafmase. M e a s u r e of (Chase) .................... LysozymeActivzty. Me& for Determination of (Grossowicz and Ariel) Magnesium Estimation. in Biological MatetiaD (Alcwk and Macintyre) MagnctiC Resonance. Biochemical App1icaiion.s of (Jardeuky and Jardetzky) .................................... Mammalian Cells. Dmnly Cradieni Electrophoresis of (Tulp) ........... Moss Spectrometty. Anulysir of Sieroids by (Gasdell) ................... Moss Specirometty. Fie12 Desorpiim: Appkation in Biochemical Anolysir (Schutten) ................................................... Mass Spectrometv in the Deierminatiun Df Structure of Certain Natural Products Containing Sugars (Hanessian) ......................... Mehmqtes. Darkening and Lighning. Frog Skin Assayfor (Lerner and Wright) ......................................... Metabolic Pathmys. Altmurtive. Estimation o f Magmtudes of (Kopin) ... Metabolism. A d + of Phenolic Compounds of Interest in (Bray and Thorp) .......................................... Metal ByFfers. Application. in B i o c h i s i t y (Raafiaub) ................ Metal Indicaiurs. AppliMfiMis. in Biocfrmtiriry (Raaflaub) ............. Metal-Protein Complexes. Analysis of (Hughes and Klotz) ............. Microbiabgad Assay of Antibiotics (Kersey and Fink) ................ Microbiobgical Assay of VitnwunBln (Hoff-Jorgensen) ............... Microbiological Assay of VitaminB 12 (Skeggs) ....................... Microbwbgical DeiermiMtMn of Vitamin B6 (Storvick. Benson. Edwards. and Woodring) .................... Muropariiculatc Gel Chromatography Accehated by Ceninfigal Force and Pressure (Ribi. Parker. and Milner) ............................. Mobthy. DeiermiMtMn by Zone Electr@hor& at C m t n n t Current (Wddmann-Meyer) .......................................... Moleculur S i x . EstinrcrCirm of. and Moleculur Weights of Biobgical Compounds by Gel F&& (Andrews) .......................... M w h i n e . and Relotcd Analgesics. A d p i s by Gas Phase M e W (Thtnot and Haegele) ........................................ Mucopolysacclwkk-s.Sqated. DetewniMtMn of (Jaques) .............. Negative-Ion Moss Spectromeiry. Fused-Silica CapilLaty Gas Chronurtogaphy of Neurotransmitters and Related Compounds ( F a d and Barchas) .......................................... Neuraminu (Sialic) A d . Isoldirm and Deiermtnaiiun of (Whitehouse and Zilliken) .................................... Nitrogen. DeunniMtimr in Biobgical MaktiaLc (Jacobs) ............... Nitrogemus Compounds. Basic. of TmiCobgacal Importance. Analysis 4 (Curry) ..................... ............................. N o r a d r d n e . Chemical Determination. in Body Flu& and Tissues (Persky) .....................................................

.

30

1 2

199 145 459 113

25

303

17 8 29 14

55 61 435 1

9 30 29

235 141 385

24

313

19

105

8 11

295 247

1 3 3 3 1 1 14

27 301 301 265 53 81 53

12

183

22

355

13

47

18

1

24 24

1 203

29

325

8 13

199 241

7

39

2

57

7

~

CUMULATIVE SUBJECT INDEX. VOLUMES 1-30 AND SUPPLEMENT Nucleic Acid. Strutture. X-ray oij'@ction in the Study of (Holmes and Blow) .......................................... Nuckic A d . Chemical DetenninaciOn of (Webb and Levy) ........... Nucleic A d . The Determination of (Munro and Fleck) .............. Nucleic A d . Estimation (Volkin and Cohn) ....................... Nuckic A d and Their Dnivdivcs. Mierobiobgical Assay of (Miller) .... Nucleic A d of Various ConformationalF a n s and Measurement of Their Associated Enzymes. w e of Ethidium Bromidefor Separation and Detenninution o f (Le Pecq) ..................................... Nucle& and Nucbotidcs and Their P a r d Bases (IJ an Amlyrical and Investigative Tool. Pokarografihy and Voltummehy of (Elving. OReilly. and Schmakel) .............................. Optical R o w Dispersh. Application of. and Circular Dichroism to the Study of B i o p o l (Tinoco. ~ Jr.) ............................... Organic P h p h Compounds. Determinution of. by P h p h a t e Analysis (Lindberg and Emster) ....................................... . . Pnialete. Use of. in Biochemical Analysis (Dyer) ........... oxrdatrons. Oxygen Electrode Measuremen& in Biochemical Analysis (Lessler and Brierley) ........................................ Paper Chromatogram. Direct Scanning of. for Quuntiidve Estimaticms (Bush) ...................................................... Paper Chromatography. for Analysis ofMiitures o f Sugan (Hough) ..... Partition Mclhodc for Fractionation of Cell Partkles and Macromole& (Albertsson} ................................................. Peptide Chroma&graphy. Autmnaeic (Jones) ......................... Peptide Mapping of Proteins (James) ............................... Peptides. Separation and Determinution. b Gas-Llquul Chromatography (Weinstein) .................................................. Pep&. T m t n a l and Sequence Studies in. Recent Developmtntsin Techniquesfor (Fraenkel.Conrat. Harris. and Levy) .............. Peptides and Amino A d in N m l Human Urine. Separation and @anttarion of (Lou and Hamilton) ............................ Pniodatc 0dahn.s. Use of. in Biochemical Analysis (Dyer) ........... Peroxiduses. Assay of (Maehly and Chance) ........................ Phase PartifimtiA Method for Purijication and Analysis of CeU O r g a n e k and Membrane Vesic&s(Albertsson. Andersson. Larsson. and Akerlund) ...................................... Phase Pmtition. I&action Between Biumoleculrr Studied by (Albertsson) Phenolic Cmpoundr of Interest in Metabolism (Bray and Thorp) ...... Phmylalaninc and Tyosine in Blood. The Measurement o f (Robins) ..... pH Gradients. IsoelectriC F&g in-A Techniquefor Fractiona&m and C h a r a c e of Amphlytcs (Haglund) ........................ pH Jump. The: Macromolecules and Solutions by a her-Induced. Ultrashort. Proton Puke. Probing of-Theq and Application in Biochemistry (Gutman) ........................................ pH and Similar Variables. Recent Developnuntr in Control o f uames and Lumry) .......................................... pH-Stat and I& Use in Biochemistry (Jacobson.LRoNs. Linderstrbm.Lang. and Ottesen) .............................. P h p h d c Analysis. D e t e d n & m o f Organic P h o s p h Compound by (Lindberg and Emster) .......................................

373

13 6 14 1 6

113 1 113 287 31

20

41

21

287

18

81

3 3

1 111

17

1

11 1

149 205

10 18 26

229 205 165

14

203

2

359

25

3

1

203 111 357

28 29 1 17

115 1 27 287

19

1

30

1

29

137

4

171

3

1

374 CUMULATIVE SUBJECT INDEX. VOLUMES 1-30 AND SUPPLEMENT Phpholrpaccs. A. C. and D. DetmniMtion of Ihe Actiw of. (Grossman. Oestreicher. and Singer) .......................... Phphoritnehy. a an Analytical Approach in Biochmrtstry (Winefordner. McCarthy. and St.John) ........................ P h p h Compounds. Organic. Determi& of, by Phosphate Analyses (Lindberg and Ernster) ....................................... Ph&mwtty. Flame. Principles and A p p l i c h of (Margoshes and Vallee) ...................................... Phytclu and Inositol PhosphatcJ. the Delmnination of (Oberleas) ........ Plant Honnmrcs. Analysis of (Bentley) ............................. Plasma. Determination of 17.21.Dihydrq.2 O.Ketas&& in (Silber and Porter) ........................................... P&ma Insulin. lmmunwsray of (Yalow and Berson) ................ Polarographic Analysis of Proteins. Amino A d . and Other Compounds by Means of the Brdifka R e a c h (Miiller) .......................... Polarographic Oxygen Senson. Adaptation of. f m Biochemical Assays (Lessler) .................................................... Polysaccharidcs. Acidic. from Tissues. Aliphatic AmmoniumS a h in the Assay of (Scott) ............................................... Polysaccharides. End Croup Analysis of (Smith and Montgomery) ..... Polysaccharides. Sugars in. New C o h Reactionsfor Determination of (Dische) ................... .............................. Polpnsaturated Fatty A d . Measurement of (Holman) ............... P-hyrins in Biological Makrials. Determination of (Schwartz.Berg. Bossenmaier. and Dinsmore) ................................. Prostaglandins. Separation. Iahyication. and Estimatia of (Shaw and Ramwell) ......................................... Protein. S h t u r e . X-ray D j f f r a c h in the Study of (Holmes and Blow) Protein. Terminal and Sequence Studies in. Recent Developmen& in Techniques for (FraenkeLConrat.Hams. and Levy) .............. Protein-Nucleic Acid and Proiein-Protein Complcxcs by Djffer&l A r e a (Bosshard) ........ Chemical ModjFcation. Mapping of C&t Proh’ns. Analysis by Means of Brdi€ha Reactiun (Miiller) .............. Proteins. Bark. Preparation and Analysts of (Lindh and Brantmark) ... Proteins. Mitochondnal Membrane. Immunological TechnipucSfor St& on the Bwgmsts of (Werner and Sebald) ........................ ProtcinC Polarography OJ Analytical Principles and Applications in B i o l o g d and Clinical Chtmtstly (Homolka) ...................... Proteins. Reversible Dmaturation of. Methodr of Study and IntngrctatMn of Dato for (Hermans. Jr.) ..................................... P r o h n Sequence Analysis Solid Phase Methodr i n (Laursen and Machleidt) ..................................... Protcolytic Enzymes. Assay of (Davis and Smith) ..................... PunjicatiOn of BwbgicaUy Active Compounds AffinttyChromatography. The Purines. New Methodr for Purifcation and Separation of (Bergmann and Dikstein) ..................................... Quantitative Marc Spectrometric Analy~ts:Chemical and Biological Applicotiotrc. Inteqated Ion-Current (IIC) Techniqu of (Majer and Boulton) ......................................... Radioactive Iodine Compounds. frmn Thyroid Gland and Body Fluids. Chromatographic Analysis of (Roche. Lissitzky. and Michel) ........ Radioimmunwssay of Polypeptide Hormones and Enzymes (Felber) ......

.

.

22

177

15

369

3

1

3 20 9

353 87 75

4 12

139 69

11

329

28

175

a 3

145 153

2 4

313 99

a

221

17 13

325 113

2

359

25 11 14

273 329 79

27

109

19

435

13

81

26 2

201 215

6

79

21

467

1

243 1

22

CUMULATIVE SUBJECT INDEX. VOLUMES 1-30 AND SUPPLEMENT Radimespirmtry (Wang) ........................................ Raffinose. Determination in Plant Pr0dut.s (de Whalley and Gross) .... Rw.ns, Ion Exchange. M e a s u r d of Complex Ion Stability. by Use of (Schubert) .................................................. Resonance. Magnetic. Biochemical Applications of Uardetzky and Jardetzky) .................................... Ribmulease. Chracterizatim of. and Determination of Its Activity (Josefsson and Lagerstedt) .................................... RM Treatment. Applications in Chromatographic Analysis (Bush) ........ R ~ Treatment. A Applications in Chromatographic Analysis. Erratum (Bush) ...................................................... Selenium in Biological Materials. Determination of (Olson Palmer. and Whitehead) .............................. Serine. Determination of, in Biological Systems.(Frisell and Mackenzie) .... Serine. Determination of, in L i e s (McKibbin) ...................... Serotonin: T h Assay of Hydroxyindole Compoundsand Their B i o p t h t i c Enzymes (Lovenberg and Engelman) ........................... Serotonin and Related Metabolites. Enzymes. and Drugs. Assay of (Udenfriend. Weissbach. and Brodie) .......................... Serum Acid Phphatases. Determinations (Fishman and Davidson) ..... Serum Glycoproteins. Determination of (Winzler) ...... .............. Serum Lipoprotkns. Uhracentnfugal Analysis of (de Lalla and Gofman) -SHGroups in Protknr. Determination of (Benesch and Benesch) ..... Siulic Acids. see Neuraminu Acids Sodiumand Potarrium. Measurementsof, by G h s Electrodes (Friedman) ... Solid P h e Immunoclssay. Use of Magnetizable Particles in (Pourfarzaneh. Kamel. Landon. and Dawes) .................... Spectrometv. Infrared. Analysis of Steroids by (Rosenkrantz) ........... Spectromeq. Principles and Applications (Margoshes and Vallee) ...... Spectrometry. X-ray Emission. Determination of Elements by (Natelson and Whitford) ..................................... Spectrophotometric Assay of Cytochrume c Oxidase (Smith) .............. Spectrophotometric OxyhmwglobinMethod. Meamrement of Oxygen Consumption by (Barzu) ....................................... Spectrophotumetty. Ultraviolet. Enzymic Micro Determination of Uric A d . Hypoxanthine. Xanthine. Adenine. and Xanthoptm’ne by (Plesner and Kalckar) ........................................ Spectrophotometry of Opaque Biological Materials;RefEection Methodr (Shibata) .................................................... Spectrophotometty of Translucent Biological Materials; Opal Glars Method (Shibata) .................................................... Stamhrh. Biological. in Biochemical Analysis (Humphrey. Long. and Perry) .................................................. Steady State Kinetics of Oxygen Uptake by Biochemical Samples. Polarographic Measurement of (Degn. Lundsgaard. Peterson and Ormicki) ....................................... Steroid Hormones, Enzymic Analysis of (Talalay) ..................... Steroid. Adrenal. in Blood. Analysis by Counlercurrent Distribution (Carstensen) ................................................. Steroids. Analysis by Infrared Spectrometry (Rosenkrantz) .............. Stem&. Nmer Developments in the Analysis of. by Gas-Chromatography (Wotiz and Clark) ............................................

.

375

15 1

311 307

3

247

9

235

9 13

39 357

14

497

21 6 7

39 63 111

SUPP.

1

6 4 2 1 10

95 257 279 459 43

10

71

28 2 3

267 1 353

12 2

1 427

30

227

6

97

9

217

7

77

5

65

26 8

47 119

9 2

227 1

18

339

376 CUMULATIVE SUBJECT INDEX. VOLUMES 1-30 AND SUPPLEMENT Steroids. Newer Developnrents in the Gas Chromabgraphic Determination of (Kuksis) ..................................................... Steroids. Separation and DetmniMtMn. by Gas Chromatography (Homing. Vanden Heuvel. and Creech) ................................. Steroids o f the Adrenal Gland. Chrvmahgraphic Separation (Haines and Karnemaat) ..................................... Stoppcd-Flow Method. Recent Deyelopmcnlc in. For the Study of Fast R e a c h (Hiromi) ........................................... Subzero Temprraiures in Biochemistry:S h R e a c h . The Use of (Douzou) ................................................... Succinic Dehydrogetwe Activrty. Determination of (Singer and Kearney) Sugars. A d y s i s of Mixtures. by Paper and CeUulare Column Chromatography (Hough) ...................................... Sugars. the Determination of Structure of Certain Natural Products Confuining Sugam (Hanessian) ................................. Sugars. in Polysaccharides. Detmnination. New C o b React- for (Dische) ..................................................... Su@ases. Assay (Dodgson and Spencer) .......................... Suljhydryl Groups. Detffminorionin Biological Substances (Chinard and Hellerman) .................................... Temperature-Jump Method for Measuring the R& of Fast R e a c h . a Practical Guide to (Yapel and Lumry) ........................... Thmnodynanric Flow Me& in Biochemistry: Caloritnctty. Dntrimetry and Dilatonrclty (Jolicoeur) ........................................ Thiamine. M e w for the Detmmhation of (Mickelsen and Yamamoto) Thiocfic Acid. Assay o f (Stokstad. Seaman. Davis. and Hutner) ....... Thyroid Gland. Chrmabgraphic Analysis of RadiwcfiveIodine Compounds from (Roche. Lissitzky. and Michel) ............................ TirSus. Aliphatic AmmoniumS& in the Assay of Acidic Polysaccharides from (Scott) .................................................. Tissues. Body. Chemical DetenninntMn of Adrenalim and Noradrenahne in (Persky) ..................................................... Tissues. Determidion of Ethyl Alcohol in (Lundquist) ................ Trace Elnnent Analysis. C o n t a m i e in. and Its Control (Thiers) ..... Transaminuse. Deknnination of (Aspen and Meister) ................ Uhqumme. Detmnination of (Crane and Dilley) .................... UDP-Enzyme Systam. Measurements of (Pontis and Leloir) ........... Ultraccntr@gal Analysk of Serum Lipoproteins (de Lalla and Gofman) Uhajiltrr Membranes in Biochemistry (Jacobs) ....................... Ultraviolet Spccbophotometry. E n z p k Micro Deknninations of Urit Acid. Hypoxanthine. X a h n e . Adenine. and Xadwpt.e&e by (Plesner and Kalckar) ........................................ Urea. Ammonk. and Ureate. The Determination of (Kaplan) ........... Urea. Gel Electrophoresis in B$fm Containing (Poulik) .............. Uric Acid. Enzymic Micro DelmniMtirmc. by Ultraviolet Spectrophotometry (Plesner and Kalckar) ........................................ Urinary Ne&al 17.Ke&steroids. Assay of (Engel) .................... Urine. Deknninaaon of 17.21 -Dihydrvqy-20-Ke&ster& in (Siber and Porter) ........................................... Urine. Human. Chemicut Determanation o j Estrogens in (Bauld and Greenway) .......................................

14

325

11

69

1

171

26

137

22 4

401 307

1

205

19

105

2 4

313 211

1

1

20

169

27 6 3

171 191 23

1

243

8

145

2 7 5 6 10 1 22

57 217 273 131 279 107 459 307

3 17 14

97 311 455

3 1

97 479

4

139

5

337

11

CUMULATIVE SUBJECT INDEX. VOLUMES 1-30 AND SUPPLEMENT Visual Biochemistly: New Insight into Structure and Function of the Genome (Vollenweider) ....................................... VitaminA. Determination of (Embree. Ames. Lehman. and Harris) ... VitaminA and Carotenoids. in Blood and Tissue.Micro&tennination of (McLaren. Read. Awdeh. and Tchalian) ....................... VitaminBe. Chemical and Microbiological Detennination of (Storvick. Benson. Edwards. and Woodring) ............................. VitaminB I Z .Microbiological Assay of (Hoff-Jorgensen) .............. VitaminBIZ.Microbiological Assay of (Skeggs) ...................... VitaminE Determination (Lehman) ................................ Vitamins. Infrared Analysis of (Rosenkrantz) ........................ Xanthine. EnzymicMicro Determination. by Ultraviolet Spectr-Ophotomehy (Plesner and Kalckar) ........................................ Xanthopterine. Enzymic Micro Determinations. UltravioLt Spectmpiwtometq (Plesner and Kalckar) ......................... X-Ray Diffraction. in the Study of Protein and Nwlkc Acid Structure (Holmes and Blow) .......................................... X-Ray D$fractk?n Analysis. The Growth and Preliminary Investigation of Protkn Nwlkc Acid Crystals for ................................. X-Ray Emission Spectrometry. Detennination of Elements by (Natelson and Whitford) ..................................... Zinc. DetenniMcion OJ in Biological Materials (Malmstrom) ........... Zone Electr@hesis (Kunkel) ..................................... Zone ELctrophuresis. at Constant Cuwent. Mobility Determination by (Waldmann-Meyer) ..........................................

377

28 4

201 43

15

1

12 1 14 2 5

183 81 53 153 407

3

97

3

97

13

113

23

243

12 3 1

1 327 141

13

47