Electrochemical Studies of Biological Systems Donald T. Sawyer, EDITOR University of
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Electrochemical Studies of Biological Systems Donald T. Sawyer, EDITOR University of
California
A symposium sponsored by the Division of Analytical Chemistry at the 172nd Meeting of the American Chemical Society San Francisco, Calif., August 30, 1976
ACS SYMPOSIUM SERIES 38
AMERICAN
CHEMICAL
SOCIETY
WASHINGTON, D. C. 1977
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
Library of Congress Œ
Data
Electrochemical studies of biological systems. (ACS symposium series; 38) Includes bibliographical references and index. ISSN 0097-6156 1. Electrochemical analysis—Congresses. 2. Biological chemistry—Congresses. I. Sawyer, Donald T . II. American Chemical Society. Division of Analytical Chemistry. III. Series: American Chemical Society. ACS symposium series; 38. QD115.E524 ISBN 0-8412-0361-X
Copyright ©
574.1'9283 ACSMC 8
76-30831 38 1-216
1977
American Chemical Society A l l Rights Reserved. N o part of this book may be reproduced or transmitted in any form or by any means—graphic, electronic, including photocopying, recording, taping, or information storage and retrieval systems—without written permission from the American Chemical Society. PRINTED IN T H E U N I T E D
STATES
OF
AMERICA
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
ACS Symposium Series Robert F. Gould, Editor
Advisory Board Donald G .
Crosby
Jeremiah P. Freeman E. Desmond Robert A .
Goddard Hofstader
J o h n L. Margrave N i n a I. M c C l e l l a n d J o h n B. Pfeiffer Joseph V . Rodricks A l a n C . Sartorelli Raymond B. Seymour Roy L. Aaron
Whistler Wold
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
FOREWORD The A C S S Y M P O S I U
a medium for publishin format of the SERIES parallels that of the continuing A D V A N C E S IN C H E M I S T R Y SERIES except that in order to save time the papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form.
As a further
means of saving time, the papers are not edited or reviewed except by the symposium chairman, who becomes editor of the book.
Papers published in the A C S S Y M P O S I U M
SERIES
are original contributions not published elsewhere in whole or major part and include reports of research as well as reviews since symposia may embrace both types of presentation.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
PREFACE electrochemistry has enjoyed a renaissance
during the past decade
because of its use for chemical characterization. In particular, the subdisciplines of organic, inorganic, and biological chemistry have found electrochemical methods uniquely effective for determining the stoichiometrics, thermodynamics, and kinetics of electron transfer reactions. Although cyclic voltammetry and controlled potential electrolysis are by far the most used electrochemical techniques, a number of new methodologies that combine electrochemical and spectroscopic measurements have been developed in recen This symposium was trochemical methods for the characterization of biological systems needs to be brought to the attention of chemists and biochemists.
M u c h of
biology and biochemistry involves oxidation-reduction processes, atomtransfer reactions, and electron-transfer reactions.
Because the theory
and principles of electrochemistry are concerned with the same kinds of processes, as well as with the thermodynamics and kinetics of heterogeneous redox processes, substantial synergistic benefits can result from a coordinated, rational application of electrochemical principles
and
theories to the electron-transfer and oxidation-reduction chemistry of biology. T h e twelve papers of the symposium provide a representative cross section of the kinds of electrochemical methodologies that are used to study biological systems.
They also illustrate the kinds of biological
problems that are being studied by such methods.
Beyond cyclic volt-
ammetry and controlled potential coulometry, the use of optically transparent thin-layer electrodes
( O T T L E ) , rotating ring-disc enzyme elec-
trodes, mediator titrants, differential capacitance and phenomena,
and differential
pulse
polarography
are
electrocapillary discussed.
The
applications range from the analysis of N T A and E D T A in water samples to the characterization of the redox chemistry for several metalloproteins. Several chapters emphasize the development of improved electrochemical techniques and instrumentation for the study of biological systems. However, the major emphasis of the papers is the study of the redox properties
of model compounds
for biological systems.
The
specific
systems include vitamin B i , cytochrome c, cytochrome c oxidase, metal 2
porphyrins, nitrogenase, mitochondrial superoxide dismutase, purines and pyrimidines, and a model for a mammalian heart.
vii
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
The assistance of H . B. Mark, Jr., and G . S. Wilson, who chaired the two sessions of the symposium, is gratefully acknowledged.
M y sincere
thanks to John Miller, Chairman of the Analytical Chemistry Division, for his support and encouragement in the organization of the symposium and to Marian M a n n for her assistance with the correspondence
and
manuscript preparation. Department of Chemistry
DONALD
T.
SAWYER
University of California Riverside, Calif. 92502 November 18, 1976
viii
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
1 Spectroelectrochemical
Investigation
of
Vitamin
Β
12
and Related Cobalamins HARRY B. MARK, JR., and THOMAS M. KENYHERCZ Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 45221 PETER T. KISSINGER Purdue University, W. Lafayette, Ind. 47907
This paper discusses three aspects of the spectroelectro chemical study of Vitamin time resolved spectral study mins u n d e r various experimental conditions; (ii) the electro chemical behavior of the cob(III)alamins, a n d (iii) the electro chemical behavior of 5'-deoxyadenosylcobalamin. Several pre viously unknown features concerning the redox chemistry of these unusual but important complexes are reported. The
Autooxidation
of
B s Under Various Conditions 12
Recent spectroelectrochemical investigations of the oxida tion of cob(I)alamins to cob(III)alamins in various media has yielded the previously unobserved sequence of intermediates and steps involved in the mechanism of this biologically important reaction. This sequence of intermediates disagrees with pre viously speculated mechanisms . The time resolved visible-UV spectra for the reoxidation of the electroreduced Vitamin B type compounds: cyanocobalamin (B ), aquocobalamin (B ) and dicyanocobalamin (B -CN) are reported below. The autooxidation of the cob(I)alamins were carried out under both air and inert argon atmospheres, the electrochemical reoxidation was also studied under these conditions. Mechanisms consistent with the qualitative kinetic data obtained from time resolved spectra are presented. The r e c e n t l y d e v e l o p e d m e r c u r y c o a t e d n i c k e l m i n i g r i d s y s t e m was e m p l o y e d i n a t h i n l a y e r e l e c t r o l y s i s c e l l a s a n o p t i c a l l y t r a n s p a r e n t e l e c t r o d e , Hg-Ni 0TTLE. L L = U T h e Hg-Ni 0TTLE c e l l was m o u n t e d i n a c o m p u t e r i z e d H a r r i c k r a p i d s c a n n i n g d u a l t e a m spectrophotometer,ϋ w h i l e a l l experimental p r o c e d u r e s , i n t e r f a c e design, e l e c t r o c h e m i c a l i n s t r u m e n t a t i o n and computer programming i n c l u d i n g data a c q u i s i t i o n , p r o c e s s i n g a n d r e d u c t i o n have been described p r e v i o u s l y . ^ A l l s o l u t i o n s (except that noted i n F i g u r e 2 ) w e r e ImM i n c o b a l a m i n a n d 1.0M i n N a S 0 i * a s t h e s u p porting e l e c t r o l y t e (the solutions f o r the Bi -CN experiments 1
2
2-8
12
12
12a
12
2
2
1
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
2
ELECTROCHEMICAL STUDIES OF BIOLOGICAL SYSTEMS
w e r e a l s o 0.1M i n N a C N ) . T h e s o l u t i o n s w e r e i n t r o d u c e d i n t o t h e OTTLE c e l l w i t h c o b a l t i n t h e +3 v a l e n c e s t a t e . T h e s o l u t i o n s were t h e n p o t e n t i o s t a t e d a t -1.0 v o l t s v s . SCE u n t i l t h e s p e c t r a changed completely t o t h a t o f t h e Co(I) species » ( s e et h e f i r s t spectrum o f F i g u r e 1) and remained c o n s t a n t ( f o r a p p r o x i mately o n e - h a l f h o u r ) . The c o b ( I ) a l a m i n s o l u t i o n s were a l l o w e d to undergo a u t o o x i d a t i o n i n e i t h e r t h e presence o f a i r d i f f u s i n g i n t o t h e OTTLE c e l l o r u n d e r a n i n e r t a r g o n a t m o s p h e r e . T h e p o t e n t i o s t a t was d i s c o n n e c t e d d u r i n g t h e s e a u t o c a t a l y s i s e x p e r i m e n t s . D u r i n g e l e c t r o r e o x i d a t i o n s t e p - w i s e ( 5 0 mv i n c r e m e n t ) p o t e n t i o s t a t i c p o l a r i z a t i o n ( p o t e n t i o s t a t e d a t each p o t e n t i a l u n t i l s p e c t r a ceased changing) under an i n e r t argon atmosphere was e m p l o y e d . C u r v e A o f F i g u r e 2 shows t h e t y p i c a l t r a n s i e n t s p e c t r u m o f a p a r t i a l l y a i r reoxidized s o l u t i o n o fcob(I)alamin while curve Β i s t h e spectrum o f aquocobalamin The p e a k s o f i n t e r e s t t h e o x i d a t i o n s e q u e n c e o c c u r a t 4 1 0 nm a n d 4 7 5 nm. D u r i n g t h e large scale preparation o fB i t was o b s e r v e d t h a t t h e r e l a t i v e r a t i o o f t h e 4 1 0 a n d 4 7 5 nm p e a k s v a r i e d m a r k e d l y depending on t h e r a t e a t w h i c h o x y g e n was i n t r o d u c e d i n t o t h e c o b ( I ) a l a m i n s o l u t i o n . F o r example, curve A o f F i g u r e 2 i s t h e s p e c t r a ob t a i n e d on bubbling oxygen through a r a p i d l y s t i r r e d B solu t i o n ( t h e 4 1 0 nm p e a k i s much l a r g e r t h a n t h e 4 7 5 nm p e a k ) . How e v e r , t h e 4 7 5 nm p e a k s i n t h e c u r v e s shown i n F i g u r e 3 a r e l a r g e r t h a n t h e 4 1 0 nm p e a k s . Inthe l a t t e r case, a i r d i f f u s e s i n t o the B s o l u t i o n s l o w l y f r o m t h e e d g e s o f t h e OTTLE c e l l . Thus i t i s f e l t t h a t t h e 4 1 0 a n d 4 7 5 nm p e a k s r e p r e s e n t t w o d i f f e r e n t s p e c i e s even though p r e v i o u s workers have r e p o r t e d both peaks a s being c h a r a c t e r i s t i c o fthe s o - c a l l e d B ! The s p e c t r a o b t a i n e d f o l l o w i n g t h e e x h a u s t i v e r e d u c t i o n o f B i , B , a n d B - C N a t - 1 . 0 v o l t s v s . SCE w e r e i d e n t i c a l c o b (I)alamin species corresponding t othose p r e v i o u s l y designated B s . Though t h e e x a c t c o o r d i n a t i o n geometry f o r c o b ( I ) a l a m i n i s unknown, i t has been s u g g e s t e d t h a t t h e b e n z i m i d a z o l e i s i n a base-off c o n f i g u r a t i o n — w i t h water molecules occupying each of the axial positions. As B a n d B appear t o r e o x i d i z e a t comparable r a t e s and have s i m i l a r time r e s o l v e d s p e c t r a l c h a r a c t e r i s t i c s , a l l argu m e n t s made f o r B a r e e q u a l l y a p p l i c a b l e t o B i . A l s o , i t w a s found t h a t t h e time r e s o l v e d s p e c t r a l sequences and r a t e s o f peak c h a n g e s w e r e v i r t u a l l y t h e same i n t h e p r e s e n c e o f a i r o r a r g o n . The t i m e r e s o l v e d s p e c t r a f o r t h e a i r r e o x i d a t i o n o f a c o b ( I ) alamin s o l u t i o n obtained by the exhaustive reduction o f cyanoc o b ( I I I ) a l a m i n i s shown i n F i g u r e s 1 a n d 3. F i g u r e 1 s h o w s t h a t t h e c o b ( I ) a l a m i n , a s m o n i t o r e d b y t h e 3 8 5 nm p e a k , i s v i r t u a l l y r e o x i d i z e d c o m p l e t e l y t o a c o b ( I I ) a l a m i n i n t h e f i r s t 100 seconds. The p e a k w h i c h d e v e l o p s a t 4 7 5 nm c o r r e s p o n d s t o a c o b ( I I ) a l a m i n , — ( d e s i g n a t e d h e r e a s B ) g r o w s t o a maximum i n t h e f i r s t 400 s e c o n d s and t h e n s l o w l y d e c r e a s e s f i n a l l y v a n i s h i n g a t a b o u t i 2 a
1 2 s
1 2 S
i 2 r
2
1 2 a
i 2
i 2
i 2
i 2 a
i 2
2 a
i 2 r
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
Vitamin
MARK E T AL.
o.o Ll 280
I
1
320
360
1
B
12
1
and Related
I
400 440 480 WAVELENGTH . Ν M
Cobalamins
1
1
*-
520
560
600
ο·β
Figure 1. Time-resolved spectra for the reoxidation of cob(I)alamin to cob(II)ahmin in 1.0M lSla S0 at pH = 7.0 in 0-400 sec 2
300
350
400
ll
450 WAVELENGTH
500
550
600
650
, NM
Figure 2. Spectra of the air-reoxidized product formed from the electroreduction of the cyanocob(IU)alamin in 0.1M NaN0 . A, partially reoxidized cob(II)ahmin; B, totally reoxidized cob(IH)alamin, aquocob(III)alamin. 3
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
4
ELECTROCHEMICAL STUDIES OF BIOLOGICAL SYSTEMS
1
5 χ 10 * s e c o n d s . T h e c y a n o c o b ( I I I ) a l a m i n p e a k o f 3 6 0 nm w h i c h b e g i n s t o d e v e l o p a t 10 s e c o n d s i s c o m p l e t e l y r e c o n v e r t e d t o Bi2 b y 1 0 s e c o n d s ( a s s e e n i n F i g u r e 3 ) . T h e p r e v i o u s l y u n r e p o r t e d i n t e r m e d i a t e e x h i b i t s a 4 1 0 nm p e a k , w h i c h o c c u r s i n t h e same q u a l i t a t i v e t i m e p e r i o d o f t h e c h a r a c t e r i s t i c 4 7 5 nm c o b ( I I ) a l a m i n p e a k , a p p e a r s i n 2 0 0 s e c o n d s , r e a c h e s a maximum b y 5 χ 1 0 s e c o n d s a n d h a s d i s a p p e a r e d b y a b o u t 2 χ 10 s e c o n d s . As t h e r e a r e n o p e a k s i n t h e r e g i o n o f 3 7 0 nm w h i c h w o u l d i d e n t i f y e i t h e r Co(I) o r C o ( I I I ) s p e c i e s ^ during t h i s time i n t e r v a l , i t i s f e l t t h a t t h e p r o n o u n c e d 4 1 0 nm p e a k i n d i c a t e s a s e c o n d , d i f f e r e n t c o b ( I I ) a l a m i n i n t e r m e d i a t e which w i l l be i d e n t i f i e d as Bi2r- A s m e n t i o n e d p r e v i o u s l y , t h e t i m e r e s o l v e d s p e c t r a f o r t h e a i r r e o x i d a t i o n o f cob(I)alamin obtained by t h e exhaus t i v e r e d u c t i o n o f a q u o c o b ( I I I ) a l a m i n , i s q u a l i t a t i v e l y t h e same as r e p r e s e n t e d i n F i g u r e s 1 and 3 However t h e a i r r e o x i d a t i o n of cob(I)alamin obtaine cob(III)alamin (in th q u i t e d i f f e r e n t a s shown i n F i g u r e 4. F i r s t o f a l l , d i c y a n o c o b ( I I I ) alamin i s t o t a l l y regenerated i n l e s s than 400 seconds. The i n c r e a s e / d e c r e a s e i n t h e 2 9 0 nm b a n d , t h e r i s e a n d f a l l o f t h e 475 nm ( c o b ( I I ) a l a m i n ) p e a k , t h e f i n a l r i s e o f t h e 3 6 8 nm p e a k p l u s t h e t o t a l l a c k o f a 4 1 0 nm p e a k i n d i c a t e s t h a t t h e r e o x i d a t i o n o f c o b ( I ) a l a m i n i n t h e presence o f excess c y a n i d e goes through only a c o b ( I I ) a l a m i n , B type i n t e r m e d i a t e . Electrochemical r e o x i d a t i o n o f cob(I)alamin, obtained by the e l e c t r o r e d u c t i o n o f B12 u n d e r a n a r g o n a t m o s p h e r e , g o e s t h r o u g h b o t h t h e B i 2 r ( 4 7 5 nm) a n d B i ( 4 1 0 a n d 4 7 5 nm) i n t e r m e d i a t e s i n t h e p o t e n t i a l r e g i o n f r o m - 0 . 6 0 t o -0.01 v o l t s v s . SCE y i e l d i n g B12 a t +0.10 v o l t s v s . S C E . T h e c o n d i t i o n s o f t h e e l e c t r o c h e m i c a l r e o x i d a t i o n e x p e r i m e n t s i n d i c a t e t h a t t h e 4 1 0 nm p e a k i s not i n d i c a t i v e o f an oxygen adduct type o f cobalamin s p e c i e s . The t i m e r e s o l v e d a u t o o x i d a t i o n s p e c t r a o f a c o b ( I ) i n i m i d e — (no b e n z i m i d a z o l e m o i e t y o n t h e c o r r i n r i n g s y s t e m ) w a s a l s o examined. The a b s o r b a n c e - p o t e n t i a l r e d u c t i o n c h a r a c t e r i s t i c s o f the c y a n o a q u o c o b ( I I I ) i n i m i d e — s t a r t i n g m a t e r i a l and t h e r a t e o f autooxidation a r e p a r a l l e l t o those o f Vitamin B itself indi c a t i n g t h a t t h e l a c k o f t h e b e n z i m i d a z o l e moiety has n o t appre c i a b l y a l t e r e d t h e redox p r o p e r t i e s o f t h e c e n t r a l c o b a l t i o n . However, t h e time r e s o l v e d a u t o o x i d a t i o n s p e c t r a o f t h e c o b ( I ) i n i m i d e d o n o t e x h i b i t a 4 1 0 nm p e a k . As v a r i a t i o n o f p e a k s i n t h e 4 0 0 - 5 0 0 nm r e g i o n h a v e p r e v i o u s l y been a s s o c i a t e d w i t h changes i n t h e a x i a l l i g a n d s , — i t i s a t t r a c t i v e t o s p e c u l a t e a t t h e p o i n t t h a t t h e two c o b ( I I ) a l a m i n s represent c o n f i g u r a t i o n s where t h e benzimidazole i s e i t h e r c o o r d i n a t e d t o t h e C o ( I I ) , a base-on form, o r where t h e benzimi d a z o l e has been r e p l a c e d by a w a t e r i n t h e y - a x i a l p o s i t i o n , a b a s e - o f f f o r m . T h o u g h t h e p o s s i b i l i t y e x i s t s t h a t t h e r e i s some a l t e r a t i o n i n t h e c o r r i n r i n g s t r u c t u r e could a l s o account f o r the observed behavior, i t i s f e l t t h a t redox changes i n t h e r i n g would be i r r e v e r s i b l e . The f a c t t h a t t h e c o b i n i m i d e and k
5
3
h
1
2
r
2 r
i 2
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
Vitamin
MARK E T AL.
B
îg
and Related
Cobalamins
5.0
0.0
I
1 1 I I I I I I \L 2 . · 2 * > 3 2 O M 0 4 0 0 4 4 O 4 t 0 5 2 O M 0 t 0 0 WAVELENGTH. NM
Figure 3. Time-resolved spectra from the reoxidation of cob(H)ahmin to a cob(III)alamin in 1.0M Na SO at pH = 7.0 in 630-70,000 sec 2
0.0
I
I
280
I
I
320
360
I
1
1
400 440 4*0 WAVELENGTH . NM
Il
1
1
520
560
*- 0.6 600
Figure 4. Time-resolved spectra for the reoxidation of cob(I)alamin to dicyanocob(Ul)alamin in 1.0M Na SO^ and 0.1M NaCN at pH = 11.0 in 0-410 sec 2
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
6
ELECTROCHEMICAL STUDIES O F BIOLOGICAL SYSTEMS
d i c y a n o c o b a l a m i n e x p e r i m e n t s show n o 4 1 0 nm b a n d i s c o n s i s t e n t with the base-on/base-off suggestion. Furthermore, i f t h i s i s c o r r e c t , then t h e B l r would be t h e base-on c o n f i g u r a t i o n and B r would be t h e b a s e - o f f form. Q u a l i t a t i v e l y , t h e time resolved spectra i n d i c a t e that t h e reoxidation o f cob(I)alamin i n the presence o fstoichiometric o r l e s s a m o u n t s o f CN~ f o l l o w s t h e r e a c t i o n s c h e m e i l l u s t r a t e d below. As p r e v i o u s l y m e n t i o n e d , t h e p r e s e n c e o f e x c e s s c y a n i d e i o n s a f f e c t s t h e r e o x i d a t i o n scheme s u c h t h a t e i t h e r t h e B J species i s n o t formed o r t h a t t h e o x i d a t i o n o f t h e B species i s kineti c a l l y favored. I t has been p o s t u l a t e d by o t h e r s — t h a t c o b ( I ) alamin autooxidizes t o cob(II)alamin with t h e e v o l u t i o n o f hydro gen a n d t h a t c o b ( I I ) a l a m i n s d i s p r o p o r t i o n a t e i n t h e m e c h a n i s m t o form cob(I) and c o b ( I I I ) alamin s p e c i e s . Under present e x p e r i mental c o n d i t i o n s t h i alamin i s not d i r e c t l c o n c l u s i o n s o f B i r k e e t a l Λ who e s t i m a t e d t h a t t h e t h e r m o d y namic and k i n e t i c parameters f o r such a d i s p r o p o r t i o n a t i o n a r e very unfavorable. However, t h e time r e s o l v e d s p e c t r a l sequence observed does n o t r u l e o u t t h e p o s s i b i l i t y t h a t B undergoes disproportionation. I f the rate o fdisproportionation o f B i s very slow compared t o t h e r a t e o f o x i d a t i o n o f B i t h e same time r e s o l v e d s p e c t r a would be o b t a i n e d . I t i s interesting to n o t e t h a t we d o n o t f i n d a n y d i r e c t e v i d e n c e f o r H e v o l u t i o n w h i c h i s e a s i l y o b s e r v e d ( t r a p p e d s m a l l b u b b l e s ) i n t h e OTTLE c e l l f o r s y s t e m s w h e r e i t o c c u r s . We h a v e b e e n u n a b l e t o i d e n t i f y t h e o x i d i z i n g agent(s) thus f a r . F u r t h e r m o r e , i t has n o t been p o s s i b l e t o c a l c u l a t e m e a n i n g f u l k i n e t i c p a r a m e t e r s f r o m the time and p o t e n t i a l r e s o l v e d s p e c t r a a s no q u a n t i t a t i v e d i f f u s i o n model has been p o s t u l a t e d . T h e r e f o r e , c a l c u l a t i o n s o f d i f f u s i o n i n t h e OTTLE t y p e c e l l c a n n o t b e made. F u r t h e r m o r e , we c a n n o t t e l l i f B i oxidizes directly t o both B and B J r a t d i f f e r e n t r a t e s o r t h a t i f B J results simply from a r a p i d e q u i l i b r i u m with B as i l l u s t r a t e d by the d o t t e d a r r o w i n t h e p r o p o s e d m e c h a n i s m u ( C o b a l t +2 c o m p l e x o f t h i s t y p e a r e a l w a y s l a b i l e . — ) T h e same a r g u m e n t a p p l i e s t o t h e interpretation o fB and B ' o x i d i z i n g t o c o b ( I I I ) a l a m i n . However, i t does appear t h a t t h e r e o x i d a t i o n o f B t o dicyanoc o b ( I I I ) a l a m i n o c c u r s much m o r e r a p i d l y t h a n t h e r e o x i d a t i o n o f either B or B l t o B o r B . D i f f u s i o n s t u d i e s a r e now i n p r o g r e s s a s w e l l a s a s i m i l a r s t u d y w i t h m e t h y l a n d 5'-deoxyadenosylcob(III)alamin. Q u a n t i t a t i v e studies o f the chemical and e l e c t r o c h e m i c a l o x i d a t i o n k i n e t i c s a n d m e c h a n i s m s w i l l b e reported a t a f u t u r e date. 2
i 2
2 r
i 2 r
1
2
r
i 2 r
2 S
2
2 S
i 2 r
2
2 r
i 2 r
i 2 r
i 2 r
1
1
The
2
r
2 r
Electrochemical
1 2
Behavior
2
r
i 2 a
of Cob(III)alamins
The e l e c t r o c h e m i c a l b e h a v i o r o f v i t a m i n B (cyanocob(III)a l a m i n ) and r e l a t e d c o b a l a m i n compounds i n aqueous media i s o f i 2
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
1.
MARK E T A L .
Vitamin
B
12
and Related
7
Cobalamins
importance f o r e l u c i d a t i n g t h e biomechanistic r e a c t i o n sequences which i n v o l v e cobalamin species. There has been c o n s i d e r a b l e study o f the redox processes o f cobalamins using the convention al e l e c t r o a n a l y t i c a ltechniques o f p o l a r o q r a p h y , * " coulom e t r y , * * * and c y c l i c v o l t a m m e t r y , * _ and d i v e r s e working e l e c t r o d e m a t e r i a l s such a s m e r c u r y * ' ~ and p l a t i n u m . i * H o w e v e r , t h e i n t e r p r e t a t i o n o f t h e e l e c t r o c h e m i c a l d a t a t o unam b i g u o u s l y determine even t h e most fundamental parameters such a s t h e t h e r m o d y n a m i c r e d o x p o t e n t i a l s , t h e n u m b e r o f e l e c t r o n s (nvalues) i n v o l v e d i n t h e e l e c t r o n t r a n s f e r s t e p s , and t h e sequence o f s t e p s i n t h e mechanism has n o t been p o s s i b l e because o f numer ous c o m p l i c a t i n g c o n d i t i o n s . The c o m p l i c a t i o n s encompass s t r o n g a d s o r p t i o n o f both r e a c t a n t and product, i r r e v e r s i b i l i t y o f t h e r e d o x r e a c t i o n s , unusual medium e f f e c t s i n v o l v i n g t h e s o l v e n t system and t h e s u p p o r t i n g e l e c t r o l y t e and marked v a r i a t i o n o f electrode kinetics wit new t e c h n i q u e s e m p l o y i n t h i n l a y e r e l e c t r o l y s i s c e l l s — h a v e been d e v e l o p e d w h i c h have proved u s e f u l t o t h e study o f t h e b a s i c redox p r o p e r t i e s o f c y t o chrome c . - ^ This paper r e p o r t s t h e r e s u l t s obtained by using t h i n l a y e r m i n i g r i d electrode c e l l s t o study the electrochemical and s p e c t r o e l e c t r o c h e m i c a l b e h a v i o r o f c y a n o c o b a l a m i n ( B ) , aquocobalamin ( B i ) , and dicyanocobalamin (Bi -CN). 9
3
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3 2
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S p e c t r o e l e c t r o c h e m i s t r y o f t h e Cobalamin Systems. As changes i n t h e valence o f c o b a l t , t h e c e n t r a l metal i o n o f t h e cobalamins, a r e r e f l e c t e d by d i s t i n c t changes i n t h e v i s i b l e ab s o r p t i o n s p e c t r a , a c o u p l i n g o f e l e c t r o c h e m i c a l and s p e c t r o s c o p i c m e a s u r e m e n t s was p e r f o r m e d t o e l u c i d a t e t h e r e d o x b e h a v i o r o f t h e cobalamins. S p e c t r o e l e c t r o c h e m i c a l experiments were c a r r i e d o u t u s i n g t h e o p t i c a l l y t r a n s p a r e n t t h i n layer e l e c t r o c h e m i c a l c e l l s (OTTLE) i n t h e presence and a b s e n c e o f t h e e l e c t r o n t r a n s f e r mediator, 2 , 6 - d i c h l o r o p h e n o l i n d o p h e n o l T h e cobalamin-containi n g OTTLE c e l l s w e r e p o t e n t i o s t a t e d w h i l e t h e o p t i c a l a b s o r b a n c e o f a peak o f i n t e r e s t and c u r r e n t l e v e l s were monitored. When both t h e a b s o r b a n c e stopped changing and t h e c u r r e n t l e v e l s had f a l l e n t o e s s e n t i a l l y z e r o (<. 0.1 μ Α ) , t h e s p e c t r u m o f t h e s o l u t i o n i n t h e OTTLE c e l l was r e c o r d e d . F i g u r e 5 s h o w s how t h e spectra of a B solution varies asthe applied potential i s changed. C u r v e 1 o f F i g u r e 5 f o r w h i c h t h e Hg-Ni m i n i g r i d e l e c t r o d e was p o t e n t i o s t a t e d a t 0.00 V i s a t y p i c a l s p e c t r u m f o r B (a c o b ( I I I ) a l a m i n ) w i t h c h a r a c t e r i s t i c p e a k s a t 520 and 550 n m . ^ The s p e c t r u m o b t a i n e d b y p o t e n t i o s t a t i n g a t - 0 . 6 0 0 V ( c u r v e 2 , F i g u r e 5 ) shows t h a t t h e c o n c e n t r a t i o n o f t h e c o b ( I I I ) a l a m i n s i s d e c r e a s i n g a s s e e n b y t h e d e c r e a s e i n t h e 5 2 0 - a n d 550-nm p e a k s and t h e d e v e l o p m e n t o f a new p e a k a t 4 7 5 nm. T h i s 475-nm p e a k i s t y p i c a l o f t h a t r e p o r t e d f o r B , a c o b ( I I ) a l a m i n s p e c i e s . ^ On p o t e n t i o s t a t i n g a t -1.0 V, t h e s p e c t r u m o b t a i n e d m a t c h e s t h a t o b t a i n e d b y o t h e r workers »**° f o r B i , a c o b ( I ) a l a m i n s p e c i e s w i t h a w e a k l y a b s o r b i n g b r o a d p e a k a t 5 6 0 nm a n d a t a p e r e d i 2
1 2
X 2 r
26
2 S
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
ELECTROCHEMICAL STUDIES O F BIOLOGICAL SYSTEMS
8
s h o u l d e r i n t h e r e g i o n o f 460 nm. The q u a n t i t a t i v e c h a n g e i n t h e v a r i o u s p e a k a b s o r b a n c e v a l u e s a s a f u n c t i o n o f a p p l i e d , p o t e n t i a l f o r B , B i , and B i - C N a r e shown i n F i g u r e s 6 t h r o u g h 13. C u r v e A o f F i g u r e 2 s h o w s t h e e f f e c t o f p o t e n t i a l a t a Hg-Ni m i n i g r i d e l e c t r o d e o n the a b s o r b a n c e o f t h e 550-nm p e a k o f B a s i t i s r e d u c e d . The B i i s t o t a l l y r e d u c e d t o a c o b ( I I ) a l a m i n o v e r a r e l a t i v e l y n a r r o w po t e n t i a l r a n g e ( a p p r o x i m a t e l y 200 mV) w i t h a n a b s o r b a n c e " h a l f w a v e p o t e n t i a l " o f a b o u t -0.63 V. T h e s l i g h t i n c r e a s e i n t h e a b s o r b a n c e b e t w e e n -0.8 a n d -1.0 V i s t h e r e s u l t o f t h e f u r t h e r r e d u c t i o n o f c o b ( I I ) a l a m i n t o c o b ( I ) a l a m i n w h i c h would be ex p e c t e d , a s B , a c o b ( I ) a l a m i n , e x h i b i t s a broad peak i n the r e g i o n o f 560 n m . ^ The e f f e c t o f p o t e n t i a l o n t h e a b s o r b a n c e a t 550 nm f o r t h e r e o x i d a t i o n o f c y a n o c o b ( I ) a l a m i n i s shown b y c u r v e Β o f F i g u r e 6. The q u a n t i t a t i v e r e o x i d a t i o n o f t h e c o b ( I ) - t o cob(II)alamin occurs ove V) a s shown i n F i g u r e 6 s e r v e d i n c u r v e s A and Β o f F i g u r e 7 ( a b s o r b a n c e v s . p o t e n t i a l c u r v e s f o r t h e 475-nm p e a k ; t h e c o b ( I I ) a l a m i n i n t h e same p o t e n t i a l r e g i o n . However, the r e o x i d a t i o n o f the c o b ( I I ) a l a m i n t o B o c c u r s o n l y when t h e p o t e n t i a l s a r e 400 mV p o s i t i v e t o t h o s e o f the r e d u c t i o n p o t e n t i a l s a s can be seen from the h y s t e r e s i s i n c u r v e s A and Β o f b o t h F i g u r e s 6 and 7 i n t h e -0.1 t o -0.7 V r a n g e . A s h o r t e r p o t e n t i a l s c a n , -1.0 t o -0.8 V , OTTLE e x p e r i m e n t w i t h t h e same c o n d i t i o n s a s F i g u r e 7 ( g o i n g o n l y t o t h e c o b ( I I ) a l a m i n ) s h o w e d t h e e x a c t same h y s t e r e s i s . I t i s i m p o r t a n t t o n o t e t h a t B a p p e a r s t o be c o m p l e t e l y r e g e n e r a t e d a s t h e op t i c a l absorbance e v e n t u a l l y r e t u r n s t o i t s i n i t i a l value (seen i n F i g u r e 6 ) . F i g u r e 7, h o w e v e r , a p p e a r s t o b e c o n t r a d i c t o r y w i t h r e s p e c t to the r e o x i d a t i o n p a r t o fthe above e x p l a n a t i o n . I f t h e 475-nm p e a k , c o r r e s p o n d i n g t o c o b ( I I ) a l a m i n f o r m a t i o n , i s t o be used a s a n a c c u r a t e i n d i c a t o r o f c o b ( I ) - , c o b ( I I ) - , o r v a r i o u s c o b ( I I I ) a l a m i n s p e c i e s b e i n g p r e s e n t , t h e n i t w o u l d seem t h a t i n a d d i t i o n t o r e f o r m i n g B , a n o t h e r c o b ( I I I ) a l a m i n may a l s o have been formed. S p e c t r a l d a t a o b t a i n e d o n the s u b s e q u e n t r e d u c t i o n o f the cobalamin formed f o l l o w i n g the r e o x i d a t i o n o f B ( c u r v e B, F i g u r e 7 ) , i n d i c a t e s a s l i g h t r i s e i n t h e i n i t i a l p o r t i o n o f t h e c o b a l a m i n a b s o r b a n c e - p o t e n t i a l wave ( m o n i t o r e d b y t h e d e v e l o p m e n t o f t h e 475-nm p e a k ) . The m a g n i t u d e o f t h i s a b sorbance-potential r i s e remains constant as the cobalamin i s re c y c l e d p o t e n t i o s t a t i c a l l y . T h e i n i t i a l r i s e d o e s n o t become a n appreciable p o r t i o n o f the B i absorbance-potential curve during t h e p o t e n t i o s t a t i c c y c l i n g p r o c e s s , b u t d o e s r e s e m b l e t h e be havior o fB shown i n c u r v e A o f F i g u r e 11. The same s e t o f e x p e r i m e n t s w a s p e r f o r m e d f o r B i - C N a t a Hg-Ni e l e c t r o d e . A s can be seen from F i g u r e s 8 and 9 d i c y a n o c o b alamin behaves q u i t e s i m i l a r l y to B i , the o n l y d i f f e r e n c e being the p o t e n t i a l r e g i o n where B - C N r e o x i d a t i o n o c c r s . The hys t e r e s i s i n the dicyanocobalamin r e o x i d a t i o n i s s i g n i f i c a n t l y l e s s than f o r B ( o n l y a b o u t 180 mV d i f f e r e n c e i n t h e h a l f - a b s o r b a n c e i 2
2 a
2
i 2
2
1 2 S
1 2
i 2
X 2
i 2
2
i 2 a
2
2
i 2
i 2
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
1.
Vitamin
MARK E T A L .
B
12
and Related
9
Cobahmins
Figure 5. Spectropotentiostatic curves for the re duction of B in a Hg-Ni OTTLE. vs. SCE; 2, B potentiostated at —0.600 V vs. SCE; 3, B potentiostated at -0.660 V vs. SCE; 4, B potentiostated at —1.000 V vs. SCE. 12
12
12
a* Y
,08
k
L
ι
0,2
0
ι
ι
-0.2
ι
-0,4
Potential
Volts
-0,6 vs
ι
-0,8
1—
-M>
SCE
Figure 6. Potential-absorbance curves for the reduction (A) and oxidation (B) of 1.2 mM B monitored at 550 nm. 1.0 M Na SO^; 0.1 Μ NaN0 ; pH 7.0; Hg-Ni minigrid; cell thickness, 0.017 cm. 12
2
3
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
ELECTROCHEMICAL STUDIES O F BIOLOGICAL SYSTEMS
J
I
Ο
I
Ι
-0.
Figure 7. Potential-absorbance curves for the re duction (A) and oxidation (B) of 1.2 mM Β i2 momtored at 475 nm. 1.0 M Na SO>; 0.1 M NaNO ; pH 7.0; Hg-Ni minigrid; cell thickness, 0.017 cm. 2
Potential
s
Volts
vs
SCE
Figure 8. Potential-absorbance curves for the reduction (A) and oxidation (B) of 1.2 mM B CN monitored at 580 nm. 1.0 M Na S0 ; O.J M KCN; pH 10.4; Hg-Ni minigrid; cell thickness, 0.017 cm. 12
2
4
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
1.
MARK E T A L .
Vitamin
B
and Related
12
11
Cobalamins
p o t e n t i a l s f o r B12-CN r e d u c t i o n a n d r e o x i d a t i o n c u r v e s i n F i g u r e s 8 and 9 ) . Vitamin B was a l s o i n v e s t i g a t e d a t a H g - N i e l e c t r o d e i n a s i m i l a r set of experiments. C u r v e s A o f F i g u r e 1 0 ( t h e 530-nm p e a k ) a n d F i g u r e 11 ( t h e 475-nm p e a k ) show t h a t B undergoes an u n u s u a l t w o - s t e p p r o c e s s , a s i l l u s t r a t e d b y t h e b r e a k s a t a b o u t -0.06 and -0.65 V , b e f o r e c o m p l e t e c o n v e r s i o n t o a c o b ( I I ) a l a m i n s p e c i e s . C u r v e A o f F i g u r e s 1 0 a n d 11 shows t h a t t h e c o b ( I l ) a l a m i n i s then reduced to cob(I)alamin a s the p o t e n t i a l i n c r e a s e s f r o m -0.8 t o -1.0 V. On r e o x i d a t i o n , c u r v e Β o f F i g u r e 1 1 , t h e c o b ( I ) a l a m i n i s reversibly reoxidized to cob(II)alamin o v e r t h e same p o t e n t i a l r a n g e a s i n t h e negative scan. H o w e v e r , t h e Β c u r v e s i n b o t h F i g u r e s 1 0 and 11 i n d i c a t e t h a t t h e r e o x i dation which corresponds to a q u a n t i t a t i v e regeneration of B i a from the c o b ( I I ) a l a m i n s p e c i e s i s a s i n g l e step process which oddly occurs at a p o t e n t i a ( c a . -50 mV).u To c h e c k t h e u n i q u e s p e c t r o e l e c t r o c h e m i c a l p r o p e r t i e s o f B , other samples of B from d i f f e r e n t s o u r c e s and p r e p a r a t i o n s were examined, and, a l s o , the e l e c t r o c h e m i c a l p r e p a r a t i o n was r e c y c l e d a n u m b e r o f t i m e s . The s p e c t r o e l e c t r o c h e m i c a l b e havior at a p a r t i c u l a r wavelength f o r B from the v a r i o u s pre p a r a t i o n s gave s p e c t r o p o t e n t i o s t a t i c curves(OTTLEgrams) i d e n t i c a l with those presented herein. A l s o , s p e c t r o p o t e n t i o s t a t i c c y c l i n g of B gave r e p r o d u c i b l e s e t s of c u r v e s . I t i s i n t e r e s t i n g t h a t the a b s o r b a n c e - p o t e n t i a l waves f o r B i n t h e OTTLE e x p e r i m e n t s do n o t c o r r e s p o n d t o a n y p e a k s i n t h e c y c l i c v o l t a m m o g r a m o f B a t t h e same e l e c t r o d e . H o w e v e r , t h e t h r e e absorbance-poten t i a l "waves" f o r the r e d u c t i o n o f B i do c o r r e l a t e reasonably w e l l w i t h t h e t h r e e waves o b s e r v e d i n the p r e v i o u s l y r e p o r t e d polarography of B . > * . To understand the unusual two-step process i n the reduction of B t o a c o b ( I I ) a l a m i n s p e c i e s and to determine i f the e l e c t r o d e i t s e l f i s p l a y i n g a r o l e i n the e l e c t r o n t r a n s f e r k i n e t i c s , the mediator 2,6-dichlorophenolindop h e n o l was u s e d i n c o n j u n c t i o n w i t h t h e A u m i n i g r i d e l e c t r o d e . — The m e d i a t o r f u n c t i o n s a s t h e p r i m a r y e l e c t r o n t r a n s f e r a g e n t b e t w e e n t h e e l e c t r o d e and a r e d o x s y s t e m t h a t has v e r y s l o w heterogeneous e l e c t r o n t r a n s f e r r a t e s . Thus, the mediator a c c e l erates the o v e r a l l e l e c t r o c h e m i c a l r e a c t i o n of the system of i n t e r e s t . The c h o i c e o f t h i s m e d i a t o r was d e t e r m i n e d b y t h e po t e n t i a l r e g i o n o f i n t e r e s t i n t h i s c a s e (+0.2 t o -0.2 V v s . S C E ) . The A u m i n i g r i d e l e c t r o d e was u s e d t o e l i m i n a t e t h e p o s s i b i l i t y of o x i d a t i o n of the working e l e c t r o d e m a t e r i a l i n t h i s p o t e n t i a l r e g i o n and b e c a u s e t h e c y c l i c v o l t a m m o g r a m s o f B exhibited a m o r e w e l l - d e f i n e d wave a t a n i n t e r m e d i a t e p o t e n t i a l , a n d B appeared t o be l e s s s t r o n g l y adsorbed on the Au e l e c t r o d e . The a b s o r b a n c e c h a n g e s o f t h e 525-nm ( B i ) and 475-nm ( B ) b a n d s a s a f u n c t i o n o f t h e a p p l i e d p o t e n t i a l a r e shown i n F i g u r e s 1 2 a n d 1 3 r e s p e c t i v e l y . C u r v e A o f F i g u r e 1 2 s h o w s o n l y one " w a v e " w i t h a h a l f - a b s o r b a n c e p o t e n t i a l o f -0.15 V i n t h e +0.2 t o -0.6 V i 2 a
1
2
a
2
i 2 a
i 2 a
i 2 a
i 2 a
1
2
a
1 2 a
2 a
9
2 6
2
7
i 2 a
1
2
a
i 2 a
i 2 a
2 a
i 2 r
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
ELECTROCHEMICAL STUDIES O F BIOLOGICAL SYSTEMS
Potential
Volts vs SCE
Figure 9. Potential-absorbance curves for the reduction (A) and oxidation (B) of 1.2 mM B CN monitored at 475 nm. 1.0 M Na S0 ; 0.1 M KCN; pH 10.4; Hg-Ni minigrid; cell thickness, 0.017 cm. 12
2
4
Figure 10. Potential-absorbance curves for the re duction (A) and oxidation (B) of 0.9 mM Β i2a monitored at 530 nm; 1.0 M Na SO,; 0.1 M NaNO ; pH 7.01; Hg-Ni minigrid; cell thickness, 0.017 cm. 2
s
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
1.
Vitamin
MARK E T A L .
B
12
and Related
13
Cobalamins
2,08
-0.2
-0.4
Potential
.
-0.6
-as
Volts vs SCE
-1.0
Figure 11. Potential-absorbance (A) and oxidation (B) of 0.9 mM B monitored at 475 nm. 1.0 M Na SO 0.1 M NaNO ; pH 7.0; Hg-Ni minigrid; cell thickness, 0.017 cm. 12a
2
i;
s
Figure 12. Potential-absorbance curves for the reduction (A) and oxi dation (B) of 0.9 mM B monitored at 525 nm. 1.0 M Na SO 0.1 M NaNO ; pH 7.0; Au minigrid and 2,6-dichlorophenolindophenol; cell thickness, 0.021 cm. 12a 2
0 Potential .
-0.2 Volts
vs
i;
s
-0.4 SCE
.13
Λ Ο
I
09
Figure 13. Potential-absorbance curves for the reduction (A) and oxi dation (B) of 0.9 mM B monitored at 475 nm. 1.0 M Na SO>; 0.1 M NaN0 ; pH 7.0; Au minigrid and 2,6-dichlorophenolindophenol; cell thickness, 0.021 cm. 12a 2
,07
3
0.2
0 Potential
-0.2 Volts
-0.4 vt
SCE
-0.6
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
ELECTROCHEMICAL STUDIES OF BIOLOGICAL SYSTEMS
14
p o t e n t i a l r e g i o n s c a n n e d . From c u r v e Β o f F i g u r e 12 i t c a n b e seen t h a t the produced B i s totally reoxidized t oB a with l i t t l e h y s t e r e s i s ( h a l f - a b s o r b a n c e p o t e n t i a l o f a b o u t -0.09 V f o r t h e r e o x i d a t i o n ) i n t h e p r o c e s s . T h e c h a n g e s i n t h e 475-nm a b s o r b a n c e p e a k ( F i g u r e 13) a g a i n i n d i c a t e o n l y one "wave" f o r t h e g e n e r a t i o n and s u b s e q u e n t r e o x i d a t i o n o f t h e B r w i t h h a l f - a b sorbance p o t e n t i a l s which correspond f a v o r a b l y t o those o f the B " w a v e s " i n F i g u r e 12. T h u s , t h e m e d i a t o r - A u e l e c t r o d e s y s tem r e f l e c t s a m o r e t y p i c a l r e d o x b e h a v i o r a s i t e l i m i n a t e s t h e unusual h y s t e r e s i s e f f e c t where the r e o x i d a t i o n o f B from B occurred a t p o t e n t i a l s negative t o the i n i t i a l reduction process ( s e e F i g u r e s 1 0 and 1 1 ) . H o w e v e r , a n e x a m i n a t i o n o f t h e m a g n i t u d e o f t h e a b s o r b a n c e c h a n g e o f b o t h t h e 5 2 5 - a n d 475-nm p e a k s s h o w s t h a t i t i s e x a c t l y t h e same a s t h a t f o r t h e f i r s t a b s o r bance waves f o r t h e Hg-Ni e l e c t r o d e — no m e d i a t o r system (see F i g u r e s 1 0 and 1 1 ) , i n d i c a t i n B i s only p a r t i a l l The t o t a l s p e c t r u m o f t h e s o l u t i o n p o t e n t i o s t a t e d a t -0.6 V a l s o i n d i c a t e s t h a t part o f the B ( a p p r o x i m a t e l y 35%) i s u n r e a c t e d . The same r e s u l t w a s a l s o o b t a i n e d f r o m t h e η - v a l u e s t u d i e s ( T a b l e I I I ) a t b o t h t h e H g - N i and A u m i n i g r i d e l e c t r o d e s . T h u s , t h e unusual two p o t e n t i a l processes necessary t o t o t a l l y reduce B appear t o be independent o f both working e l e c t r o d e m a t e r i a l a n d mediator p a r t i c i p a t i o n . Neither the spectra f o r B o r B -CN showed any s i g n i f i c a n t r e d u c t i o n e m p l o y i n g Au m i n i g r e d - m e d i a t o r s y s t e m . No s a t i s f a c t o r y m e d i a t o r w i t h t h e n e c e s s a r y o p t i c a l a n d p o t e n t i a l c h a r a c t e r i s t i c s t o e x p l o r e t h e -0.6 t o -1.0 V p o t e n t i a l a b s o r b a n c e b e h a v i o r a t a H g - N i m i n i g r i d e l e c t r o d e has b e e n f o u n d to date. The h a l f - a b s o r b a n c e p o t e n t i a l s f o r t h e c o b a l a m i n s p e c i e s i l l u s t r a t e d i n F i g u r e s 6 through 13 a r e p r e s e n t e d i n T a b l e I . i 2 r
1 2
1 2
1 2 a
1 2 a
1
2
r
1 2 a
i 2 a
i 2 a
i 2
i 2
η-Value Determination. C o n t r o l l e d p o t e n t i a l coulometry with a t h i n l a y e r m i n i g r i d e l e c t r o d e system^°ιM was used t o determine t h e number o f e l e c t r o n s ( η - v a l u e ) f o r v a r i o u s waves found i n t h e c y c l i c voltammograms o f each o f the c o b a l a m i n s . ^ A typical charge vs. time curve f o r B i s shown i n F i g u r e 14. I t w a s n e c e s s a r y t o e x t r a p o l a t e t h e f i n a l s l o p i n g p o r t i o n o f t h e Q-t curve back t o t = 0 t o c o r r e c t f o r edge e f f e c t s i n h e r e n t i n the t h i n l a y e r c e l l s y s t e m . ^ - The m e t h o d f o r c o r r e c t i o n and c a l c u l a t i o n o f η - v a l u e s f o r c h a r g i n g and r e s i d u a l c u r r e n t b y r e p e a t i n g t h e e x p e r i m e n t o n t h e s u p p o r t i n g e l e c t r o l y t e has b e e n d e scribed p r e v i o u s l y . T h e η-values, as well as the i n i t i a l and f i n a l v a l u e s o f t h e a p p l i e d p o t e n t i a l s t e p s , a r e shown i n T a b l e I I . F o r t h e t h r e e c o b ( I I I ) a l a m i n s y s t e m s u s i n g t h e Hg-Ni m i n i g r i d e l e c t r o d e , o n l y one r e d u c t i o n w a v e i s o b s e r v e d i n t h e -1.0 V v s . SCE p o t e n t i a l r e g i o n and t h e η - v a l u e o b t a i n e d i n e a c h c a s e f r o m t h e Q v s . t d a t a w a s e f f e c t i v e l y two ( 2 ) y i e l d i n g a c o b ( I ) a l a m i n p r o d u c t i n each c a s e w h i c h c o n f i r m s p o l a r o g r a p h i c and other p r e v i o u s l y reported r e s u l t s * * " As expected, the 1 2
2
8
9
2 2
3 7
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
Vitamin
MARK E T A L .
Table I .
B
12
and Related
Cobalamins
Half-Absorbance Potentials*
Monitor^ Working ed electrodes system Cobalamin Reduction Oxidation wavelength (OTTLE) species mV vs. SCE mV vs. SCE (nm) Hg-Ni
B, "
Hg-Ni
B i 2
Hg-Ni Hg-Ni
Bi2-CN*B, -C!N^
Hg-Ni
B,2a^
Hg-Ni
Au Au Au Au
+ + + +
mediator mediator mediator mediator
2
W
C
2
-625 (-875) -625 -875 -850 -825 -910 -60 -635
-180 (-880) -185 -875 -690 -689 -910
-634 -880
-176 -878
— —
— —
550 475 580 475 530
-188
B
B, " Bi -CN^ 2
C
2
2a
525 475
-93 -110
-155 -140
B, ^
a
The cobalamin concentration is 1 mM. It should be pointed out that no relationship between the half-absorbance potentials and the reversible potentials for these species exists at this time. Supporting electrolyte = 1.0 M Na2S0 . < Supporting electrob
4
\Λ \Λ \λ
\ 0J
300
Time
Figure 14. Charge-time curve for the application of a potential step from 0.000 to -0.970 to +0.100 V vs. SCE at a Hg-Ni OTTLE. (A) background, 1.0 M Na SO 0.1 M NaNOs. (B) B , 0.6 mM B , 1.0 M Na SO 0.1 M NaN0 . 2
lt
12
2
i>
h
3
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
16
ELECTROCHEMICAL STUDIES OF BIOLOGICAL SYSTEMS
background breakdown p o t e n t i a l s h i f t s p o s i t i v e on t h e Au m i n i g r i d e l e c t r o d e a n d o v e r l a p s t h e C o ( I I I ) - C o ( I ) wave o b s e r v e d o n t h e H g - N i e l e c t r o d e . On A u e l e c t r o d e s , B and B i - C N e x h i b i t s a s m a l l prewave a t -0.3 V a n d a broad i r r e v e r s i b l e a p p e a r i n g wave a t a b o u t - 0 . 8 V. T h e B e x h i b i t s a s i n g l e very broad p o o r l y d e f i n e d w a v e w i t h a p e a k p o t e n t i a l a t a b o u t - 0 . 5 V. P o t e n t i a l step experiments w i t h B -CN gave f r a c t i o n a l η-values r e g a r d l e s s of t h e magnitude o f t h e f i r s t a p p l i e d p o t e n t i a l , t h e meaning o f which c o u l d not be i n t e r p r e t e d from t h e e l e c t r o c h e m i c a l data. The η - v a l u e f o r B o n a p o t e n t i a l s t e p t o -0.6 V a l s o y i e l d e d a f r a c t i o n a l v a l u e o f a b o u t 0.65. I t h a s b e e n p r e v i o u s l y r e p o r t e d b y some w o r k e r s t h a t o n l y B can be c o u l o m e t r i c a l l y r e duced t o B ( c o b ( I I ) a l a m i n ) i n a o n e - e l e c t r o n s t e p a t a mer cury electrode a t intermediate p o t e n t i a l s ^ A t a Hg-Ni m i n i g r i d , t h e three c o b ( I I I ) a l a m i n s were c o u l o m e t r i c a l l y reduced t o c o b ( I ) a l a m i n a t -0.97 and t h e Q v s . t c u r v e a r e o x i d a t i o n η-value equal t o 2 found which i n d i c a t e s a v i r t u a l l y q u a n t i t a t i v e r e o x i d a t i o n t o B i . F r a c t i o n a l η-values ob tained f o r B and B i - C N d e r i v e d cob(I)alamins i n d i c a t e s t h a t o n l y p a r t o f these c o b ( I I I ) a l a m i n s a r e regeneraged even a t p o s i t i v e p o t e n t i a l s . However, these r e o x i d a t i o n η-values a r e d i f f i c u l t t o i n t e r p r e t as complicating e f f e c t s a r i s e from the i n t e r f e r i n g m e r c u r y ( I I ) c y a n i d e s p e c i e s w h i c h f o r m i n some c a s e s . A t t h e Au m i n i g r i d e l e c t r o d e s o n l y p a r t o f t h e c o b ( I I I ) a l a m i n s are reduced a s e x p l a i n e d above; however, i t appears from t h e r e o x i d a t i o n η-values t h a t t h e f r a c t i o n reduced i s q u a n t i t a t i v e l y regenerated a tp o s i t i v e p o t e n t i a l s . Thea b i l i t y o f the base-off cobalamin t o form complexes w i t h metal ions a l s o obscures t h e issue.~ F u r t h e r η - v a l u e i n f o r m a t i o n was o b t a i n e d b y f i x e d wave length o p t i c a l monitoring techniques coupled with c o n t r o l l e d p o t e n t i a l coulometry t odetermine η-values f o r appropriate redox processes i n v o l v i n g vitamin B . As mentioned p r e v i o u s l y B was c h o s e n f o r t h i s i n v e s t i g a t i o n a s e a r l i e r s t u d i e s h a d s u g gested that B underwent o n l y a s i n g l e two-electron r e d u c t i o n s t e p . * - . T h e m o n i t o r i n g w a v e l e n g t h o f 4 7 5 nm was c h o s e n a s t h i s peak i s i n d i c a t i v e o f t h e presence ( o r absence) o f a c o b ( I l ) a l a m i n . Monitoring t h i s wavelength, while c o u l o m e t r i c a l l y t h e number o f e l e c t r o n s t r a n s f e r r e d t o t h e c o b a l a m i n i n t h e p r o c e s s i s m e a s u r e d , y i e l d s t h e η - v a l u e f o r each s t e p o f t h e mech anism. Table I I I summarizes t h e r e s u l t s o ft h i s s p e c t r o e l e c t r o chemical study. I t i s e v i d e n t from t h e growth and decay o f t h e 475-nm p e a k t h a t a o n e - e l e c t r o n r e d u c t i o n d o e s o c c u r a t i n t e r mediate p o t e n t i a l s and t h a t t h i s s p e c i e s can undergo a f u r t h e r o n e - e l e c t r o n t r a n s f e r t o form c o b ( I ) a l a m i n . The η - v a l u e i n t h i s case cannot be determined d i r e c t l y because o f i n t e r f e r e n c e from background. This cob(I)alamin i s r e a d i l y r e o x i d i z e d t o a cob( I l ) a l a m i n ; η value equals one. E x i s t i n g experimental c o n d i t i o n s again d i d not allow f o r an accurate determination o f the n-value 1 2
1
2
2
a
1 2
X 2 a
1 2 a
x 2 r
2 6
3 1
2
i 2 a
2
i 2
i 2
9
22
33
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
i 2
1.
MARK E T A L .
Table I I .
Vitamin
B
12
and Related Cobalamins
17
n- • Value Results
Minigrid working electrode system
Potential step region mV vs. SCE
No. of electrons = η
Species
Reduction Coulometry 0 to -970 B -CN*>
Hg-Ni
1.90 2.00 1.96 0.013 0.126 1.37 0.65
12
B,2a*'
0 to -500 +300 to -400 + 100 to-1000 +300 to -600
Au
B,2 a
C
C
e
B -CN -* B -CN > 12
Û
12
B,2a*'
C
Hg-Ni Bi2-CN *
0.51 0.38 0.013 0.125 1.40 0.65
Bl2a
-500 toO -400 to +300 -1000 to+100 -600 to +300
Au
B,2 ' a
c
Bi2-CN '* fl
B -CN -
a b
X2
Bi2a ' f l
C
a
b
Supporting electrolyte = 1.0 M Na S0 . Supporting electrolyte = 0.1 M KCN. Supporting electrolyte = 0.1 M NaN0 . The cobalamin concentration is 1 mM. 2
4
c
3
Table I I I .
Spectropotential Step Λ-Values for B12
Working electrode system (OTTLE) Hg-Ni
a
Potential step V vs. SCE To From Rest -0.755 Rest -0.755 -1.000 -0.755
-0.755 0.200 -0.755 -1.000 -0.755 0.200
Monitored wavelength No. of electrons (nm) 475 475 475 475 475 475
0.98 a
0.99 0.93 1.04 a
Catalytic process, η > 2.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
18
ELECTROCHEMICAL STUDIES OF BIOLOGICAL SYSTEMS
for
the r e o x i d a t i o n to a c o b ( I I I ) a l a m i n .
Conclusions. T h e r e s u l t s d e s c r i b e d a b o v e show t h a t , i n s p i t e o f the f a c t t h a t the e l e c t r o k i n e t i c data are vary compli c a t e d , u n u s u a l , and v i r t u a l l y i m p o s s i b l e t o i n t e r p r e t m e c h a n i s t i c a l l y , the o p t i c a l monitoring o f the s o l u t i o n composition using t h e OTTLE t e c h n i q u e g i v e s a g o o d p i c t u r e o f t h e n e t o r o v e r a l l redox r e a c t i o n s that take place. The f i r s t o b s e r v a t i o n o f s i g n i f i c a n c e i s t h a t a l l t h r e e c o b ( I I I ) a l a m i n s ( B , B12-CN, and B a ) u n d e r g o a q u a n t i t a t i v e o n e - e l e c t r o n r e d u c t i o n t o e i t h e r t h e same o r s i m i l a r c o b ( I I ) a l a m i n ( B i ) s p e c i e s a t i n t e r m e d i a t e p o t e n t i a l s i n t h e 0.0 t o -0.8 V range. Previous electrochemical s t u d i e s by other groups had claimed that only B c o u l d be reduced t o Β at intermediate p o t e n t i a l s . » ~ A s t h e p o l a r o g r a p h i c and c y c l i c v o l t a m m e t r i c s t u d i e s d i d not i n d i c a t range f o r B o r B -CN attempted c o u l o m e t r i c r e d u c t i o n s a t such p o t e n t i a l s . However, t h e OTTLE r e s u l t s c l e a r l y show t h a t t h e o n e - e l e c t r o n r e a c t i o n i s common t o a l l t h e s p e c i e s b u t t h a t i n t h e c a s e o f B a n d B - C N the k i n e t i c s o f the r e a c t i o n i s u n u s u a l l y slow even w i t h r e s p e c t t o t h e s l o w s c a n r a t e s e m p l o y e d i n p o l a r o g r a p h y and t h e c y c l i c voltammetry reported here. These one-electron processes f o r B a n d B - C N show u p o n l y d u r i n g p o i n t - b y - p o i n t p o t e n t i o s t a t i c OTTLE t e c h n i q u e s . The r e a s o n f o r t h e e x t r e m e l y s l o w k i n e t i c s o f t h i s o n e - e l e c t r o n r e a c t i o n has n o t b e e n e l u c i d a t e d a t t h i s t i m e . The e l e c t r o n t r a n s f e r r a t e i s f a s t e n o u g h f o r w a v e s t o b e ob served p o l a r o g r a p h i c a l l y or w i t h c y c l i c voltammetry only i n the Β c a s e . U n d e r t h e same c o n d i t i o n s t h e f u r t h e r r e d u c t i o n o f a l l the cobalamin systems from the Co(II) t o Co(I) o x i d a t i o n s t a t e was q u a n t i t a t i v e a n d " r e v e r s i b l e " . The a p p a r e n t h y s t e r e s i s i n v o l v i n g C o ( I I ) to C o ( I I I ) cobalamins i s not p r e s e n t l y w e l l u n d e r s t o o d b u t may r e s u l t f r o m c h e m i c a l r e a c t i o n s i n v o l v e d i n t h e mechanism. It i s i n t e r e s t i n g to note that B -CN i s t o t a l l y re-formed ( s h o w n i n c u r v e B, F i g u r e 9 ) w h i l e c y a n o c o b ( I ) a l a m i n d o e s n o t completely reoxidize to B . T h i s s u g g e s t s t h a t B and B i - C N may r e o x i d i z e b y s e p a r a t e p a t h w a y s . B e c a u s e o f t h e m a g n i t u d e o f the i r r e v e r s i b i l i t y o fthe B r e d o x c o u p l e and a l s o t h e f a c t that B i s n o t t o t a l l y r e - f o r m e d (some B i appears t o be a minor r e o x i d a t i o n product), i t i s thought that on electrochemical reoxidation that B i s t h e i n i t i a l p r o d u c t f o r m e d and t h a t B subsequently forms on a l i g a n d exchange r e a c t i o n i n v o l v i n g the cyanide i n s o l u t i o n ( i n i t i a l l y r e l e a s e d i n t o the s o l u t i o n phase during the reduction o f B t o B i r » a s shown b y t h e f a c t t h a t a -0.1 t o -0.8 V OTTLE e x p e r i m e n t ( c o b ( I I I ) a l a m i n cob(II)alamin) with vitamin B shows t h e same l a r g e i r r e v e r s i b i l i t y i n d i c a t i n g t h a t t h e CN" i s l o s t i n t h e f i r s t r e d u c t i o n s t e p ) . T h i s l i g a n d exchange r e a c t i o n o f B i w i t h C N " has b e e n shown t o b e v e r y f a s t . — However, the net r a t e i s slow because o f the d i l u t e i 2
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In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
1.
Vitamin
MARK E T AL.
B
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and Related
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s o l u t i o n s employed. The c o m p l e t e r e g e n e r a t i o n o f B i s not pos s i b l e a s some CN' i s l o s t , p r o b a b l y t h r o u g h t h e f o r m a t i o n o f s t a b l e m e r c u r y ( I I ) cyanide complexes. I t was n o t e d t h a t t h e p e r cent recovery increased on a d d i t i o n of excess cyanide which i s c o n s i s t e n t w i t h t h i s i n t e r p r e t a t i o n . With r e s p e c t to B -CN, the f i n a l p r o d u c t i s formed d i r e c t l y upon r e o x i d a t i o n o r t h e f o l l o w up l i g a n d e x c h a n g e r e a c t i o n b e t w e e n t h e c o n c e n t r a t e d c y a n i d e s o l u t i o n a n d t h e B , f o r m e d b y t h e l o s s o f one e l e c t r o n f r o m B (with water molecules i n the a x i a l p o s i t i o n s ^ - ) , i s very fast. At t h i s time i t i s i m p o s s i b l e t o d i s t i n g u i s h between t h e s e two m e c h a n i s m s f o r t h e r e o x i d a t i o n o f B - C N . H o w e v e r , i t s h o u l d be n o t e d t h a t t h e B s p e c t r a ( a s i n d i c a t e d b y t h e 475-nm p e a k ) i n b o t h B and B - C N r e a c t i o n s a r e v i r t u a l l y i d e n t i c a l . P e r h a p s t h e m o s t u n u s u a l and d i f f i c u l t t o u n d e r s t a n d r e s u l t is the observation of f o r the reduction of B and t h e a p p a r e n t η - v a l u e d a t a i n d i c a t e t h a t B convertst o B ( a b o u t 65%) a t p o t e n t i a l s a r o u n d -0.05 V a t b o t h t h e H g - N i and Au e l e c t r o d e s w h i l e i t i s n e c e s s a r y t o r a i s e t h e p o t e n t i a l t o g r e a t e r t h a n -0.6 V w h e r e t h e s e c o n d w a v e c o r r e s p o n d i n g t o t h e r e d u c t i o n o f the r e m a i n i n g 35% o f the B i s o b s e r v e d . The m o s t obvious c o n c l u s i o n t h a t f i t s the data q u a l i t a t i v e l y i s t h a t the B e m p l o y e d i n t h e s e e x p e r i m e n t s was i m p u r e a n d c o n t a i n e d a b o u t 35% o f B itself (B was p r e p a r e d f r o m B ) . However, a s p o i n t e d o u t a b o v e , we f o u n d t h a t a l l b a t c h e s o f B gave the same r e s u l t s w h i c h a g a i n w o u l d n o t b e e x p e c t e d t o r e m a i n c o n s t a n t i f the various synthesis routes y i e l d e d only p a r t i a l conversion. A l s o t h e s p e c t r a l and p o l a r o g r a p h i c p r o p e r t i e s d o n o t s u g g e s t t h a t any a p p r e c i a b l e c o n c e n t r a t i o n o f B r e m a i n u n c o n v e r t e d and a l s o a r e i d e n t i c a l w i t h t h e s p e c t r a and p o l a r o g r a p h i c p r o p e r t i e s of vitamin B produced by the t o t a l l y d i f f e r e n t procedures. Furthermore, there i s considerable other i n d i r e c t evidence t h a t t h e r e i s no s i g n i f i c a n t u n c o n v e r t e d B i n the B samples. Note f i r s t o f a l l t h a t t h e r e i s no-0.6 V p o l a r o g r a p h i c wave f o r B t h a t c o r r e s p o n d s t o t h e wave f o r t h i s s e c o n d B species. ( I t i s i n t e r e s t i n g to note t h a t previous p o l a r o g r a p h i c s t u d i e s had r e f e r r e d t o t h e w a v e a t -0.6 V a s a n i m p u r i t y . ) > * * A l though the c y c l i c voltammogram f o r B does e x h i b i t an a n o d i c p e a k a t -0.28 V w h i c h c o u l d b e i n d i c a t i v e o f Hg o x i d a t i o n i n t h e p r e s e n c e o f a c o m p l e x i n g l i g a n d , t h i s w a v e i s a b o u t 5 0 mV p o s i t i v e t o t h e peak c o r r e s p o n d i n g t o m e r c u r y - c y a n i d e f o r m a t i o n i n the B c y c l i c voltammogram and t h e r e i s no c o r r e s p o n d i n g c a t h o d i c sweeps o f B i t s e l f . F i n a l l y high p r e s s u r e l i q u i d chroma t o g r a p h y ( u s i n g a m i x t u r e o f e i t h e r 80% i s o p r o p y l a l c o h o l and 20% w a t e r , o r 65% methanol and 35% w a t e r , a t 2000 p s i o n a n A m i n e x A - 4 c o l u m n , w i t h d e t e c t o r w a v e l e n g t h s e t a t λ 360 nm) o n Bi h a s e x h i b i t e d two c l o s e l y s p a c e d y e t d i s t i n c t p e a k s b o t h w i t h r e t e n t i o n times t h a t are d i f f e r e n t than B . Also, a thin l a y e r c h r o m a t o g r a p h i c c o m p a r i s o n o f B and B u s i n g a 65% 1 2
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In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
ELECTROCHEMICAL
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STUDIES O F BIOLOGICAL
SYSTEMS
m e t h a n o l a n d 3 5 % w a t e r s o l v e n t s y s t e m has shown t h a t B and Bi2a have r e l a t i v e f r o n t s , t h o u g h no s e p a r a t i o n o f B a itself was o b s e r v e d . I t i s f e l t . t h a t r i n g s t r u c t u r e d i f f e r e n c e s would n o t a c c o u n t f o r t h e two u n i q u e B a s p e c i e s , a s t h e B a p r e p a r e d f r o m t h r e e t e c h n i q u e s ( p o t e n t i o s t a t i c , b i o l o g i c a l and c h e m i c a l ) would not g i v e i d e n t i c a l 65/35 r a t i o o f c o n c e n t r a t i o n s . Moreo v e r , i t i s h a r d t o u n d e r s t a n d how two d i f f e r e n t r i n g s , w h i c h w o u l d b e e x p e c t e d t o b e common t o a l l c o b a l a m i n s , e x h i b i t d r a s t i c reduction potential differences for B and n o t f o r B o r B CN. T h u s , i t i s a t t r a c t i v e t o s p e c u l a t e t h a t t h e two s p e c i e s r e present d i f f e r e n c e s i n a x i a l l i g a n d c o n f i g u r a t i o n . The simplest a n s w e r w o u l d b e t h a t one o f t h e B i species c o n t a i n s water molec u l e s i n t h e X and Y p o s i t i o n s ( t h e " b a s e - o f f " f o r m ) w h i l e t h e o t h e r i s i n t h e c o n f i g u r a t i o n w i t h one w a t e r i n t h e X p o s i t i o n and t h e 5 , 6 - d i m e t h y l b e n z i m i d a z o l e i n t h e Y p o s i t i o n ( t h e " b a s e on" f o r m ) . T h e s p e c t r o e l e c t r o c h e m i c a t h a t t h e two B specie T h u s i u s has shown t h a t t h e X p o s i t i o n o f B i s very l a b i l e ( r a t e c o n s t a n t s o f about 170-2300 M - s " ) . — However, no measurem e n t s h a v e b e e n made o n t h e Y p o s i t i o n b e n z i m i d a z o l e - H 0 e x c h a n g e r a t e s . — I t i s p o s s i b l e t h a t t h i s exchange c o u l d be very slow. The f a c t t h a t t h e d i a q u o c o b ( I I I ) i n a m i d e ( h a v i n g n o b e n z i m i d a z o l e a t t a c h e d t o t h e c o r r i n r i n g s i d e c h a i n ) has b e e n r e p o r t e d t o b e d i f f i c u l t t o r e d u c e ( t f w — -0.7 V ) — i s c o n s i s t e n t b u t n o t p r o o f of the "base-on"-"base-off" explanation. This f a c t suggests that t h e " b a s e - o n " a q u o c o b ( I I I ) a l a m i n f o r m has a c o n f i g u r a t i o n f a v o r a b l e t o r e d u c t i o n ( t h e -0.15 V w a v e ) and t h e " b a s e - o f f " f o r m which would c l o s e l y correspond t o a diaquocob(III)inamide c o n f i g u r a t i o n i s d i f f i c u l t t o r e d u c t (-0.6 V w a v e ) . - ^ H o w e v e r , r e c e n t s p e c t r o e l e c t r o c h e m i c a l s t u d i e s b y L e x a and S a v e a n t ^ - h a v e shown that a tplatinum grid electrodes B does not e x h i b i t t h i s two w a v e o n e - e l e c t r o n b e h a v i o r . O n l y one w a v e i s o b s e r v e d a t a b o u t 0.0 V. F u r t h e r m o r e , t h e y h a v e a l s o shown t h a t t h e d i a q u o c o b ( I l l ) i n a m i d e o x i d i z e s m e r c u r y m e t a l s p o n t a n e o u s l y and f o r m s t h e c o b ( I I ) i n a m i d e . Thus, t h e r e p o r t e d v a l u e s f o r t h i s compound * a r e i n c o r r e c t and r e a l l y c o r r e s p o n d t o t h e s u b s e q u e n t e l e c t r o chemical r e d u c t i o n o f the Cob(II)inamide. Thus, i t appears t h a t t h e m e r c u r y e l e c t r o d e i s i n some w a y t h e c a u s e o f t h i s two w a v e one-electron observation. I t i s o b v i o u s t h a t t h e r e a r e many u n a n s w e r e d q u e s t i o n s c o n c e r n i n g r a t e s o f the m i c r o s c o p i c precesses i n v o l v e d i n the redox c h e m i s t r y o f cobalamin complexes. However, the macroscopic r e sultant e f f e c t o felectrode potential i n s o l u t i o n composition i s now w e l l d e f i n e d . W i t h t h i s b a s i c o v e r a l l m e c h a n i s t i c i n f o r m a t i o n , a m o r e c o m p r e h a n s i v e s t u d y o f t h e e l e c t r o d e k i n e t i c s and time resolved s p e c t r a l s t u d i e s on p o t e n t i a l step experiments on t h e s e a n d o t h e r c o b a l a m i n s u n d e r v a r i a b l e c o n d i t i o n s o f pH, s u p p o r t i n g e l e c t r o l y t e , and e l e c t r o d e m a t e r i a l may e l u c i d a t e a l l t h e steps i n the o v e r a l l mechanism. i 2
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In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
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B
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The E l e c t r o c h e m i c a l B e h a v i o r o f 5 ' - D e o x y a d e n o s y l - C o b a l a m i n enzyme B i T T
(Co-
The u n d e r s t a n d i n g o f t h e r e d o x p r o c e s s e s o f t h e c o b a l a m i n coenzyme, 5'-deoxyadenosylcobalamin (coenzyme B o r A d e n - B ) , is fundamental i n c o n s t r u c t i n g a v a l i d r e a c t i o n sequence f o r cob a l a m i n compounds i n n a t u r e . H i l l O f p a r t i c u l a r i m p o r t a n c e i s t h e o x i d a t i o n s t a t e o f c o b a l t i n A d e n - B and i t s i n f l u e n c e o n the r e a c t i v i t y of the cobalamin species. As a matter of notat i o n a l e a s e and t o m a i n t a i n c o n s i s t e n c y w i t h e x i s t i n g v i t a m i n B c o n c e p t s , t h e c o b a l a m i n c o e n z y m e has b e e n p r i m a r i l y i n t e r preted i n terms of a C o and a n a d e n s y l c a r b a n i o n . — R e d o x mesomers c o n s i s t i n g o f a C o and a n a d e n o s y l r a d i c a l o r a C o and a n a d e n o s y l c a r b o n i u m i o n h a v e a l s o b e e n f o r m a l l y c o n s i d e r e d . A l t h o u g h numerous p u b l i c a t i o n s have assumed the c o b a l t t o be i n t h e +3 o x i d a t i o n s t a t e , i s a v a i l a b l e . Some e v i d e n c t h e c o b a l t o f t h e c o b a l a m i n s h o u l d b e c o n s i d e r e d as a d i v a l e n t species.^-Z! S i m i l a r e l e c t r o c h e m i c a l and s p e c t r o e l e c t r o c h e m i c a l t e c h n i ques u t i l i z i n g a n amalgamated g o l d m i n i g r i d e l e c t r o d e i n a t h i n l a y e r c o n f i g u r a t i o n have been employed t o examine t h e r e d o x sequence o f 5'-deoxyadenosylcobalamin. This i n v e s t i g a t i o n suggests t h a t the cobalamin coenzyme undergoes a s i n g l e e l e c t r o n r e d u c t i o n to form v i t a m i n B ç , i n d i c a t i n g cleavage of the c o b a l t - c a r b o n b o n d . The r e o x i d a t i o n o f t h e r e d u c e d s y s t e m c o n t a i n i n g t h e B ^ o c c u r s v i a two c o n s e c u t i v e s i n g l e e l e c t r o n t r a n s f e r s r e s u l t i n g i n the q u a n t i t a t i v e formation of vitamin B , aquocobalamin. High performance l i q u i d chromatography confirmed t h a t 5'-deoxyadenos i n e i s the u l t i m a t e form of the cleaved 5'-deoxyadenosyl moiety. These r e s u l t s i n d i c a t e t h a t 5'-deoxyadenosyl-cobalamin i s reduced by a s i n g l e e l e c t r o n to form the c o b ( I ) a l a m i n , B , w h i c h i s t h e common r e d u c t i o n p r o d u c t o f a l l c o b a l a m i n s p e c i e s . — The f o r m a t i o n o f t h e c o b ( I ) a l a m i n , B , f r o m A d e n - B i s s i g n i f i c a n t as numerous p u b l i c a t i o n s ^ have a l l u d e d t o t h e f a c t t h a t due t o t h e h i g h n u c l e o p h i l i c i t y o f t h e c o b ( I ) a l a m i n s p e c i e s i t may b e t h e b i o l o g i c a l l y a c t i v e f o r m o f t h e c o e n z y m e . The r e s u l t reported herein i s the f i r s t electrochemical evidence f o r the f o r m a t i o n o f the c o b ( I ) a l a m i n from the cobalamin coenzyme. That B can be c o m p l e t e l y r e o x i d i z e d t o B , a c o b ( I I I ) a l a m i n , i s s i g n i f i c a n t i n t h a t e x i s t i n g c y c l i c biochemical mechanisms i n volve Aden-B a s the i n i t i a l l y r e a c t i v e s p e c i e s . ^ Furthermore, r i b o n u c l e o t i d e r e d u c t a s e has shown s p e c i f i c a c t i v i t y t o w a r d B s and A d e n - B i n t h e p r e s e n c e o f ô'-deoxyadenosine.^- -»- -!*1 2
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1 2
Acknowledgments T h i s r e s e a r c h was s u p p o r t e d i n p a r t b y t h e N a t i o n a l S c i e n c e F o u n d a t i o n , NSF CHE76-04321 and t h e N a t i o n a l I n s t i t u t e s o f H e a l t h , GM-22713-01.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
22
electrochemical studies of biological systems
Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
14. 15.
16. 17. 18. 19. 20.
Pratt, J. M., "Inorganic Chemistry of Vitamin B ," Academic Press, New York, Ν. Y., 1972, pp. 15-17. Huennekens, F. M., in "Biological Oxidations," Thomas P. Singer, Ed., Interscience Publishers, New York, Ν. Y., pp. 482-502. Tackett, S. L . , Collat, J. W., and Abbot, J. C., Biochemis try, (1963), 2, 919. Collat, J. W. and Abbot, J. C., J. Amer. Chem. Soc., (1964), 86, 2308. Schrauzer, G. N., Deutsch, E. and Windgassen, R. J., J. Amer. Chem. Soc., (1968), 90, 2441. Yamada, R., Shimizu, S. and Fukui, S., Biochemistry, (1968), 2, (7), 1713. Rudiger, H., Eur Birke, R. L . , Brydon, , Boyle, , Chem., (1974), 52, 237. Kenyhercz, T. M. and Mark, Jr., Η. B., Anal. Lett., (1974), 7, 1. Heineman, W. R., Norris, B. J. and Goelz, J. Anal. Chem., (1975), 47, 79. Heineman, W. R., DeAngelis, T. P. and Goelz, J., Anal. Chem., (1975), 47, 1364 Kenyhercz, Τ. Μ., DeAngelis, T. P., Norris, B. J., Heineman, W. R. and Mark, Jr., H. B., J. Amer. Chem. Soc., (1975), 98, 2469. However, experiments were performed with the minigrid elec trode area increased to occupy the entire cell volume to determine if the edge effects from the diffusion of unreduced cob(III)alamins from solution not in immediate contact with the minigrid affected the time resolved spectra. No signifi cant difference was observed. Strojek, J. W., Gruver, G. and Kuwana, T., Anal. Chem., (1969), 41, 481. Mark, Jr., H. B., Wilson, R. M., Miller, T. L . , Atkinson, T. V., Yacynych, A. M., and Woods, H., "The On-Line Computer in New Problems in Spectroscopy: Applications to Rapid Scanning Spectroelectrochemical Experiments and Time Resolved Phosphorescence Studies" in "Information Chemistry; Computer Assisted Chemical Research Design," S. Fujiwara and H. B. Mark, Jr., Eds., University of Tokyo Press, Tokyo, Japan, 1975, pp. 3-28. Beaven, G. H. and Johnson, Ε. Α., Nature, (1955), 176, 1264. Ref. 1, p. 184. Ref. 1, p. 20 to 27. Ref. 1, p. 55 The cyanoaquocol(III)inimide was prepared by a previously described method; Ref. 1, p. 294. 12
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
1.MARKETAL. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41.
23
Vitamin B and Related Cobalamins 12
Basolo, F. and Pearson, R. G., "Mechanisms of Inorganic Re actions," John Wiley and Sons, Inc., New York, Ν. Y . , pp. 141-144. Diehl, H., Sealock, R. R., and Morrison, J., Iowa State Coll. J. S c i . , (1950), 24, 433. Diehl, H., Morrison, J . I., and R. R. Sealock, Experientia, (1951), 7, 60. Diehl, J., and Morrison, J . I., Rec. Chem. Prog. (1972), 31, 15. Boos, R. N., Carr, J . E . , and Conn, J . B., Science, (1953), 117, 603. Jaselskis, B. and Diehl, H., J. Am. Chem. Soc., (1959), 81, 4345. Jaselskis, B. and Diehl, H . , J . Am. Chem. Soc., (1958), 80, 2147. Collat, J . W., an (1962), 4, 59. Tackett, S. L . , Ph.D. Thesis, Ohio State University, 1962. Kratochvil, B., and Diehl, H., Talanta, (1966), 13, 1013. Hogenkamp, H. P. C. and Holmes, S., Biochemistry (l970), 9, 1888. Lexa, D. and L'hoste, J . M., in "Biological Aspects of Elec trochemistry," G. Milazzo, P. E. Jones, and L. Rampazzo, Ed., Birkhauser Verlag, Stuttgart, 1971, pp. 395-404. Abd-el-Nabey, Β. Α., J . Electroanal. Chem., (1974), 53, 17. Das, P. K. et al., Biochim. Biophys. Acta., (1967), 141, 644. Tackett, S. L. and Ide, J . W., J . Electroanal. Chem. (l971 ), 30, 510. Swetik, P. G., and Brown, D. G., J. Electroanal. Chem., (1974), 51, 433. Kenyhercz, T. M. and Mark, J r . , Η. B., in preparation. Murray, R. W., Heineman, W. R., and O'Dom, G. W., Anal. Chem., (1967), 39, 1666. Provided by Dr. E. A. Deutsch, Department of Chemistry, University of Cincinnati. Beaven, G. H. and Johnson, Ε. Α., Nature (London), (1955), 176, 1264. It should be pointed out the reaction B = [H ] B (hydroxycob(III)alamin) has a pk of 7.8 and a more negative reduction potential than B : H. O. A. H i l l , "Inorganic Biochemistry," Vol. 2, G. Eichcon, Ed., Elsevier, New York, Ν. Y., 1973, Chapter 30. This proton equilibrium is un doubtedly fast and, thus, only the reduction of the B will be observed in these OTTLE experiments. McDuffie, B., Anderson, L. B., and Reilley, C. N., Anal. Chem., (1966), 38, 883. Cotton, F. A. and Wilkinson, G., "Advanced Inorganic Chemis try," 3d ed., Interscience, New York, Ν. Y., 1972, p. 519. Thusius, D. J . Am. Chem. Soc., (1971), 93, 2629. +
+
12a
12b
a
12a
12s
42. 43. 44.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
24 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72.
ELECTROCHEMICAL STUDIES OF BIOLOGICAL SYS Kenyhercz, T. M., Yacynych, Α. Μ., and Mark, J r . , H. B., Anal. Lett., (1976), 9, 203. Reference 6, p. 4346. Reference 11, p. 1889. Lexa, D. and Saveant, J . M., University of Paris, Private Communication, 1976. Babior, Β. M. in "Cobalamin: Biochemistry and Pathophysiol ogy," Β. M. Babior, Ed., John Wiley and Sons, New York, N.Y., 1975, p. 141. Pratt, J . M., "Inorganic Chemistry of Vitamin B12," Academic Press, London, 1972, p. 296. Mahler, H. R. and Cordes, Ε. Η., "Biological Chemistry," Harper and Row, Evanston, Ill., 1971, p. 427. H i l l , J . Α., Pratt, J . M., and Williams, R. J. P., J . Theor. Biol., (1962), 3, 423 Hogenkamp, H. P. C . Biochem. Biophys. H i l l , J . Α., Pratt, J . M. and Williams, R. J . P., J . Chem. Soc., (1964), 5149. Pratt, J . M., J . Chem. Soc., (1964), 5154. Huennekens, F. M. in "Biological Oxidations," T. P. Singer, Ed., John Wiley and Sons, New York, Ν. Y . , 1968, p. 483. Hogenkamp, H. P. C. and Holmes, S., Biochem., (1970), 9, 1889. Cotton, F. A. and Wilkinson, G., "Advanced Inorganic Chemis try," John Wiley and Sons, New York, Ν. Y. 1972, p. 888. Hughes, M. N., "The Inorganic Chemistry of Biological Pro cesses," John Wiley and Sons, New York, Ν. Y . , 1974, p. 187. Costa, C . , Puzeddu, A. and Reisenhofer, E . , Bioelectrochem. Bioenerg., (1974), 1, 29. H i l l , H. A. O. in "Inorganic Biochemistry," G. L. Eichhoren, Ed., Elsevier, New York, Ν. Y . , 1975, p. 1076. Babior, Β. M., Acc. Chem. Res., (1975), 8, 378. Abeles, R. H. and Dolphin, D., Acc. Chem. Res., (1976), 9, 114. Seki, H., Shida, T . , and Imamura, Μ., Biochem. Acta., (1974), 372, 106. H i l l , H. A. O., Pratt, J . M. and Williams, R. J . P., Disc. Farad. Soc., (1969), 16S. Kratochvil, B. and Diehl, H., Talanta, (1966), 13, 1013. Nowick, L. and Pawelkiewicz, Bull. Acad. Pol. Sci. C l . II., (1960), 17., 433. Johnson, A. W. and Shaw, N., Proc. Chem. Soc., (1960), 420. Bernhauser, K., Gaiser, P., Muller, O., Muller, E . , and Gunter, F . , Biochem. (1961), 333, 560. Johnson, A. W., Mervyn, L., Shaw, N., and Smith, E. L., J . Chem. Soc., (1963), 4146. White, Α., Handler, P., and Smith, E. L., "Principles of Biochemistry," McGraw H i l l , St. Louis, Mo., 1973, p. 1173. Schrauzer, G. N. and Sibert, J . W., J . Amer. Chem. Soc., (1970), 92, 1022, and references therein.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
1.MARKETAL. 73. 74.
Vitamin B and Related Cobalamins 12
25
Hamilton, Α., Yamada, R., Blakley, R. L., Hogenkamp, H.P.C., Looney, F. D., and Winfield, M. E., Biochem. (1971), 10, 347. Tamao, Y. and Blakley, R. L., Biochem., (1973), 12, 24.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
2 Bioelectrochemical Modelling of Cytochrome c CHARLES C. Y. TING and JOSEPH JORDAN 152 Davey Laboratory, Department of Chemistry, Pennsylvania State University, University Park, Penn. 16802 MAURICE GROSS Laboratoire d'Electrochimie et Chimie-Physique du Corps Solide, Université Louis Pasteur, BP 296 R/8, 67008 Strasbroug, France Numerous papers have in recent years been devoted to electrochemical studies of porphyrin cally, an investigation chrome c) is conspicuous by its absence. Heme c was first prepared from the naturally occurring protein in a classical piece of work by Theorell (1). Subsequently both heme c and its equatorial ligand (porphyrin c) became accessible by the synthetic route (2,3) outlined in Figure 1. The salient feature of porphyrin c is the bis-cysteinated substitution on the ring, which is unique in cytochrome c among hemoproteins (4). The corresponding substituents in hemoglobin and myoglobin are vinyl groups (5,6). In the present paper, we report some preliminary findings on the electrochemical behavior of porphyrin c and heme c. Experimental Materials. Porphyrin c and heme c were synthesized ad hoc using procedures referred to earlier in this write-up. Yields and elemental analyses are summarized in Table I. Table I Compound
Mol. % 1 Wt. Yield
Porphyrin c 805.0 40 Heme c 917.8 90
% Theory 0 6.08
Elemental Analysis Fe %S % Ν Actual Theory Actual Theory Actual 8.05 10.44 10.10 0.22 7.97 8.94 9.16 6.05 6.99 6.81
Authenticity was verified with the aid of the spectra illustrated in Figure 2 recorded with the aid of a Bausch and Lomb 505 Spectro photometer and quartz cells whose optical pathlength was 1 and 0.1 cm. P.C. Polarography. Current-voltage curves were recorded at a conventional (dropping mercury electrode (dme) which had 26
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
2.
TING E T A L .
Bioelectrochemical
the f o l l o w i n g c h a r a c t e r i s t i c s : c i r c u i t ) = 4.55 s e c o n d s .
Modelling
m
=
1.66
of Cytochrome
c
27
mg p e r s e c o n d ; t ( o p e n
C y c l i c V o l t a m m e t r y . K e m u l a ' s h a n g i n g chrop m e r c u r y e l e c t r o d e (hdme) s e r v e d a s i n d i c a t o r e l e c t r o d e . P o t e n t i a l s c a n r a t e s i n a r a n g e b e t w e e n 0.01 and 5 0 v o l t * s e c " w e r e u s e d . 1
Coulometry. Current-time i n t e g r a l s were determined a t appro p r i a t e c o n t r o l l e d p o t e n t i a l s , corresponding to well-defined polar o g r a p h i c d i f f u s i o n c u r r e n t d o m a i n s . The c a t h o d e was a m e r c u r y p o o l o f 2.60 s q . cm. Instrumentation, Solvents, Supporting E l e c t r o l y t e s , etc. A l l e x p e r i m e n t s w e r e c a r r i e d o u t a t 25°C. On s o l u b i l i t y c o n s i d e r a t i o n s DMF and w a t e r w e r e u s e d a s s o l v e n t s f o r heme c and p o r p h y r i n c r e s p e c t i v e l y . 0.1 M l y t e i n a l l experiments t h r o u g h o u t , u s i n g a s a t u r a t e d a q u e o u s £alomel r e f e r e n c e e l e c t r o d e (SCE) a n d a p l a t i n u m f o i l a u x i l i a r y c o u n t e r e l e c t r o d e . A l l e l e c t r o c h e m i c a l measurements were performed w i t h the a i d o f a m u l t i purpose instrument equipped w i t h advanced s o l i d s t a t e o p e r a t i o n a l a m p l i f i e r a n d f e e d b a c k c i r c u i t s , v i z , t h e M o d e l 170 E l e c t r o c h e m i c a l S y s t e m s u p p l i e d by £ r i n c e t o n A p p l i e d R e s e a r c h (PAR) C o r p o r a t i o n , P r i n c e t o n , N.J. O u t p u t s i g n a l s were a u t o m a t i c a l l y c o r r e c t e d f o r i R d r o p s a n d r e c o r d e d o n a b u i l t - i n X-Y pen r e c o r d e r a n d / o r w i t h t h e a i d o f a d i g i t a l o s c i l l o s c o p e ( M o d e l 1090 w i t h M o d e l 9 0 p l u g - i n u n i t , N i c o l e t Instrument Corporation, Madison, Wisconsin). The s c o p e was e q u i p p e d w i t h a mi n i - c o m p u t e r w h i c h had c a p a b i l i t i e s o f s t o r i n g d a t a i n a 4 0 9 6 χ 4 0 9 6 a r r a y memory a s f a s t as 5 y s e c p e r d a t a p o i n t . The c o u p l i n g o f t h e PAR i n s t r u m e n t w i t h t h e o s c i l l o s c o p e a l l o w e d measurement o f f a s t l i n e a r sweep voltammograms w i t h an a c c u r a c y o f 1 p e r c e n t . Whenever a p p r o p r i a t e , t o t a l c u r r e n t s were c o r r e c t e d f o r r e s i d ual c u r r e n t s to y i e l d the corresponding f a r a d a i c c u r r e n t s . Poten t i a l s are r e p o r t e d i n accordance w i t h the Stockholm Sign Conven t i o n o f t h e I n t e r n a t i o n a l U n i o n o f P u r e and A p p l i e d C h e m i s t r y ( 7 ) , i . e . , t h e more c a t h o d i c ( r e d u c i n g ) a p o t e n t i a l t h e more n e g a t i v e i t s assignment. R e s u l t s and
Discussion
Experimental 1.
f i n d i n g s are summaried below.
E l e c t r o a n a l y t i c a l Chemistry of Porphyrin c. D.C. p o l a r o g r a m s o f p o r p h y r i n c y i e l d e d two c a t h o d i c w a v e s w i t h w e l l - d e f i n e d l i m i t i n g c u r r e n t s whose c h a r a c t e r i s t i c s a r e l i s t e d i n Table I I .
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
28
ELECTROCHEMICAL STUDIES O F BIOLOGICAL SYSTEMS
Table I I Half-Wave Limiting Current Potential P o t e n t i a l Domain J idt (faradays-mole ) ( v o l t vs. SCE) (volt) - 1
-0.525 -0.730
-0.56 -0.88
2.02 ± 0.04 3.94 ± 0.02
t o -0.62 t o -1.0
Wave A n a l y s i s Slope (volt)
7
0.030 0.054
* S l o p e o f p l o t o f l o g [ ( i - i ) / i ] v e r s u s E. H
2. 3.
4.
Coulometry a t -0.615 v o l t v e r s u s SCE ( o n t h e f i r s t l i m i t i n g current plateau) substantiated a two-electron transfer i . e . two f a r a d a y s p e r m o l Coulometry a t -0.94 g current plateau) i n d i c a t e d t h e occurrence o f an o v e r a l l f o u r e l e c t r o n t r a n s f e r , i . e . , a t o t a l o f f o u r f a r a d a y s p e r mole o f porphyrin c. C y c l i c V o l t a m m e t r y a t t h e hdme y i e l d e d t h e f o l l o w i n g r e s u l t s . ( a ) A t r e l a t i v e l y f a s t p o t e n t i a l s c a n r a t e s (10
p
p
E l e c t r o a n a l y t i c a l C h e m i s t r y o f Heme c . Upon i n s e r t i o n o f i r o n i n t o p o r p h y r i n c, t h e p o l a r o g r a p h i c and v o l t a m m e t r i c r e s u l t s under went a d r a s t i c change. I n l i e u o f two d i s c r e t e t w o - e l e c t r o n t r a n s fer waves, o n l y a s i n g l e q u a s i r e v e r s i b l e o n e - e l e c t r o n t r a n s f e r was o b s e r v e d , b o t h a t t h e d r o p p i n g m e r c u r y e l e c t r o d e b y c l a s s i c a l d . c . p o l a r o g r a p h y a n d a t t h e hdme b y c y c l i c v o l t a m m e t r y a t a l l scan r a t e s . The c o r r e s p o n d i n g half-wave p o t e n t i a l was -0.340 v o l t which i s c o m p a t i b l e w i t h e x p e c t a t i o n s based on comparable f e r r i ferroheme redox p o t e n t i a l s (8,9). A summary o f t h e i n f o r m a t i o n w h i c h t r a n s p i r e d i s p r e s e n t e d i n Table I I I .
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
Bioelectrochemical
TING E T A L .
CH CH C0 H 2
2
2
Hemin
x
Modelling
of Cytochrome
c
CH CH C0 H 2
2
2
Chloride Cysteine
SCH CH(NH ) C 0 H
SCH CH(NH )C0 H 2
2
CH
2
2
3
I60°C
·.
SCH
2
ι
2
CH
CH
3
CH CH C0 H 'CH CH C0 H 2
Heme c
2
2
Porphyrin
Figure 1.
2
2
2
c
Synthesis of porphyrin c and heme c χ
0.9 0.8
porphyrin c in water
660
640
620
600
580
560
540
520
500 480 '
4304ÏC)
390
X (nm) Figure 2.
Spectra of porphyrin c and heme c in the presence of 0.1 M perchloric acid
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
ELECTROCHEMICAL STUDIES O F BIOLOGICAL SYSTEMS
SCH CH(NH )C0 H 2
2
2
SCH CH(NH ) C 0 H 2
2
2
Figure 3. Electrochemical reaction mechanisms. A, conversion of porphyrin c (I) to corresponding porphomethene moiety (II); B, electrooxidationr-reduction of heme c.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
2. ting et al.
Bioelectrochemical Modelling of Cytochrome c
31
Table III Synopsis of the Electroanalytical Behavior of Porphyrin cand Heme c Number of Number Type of Electroactive Total Discrete Electron Electron Transfer of faradays Moiety Transfer Steps per mole Porphyrin c
4
Heme c
1
2 steps of 2 electrons each 1
EC Quasi-reversible
By analogy with known mechanisms previously substantiated for other porphyrins (10), we postulate that porphyrin c was electroreduced via two successive two-electron transfer sequences, yield ing a porphomethene product as shown in Figure 2 Part A Each two-electron-transfer ste tion. In contradistinction, porphyri blocked all the electron transfer sites operative in porphyrin c and converted the corresponding metalloporphyrin (heme c) into a single-electron donor-acceptor system as illustrated in Figure 3, Part B. Acknowledgments The work described in this communication was supported by the Centre National de la Recherche Scientifique (CNRS, France), by Research Grant HL 02342 from the National Heart, Lung and Blood Institute, National Institutes of Health, United States Public Health Service and by Research Grant RG 764 from the North Atlan tic Treaty Organization (NATO). Literature Cited 1.
Falk, J . E . , "Porphyrins and Metalloporphyrins", p 95, Else vier, Amsterdam, 1964. 2. Neilands, J.B., Tuppy, H., Biochim. Biophys. Acta, (1960), 38, 351. 3. Gnichtel, H., Lautsch, W., Ber., (1965), 98, 1647. 4. Dickerson, R.E., Takano, T., Eisenberg, D., Kallai, O.B., Sam son, L., Cooper, Α., Margoliash, E., J. Biol. Chem., (1971), 246, 1511. 5. Perutz, M.F., Nature, (1970), 228, 726. 6. Kendrew, J.C., Dickerson, R.E., Strandberg, B.E., Hart, R.G., Davies, D.R., Phillips, D.C., Shore, V.C., Nature, (1960), 185, 422. 7. McGlashan, M.L., Pure and Appl. Chem., (1970), 21 (1), 3. 8. Feinberg, B.A., Gross, M., Kadish, K.M., Marano, R.S., Pace, S.J., Jordan, J., Bioelectrochem. Bioenerg., (1974), 1, 73. 9. Betso, S.R., Klapper, M.H., Anderson, L.B., J. Am. Chem. Soc., (1972), 94, 8197. 10. Neri, B.P., Wilson, G.S., Anal. Chem. (1972), 44, 1002.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
3 Control of the Potentials of Metal Ion Couples in Complexes of Macrocyclic Ligands by Ligand Structural Modifications DARYLE H. BUSCH, DALE G. PILLSBURY, FRANK V. LOVECCHIO, A. MARTIN TAIT, YANN HUNG, SUSAN JACKELS, MARY C. RAKOWSKI, WAYNE P. SCHAMMEL, and L. Y. MARTIN Evans Chemical Laboratory, The Ohio State University, 88 W. 18th Avenue, Columbus, Ohio 43210 The functions of heme-proteins in their natural systems may all be regarded as related to oxidatio eristic feature of these substance vary broadly with the specific heme protein and the potentials for the Fe2 /Fe3 couple vary correspondingly over a substantial range of values. Much of the interest in the metal complexes of synthetic macrocyclic ligands is understandably related to their structural similarity to the heme prosthetic group. Complexes with synthetic macrocyclic ligands have provided uniquely convenient systems for evaluation of the dependence of oxidation reduction properties of metal chelates on the detailed structure of the ligand while maintaining a given metal ion in an approximately constant coordination geometry. The complexes of tetraaza tetradentate macrocycles have figured most heavily in these studies and are the subject of this report. A variety of kinds of processes are possible, depending primarily on the metal ion, the ligand and the solvent, These include oxidation and reduction of the central metal ion, various oxidation and reduction reactions of the ligand and processes which involve both the metal center and the ligand. Attention is focused here on the first category of processes. For a large number of macrocyclic ligands, it has been possible to identify electrode processes that are attributable to the same metal ion couple. Figure 1 illustrates the range of E values found for the Ni /Ni couple. The majority of the entries (vida infra) in Figure i involve the results of measurements in acetonitrile solutions against an Ag/Ag (0. 1 M) reference electrode using 0. 1 M (n-Bu) NBF supporting electrolyte. In a few cases, especially the porphyrins, literature values have been adapted (1) to the same conditions by empirical corrections. The Ni2 /Ni3 couple has been confirmed for the processes by esr examination of the product of oxidation of the Ni2 starting material in many cases (2). The most striking aspect of the data summarized +
+
1/2
2
3
+
4
+
+
+
32
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
4
3.
BUSCH E T AL.
Potentials of Metal Ion Couples in Complexes
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
33
34
ELECTROCHEMICAL STUDIES O F BIOLOGICAL SYSTEMS
in Figure 1 is the remarkable range of electrode potentials that can be realized for this single metal ion couple by variations in ligand s t r u c ture. C l e a r l y , the means are at hand to generate almost any desired value of E j / for a given metal ion couple within a range approaching 2 volts. The analysis of the structure-potential relationship that follows shows in detail how this can be accomplished. 2
Complexes With Saturated Neutral Tetraaza Ligands. The ligands of Figure 2 all contain four secondary amine donors as members of saturated tetradentate ligands. The structures vary in ring size and in the numbers of methyl substituents on the rings. In the case of the C o / C o couple (3) it has been possible to examine the effect of r i n g size in detail using ligands 1 through 4 of Figure 2 and E / values are listed in Table I. Theoretically, these ligands can chelate in a variety of configurationally isomeric forms depending on the chiralities of the four nitrogen donors. Th produce only one structure in the complexes t r a n s - C o ( M A C ) C l ^ , while [l5]aneN and [l6]aneN yield two configurational isomers (3,7). Thus, the four rings yield 6 compounds for correlation between ring size and oxidation reduction behavior. The configurations of the two isomers have been established for [l5]aneN by nmr combined with chemical properties and quantitative conformational analysis (3,7). These two isomers are represented by structures 1 and 2 below (isomer I of Table I has structure 1; isomer II, structure 2). T h i s representation indicates the chirality of a nitrogen atom by the + or - sign. A plus means the hydrogen on that nitrogen is above the plane of coordination of the four nitrogens of the macrocycle; minus means it is below that plane. The numerals 2 and 3 identify the chelate rings by indicating how many carbon atoms are in each particular chelate r i n g . With a sophisticated computer program, the relative sizes of the metal ion sites inside the macrocyclic ligands have been estimated. The programs are based on the classic model of the strained molecule (the ligand (4,5)), and they may be used to predict the least strained conformations of the ligands with their donors appropriately arrayed for chelation (6). A v e r y useful parameter derived from such calculations is the ideal M - N distance, a quantity that has a characteristic value for each macrocycle in each configuration. These values are also included in Table I. Since it has not been possible to definitely assign the configurations of the isomers of trans-Co([l6]aneN^)Cl t and since the E / values for the isomers are very s i m i l a r only the value of ideal M - N for the most stable configuration is included in the table. Within the constraints of the model, it has been concluded that the relative values of ideal M - N are meaningful while the absolute values are probably displaced slightly from the unavailable true values. A calibration of these values has been made (5,7) using the d-d electronic spectra of the complexes and the 2 +
3 +
t
2
+
4
4
4
9
t
2
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
3.
BUSCH E T AL.
Potentials of Metal Ion Couples in Complexes
35
ο
8
I g.Ι
II 8 ° Q
ci
ε SUD
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
36
ELECTROCHEMICAL STUDIES OF BIOLOGICAL SYSTEMS
Table I. Half-Wave Potentials for the Reduction of t r a n s - C o ( [ l 3 - l 6 ] a n e N ) C 1 i n Acetonitrile Solution; Ag/AgN0 (.1M) Reference Electrode, 0.1 M n-Bu4NBF Supporting Electrolyte. Ε j / ^ (volts) a D q y (cm" ) Ligand (isomer) Ideal M - N (A( [l3]aneN 2750 -0.66 r 1.92 [l4]aneN 2.07 2480 -0.69 r [l5]aneN (D 2303 -0.38 r 2.28 [l5]aneN (ID -0.47 qr 2421 2.23 [16]aneN (D 2249 -0.15 i 2.38 [!6]aneN (ID -0.11 i 2341 4
2
+
3
4
x
1
4
4
4
4
4
4
Abbreviations:
r , reversible; q r , quasi-reversible;
i , irreversible.
derived ligand field parameter DqXY that measures the ligand field strength of the nitrogen donor reported in the last colum p r i m a r y and secondary amine donors bound to C o is ~2500 c m " , it has been concluded that [l4]aneN binds to C o in the least strained manner (it exhibits a normal D q * ^ of 2480 c m " ) ; thus, the 14-membered ring fits C o best. The range of C o - N distances with the usual saturated nitrogen-containing ligands is 1.94 to 2.03 A (8). Thus, the calculated ideal M - N distance is high by about 0,1 A. Recognizing this limitation, ideal M - N is taken as a parameter having a best-fit value for C o - N of 2.07. 3 +
1
3 +
4
1
3 +
3 +
Returning to Table I and the E / values therein, it is apparent that the value of E / is most negative for the complex in which the m a c r o cycle fits the metal ion best. A s the misfit increases, it becomes more difficult to p r o d u c e C o ; i . e . , easier to reduce t r a n s - C o ( M A C ) C l . T h i s i s true even for [l3]aneN , which because of its small ideal M - N might have been expected to fit C o better than C o . T h i s emphasizes the fact that the relationship between ring size and redox potential is more complex than the obvious effect associated with the fact that as the oxidation state of an ion increases its size usually decreases. The data presented in Table I suggests that E j / correlates with the strain energy of the C o complex, for this does indeed increase as the misfit increases between the ideal M - N and the usual M - N distance. T h i s i s rationalized by assuming that the reduction of C o to C o is a c c o m panied by a relief in the strain energy of the macrocyclic ligand. T h i s , in turn, is consistent with the fact that the cobalt(ID complexes of tetraaza macrocyclic ligands are relatively labile and commonly exhibit reduced coordination numbers and distorted geometries. t
t
2
2
3+
2
+
4
3 +
2 +
2
3 +
3 +
2 +
O n the basis of more limited data a related behavior has been reported for the N i / N i couple (Table D) (2). The 15-membered ring fits the larger, high spin N i ion best and this is marked by an enhanc ed difficulty in the oxidation of Ni([i5]aneN ) over Ni([i4]aneN4) of 2 +
3 +
2 +
4
2+
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
2+
3.
Potentials
BUSCH E T A L .
of Metal
Ion
Couples
in
37
Complexes
Table Π. Half-Wave Potentials for the Oxidation of N i ( M A C ) in A c e t o nitrile Solution, A g / A g N 0 (0.1 M) Reference Electrode, 0.1 M n - B u N B F Supporting Electrolyte. E i £ (volts) a Ligand Ideal M - N (lb Dq*y (cm-i) net 2 [l3]aneN 1.92 +0.7-0.9 i 2 1480 2.07 [l4]aneN +0.67 r 2 1250 [l5]aneN 2.28 +0.90 r 2 1110 [l6]aneN 2.38 b 2
3
4
4
4
4
4
4
Me [14laneN Me [14]aneN t-Me [i4]aneN c-Me [i4]aneN t-Me [i6]aneN 2
4
+0.68 r
4
+0.71 r
4
+0.8
4
+0.86 r
2
1471
4
2
6
6
9
6
~+1.3
4
2
i
Abbreviations: r , r e v e r s i b l e ; q r , quasi-reversible; i , i r r e v e r s i b l e . 'The oxidation wave is ill-defined. some 230 m V . Comparing ligands of s i m i l a r extents of substitution, Ni(Me [i6]aneN ) i s much more difficult to oxidize than is N i ( M e [ i 4 j a n e N ) (a difference of about 400 mV). A more common variety of steric strain is evident upon comparison of the behavior of the N i complexes of Me [l4laneN and [i4]aneN (2). The presence of a gem-dimethyl group in the former, combined with the most stable ring conformations, assures that two of the C H groups are oriented axially. T h i s leads to strong repulsions between these methyls and the monodentate solvent molecules coordinated to N i above and below the plane of the macrocycle (2). Since the corresponding N i complex is 4-coordinate and square planar, that interaction occurs only for the oxidized form of the complex. The resulting de stabilization of the trivalent state causes a 200 m V increase for E j / for the N i / N i couple from +0.67 V to 0.87 V . The reliability of this conclusion is demonstrated by the fact that the cis and trans isomers (Figure 2) of Me [i4]aneN give closely s i m i l a r values for E / (0.86 and 0.87, respectively). F u r t h e r , Me [i4]aneN and Me [l4]aneN , which do not contain gem-dimethyl groups in their 6-membered chelate r i n g s , give i / 2 values very close to that of the unsubstituted ligand [l4]aneN . 6
4
4
2
6
2+
2 +
6
4
4
3
3 +
2 +
2 +
2
6
4
x
2
4
4
3 +
2
4
E
4
Complexes With Unsaturated Neutral Ligands. Figure 3 presents the structures of a family of closely related macrocyclic ligands. A l l are 14-membered rings and most have six methyl groups, three on each of their six-membered chelate r i n g s . The unsaturated linkages are a l l
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
38
ELECTROCHEMICAL STUDIES O F BIOLOGICAL SYSTEMS
associated with nitrogen donors and both the number of such linkages and their positions vary. The N i complexes are a l l low spin and 4-coordinate, and they therefore provide a simple set of species suitable for assessing the effect of unsaturated vs saturated nitrogen donors on the relative s t a b i l i ties of the various oxidation states. The change i n ligand field strength upon substitution of unsaturated nitrogens into the structures is indicated by D q * ^ . In general, increased unsaturation enhances the ligand field strength toward high spin N i (such measurements are only feasible with the high spin species) (10). Only the species containing conjugated O-diimine groupings (structures 13, 15, 16, 17, and 18) add c o m p l i c a tions (2). In structures of this type, the first electron added to N i ( M A C ) ^ goes into a ττ ligand orbital. In a l l other cases, one-electron reduction produces a N i complex and one-electron oxidation produces a Ni species in all case cesses occur only in the voltammograms of N i complexes whose ligands involve a-diimine linkages. Most generally, the advent of unsaturation in the ligands favors reduction and causes oxidation to become more difficult. In order to consider these effects more quanti tatively, it is necessary to r e c a l l the results of the preceding section. Ni(Me [i4]4, i i - d i e n e N ) must be compared with Ni(Me [i4]aneN4) and N i ( M e [ i 4 ] i , 3 - d i e n e N ) with N i ^ J a n e N ^ * because of the effect of axial C H groups. F r o m these comparisons it is evident that the substitution of 2 isolated imines for 2 amines in the structure increases Ej/2 for oxidation of N i by about 100 m V . F o r the conjugated case, the substitution of two imines increases E / for the same couple by about 200 m V . Thus, the ot-diimine linkage stabilizes the lower state more greatly than does a pair of isolated imines. In a strictly parallel manner, N i ( M e [ l 4 ] l , 4 , 8 , i i - t e t r a e n e N ^ * can be compared with N i ( M e [ i 4 ] a n e N ) or with Ni(Me [i4]4, l i - d i e n e N ^ * . It foUows that the substitution of 4 isolated imines for amines increases by 180 mV and the substitution of the remaining two amines in M e [ i 4 ] 4 , 1 1 dieneN by imines increases E / by 70mV. F r o m these considerations we offer the generalization that E / is increased by about 40-45 m V (call it 43 mV) for each amine that is replaced by an imine, so long as the imines are not conjugated. The corresponding reasoning for the conjugated case leads to the figure of +170 m V as the increase in E / for the oxidation of N i ( M A C ) to the N i complex when a pair of amines is replaced by a conjugated α-diimine grouping. It has been pointed out that these incremental changes in E / for the 2 +
2 +
1
3 +
2 +
6
4
2 +
2
2+
6
4
2+
2 4
3
2 +
t
24
6
6
2
4
2+
2
6
6
4
1
2
t
2
t
2 +
2
3+
t
2
N i
2+/
N i
3 +
couple are roughly additive so that the E / can be predicted to a fair degree of approximation for related structures (2). A s i m i l a r treat ment for the reduction of N i ( M A C ) is less appropriate for two reasons. The v e r y negative reductive electrode processes are not generally so x
2
2 +
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
4
4
2
0
0
2
i , irreversible.
Me [i4]l,3,8,10tetraeneN
Me [i4]i,3,8,i0tetraeneN
Me [i4]l,3,7,litetraeneN
Abbreviations:
6
4
6
6
4
4
4
4
4
Me [l4]l,3,7,l0tetraeneN
6
4
4
1
6
6
Me [l4]l,4,8,iitetraeneN
0
2
3
4
2
4
4
2
0
2
0
2
0
No. of Conjugated Imines
Me [i4]i,3,8trieneN
4
No. of Isolated Imines
Me [i4]i,4,iltrieneN
2
Me [i4]i,3-dieneN
6
Me [l4]4,ii-dieneN
Ligand
4
8,
2
+
+
+
2 +
—
—
—
—
—
+
2 -+0
Ni-
—
—
—
1553
1569
2 +
J
+1.05 -0.76 -1.62
1
2 +
+
2 +
3
+0.72 - 1 . 3 7
2
+0.13-1.32
—
—
+0.05 - 1 . 1 7
+0.13 - i . 6 9
χ
2940
2790
+
1
2780
Q
7
—
+0.12-0.72-1.612960
+0.89 - 0 . 8 0 - 1 . 4 1 - 1 . 8 3 —
+0.82 -1.18
+0.59 - 2 . O i
+0.76 - 1 . 3 3 - 1 . 8 5 —
+0.51 - 2 . Oi
+0.44 - 2 . i i
+
l / 2
2 +
Dcf E ( V ) for F e Ε / ( ν ) for Co-}. Dq"* (cm" ) (MAC) ( M A C ) ( C H o C N | (cm" ) Ni 2* 3 2 t l 2 t p 2 V l 3 - 2 + 3 * 1 * 3 - 0 Ç 3
+1.00 -0.82 - 1 . 1 5 1767
+1.05 - 1 . 3 5
—
+0.86 -1.16
+0.98 - 1 . 5 7
+
(MAC) 2 -»3 2 - l
t
E / (V) for
2 +
Table III. Half-Wave Potentials for the Complexes of F e , C o , and N i w i t h Unsaturated Tetraaza F o u r teen-Membered Neutral M a c r o c y c l i c Ligands in Acetonitrile Solution, A g / A g N 0 (0.1 M) Reference Electrode, 0.1 M n - B u N B F Supporting Electrolyte.
40
ELECTROCHEMICAL STUDIES O F BIOLOGICAL SYSTEMS
well behaved and the added electron is centered on the metal atom i n some cases and on the ligand i n others. In most general terms, the redox behavior of the iron (H) comple xes (1_1) parallels that of the nickel derivatives that have just been considered. Increasing unsaturation favors the lower state and the presence of α-diimine linkages is accompanied by multiple reduction processes. There are, however, significant differences. The F e 2 / Fe couple is substantially more sensitive to unsaturation than the N i / N i couple (Table ID) and the one-electron reduction product of F e ( M A C ) is best formulated as an F e derivative even in the c o m p lexes of ligands containing a-diimine linkages (12). T h i s has been thoroughly demonstrated by the isolation and characterization of the salts of the complex F e ( M e [ i 4 ] i , 3,8, lO-tetraeneN^ . It should be emphasized that a l l of the iron(II) complexes are solvated in acetonit r i l e solutions (13). In orde for F e complexes with several saturated tetraaza macrocycles are summarized in Table IV. +
3 +
2 +
3 +
2 +
1 +
1
6
2 +
Table IV. Half-Wave Potentials for the Complexes of F e with Saturated Tetraaza M a c r o c y c l i c Ligands in Acetonitrile Solution, A g / A g N 0 (0.1 M) Reference Electrode, 0. i M n - B i ^ N B F ^ Supporting E l e c trolyte. 2 t
3
Ej/ Ligand
+2 ->
[i4]aneN [i5]aneN [i6]aneN Me [i4]aneN Me [l4]aneN 4
4
4
6
4
(volts)^
+3
+2
+.24 + .49 qr +.66 i + .27 +.38
4
2
2
-
+1
-2.3 i -2.1i -2.2 i -2.1 i
Abbreviations: q r , quasi-reversible; i , i r r e v e r s i b l e . F r o m Table IV the same effects are evident for iron complexes as for nickel complexes; i . e . , increasing ring size favors the lower state as does the presence of axial C H groups. Now, comparing these values (Table TV) with those in Table ΙΠ for unsaturated ligands, the following results are found (averaged to fit the several cases r e a s o n à ably well) : Et/2 = +140 m V for a pair of axial C H groups, +300 m V for an α-diimine group, and +50 mV for one isolated C=N. F r o m these values, it i s apparent that the enhanced sensitivity of the F e / F e couple to thejpresence of ligand unsaturation resides in the rather large effect of the α-diimine group. T h i s can be attributed to the special stability of the Fe -a-diimine r i n g structure (15). 3
3
2 +
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
3 +
3.
Potentials
BUSCH E T A L .
of Metal
Ion
Couples
in
41
Complexes
In view of the systematic structure-potential relationships observed for the N i / N i and F e / F e couples, it i s surprising that the C o / C o couple is insensitive to variations in the degree of ligand unsatura tion (16). A s i m i l a r observation (17) has been made on the complexes Co(MAC)(H 0) . T h i s is perhaps the result of the fact that the orbital receiving the added electron (d 2) is oriented on the ζ axis and relatively unaffected by the in-plane ligand field. F u r t h e r , since the unsaturated macrocyclic ligands are relatively inflexible, as compared to the satu rated tetraaza macrocyclic ligands discussed e a r l i e r , little rearrange ment is likely to occur upon reduction. The studies by Endicott et al (18) on the electrode potentials of the C o / C o couple in complexes containing a constant macrocyclic ligand and a variety of axial ligands supports this view. A strong dependence of the potential on the nature of the monodentate axial related to the ligand fiel is easily rationalized if the receptor orbital on C o is antibonding σ 2 +
3 +
2 +
3+
2 +
3 +
2
2
3
+
z
3 +
2 +
3 +
+ The C o ^ / C o couple is responsive to the extent and nature of the unsaturation of the ligands listed in Table HI. E / increases from - 2 . 3 V for [i4]aneN (7) to - 1 . 6 9 V for Me [14]4, l l - d i e n e N and -1.32 V for M e [ l 4 ] i , 4 , 8 , i l - t e t r a e n e N (Table ID). The effect of α-diimines is more extreme with E i / for the Me [14]i, 3-dieneN derivative being - 1 . 1 7 V and that for M e [ i 4 ] l , 3,8, iO-tetraeneN being -0.72 V . Since the v e r y negative E / for the saturated ligand system bears a large uncertainty, the magnitudes of these shifts are assessed by comparing values for the unsaturated ligand derivatives. O n that basis, Δ Ε ^ « +190 m V for each isolated C=N group and ~450 mV for each oediimine grouping. The greater sensitivity for the C o / C o couple as c o m pared to the isoelectronic N i / N i couple is attributed to the lower valence states involved in the former case. If back-bonding is a c o n s i deration it should be more important for lower states. Complexes With Anionic Tetraaza M a c r o c y c l i c Ligands. Ligands of general structures 3 and 4 have been utilized to characterize the oxidation-reduction behavior of complexes with macrocyclic anionic ligands. The N i complexes of ligands of structure 3 exhibit i r r e v e r sible oxidations at modest potentials (Ej/ - +.27 V for X = (CH ) and +.23 V for X = (CH ) and v e r y cathodic reductions (-2.30 V and -2.34 V, respectively (2)). These data show that the presence of charge on the ligand facilitates the oxidation process since the least positive values observed for this couple with neutral macrocyclic ligands is some 400 m V more positive. A l s o , reductions are made extremely unfavor able and the effect of ring size on E / has become v e r y s m a l l . The complexes of structure 4, having Ζ = Η and ring sizes of 14, 15, and 16 members, show i r r e v e r s i b l e oxidations at E / values of approximately ( d
z
2 K
1
t
4
2
6
6
4
4
2
2
4
4
t
4
2
2 +
3 +
1 +
2 +
2 +
2
2
2
3
x
2
t
2
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
2
42
ELECTROCHEMICAL STUDIES O F BIOLOGICAL SYSTEMS
-0.34 V , -0.39 V and - 0 . 2 9 V , respectively. Not only are these values extremely cathodic for the N i / N i couple, but greater uncertainties arise in their values because of their i r r e v e r s i b i l i t y . These uncertain ties and the proximity of their approximate E ^ values,suggest further that the oxidation reaction is not sensitive to ring size among these structures. T h i s insensitivity to ring size issiipported by data for Ni complexes of structure 4 having Ζ = - C ^ ç (Table V (2)). 2 +
3 +
2 +
H
The data of Table V has been obtained under Ihe same conditions as those for all the systems described above (19). A s w i l l be apparent, shortly, the derivatives of the ligands having stmcture 4 are more readily studied in a different solvent; however, the limited data available f r o m measurements in acetonitrile solutions is quite revealing. E a r l y Table V .
Half-Wave Potentials for N i
2 +
Complexes of Dianionic Ligands
of Structure 4, Having Differen Ag/AgN0
3
(0.1 M) Reference Electrode, 0.1 M n - B u 4 N B F
Supporting
4
Electrolyte. E A(volts) MÎT. .Mi T. 2+^ NiL-»NiL 1
Ring Size
N i L - N i L4T" *. "
Substituent
£
2
NiL -
NiL
15
H
-0.39 i
+1.77 i
-2.77 i
15
- C ^ ™
+0.26
+0.9
-2.53 i
14
~C^°
+0.25
+0.97 i
+0.24
+0.85 i
-2.38 q r
+0.27
+0.89 i
-2.39 qr
<~Ή 1 5
i
3
-Ccif^v υ '^-<0>CH
15
-C^L
1 5
-°<<2>N0
15
-N0
:
3
- 1 42
Abbreviations:
2
2
*°·
3 7
+0.64
i , irreversible; qr,
+
0
·
9
6
+1.08
-lissqr -2.01 q r _ ^ 2
g
quasi-reversible.
studies (2) were c a r r i e d out on the 14- and 15-membered ring species of structure 4 having acetyl groups as their substituents, Z . Product studies (2) showed that the f i r s t oxidation reaction yields a square planar Ni complex. T h i s process is electrochemically reversible for a l l the complexes of Table V except the parent compound (Ζ = H). In that case, it is probable that oxidation at the metal center is quickly followed by one or more ligand reactions. It has been shown that complexes having 3 +
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
3.
BUSCH E T A L .
Potentials
of Metal
Ion
Couples
in
43
Complexes
chelate rings of this class undergo a coupling reaction upon oxidation (20,21). Though the absolute value for E / suffers from considerable uncer tainty, it remains clear that the f i r s t oxidation reaction of the complex with the unsubstituted ligand occurs at a highly cathodic potential. Further, the replacement of the hydrogen atom by electron withdrawing substituents shifts the potential for this process dramatically in the anodic direction. In fact, the range of E / values reported for different derivatives in Table V spans approximately one volt. Thus, the effect of substituents on the metal-centered oxidation is truly remarkable. The magnitude of incremental change in E / with substituent correlates linearly with the Hammet (22) substituent constants ( a J for the three benzoyl derivatives (the slope ( p ) =0.138). T h i s emphasizes the fact that the substituent effect is felt directly by the metal ion. t
2
t
2
x
2
It has been possible t convincingly using data obtained on dmf solutions (Table VI,(19)).
As
Table VI. Half-Wave Potentials for N i Complexes of Dianionic Ligands of Structure 4, Having Different Substituents (Z), in Dimethylformamide Solutions, A g / A g N 0 (0.1 M) Reference Electrode, 0. i M n - B u 4 N B F Supporting Electrolyte. 2
3
Compound Number
4
Substituent
Oxidation Reduction Ε / (volts) E / (volts) a
Q
2
λ
1
"
2
-H
3
CH Cc;
4
- C ^ L „ ^NH-OhC H
C H
2 ^0C H
C H
C
2
; -H
3
TT
10
7
5
0
t
(l)-0.39 (2) +0.29 i (l)-0.36i (2)+0.39i (1) -0.14 i (2)+0.5li (i)-K).03
°
β
Η
5
(L 3
-N0
2
2
d - d Band v(kK) (ε)
-2.92 qr
17.02 (224)
-2.83 qr
17.15(169)
-2.64 q r - 2 . 5 9 qr
18.32 (197) 18.66 (285)
-2.42 qr
19.27 (433)
(D+0.22 (2)+0.64 i
-2.52 qr
19.30 (330)
(i)+0.42 (2) +1.04 i
(i)-2.00 (2)-2.60 i
19.80 (922)
(
2
)
4
O
#
5
5
i
(1) +0.2i
-8.
a
(2)+0.64 i
Abbreviations: q r , quasireversible; i , i r r e v e r s i b l e . stated e a r l i e r , the f i r s t oxidation wave has been assigned to the N i / Ni couple. It is generally well behaved except, again, for those c o m 2 +
3 +
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
44
ELECTROCHEMICAL STUDIES OF BIOLOGICAL SYSTEMS
pounds having hydrogen atoms on the γ-carbons. The second oxidation has been attributed to ligand oxidation (2), a reaction that has been well demonstrated using chemical oxidizing agents (23,24). These data i n d i cate that electron withdrawing substituents are highly effective at produ cing more positive potentials while alkyl substituents produce potentials at least as negative as that of the parent compound (Z = H). The total range of potentials spanned by these substituted derivatives in dmf i s 0.81 V . The responsiveness of E 0 to substitution is shown most c l e a r ly by the linear free energy relationship of Figure 4. E / correlates well with σ , Op", and o and the quality of the correlation is displayed in Table VII. The correlations with σ and σ ~ are equally good and t
ρ
ρ
Table VII.
2
m
ρ
Linear F r e e Energy C o r r e l a t i o n for
σ σ~ Ρ Ρ ηι
ΡΕ ι A .645 1.013 1.016
σ
σ
E/. t
2
(Εί/ )σ=0
r
-.345 -.331
.993 .978
" T h e appropriate equation is
E
*
=
Ρ
Ε
ι / 2
σ
+
(
Ε
^σ=0
that with o is not much weaker. T h i s suggests that resonance i n t e r actions are not principally responsible for the correlation. These data establish the fact that the effects of substituents on ligands of structure 4 are transmitted strongly to the metal ion and that the redox properties of the metal ion can be controlled with some p r e c i sion by the choice of substituents. These relationships are supported further by the parallel correlation of the energies (cm" ) of the lowenergy electronic transitions of these complexes (Table VI) with the same sets of substituent constants (Table VIII, Figure 5). These spec tral bands are presumed to involve d-d transitions because they fall in the usual range and exhibit extinction coefficients that are typical of such electronic processes for low spin, square planar Ni** complexes (25). The quality of the correlations follows that for E / very closely: Op" Op > o . Assuming the spectral bands to be of d-d origin, ligand field strength varies with band position. It follows that the substi tuent effect is exerted by alterations in ligand field strength, a r e l a t i o n ship that is certainly not unexpected. A s one looks toward possible applications of these results in the design of biochemical models, E j / is a functional property; i . e . , the ability of the metal ion to function in certain model systems will depend on achieving appropriate values. On the other hand, electronic m
1
t
w
2
m
2
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
BUSCH E T A L .
Potentials
of Metal
Ion
Couples
in
Complexes
NT
(10)
(11)
Me [14]4,ll-dieneN 6
Me [l4]l,3-dieneN
4
2
Me [l4]l,4,11-triene-
4
6
XX
" Π 5
XX (13)
(15)
(14)
Me [l4]l,3,8-trieneN 6
Λ
A
N
(17) Me [l4]l,3,8,l0-tetraeneN
Figure S.
(18)
Me ll4]l,3,8,10-tetraeneN
6
4
4
4
Me [l4]l, 3,7,11-tetraeneN G
4
Unsaturated tetraaza fourteen-membered neutral macrocyclic ligands
*4
-.20
-.30
-.40
—I .1
Figure 4.
1 .2
1 .3
Correlation of
1 .4
1 .5
h— .6
1.' 2
i l l 1Î3 l!«
tm'ih σ„" /or nickel complexes listed in Table 6
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
46
ELECTROCHEMICAL STUDIES OF BIOLOGICAL SYSTEMS
transition energies and ligand field strengths are diagnostic of the e l e c tronic structure of the metal ion. It is significant that the functional property and the diagnostic property both respond linearly to substituent effects. Table VIII. Linear F r e e Energy C o r r e l a t i o n for the Low Energy E l e c tronic Spectral Bonds. σ
r
( Η-Η)Π=0 Ε
Op Om
.991 .987 .958
17.243 17.308 17.371
2.205 3.508 3.947
VF
^ h e appropriate equation is
E
d-d
Pd-d "
=
0
+
( Ε
ά-^σ=θ
The ligands to which the most attention has been devoted here are of special interest in on-going programs directed toward the developing of relatively uncomplicated molecules and complex ions that can m i m i c the function of heme proteins (19, 23,, 26). P r o g r e s s in that direction has included the syntheses of the i r o n complexes of ligands of structure 4 as the prosthetic groups of such model systems (27,28). The electrochemical behavior of these species is shown in Table ΓΧ. The complexes with the Unsubstituted ligands are extremely sensitive to oxidation and several ligand oxidation waves are observed at E ^ values more positive than the highly coulombic F e / F e couple. The oxida tion wave associated with the F e / F e couple was identified by esr 2 +
2 +
3 +
3 +
Table LX. Half-Wave Potentials for the Iron(D) Complexes of Ligands of Structure 4, in Acetonitrile Solution, A g / A g N 0 (0.1 M) Reference E l e c trode, 0.1 M n - B u 4 N B F Supporting Electrolyte. 3
4
Complex
|
Fe(Me [l4]tetraenatoN ) Fe(Me [l5]tetraenatoN4) Fe(Me [i5]tetraenatoN4) Fe(Me [l6]tetraenatoN ) 2
4
2
6
2
ΕOxidations Fe^ /F& Ligand -0.77 +0.02 i -0.89 +0.03 i -0.90 +0.30 i -0.83 i - 1 . 2 i
4
(volts) ~0.7 -0.7 -0.8
Ε R e d u c tions (volts) -1.2 -2.38 ~i.2 - 2 . 4 7 qr -2.57
measurements. The extremely cathodic values for this couple (-0.7 - 0 . 9 V) are consistent with the data reported above for the N i
2+/
N i
-
3 +
couple. The ligand oxidations occurring at low positive potentials give well-defined but i r r e v e r s i b l e waves while the additional oxidation waves are poorly defined. The 16-membered ring derivative fails to
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
3.
Potentials of Metal
BUSCH E T AL.
Ion Couples
in
47
Complexes
exhibit the low energy ligand oxidation. The electrochemical behavior of this species is also unusual in that the F e / F e couple is irreversible and esr studies on the F e / F e oxidation product failed to reveal a signal for F e . It is suspected that the f i r s t ligand oxidation is coupled to the metal oxidation i n this case. Such processes are well documented from synthetic studies on i r o n complexes (29). The complex denoted in Table IX as Fe(Me [l5]tetraenatoN ) has its four additional methyl groups as substituents on the carbon atoms of the five-membered chelate rings. 2 +
2 +
3 +
3 +
3 +
6
4
Studies on i r o n complexes of substituted ligands of structure 4 remain in the synthetic stages at this point; however, the opportunities that these investigations should provide can be anticipated by a number of results that are presently available. In order for an i r o n atom to function in a heme protein, two general structural assemblages must meet c r i t i c a l requirements. The f i r s t is the coordination sphere of the metal ion, which must caus fall in useable ranges. The second involves the noncoordinated s t r u c tural a r r a y in the vicinity of the metal site—we refer to this as the associated proximate structure (APS). The latter may serve one or more of several functions, such as providing propitiously oriented func tional groups or determining the polarity in that region. F o r example, to facilitate 0 transport by F e , it is generally presumed that the A P S must both provide a nonpolar, aprotic environment and prevent the close approach of two iron atoms. In initial experiments designed to provide a suitable A P S while using the substituent-based control of E i / described immediately above, we have formed a cholesterol derivative (30) of the nickel complex Ni(Me [l5ltetraenatoN4) by first forming a derivative with the substituents Ζ (structure 4) as succinoyl half-esters p L „ ~ „ Α^^ττ and then c a r r y i n g out a trans-esterincation reaction with cholesterol (structure 5). It would be expected that this large hydrophobic moiety would orient itself over the metal ion in aqueous or other highly polar media, forming a nonpolar umbrella (the APS) in the vicinity of the metal ion. The nickel(D) complex having this bulky substituent has been thoroughly characterized (29) and it exhibits electrochemical properties closely s i m i l a r to those of the complex having simple acetyl substituents. Oxidations: Εχ/ = +0.21 V ; +.67 V(i); reduction: -2.48 V(qr)—compare to Table VI. T h i s c l e a r l y establishes the opportunity to design structures having both the requisite coordination spheres and associated proximate structures to m i m i c heme proteins with totally synthetic systems. 2 +
2
2
2
ù
2
A closer approach to the totally synthetic analogue of an heme p r o t e i n — 0 binding site, under study in these laboratories (31), makes use of a more complicated modification of structure 4. The final p r o duct contains a ligand having an organic moiety sheltering the metal ion site but fixed in position by two points of attachment (structure 6). 2
American Chemfcai S o c i e t y Library
1155
16th St. N. VY.
In Electrochemical Studies of Biological Systems; Sawyer, D.; Washington, D. C.Chemical 20036 ACS Symposium Series; American Society: Washington, DC, 1977.
48
ELECTROCHEMICAL STUDIES O F BIOLOGICAL SYSTEMS
^ !.7 1.6 1.5 1.4 1.3 1.2
N
(H 0) 2
PC
6
P450
Hb Mb
ι:
I.I 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 Η H-*-T+ •+—J—I 1 h
) V
(CN)6 1
0 -0.1-0.2 0.3 0.4-0^
TPP(CI)
J HPP
" [l6]-m-xyL"
Μ '3
Γ
J-2py
Η i^J|H Figure 6. Half-wave potentials for the Fe /Fe couple in selected complexes adjusted to SHE reference electrode 2+
3+
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
3. BUCH ET AL. Potentials of Metal Ion Couples in Complexes Structure 6 as drawn is misleading in that it does not properly represent the orientation of the m-xylyl group. This group is constrained to occu py a position above the metal ion site producing a hydrophobic cavity (the dry cave ) in that location. The coordination site on the opposite side of the coordination plane is available for binding of monodentate ligands and the N-methylimidazole adduct has been characterized (31). The Fe /Fe couple for this species displays an E / of -0.36 V (vs Ag/AgN0 (0.1 M) in acetonitrile (usual conditions). Figure 6 shows how this process for the new complex, identified as [l6]-m-xyl , relates to those for a number of heme proteins, the common iron comp lexes and a few other complexes with macrocyclic ligands. It is parti cularly encouraging that the E / value for this first totally synthetic dry cave complex of iron(E) falls in close proximity to that of hemoglo bin. For this comparison Ag/AgN0 (0.1 M) referenc standard aqueous hydrogen electrode by the addition of 0.58 V (1). Preliminary evidence (30) indicates that the dry cave complex does interact with the small molecules CO and 0 . M
M
2+
3+
t
2
3
M
t
tf
2
3
2
1.
LITERATURE CITED To adjust E / from SCE to Ag/AgNO (0.1 M) reference electrode in acetonitrile, 0.334 V is subtracted (Mann, C., and Barnes, Κ., "Electrochemical Reactions in Nonaqueous Systems, " Marcel Dekker, New York, 1970). Normal hydrogen electrode is not stable in acetonitrile but appropriate adjustment from Ag/AgNO (0.1 M) to SHE appears to involve addition of 0. 58 to 0.63 V (Butler, J. Ν., Advan. Electrochemical Engr., (1970), 7, 87). To adjust from SCE in dmf to Ag/AgNO (0.1 M) in acetonitrile, subtract 0.354 V (Butler, p. 135 and above). To adjust from SCE in dmso to Ag/AgNO (0.1 M) in acetonitrile, add 0.016 volt (Mann and Barnes). To adjust from SCE in benzonitrile to Ag/AgNO (0.1 M) subtract 0.454 volt (Mann and Barnes, p. 479). Lovecchio, F. V., Gore, E. S., and Busch, D. H., J. Am. Chem. Soc., (1972), 96, 3109. Hung, Υ., Ph.D. Thesis, The Ohio State University, 1976. DeHayes, L. J., and Busch, D. Η., Inorg. Chem., (1973), 12, 1505. DeHayes, L. J., and Busch, D. Η., Inorg. Chem., (1973), 12, 2010. Martin, L. Υ., DeHayes, L. J., Zompa, L. J., and Busch, D.H., J. Am. Chem. Soc., (1974), 96, 4046. Hung, Y., Jackels, S., and Busch, D.H., submitted for publica tion. 1 2
3
3
3
3
3
2. 3. 4. 5. 6. 7.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
4
50 ELECTROCHEMICAL STUDIES OF BIOLOGICAL SYTEMS 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.
Saito, Υ., "Spectroscopy and Structure of Metal Chelate Com pounds," Ed. by K. Nakamoto and P. J. McCarthy, John Wiley, New York, 1968. Olson, D.C., and Vasilevskis, J . , Inorg. Chem., (1969), 8, 1611. Busch, D.H., Helv. Chim. Acta, (1967), Werner Commemoration Volume, 174. Dabrowiak, J . C . , Lovecchio, F. V., Goedken, V. L., and Busch, D. H., J . Am. Chem. Soc., (1972), 94, 5502. Rakowski, M.C., and Busch, D.H., J. Am. Chem. Soc., (1975), 97, 2570. Watkins, D. D., Riley, D.P., Stone, J.Α., and Busch, D. Η., Inorg. Chem., (1976), 15, 387. Rakowski, M.C., Ph.D. Thesis, The Ohio State University, 1974. Figgins, P. E., an 2236. Tait, A.M., Lovecchio, F.V., and Busch, D. Η., submitted for publication. Rillema, D. P., Endicott, J. F . , and Patel, R. C., J. Am. Chem. Soc., (1972), 94, 394. Rillema, D. P., Endicott, J. F . , and Popaconstantineu, E., Inorg. Chem., (1971), 10, 1739. Pillsbury, D. G., and Busch, D. H., submitted for publication. Cunningham, J.Α., and Sievers, R.E., J. Am. Chem. Soc., (1973), 95, 7183. Dabrowiak, J.C., private communication. Gordon, A . J . , and Ford, R.A., "The Chemist's Companion," John Wiley, New York, pp. 144-153, 1972. Truex, T., and Holm, R. H., J. Am. Chem. Soc., (1972), 94, 4529. Hipp, C. J., Lindoy, L. F . , and Busch, D.H., Inorg. Chem., (1972), 11, 1988. Lever, A.B.P., "Inorganic Electronic Spectroscopy," Elsevier Publishing Co., Amsterdam, 1968. Koch, S., Tang, S.E., and Holm, R.H., J. Am. Chem. Soc., (1975), 97, 914. Riley, D.P., Stone, J.Α., and Busch, D.H., J. Am. Chem. Soc., in press. Koch, S., Holm, R.H., and Frankel, R., J. Am. Chem. Soc., (1975), 97, 6714. Dabrowiak, J.C., and Busch, D.H., Inorg. Chem., (1975), 14, 1881 and references therein. Pillsbury, D.G., and Busch, D.H., unpublished results. Schammel, W. P., Ph.D. Thesis, The Ohio State University, 1976
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
4 Electrochemical Studies on the Thermodynamics of Electron Transfer and Ligand Binding of Several Metalloporphyrins in Aprotic Solvents Κ. M. KADISH,* L. K. THOMPSON, D. BEROIZ, and L. A. BOTTOMLEY Department of Chemistry, California State University, Fullerton, Calif. 92634 The utility of electrochemistry as a tool for studying physical properties o extensively during th known that the half wave potentials for metalloporphyrin electrooxidation-reduction are directly influenced by the number and type of complexed axial ligands and that these may, in some instances, be related to the dioxygen carrying ability of the M(II) metalloporphyrin where M is Co(3-6), Fe (7-9) or Mn (10). Enthalpy and entropy values for complexation of cobalt(II) (4,11,12) and iron(II) (13) porphyrins, by several Lewis bases have been reported, but similar data for Lewis base complexation is not available for the oxidized cobalt(III) and iron(III) species. This data is of some interest in that changes of solva tion and/or ligand binding, concomitant with electron transfer, may produce large entropie effects which would significantly shift the half wave potentials as a function of temperature and could alter reported relationships between half wave potentials at 20°C and stability constants of dioxygen complexes elucidated at reduced temperatures. Therefore, we have undertaken in our laboratory a systematic study of the thermodynamics associated with electron transfer to and from metalloporphyrins in nonaqueous media. In this paper we present initial results on the entropy changes associated with π cation and π anion radical reactions of several porphyrin complex es. We have compared these to entropy changes for electron trans fer to and from the central metal of Co(II)TPP and Fe(II)TPP in bonding and nonbonding solvents. Thermodynamic data is also presented for Lewis base addition to Co(II)TPP and Fe(II)TPP to form the mono and bis pyridine complexes, respectively. The method of investigation consisted of measuring each reversible half wave potential, E , at several temperatures, and from a plot of E½vs T, the entropy was approximated utilizing the Gibbs-Helmhoitz equation: (14) ½
*Present address: Department of Chemistry, University of Houston, Houston, Texas 77004 51
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
52
ELECTROCHEMICAL STUDIES OF BIOLOGICAL SYSTEMS
(Δτ)
τ
Ρ
where A s , ΔΗ and ΔΘ are the entropy, enthalpy and f r e e energy of the e l e c t r o n t r a n s f e r step and E i s the polarographic h a l f wave potential. The v a l i d i t y of t h i s e q u a t i o n i n v o l v e s the assumption t h a t E° - E as measured by c y c l i c voltammetry, t h a t the d i f f u s i o n coeffièients of the o x i d i z e d and reduced forms of the comp l e x are about equal and t h a t the ambient pressure i s constant. For the compounds and c o n d i t i o n s of t h i s study, a l l of these assumptions are v a l i d . The h a l f wave p o t e n t i a l s are a l s o a f u n c t i o n o f a x i a l l i g a n d complexation of both the o x i d i z e d and reduced species and may be d e s c r i b e d by equation 2 f o r e l e c t r o r e d u c t i o n s . (15) ±
2
±
ζ— \ V c
p-q K~~ ~ nF red (E ) and (E ) are the h a l f wave p o t e n t i a l s of the complexed and uncomplexed èxidized s p e c i e s , r e s p e c t i v e l y ; and are the formation constants o f the o x i d i z e d and reducect complex, (L) i s the f r e e concentration of the complexing l i g a n d , ρ and q are the number of l i g a n d s bound t o the o x i d i z e d and reduced s p e c i e s , and η i s the number o f e l e c t r o n s t r a n s f e r r e d i n the d i f f u s i o n c o n t r o l led reaction. I t i s seen from equation 2 t h a t the more s t a b l e the o x i d i z e d complex, i . e . the l a r g e r the Κ r e l a t i v e to , the more cathodic w i l l be i t s reduction°potential. The Êalf wave potent i a l should a l s o s h i f t with changing concentration of the l i g a n d by -(p-q)RT/nF l n ( L ) . T h i s r e l a t i o n s h i p allows us to determine the c o o r d i n a t i o n number o f the complex a f t e r we have c a l c u l a t e d the number o f e l e c t r o n s t r a n s f e r r e d . I n s e r t i o n of ρ and q i n t o equation 2 w i l l y i e l d the formation constant, which, when d e t e r mined a t s e v e r a l temperatures, then allows us to c a l c u l a t e ΔΗ and Δβ f o r l i g a n d b i n d i n g using the Van't Hoff equation. (14) (
(
-
±
,„ . R V s " ÏÎF
_
l n ( L )
( 2 )
±
r
Experimental Chemicals. The porphyrins CoTPP, FeTPPCl, H TPP, ZnTPP, MgTPP, and MnTPPCl were purchased from Strem Chemical Inc. (Danv e r s , Mass.) and were used as r e c e i v e d . Reagent grade N,Ndimethylformamide (DMF) and b u t y r o n i t r i l e were d r i e d over a c t i v a ted 4A molecular s i e v e s before use. Other solvents were s p e c t r a l or reagent grade and were used as r e c e i v e d . The supporting e l e c t r o l y t e , tetrabutylammonium p e r c h l o r a t e (TBAP), was r e c r y s t a l l i z e d twice from absolute methanol and d r i e d a t reduced pressure over Pi+OIQ. 2
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
4.
KADISH E T AL.
Metalloporphyrins
in Aprotic
Solvents
53
Instrumentation and Data A n a l y s i s . C y c l i c voltammograms were obtained on a PAR 174 p o l a r o g r a p h i c analyzer i n c o n j u n c t i o n with an X-Y r e c o r d e r . Measurements were made i n a Brinkman Model EA 875-5 e l e c t r o c h e m i c a l c e l l which was immersed i n a 400 ml Dewar or a 1000 ml beaker and allowed t o e q u i l i b r a t e before commensing the experiments. Temperatures below 0°C were achieved through the use o f s l u s h baths c o n t a i n i n g KC1 and i c e . Temperatures above 0°C were maintained by immersion of the c e l l i n t o a temperature bath which was accurate t o 0.5° during the time of the measure ment. A three e l e c t r o d e system c o n s i s t i n g o f two platinum e l e c trodes and a standard calomel e l e c t r o d e (SCE) was used. The SCE was separated from the bulk of the s o l u t i o n by a double bridge con t a i n i n g f i n e g l a s s f r i t s and f i l l e d with s o l v e n t and supporting e l e c t r o l y t e . S o l u t i o n s i n the bridge were changed p e r i o d i c a l l y to a v o i d aqueous contaminatio e l e c t r o d e was p l a c e d i n t e q u i l i b r a t i o n i n the e l e c t r o c h e m i c a l c e l l and removed a f t e r each scan i n order t o r e - e q u i l i b r a t e a t room temperature f o r 5 minutes between runs. The p o t e n t i a l was p e r i o d i c a l l y checked a g a i n s t a second SCE and was found not t o vary by more than lmV between temperature t r i a l s . S o l u t i o n s were purged of oxygen by degassing with p u r i f i e d n i t r o g e n before running c y c l i c voltammograms. A f t e r d e a e r a t i o n , a blanket of n i t r o g e n was kept over the s o l u t i o n . The h a l f wave p o t e n t i a l s were measured as the average of the anodic and the cathodic peak p o t e n t i a l s and As was c a l c u l a t e d by means o f a l e a s t squares b e s t f i t program which analyzed dE /dT. Calcula t i o n s of As are based on the average of 3 - 5 measurements. A H , A s , and AG from the Van't Hoff p l o t s were a l s o analyzed by a l e a s t squares b e s t f i t program. U n c e r t a i n t i e s are expressed as the r e l a t i v e average d e v i a t i o n o f the measurements. ±
RESULTS AND
DISCUSSION
Entropy of E l e c t r o n T r a n s f e r Reactions at Porphyrin Ring. A t y p i c a l c y c l i c voltammogram a t 295°K i s shown i n F i g u r e 1 f o r ZnTPP i n b u t y r o n i t r i l e . The four e l e c t r o d e r e a c t i o n s are a l l r e v e r s i b l e and have been shown t o correspond to formation of a π anion r a d i c a l and d i a n i o n product a t -1.32 V and -1.71 V(16) and a π c a t i o n r a d i c a l and d i c a t i o n a t +0.81 V and +1.10 V.(17). The p o t e n t i a l s f o r each r e a c t i o n were not constant but s h i f t e d along the p o t e n t i a l a x i s as a l i n e a r f u n c t i o n o f temperature. A p l o t of i YJL constructed (Figure 2) and u s i n g equation 1, As f o r trie i n i t i a l one e l e c t r o n a d d i t i o n and one e l e c t r o n a b s t r a c t i o n from the n e u t r a l ZnTPP was c a l c u l a t e d as 9.3±1.5 eu and 4.0±0.3 eu, r e s p e c t i v e l y . Entropy changes f o r other π r a d i c a l r e a c t i o n s were c a l c u l a t e d and are summarized i n Table I. E
T
w
a
s
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
ELECTROCHEMICAL STUDIES O F BIOLOGICAL SYSTEMS
Li 1.6
I 1.2
I 0.8
I 0.4
I 0.0
POTENTIAL Figure 1.
-0.4 (VOLTS
-0.8
-1.2
-1.6
VS SCE)
Cyclic voltammogram of ZnTPP in butyronitrile, 0.1 M
b 260
I 270
I I I I I 280 290 300 3 1 0 TEMPERATURE
I 320
L_ 330
TBAP
3 40
( ° K)
Figure 2. Half wave potential as a function of temperature for the four electrode reactions of ZnTPP in butyronitrile, 0.1 M TBAP
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
-2.0
4.
Metalloporphyrins
KADISH E T A L .
in Aptotic
55
Solvents
Table I Entropy data f o r o x i d a t i o n o r r e d u c t i o n o f a n e u t r a l porphyrin r i n g t o y i e l d π anion and π c a t i o n r a d i c a l s i n b u t y r o n i t r i l e , 0.1M TBAP
Porphyrin Ε,
Ring Oxidation (a) (V) AS (eu)
Ring Reduction(b) Ε, (V) AS (eu)
H TPP
1.09
5.0 ± 0.6
-1.14
-3.3 ± 0.2
Zn(II)TPP
0.81
4.0 ± 0.3
-1.35
9.3 ± 1.5
Mg(II)TPP
0.69
Mn(II)TPP
(c)
-1.50
-6.8 ± 2.0
2
(a) (b) (c)
13. (c)
Corresponds t o MTPP + MTPP * + e Corresponds t o MTPP + e JMTPP~" Oxidation o f c e n t r a l metal occurs before r i n g o x i d a t i o n
Provided t h a t the i n i t i a l reactant i s n e u t r a l and the entropy change due t o the e l e c t r o n t r a n s f e r step i s l a r g e r than t h a t due to s o l v e n t r e o r g a n i z a t i o n and change i n l i g a n d c o o r d i n a t i o n , i t w i l l be observed t h a t As i s p o s i t i v e f o r o x i d a t i o n s and negative f o r reductions(180 · Although the data i n Table I i s l i m i t e d t o only a few compounds, the f o l l o w i n g observations may be made: porphyrin r i n g o x i d a t i o n s t o y i e l d π c a t i o n r a d i c a l s i n v o l v e a p o s i t i v e entropy change, while porphyrin r i n g reductions t o y i e l d π anion r a d i c a l s , with the exception o f ZnTPP, i n v o l v e a negative entropy change. S i m i l a r p o s i t i v e values o f As were obtained f o r r e d u c t i o n o f ZnTPP i n DMF and DMSO. The As = 9.3±1.5 eu i n b u t y r o n i t r i l e represents an increase o f 13 t o 16 eu over the other n e u t r a l porphyrin reductions i n Table I , and might be accounted f o r by l o s s o f an a x i a l l i g a n d and a change from 5 t o 4 coordinate geometry upon r e d u c t i o n . An a l t e r n a t e e x p l a n t i o n o f the l a r g e p o s i t i v e As might be t h a t ZnTPP e x i s t s as a l o o s e l y h e l d dimer i n b u t y r o n i t r i l e which s p l i t s on r e d u c t i o n t o y i e l d the π anion r a d i c a l . However, t h i s explanation does not seem l i k e l y s i n c e no evidence f o r d i m e r i z a t i o n has been observed f o r n e u t r a l ZnTPP, which has been e x t e n s i v e l y s t u d i e d by s p e c t r o s c o p i c methods (19,20) and e l e c t r o c h e m i c a l methodologies(16,17). In c o n t r a s t , however, z i n c e t i o p o r p h y r i n and z i n c o c t a e t h y l p o r p h y r i n r e a d i l y form dimers a t low temperature i n non-bonding s o l v e n t s and i n
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
56
ELECTROCHEMICAL STUDIES O F BIOLOGICAL SYSTEMS
methyleyelohexane a A s o f -17.8 and -21.5 eu has been c a l c u l a t e d f o r d i m e r i z a t i o n o f these complexes.(21) E l e c t r o d e Reactions o f CoTPP. Entropy changes are summarized i n Table I I f o r the one e l e c t r o n reduction o f CoTPP t o y i e l d a n e g a t i v e l y charged Co(I) complex. Co(II)TPP + e J [Co(I)TPP]"
(3)
as w e l l as f o r the s i n g l e e l e c t r o n o x i d a t i o n t o y i e l d a p o s i t i v e l y charged Co(III) complex. Co(II)TPP J
[Co(III)TPP]
+
+ e
(4)
In the absence o f nitrogenous bases, the h a l f wave potent i a l for reaction 3 i s t i o n s . H a l f wave p o t e n t i a l than 80 mV on going from a solvent o f low complexing a b i l i t y such as b u t y r o n i t r i l e o r C H 2 C I 2 t o a solvent o f higher complexing strength such as DMF o r DMSO. (22_) A d d i t i o n s o f b u t y r o n i t r i l e t o toluene s o l u t i o n s o f Co(II)TPP do not r e s u l t i n s p e c t r a l changes i n d i c a t i v e o f complexation(23), while t i t r a t i o n o f DMSO i n t o b u t y r o n i t r i l e s o l u t i o n s o f Co(II)TPP does not change the h a l f wave p o t e n t i a l f o r Co(II) r e d u c t i o n . Both o f these experiments i n d i c a t e t h a t a change i n solvent c o o r d i n a t i o n does not occur a t the a x i a l p o s i t i o n o f c o b a l t ( I I ) . Only i n the presence o f strong l i g a n d s w i l l cobalt(II)TPP r e a c t t o form mono and b i s l i g a n d adducts. These i n t e r p r e t a t i o n s are confirmed by the data i n Table I I . Reduction o f Co(II)TPP i n e i t h e r b u t y r o n i t r i l e o r DMSO gives a zero o r small p o s i t i v e A s f o r r e a c t i o n 3, while i n DMSO, which was 1.13 M i n p y r i d i n e A s = 16.2 ± 2.4 eu. Since Co(I)TPP does not bind a x i a l l i g a n d s ( 2 4 ) , a p o s i t i v e As i s i n d i c a t i v e o f l i g a n d r e l e a s e upon reduction o f Co(II). P y r i d i n e t i t r a t i o n s o f Co(II)TPP - DMSO s o l u t i o n s , when followed e l e c t r o c h e m i c a l l y , gave a A E / A l o g ( p y r i d i n e ) = -60mV between 0.5 and 3 M pyridine.(25) This slope i s a c l e a r i n d i c a t i o n t h a t one more p y r i d i n e i s complexed by Co(II) than by C o ( I ) , so t h a t the e l e c t r o d e r e a c t i o n i n DMSO, 1.13 M p y r i d i n e may be formulated a s : ±
e
[Co(II)TPP Py] + e J [Co(I)TPP]"
+ Py
(5)
f o r which the o v e r a l l A s = 16.2 ± 2.4 eu. This i n c l u d e s both the e l e c t r o n t r a n s f e r step and the coupled l i g a n d d i s s o c i a t i o n . Combining the inverse o f r e a c t i o n 5 with r e a c t i o n 3 y i e l d s by a H e s s s law r e l a t i o n s h i p the o v e r a l l r e a c t i o n f o r l i g a n d a d d i t i o n given by equation 6, 1
Co(II)TPP + Py J Co(II)TPP-Py;
(6)
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
4.
Metalloporphyrins
KADISH E T A L .
in Aprotic
Solvents
57
Table I I Entropy data f o r metal redox r e a c t i o n o f Co(II)TPP i n s e v e r a l s o l v e n t systems.
AS, eu Solvent
Co(II) j C o ( I )
Butyronitrile
0.0 ± 2
DMSO
6.2 ± 0.9
Py-DMSO
(a) (b) (c) (d)
(c)
( a )
Co(II) J C o ( I I I )
( b )
(d) -35.0 ± 1.6
16.
[Co(II)TPP]° + e J [Co(I)TPP] [Co(II)TPP]° £ [Co(III)TPP] + e 1.13 M p y r i d i n e i n DMSO 111 d e f i n e d e l e c t r o d e r e a c t i o n
Table I I I Thermodynamic data f o r a d d i t i o n o f p y r i d i n e t o c o b a l t ( I I ) t e t r a phenylporphrin according t o the r e a c t i o n : Co(II)TPP + Py J Co(II)TPP*Py
A G
Δ Η
298°
As<
Solvent
kcal/mole
kcal/mole
DMSO
-1.50±0.06
-4.510.6
-10.011.7
Toluene
-3.66±0.05
-8.5±1
-16±4
(a) (b)
(c)
eu
b )
eu
-10.0±2.6
Obtained from i n t e r c e p t o f l o g Κ vs — p l o t . Obtained from H e s s s Law c a l c u l a t i o n i n v o l v i n g e l e c t r o n t r a n s f e r r e a c t i o n s o f Co(II)TPP i n the presence and absence of p y r i d i n e F. A. Walker, J . Amer. Chem. S o c , 95, 1150 (1973) 1
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
58
ELECTROCHEMICAL STUDIES O F BIOLOGICAL
SYSTEMS
f o r which A S = -10.0 ± 2.6 eu. This i s l i s t e d i n Table I I I . In c o n t r a s t t o the i n v a r i a n c e o f h a l f wave p o t e n t i a l s f o r Co(II)TPP r e d u c t i o n (Reaction 3), o x i d a t i o n t o y i e l d Co(III) (Reaction 4) i s markedly dependent on solvent(22,26,27). Changi n g from a r e l a t i v e l y low d i e l e c t r i c constant s o l v e n t such as b e n z o n i t r i l e t o a s o l v e n t o f higher d i e l e c t r i c constant such as DMSO produces a cathodic s h i f t o f over 350 mV.{22) This e f f e c t i s even more dramatic on going from b e n z o n i t r i l e t o p y r i d i n e where h a l f wave p o t e n t i a l s w i l l s h i f t by more than 600 mV on changing s o l v e n t s . The ease o f Co(II) o x i d a t i o n has been shown t o p a r a l l e l the c o o r d i n a t i n g a b i l i t y o f the s o l v e n t , i n d i c a t i n g a s t a b i l i z a t i o n o f c o b a l t ( I I I ) with a x i a l ligands.(22) The entropy data o f Table I I bear t h i s out. The negative entropy changes are l a r g e f o r o x i d a t i o n o f Co(II) t o Co(III) i n both DMSO and DMSO-pyridine mixtures, and can best s o l v e n t molecules. Thi Manassen(27) and was confirmed i n our l a b o r a t o r y by monitoring the h a l f wave p o t e n t i a l s f o r r e a c t i o n 3 during a DMSO t i t r a t i o n o f Co(II)TPP i n b u t y r o n i t r i l e . From 0 t o 10 M^DMSO the o x i d a t i o n p o t e n t i a l s s h i f t e d only s l i g h t l y . Above 10 M and up t o 1M DMSO the observed s h i f t was -68mV/log(DMSO). Above 1M DMSO t h i s s h i f t i n c r e a s e d t o -116mV/log(DMSO). Based on these r e s u l t s , the f o l l o w i n g f o r m u l a t i o n i s given f o r complexation o f Co(III) by DMSO: 2
[Co (II) TPP] °
+
+ 2 DMSO J [Co (III) TPP-DMSO^ + e
f o r which a A s = -35.0
(7)
± 1.6 eu i s c a l c u l a t e d .
Thermodynamics o f Co(II)TPP Complexation with P y r i d i n e i n DMSO. U t i l i z a t i o n o f equation 2 permits c a l c u l a t i o n o f both the s t a b i l i t y constant and formula f o r the b i n d i n g o f Co(II)TPP by p y r i d i n e i n DMSO. E l e c t r o c h e m i c a l l y followed t i t r a t i o n s o f CoTPP with p y r i d i n e show t h a t one l i g a n d i s bound t o C o ( I I ) , while zero are bound t o Co(I).(26) At 298°K a AG = -1.50±0.06 Kcal/mole was c a l c u l a t e d f o r the mono p y r i d i n e complex. The temperature was v a r i e d , and from the r e s u l t i n g Van't Hoff p l o t i n F i g u r e 3, a A H = -4.5 ± 0.6 kcal/mole and As = -10.0 ± 1.7 eu c a l c u l a t e d f o r p y r i d i n e a d d i t i o n according t o r e a c t i o n 6. (These thermodynamic values are l i s t e d i n Table I I I . ) The A s agrees, w i t h i n experiment a l e r r o r , with the As = -10.0 ± 2.6 eu c a l c u l a t e d from combinat i o n o f r e a c t i o n s 3 and 5. I t i s a l s o w i t h i n the range o f AS c a l c u l a t e d i n toluene by Walker.(11) In c o n t r a s t , however, the enthalpy change, AH, i s smaller by 4.0 kcal/mole from t h a t o b t a i n ed i n toluene and would account f o r the l a r g e d i f f e r e n c e s i n AG between s o l v e n t s . E l e c t r o d e Reaction o f FeTPPCl. I t i s o f some i n t e r e s t t o draw p a r a l l e l s between the o x i d a t i o n s o f Co(II)TPP and Fe(II)TPP.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
4.
KADISH E T A L .
Metalloporphyrins
Iron tetraphenylporphyrin the c e n t r a l metal,
in Aprotic
59
Solvents
w i l l undergo two e l e c t r o d e r e a c t i o n s a t
[Fe(II)TPP]° + e J [Fe(I)TPP]"~
(8)
and [Fe(II)TPP]° £ [ F e ( I I I ) T P P ]
+
+ e
(9)
Both r e a c t i o n s , and e s p e c i a l l y r e a c t i o n 9, are h i g h l y dependent on the c o o r d i n a t i n g a b i l i t y o f the s o l v e n t . For r e a c t i o n 9 the h a l f wave p o t e n t i a l s are observed t o s h i f t a n o d i c a l l y by up t o 200 mV on going from a non-coordinating solvent such as methylene c h l o r i d e or b u t y r o n i t r i l e t o a c o o r d i n a t i n g solvent such as DMF, DMA o r DMSO.(28) This has been accounted f o r by an increased s t a b i l i t y o f Fe(II)TPP Since i r o n ( I ) doe temperature i t can be p r e d i c t e d t h a t r e d u c t i o n of Fe(II)TPP should be accompanied by a p o s i t i v e entropy change due p r i m a r i l y to l i g a n d d i s s o c i a t i o n . This i s e x a c t l y what i s observed i n Table IV. Table IV Entropy data f o r metal redox r e a c t i o n s o f Fe(II)TPP i n s e v e r a l s o l v e n t systems.
AS, eu Solvent
Fe(II) + F e ( I )
Butyronitrile
( a )
Fe(II)
jFe(III)
7.5 + 0.5
-2.5
+ 1.0
-7.6
± 0.7
DMF
12.6
+ 1.0
DMSO
24.6
± 1.0
27.2
± 2.0
-0.6
+ 0.5
29.5
± 1.8
-0.5
± 0.5
39.4
± 2.3
15.3
± 3.2
(c) Py-Butyronitrile Py-DMF
(C)
Py-DMS0*
C)
3.5 + 0.2
+e (a) (b) (c)
[Fe(II)TPP]° + e+[Fe(I)TPP] [Fe(II)TPP]°j[Fe(III)TPP]+ 2.06 M p y r i d i n e
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
60
ELECTROCHEMICAL STUDIES O F BIOLOGICAL SYSTEMS
In DMF-pyridine s o l u t i o n s the e l e c t r o d e [Fe(II)TPP-Py ]° + e J [Fe(I)TPP]~ 2
reaction i s + 2Py
(10)
f o r which AS = 29.5 ± 1 . 8 eu. Reversing the s i g n on r e a c t i o n 10 and adding t o r e a c t i o n 11 i n DMF e
[Fe(II)TPP DMF ]° + e J [Fe(I)TPP]~ x
+ xDMF
(11)
y i e l d s the o v e r a l l r e a c t i o n 12 f o r formation o f a b i s p y r i d i n e adduct. [Fe(II)TPP»DMF ]° + 2Py J [Fe(II)ΤΡΡ·Ρν ] x
0
2
+ xDMF
(12)
f o r which A s = -16.9 ± 2.0 eu. Entropy c a l c u l a t i o n s f o r t h i s r e a c t i o n i n DMF as w e l i n Table V. Table V Thermodynamic data f o r additon o f p y r i d i n e t o i r o n phenylporphyrin according t o the r e a c t i o n :
III)tetra-
Fe(II)TPP + 2 Py -> Fe(II)TPP-Py,
Δ
G
Δ Η
298°
Δ S
As
( a )
(
b
)
eu
Solvent
kcal/mole
DMSO
-3.6±0.2
-8.8±0.5
-17.411.2
-14.812.5
DMF
-7.3±0.2
-13.010.8
-19.212.0
-16.912.0
Butyronitrile
-8.9±0.1
-15.610.4
-22.511.8
-19.712.1
(a) (b)
kcal/mole
eu
1 Obtained from i n t e r c e p t o f l o g Κ vs_ Τ p l o t . Obtained from H e s s s law c a l c u l a t i o n i n v o l v i n g e l e c t r o n t r a n s f e r r e a c t i o n o f Fe(II)TPP i n the presence and absence of pyridine. 1
A t y p i c a l p l o t o f dE /dT f o r r e a c t i o n s 10 and 11 i s shown i n F i g u r e 4 and A s c a l c u l a t e d from these p l o t s i s l i s t e d i n Table V for several solvents. A l l entropy changes f o r Fe(II) complexa t i o n i n Table V are w i t h i n the range reported f o r other i r o n ( I I ) p o r p h y r i n s , but, as has been p o i n t e d out, As (as w e l l as AH) f o r b i n d i n g o f 2 p y r i d i n e molecules i s extremely s o l v e n t depenent.(13) x
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
KADISH E T A L .
Metalloporphyrins
in Aprotic
Solvents
[ C o l E D T P P · py]
_J 2.8
I 2.9
l 3.0
3.1
l/T Figure 3.
3.2
3.3 3
ο
X 10 ( κ
3.4
_ 1
3.5
)
Vant H off plot for pyridine binding by Co(II)TPP in DMSO, 0.1M TBAP
L 260
I
I
I
270
280
290
TEMPERATURE Figure 4.
L_ 300
< ° K)
Half wave potential as a function of temperature for Reactions 10 and 11
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
ELECTROCHEMICAL STUDIES O F BIOLOGICAL SYSTEMS
1/T Figure 5.
X
10
3
(°K" ) 1
Van t H off plot for pyridine binding by Fe(II)TPP in DMF, 0.1M TBAP
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
4. KADisH et al.
Metaîloporphyrins in Aprotic Solvents
63
In contrast to Co(II)TPP, AS for oxidation of Fe(II) to Fe(III) (see Table IV) is quite small in pyridine-DMF and pyridine-butyronitrile mixtures where both iron(II) and iron(III) form bis pyridine adducts. As can, in this case, be assigned entirely to the electron transfer step without contribution from changes in axial ligand coordination. The positive AS = 15.3 ± 3.2 eu in Py-DMSO implies a reduction in coordination on going from Fe(II) to Fe(III). Likewise, the small entropy changes in pyridine free solutions imply an identical coordination number of both the oxidized and the reduced forms of the complex. In order to corroborate the Hess s law determination for complexation with iron(II), as has been done for Co(II)TPP, we have calculated As directly from the temperature dependence of the measured stability constants. Calculations utilizing equation 2 gave aK= 2.2 χ 10 at 298° for FeTPP»Py in DMF and a similar Κ = 3.2 χ 10 varied and from the slop AH and As were calculated, and listed in Table V. No difference appears to exist between entropy changes calculated by the differ ent methods. 1
5
2
6
Acknowledgements. The support of Research Corporation is greatfully acknowledged. Literature Cited 1.
J.-H. Fuhrhop in "Structure and Bonding", Vol. 18, J. D. Dunitz, Ed., Springer-Verlag, New York, 1974. 2. Κ. M. Smith "Porphyrins and Metalloporphyrins", Elsevier Scientific Publishing Co., New York, Ν. Υ., 1975, chapter 14. 3. M. J. Carter, D. P. Rillema and F. Basolo, J. Amer. Chem. Soc., 96, 392 (1974). 4. H. C. Stynes and J. A. Ibers, J. Amer. Chem. Soc., 94, 1559 (1972). 5. F. A. Walker, J. Amer. Chem. Soc., 95, 1154 (1973). 6. D. V. Stynes, H. Stynes, J. A. Ibers and B. R. James, J. Amer. Chem. Soc., 95, 1142 (1973). 7. C. J. Weschler, D. C. Anderson and F. Basolo, J. Amer. Chem. Soc., 97, 6707 (1975). 8. C. J. Weschler, D. C. Anderson and F. Basolo, J. Amer. Chem. Soc., 96, 5599 (1974). 9. C. K. Chang and T. G. Traylor, Proc. Nat. Acad. Sci., U.S.A., 72, 1177 (1975). 10. C. J. Weschler, Β. M. Hoffman and F. Basolo, J. Amer. Chem. Soc., 97, 5278 (1975). 11. F. A. Walker, J. Amer. Chem. Soc., 95, 1150 (1973). 12. D. V. Stynes, H. C. Stynes, B. R. James and J. A. Ibers, J. Amer. Chem. Soc., 95, 1797 (1973).
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
64 ELECTROCHEMICAL STUDIES OF BIOLOGICAL SYSTEMS 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
S. J. Cole, G. C. Curthoys and E. A. Magnusson, J. Amer. Chem. Soc., 92, 2991 (1970), ibid, 92, 2153 (1971). W. J. Moore, "Physical Chemistry", Prentice Hall Inc., Englewood Cliffs, N.J., 1972. I. M. Kolthoff and J.J. Lingane, "Polarography", Vol. 1, Interscience Publishers, New York, Ν. Y., 1952, chapter 12. R. H. Felton and H. Linschitz, J. Amer. Chem. Soc., 88, 1113 (1966). J. Fajer, D. C. Borg, A. Forman, D. Dolphin and R. H. Felton, J. Amer. Chem. Soc., 92, 3451 (1970). R. P. Van Duyne and C. N. Reilley, Anal. Chem., 44, 142 (1972). D. J. Quimby and F. R. Longo, J. Amer. Chem. Soc., 97, 5111 (1975). D. Dolphin, R. H. Felton, D. C. Borg and J. Fajer, J. Amer. Chem. Soc. K. A. Zachariass 22, 527 (1973). F. A. Walker, D. Beroiz, and Κ. M. Kadish, J . Amer. Chem. Soc., 98 3484 (1976). F. A. Walker, private communication. D. Lexa and J. M. Lhoste, Experimentia Suppl. 18, 395 (1971). D. Beroiz and Κ. M. Kadish and L. Bottomley, manuscript in preparation. L. A. Truxillo and D. G. Davis, Anal. Chem., 47, 2260 (1975). J. Manassen, Isr. J. Chem., 12, 1059 (1974). Κ. M. Kadish, M. M. Morrison, L. A. Constant, L. Dickens and D. G. Davis, J. Amer. Chem. Soc., in press.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
5 Electrochemical Investigations of the Redox Properties of a N-Bridged Dimer, μ-Nitrido-bis[α,β,γ,δtetraphenylporphyriniron], in Nonaqueous Media Κ. M. KADISH* and J. S. CHENG Department of Chemistry, California State University, Fullerton, Calif. 92634 I. A. COHEN and D. SUMMERVILLE Brooklyn College of the City University of New York, Brooklyn, Ν. Y. 11210 Investigations of iron porphyrin redox properties in nonaque ous media have led, in several previously unreporte Iron(I) porphyrins have been characterized both chemically(2) and electrochemically(3,4) while electrochemical studies of porphyrin πcation radicals and dications have led to the proposed assign ment of quadrivalent heme iron(5,6). This latter oxidation state was generated by electrooxidation of either monomeric or dimeric iron(III) complexes of octaethyl or tetraphenylporphyrin(5,6). With the μ-οxο dimers, only one of the two iron atoms was oxidized to yield a mixed oxidation state Fe(III)-Fe(IV) dimer. Electroreduction of μ-οxο-bis-[tetraphenylporphinatoiron(III)], (FeTPP)O, in DMF has also been shown to yield a mixed Fe(III)-Fe(II) complex, which was characterized by e.s.r. at low temperature(4) before dissociation to [Fe(I)TPP]. Recently, synthesis of the first stable nonintegral or mixed oxidation state iron porphyrin dimer was reported by Summerville and Cohen(7). This is a nitrogen-bridged species, μ-nitrido-bis[α,β,γ,δ-tetraphenylporphinatoiron], written as (FeTPP)N, and similar to, but not isoelectronic with, (FeTPP)O. In neutral (FeTPP)O, the formal oxidation state on both irons is +3. In (FeTPP)N, however, the extra negative charge on the bridging atom Ν , when compared to Ο , leads to the average iron oxida tion state +3½. The neutral 17 valence electron nitrido complex is thus isoelectronic with the cationic species [(FeTPP)O] characterized by Felton(5,6) while the reduced nitrido complex, [(FeTPP)N] , is isoelectronic with the well characterized 18 valence electron (FeTPP) O.(4,9-12) Comparisons of the physical properties (7) and X-Ray struc ture (8) of the neutral (FeTPP)N and (FeTPP)O have recently been made. The most outstanding difference between these two systems is the extent of antiferromagnetic coupling between the iron atoms accross the bridge. Whereas (FeTPP)N is a completely coupled dimer, (FeTPP)O is only weakly magnetically coupled. Because of the relationship between spin coupling and bridge *Present address: Department of Chemistry, University of Houston, Houston, Texas 77004 65 2
2
2
2
2
3
2
+
2
2
2
2
2
2
2
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
66
ELECTROCHEMICAL
STUDIES OF BIOLOGICAL
SYSTEMS
mediated e l e c t r o n t r a n s f e r and i t s relevance to b i o l o g i c a l e l e c t r o n t r a n s p o r t i n cytochrome oxidase, we wish to compare a s e r i e s o f i s o e l e c t r o n i c hemin dimers u t i l i z i n g d i f f e r e n t b r i d g i n g groups. In t h i s paper, we r e p o r t the e l e c t r o n t r a n s f e r p r o p e r t i e s of μ - n i t r i d o - b i s [α,3,γ,ό-tetraphenylporphinatoiron] i n methylene chloride. Experimental Chemicals. A l l s o l v e n t s and chemicals were reagent grade and were used without f u r t h e r p u r i f i c a t i o n . The supporting e l e c t r o l y t e , tetrabutylammonium p e r c h l o r a t e (TBAP) was r e c r y s t a l l i z e d from absolute methanol and was d r i e d at reduced pressure over Ρι+Oio. (FeTPP) 2Ο was purchased from Strem Chemical Inc. (Danvers, Mass.) and was used as r e c e i v e d . (FeTPP)2N was synthe s i z e d from TPPFeN3 as describe Cohen (7) . E l e c t r o c h e m i c a l Measurements. A l l polarographic measurements were made on a PAR Model 174 Polarographic Analyzer u t i l i z i n g a three e l e c t r o d e system. The working e l e c t r o d e and counter e l e c trode were platinum and a commercial calomel e l e c t r o d e was u t i l i z ed as the reference e l e c t r o d e . This was separated from the bulk of the s o l u t i o n by a bridge f i l l e d with the same solvent and supporting e l e c t r o l y t e . Porphyrin concentrations were between 10 and 10 ** M. The o v e r a l l number of e l e c t r o n s (faradays per mole of i r o n monomer) was determined by c o n t r o l l e d p o t e n t i a l coulometry u t i l i z i n g a PAR Model 173 P o t e n t i o s t a t . E l e c t r o n i c i n t e g r a t i o n o f the current-time curve was achieved using a PAR Model 179 i n t e g r a t o r i n conjunction with the Model 173 P o t e n t i o s t a t . The Coulometric c e l l was s i m i l a r to t h a t used f o r c y c l i c voltammetry. A l a r g e c o i l e d platinum wire served as the anode and was separated from the cathodic compartment by means of a f r i t t e d d i s k . A platinum mesh e l e c t r o d e was used as the cathode and a saturated calomel e l e c t r o d e was the reference e l e c t r o d e . S t i r r i n g of the s o l u t i o n was achieved by means of a magnetic s t i r r i n g bar. Dearation of the s o l u t i o n was performed before commencing the experiment and a stream of high p u r i t y argon was passed above the s o l u t i o n throughout the experiment. A l l e x p e r i ments were c a r r i e d out i n a c o n t r o l l e d temperature room of 20±0.5° and a l l p o t e n t i a l s are reported vs_ the saturated calomel e l e c t r o d e (SCE). 3
O p t i c a l Spectroscopy. The e l e c t r o l y s i s of (FeTPP)2N was followed o p t i c a l l y using a Cary 15 Spectrophotometer. A s p e c i a l l y constructed quartz flow c e l l of path length 0.90 cm was used, which was attached t o the e l e c t r o l y s i s c e l l and c o u l d be removed f o r i n s e r t i o n i n t o the Cary 15. For the n e u t r a l s p e c i e s , quartz spectrophotometric c e l l s of path length 1.00 and 0.10 cm were also u t i l i z e d .
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
5.
KADisH E T A L .
N-BHdged
Dimer
in Nonaqueous
Media
67
Results C y c l i c Voltammetry and D i f f e r e n t i a l Pulse Polarography. The e l e c t r o c h e m i c a l r e d u c t i o n o f (FeTPP) N proceeds i n s e v e r a l d i s c r e t e steps without d e s t r o y i n g the porphyrin r i n g . In F i g u r e l a i s shown a c y c l i c voltammogram o f (FeTPP) N obtained i n CH C1 . A l s o shown i n t h i s f i g u r e are c y c l i c voltammograms o f (FeTPP) 0 and FeTPPCl i n the same s o l v e n t . F o r comparison, d i f f e r e n t i a l pulse polarograms are shown overlapping the c y c l i c voltammograms. The advantage o f d i f f e r e n t i a l pulse polarography i s t h a t peak c u r r e n t height may be a c c u r a t e l y measured f o r c l o s e l y overlapping r e a c t i o n s which cannot be analyzed by c y c l i c voltammetry. In a d d i t i o n , h a l f wave p o t e n t i a l s are r e a d i l y obtainable from the p o s i t i o n o f the peak ( E ~E a t small modulation amplitudes (13)). H a l f wave p o t e n t i a l s i n è H c ï were i d e n t i c a l by each method and are summarized i n Tabl 2
2
2
2
2
x
2
2
Table I H a l f Wave P o t e n t i a l s f o r E l e c t r o o x i d a t i o n - R e d u c t i o n o f Several S i m i l a r Porphyrins a t a Platinum E l e c t r o d e i n C H C l , 0.1M TBAP 2
2
Half Wave P o t e n t i a l ( v o l t s v s . SCE) (a) Reaction Compound
(4)
(3)
(2)
(1)
(5)
(FeTPP) N
1.76
1.51
1.15
0.15
-1.21
(FeTPP) 0
—
1.45
1.09
0.84
-1.17
—
1.63
1.42
1.14
-0.32
2
2
FeTPPCl
(a)
See F i g u r e 1 and t e x t f o r i d e n t i f i c a t i o n o f each peak
In order t o i n v e s t i g a t e each r e a c t i o n process o f (FeTPP) N s e p a r a t e l y and t o determine the existence o f any chemical react i o n s coupled t o the e l e c t r o n t r a n s f e r , c y c l i c voltammograms were taken over v a r i o u s sweep ranges. The p o t e n t i a l was i n i t i a l l y s e t at 0.4 V and scanned i n s u c c e s s i v e l y l a r g e r increments f i r s t up to -1.6 V i n a cathodic d i r e c t i o n and then up t o +1.9 V i n an anodic d i r e c t i o n . F o r e i t h e r s i n g l e o r m u l t i p l e scans between the range o f +1.9 V and -1.6 V, a d i f f u s i o n c o n t r o l l e d r e d u c t i o n and r e o x i d a t i o n was i n v a r i a b l y observed a t 0.15 V i n methylene c h l o r ide. T h i s i s l a b e l e d peak 1 i n F i g u r e l a and can be assigned t o 2
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
ELECTROCHEMICAL STUDIES O F BIOLOGICAL SYSTEMS
(a) ( F e T P P ) N
-
2
L
300
1.6 0
0.8 0 POTENTIAL
0.0 0 (VOLTS
μA
-0.8 0
VS S C E )
Figure 1. Cyclic voltammograms obtained at 100 mV'/sec on a platinum electrode ( ) and differential pulse polarograms at 2mV/sec, modulation amplitude 25mV/sec on a platinum electrode ( ) for (a) (FeTPP) N; (b) (FeTPP) 0; and (c) FeTPPCl in CH Cl 0.1U TBAP 2
z
2
2y
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
5.
KADISH E T A L .
the
N-Bridged
Dimer
in Nonaqueous
69
Media
transition: [TPPFe(III)-N-Fe(IV)TPP]° +e- J
[TPPFe(III)-N-Fe(III)TPP]"
T h i s same r e a c t i o n was observed a t a s i m i l a r p o t e n t i a l i n benzon i t r i l e (Table II) but i n p y r i d i n e t h i s r e d u c t i o n peak was s h i f t e d c a t h o d i c a l l y i n p o t e n t i a l t o -0.26 V. This was not due t o the r e d u c t i o n o f FeTPPCl*py2 which i s reduced a t 0.18 and -1.38 V i n neat p y r i d i n e (14). No f u r t h e r r e d u c t i o n was observed i n any range o f scans up t o cathodic p o t e n t i a l s o f a t l e a s t -1.1 V. Table I I H a l f Wave P o t e n t i a l s f o r Oxidation-Reduction s e v e r a l s o l v e n t s , 0.1M TBAP
o f (FeTPP)2N i n
H a l f Wav So1vent (a) Reaction (2)
(1)
(5)
1.15
0.15
-1.21
1.22
1.06
0.16
-1.17
(b)
(b)
(4)
(3)
1.76
1.51
Benzonitrile
(b)
Pyridine
(b)
CH C1 2
2
(a)
See F i g u r e l a f o r C H C 1
(b)
Beyond p o t e n t i a l range o f s o l v e n t
2
-0.26
-1.15
2
When the scan was extended t o -1.21 V a second r e d u c t i o n peak (5 o f F i g u r e l a ) was obtained. This i s a t almost an i d e n t i c a l p o t e n t i a l t o the f i r s t r e d u c t i o n o f (FeTPP) 0 a t -1.17 V i n CH2CI2 and the second r e d u c t i o n o f FeTPPCl a t -1.08 V. No f u r t h e r r e d u c t i o n was observed up t o the s o l v e n t l i m i t o f -1.7 V. Currents and h a l f wave p o t e n t i a l s f o r peaks 1,2 and 5 o f (FeTPP)2N are l i s t e d i n Table I I I f o r slow scan r a t e s . As seen from t h i s t a b l e the i n v a r i a n c e o f peak c u r r e n t with the square r o o t o f the scan r a t e , as w e l l as the constant h a l f wave p o t e n t i a l , indicates clearly a diffusion controlled electron transfer f o r each r e a c t i o n . Peak c u r r e n t s from d i f f e r e n t i a l p u l s e polarograms were constant f o r each r e a c t i o n o f (FeTPP)2N, and p o l a r o g r a p h i c wave a n a l y s i s gave a slope o f 63mV, i n d i c a t i n g again a s i n g l e e l e c t r o n t r a n s f e r step. 2
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
0.148
0.148
0.148
0.146
50
100
200
500
See f i g u r e l a .
0.148
20
(a)
0.147
(Volt)
10
(mV/sec)
Scan Rate
p,c
40.8
26.8
19.2
13.2
8.4
6.0
(μΑ)
i
(D
p,c'
1.82
1.90
1.92
1.88
1.88
1.90
2
mV
±
2
A* 1
]ΐΡί· sec
i
-1.216
-1.210
-1.210
-1.213
-1.211
-
(Volt)
2
E,
35.0
23.1
17.5
12.2
7.5
-
p,c (μΑ)
i
Reaction
-4 χ 10 M (FeTPP) Ν i n 0.1M TBAP i n Methylene C h l o r i d e .
2
1.57
1.63
1.75
1.73
1.68
-
2
mV
2
/v " p,c'
\iA*sec
i
C5)
p,a
24.4
-
-
17.6
12.4
8.4
-
(μΑ)
i
+1.144
+1.144
+1.147
+1.149
-
(Volt)
2
E,
(2)
-
1.73
1.76
1.75
1.88
-
mV
2
2
2
1 /v P,a ι )iA*sec
i
Scan Rate Dependence o f Half Wave P o t e n t i a l and Peak Current f o r Three E l e c t r o d e Reactions o f 8.14
Table I I I
5.
KADISH E T A L .
N-Bridged
Dimer
in Nonaqueous
71
Media
Controlled Potential E l e c t r o l y s i s . In order t o i d e n t i f y the products o f each e l e c t r o d e r e a c t i o n , the n e u t r a l species was both e l e c t r o r e d u c e d and e l e c t r o o x i d i z e d a t c o n t r o l l e d p o t e n t i a l and the number o f coulombs recorded by i n t e g r a t i o n o f the r e s u l t i n g current-time curve. The voltammogram before e l e c t r o l y s i s i s shown i n F i g u r e l a . The p o t e n t i a l was then s e t a t -0.6 V. T h i s i s on a p l a t e a u o f the f i r s t r e d u c t i o n wave but 600mV anodic o f the second r e d u c t i o n . C o n t r o l l e d p o t e n t i a l e l e c t r o l y s i s was complete a f t e r 10 minutes a t t h i s p o t e n t i a l and y i e l d e d an η = 0.50 electrons/monomeric u n i t . The p o t e n t i a l was then s e t t o 1.4 V and (FeTPP)2N was reduced a t a c o n t r o l l e d p o t e n t i a l . The 60mV s e p a r a t i o n o f the cathodic and anodic peak on the c y c l i c voltammogram (Figure 1) as w e l l as p o l a r o g r a p h i c wave a n a l y s i s i n d i c a t e a r e v e r s i b l e one e l e c t r o n t r a n s f e r r e a c t i o n a t =-1.21 V. However, c o n t r o l l e d p o t e n t i a l r e d u c t i o n d i d not y i e l d c u r r e n t time curves i n d i c a t i v e gave evidence o f a chemica t r a n s f e r step. C a l c u l a t i o n s o f η f o r t h i s step were not reproduc i b l e , suggesting t h a t the dimer was cleaved d u r i n g e l e c t r o r e d u c tion. E l e c t r o o x i d a t i o n s were performed a t +1.13, +1.65 and +1.90 V. In each case a w e l l d e f i n e d i n t e g r a t e d current-time curve was obtained, with a n=0.5, 0.99 and 1.35 electrons/monomeric u n i t as the s o l u t i o n c o l o r changed from brown t o green. Values o f η a t each p o t e n t i a l are summarized i n Table IV. Table IV C o n t r o l l e d P o t e n t i a l E l e c t r o l y s i s and Coulometry o f C H
2
C 1
(FeTPP)in
2
Potential(v)
Electrons Transfered Reaction
(b) -0.60 (c) 1.30 (d) 1.65 (e) 1.90
(a) (b) (c) (d) (e)
Values 750 mV 150 mV 140 mV 140 mV
ο
0.50 0.50 0.99 1.35
_
[Fe(IV)-N-Fe(III)]+e [Fe (III) -N-Fe ( H f ^ l ~_ [Fe(IV)-N-Fe(III)]Sj[Fe(IV) -N-Fe(III)].-+e , J [ Fe(IV)-N-Fe(III)] +2e" [Fe(IV)-N-Fe(III)], [Fe(IV)-N-Fe(III)] ^ [ F e ( I V ) - N - F e ( I I I ) ] +3e 0
given as electrons/monomeric u n i t more c a t h o d i c than Reaction CD more anodic than Reaction (2) more anodic than Reaction (3) more anodic than Reaction (4)
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
3
72
ELECTROCHEMICAL
STUDIES OF BIOLOGICAL
SYSTEMS
O p t i c a l Spectra. Before e l e c t r o l y s i s the spectrum of (FeTPP) N c o n s i s t e d of a s p l i t Soret Band i n the UV region (λ = 408 and 385 nm) and two weaker bands i n the v i s i b l e r e g i o n (λ = 625 and 532 nm). A methylene c h l o r i d e s o l u t i o n of (FeTPP) N was s t a b l e i n a i r and showed no change i n s p e c t r a l p r o p e r t i e s f o r s e v e r a l days. Values of these molar a b s o r p t i v i t i e s are l i s t e d i n Table V, Group I. For comparison we h a v e a l s o l i s t e d λ and ε f o r the spectrum of i s o e l e c t r o n i c [(FeTPP) 0] obtained by Felton.(6) O p t i c a l s p e c t r a of the s i n g l y reduced species (Table 5, Group 2) show the f e a t u r e s of an u n s p l i t Soret band (λ = 396 nm) which i s of approximately the same molar a b s o r p t i v i t y as t h a t f o r the i s o e l e c t r o n i c (FeTPP) 0 but s h i f t e d toward the blue. There i s no s t r u c t u r e d absorption of [(FeTPP) N] i n the 500-700 nm region. T h i s species was s t a b l e i n the presence of 0 f o r over 24 hours and was unchanged from t h a t obtained under an i n s e r t argon atmosphere. Reoxidation i n a l s t a r t i n g spectrum and (FeTPP) 0 are d i s p l a y e d i n F i g u r e s 2 and 3. As p r e v i o u s l y mentioned, r e d u c t i o n at -1.4 V d i d not proceed i n a s i n g l e step and y i e l d e d , i n a l l cases, an ultimate monomeric product a f t e r s e v e r a l hours. Products of the o x i d i z e d species were a l s o unstable i n the time i n t e r v a l o f t h e experiment and the s p e c t r a resembled those reported f o r FeTPP .(€0 2
2
+
2
2
2
2
2
+
2
D i s c u s s i o n of
Results
Based on the data, the e l e c t r o o x i d a t i o n - r e d u c t i o n of (FeTPP) N can be accounted f o r by the f o l l o w i n g mechanism: 2
[TPPFe
IIl2
= 1.15
-N-Fei n * -
TPP]
\
V
2
[TPPFe
Ii:E
IV
-N-Fe TPP]
- If * e
e
1.51
[TPPFe
+
V
+e
^ III „ IV _+2 [TPPFe rPPFe -N-Fe TPP] r m T %
XT
11
m
-It [TPPFe
I I I
[TPPFe
= 0.15 -N-Fe
I I I
TPP]"
= -1.21 I I I
-N-Fe
I I I
V 1
V
TPP]"
2
f u r t h e r r e d u c t i o n products I I I
I V
-N-Fe TPP]
+ 3
The i n i t i a l r e d u c t i o n of (FeTPP) N i s q u i t e f a c i l e and y i e l d s i n i t i a l l y an i r o n ( I I I ) dimer i s o e l e c t r o n i c with (FeTPP) 0. Further r e d u c t i o n of the dimer occurs a t -1.21 V by a s i n g l e e l e c t r o n t r a n s f e r step and y i e l d s a product assigned not as a mixed F e ( I I I ) , Fe(II) dimer, but r a t h e r as a dimeric i r o n ( I I I ) anion r a d i c a l . 2
2
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
e
e
(a)
III
II
Group
region.
11.3
417
510 -4 ε x 10
1.34
10.0
0.71
4
ε x 1θ" 0.43
9.40 408
396
8.50
562
(a)
(a)
408
408 10.7
532 0.86
.-4 ε x 10 λ 613
-4 ε χ ίο
λ ε χ ίο" λ
No s p e c t r a l d e t a i l i n v i s i b l e
Fe(III)TPPCl
[Fe(III)-0-Fe(III)] '
[Fe(III)-N-Fe(III)]
[Fe(IV)-0-Fe(III)]
[Fe(IV)-N-Fe(III)]'
Compound
6.34
378
-
11.3
385
3.60
327
This work
Reference
Réf. 6
This work
This work
Réf. 6
Absorbance Maxima (nm) and Molar A b s o r p t i v i t i e s ( l m o l e cm ) f o r Several Iron Tetraphenylporphyrin Complexes i n Methylene C h l o r i d e . Values o f Molar A b s o r p t i v i t i e s Are C a l c u l a t e d Per Mole o f Iron.
Table V
ELECTROCHEMICAL STUDIES O F BIOLOGICAL SYSTEMS
12h
400
500
600
700
λ , nm Figure 2. Spectra of (FeTPP) N first reduction product in CH Cl , 0.1M TBAP. (FeTPP) N before electrolysis ( ); after controlled potential reduction at -0.60 V to yield [(FeTPP) N]~ ( ). The molar absorptivity is per monomeric iron. 2
2
2
2
t
12
10 \
(FeTPP) 0
/V \
2
- /A
8
// / / > / / / / / ι I
β-
4
-1
\
1
s
/
\\ 1
\\
(FeTPP) N" 2
1 \ \ \ \ \ \ \
1
\ \ \ \ \ \
\\ \\ \\ \\ \ χ
2~
\\
0
1
400
1
500
600
700
λ, ηm Figure 3. (FeTPP) 0 2
Spectra of [(FeTPP) N]~ ( ) and the isoelectronic ( ; in CH Cl , 0.1M TBAP. The molar absorptivity is per monomeric iron. 2
2
2
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
5.
N-Bridged
KADISH E T A L .
Dimer
in Nonaqueous
75
Media
O x i d a t i o n o f (FeTPP)2N occurs i n three w e l l d e f i n e d s i n g l e e l e c t r o n t r a n s f e r steps (see f i g u r e 1). We have chosen t o a s s i g n the r e a c t i o n as o c c u r i n g a t the porphyrin r i n g to y i e l d a c a t i o n r a d i c a l r a t h e r than a t the c e n t r a l metal t o y i e l d an F e ( I V ) , Fe(IV) dimer. The reasons f o r these assignments are based only on p o t e n t i a l s o f the redox r e a c t i o n s and w i l l be d i s c u s s e d i n the following sections. The s t a r t i n g m a t e r i a l (FeTPP)2N has been w e l l c h a r a c t e r i z e d from Môssbauer data and magnetic s u s c e p t i b i l i t y measurements. At room temperature the Môssbauer spectrum i s sharp and symmetrical and a t 80°K shows only a s l i g h t asymmetry. Magnetic s u s c e p t i b i l i t y measurements between 80 Κ and 300 Κ i n d i c a t e a simple paramag n e t i c s p e c i e s with μ = 2.04BM per (FeTPP)2N. Since the Môssbauer data presents a temperature independent doublet t h i s would i n d i c a t e t h a t e i t h e r the two i r o n atoms are i n the same o x i d a t i o n s t a t e (3i) an e x i s t s the occurance o centers (<10 sec) as shown below. 7
[TPPFe(III)-N-Fe(IV)TPP]
£
[TPPFe(IV)-N-Fe(III)TPP]
The equivalence of the two i r o n atoms has been confirmed by the X-ray s t r u c t u r a l r e s u l t s (8) and an average o x i d a t i o n s t a t e g r e a t e r then +III i s c o n s i s t e n t with the observed Môssbauer isomer s h i f t f o r (FeTPP) 2 N ( 7 ^ . Thus the formulation of i r o n (3{) i s prefered. On the other hand, the i s o e l e c t r i c c a t i o n [(FeTPP)2O] has been shown t o have a temperature dependent moment s i m i l a r t o (FeTPP)2O, and might be represented as having two non-equivalent i r o n atoms [Fe(III) and Fe(IV)] which do not exchange r a p i d l y , or two e q u i v a l e n t i r o n atoms which are not s t r o n g l y m a g n e t i c a l l y coupled. Thus, i t i s of some i n t e r e s t to compare both o x i d a t i o n and r e d u c t i o n p o t e n t i a l s f o r r e a c t i o n s o f two i s o e l e c t r o n i c i r o n ( I V ) dimers, [(FeTPP) 0] and (FeTPP)2N (Reaction 2), and (FeTPP) 0 and [(FeTPP) N]"(Reaction 5). As seen from Table I, the h a l f wave p o t e n t i a l s f o r o x i d a t i o n o f an F e ( I V ) , F e ( I I I ) dimer (Reaction 2^ are about equal f o r the i s o e l e c t r o n i c (FeTPP)2N and [ (FeTPP)2O] . The former i s o x i d i z e d at 1.15 V while the l a t t e r r e a c t i o n occurs a t 1.09 V. T h i s 60 mV d i f f e r e n c e i s not l a r g e and the h a l f wave p o t e n t i a l s do not seem t o b e g r e a t l y i n f l u e n c e d by the e x t r a p o s i t i v e charge on [(FeTPP) 0] . Likewise, the i s o e l e c t r o n i c [ ( F e T P P ) 2 N ] " and (FeTPP)2O have almost i d e n t i c a l r e d u c t i o n p o t e n t i a l s with the former being measured a t -1.21 V and the l a t t e r a t -1.17 V. Again, t h i s 40 mV d i f f e r e n c e i s not l a r g e and can be accounted f o r by the e x t r a negative charge on [(FeTPP)2N] which r e t a r d s s l i g h t l y the r e d u c t i o n when compared t o the n e u t r a l (FeTPP)2O. In marked c o n t r a s t , however, the p o t e n t i a l s f o r r e d u c t i o n at the c e n t r a l metal of the F e ( I I I ) - F e ( I V ) dimer are extremely s e n s i t i v e to the b r i d g i n g atom. The range of s t a b i l i t y of the +
2
2
2
+
2
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
76
ELECTROCHEMICAL STUDIES OF BIOLOGICAL
Fe(III)-Fe(IV) dimers vs the Fe(III)-Fe (II) dimers indicates that the Ν bridge stabilizes the Fe(III), Fe(IV) oxidation state by almost 700mV when compared to the Ο bridge. This is consistant with the properties of Ν vs Ο as the bridging atom. The greater negative charge on the Ν bridge, the larger size of Ν and the greater magnitude of π bonding with Ν , all favor the higher oxidation state of the iron atoms in the Ν bridged dimer when compared to the Ο bçidged dimer. X-Ray data shows that the Fe-N distance is 1.6605A in (FeTPP)N which is considerably shorter than the 1.763 A in (FeTPP) 0. This is consistant with the infrared data which indicates a greater degree of π charge delocalization for Fe-N-Fe vs Fe-O-Fe. However, despite this difference between bridging atoms, the potential difference, Δ, between reactions (2) and (5) are about equal with Δ = 2.36 V for (FeTPP)N and Δ = 2.26 V for (FeTPP) 0. This is withi been reported for a serie of octaethyl and tetraphenylporphyrin(14,15) complexes independent of changes in central metal oxidation state. Accordingly, we would like to propose that the electrode reactions (2) and (5) correspond to the formation of the cation radical and anion radical, respectively. The assignment of a cation radical product has been previously reported (_5,6J while that of an anion radical product differs from an earlier characterization of [(FeTPP)^O] in DMF (4). In this solvent, an Fe(III), Fe(II) dimer was observed. Further studies aimed at characterization of the reduction products of (FeTPP)0 and (FeTPP)N in several solvents are now in progress. 3
2
3
3
3
o
2
2
2
2
2
2
Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9.
(a) California State University, Fullerton (b) Brooklyn College Cohen, I. A., Ostfeld, D., and Liechtenstein, B., J. Amer. Chem. Soc., (1972) 94, 4522. Lexa, D., Momenteau, Μ., and Mispelter, J., Biochim. Biophys. Acta., (1974), 338, 151. Kadish, Κ. Μ., Larson, G., Lexa, D., and Momenteau, M., J. Amer. Chem. Soc., (1975), 97, 282. Felton, R. H., Owen, G. S., Dolphin, D. and Fajer, T., J. Amer. Chem. Soc., (1971), 93, 6332. Felton, R. Η., Owen, G. S., Dolphin, D., Forman, Α., Borg, D. C., and Fajer, T., Ann. N.Y. Acad. Sci., (1973), 206, 504. Summerville, D. A. and Cohen, I. Α., J. Amer. Chem. Soc., (1976), 98, 1747. Scheldt, W. R., Summerville, D. A. and Cohen, I. Α., J. Amer. Chem. Soc., (1976), 98, 6623. Torrens, Μ. Α., Straub, D. K. and Epstein, L. M., J. Amer. Chem. Soc., (1972), 94, 4160.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
5.KADISHETAL. N-Bridged Dimer in Nonaqueous Media 10. 11. 12. 13. 14. 15. 16.
77
Cohen, I. Α., J. Amer. Chem. Soc., (1969), 91, 1980. Hoffman, Α. Β., Collins, D. Μ., Day, V. W., Fleischer, E. B., Srivastara, T. S. and Hoard, J. L., J. Amer. Chem. Soc., (1972), 94, 3620. LaMar, G. Ν., Eaton, G. R., Holm, R. H. and Walker, F. Α., J. Amer. Chem. Soc., (1972), 94, 3620. J. H. Christie, J. Osteryoung and R. A. Osteryoung, Anal. Chem., (1973), 415, 210. Morrison, M. and Kadish, Κ., unpublished results. Fuhrhop, J. Η., Kadish, R. M. and Davis, D. G., J. Amer. Chem. Soc., (1973), 95, 5140. Kadish, Κ. Μ., Davis, D. G. and Fuhrhop, J. Η., Angew. Chem. (Int. Edit.), (1972), 11, 1014.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
6 Electrochemically Catalyzed Reduction of Nitrogenase Substrates by Binuclear Molybdenum(V) Complexes FRANKLIN A. SCHULTZ, DEBRA A. LEDWITH, and LOUIS O. LEAZENBEE Department of Chemistry, Florida Atlantic University, Boca Raton, Fla. 33431
A widely cited model for nitrogenase enzyme (1) is based on the binuclear di-μ-oxo-bridge Na2Mo2O4(Cys)2(2).Solution reducing agent (NaBH or Na2S2O ) catalytically reduce the enzyme substrates dinitrogen, acetylene, nitriles, and isonitriles in mildly alkaline (pH 7-12) aqueous media. The mechanism for these catalytic reductions (see Scheme I i n ref. 3) i s proposed to consist of the following sequence of reactions: dissociation of the binuclear Mo(V) complex, reduction to a monomeric Mo(IV) species, binding and reduction of substrate, and completion of the catalytic cycle by reduction of the oxidized catalyst with BH4-. 4
4
1/2 Mo O (Cys)2 2
Mo(V)-Cys
2-
4
-
(1)
-
Mo(V)-Cys + e (BH4 ) -> Mo(IV)-Cys Mo(IV)-Cys + C H 2
(2)
Mo(IV)-Cys-C H 2
2
(3)
2
Mo(IV)-Cys-C H + 2H+->Mo(VI)-Cys + C H 2
2
2
(4)
4
Mo(VI)-Cys + 2e-(BH -) -> Mo(IV)-Cys
(5)
4
Despite the success of this model i n simulating many of the reactions of nitrogenase enzyme, relatively little is known about the oxidation-reduction chemistry of the binuclear Mo O4 center and the means by which an active catalyst is generated from this species. A major research objective i n our laboratory has been to characterize the electrochemical behavior of binuclear molybdenum complexes. We have recently reported detailed electrode reaction mechanism studies of the principal nitrogenase model compound, Na Mo O4(Cys) (4), and its EDTA analog, NaMoO(EDTA) (5), in aqueous borate, phosphate, and ammonia buffers. This work is now being extended to a series of cysteine and EDTA complexes containing μ-οχο-μ-sulfido (Mo O3S ) and 2+
2
2
2
2
2
2
4
2+
2
78
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
6.
SCHULTZ E T A L .
Reduction
of Nitrogenase
79
Substrates
2+
and d i - y - s u l f i d o (Mo20 S2 ) bridged Mo(V) cores (Figure 1) ( 6 ) . Not s u r p r i s i n g l y , we a l s o have found that e l e c t r o c h e m i c a l r e d u c t i o n of Na Mo20i (Cys) and other oxo- and s u l f i d o - b r i d g e d Mo(V) species i n the presence o f acetylene leads to c a t a l y t i c r e d u c t i o n of t h i s substrate (7). Aside from demonstrating that c a t a l y s i s can be i n i t i a t e d e l e c t r o c h e m i c a l l y , two o b j e c t i v e s i n t h i s work have been 1) to use information from e l e c t r o d e r e a c t i o n mechanism s t u d i e s to help determine the nature and o x i d a t i o n s t a t e of the a c t i v e c a t a l y s t , and 2) to use c o n t r o l of e l e c t r o c h e m i c a l v a r i a b l e s and s o l u t i o n c o n d i t i o n s to provide i n s i g h t to the mechanism of c a t a l y s i s . F o r the oxo- and s u l f i d o - b r i d g e d Mo(V) complexes features such as l i g a n d and b r i d g i n g atom s i g n i f i c a n t l y i n f l u e n c e the e l e c t r o c h e m i c a l and c a t a l y t i c p r o p e r t i e s of the b i n u c l e a r center. The r e s u l t s of these s t u d i e s provide a general framework f o r understanding the mode of production of a c t i v e c a t a l y s t s from b i n u c l e a which these species c a t a l y z substrates. 2
2
f
2
E l e c t r o c h e m i s t r y o f Oxo- and S u l f i d o - B r i d g e d Complexes of Mo(V) A l l of the oxo- and s u l f i d o - b r i d g e d Mo(V)-cysteine and EDTA complexes are reduced to b i n u c l e a r Mo(III) products i n a s i n g l e , d i f f u s i o n - c o n t r o l l e d step a t ca. -1.1 to -1.3 V vs. SCE i n 0.1 F_ Νβ2Βι θ7· Some experimental r e s u l t s are shown i n Figures 2 and 3 and Table I . C o n t r o l l e d p o t e n t i a l coulometry and compara t i v e voltammetric and chronoamperometric current measurements confirm that four e l e c t r o n s are t r a n s f e r r e d i n the r e d u c t i o n of each complex. The Mo(111)2 e l e c t r o d e products are r e o x i d i z e d to Mo(V) species at p o t e n t i a l s 200-500 mV p o s i t i v e of the i n i t i a l r e d u c t i o n peak. The q u a s i r e v e r s i b l e character of t h i s e l e c t r o n t r a n s f e r process i s dependent upon both the s t r u c t u r e of the complex and the composition of the b u f f e r i n g medium. Several important e f f e c t s of l i g a n d and bridge atom s u b s t i t u t i o n are apparent from the e l e c t r o c h e m i c a l data. Replacement of 0 by one or two b r i d g i n g S atoms g r e a t l y increases the r e v e r s i b i l i t y o f the Mo(V) /Mo(111)2 e l e c t r o n t r a n s f e r process, as i n d i c a t e d by the decrease i n ΔΕ . S u l f u r b r i d g i n g atoms a l s o i n f l u e n c e the s t a b i l i t y of t h i t i m e r i c Mo(III) e l e c t r o d e products. The reverse peak currents shown i n the c y c l i c voltam metric experiments i n F i g u r e 2 i n d i c a t e that the s u l f i d o - b r i d g e d M o ( I I I ) - c y s t e i n e products a r e l e s s s t a b l e than the di-μ-οχο analog, and that the r a t e o f decomposition increases i n the order: Mo 0 < Mo 0 S < Mo 0 S +. A l l of the oxo- and s u l f i d o - b r i d g e d Mo(III) -EDTA products a r e s t a b l e on the time s c a l e of c y c l i c voltammetry (Figure 3). The Μο 0ι+ (EDTA) *" complex can be c a r r i e d through a complete coulometric r e d u c t i o n and r e o x i d a t i o n c y c l e with i t s oxo-bridged s t r u c t u r e i n t a c t (5). However, changes i n absorption s p e c t r a f o l l o w i n g reduction of M02O2S2(EDTA) " i n d i c a t e that i t s r e d u c t i o n product undergoes a +
2
2
e
2 +
2
i +
2 +
2
3
2
2
2
2
2
2
2
2
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
ELECTROCHEMICAL STUDIES O F BIOLOGICAL SYSTEMS
80
Mo 0 X (cysteine) 2
2
2
2
.2-
Ο
Figure 1. Structures of the bi nuclear oxo- and sulfido-bridged molybdenum(V)-cysteine and EDTA complexes
Figure 2. Cyclic voltammetric curves for reduction of ImM oxo- and sulfido-bridged Mo(V)-cysteine complexes at a H g elec trode in 0.1F Na Bj,0 . Scan rate = 0.1 V/sec. Inset, 2c: scan rate = 20 V'/sec. 2
0
Mo 0 X (EDTA) 2
2
X=SorO
2
7
E,V vs SCE
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977. 1 / 2
/Τ7
/v
C λ
k
l
2
3
2
2
2
1
(46.6)
100
7
0.9
0.004
(small)
(stable product) (stable product)
2
2
4
From references (4) , (5) and ( 6 ) . ^Data recorded at ν = 0. 1 V/s i n 0.1 F N a B 0 at a hanging Values i n parentheses recorded at Hg drop e l e c t r o d e (A = 0.022 cm ); p o t e n t i a l s i n V vs. SCE. ν = 20 V/s.
2
Na Mo 0 S (Cys)
2
(0.51)
(0.096)
-1.29(sh) (-1.39)
i+
2
49.2
0.296
0.079
-1.213
2
2
Na Mo 0 S(Cys)
2
2
2
57.0
0.522
0.056
-1.310
Na Mo 0 (Cys)
2
2
>0
57.9
0.170
0.055
-1.068
Na Mo 0 S (EDTA)
2
0
60.1
0
0.172
3
2
0.052
2
2
-1.079
2
2
Na Mo 0 S (EDTA)
c
A
Ρ , 1/2 1/2 „ (μΑ s /V mM) ( s " )
i
59.1
Ρ
0.382
ΔΕ
0.053
P
-1.248
E
pc" /2
N a M o 0 (EDTA)
E
(V)
pc (V)
Ε
Voltammetric Data f o r Reduction of Oxo- and S u l f i d o - B r i d g e d Mo(V) Complexes
(V)
Compound
Table I.
ELECTROCHEMICAL
82
STUDIES OF BIOLOGICAL
SYSTEMS
slow d i s s o c i a t i o n r e a c t i o n . Some d i s s o c i a t i o n a l s o may occur f o l l o w i n g reduction of Mo 0 S(EDTA) ~. The dimeric Mo(III)-EDTA products are c l e a r l y more s t a b l e than the analogous c y s t e i n e compounds. We b e l i e v e t h i s greater s t a b i l i t y i s due p r i m a r i l y to the f a c t that EDTA bridges both Mo centers and thereby i n creases the i n t e g r i t y of the b i n u c l e a r u n i t . Another feature observed during e l e c t r o c h e m i c a l experiments i s that decomposition of the i n i t i a l Mo ( I I I ) - c y s t e i n e e l e c t r o d e products leads to species which c a t a l y z e H r e d u c t i o n at the mercury e l e c t r o d e . T h i s behavior i s p a r t i c u l a r l y pronounced f o r M o 0 S 2 ( C y s ) ~ , i n which case the voltammetric wave i s observed as a shoulder on the background discharge of H+. However, the c h a r a c t e r i s t i c M o ( V ) / M o ( I I I ) redox process f o r t h i s compound i s apparent at f a s t e r scan rates (Inset, F i g u r e 2c) where d i s s o c i a t i o n of the Mo (III)2 product i s l e s s extensive A mechanism f o r e l e c t r o c h e m i c a Mo(V) complexes i s show b u f f e r e f f e c t s has shown that the i n i t i a l step i n the r e a c t i o n i s a concerted 4-electron/4-proton t r a n s f e r i n which protonated b u f f e r species are involved i n the t r a n s i t i o n s t a t e of the e l e c t r o d e r e a c t i o n (4, .5)· h e protonated b u f f e r species probably i n t e r a c t with the terminal oxo groups of the Mo^^ "*" u n i t and f a c i l i t a t e coupled e l e c t r o n - p r o t o n t r a n s f e r to produce c o o r d i nated aquo groups i n the Mo (III) products. The b u f f e r species apparently r e p l a c e water molecules w i t h i n the Mo (III) coordina t i o n sphere soon a f t e r r e d u c t i o n of the Mo(V) dimer. For example, the s t a b l e oxo-bridged Mo (III) -EDTA products d i s p l a y v i s i b l e absorption bands which s h i f t with changes i n b u f f e r medium (5), and a b i n u c l e a r Mo(III) complex r e c e n t l y has been i s o l a t e d i n which acetate, EDTA, and oxo groups simultaneously bridge the two Mo atoms (8). S i m i l a r b r i d g i n g by borate or phosphate oxyanions (A) i s represented i n the e l e c t r o d e products i n F i g u r e 4. D i s s o c i a t i o n of the b i n u c l e a r Mo (III) products to c a t a l y t i c a l l y a c t i v e species i n v o l v e s a complicated s e r i e s of r e a c t i o n s . The process appears to be i n t r a m o l e c u l a r and to i n v o l v e a b u f f e r coordinated e l e c t r o d e product, s i n c e the r a t e of d i s s o c i a t i o n depends on b u f f e r type but not on pH, b u f f e r c o n c e n t r a t i o n , or a d d i t i o n of n u c l e o p h i l e s . For Μ ο 0 ( C y s ) " " i t has been deter mined that the rate-determining step i n v o l v e s cleavage of one of the μ-οχο bridge bonds to form a mono-oxo-bridged s p e c i e s . The l a t t e r species i s observed as a second anodic peak at slow voltam metric scan r a t e s , but vanishes a f t e r t o t a l e l e c t r o l y s i s . Further steps i n the sequence have not been f u l l y d e l i n e a t e d , but appear at l e a s t to i n v o l v e r e a c t i o n to a f u r t h e r Mo (III) dimer (bridged s o l e l y by oxo group or b u f f e r anion) i n e q u i l i b r i u m with Mo(III) monomer (which i s apparent from c a t a l y t i c s t u d i e s ) . The u l t i m a t e e l e c t r o d e r e a c t i o n products of Μ ο 0 ^ ( C y s ) ~ are n o n - e l e c t r o a c t i v e and have not been s u c c e s s f u l l y c h a r a c t e r i z e d to date. However, three bands (2 brown, 1 green) can be resolved by g e l column 2
2
3
2
+
2
2
2
2
2
T
2
2
2
2
4
2
2
2
2
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
2
6.
Reduction
SCHULTZ E T A L .
I -0.6
1
1
.
.
1
-0.8
I
of Nitrogenase
ι
I
-1.2
-1.0
-1.4
83
Substrates
Figure 3. Cyclic voltammetric curves for reduction of ImM oxo- and sulfido-bridged Mo(V)-EDTA complexes at a H g elec trode in 0.1F Na Bfi . Scan rate = 0.1 \ /sec. 2
7
7
E,VvsSCE
A" Ή
.Mo + 4HA + 4e" ^
? / 0 \ ? eq Mo^ Mo ^ : M o ^
Η
V ^ι
Mo ^ 0 ' ^Mo • 3/Γ
K
A"
Λ .0^ Mo;
Mo + 2HJ0
.Mo
T J Q * " Μσ H
Figure 4.
2°
OH
MO HO
further ^ Mo(m)dimer
2Mo(m)-cys
Proposed mechanism for electrochemical reduction of bi nuclear molybdenum(V) complexes
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
84
ELECTROCHEMICAL
STUDIES O F BIOLOGICAL
SYSTEMS
chromatography f o l l o w i n g exhaustive e l e c t r o l y s i s of Mo20i (Cys)2 · A mixture of products i s c o n s i s t e n t with the r e a c t i o n scheme shown i n F i g u r e 4. The μ-οχο-μ-sulfido- and d i ^ - s u l f i d o - b r i d g e d complexes appear to undergo s i m i l a r d i s s o c i a t i o n r e a c t i o n s f o l l o w i n g reduction to the Mo (III) s t a t e . Table I shows estimated values of the d i s s o c i a t i o n r a t e constant, k}, f o r a l l compounds i n 0.1 F Na2Bi 0 . These estimates were made by c y c l i c voltammetry (9) or double p o t e n t i a l - s t e p chronocoulometry (10) assuming a pseudo f i r s t - o r d e r d i s s o c i a t i o n r e a c t i o n (EC mechanism) with k = /(l + ) £ k j . More d e t a i l e d s t u d i e s of the d i s s o c i a t i o n michanism Ire c u r r e n t l y underway i n our l a b o r a t o r y . As demonstrated i n the f o l l o w i n g s e c t i o n , t h i s d i s s o c i a t i o n r e a c t i o n plays a key r o l e i n the generation of c a t a l y t i c a l l y a c t i v e s p e c i e s . +
t
7
Qfe
e
E l e c t r o c a t a l y t i c Reductio Previous s t u d i e s of the chemical model system GL,3) have f a i l e d to answer a number of important questions regarding the nature of the a c t i v e c a t a l y s t and the mechanism of c a t a l y t i c substrate reduction. In attempting to answer the l a t t e r two p o i n t s we have decided to i n v e s t i g a t e i n d e t a i l the e l e c t r o c a t a l y t i c r e d u c t i o n of a s i n g l e s u b s t r a t e , acetylene, rather than to survey the behavior of a l l known substrates of the system. T h i s choice i s d i c t a t e d l a r g e l y by the f a c t that acetylene provides more e a s i l y assayed products and i s reduced more r a p i d l y i n the model system than the true b i o l o g i c a l sub s t r a t e , d i n i t r o g e n . While i n v e s t i g a t i o n s with N w i l l provide the u l t i m a t e b i o l o g i c a l relevance, our experiments with ΰ Η have provided s i g n i f i c a n t information regarding the o x i d a t i o n s t a t e and p r o p e r t i e s of the reduced Mo c a t a l y s t s . Our experiments a r e c a r r i e d out by c o n t r o l l e d p o t e n t i a l coulometry at a s t i r r e d Hg pool cathode i n a sealed c e l l equipped with gas sampling p o r t s (7). Vapor phase samples are withdrawn p e r i o d i c a l l y f o r gas chromatographic a n a l y s i s on Porapak N. Two procedures a r e used to study the e l e c t r o c a t a l y t i c reduction: (A) a s o l u t i o n of Mo20i (Cys)2 "" i s reduced d i r e c t l y under 1 atm of C2H2; (B) a s o l u t i o n of Mo20 (Cys)2 ~ i s prereduced to the Mo (III) s t a t e , the c e l l i s purged with 1 atm C H , and p o t e n t i a l i s r e a p p l i e d . The l a t t e r procedure i s more convenient because r a t e p l o t s are i n i t i a l l y l i n e a r and i n t e r s e c t the o r i g i n . In procedure A, 20-30 minutes pass before mass transport c o n t r o l l e d r e d u c t i o n of the complex i s complete and acetylene i s reduced a t a constant r a t e . T y p i c a l behavior of the e l e c t r o c h e m i c a l p a r t of the e x p e r i ment i s shown i n F i g u r e 5 using procedure Β f o r the r e d u c t i o n of C2H2 with Mo20i (Cys)2 ~ as c a t a l y s t . During c o n t r o l l e d p o t e n t i a l r e d u c t i o n of the complex at -1.40 V, current f a l l s to a r e l a t i v e l y high steady-state value (5-10 mA) due to r e d u c t i o n of hydrogen ion at the e l e c t r o d e s u r f a c e c a t a l y z e d by the u l t i m a t e e l e c t r o d e 2
2
2
+
2
I+
2
2
2
+
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
2
6.
scHULTZ E T AL.
Reduction
of Nitrogenase
85
Substrates
T n e
products of Mo20i+(Cys) · magnitude of the H reduction current i s p r o p o r t i o n a l to the square-root of i n i t i a l dimer con c e n t r a t i o n , thus i n d i c a t i n g c a t a l y s i s by a monomeric s p e c i e s . A f t e r the reduced complex i s purged with acetylene and p o t e n t i a l i s r e a p p l i e d , the current r i s e s to a much higher steady-state value than during the previous e l e c t r o l y s i s . The magnitude of t h i s current a l s o increases with negative p o t e n t i a l f o l l o w i n g an i n i t i a l reduction at -1.40 V. A p p l i c a t i o n of p o t e n t i a l to the reduced molybdenum s o l u t i o n under 1 atm C2H2 r e s u l t s i n the reduction of acetylene by an apparent f i r s t - o r d e r process ( i . e . , l o g [ C H ] = - k t + const.) and l i n e a r production of ethylene and ethane over a 2-3 hour p e r i o d . D i s t r i b u t i o n of these species during a t y p i c a l e x p e r i ment i s shown i n Figure 6. The C^^iC^^ P °duct r a t i o i s about 4 or 5:1 and v a r i e s only s l i g h t l y with changes i n experimental c o n d i t i o n s . A considerabl concurrently with acetylen Figure 6 r e v e a l s that amounts of C^H^ and C H produced do not equal the quantity of C H reduced. An a d d i t i o n a l hydrocarbon product i s 1,3-butadiene (C^Hg), which i s produced at about 40% the l e v e l of ethylene ( i . e . , C H :C H :C Hg^5:2:l) . Butadiene a l s o has been discovered to be the major product of acetylene reduction i n the Μο 0^(Cys) "702^/6^"chemical model system (11), but i t i s not produced upon reduction of C H2 by the enzyme. With i n c l u s i o n of C^Hg a s u i t a b l e hydrocarbon balance i s obtained for the chemical model system (11), but the product balance f o r our e l e c t r o c a t a l y t i c system f a l l s as much as 60% short of the quantity of acetylene reduced. Thus, a d d i t i o n a l and as yet un detected products must be produced i n the e l e c t r o c h e m i c a l system. We d i d not discover the presence of butadiene or the discrepancy i n hydrocarbon balance e a r l y enough to take these f a c t o r s i n t o account i n a l l aspects of our i n v e s t i g a t i o n . Consequently, most r e s u l t s f o r the e l e c t r o c a t a l y t i c system are based e i t h e r on the r a t e of C H2 reduction or the r a t e of C ^ and C2H production. Our present r e s u l t s provide strong evidence that a monomeric Mo (III) complex i s the c a t a l y t i c a l l y a c t i v e species i n chemical models f o r nitrogenase based on molybdenum-sulfhydryl complexes. The e l e c t r o d e r e a c t i o n mechanism s t u d i e s of Μ θ 2 θ ( C y s ) 2 ~ and the analogous oxo- and s u l f i d o - b r i d g e d complexes with c y s t e i n e and EDTA e s t a b l i s h that these compounds are reduced d i r e c t l y to Mo (III) products with no evidence of the intermediate Mo(IV) o x i d a t i o n s t a t e . Involvement of Mo (III) i s e s t a b l i s h e d by the f a c t that C2H2 reduction occurs subsequent to the reduction of these complexes by two e l e c t r o n s per molybdenum. In a d d i t i o n , a p p l i c a t i o n of negative p o t e n t i a l to s o l u t i o n s containing equimolar Mo(III) (added as K M o C l ) and c y s t e i n e c a t a l y z e s acetylene r e d u c t i o n at approximately the same r a t e (see Table I I , procedure C). I t i s not l i k e l y that reduction proceeds beyond Mo(III) i n formation of the a c t i v e c a t a l y s t , because e l e c t r o c h e m i c a l l y r e duced s o l u t i o n s of M02O4(Cys)2 ~ or s o l u t i o n s of Mo(III) plus 2
2
2
r
2
2
6
2
2
I+
lf
6
2
2
2
2
2
2
2
g
4
3
2
6
2
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
86
ELECTROCHEMICAL STUDIES O F BIOLOGICAL SYSTEMS
120 100
CH 2
- 1.70V
2
-1.60 "
< Ε 60 Figure 5. Current-time curves ob served during electrocatalytic reduc tion of acetylene using procedure B. Experimental conditions: 1.70mM Na Mo 0 (Cys) 0.1F Ν α Β 0 , cell purged with 1 atm C H and potential reapplied as indicated.
ce
=> 40 ο —1.40V— 20
2
2
ll
2)
2
2
4
f Γ ^
-1.50 "
_____
7
50
2
100 TIME,min
100 TIME.min. Figure 6. Product-time and reactant-time behavior during electrocatalytic reduction of acetylene using procedure B. Experimental conditions: 1.70mM -1.40 V, 0.1F Na B 0 , Na Mo O (Cys) , 1 atm C H . 2
2
i
2
2
2
h
7
2
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
-140 -130 150
6.
Reduction
SCHULTZ E T A L .
Table I I .
a
Substrates
87
E l e c t r o c a t a l y t i c Reduction of Acetylene with Na Mo 0i (Cys)2 2
Proce dure
of Nitrogenase
2
+
Ε
C Hi . :
Rate
Cone.
2
4
app (μπιοί C Hi / min)
C H
(V)
(mM)
A A A
-1.40 -1.40 -1.40
1.00 1.00
9.6 9.2
0.204 0.264
4.8 4.9
Β Β Β Β
-1.40 -1.40 -1.40 -1.40
0.30 1.00 1.70 3.00
9.2 9.2 9.2 9.2
0.100 0.195 0.240 0.313
5.0 4.5 4.9 4.7
Β Β Β Β Β
-1.30 -1.40 -1.50 -1.60 -1.70
1.70 1.70 1.70 1.70 1.70
9.2 9.2 9.2 9.2 9.2
0.136 0.240 0.353 0.498 0.708
3.9 4.9 3.9 4.2 4.2
Β Β
-1.35 -1.27
1.00 1.00
8.3(P) 9.2(A)
0.139 0.106
2.4 2.9
C
-1.40
1.00
9.2
0.133
4.7
D
no Ε
1.00
9.2
0.003
-
2
+
2
6
P r o c e d u r e s : A. Complex reduced under 1 atm C H B. Complex prereduced at -1.40 V, c e l l purged with 1 atm C H , p o t e n t i a l r e a p p l i e d as i n d i c a t e d C. P o t e n t i a l a p p l i e d to 2mM K M o C l + 2mM c y s t e i n e under 1 atm C H D. Complex prereduced at -1.40 V, purged with 1 atm C H , and allowed to stand without p o t e n t i a l applied 2
2
2
3
2
2
2
6
2
2
b A l l s o l u t i o n s contain 0.1 F N a B 0 and A = ammonia (0.25 F) 2
i+
7
except Ρ = phosphate (0.5 F)
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
88
ELECTROCHEMICAL
STUDIES O F BIOLOGICAL
SYSTEMS
c y s t e i n e e x h i b i t no voltammetric r e d u c t i o n peaks. A number of c o n t r o l experiments have been performed and demonstrate that Mo(III) plus an a d d i t i o n a l source of e l e c t r o n s (or Η ) i s r e q u i r e d f o r C2H2 r e d u c t i o n . For example, e l e c t r o l y s i s of s o l e l y C H2 at 1 atm i n borate b u f f e r at very negative p o t e n t i a l s , even i n the presence of t ^ - e v o l v i n g c a t a l y s t s such as c y s t e i n e , produces no reduced hydrocarbons. I f the Μθ2θ^(Cys)2 ~ complex i s reduced e l e c t r o c h e m i c a l l y , purged w i t h 1 atm C2H2, and allowed to stand, only minimal C R^ i s produced (Table I I , procedure D). When p o t e n t i a l i s r e a p p l i e d , c a t a l y s i s resumes at the normal r a t e . A sampling of data i s shown i n Table I I i l l u s t r a t i n g the e f f e c t s of various experimental c o n d i t i o n s and components on the e l e c t r o c a t a l y t i c r e d u c t i o n of acetylene. Notable f a c t o r s which i n c r e a s e the r a t e of C H.i+ production are decreasing pH, i n c r e a s ing concentration of complex S e v e r a l experiments ru that the r a t e of C U^ production i s decreased s l i g h t l y r e l a t i v e to borate b u f f e r . The e f f e c t of Μο 0ι (Cys) concentration on the rates of C E^ and C H production i s shown i n F i g u r e 7. These squareroot dependences on i n i t i a l concentration of complex demonstrate that the a c t i v e c a t a l y s t i s a monomeric species i n e q u i l i b r i u m w i t h a l a r g e r f r a c t i o n of dimeric m a t e r i a l . The same r e s u l t i s found i n the chemical model system regarding C E^ production (3,12) , but a l i n e a r dependence of C ]i^ formation on [Mo20i (Cys)2 ~] has not been noted p r e v i o u s l y . Production of a monomeric c a t a l y s t could be achieved through a sequence of chemical steps as o u t l i n e d i n Figure 4. Although we have not confirmed t h i s mechanism i n the d e t a i l depicted, s e q u e n t i a l d i s s o c i a t i o n of the i n i t i a l Mo ( I I I ) e l e c t r o d e product through one or more dimeric intermediates i s c o n s i s t e n t with the electrochem i c a l r e s u l t s , observation of s e v e r a l species i n product i s o l a t i o n attempts, and observation of an e q u i l i b r i u m c o n c e n t r a t i o n of monomeric c a t a l y s t . D i s s o c i a t i o n of Mo(III) atoms f o l l o w i n g r e d u c t i o n of the b i nuclear center i s c l e a r l y an important step i n the generation of an a c t i v e c a t a l y s t . For t h i s reason the e n t i r e s e r i e s of oxoand s u l f i d o - b r i d g e d Mo(V)-cysteine and EDTA complexes described e a r l i e r has been examined i n the acetylene r e d u c t i o n experiment. Results are shown i n Table I I I , and i n d i c a t e that s u l f u r b r i d g i n g atoms and l i g a n d play an important r o l e i n producing an a c t i v e c a t a l y s t and i n the r a t e and mechanism of C H2 r e d u c t i o n . The s u l f i d o - b r i d g e d c y s t e i n e complexes, Mo20 S(Cys)2 ~ and M02O2S2(Cys)2 ~, are reduced to Mo ( I I I ) products which d i s s o c i a t e r a p i d l y on the voltammetric time s c a l e . These products, however, provide only a marginal i n c r e a s e i n the r a t e of C H2 r e d u c t i o n and s i m i l a r G ^ H k ^ H g r a t i o s of 4 or 5:1. The di-μ-οχο EDTA complex, Μ ο 0 ^ ( E D T A ) * - , i s reduced to a s t a b l e b i n u c l e a r product which i s completely i n e f f e c t i v e i n acetylene r e d u c t i o n . 2
2
2
2
2
2
2
2
2
+
6
2
2
2
1
2
+
2
2
2
3
2
2
2
2
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
6.
SCHULTZ E T A L .
Table I I I .
Reduction
of Nitrogenase
E l e c t r o c a t a l y t i c Reduction of Acetylene with Various Oxo- and S u l f i d o - B r i d g e d Molybdenum(V) Complexes
Compound
Rate (ymol C Hi + C H /min)
C Hi : C Hg
0.302
3.9
0.323
4.0
0.324
4.8
2
+
Na Mo 0i (Cys)2 2
2
t
Na Mo 0 S(Cys) 2
2
3
2
Na Mo 0 S (Cys) 2
2
2
2
2
Na Mo 0 (EDTA) 2
2
89
Substrates
2
2
+
-
0.0
4
2
6
Na Mo 0 S(EDTA) 2
2
3
Na Mo 0 S (EDTA) 2
2
2
0.33
0.330
2
a) A l l s o l u t i o n s contain 1.70 mM complex i n 0.1 F Na Bi 0y, Ε -1.40 V, 1 atm C H , procedure Β (see footnote a, Table i f ? ? 2
2
+
2
2
Mo 0 S(EDTA) "", which shows some a c t i v i t y towards C H r e d u c t i o n , may d i s s o c i a t e s l i g h t l y a f t e r e l e c t r o c h e m i c a l reduction. The d i - y - s u l f i d o complex, M o 0 S ( E D T A ) ~ however, d i s s o c i a t e s slowly but q u i t e e v i d e n t l y f o l l o w i n g i t s e l e c t r o c h e m i c a l reduction. This reduced m a t e r i a l c a t a l y z e s production of C H and C H at a r a t e equal to the corresponding c y s t e i n e compound and y i e l d s an i n v e r t e d product r a t i o of 0 Η β : 0 Η ι = 3:1. F i g u r e 8 shows the s i g n i f i c a n t e f f e c t of e l e c t r o d e p o t e n t i a l on the e l e c t r o c a t a l y t i c process. The rates of 0 Η ^ and C Hg pro duction and acetylene r e d u c t i o n increase e x p o n e n t i a l l y with poten t i a l , as does the steady-state current f o l l o w i n g a p p l i c a t i o n of p o t e n t i a l to an acetylene-purged s o l u t i o n . One experimental ob s e r v a t i o n which p a r a l l e l s t h i s behavior i s the c a t a l y t i c evolu t i o n of H at the mercury e l e c t r o d e , which occurs concurrently with acetylene reduction and a l s o i n the presence of the Mo ( I I I ) e l e c t r o d e products alone. During a t y p i c a l experiment hydrogen e v o l u t i o n may account f o r 30-40% of the t o t a l coulombs passed, the remainder going to C H r e d u c t i o n . We b e l i e v e i t i s premature to dismiss hydrogen e v o l u t i o n as an experimental a r t i f a c t . Evolu t i o n of H i n the absence of substrates other than IT*" i s an im portant feature of the chemical model system (3,12) and of n i t r o genase enzyme (13). In the chemical model, f o r example: (a) NaBH^ i s a more e f f e c t i v e reductant than N a S 0 i and a l s o c a t a l yzes H e v o l u t i o n more s t r o n g l y (3), and (b) a d d i t i o n of c o c a t a l y s t s such as methyl v i o l o g e n (14) and F e ^ S ^ ( S R ) c l u s t e r s 2
3
2
2
2
2
2
2
9
2
2
2
l+
2
+
2
2
2
6
2
2
2
2
+
2
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
2
90
ELECTROCHEMICAL STUDIES OF BIOLOGICAL SYSTEMS
Figure /. Rates of ethylene and ethane production as a function of square root of Na Mo 0 (Cys) concentration. Other experimental as in Figure 6. 2
2
Jf
2
Figure 8. Effect of electrode potential on catalytic current, acetylene reduction rate, and ethylene and ethane production rates during electrocatalytic reduction of C H using procedure B. Experimental conditions as in Figure 5. 2
2
E.Vvs. SCE
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
SCHULTZ E T A L .
6.
Reduction
of Nitrogenase
91
Substrates
(12,15), which i n c r e a s e the C2H2 r e d u c t i o n r a t e , a l s o s t i m u l a t e the production of H . The e f f e c t of e l e c t r o d e p o t e n t i a l i n d i c a t e s that an important r e l a t i o n s h i p e x i s t s between H2 e v o l u t i o n and C H r e d u c t i o n i n the e l e c t r o c a t a l y t i c system. C l e a r l y , an e l e c trode a c t i v a t e d rather than a bulk s o l u t i o n process i s i n v o l v e d i n the rate-determining step of acetylene reduction. Four p o s s i b l e mechanisms which have been considered f o r the e l e c t r o c h e m i c a l l y c a t a l y z e d r e d u c t i o n of acetylene are l i s t e d i n Table IV. In each case formation of a Mo(III)-C2H2 adduct i s assumed to be the i n i t i a l step i n the mechanism, because the requirement of molybdenum i n d i c a t e s that some i n t e r a c t i o n between the reduced Mo species and acetylene must occur during c a t a l y s i s . I t i s not p o s s i b l e to confirm or r e j e c t any of these mechanisms with c e r t a i n t y . Mechanism D, however, i s c o n t r a d i c t e d l e a s t by present evidence. T h i s mechanism i s viewed as an e l e c t r o c a t a l y t i c hydrogénation i n whic c a t a l y s t i s to bind acetylen produced at the e l e c t r o d e surface. Hydrogen atoms are the product of hydrogen i o n r e d u c t i o n catalyzed by the Mo(III)-cysteine 2
2
2
Table IV.
P o s s i b l e Mechanisms f o r E l e c t r o c a t a l y t i c of Acetylene
Mo (III)
A.
2
2
Mo(V)
+ 2H
+
+ 2e~
+ 2H
+
+ 2e"
H-MoUlD-C^
+ H+
followed
2
+
Mo(V)
+ C^fy
£
Mo (III)
+
Mo (III)
+ C
+
H-MoUH)-^^ +
+
Mo (III)
+
H+
C^H,
E l e c t r o c a t a l y t i c Hydrogénation j.Mo(III)-Cys + 2H+ + 2e"
Ο Γ
2
Homogeneous Hydrogénation Mo ( I I I ) - C ^ +
D.
Mo(III)-C H
E l e c t r o c h e m i c a l Reduction of Adduct Mo ( I I D - C ^
C.
?
Homogeneous Redox Reaction Mo(III)-C H
B.
+ C ^
Reduction
+
Mo(III)-Cys +
Mo(III)-C H, + 2H+ + 2e"
->
Mo(III)-C H
Modll)-^!^
+
Mo(III) +
2
+ 2H-
2
2H+
2
2H-
C> \ 2
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
by:
92
ELECTROCHEMICAL
STUDIES OF BIOLOGICAL
SYSTEMS
complex with or without C H bound to i t . This process i s much the same as the t r a d i t i o n a l e v o l u t i o n of H at mercury e l e c t r o d e s c a t a l y z e d by t r a n s i t i o n metal complexes with s u l f u r - c o n t a i n i n g ligands (16), and would e x h i b i t an exponential dependence on p o t e n t i a l . C a t a l y s i s presumably occurs when a M o ( I I I ) - c o o r d i nated s u l f u r atom (from e i t h e r c y s t e i n e l i g a n d or b r i d g i n g S group) i s protonated to form an -SH+ species and then reduced: 2
2
2
Mo(III)-S:
+ H+
Mo(III)-SH+ + e"
Mo(III)-SH+
(6)
+ Mo(III)-SH-
(7)
These species could r e a c t to form
H
2
2 Mo(III)-SH- -> 2 Mo(III)-S:
+ H
(8)
2
or, i n the presence of 2 Mo(III)-SH- + M o ( I I I ) - C H 2
+ 2 Mo(III)-S:
2
+ Mo(III) + 0 Η 2
4
(9)
The a l t e r n a t i v e mechanisms i n Table IV are c o n t r a d i c t e d by at l e a s t one piece of experimental evidence. In mechanisms A and Β the regeneration of c a t a l y s t and r e d u c t i o n of adduct are accomplished through d i f f u s i o n - l i m i t e d e l e c t r o c h e m i c a l processes. I t i s d i f f i c u l t to imagine how these e l e c t r o c h e m i c a l steps could be so i r r e v e r s i b l e as to d i s p l a y the observed p o t e n t i a l depen dence over a range of 400-500 mV. Mechanism A a l s o i s discounted by the f a c t that a d d i t i o n of C H to s o l u t i o n s of Mo (III) does not lead to s u b s t a n t i a l formation of 0 Η . Mechanism C i s s i m i l a r to known homogeneous hydrogénation r e a c t i o n s c a t a l y z e d by t r a n s i t i o n metal complexes (17). A s i m i l a r mechanism i n v o l v i n g a h y d r i d i c intermediate has been suggested f o r the chemical system (18). T h i s mechanism does not seem a t t r a c t i v e i n the e l e c t r o c a t a l y t i c system, because, as the quantity of H i n the c e l l i s constantly i n c r e a s i n g during e l e c t r o l y s i s , the r a t e of c a t a l y t i c acetylene r e d u c t i o n remains constant. Ethane and 1,3-butadiene are two a d d i t i o n a l products of e l e c t r o c a t a l y t i c acetylene r e d u c t i o n . Ethane i s produced i n constant p r o p o r t i o n to ethylene under a v a r i e t y of experimental c o n d i t i o n s , and ethylene i t s e l f i s not reduced i n the c a t a l y t i c system. Therefore, a separate b i n d i n g r e a c t i o n between Mo(III) and ethylene does not take p l a c e before r e d u c t i o n to C Hg. A l s o , i t i s not l i k e l y that a dimeric Mo (III) species i s r e s p o n s i b l e f o r r e d u c t i o n of C H to C H because ethane formation i s l i n e a r l y dependent on [ M o 0 i ( C y s ) ~ ] / (Figure 7) and the Mo(III) dimer produced by r e d u c t i o n of M o 0 ( E D T A ) i s completely i n a c t i v e i n acetylene r e d u c t i o n (Table I I I ) . We b e l i e v e i t i s more l i k e l y that about 20-25% of the time two a d d i t i o n a l hydrogen atoms are i n s e r t e d i n t o a bound acetylene molecule before d i s s o c i a t i o n from the Mo (III) c a t a l y s t takes p l a c e : 2
2
2
4
2
2
2
2
2
6
2
2
+
1
2
2
2-
2
t+
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
6.
SCHULTZ E T A L .
Reduction
Mo(III)-C H 2
2
of Nitrogenase
+ 2H. + Mo(III)-C Hi 2
(10)
+
Mo(III)-C H^ + 2H- + Mo(III) + C H 2
2
Formation of butadiene can occur i f C H M o ( I I I ) - C H adduct before r e d u c t i o n . 2
2
93
Substrates
2
(11)
6
i n t e r a c t s with the
2
-, CH
CH + HI . . . CH
Mo(III)^|| ^CH
OTT+ η — 8
'
> Mo (III) + H C=CH-CH=CH 2
2
(12)
Subsequent polymerization r e a c t i o n s or e l e c t r o c h e m i c a l r e d u c t i o n of butadiene could e x p l a i n the i n a b i l i t y to o b t a i n a t o t a l hydro carbon balance i n the e l e c t r o c h e m i c a l system. Reaction 12 could w e l l be favored a t the high C H p a r t i a l pressures used i n t h i s work. The e f f e c t of acetylen t r i b u t i o n has not been chemical c a t a l y t i c systems. Discussion Several years" study of these model systems has impressed upon us the complicated nature of molybdenum s o l u t i o n chemistry. S p e c i f i c and unique e f f e c t s of b r i d g i n g atom, l i g a n d , s o l u t i o n environment, and b u f f e r s a l t are evident i n the chemical, e l e c trochemical, and c a t a l y t i c p r o p e r t i e s of these compounds. Such complexity, incompletely understood, makes the e x t r a p o l a t i o n of r e s u l t s to an e q u a l l y complicated b i o l o g i c a l system somewhat tenuous. However, we b e l i e v e our e l e c t r o c h e m i c a l s t u d i e s have provided r e s u l t s which are d i r e c t l y u s e f u l i n understanding the behavior of chemical models f o r nitrogenase based on molybdenums u l f h y d r y l complexes. These r e s u l t s a l s o c o n t r i b u t e to the general knowledge of molybdenum chemistry which i s necessary i n i n t e r p r e t i n g the behavior of the enzyme. A number of s i g n i f i c a n t p o i n t s emanating from these e l e c t r o c h e m i c a l s t u d i e s are d i s c u s sed below. 1. Molybdenum Oxidation State. A l l b i n u c l e a r Mo(V) compounds we have examined undergo Mo(V) ->Mo(III) reduction under aqueous s o l u t i o n c o n d i t i o n s comparable t o those used i n s t u d i e s of the chemical model and the enzyme i t s e l f . There i s no evidence o f the intermediate Mo(IV) o x i d a t i o n s t a t e . I t seems l i k e l y , there f o r e , that Mo (III) and not Mo(IV) i s the o x i d a t i o n s t a t e of the a c t i v e c a t a l y s t i n the Mo-cysteine model system. Despite recent comments to the contrary (19), we a l s o b e l i e v e that Mo (III) i s a strong candidate f o r the o x i d a t i o n s t a t e of the reduced Mo center i n nitrogenase. Other evidence a l s o supports the p o s s i b l e impor tance of Mo (III) i n the molybdenum-containing reductases: the r e d u c t i o n of N and C H c a t a l y z e d by i n o r g a n i c Mo (III) species over a range of temperatures and pressures (20,21), c o n s t r u c t i o n 2
2
2
2
2
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
94
ELECTROCHEMICAL
STUDIES OF BIOLOGICAL
SYSTEMS
of a s u c c e s s f u l model f o r n i t r a t e reduction based on the hexaaquomolybdenum(III) c a t i o n , M o ( H 2 0 ) (22), and t e n t a t i v e obser v a t i o n of Mo(III) epr s i g n a l s i n n i t r a t e reductase (23-25). Evidence f o r the molybdenum o x i d a t i o n s t a t e i n reduced n i t r o genase may be provided by x-ray absorption edge spectroscopy (26), but d e f i n i t i v e r e s u l t s are not yet a v a i l a b l e . 3+
6
2. Coupled Electron-Proton T r a n s f e r . Reduction of the b i nuclear complexes proceeds by coupled t r a n s f e r of four e l e c t r o n s and four protons i n a s i n g l e step. This observation i s s i g n i f i cant i n view of a recent proposal (27) that coupled e l e c t r o n proton t r a n s f e r i n m u l t i p l e s of two i s an important feature of molybdenum-containing enzymes. Furthermore, the unique b u f f e r e f f e c t s observed i n the e l e c t r o d e r e a c t i o n mechanism suggest that oxomolybdenum species may f a c i l i t a t e proton t r a n s f e r to or from substrates i n a h i g h l 3. D i s s o c i a t i o n of the B i n u c l e a r Center. D i s s o c i a t i o n of Mo(III) atoms f o l l o w i n g r e d u c t i o n of the b i n u c l e a r Mo(V) u n i t appears to be an e s s e n t i a l step i n the generation of an a c t i v e c a t a l y s t . Only those compounds which show evidence of d i s s o c i a t i o n f o l l o w i n g e l e c t r o c h e m i c a l reduction are e f f e c t i v e i n the c a t a l y t i c reduction of C H and H+. On the other hand, the dimeric Mo (III) r e d u c t i o n product of M02O4(EDTA) ~ does not c a t a l y z e r e d u c t i o n of C2H2, even though i t i s an extremely strong reducing agent (Ε°' = -1.06 V vs. SCE i n 0.1 F N a B i 0 ) . Also, i t i s apparent that d i s s o c i a t i o n of the b i n u c l e a r center occurs a f t e r , not p r i o r to Ç3), r e d u c t i o n to the Mo(III) s t a t e , and that s u l f u r b r i d g i n g atoms and ligands which do not bridge the two Mo centers i n c r e a s e t h i s tendency f o r d i s s o c i a t i o n . 2
2
2
2
+
7
4. S u l f u r B r i d g i n g Atoms. S u l f u r b r i d g i n g atoms i n c r e a s e the r e v e r s i b i l i t y of e l e c t r o n t r a n s f e r i n the b i n u c l e a r Mo(V) center and the ease of d i s s o c i a t i o n of b i n u c l e a r Mo(III) u n i t s . Both features enhance the c a t a l y t i c p r o p e r t i e s of the compounds we have studied. Thus, s u l f u r b r i d g i n g may be an important and d e s i r a b l e feature of Mo enzyme model chemistry. Several i n stances have been noted wherein s u l f i d o b r i d g i n g has imparted unusual s t a b i l i t y to b i n u c l e a r Mo(V) species (28,29), and i t has been suggested that such a feature may be undesirable i n terms of Mo enzyme model chemistry (29). In the Mo (III) o x i d a t i o n s t a t e , however, s u l f i d o b r i d g i n g enhances the r e a c t i v i t y of the b i nuclear u n i t and thus improves c a t a l y t i c a c t i v i t y . Presence of s u l f u r i n the Mo ( I I I ) c o o r d i n a t i o n sphere may i n c r e a s e the l a b i l i t y of t h i s o x i d a t i o n s t a t e and permit more f a c i l e b i n d i n g of substrates. S i m i l a r increases i n ease of s u b s t i t u t i o n promoted by t h i o l ligands have been noted r e c e n t l y i n chromium(III) chemistry (30-32).
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
95
Reduction of Nitrogenase Substrates
6. schultz et al.
5. Biological Function of Molybdenum. The primary function of the reduced molybdenum species may be simply to bind rather than transfer electrons to the substrate. In our electrochemical studies a number of mechanisms appear to be at least as probable as one involving a bulk solution Mo(V)/Mo(III) redox cycle. In the chemical model systems these alternative mechanisms could be translated to ones in which the chemical reductant [NaBH^, Na^O^ or Fe^Si^SR)^-*] transfers electrons (or H ) to molybdenum-bound C H without need of reoxidizing the Mo(III) center. In nitrogenase, a similar process can be pictured in which the molybdenum center is first reduced to its substratebinding oxidation state. Once bound, the substrate is reduced by a flow of electrons or reactive hydrogen from a proximal site (presumably Fei+S^-type ferredoxin) and then released, leaving the molybdenum site in its reduced state. Transfer of two electrons and two protons or transfe ally equivalent mechanism or bridged by an atom such as sulfur which could facilitate both proton and electron transfer. 2
2
2
Acknowledgment This research has been supported by the National Science Foundation under Grant GP-38442X. We are particularly grateful to Drs. W. E. Newton, Ε. I. Stiefel, J. W. McDonald and J. L. Corbin of the Charles F. Kettering Research Laboratory, Yellow Springs, Ohio for many fruitful discussions and for disclosing to us their discovery of butadiene product in the chemical model system prior to publication. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9.
Schrauzer, G.N., Angew. Chem. Int. Ed., (1975) 14, 514, and references therein. Kay, A. and Mitchell, P. C. H., J. Chem. Soc. A, (1970), 2421. Schrauzer, G. N. and Doemeny, P. Α., J. Amer. Chem. Soc., (1971) 93, 1608. Ott, V. R. and Schultz, F. Α., J. Electroanal. Chem., (1975) 61, 81. Ott, V. R. and Schultz, F. Α., J. Electroanal. Chem., (1975) 59, 47. Ott, V. R., Swieter, D. S. and Schultz, F. Α., manuscript in preparation. Ledwith, D. A. and Schultz, F. Α., J. Amer. Chem. Soc., (1975) 97, 6591. Kneale, G. G., Geddes, A. J., Sasaki, Y., Shibahara, T. and Sykes, A. G., J. Chem. Soc. Chem. Commun., (1975), 356. Nicholson, R. S. and Shain, I., Anal. Chem., (1964) 36, 706.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
96 10.
ELECTROCHEMICAL STUDIES OF BIOLOGICAL SYSTE Ridgway, Τ. Η., Van Duyne, R. P. and Reilley, C. N., J . Electroanal. Chem., (1972) 34, 267, 283.
11.
Corbin, J. L . , Pariyadath, N. and Stiefel, Ε. I., J. Amer. Chem. Soc., in press. 12. Tano, K. and Schrauzer, G. N., J. Amer. Chem. Soc., (1975) 97, 5404. 13. Hardy, R. W. F., Burns, R. C. and Parshall, G. W., Advan. Chem. Series, (1971) 100, 219. 14. Ichikawa, M. and Meshitsuka, S., J . Amer. Chem. Soc., (1973) 95, 3411. 15. Schrauzer, G. N., Kiefer, G. W., Tano, K. and Doemeny, P. Α., J. Amer. Chem. Soc., (1974) 96, 641. 16. Mairanovskii, S. G., "Kinetic and Catalytic Waves in Polarography," Plenum Press, New York, 1968. 17. James, B. R., "Homogeneous Hydrogenation," Wiley, New York, 1973. 18. Khrushch, A. P., Shilov Amer. Chem. Soc., (1974) 96, 4987. 19. Wentworth, R. A. D., Coordin. Chem. Rev., (1976) 18, 1. 20. Denisov, N. T., Shuvalov, V. F., Shuvalova, Ν. I., Shilova, A. K. and Shilov, A. E . , Dokl. Akad. Nauk SSSR, (1970) 195, 879. 21. Shilov, Α. Ε., Denisov, N. T., Efimov, O. Ν., Shuvalov, V. F., Shuvalova, N. D. and Shilova, Α. Κ., Nature, (1971) 231, 460. 22. Ketchum, P. Α., Taylor, R. C. and Young, D. C., Nature (1976), 259, 202. 23. Forget, P. and DerVartanian, D. V., Biochim. Biophys. Acta, (1972) 256, 600. 24. DerVartanian, D. V. and Forget, P., Biochim. Biophys. Acta, (1975) 379, 74. 25. Orme-Johnson, W. Η., Jacob, G., Henzl, M. and Averill, Β. Α., ACS Centennial Meeting, New York, 1976, Abstr. INOR-137. 26. Cramer, S. P., Eccles, T. K., Kutzler, F. W., Hodgson, K. O. and Mortenson, L. E., J. Amer. Chem. Soc., (1976) 98, 1287. 27. Stiefel, Ε. I., Proc. Nat. Acad. Sci. U.S.A., (1973) 70, 988. 28. Spivack, B. and Dori, Z., J. Chem. Soc. Chem. Commun., (1973), 909. 29. Newton, W. E., Corbin, J. L., Bravard, D. C., Searles, J. E. and McDonald, J. W., Inorg. Chem., (1974) 13, 1100. 30. Weschler, C. J. and Deutsch, Ε., Inorg. Chem., (1973) 12, 2682. 31. Ramasami, T. and Sykes, A. G., Inorg. Chem., (1976) 15, 1010. 32. Asher, L. E. and Deutsch, Ε., Inorg. Chem., (1976) 15, 1531.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
7 Manganese(II) and -(III) 8-Quinolinol Complexes. R e d o x M o d e l for M i t o c h o n d r i a l Superoxide Dismutase
JOHN K. HOWIE, MARK M. MORRISON, and DONALD T. SAWYER Department of Chemistry, University of California, Riverside, Calif. 92502
The discovery i n 1969 (1) that superoxide i o n O, i s a common r e s p i r a t o r organisms with i t s y super oxide dismutase (SOD) has revolutionized the i n t e r pretation of b i o l o g i c a l oxidation-reduction processes. A subsequent discovery was a manganese-containing version of superoxide dismutase which can be i s o l a t e d from b a c t e r i a l sources (2,3) and from mitochondria (4) as well as the o r i g i n a l l y discovered copper-zinc form from erythrocytes. L i t t l e i s known about manganese SOD. The c r y s t a l structure has not been determined and there i s still controversy as to whether the manganese SOD contains one or two manganese atoms per enzyme molecule (2,4) . The exact r o l e of the manganese atom(s) i n the enzyme, the o x i dation state(s) of the manganese atom(s), the degree of association of the two manganese atoms, i f two are indeed present, the type of ligands coordinated to the manganese atom(s), and the stereochemistry around the metal(s) are not known. Although little i s known about the structure and properties of manganese SOD, its c a t a l y t i c reactions with superoxide ion can be represented by (5) -
2
Such a mechanism requires that the manganese e x i s t i n three d i f f e r e n t oxidation states if the enzyme contains only one metal atom per molecule. However, 97
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
98
ELECTROCHEMICAL
STUDIES O F BIOLOGICAL
SYSTEMS
only two formal oxidation states are required i f the enzyme contains two metal atoms. Because the manganese-containing enzymes catalyze oxidation-reduction reactions, electrochemical methods are p a r t i c u l a r l y a t t r a c t i v e f o r the study of the redox behavior of manganese complexes, both alone and i n the presence of substrate. The further objective i s to charact e r i z e the structures and coordination chemistry of these complexes i n s o l u t i o n by use of spectroscopic as w e l l as other p h y s i c a l c h a r a c t e r i z a t i o n techniques. The goal of the present research i s the i d e n t i f i c a t i o n and c h a r a c t e r i z a t i o n of manganese complexes that mimic the enzyme i n reactions represented by Equations 1-4 and that can serve as models f o r manganese SOD. This pape ese(II) and - ( I I I model compounds. Experimental Measurements and Materials. C y c l i c voltammetric experiments were performed using a v e r s a t i l e i n s t r u ment constructed from P h i l b r i c k s o l i d - s t a t e operat i o n a l a m p l i f i e r s (6). The c o n t r o l l e d p o t e n t i a l e l e c t r o l y s i s experiments were performed using a Wenking Model 61RH potentiostat and i n t e g r a t i n g the current vs. time curve using a K&E Model 62005 compensating polar planimeter. The electrochemical c e l l employed i n a l l electrochemical experiments was described previously (7). A Beckman Model 39273 platinum i n l a y electrode was used as the working electrode f o r c y c l i c voltammetry and a platinum gauze electrode was employed as the working electrode i n the coulometric experiments. The reference electrode was composed of a Ag/AgCl electrode i n aqueous t e t r a methylammonium chloride s o l u t i o n (0.000 V vs. SCE) and a glass bridge tube which made contact with the bulk s o l u t i o n through a cracked glass-bead j u n c t i o n . The platinum f l a g a u x i l i a r y electrode was i s o l a t e d from the bulk s o l u t i o n by a f i n e porosity f r i t . Dimethyl sulfoxide (DMSO) ( J . T. Baker analyzed reagent grade) had a water content of 0.02 to 0.06% as s p e c i f i e d by the manufacturer. Pyridine (Burdick and Jackson) contained 0.009% water and a c e t o n i t r i l e (MC/B Spectroquality grade) contained a maximum of 0.02% water. The solvents were degassed with argon i n the electrochemical c e l l p r i o r to the addition of the compound to be studied. Tetraethylammonium perchlorate (TEAP) was used as the supporting e l e c t r o -
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
7.
Manganese(II)
HOWIE E T A L .
and -(HI)
8-Quinolinol
Complexes
99
l y t e i n a 50-to 100-fold excess over the concentration of the e l e c t r o a c t i v e species. S o l i d - s t a t e magnetic s u s c e p t i b i l i t y values were determined by the Guoy method and s o l u t i o n mag n e t i c s u s c e p t i b i l i t y values were determined by the nuclear magnetic resonance (nmr) method (8). Conductivity measurements were made with an Ind. Instr. Model RC16B conductivity bridge and a dip c e l l . Mole cular weight determinations were made c r y o s c o p i c a l l y i n DMSO s o l u t i o n using a Hewlett-Packard Model 2801A Quartz thermometer t o measure temperature changes. The apparatus was c a l i b r a t e d with b e n z i l and a l l s o l u tions were prepared from the same f r e s h l y opened b o t t l e of DMSO. The solutions were protected from contamination by water i n the a i r by an atmosphere of dry argon. 8-Quinolinol wa ous acetate tetrahydrate, Mn (OAc) ·4Η2θ, was ob tained from A l f a . Methanol was reagent grade and used without further p u r i f i c a t i o n . Argon was dried by passing i t through a column packed with Aquasorb (Mallinckrodt). 1.00 M HC10 i n water and 1.42 M tetraethylammonium hydroxide i n methanol (Eastman) were used i n the electrochemical experiments where hydrogen and hydroxide ions were employed. II
2
4
Preparation of the Complexes 1.
Bis£8-quinolinolato)manganese(II) dihydrate, Mn Q *2H20. The compound was prepared by the r e a c t i o n between 10 g (0.041 mole) Μη (0Αο)2· 4H20 and 11.8 g (0.082 mole) HQ i n 250 ml of deaerated 1:1 methanol/water. The yellow product was f i l t e r e d under argon, washed with deaerated water and methanol, and d r i e d i n vacuo at room temperature f o r 2 nr. Elemental a n a l y s i s : Calcd. for M n C N H 0 : Mn, 14.49; C, 57.00; N, 7.39; and H, 4.26. Found: Mn, 14.27; C, 56.63; N, 7.04; and H, 4.31. II
2
ΑΙ
18
2.
2
16
4
μ-0xo-bis(8-quinolinQlato-8-quinolinol)manganese(III) dimethanol, Mni 0Q (HQ) -2CH 0H. The compound was prepared by the r e a c t i o n of a i r with a saturated s o l u t i o n of Mn Qo*2H 0 i n 1:1 meth anol/ water. The product which formed as black c r y s t a l s was f i l t e r e d , washed with water and methanol, and dried i n vacuo at room temperature for 2 nr. Elemental a n a l y s i s : Calcd. f o r M n C N H O : Mn, 10.39; C, 63.64; N, 7.95; and H, 4.38. Found: Mn, 10.14; C, 63.70; N, 7.76; II
4
2
3
2
2
56
6
46
g
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
100
ELECTROCHEMICAL STUDIES O F BIOLOGICAL SYSTEMS
and H, 4.30. I][
3.
Bis(8-quinolinolato)magnesixim(II) , Mg Q , and b i s ( 8 - q u i n o l i n o l a t o ) z i n c ( I I ) , Zn Q - Both of the compounds were prepared by the procedure used to synthesize Mn Q -2H 0. 2
2
I]C
2
4.
2
Superoxide ion, 0 ". Superoxide ion was generated i n s i t u i n DMSO and pyridine solutions by cont r o l l e d p o t e n t i a l e l e c t r o l y s i s at -1.00 V at a gold f o i l electrode of oxygen-saturated s o l u t i o n s . The solutions were degassed with argon, prereduced at -1.00 V f o r 10 min, and then saturated with oxygen. The oxygen flow was continued throughout the e l e c t r o l y s i s . To avoid the formation of protons during e l e c t r o l y s i which migrat int the working electrod electrode compartmen t i o n of tetraethylammonium hydroxide i n water. The solutions were degassed with argon p r i o r to use. 2
Results D i s s o c i a t i o n and Magnetic S u s c e p t i b i l i t i e s of Manganese Complexes. Conductance measurements i n d i cate that MnilQ «2H 0 i n DMSO s o l u t i o n i s about 10% dissociated into a 1-to-l e l e c t r o l y t e and that s o l u tions of Mni 0Q (HQ) '2CH 0H are not dissociated into ionically-cénducting species. The molecular weight determinations indicate that MnQ *2H 0 i s about 80% dissociated into MnQ and H 0. The manganese(III) 8-quinolinol complex contains a high spin d manganese ion i n the s o l i d state (μ=5.0+0.1 B.M.), but i n DMSO s o l u t i o n the complex exhibits a decreased magnetic moment ( f X 4.56+ 0.10 B.M.) The magnetic moment i s close t o the spin only value i n a c e t o n i t r i l e s o l u t i o n ^ =4.8+0.2 B.M.) and i n pyridine s o l u t i o n ( μ = 4 . 9 1 + 0 . 0 7 B.M.). 2
2
i:i:
4
9
3
2
2
2
2
4
=
c o r r
οθΓΓ
Electrochemistry of Mn(II)- and Mn(III)-8-Quinoli n o l Complexes! C y c l i c voltammograms of MnilQ -2H 0 and Mn OQ (HQ) i n DMSO s o l u t i o n appear i n Figure 1. The two compounds share common redox products. Mn Q *2H 0 i s not reduced but i s oxidized at +0.16 and +0.75 V. The +0.16 V peak i s a r e v e r s i b l e one-electron per manganese oxidation based on peak currents; however, c o n t r o l l e d p o t e n t i a l coulometry at +0.25 V reveals that on the longer coulometric time scale («~-*20 min) the oxidation i s only a 0.5 e l e c t r o n process. I f an equivalent of hydroxide ions i s added 2
2
4
2
II
2
2
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
2
7.
HOWIE E T AL.
Manganese(II)
and -(III) 8-Quinolinol
Complexes
101
at t h i s point and the e l e c t r o l y s i s i s continued, another 0.25 e l e c t r o n per manganese atom i s trans ferred. Reversing the c y c l i c voltammetric scan d i r e c t i o n a f t e r the oxidation at +0.16 V reveals another catho dic peak at -0.31 V. This peak i s observed also with Mn* ^0Q4(BQ)2both complexes, c o n t r o l l e d poten t i a l coulometry at -0.50 V indicates that t h i s i s a one-electron (per manganese atom) reduction. For Mn^ *0Q (HQ)2> as seen by c y c l i c voltammetry, most of the reduction product formed at -0.50 V i s reoxidized at -0.16 V with a small a d d i t i o n a l amount being r e o x i dized a t +0.16 V. For Un Q ' 2° P y electrolyzed at +0.25 V, the s i t u a t i o n i s reversed with respect to the product y i e l d s at -0.16 and +0.16 V Addition of 1 equivalen manganese atoms) t a f t e r e l e c t r o l y s i s at -0.5 V y i e l d s c y c l i c voltammo grams i d e n t i c a l to those observed a f t e r the e l e c t r o l y s i s sequence described above f o r solutions of Mn. Q2. MniiQ has a second large anodic peak at +0.75 V which i s also present i n solutions of Mn *0Q (HQ>2· The oxidation i s i r r e v e r s i b l e i n DMSO and pyridine but q u a s i - r e v e r s i b l e i n a c e t o n i t r i l e . Again, t h i s appears to be a one-electron per manganese oxidation based on peak currents. Electrode f i l m i n g by the oxidation product precluded confirmation by c o n t r o l l e d p o t e n t i a l coulometry. When one equivalent of protons i s added to a solu t i o n of Mn Q , both anodic peak currents are reduced by one h a l f ana a new cathodic peak appears at -1.85 V which corresponds to the reduction of the hydroxy1 protons of free HQ (7). When another equivalent of protons i s added, the anodic peaks disappear e n t i r e l y and the only peak which remains i s the cathodic peak at -1.85 V. Addition of OH" ions to solutions of Mn**Q2 also decreases the o r i g i n a l anodic peak currents and a new anodic peak appears at +0^08 V which corresponds to the oxidation of free Q~ ions to dimeric Q2 Addition of one equivalent of protons per mole of manganese atoms to solutions of Mn|- 0Q (HQ) s l i g h t l y decreases the peak current f o r the reduction at -0.31 V and reveals a new small cathodic peak at +0.11 V. (The peak at +Q-H V i s a l s o observed as part of the r e v e r s i b l e MniiQ re-reduction following the oxidation at +0.16 V.) Addition of one equivalent of 0ΗΓ ions per mole of manganese to solutions of Mni 0Q (HQ) r e s u l t s i n a spontaneous chemical reduction, a decrease i n the o r i g i n a l cathodic peak at -0.31 V, F
o
r
1
4
I1
2E
9
r e v i o u s l
2
2
2
4
II
2
CI
4
2
2
i:[
4
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
2
ELECTROCHEMICAL STUDIES O F BIOLOGICAL SYSTEMS
102
and the formation of a new anodic peak at -0.16 V. Reactions of Mn(II)- and Mn(III)-8-Quinolinol Complexes with 0 ~ , 0 , and HgC^. Figure 2 i l l u s 2
2
t r a t e s c y c l i c voltammograms i n DMSO s o l u t i o n at a platinum electrode f o r a) 1.3 mM 0 ~ , b) 1 mM Mn Q -2H 0, and c) a mixture of 1.3 mM 0 " and 1 mM M n Q * 2 H 0 ten seconds a f t e r mixing. C l e a r l y a l l of the 0 " i s decomposed within the time i t takes to record the f i r s t c y c l i c voltammogram, and the complex remains i n i t s i n i t i a l oxidation state and i s not appreciably decomposed. The s o l u t i o n does not change c o l o r during the r e a c t i o n . In addition there are cathodic peaks at -0.83 V and -1.30 V, which c o r r e s pond to the reductio and an enhancement The r e l a t i v e y i e l d s of 0 and H 0 vary but are between 50 and 75% of the t h e o r e t i c a l values. A f t e r standing f o r 30 minutes the c y c l i c voltammograms look s i m i l a r to those obtained f o r solutions of pure i 0 Q ( H Q ) . At higher 0 Q ~ to Mn Qo*2H 0 concent r a t i o n r a t i o s there i s 50 to 80% immediate decompos i t i o n , but then the r a t e of decomposition decreases. A large anodic peak appears at +0.08 V which c o r r e s ponds to Q" oxidation. Mn Q3" (prepared i n s i t u by e l e c t r o l y s i s of Mn| OQ (HQ) solutions at -0.50 V) and M n 0 Q 4 ( H Q ) a l s o react with 0o" to form 0 and H2O0 but at a slower rate than Mn- Q »2H 0. In addition, Mn Q *2H 0 reacts slowly with both 0 and H 2 0 to y i e l d solutions which have c y c l i c voltammograms s i m i l a r to those of M n i 0 Q ( H Q ) . I t i s noteworthy that Mni 0Q (HQ) does not react with e i t h e r 0 or H 02 i n DMSO s o l u t i o n . When 0.39 mM 0 ~ and 1.25 mM Mn^Q^HgO are mixed i n p y r i d i n e tne r e s u l t s are q u a l i t a t i v e l y s i m i l a r t o those obtained i n DMSO s o l u t i o n . Hydrogen peroxide formation, however, i s more c l e a r l y v i s i b l e and the y i e l d s of 0« and H 0 are somewhat higher. At higher 0 "-to-MnÎÏQ *2H 0 concentration r a t i o s , although the decomposition of 0 ~ remains rapid, the complex i s destroyed as indicated by the absence of a l l peaks assignable to manganese species. C y c l i c voltammograms recorded a f t e r the r e a c t i o n show only 2
l:[
2
2
2
II
2
2
2
2
M n
2
2
11
I I
4
2
2
II
4
2
2
2
2
T
LI
2
2
i:i
2
2
2
2
II
4
2
Il:
4
2
9
2
2
2
2
2
2
2
0
2
and H o 0 . 2
Μη ^θ2·2Η2θ a l s o reacts slowly with H 0 i n pyridine to give solutions which a f t e r degassing with argon give c y c l i c voltammograms s i m i l a r to M n ^ 0 Q (HQ>2. However, Mn ^Q2»2H20 reacts very r a p i d l y with Ι
2
2
II
I
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
4
7.
HOWIE E T AL.
Manganese(II)
and -(III) 8-Quinolinol
Complexes
103
0 i n pyridine to give a s o l u t i o n with c y c l i c voltam mograms devoid of Μη(II) or Mn(III) redox a c t i v i t y . This indicates the formation of an insoluble or e l e c trochemically i n a c t i v e species. The c y c l i c voltammetric experiments d e t a i l e d i n Figure 2 were also c a r r i e d out using M g ^ and Z n ^ i n place of Mn* Q *2H 0. In both cases the rate of decomposition f o r 0 "" i s as slow as i t i s f o r DMSO solutions without added metal complex (about 5-15% per hour). 2
1 1
1 1
I
2
2
2
Discussion and Conclusions Structure of the Complexes i n Solution. The conductance and molecular weight data indicate that MnllQg-HgO i n DMSO different equilibria y i e l d s about 80% M n Î Q and HgO, and a ligand transf e r or hydrolysis whicn y i e l d s about 10% of some charged species. The electrochemical data to be d i s cussed l a t e r support the formulation of the charged species as Mn Q and Mn Q . The manganese(III)-8-quinolinol complex also undergoes s t r u c t u r a l changes upon d i s s o l u t i o n into DMSO as i l l u s t r a t e d by i t s decrease i n magnetic moment. Because a l l known Mn(III)-DMSO complexes are high-spin (9), solvent e f f e c t s are u n l i k e l y to cause spin p a i r i n g . The decreased value of the magnetic moment implies some antiferromagnetic coupling of the type that would be expected f o r μ-οχο bridged species or d i ^ - h y d r o x o bridged species, but i s weaker than that observed f o r di-μ-οχο bridged species (10,11). The decrease i n magnetic moment i n the Mn(IlTT-8q u i n o l i n o l complex i s comparable to that observed f o r μ-οχο-bis(tetraphenylporphinato)dimanganese(III,III) where the s o l i d state magnetic moment at 295 °K i s reported as 4.12 B.M. (12). In contrast to the magnetic moment i n DMSO s o l u t i o n , the near t h e o r e t i c a l value f o r the mangane s e ( I I I ) - 8 - q u i n o l i n o l complex i n a c e t o n i t r i l e and p y r i dine solutions suggests monomeric structure i n aceto n i t r i l e and pyridine. The di-μ-οχο bridged manganese(IV) 1,10-phenant h r o l i n e complex, Mn 0 (1,10-phen)^(ClO^)^, has a s o l i d - s t a t e magnetic moment of 1.86 B.M. per manganese ion (10). This value corresponds to one unpaired electron per manganese ion rather than the expected three unpaired electrons. Unfortunately, the compound i s not soluble enough t o permit measurement of i t s s o l u t i o n magnetic moment. The one-electron I
2
II
+
II
3
v
2
2
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
ELECTROCHEMICAL STUDIES OF BIOLOGICAL SYSTEMS
104
Figure 2. Cyclic voltammograms in 0.1M TEAP-DMSO at a Ft electrode of (a) 1.32mU 0 (b) lOOmM Mn Q - 2H 0, and (c) a mixture of 1.32mU Of and l.OOmU Mn Q · 2H 0. Scan rate, 0.1 V s~*. 2y
n
2
2
11
2
2
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
7.
HOWIE
Manganese(II)
E T A L .
and -(III) 8-Quinolinol
105
Complexes
IV
reduction product of t h i s dimer, MnJH" 0 (1,10-phen^~ ( C 1 0 ) , i s soluble, however, and has a magnetic moment of 1.56 B.M. per manganese ion i n a c e t o n i t r i l e s o l u t i o n (11). This value corresponds to an average of less than one unpaired electron per mangan ese instead of the expected 3.5. Similar r e s u l t s have been obtained f o r the di-μ-οχο manganese(III-IV) b i p y r i d y l complex Mn* ~ 0 ( b i p y ) ~ (ClO.)o* magnetic moment i n a c e t o n i t r i l e s o l u t i o n of 1.81 B.M. per manganese (11) i s again close to the spin-only value of one unpaired electron per manganese. These r e s u l t s imply strong anti-ferromagnetic coupling across the dioxo bridge and confirm the s t a b i l i t y of oxo-bridged species i n s o l u t i o n . The elemental analysis and s o l i d - s t a t e magnetic moment of the manganese(III)-8-quinolino imply that the comple chelate, Mn Q -jHgO-CHoOH, i n the s o l i d state and becomes a bridged dimer i n DMSO s o l u t i o n . There are several possible dimeric structures: a di-μ-οχο bridge, a μ-οχο bridge, or a d i ^ - h y d r o x o bridge. The dioxo bridged structure can be ruled out because of the lack of strong antiferromagnetic coupling. The d i ^ - h y d r o x y species, Mn£**(OH) Q (HQ) , and the μ-οχο species, Mn| OQ (HQ) , are a l t e r n a t i v e s . Magnetic behavior f o r these species cannot be predicted because w e l l characterized manganese(III) dimers with these types of bridging are unknown. The mono-oxo-bridged formulation i s a t t r a c t i v e , however, because the r e l a t e d i r o n ( I I I ) - 8 - q u i n o l i n o l complex, Fe Q *^H 0, becomes mono-oxo bridged i n DMSO s o l u t i o n (13). Although the structure of the complex i s s t i l l i n doubt, the mono-oxo bridged formulation i s reasonable and w i l l be used throughout the remaining discussion. 2
4
3
Ii:
IV
4
T
h
e
Ii:i
3
2
4
2
II
4
2
3
2
Redox Properties of Mn(II)- and Mn(III)-8QuinoTinol Complexes. A s e l f - c o n s i s t e n t redox mechanism f o r these compounds appears i n Table I. The Μ η ^ · 2 Η 0 complex i s not reducible but i s r e v e r s i b l y oxidized to M n Q at +0.16 V. Most of the oxidized species ends up as Mn^ OQ (HQ) . Mn Q therefore must undergo a h y d r o l y t i c reaction with r e s i d u a l water to form Mn Q (0H) and H . The IT ions th^it are formed attack unoxidized M n ^ to form Mn -Q and HQ. This r e a c t i o n has been confirmed by the experiments i n which H ion i s added to solutions of mi Q . The l i b e r a t e d HQ reacts with Mn Q (0H) to form Bn^^OQ^ (HQ) and H 0. Furthermore, c o n t r o l l e d ι:ι
2
2
IIi:
+
2
1
+
4
1IA
2
2
+
2
1 1
I3
+
iI
Ii:E
2
2
9
9
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
lJ
2
2
3
1 1
2
^
4
2
4
2
II
Mn^ 0Q (HQ)
4
3
2
II
AiA
Mn^ 0Q (HQ)
2
2
Q (OH) + 2HQ
+
+ H0
+ H
+
Q
JI
+ HQ
+
i:i
3
1 1
4
+ 20H"
2
II
3
2
2+
II
+ 2e~
» Mn Q " + Mn Q
4
^ = ± Mn 0Q (HQ)
V
2
* M n ^ O Q ^ H Q ^ + 1^0
+
2
3
Q (0H) + H
Mn Q
> Mn
b
0Q (HQ) + 2 e ~ ^ = ± 2Mn Q " + B^O τττ , „ DMSO _ τττ 2 M n Q (solid) + H^0 ^ = = i M n J O Q ( H Q )
Mn
I]CI
Ii:
2Mn
Mn ^
2
III
Q
I I I
Mn
1
^ + HQ
3
d
2
+ 2H 0
Mn ^" " + e"
Mn
1 1
±A
2
Mn"Q' + Mn II _ > Mn Q
= ^
11
1
+
^
II
Mn Q
Mn ^
Mn Q " + H
ir
A1
2Mn -Q II Mn Q + Q
11
m 0%-2^0
+
+ Q
2
e
+ HgO
+0.70
-0.31
+0.11
+0.75
-0.16
+0.16
V vs. SCE Ε Ε pc pa
Manganese(II) and - ( I I I ) 8-Quinolinol Complexes*
Redox Reactions and Voltammetric Peak P o t e n t i a l s f o r
Table I
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
a platinum electrode
1
(scan rate, 0.1 V s"" ) i n 0.1 M TEAP/DMSO.
A c e t o n i t r i l e solution.
e
2
i n s i t u by the reduction of HQ.
9
Q~ was prepared electrochemically
I3[
d
AA
C y c l i c voltammetry indicates that 1 mM solutions of Mn Q -2H 0 i n DMSO contain about 10% Mn Qo , and conductance measurements indicate that such solutions d i s s o c i a t e about 10% into a 1-to-l e l e c t r o l y t e .
Cryoscopic molecular weight determination i n DMSO indicates compound i s 80% dissociated.
At
c
a
ELECTROCHEMICAL STUDIES O F BIOLOGICAL SYSTEMS
108
p o t e n t i a l coulometry of MnIlQ at +0.25 V indicates that 0.5 equivalent of electrons i s transferred per mole of complex instead of 1. This i s consistent with the o v e r a l l reaction 2
11
2 Mn Q
2
+ Mn Q + l e " ( 5 )
II
+JH 0
>iMn^ OQ (HQ)
2
4
I][
2
+
i:i
Mn* 0Q (HQ) i s reduced, i n the absence of acid, by a one electron-per-manganese i o n process at -0.31 V t o form MniiQ " and H 0. The electron stoichiometry has been confirmed by c o n t r o l l e d p o t e n t i a l coulometry at -0.50 V. The formulation of the reduced species as Mn^Qo" i s supported by experiments i n which Q~ i o n i s eleçtrochemically generated i n s i t u i n the presence of Mn Q . The f i r s t anodic peak s h i f t s from +0.16 to -0.16 V, the p o t e n t i a a f t e r reduction of H ions are present, Mn Q i s converted t o Mn *^ and HQ. The anodic peak corresponding to the reoxidat i o n of the product species s h i f t s back to +0.16 V and a cathodic peak appears at -1.85 V due t o HQ reduc tion. The Mn OQ (HQ) complex i s i r r e v e r s i b l y o x i dized at +0.75 V to form a manganese(IV) species which immediately oxidizes the solvent, the r e s i d u a l water present i n the solvent, or i t s own ligands, and forms a mixture of the two manganese(III) complexes again. F i n a l l y , Mnî fOQ (HQ) i s chemically reduced i n the presence of OH ions to give a species which i s oxidized at -0.16 V. This p o t e n t i a l corresponds to the oxidation of Mn Q "". The reducing agent i s probably Q~ which i s oxidized to Q . The Mn Q complex also i s highly susceptible to n u c l e o p h i l i c d i s placement of the Q" ligands by OH"ions. In addition, Mn^iQo reacts slowly with both 0 and H 0 and, based on the peak p o t e n t i a l s , the product species of the r e a c t i o n i s M n | 0 Q ( H Q ) « . The extra HQ ligands must of necessity come from Mn^Qg so the other product of the r e a c t i o n i s Mn Q . 4
2
3
2
i:[
2
+
I3:
2
4
11
2
I
i
4
2
i:t
3
2
2
2
2
2
II
4
lA
+
Reaction of the Complexes with 0 ~. 2
ese ( iTf-S^quIno^
The mangan-
and
MniiQ3, and the manganese(III)-8-quinolinol complex, Mn| 0Q (HQ) *2CH 0H, i n DMSO s o l u t i o n represent a system that undergoes oxidation-reduction chemistry which p a r a l l e l s much of that observed f o r mitochond r i a l superoxide dismutase. Mn Q accelerates the decomposition of 0 ~ to form nearly stoichiometric amounts of the correct products, 0 and a mixture of II
4
2
3
2
2
9
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
7.
Manganese(II)
H O W I E E T AL.
and -(III) 8-Quinolinol
Complexes
109
H 0 and HO ". The increased peak current that appears t o be associated with the oxidation of Mn Q at +0.16 V probably i s due to the coincident oxida t i o n of the decomposition product, H0 " to 0 . When the scan i s reversed a f t e r the anodic peak at +0.16 V the reduction peak at -0.83 V i s enhanced. The c y c l i c voltammograms and the s o l u t i o n c o l o r i n d i c a t e that the complex i t s e l f i s i n i t s i n i t i a l oxidation state and i s not appreciably decomposed. The c a t a l y t i c properties of the system are com p l i c a t e d by the f a c t that the only source of protons for H O " and H 0 formation i s H^O. The OH" ions thus generated attack the c a t a l y s t to form what appears to be an i n a c t i v e species. When the c a t a l y s t i s used at lower concentrations (0.5 mM or l e s s ) the decomposition of 0 quickly, presumably decompo s i t i o n of the c a t a l y s t , and also because the i n creasing b a s i c i t y of the medium decreases the proton a c t i v i t y , which i n turn i n h i b i t s peroxide formation. In addition, the 0« and H 0 which are formed react with M n ^ to form Mn^^Q^iHQ)^. Mn^ 0Q (HQ) and Mn Q ~ a l s o react with 0 ~ to give 0 and H 6 , although more slowly. Based on our observations, a reasonable mechanism f o r the Q 2 t a l y z e d disproportionation of 0 "" appears to be 2
2
ft
II
2
o
2
Λ
2
2
2
1 1
l:f
JÎ
4
2
2
2
M n I I
2
c a
2
II
Mn Q (H 0) 2
Mn
II]C
2
2
+ 0 ~
^Mn
2
III
Q ( 0 H ) ( H 0 ) + OH" 2
2
2
I I
Q (0 H)(H 0)+0 ^ 2 ^ M n Q ( H 0 ) 2
2
2
2
2
2
2
+ 0
2
(6)
+ H0 " (7) 2
with secondary reactions η
Μη <3 ~ + 0 " 3
Mn
i:EI
^Mn
2
2
Q ( 0 H ) ~ + OH" 3
2
Ii:
Q (0 H)" + 0 " 3
II]C
^Mn Q "" + 0
2
3
2
+ H0 " 2
(8) (9)
The most remarkable feature of t h i s model i s the apparent a b i l i t y of a manganese(II) complex to reduce Oo" to H0 ". Such a process, on the basis of the electrochemical peak p o t e n t i a l s f o r the i n d i v i d u a l components, Figure 1, appears t o be thermodynamically impossible. Apparently Equation 5 i s favored as a r e s u l t of the strong i n t e r a c t i o n of the product species, M n and H0 ~. Several unsuccessful attempts have been made to i s o l a t e a manganese(III) 8-quinolinol-peroxide complex. The following two reactions i n DMSO with 2
1 1 1
o
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
ELECTROCHEMICAL STUDIES OF BIOLOGICAL SYSTEMS
110
the indicated reaction stoichiometries II
6Mn Q
2
+ 20
>Mn
+ 3H 0
2
2
2Mn Mn
Q
2
+ H 0 2
+0.25V 2
^Mn
Ii:E
III
II 2
OQ (HQ) 4
2
+ I]C
Q (H0 ) + 2Mn Q(OH) (10) 2
2
Q (H0 ) + H 2
+
+ e"
2
(ll)
were studied by c y c l i c voltammetry. Both solutions e x h i b i t an a d d i t i o n a l cathodic peak at -0.75 V (besides the peak at -0.31 V f o r Mné OQ (HQo) which may r e s u l t from the reduction of MniHQ (H0 ; or of bound peroxide ions. The apparent oxidation by 0 ~ ion (generated from oxygen plus xanthine-xanthine oxidase or from i l l u m i n a t i o n of spinac Mn(III) i n the presenc pyrophosphat system was reported recently (14) . However, the authors conclude that the manganese(II)-pyrophosphate complex does not act as a disproportionation c a t a l y s t f o r Oo~ ions, and imply that the manganese(III)-pyrophosphate does not oxidize 0 ~ ion but does oxidize H 0 . This i s i n sharp contrast to the r e s u l t s of the present study (Equations 6-9). Although the F r i d o v i c h mechanism (Equations 1-4) invokes a t h i r d oxidation state f o r the enzyme to r a t i o n a l i z e the k i n e t i c r e s u l t s , there i s another possible explanation. The l e s s c a t a l y t i c state of the enzyme may simply be a d i f f e r e n t h y d r o l y t i c species. Because protons also catalyze the disproportiona t i o n of 0 ~ ions and coordinated DMSO might serve as a proton source, the c y c l i c voltammetric experiments d e t a i l e d i n Figure 2 have been made i n p y r i d i n e . The r e s u l t s are q u a l i t a t i v e l y the same. Combination of 1.25 mM Mn Qo with 0.39 mM On" immediately destroys a l l of the 0 ~ and y i e l d s a s o l u t i o n that contains Mn Q , 0 , and Hr>0 i n amounts equivalent to 70-90% e f f i c i e n c y f o r Equations 6-9. At higher 0 "-to-Mn Qo 2H 0 r a t i o s the complex i s destroyed. The f a c t that the complex i s more e a s i l y destroyed i n pyridine s o l u t i o n during the c a t a l y t i c r e a c t i o n can be explained on the basis of the greatly accelerated r e a c t i o n of one of the products, 0 , with M n ^ . Since separate experiments have shown that 0 reacts r a p i d l y with Mn Q i n pyridine to form an exectrochemically i n a c t i v e species, i t i s expected that the c a t a l y s t w i l l disappear when the 0 ~ to Mn Q concentration r a t i o becomes higher, and thus the catal y t i c a l l y generated oxygen concentration increases. I3:
4
2
2
2
2
2
2
2
I3:
2
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e
2
1 1
2
2
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2
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In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
7.
Manganese(II)
HOWIE E T AL.
and -(HI)
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Another possible i n t e r p r e t a t i o n of the r e s u l t s i s that the M n ^ complex acts as a Lewis a c i d to catalyze the disproportionation of 0 ~ ions. Charge n e u t r a l i z a t i o n of one Og" ion by the Lewis a c i d would make i t e l e c t r o s t a t i c a l l y easier f o r 0 ~ c o l l i s i o n s and electron transfer to occur. However, when the experiment summarized i n Figure 2 i s repeated with Mg Q or Zn Q substituted f o r M.n Q , the rate of decomposition f o r 0 ~ i s not any f a s t e r than the normal decomposition rate i n DMSO. This supports the conclusion that Mn Q acts as a redox c a t a l y s t . The present study i s being extended to determine what e f f e c t s the ligands have on both the redox chem i s t r y of the complexes and t h e i r a b i l i t y to catalyze the decomposition of superoxide i o n . In p a r t i c u l a r , ligands are being sough ganese (II) complexe ment by OH" ions and which w i l l accelerate the reac t i o n i n Equation 6. We also are t r y i n g t o f i n d a s u i t a b l e buffer system to f a c i l i t a t e the formation of hydrogen peroxide and prevent the ultimate destruc t i o n of the c a t a l y s t . Such a system w i l l make i t possible to evaluate the k i n e t i c parameters f o r the various reactions. 1 1
2
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13:
11
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Acknowledgment This work was supported by the U. S. Public Health Service-NIH under Grant No. GM 22761. L i t e r a t u r e Cited 1. J . M. McCord and I. Fridovich, J . Biol. Chem. (1969), 244, 6049. 2. Β. B. Keele, J r . , J . M. McCord, and I. Fridovich, J . B i o l . Chem. (1970), 245, 6176. 3. P. G. Vance, Β. B. Keele, J r . , and Κ. V. Rajagopalan, J . Biol. Chem. (1972), 247, 4782. 4. R. A. Weisiger and I. Fridovich, J . Biol. Chem. (1973), 248, 3582. 5. M. Pick, J . Rabani, F. Yost, and I. Fridovich, J. Am. Chem. Soc. (1974), 96, 7329. 6. A. D. Goolsby and D. T. Sawyer, Anal. Chem. (1967), 39, 411. 7. A. F. I s b e l l , J r . , and D. T. Sawyer, Inorg. Chem. (1971), 10, 2449. 8. D. F. Evans, J . Chem. Soc. (1959), 2003. 9. C. P. Prabhakaran and C. C. P r a t e l , J . Inorg. Nucl. Chem. (1968), 30, 867.
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112 10. 11. 12. 13. 14.
ELECTROCHEMICAL STUDIES OF BIOLOGICAL H. A. Goodwin and R. N. Sylva, Aust. J . Chem. (1967), 20, 629. M. M. Morrison and D. T. Sawyer, submitted to J . Am. Chem. Soc. (1976). Ε. B. Fleischer, J . M. Palmer, T. S. Srivastava, and A. Chatterjee, J . Am. Chem. Soc. (1971), 93, 3162. T. L . Riechel and D. T. Sawyer, submitted to Inorganic Chemistry (1976). T. Kono, M. Takahashi, and K. Asada, Arch. Biochem. Biophys. (1976), 174, 454.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
8 Interfacial Behavior of Biologically Important Purines at the Mercury Solution Interface H . K I N O S H I T A , S. D . C H R I S T I A N , M . H . K I M , J. G . B A K E R , and GLENN D R Y H U R S T Department of
Chemistry, University of Oklahoma, Norman, Okla. 73019
Over the p a s t l increasin bod o f e v i d e n c e has s u g g e s t e cleic acids with electrically charge play an i m p o r t a n t r o l e i n many b a s i c biological processes. However, the p h y s i c s and c h e m i s t r y o f biodynamic m o l e c u l e s adsorbed a t m e m b r a n e - f l u i d i n t e r f a c e s i n living organisms are not at all w e l l u n d e r s t o o d . Basic studies of interfacial phenomena in vivo are rendered difficult by the m u l t i t u d e o f s u r f a c e active compounds which e x i s t i n biological fluids and the virtual impossibility of identifying specific i n t e r a c t i o n s between a g i v e n a d s o r b a t e m o l e c u l e and a c t i v e s i t e s on a biological interface. Electric fields have been known f o r some time t o i n f l u e n c e the c o n f o r m a t i o n o f v a r i o u s n a t u r a l and biosynthetic polynucleotides in solution. F o r example, Hill (1) has c a l c u l a t e d t h a t h i g h electric fields c o u l d b r i n g about s e p a r a t i o n o f the two m o l e c u l a r c h a i n s o f n u c l e o t i d e s i n DNA. Based on birefringence measurements, it has been demonstrated (2) t h a t in high intensity electrical fields (>10 V c m ) DNA first aggregates and then undergoes a s t r u c t u r a l transition in which the a n g l e s o f the p u r i n e and p y r i m i d i n e bases w i t h r e s p e c t t o the h e l i x - a x i s . , are altered. In e l e c t r i c f i e l d s o f about 2x10 VcnT r i b o s o m a l RNA and p o l y n u c l e o t i d e s such as p o l y ( A ) . 2 p o l y ( U ) appear t o undergo a t r a n s i e n t o p e n i n g o f base p a i r s f o l l o w e d by o n l y p a r t i a l r e a s s o c i a t i o n o f the unfolded regions (3). Such s t r u c t u r a l e f f e c t s o f e l e c t r i c a l f i e l d s on n a t u r a l p o l y n u c l e o t i d e s have been i m p l i c a t e d i n the mechanisms o f nerve impulse t r a n s m i s s i o n and i n f o r m a t i o n s t o r a g e i n the c e n t r a l nervous system (2), perhaps as an i n i t i a l step i n the r e c o r d i n g o f b i o l o g i c a l memory ( 3 - 6 ) . 4
- 1
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The p o t e n t i a l s t h a t e x i s t a t c e r t a i n b i o l o g i c a l membranes s u c h a s a c e l l membrane a r e t h o u g h t t o be of the order of 0.1V. In a b i o l o g i c a l f l u i d h a v i n g an i o n i c s t r e n g t h o f 0 , 1 t o 0 . 2 t h i s p o t e n t i a l w o u l d e x t e n d o v e r d i s t a n c e s o f 1 0 - 1 0 0 ^ (1^. Tljiis c o r r e s p o n d s t o e l e c t r i c f i e l d s o f 10 - 1 0 Vcm . Clearly, i f a b i o p o l y m e r s u c h as DNA o r RNA i s p r e s e n t i n c l o s e p r o x i m i t y t o s u c h a b i o s u r f a c e t h e n i t seems quite reasonable to suggest that macromolecular s t r u c t u r a l t r a n s i t i o n s might occur. In f a c t , i n l i v i n g o r g a n i s m s DNA, f o r e x a m p l e , i s p a r t i a l l y a s s o c i a t e d w i t h t h e n u c l e a r o r c y t o p l a s m i c membrane, o r w i t h the i n t e r f a c e o f the n u c l e o l u s (8-11). A theory has been advanced t h a t r e p l i c a t i o n c o u l d b e g i n a t the l e v e l of the n u c l e a r o r c e l l u l a r w a l l (10-12). Indeed, H i l l (1_) h a s and t h e i r v a r i a t i o n a c t as t h e t r i g g e r f o r d i v i s i o n o f g e n e t i c m a t e r i a l i n the c e l l p r i o r to s e l f - d u p l i c a t i o n . A very i n t e r e s t i n g aspect of e l e c t r i c a l a c t i v i t y a s s o c i a t e d w i t h b i o l o g i c a l processes i s the e x i s t e n c e o f the p o t e n t i a l o f i n j u r y at a trauma s i t e . A sign i f i c a n t o b s e r v a t i o n i s t h a t the i n j u r y p o t e n t i a l follows a d i f f e r e n t time course i n the h e a l i n g o f , for example, a limb amputation i n the case of a s p e c i e s w h i c h c a n r e g e n e r a t e t h e l i m b a s o p p o s e d t o one w h i c h e x h i b i t s o n l y s c a r f o r m a t i o n (13, 1 4 ) . I t has been found t h a t i m p l a n t a t i o n o f s m a l l e l e c t r o d e s at the i n j u r y s i t e i n a nonregenerating s p e c i e s (forci n g the i n j u r y p o t e n t i a l to approximate t h a t of a r e generating species) causes at l e a s t p a r t i a l limb r e g e n e r a t i o n even i n a complex s p e c i e s s u c h as t h e rat (15,16). T h i s i m p l i e s t h a t fundamental b i o l o g i cal processes ( u l t i m a t e l y a t t h e g e n e t i c l e v e l ) may be c o n t r o l l e d b y t h e n a t u r a l o r a r t i f i c i a l l y a p p l i e d e l e c t r i c a l environment at a t i s s u e r e p a i r s i t e . The application of small, l o c a l l y applied e l e c t r i c fields h a s b e e n u s e d f o r t h e s t i m u l a t i o n o f b o n e h e a l i n g as a v e r y p r a c t i c a l outcome o f s u c h s t u d i e s . Cope ( 1 7 - 2 4 ) h a s p r e s e n t e d some c o n v i n c i n g a r g u ments t h a t a c e l l s u r f a c e - b i o l o g i c a l f l u i d i n t e r f a c e may be r e g a r d e d as b e i n g v e r y s i m i l a r t o a l i q u i d s o l i d i n t e r f a c e which e x h i b i t s e l e c t r i c a l behavior a n a l o g o u s t o t h a t o c c u r r i n g a t an e l e c t r o d e - s o l u t i o n interface. An e l e c t r o d e - s o l u t i o n i n t e r f a c e i s generally c h a r a c t e r i z e d by a w e l l - d e f i n e d e l e c t r i c a l d o u b l e l a y e r b o u n d e d o n one s i d e b y t h e e l e c t r o d e surface and on t h e o t h e r s i d e by an i o n i c l a y e r a c r o s s w h i c h a r e l a t i v e l y high e l e c t r i c a l f i e l d develops (up t o
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ca. 10 V cm ) . This c o n s t i t u t e s the s o - c a l l e d i n n e r o r compact double l a y e r , the w i d t h o f which i s o n l y a few a t o m i c d i a m e t e r s . On t h e b o u n d a r y b e t w e e n the i n n e r and the d i f f u s e d o u b l e l a y e r s the f i e l d s t r e n g t h h a s o n l y a b o u t 1/10 o f i t s o r i g i n a l v a l u e , and i t t h e n d e c r e a s e s i n the d i f f u s e double l a y e r t o v i r t u a l l y zero. I n a medium o f i o n i c s t r e n g t h c a . 0. 1-0.2 ( t y p i c a l o f b i o l o g i c a l f l u i d s } the depth o f t h e d i f f u s e d o u b l e l a y e r i s a b o u t 100& ( 2 5 ) . An e x a c t l y s i m i l a r l y structured region exists at a c e l l membrane-biological f l u i d interface (26). A t p h y s i o l o g i c a l b u l k - p h a s e pH v a l u e o f 7.2 a l l mammalian c e l l s so f a r e x a m i n e d c a r r y a n e t n e g a t i v e charge at t h e i r s u r f a c e s . However, the s u r f a c e p o t e n t i a l of a c e l l i s not c o n s t a n t but can undergo some r a t h e r d r a m a t i i s o l a t e d f r o m t h e r e g e n e r a t i n g l i v e r s o f r a t s some days a f t e r p a r t i a l hepatectomy and c e l l s from n e o n a t e s have s i g n i f i c a n t l y h i g h e r e l e c t r o p h o r e t i c mob i l i t i e s t h a n l i v e r c e l l s from normal a d u l t s (21), 1. e . , c e l l p r o l i f e r a t i o n i s a s s o c i a t e d w i t h i n c r e a s e d net surface n e g a t i v i t y . S i m i l a r l y , the e l e c t r o p h o r e t i c m o b i l i t i e s o f c e r t a i n tumor c e l l s i n c r e a s e w i t h g r o w t h r a t e (28!) . A t the time o f m i t o s i s a v e r y s i g n i f i c a n t i n c r e a s e i n n e t s u r f a c e n e g a t i v i t y has been o b s e r v e d i n v a r i o u s t y p e s o f c u l t u r e d tumor c e l l s (29, 3 0 ) . I n d e e d , Ambrose e t a l . (31, 3 2 ) , have n o t e d a c o r r e l a t i o n between m a l i g n a n c y and i n c r e a s e d c e l l surface n e g a t i v i t y , although t h i s i s c e r t a i n l y not t h o u g h t t o be a u n i v e r s a l c o r r e l a t i o n (33). A s u b s t a n t i a l amount o f e v i d e n c e i s b e i n g d e v e l oped which i n d i c a t e s t h a t i n t e r a c t i o n s w i t h b i o l o g i c a l i n t e r f a c e s i s a p r e r e q u i s i t e f o r the m a n i f e s t a t i o n of the b i o l o g i c a l e f f e c t s of p o l y n u c l e o t i d e s i n mammalian c e l l s y s t e m s i n v i v o and i n v i t r o . Thus, F i e l d e t a l . (34) h a v e r e p o r t e d t h a t RNA d o u b l e s t r a n d s , and p a r t i c u l a r l y p o l y (I), poly(C) induce i n t e r f e r o n f o r m a t i o n i n mammalian c e l l s . Subsequently, S c h e l l (15) h a s shown t h a t p o l y (I) . p o l y (C) i s a d s o r b e d t o t h e o u t s i d e o f t h e c e l l f o l l o w e d by s t r a n d s e p a r a t i o n a n d u l t i m a t e l y by i n t e r f e r o n f o r m a t i o n . I t h a s f u r t h e r b e e n s u g g e s t e d (36) that othex b i o l o g i c a l e f f e c t s o f p o l y n u c l e o t i d e s , s u c h as adjuvant e f f e c t s and enzyme i n h i b i t i o n / a c t i v a t i o n r e q u i r e s i n t e r a c t i o n of the p o l y n u c l e o t i d e with the charged c e l l surface. T h u s , i n summary, t h e s u r f a c e s o f m a m m a l i a n c e l l s a n d o t h e r b i o l o g i c a l membranes c a r r y a n a p preciable electrical potential. The e l e c t r i c a l d o u b l e - l a y e r formed i n the immediate v i c i n i t y o f a
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charged membrane-biological f l u i d i n t e r f a c e i s essent i a l l y i d e n t i c a l t o t h a t f o r m e d a t an e l e c t r o d e surface. The p o t e n t i a l o f a c e l l membrane s u r f a c e is known t o a l t e r when p r o c e s s e s s u c h a s c e l l regenerat i o n o c c u r o r , o f t e n , when a c e l l b e c o m e s m a l i g n a n t and p a r t i c u l a r l y a t the time o f m i t o s i s . The p o t e n t i a l of i n j u r y at a s i t e o f trauma i s c l e a r l y i m p l i c a t e d i n c o n t r o l of the t i s s u e r e g e n e r a t i o n p r o c e s s . The e l e c t r i c a l f i e l d s i n t h e i m m e d i a t e v j c i n j t y o f a ^ c e l l s u r f a c e a r e p r o b a b l y v e r y l a r g e (10 - 1 0 Vcm ) although they extend over only very small d i s t a n c e s ( c a . 1 0 - 1 0 0 K) . Such i n t e n s e e l e c t r i c f i e l d s h a v e b e e n shown t o c a u s e s t r u c t u r a l t r a n s i t i o n s i n c e r t a i n n a t u r a l and b i o s y n t h e t i c p o l y n u c l e o t i d e s . In a d d i t i o n , the i n t e r a c t i o n (adsorption) of p o l y n u c l e o t i d e s at the charge a prerequisite for manifestatio e f f e c t s of the p o l y n u c l e o t i d e s . Because a charged c e l l s u r f a c e - b i o l o g i c a l fluid i n t e r f a c e i s s i m i l a r to a charged e l e c t r o d e e l e c t r o l y t e s o l u t i o n i n t e r f a c e , i t seems reasonable t h a t an u n d e r s t a n d i n g o f t h e i n t e r f a c i a l b e h a v i o r o f biomolecules at the l a t t e r i n t e r f a c e might r e v e a l s i g n i f i c a n t i n f o r m a t i o n regarding the i n t e r a c t i o n s of these molecules at b i o l o g i c a l interfaces. I t w o u l d seem t o be s e l f - e v i d e n t , h o w e v e r , t h a t i n t e r f a c i a l s t u d i e s o f n u c l e i c a c i d s and o t h e r p o l y n u c l e o t i d e s , and i n t e r p r e t a t i o n o f the d a t a so c o l l e c t e d , must r e l y on a f u n d a m e n t a l k n o w l e d g e o f t h e i n t e r f a c i a l b e h a v i o r o f the monomeric u n i t s , i.e., b a s e s , n u c l e o s i d e s and n u c l e o t i d e s . A number o f i n v e s t i g a t o r s have r e p o r t e d t h a t v a r i o u s monomeric p u r i n e and p y r i m i d i n e d e r i v a t i v e s are a d s o r b e d a t mercury e l e c t r o d e s (37-45). Such s t u d i e s , however, have g e n e r a l l y been v e r y q u a l i t a t i v e ; t h e y have r e v e a l e d v i r t u a l l y n o t h i n g about the s u r f a c e areas o c c u p i e d by t h e a d s o r b e d m o l e c u l e s and h e n c e t h e i r probable surface o r i e n t a t i o n s , the nature o f the a d s o r p t i o n i s o t h e r m s and t h e e f f e c t s o f p o t e n t i a l on the a d s o r p t i o n p r o c e s s e s , the i n t e r m o l e c u l a r i n t e r a c t i o n s between a d s o r b e d m o l e c u l e s and i n t e r a c t i o n s between the adsorbed m o l e c u l e s and the e l e c t r o d e surface. R e c e n t l y , N u r n b e r g e t a l . (46) h a v e r e p o r t e d o n t h e a d s o r p t i o n o f a d e n o s i n e and a d e n o s i n e m o n o n u c l e o t i d e s at a mercury e l e c t r o d e . Measurements o f the a m o u n t o f t h e a d e n i n e s p e c i e s a d s o r b e d w e r e accom-^ p u s h e d by use o f t h e t i m e i n t e g r a l o f t h e faradaic r e d u c t i o n peak o f t h e s e m o l e c u l e s a t a s t a t i o n a r y m e r c u r y d r o p e l e c t r o d e a t 5 ° C a n d a t pH 3 . 4 . This
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KiNosHiTA E T A L .
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117
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v o l t a m m e t r i c t e c h n i q u e , h o w e v e r , i s s u b j e c t t o many e x p e r i m e n t a l l i m i t a t i o n s and p r o b l e m s (£7). We h a v e b e g u n a s y s t e m a t i c i n v e s t i g a t i o n o f t h e i n t e r f a c i a l b e h a v i o r o f t h e p u r i n e and p y r i m i d i n e b a s e s , d e o x y n u c l e o s i d e s and d e o x y n u c l e o t i d e s found i n nucleic acids. In t h i s r e p o r t the i n t e r f a c i a l behav i o r o f v a r i o u s a d e n i n e d e r i v a t i v e s a t pH 9 a t m e r c u r y e l e c t r o d e s w i l l be d e s c r i b e d . Two m a j o r s u r f a c e elect r o c h e m i c a l t e c h n i q u e s were employed i n t h e s e studies — t h e c a p i l l a r y e l e c t r o m e t e r and d i f f e r e n t i a l capacitance measurements. Some p r e l i m i n a r y r e s u l t s u s i n g e l e c t r o e l l i p s o m e t r y w i l l a l s o be p r e s e n t e d . Experimental D i f f e r e n t i a l capacitanc o b t a i n e d by a p h a s e - s e l e c t i v e a . c . polarographic method. A Princeton Applied Research Corporation (PARC) M o d e l 174 P o l a r o g r a p h i c A n a l y z e r c o u p l e d w i t h a PARC M o d e l 1 7 4 / 5 0 AC P o l a r o g r a p h i c A n a l y z e r Interf a c e a n d a PARC M o d e l 121 L o c k - i n A m p l i f i e r / P h a s e D e t e c t o r were employed f o r d i f f e r e n t i a l capacitance measurements. A phase a n g l e o f 90° w i t h r e s p e c t t o t h e a p p l i e d a l t e r n a t i n g v o l t a g e was e m p l o y e d . At the pH v a l u e s e m p l o y e d i n t h i s s t u d y , a d e n i n e a n d i t s d e r i v a t i v e s are not e l e c t r o c h e m i c a l l y r e d u c i b l e . The dropping mercury e l e c t r o d e (DME) was s i l i c o n i z e d (39) a n d was e q u i p p e d w i t h a m e c h a n i c a l d r o p dislodger. A p o o l o f mercury i n s e r t e d a t the bottom o f a thermos t a t t e d 5ml c a p a c i t y c e l l s e r v e d a s t h e c o u n t e r e l e c trode. A saturated calomel reference electrode (SCE) was e m p l o y e d u s i n g a f i n e L u g g i n c a p i l l a r y p o s i t i o n e d c l o s e t o t h e t i p o f t h e DME. A . c . p o l a r o g r a m s were u s u a l l y o b t a i n e d a t a f r e q u e n c y o f 100Hz a n d w i t h a m o d u l a t i n g a m p l i t u d e o f lOmV p e a k - t o - p e a k . Thus, the c a p a c i t y r e s u l t s r e p o r t e d h e r e were a l l measured a t a f r e q u e n c y o f 100Hz a n d w e r e n o t e x t r a p o l a t e d to zero frequency. However, the c a p a c i t y v a l u e s were v i r t u a l l y i n d e p e n d e n t o f f r e q u e n c y b e t w e e n a b o u t 50 a n d 600 H z . A l l m e a s u r e m e n t s w e r e made w i t h o u t d a m p i n g o n t h e M o d e l 174 a n d u s i n g a c o n t r o l l e d d r o p t i m e of 2.00s. When t h e a . c . p o l a r o g r a m was r e c o r d e d o n a n X - Y r e c o r d e r ( H e w l e t t - P a c k a r d M o d e l 7001A) t h e d . c . p o t e n t i a l was s c a n n e d a t a sweep r a t e o f 0.005Vs" . H o w e v e r , i n some o f o u r l a t e r studies t h e a l t e r n a t i n g c u r r e n t was m e a s u r e d b y u s e o f a K e i t h l e y M o d e l 16 8 A u t o r a n g i n g D i g i t a l M u l t i m e t e r connected to the Y a x i s ( c u r r e n t ) o u t p u t o f t h e PARC Model 174. The c a p i l l a r y
electrometer
and
its
associated
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
118
ELECTROCHEMICAL
STUDIES
OF
BIOLOGICAL
SYSTEMS
p r e s s u r e s y s t e m i s shown s c h e m a t i c a l l y i n F i g u r e 1. The P y r e x c a p i l l a r i e s h a d a d i a m e t e r , 1mm a b o v e t h e t i p , of 0.002-0.005mm. The c a p i l l a r i e s w e r e n o r m a l l y aged f o r s e v e r a l d a y s by s o a k i n g i n d e i o n i z e d w a t e r and s u b s e q u e n t l y s t o r e d w i t h the t i p immersed i n w a t e r . A B r i n k m a n n / W e n k i n g M o d e l L T 7 3 p o t e n t i o s t a t was u s e d . The l o c a t i o n o f t h e m e r c u r y c o l u m n i n t h e c a p i l l a r y was o b s e r v e d w i t h a G a e r t n e r 2 2 0 6 - A C a t h e t o m e t e r t h r o u g h a n o p t i c a l l y f l a t P y r e x window s e a l e d o n one side of the c e l l (Figure 1). The c e l l was w a t e r j a c k e t e d and m a i n t a i n e d a t a t e m p e r a t u r e o f 25±0.1°C. The p r e s s u r e a t t h e m e r c u r y - t e s t s o l u t i o n interface was v a r i e d b y a p p l y i n g p r e s s u r e t o t h e g a s a b o v e t h e m e r c u r y b y means o f a s y r i n g e a n d two m i c r o m e t e r burets. The c o a r s e a d j u s t u t i l i z e d a 20ml p l a s t i c s y r i n g e w h i l e f i n e p r e s s u r e a d j u s t m e n t was a c c o m p l i s h e d w i t h two G i l m o n M e n s o r C o r p o r a t i o n Q u a r t z M a n o m e t e r p r e s s u r e g a u g e was u s e d t o measure t h e gas pressure. T e s t s o l u t i o n s were d e a e r a t e d w i t h n i t r o g e n for a t l e a s t 15 m i n u t e s b e f o r e m e a s u r e m e n t s w e r e c a r r i e d out. A l l p o t e n t i a l measurements u t i l i z e d a s a t u r a t e d c a l o m e l r e f e r e n c e e l e c t r o d e at 25°C. D a t a p o i n t s w e r e t a k e n a t 50mV i n t e r v a l s from -0.2V to - 1 . 8 V . A d r o p o f m e r c u r y was e x p e l l e d f r o m the c a p i l l a r y before the e l e c t r o c a p i l l a r y curve at e a c h c o n c e n t r a t i o n was m e a s u r e d . The p r e s s u r e at e a c h a p p l i e d p o t e n t i a l was t h e n a d j u s t e d t o b r i n g the mercury to the reference p o i n t i n the c a p i l l a r y (1mm f r o m t h e t i p ) . The h e i g h t o f t h e m e r c u r y c o l umn was m e a s u r e d w i t h t h e G a e r t n e r c a t h e t o m e t e r t o a p r e c i s i o n o f 0.02mm b e f o r e a n d a f t e r m e a s u r e m e n t o f each e l e c t r o c a p i l l a r y curve. A c o r r e c t i o n was a p p l i e d t o the measured p r e s s u r e f o r t h i s h e i g h t and f o r the s m a l l back p r e s s u r e o f the s o l u t i o n . The b o r a t e b u f f e r pH 9 u t i l i z e d was c o n s t i t u t e d as f o l l o w s : 17.5g N a J 0 10H 0 , 6 7 . 7 g KC1 a n d 1 6 . 8 5 m l 1M HC& d i l u t e d t o I I w i t h d e i o n i z e d w a t e r . M c l l v a i n e b u f f e r pH 7 was c o n s t i t u t e d a s f o l l o w s : 5 8 . 9 g N a H P O . 1 2 H 0 , 3.7g c i t r i c a c i d . H 0 and 5.4g KC1 d i l u t e d t o 11 w i t h w a t e r . Both of these buffer s o l u t i o n s have an i o n i c s t r e n g t h o f 0 . 5 . Sample s o l u t i o n s w e r e p r e p a r e d by d i s s o l v i n g t h e a d e n i n e s p e c i e s i n the a p p r o p r i a t e volume o f buffer. A G a e r t n e r M o d e l L 1 1 9 E l l i p s o m e t e r was e m p l o y e d for e l l i p s o m e t r i c studies. A schematic diagram of t h e a p p a r a t u s u t i l i z e d i s shown i n F i g u r e 2 . The a n g l e o f i n c i d e n c e o f t h e l i g h t beam was 7 0 ° . Test s o l u t i o n s w e r e d e a e r a t e d f o r a b o u t 30 m i n u t e s a n d a n i t r o g e n a t m o s p h e r e was m a i n t a i n e d o v e r t h e s o l u t i o n 2
4
2
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
8.
Interfacial
KiNosHiTA E T A L .
Behavior
of
119
Purines
Potentiostat
Figure 1.
System for electrocapillary measurements
Potentiostat
Figure 2.
Electroellipsometry system
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
ELECTROCHEMICAL
120
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when m e a s u r e m e n t s w e r e t a k e n . The c i r c u l a r m e r c u r y p o o l w o r k i n g e l e c t r o d e h a d an a r e a o f 5.07cm . The c u r v a t u r e o f t h e m e r c u r y s u r f a c e was r e d u c e d by p l a c i n g an amalgamated p l a t i n u m i n s e r t a r o u n d t h e p e r i p h e r y of the g o o l . A small c r o s s - s e c t i o n a l area l i g h t b e a m , 0.8mm , was u s e d t o m i n i m i z e t h e e f f e c t of c u r v a t u r e of the mercury e l e c t r o d e s u r f a c e . The p r o cedure used i n measuring t h i c k n e s s e s and changes i n t h i c k n e s s o f f i l m s w i t h t h e e l l i p s o m e t e r was t h e f o l lowing. F i r s t , with o n l y the background e l e c t r o l y t e (buffer) s o l u t i o n i n the c e l l , values o f the p o l a r i z e r a n g l e (P) p r o d u c i n g a minimum i n i n t e n s i t y o f t h e r e f l e c t e d beam w e r e d e t e r m i n e d a s a f u n c t i o n o f p o t e n t i a l i n the range - 0 . 2 t o - 1 . 6 V v s . SCE. (The a n a l y z e r a n g l e , A , was n o t v a r i e d d u r i n g t h e s e e x p e r i m e n t s s i n c e the optimum v a l u by t h e p r e s e n c e o f f i l m t h i c k and s i n c e t h e c h o i c e o f a n a l y z e r a n g l e i n t h e minimum r e g i o n d o e s n o t i n f l u e n c e t h e v a l u e o f Ρ l e a d i n g t o minimum i n t e n s i t y . ) Next, with a s o l u t i o n of a d s o r b a t e i n t h e c e l l , new v a l u e s o f Ρ p r o d u c i n g m i n i mum i n t e n s i t y w e r e d e t e r m i n e d . Values of f i l m t h i c k n e s s were c a l c u l a t e d from changes i n Ρ (at f i x e d A and f i x e d p o t e n t i a l ) by u s i n g a c o m p u t e r p r o g r a m d e v e l o p e d by M c C r a c k i n ( £ 3 ) . In p r a c t i c e , i t i s sometimes d i f f i c u l t t o r e p r o d u c e e x a c t l y t h e v a l u e s o f Ρ w h i c h p r o d u c e minimum i n t e n s i t y for a given adsorbate or background s o l u tion. However, the shapes o f the v a r i o u s Ρ v s . p o t e n t i a l c u r v e s are o r d i n a r i l y q u i t e r e p r o d u c i b l e , and i t i s p o s s i b l e t o use the e x t e n s i v e a d s o r p t i o n r e s u l t s a v a i l a b l e from the c a p a c i t a n c e and e l e c t r o c a p i l l a r y experiments to " n o r m a l i z e " the e l l i p s o m e t r i c data. Thus, simple v e r t i c a l displacement o f the o b s e r v e d Ρ v s . p o t e n t i a l c u r v e s u s u a l l y b r i n g s them i n t o c o i n c i d e n c e w i t h the background curve i n regions o f p o t e n t i a l w h e r e l i t t l e o r no a d s o r p t i o n o c c u r s . D i f f e r e n c e s between the d i s p l a c e d curves f o r the a d s o r b a t e s o l u t i o n s and t h e b a c k g r o u n d c u r v e can t h e n be a t t r i b u t e d d i r e c t l y t o t h e o p t i c a l e f f e c t o f t h e f i l m and i n t e r p r e t e d t o y i e l d f i l m t h i c k n e s s e s as a f u n c t i o n o f a d s o r b a t e c o n c e n t r a t i o n and p o t e n t i a l . Analysis
of
Capacitance
and E l e c t r o c a p i l l a r y
Data
U s i n g t^e a . c . p o l a r o g r a p h i c method the c a p a c i t a n c e , pFcm , i s r e a d i l y d e t e r m i n e d from the o b s e r v e d a l t e r n a t i n g c u r r e n t , yA, f r o m t h e e q u a t i o n : C
=
I ΔΕΑ2πί
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
(1)
8.
KINOSHITA
Interfacial
E TAL.
Behavior
121
of Purines
w h e r e ΔΕ i s t h e a m p l i t u d e o f t h e a p p l i e d a l t e r n a t i n g v o l t a g e ( V ) , A i s t h e s u r f a c e a r e a o f t h e DME (cm ) at the time the c u r r e n t i s sampled, and f i s the frequency (Hz) o f a p p l i e d a l t e r n a t i n g v o l t a g e . This e q u a t i o n i s v a l i d when t h e r e s i s t a n c e o f t h e t e s t s o l u t i o n i s s m a l l and the frequency o f the a p p l i e d a l t e r n a t i n g v o l t a g e i s low (48). The g e n e r a l p r o c e d u r e i n v o l v e d i n p r o c e s s i n g capacitance data to obtain adsorption isotherms i n volved f i r s t , c a l c u l a t i o n of surface spreading pres s u r e v a l u e s , π, f o r t h e o r g a n i c compound a t t h e m e r c u r y - s o l u t i o n i n t e r f a c e by back i n t e g r a t i o n o f capacitance data. T h e n , f r o m v a l u e s o f π a t known p o t e n t i a l , E , and c o n c e n t r a t i o n s o r a c t i v i t i e s , t h e p a r a m e t e r s i n t h e F r u m k i n a d s o r p t i o n e q u a t i o n were c a l c u l a t e d by a n o n l i n e a The b a c k i n t e g r a t i o n m e t h o d (49) r e l i e s o n t h e assumption t h a t at s u f f i c i e n t l y negative p o t e n t i a l s , c a p a c i t a n c e v e r s u s p o t e n t i a l c u r v e s (C v s . E) f o r aqueous s o l u t i o n s o f an o r g a n i c a d s o r b a t e c o i n c i d e with the background C v s . Ε curve f o r the e l e c t r o l y t e alone. T h i s c o n d i t i o n was m e t f o r a l l t h e a d e n i n e systems d e s c r i b e d h e r e , i . e . , a t p o t e n t i a l s between - 1 . 6 V a n d - 1 . 8 V t h e C v s . Ε c u r v e s become c o i n c i d e n t a n d r e m a i n s o a t e v e n more n e g a t i v e potentials. I n t e g r a t i o n o f C vs_. Ε c u r v e s f o r t h e b a c k g r o u n d electrolyte solution (i.e., v s . E) g i v e s values of charge q , r e l a t i v e q * , the charge a t the mercurye l e c t r o l y t e solution interface at the s t a r t i n g poten t i a l E * ( t h i s was t y p i c a l l y - 1 . 8 V i n t h e s e studies). Thus, Ε - q * =/ CdE (2) . E* By m e a s u r e m e n t o f a n e l e c t r o c a p i l l a r y c u r v e o f i n t e r f a c i a l t e n s i o n v e r s u s p o t e n t i a l (γ v s . E) o n t h e background e l e c t r o l y t e s o l u t i o n the vallie o f the e l e c t r o c a p i l l a r y maximum p o t e n t i a l (ECM) may b e o b tained ( i . e . , Ε at γ max.). A t t h e ECM q = 0 , h e n c e the a b s o l u t e v a l u e o f q ^ a t any p o t e n t i a l E (q^(E)) i s q
w
w
q (E)
=
w
fq (E) w
-
q*]
-
[q^ECM)
-
q * ] (3) .
From t h e measured v a l u e s o f γ (Ε) d e t e r m i n e d from e l e c t r o c a p i l l a r y curves o f the background s o l u t i o n the v a l u e o f <3 (E) may b e d e t e r m i n e d b y d i f f e r e n t i a t i o n o f γ values, i . e . , W
q
w
(E)
= dy
w
(£) .
dE
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
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C h a r g e (q (E)) v a l u e s d e t e r m i n e d by e l e c t r o c a p i l l a r y and c a p a c i t a n c e methods always a g r e e d w e l l . C a p a c i t a n c e m e a s u r e m e n t o n s o l u t i o n s o f known a c t i v i t y o f t h e o r g a n i c compounds were b a c k i n t e g r a t e d f r o m E * i n e x a c t l y t h e same f a s h i o n . Assuming at E * that q = q , then w
< W
Q
E
r
g
=
)
[
q
org
(
E
"
)
* *
]
+
V
e
* *
Φ *
Thus, absolute values of q (E) may b e o b t a i n e d . These v a l u e s a g a i n agreed we?l w i t h q (E) obtained by d i f f e r e n t i a t i o n o f e l e c t r o c a p i l l a r y â a t a ( Y vs. Ε) f o r s o l u t i o n s o f t h e o r g a n i c s p e c i e s . To o b t a i n v a l u e s o f t h e i n t e r f a c i a l t e n s i o n ( γ ( Ε ) ) i t i s necessary to perform a second i n t e g r a tion. We c a n w r i t e r
o
r
a
g
γ
-
γ*
= -/jjj*
q dE
(6) .
Now, γ * ( t h e i n t e r f a c i a l t e n s i o n a t t h e s t a r t i n g p o tential) i s known f r o m e l e c t r o c a p i l l a r y m e a s u r e m e n t s on the b a c k g r o u n d e l e c t r o l y t e s o l u t i o n ; m o r e o v e r , it does not change w i t h a d d i t i o n o f a d s o r b a t e t o the solution. T h e r e f o r e , e q (6) c a n be u s e d t o o b t a i n t h e i n t e r f a c i a l t e n s i o n as a f u n c t i o n o f b o t h Ε and a , t h e a c t i v i t y of the organic s o l u t e . The s p r e a d i n g pressure a t any s o l u t e a c t i v i t y and p o t e n t i a l i s π =
y (E) w
-
γ(Ε)
(7) ,
where γ i s the value o f γ for the background e l e c t r o l y t e s o Y u t i o n a t a = 0. With the e x c e p t i o n o f the deoxyadenosine monophosphate system (vide i n f r a ) , the a s s u m p t i o n i s made t h r o u g h o u t t h a t t h e s o l u t e a c t i v i t y c a n be r e p l a c e d b y i t s m o l a r c o n c e n t r a t i o n i n t h e d i l u t e s o l u t i o n s employed i n the study. T h e π v a l u e s may be m e a s u r e d d i r e c t l y f r o m e l e c t r o c a p i l l a r y d a t a u s i n g eq (7). Using t h i s approach, therefore, i t i s possible t o compare c h a r g e , q and s u r f a c e s p r e a d i n g pressure v a l u e s , π, a t a n y p o t e n t i a l a n d c o n c e n t r a t i o n d e r i v e d from c a p a c i t a n c e and e l e c t r o c a p i l l a r y d a t a . W i t h i n the accuracy o f our measurements, p l o t s o f π v s . Jin a f o r a l l c o m p o u n d s r e p o r t e d h e r e a t p o t e n t i a l s more n e g a t i v e t h a n c a . - 0 . 5 V w e r e s u p e r i m p o s a b l e by s i m p l e h o r i z o n t a l t r a n s l a t i o n . This implies t h a t the isotherms are congruent w i t h r e s p e c t t o p o t e n t i a l and t h a t t h e a t t r a c t i o n c o e f f i c i e n t , a, i n t h e F r u m k i n a d s o r p t i o n e q u a t i o n (i50) i s i n d e p e n d e n t o f potential (vide infra). f
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
8.
KINOSHITA
therm
Interfacial
E T AL.
The method u s e d equation θ
= Ba e
2
Behavior
to
a
fit
of
data
123
Purines
to
the
Frumkin
iso
(8)
G
i s s i m i l a r t o t h a t employed by Hansen and coworkers (51). In t h i s f i x e d p o t e n t i a l form o f the Frumkin e q u a t i o n , Θ i s the f r a c t i o n o f the e l e c t r o d e surface c o v e r e d w i t h m o n o l a y e r a d s o r b a t e , Β and α a r e c o n s t a n t s depending on the i n t e r a c t i o n s between the a d s o r b e d m o l e c u l e s and the s u r f a c e and on l a t e r a l i n t e r m o l e c u l a r i n t e r a c t i o n s between the adsorbate molecules, respectively. The c o n s t a n t Β i s d e p e n d e n t o n p o t e n t i a l a c c o r d i n g t o e q (9) (52). Β =
Β β<Ο w h e r e Tm i s t h e l i m i t i n g s u r f a c e e x c e s s o f t h e s o l u t e at f u l l monolayer coverage i n moles p e r u n i t a r e a . The t e r m Β i s the value o f Β at the e l e c t r o c a p i l l a r y maximum p o S e n t i a l f o r t h e e l e c t r o l y t e , i . e . , a t a=0. Combining equations ( £ ) a n d {9) an e q u a t i o n referred t o as t h e g e n e r a l i z e d F r u m k i n e q u a t i o n i s o b t a i n e d (eq 10). θ In
= Β ae<-^ m r
this Φ =
equation
.e
R T )
the
2
a
function
/jj q d E + C E
(Ε -
w
(10)
G
Φ is
1/2
Ε)
given
by (11)
where q i s the charge at a p o t e n t i a l Ε for background e l e c t r o l y t e s o l u t i o n , C' i s the c a p a c i t a n c e of the electrode completely covered with adsorbate monolayer (assumed c o n s t a n t ) (53) and E„ i s t h e e l e c t r o c a p i l l a r y maximum p o t e n t i a l f o r t h e m e r c u r y - s o l u t i o n i n t e r f a c e a t 0=1 m e a s u r e d r e l a t i v e t o t h e ECM f o r t h e e l e c t r o lyte alone. T h e p o t e n t i a l , E , i n e q (11) is also r e l a t i v e t o t h e ECM f o r e l e c t r o l y t e s o l u t i o n . Inte g r a t i o n o f e q (11) gives Φ = G(E)
+ C E E
-
C'E 2
N
where
G(Ε)
= γ
w
(Ο)
-
Y
w
(Ε)
2
(12)
(13).
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
ELECTROCHEMICAL
124
STUDIES O F B I O L O G I C A L
SYSTEMS
H e n c e G ( E ) may b e d e t e r m i n e d f r o m t h e d i f f e r e n c e be t w e e n t h e i n t e r f a c i a l t e n s i o n a t t h e ECM a n d t h e v a l u e a t any p o t e n t i a l Ε ( v s . ECM a t a = 0 ) . T h i s was n o r m a l l y o b t a i n e d d i r e c t l y from e l e c t r o c a p i l l a r y c u r v e s on t h e electrolyte solution. The G i b b s a d s o r p t i o n e q u a t i o n Γ = -1 RT
(14)
άτ\
din
a
may be c o m b i n e d w i t h t h e F r u m k i n i n t e g r a t e d t o g i v e an e x p r e s s i o n Θ and Γ (51). m π =
Γ RT m
e q u a t i o n (eq that relates
[-£η(1-Θ)-αΘ ]
[£] ) π to
and a,
(15). —
2
We h a v e d e v e l o p e d a n o n l i n e a r l e a s t squares method t o f i t π v a l u e s a t v a r i o u s c o n c e n t r a t i o n s and p o t e n t i a l s t o g i v e t h e b e s t v a l u e s o f Β , α, C , Γ and Ε (_54) . T h i s i s d o n e by f i r s t t a k i n g t r i a l val ues o r t h e l a t t e r 5 p a r a m e t e r s and c a l c u l a t i n g v a l u e s o f Θ f r o m e q (10) f o r each p a i r o f a. and E ^ v a l u e s by an i t e r a t i v e n u m e r i c a l m e t h o d . TÎlis s e t o f calcul a t e d Θ. v a l u e s i s t h e n u s e d t o p r e d i c t a s e t o f π. v a l u e s u s i n g eq (15). For a given set of parameters, a v a l u e o f t h e sum g squares o f the r e s i d u a l s s = Σ. ( π . - π c a l c u l a t e d ) i s then obtained. The v a l u e o f s ià minimized with respect to v a r i a t i o n of a l l five p a r a m e t e r s ; s t a n d a r d e r r o r s i n a l l the c o n s t a n t s and t h e r o o t mean s q u a r e d e v i a t i o n i n π a r e a l s o c a l c u lated. F o r m o s t o f t h e s y s t e m s , c o n v e r g e n c e was o b t a i n e d w i t h i n 5 t o 20 i t e r a t i o n c y c l e s p r o v i d e d r e a s o n a b l y good i n i t i a l e s t i m a t e s o f t h e p a r a m e t e r v a l u e s w e r e made. In t h o s e systems where e l e c t r o c a p i l l a r y a n d c a p a c i t a n c e d a t a w e r e a v a i l a b l e , i t was o f t e n u s e f u l t o f i x the Ε v a l u e , which i s estimated r e a d i l y from e l e c t r o c a p i l l a r y curves f o r l a r g e a c t i v i t i e s o f the o r g a n i c adsorbate ( i . e . , a t 0->l) . In addition, C may g e n e r a l l y be e s t i m a t e d b y m e a s u r i n g the c a p a c i t a n c e o f a n e a r l y s a t u r a t e d s o l u t i o n o f the o r g a n i c compound a t a p o t e n t i a l a t , o r v e r y c l o s e t o , t h e p o t e n t i a l o f maximum a d s o r p t i o n ( t h i s i s e a s i l y r e c o g n i z e d as t h e p o t e n t i a l a t w h i c h π r e a c h e s i t s maximum v a l u e i n π v s . Ε p l o t s p a r t i c u l a r l y i n s o l u t i o n s w h e r e 0-*l) . The l a t t e r a p p r o a c h has a l s o b e e n u s e d by Hansen and c o - w o r k e r s (55). 1
1
f
Results In
and this
Discussion study,
the
interfacial
behavior
of
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
ade-
8.
KiNosHiTA E T A L .
Interfacial
Behavior
of F urines
125
n i n e and i t s d e o x y n u c l e o s i d e and m o n o d e o x y n u c l e o t i d e s was s t u d i e d a t pH 9 i n b o r a t e b u f f e r b y u s e o f dif f e r e n t i a l c a p a c i t a n c e and e l e c t r o c a p i l l a r y s t u d i e s . I n pH 9 b o r a t e b u f f e r t h e C v s . Ε c u r v e s o f b a c k g r o u n d e l e c t r o l y t e and a l l s o l u t i o n s c o n t a i n i n g o r g a n i c a d s o r b a t e become c o i n c i d e n t a t n e g a t i v e p o t e n t i a l s , which i s a necessary c o n d i t i o n i f the b a c k - i n t e g r a t i o n o f c a p a c i t a n c e m e t h o d o f Grahame e t a l . (49) i s used. Capacitance
and E l e c t r o c a p i l l a r y
Studies
A t y p i c a l series of capacitance curves for ade n i n e i s shown i n F i g u r e 3 w h e r e i t may be s e e n t h a t up t o a c o n c e n t r a t i o n o f c a . 2-3mM a s y s t e m a t i c d e crease o f the c a p a c i t a n c e o c c u r s at around - 0 . 3 to - 0 . 7 V with a broad -1.2V. At concentration sharp decrease i n capacitance i s observed centered at -0.6V g i v i n g r i s e to a very sharply defined p i t or well. A s i m i l a r p i t i s observed i n the case of deoxyadenosine ( F i g u r e 4) a l t h o u g h f o r t h i s c o m p o u n d t h e p i t i s centered at -1.25V. The m o n o d e o x y n u c l e o t i d e s do n o t e x h i b i t a p i t a t p H 9 . Other workers have o b s e r v e d s i m i l a r p i t s a t l o w e r pH f o r many o f t h e s e t y p e s o f compounds ( 4 0 - 4 4 ) . Interpretation of c a p a c i t a n c e r e s u l t s b y t h e m e t h o d s u s e d i n t h i s s t u d y was not p o s s i b l e i n the p i t r e g i o n . However, e l e c t r o c a p i l l a r y measurements c o u l d be i n t e r p r e t e d w i t h i n t h e pit region. The a d s o r p t i o n p r o c e s s e s o c c u r r i n g a t c o n c e n t r a t i o n s b e l o w t h o s e n e c e s s a r y f o r p i t f o r m a t i o n w i l l be r e f e r r e d t o as f o r m a t i o n o f t h e f i r s t a d s o r p t i o n layer. Using the c a l c u l a t i o n a l approach o u t l i n e d e a r l i e r s u r f a c e s p r e a d i n g p r e s s u r e v a l u e s , π, v e r s u s t h e l o g arithm of a c t i v i t y (concentration) o f adenine and i t s d e o x y n u c l e o s i d e and t h r e e m o n o d e o x y n u c l e o t i d e s at v a r i o u s e l e c t r o d e p o t e n t i a l s are r e a d i l y superimpos a b l e by a b s c i s s a t r a n s l a t i o n ( F i g u r e 5 ) . The p r e c i s i o n o f t h e d a t a , e x p r e s s e d a s r o o t mean s q u a r e (rms) d e v i a t i o n s i n π i n F i g u r e 5 i s somewhat p o o r e r t h a n r e s u l t s f o r some s i m p l e a l i p h a t i c c o m p o u n d s r e p o r t e d b y B a i k e r i k a r a n d H a n s e n (56_) , who e m p l o y e d a s i m i l a r c o m p u t a t i o n a l method. T h i s i s p r o b a b l y due t o t h e g r e a t e r complexity of the molecules s t u d i e d here and, p e r h a p s , the s l i g h t l y lower p r e c i s i o n o f the a . c . p o l a r o g r a p h i c measurement o f c a p a c i t a n c e used h e r e c o m p a r e d t o t h e more c o n v e n t i o n a l b r i d g e m e t h o d s for c a p a c i t a n c e measurements. N e v e r t h e l e s s , the f a c t t h a t t h e π v s . &n a p l o t s a r e s u p e r i m p o s a b l e implies
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
ELECTROCHEMICAL
η I u
ι
_0.2
ι
-0.4
ι
-0.6
ι
ι
STUDIES O F B I O L O G I C A L S Y S T E M S
ι
-0.8 -1.0 -1.2 Potential/Volt vs SCE
ι
-1.4
1
-1.6
1
-1.8
Figure 4. Differential capacitance curves for deoxyadenosine in borate buffer pH 9 (ionic strength 0.5) at the DME. Curves obtained at 100Hz and lOmV peak-to-peak.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
1
8.
KINOSHITA
' -4
' 3
I 2
Interfacial
E T AL.
I 1
I 0
I 1
I 2
I 3
I
I
Behavior
I 4
I 3
I 2
of Purines
I 1
0
1 1
1 2
127
L 3
In a
Figure 5. Composite π vs. In a plots for (A) adenine, (B) deoxyadenosine, (C) dAMP, (D) dADP, and (E) dATP at pH 9 in borate buffer. Data points are for potentials from —1.0V to —0.4V. The rms deviation in π from the calculated curve for (A) is 0.54, (B) 1.27, (C) 0.31, (D) 0.79, and (E) 0.46 dyne cm- . The calculated curve for dAMP (C) takes into account the self-association of this compound using a sequential equilibrium model and a value of the equilibrium constant of 110 I/mol. 1
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
128
ELECTROCHEMICAL
STUDIES O F B I O L O G I C A L S Y S T E M S
that for molecules i n t h i s adenine s e r i e s the i n t e r m o l e c u l a r i n t e r a c t i o n s between a d s o r b a t e m o l e c u l e s on the e l e c t r o d e surface are e s s e n t i a l l y independent of t h e e l e c t r o d e p o t e n t i a l ( i . e . , α i n eq ( 8 ) i s i n d e pendent of potential). The F r u m k i n a d s o r p t i o n m o d e l ( w i t h c o n s t a n t a) employed i n f i t t i n g i n t e g r a t e d c a p a c i t a n c e data r e q u i r e s t h a t the l i n e a r r e l a t i o n s h i p between c h a r g e , q , a n d s u r f a c e c o v e r a g e (Θ) be v a l i d a t a n y f i x e d p o t e n t i a l a c c o r d i n g ( 5 J 7 ) t o eq ( 1 6 ) where q i s the charge at a 1
q
= q (l-e w
+ q ' (Θ)
(16)
p o t e n t i a l , E , corresponding to a completely covered monolayer of organi T h i s e q u a t i o n was t e s t e d b y p l o t t i n g q v s . v a l u e s o f 0 d e t e r m i n e d from the n o n l i n e a r l e a s t squares a n a l y s i s of π, Ε a n d a d a t a . Such p l o t s are s a t i s f a c t o r i l y l i n e a r f o r a l l of the adenine systems at p o t e n t i a l s o f - 0 . 5 V o r more n e g a t i v e . A set of q vs. 0 plots is shown i n F i g u r e 6 f o r d A T P . D e v i a t i o n s from eq ( 1 6 ) become q u i t e p r o n o u n c e d f o r d e o x y a d e n o s i n e a t p o t e n t i a l s more p o s i t i v e t h a n - 0 . 6 V . The f a c t t h a t a n o d i c d e s o r p t i o n p e a k s a r e n o t o b s e r v e d f o r any a d e n i n e spe c i e s i m p l i e s t h a t a t p o t e n t i a l s more p o s i t i v e t h a n about - 0 . 5 V the Frumkin model does not h o l d . Hansen (55) i n a recent study of the a d s o r p t i o n of v a r i o u s a r o m a t i c m o l e c u l e s has a t t r i b u t e d the s u p p r e s s i o n o f anodic d e s o r p t i o n peaks to s p e c i f i c anion a d s o r p t i o n . The c o n s i s t e n c y o f π, Ε , a r e s u l t s a n d t h e l i n e a r i t y o f q v s . 0 p l o t s a t f i x e d p o t e n t i a l s more n e g a t i v e t h a n - 0 * 5 V f o r m o s t a d e n i n e s y s t e m s makes i t u n l i k e l y t h a t there are s y s t e m a t i c e r r o r s i n the measured c a p a c i t a n c e d a t a w h i c h r e n d e r t h e d e r i v e d π and q v a l u e s unreliable. A t pH 9 a d e n i n e a n d t h e a d e n i n e m o i e t y i n i t s d e o x y n u c l e o s i d e and d e o x y n u c l e o t i d e s i s a l m o s t e n t i r e l y in i t s neutral state ( 5 8 ) . The r e s u l t s o f a n a l y s i s o f the c a p a c i t a n c e curves of a d e n i n e a r e shown i n T a b l e 1 . These r e s u l t s r e v e a l t h a t the a t t r a c t i o n c o e f f i c i e n t , a , i s s m a l l and p o s i tive i n d i c a t i n g a small l a t e r a l attractive interaction between the adsorbed adenine m o l e c u l e s . The a r e a o c c u p i e d p e r a d e n i n e m o l e c u l e on t h e e l e c t r o d e surface is 55±4A . A d e n i n e i s a p l a n a r m o l e c u l e as shown f r o m i t s c r y s t a l s t r u c t u r e ( 5 9 ) and has an a r e a o f about 4 2 A . However, c o n s i d e r i n g the u n s y m m e t r i c a l shape o f t h i s m o l e c u l e and the r e s u l t a n t d i f f i c u l t y
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
1.
-0.10±0.19
Deoxyadenosine5 -monophosphate
Borate
buffer
(see
10.811.3 -5498
-4923
-4949
4.24±0.80 4.08±0.56
-5695
-4428
15.0±3.5 e
/&/mole
1.77±0.17
xlO
Experimental)
-1.42+0.4 3
e
I
cal.
0
AG /
e
a
_
for
- 0 . 5 1 4 1 0 . 007
- 0 . 4 7 2 ± 0 . 015
-0.527±0. 005
- 0 . 5 0 6 ± 0 . 021 e
2.45
3.04
3.04
2.06
2.22
l
±
u
2
per
8017
7514
6 7 1 8
5514
5514
E
molecule
Area/8
deoxyadeno-
Γ /mole Γ Π /Ν *-ΓΙ2f\ 10 cm xl0
adenine,
-0.56010. 1
equation a t pH 9
α value
inferred
by
use
of
eq.
16.
° ° E l e c t r o c a p i l l a r y maximum p o t e n t i a l where
0 = 1
for
each
compound.
e
T h e p a r a m e t e r s f o r dAMP w e r e c a l c u l a t e d u s i n g t h e s e q u e n t i a l e q u a l e q u i l i b r i u m c o n s t a n t model d i s c u s s e d i n the t e x t and a v a l u e f o r the e q u i l i b r i u m c o n s t a n t o f 110 β / m o l e .
c
S t a n d a r d f r e e e n e r g y o f a d s o r p t i o n a t t h e e l e c t r o c a p i l l a r y maximum p o t e n t i a l f o r t h e e l e c t r o l y t e , b a s e d on i n f i n i t e d i l u t i o n s t a n d a r d s t a t e s f o r t h e a d s o r b a t e , both i n s o l u t i o n (at u n i t m o l a r i t y ) and on t h e s u r f a c e (at u n i t v a l u e o f θ ). AG° = - R T l n B , where Β i s e x p r e s s e d i n £/mole u n i t s . Ι^ΠΤ
a
1
Deoxyadenosine5 -triphosphate
1
Deoxyadenosine5 -diphosphate
-0.55
-0.52±0.44
Deoxyadenosine
1
0.54±0.21
α 3
J
1
Parameters o f the g e n e r a l i z e d Frumkin s i n e and d e o x y a d e n o s i n e - 5 - p h o s p h a t e s
Adenine
Compound
Table
130
ELECTROCHEMICAL STUDIES OF BIOLOGICAL SYSTEMS
i n p a c k i n g o n t h e s u r f a c e , we e s t i m a t e t h a t t h e a c t u a l gjjea o c c u p i e d b y o n e a d e n i n e m o l e c u l e s h o u l d be 50-6OA . T h i s i m p l i e s t h e r e f o r e t h a t i n the first adsorption r e g i o n , adenine i s adsorbed i n a f l a t ori e n t a t i o n on t h e e l e c t r o d e s u r f a c e . Any e l e c t r o d e s u r f a c e n o t c o v e r e d b y a d e n i n e w o u l d be a v a i l a b l e to water d i p o l e s or other i n o r g a n i c i o n s . In the case of deoxyadenosine, the α value shifts somewhat more n e g a t i v e c o m p a r e d t o a d e n i n e (Table 1), b u t t h e a r e a o c c u p i e d b y one m o l e c u l e i s t h e same a s for adenine. This implies that the deoxyribose group i s t i l t e d away f r o m t h e e l e c t r o d e surface. The a t t r a c t i o n c o e f f i c i e n t , a , s h i f t s s y s t e m a t i c a l l y more n e g a t i v e f o r t h e s e r i e s d A M P , d A D P , d A T P . Since the phosphate groups are e x t e n s i v e l y i o n i z e d a t pH 9 , t h i s e f f e c t i s i v e i n t e r a c t i o n betwee is expected. The a r e a o c c u p i e d p e r m o l e c u l e f o r e a c ^ adenine mononucleotide i s very s i m i l a r at c a . 70-80A (Table 1). T h i s a r e a i s o n l y a b o u t 30 p e r c e n t greater than i s observed for u n s u b s t i t u t e d adenine or deoxy a d e n o s i n e and hence i n d i c a t e s t h a t t h e phosphate g r o u p s a r e a l s o l a r g e l y d i r e c t e d away f r o m t h e e l e c trode surface. I n a l l o f t h e s y s t e m s e x c e p t dAMP, t h e a n a l y s i s o f π, Ε , a d a t a was c a r r i e d t h r o u g h a s s u m i n g t h a t c o n c e n t r a t i o n s o f t h e o r g a n i c s o l u t e c o u l d be s u b s t i t u t e d for solute a c t i v i t y . H o w e v e r , i n t h e c a s e o f dAMP, considerable negative curvature occurs i n plots of π vs. log concentration at concentrations greater than a b o u t 5mM ( F i g u r e 7 ) . The b e s t f i t o f t h e s e d a t a w i t h t h e 5 - p a r a m e t e r l e a s t s q u a r e s program (at c o n c e n t r a t i o n s up t o 20mM) l e a d s t o an a n o m a l o u s l y l a r g e surf a c e a r e a f o r dAMP i n t h e a d s o r b e d m o n o l a y e r (^ 1 2 6 A ) . A much i m p r o v e d c o r r e l a t i o n r e s u l t s i f i t i s a s s u m e d t h a t dAMP p a r t i a l l y a s s o c i a t e s a c c o r d i n g t o t h e s c h e m e : monomer + monomer = d i m e r , monomer + d i m e r = t r i m e r , monomer + t r i m e r = t e t r a m e t e r , e t c . , a n d t h a t t h e a c t i v i t y o f dAMP c a n be e q u a t e d t o t h e c o n c e n t r a t i o n o f monomer. For s i m p l i c i t y , the e q u i l i b r i u m c o n s t a n t f o r e a c h a s s o c i a t i o n s t e p has b e e n t r e a t e d as a c o n s t a n t , K, ( £ 0 ) . I n f i t t i n g t h e dAMP d a t a , Κ i s c o n s i d e r e d t o be a s i x t h a d j u s t a b l e p a r a m e t e r . The rms d e v i a t i o n i n π i s reduced to nearly h a l f i t s least squares v a l u e from the 5-parameter o p t i m i z a t i o n , and a r e a s o n a b l e v a l u e o f t h e a r e a (67±8A / m o l e c u l e ) is o b t a i n e d f o r dAMP when t h e l e a s t s q u a r e s v a l u e o f t h e a s s o c i a t i o n c o n s t a n t (K = 1 1 0 ± 2 6 i / m o l e ) i s used to c a l c u l a t e t h e a c t i v i t y o f dAMP f r o m i t s f o r m a l c o n c e n tration. T h e s o l i d c u r v e i n F i g u r e 5C was c a l c u l a t e d 2
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
KINOSHITA E T A L .
Interfacial
Behavior
of
Purines
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
132
ELECTROCHEMICAL
STUDIES O F B I O L O G I C A L
SYSTEMS
u s i n g Κ = 11 Oil/mole a n d u s i n g t h e f i v e c o n s t a n t s i n t h e g e n e r a l i z e d F r u m k i n e q u a t i o n r e p o r t e d i n T a b l e 1. We h a v e n o t b e e n a b l e t o f i n d a n y r e p o r t s o f a s s o c i a t i o n o f dAMP. However, v a p o r p r e s s u r e osmometry and sedimentation e q u i l i b r i u m experiments i n d i c a t e that adenosine-5*-monophosphate (AMP) i s h i g h l y a s s o c i a t e d i n t h e 0-5OmM c o n c e n t r a t i o n r a n g e (61,62). The d e p e n d e n c e o f a d s o r p t i o n o n p o t e n t i a l i s shown i n F i g u r e 8 where i t i s c l e a r t h a t a l l compounds e x h i b i t maximal a d s o r p t i o n at c l o s e t o - 0 . 5 V . A t more p o s i t i v e o r n e g a t i v e p o t e n t i a l s a d s o r p t i o n becomes less pronounced. These c u r v e s c o u l d have been e x t e n d e d t o about - 1 . 5 t o - 1 . 6 V where a d s o r p t i o n o f the adenine s p e c i e s no l o n g e r o c c u r s t o a n y a p p r e c i a b l e extent. The f r e e e n e r g y o f a d s o r p t i o n a t t h e E . C . M . for t h e e l e c t r o l y t e (AG v e r y s i g n i f i c a n t f e a t u r e e x c e p t an a p p r e c i a b l e de c r e a s e i n AG° b e t w e e n a d e n i n e a n d i t s deoxynucleoside and m o n o d e o x y n u c l e o t i d e s . T h e AG° v a l u e s s i m p l y i n d i c a t e an i n c r e a s e i n t h e s u r f a c e a c t i v i t y o f t h e d e o x y n u c l e o s i d e and d e o x y n u c l e o t i d e s o v e r a d e n i n e . Using a c a p i l l a r y e l e c t r o m e t e r the i n t e r f a c i a l t e n s i o n of the mercury-aqueous s o l u t i o n i n t e r f a c e for a d e n i n e and d e o x y a d e n o s i n e has been m e a s u r e d . Using t h i s t e c h n i q u e , s u r f a c e s p r e a d i n g p r e s s u r e , π, may be m e a s u r e d d i r e c t l y (eq 9 ) . Some t y p i c a l a d s o r p t i o n r e s u l t s o b t a i n e d by e l e c t r o c a p i l l a r y measurements are shown i n T a b l e 2 . These r e s u l t s a r e i n good agreement w i t h t h e v a l u e s o b t a i n e d b y t h e more i n d i r e c t c a p a c i tance method. E l e c t r o c a p i l l a r y measurements have a l s o been used t o study t h e a d s o r p t i o n o f adenine and deoxyadeno s i n e a t c o n c e n t r a t i o n s where the c a p a c i t a n c e p i t i s observed (see F i g u r e s 3 a n d 4 ) . I t has not been p o s s i b l e y e t t o deduce the d e t a i l s o f the a d s o r p t i o n p r o cesses but i t i s f a i r l y easy to determine the l i m i t i n g s l o p e o f t h e π vs_. £n a p l o t s i . e . , o b t a i n from t h e G i b b s a d s o r p t i o n e q u a t i o n (eq 1 7 ) . Typical results a r e shown i n T a b l e 3. C a l c u l a t i o n s reveal that these areas correspond c l o s e l y to those expected for a v e r t i c a l o r i e n t a t i o n of the adenine molecule at the e l e c trode surface. I t has been c o n c l u d e d t h e r e f o r e that a t c o n c e n t r a t i o n s where adenine o r d e o x y a d e n o s i n e exhi b i t a capacitance p i t there i s a rearrangement of the molecules, over a very sharply defined p o t e n t i a l range, from a f l a t arrangement on the s u r f a c e t o a p e r p e n d i c u lar arrangement. I n o r d e r t o g a i n some a d d i t i o n a l i n s i g h t s i n t o the o r i e n t a t i o n of adenine molecules w i t h r e s p e c t to t h e e l e c t r o d e s u r f a c e when i n t h e p e r p e n d i c u l a r o r i e n -
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
KiNosHiTA E T A L .
Interfacial
ι ι ι -0.5 -0.7 -0.9 Potential, Volts ys S C E
Behavior
I -ii
of
Purines
in borate buffer pH 9 at 25°C. Numbers on plots refer to the coni (mM) of the adenine species in the bulk solution.
c e n t r a t
o n
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
2.
in
Table
Figures
3 and
-1.1
4 for
to
SCE
appearance
-1.45
-0.9
Deoxyadenosine
to
-0.35
vs.
Maximum P o t e n t i a l
Adenine
See
1.
Tmax a n d a r e a o c c u p i e d p e r adenine and deoxyadenosine capacitance p i t i n borate
defined
Range/Volt
3.
are
1.21
1.26
3
l e
2
per
molecule
54±3
0
3.09
1
-0.50
xl0
-4204
2
Area/8
63±5
cnf
Γ^/mole
2.62
SCE
/
-0.54
V vs.
W
-4226
cal.
Ε
a d e n i n e and d | o x y measurements.
of
30±7
5.5±1.3
capacitance
23±3
pits,
2
per molecule
Area/8
7.2±0.9
Π Λ
/mole/ m ' -2 10 cm xlO
Γ
molecule at complete surface coverage for at p o t e n t i a l s c o r r e s p o n d i n g t o the anomolous b u f f e r pH 9.
Β xlO~ /VnK>
Compound
Table
Terms
1.49±0.33
Deoxyadenosine
a
0.49±0.12
α
AG°/
Parameters o f the g e n e r a l i z e d Frumkin e q u a t i o n for a d e n o s i n e a t pH 9 d e t e r m i n e d f r o m e l e c t r o c a p i l l a r y
Adenine
Compound
Table
8.
KINOSHITA
E T AL.
Interfacial
Behavior
of
Purines
135
t a t i o n , t h e e f f e c t o f m e t h y l a t i o n o f t h e amino group o f adenine has been s t u d i e d . Capacitance vs. potent i a l c u r v e s f o r 6 - d i m e t h y l a d e n i n e a r e shown i n F i g u r e 9. 6-Methyladenine e x h i b i t s very s i m i l a r curves with no c a p a c i t a n c e p i t . Quite c l e a r l y , s u b s t i t u t i o n of one o r b o t h amino h y d r o g e n s c o m p l e t e l y i n h i b i t s f o r m a t i o n o f the capacitance p i t s . Some t y p i c a l r e s u l t s f o r 6 - m e t h y l - a n d 6 - d i m e t h y l a d e n i n e a r e shown i n T a b l e 4. T h e g e n e r a l b e h a v i o r o f t h e s e two c o m p o u n d s i s very s i m i l a r to adenine although the area o c c u p i e d per molecule i s a l i t t l e s m a l l e r i n the case o f 6 - m e t h y l adenine. In view o f the f a c t t h a t o n l y the p u r i n e s and p y r i m i d i n e s found n a t u r a l l y i n n u c l e i c a c i d s e x h i b i t c a p a c i t a n c e p i t s (41-44) , i t has been t e n t a t i v e l y concluded t h a t the s t r u c t u r a l f u n c t i o n a l i t i e s a s s o c i a t e d w i t h th b a s e p a i r s i n n u c l e i c a c i d s ( F i g u r e 10) a r e r e s p o n s i b l e f o r b i n d i n g t o the e l e c t r o d e s u r f a c e where m o l e c u l e s are i n t h e i r p e r p e n d i c u l a r o r i e n t a t i o n , perhaps a s shown i n F i g u r e 1 1 . Ellipsometric
Studies
E l l i p s o m e t r y i s b a s e d o n t h e f a c t t h a t when e l l i p t i c a l l y p o l a r i z e d l i g h t i s i n c i d e n t on a f i l m covered, h i g h l y r e f l e c t i v e surface the p o l a r i z e d s t a t e o f the r e f l e c t e d l i g h t i s d i f f e r e n t from t h a t observed i n the case o f r e f l e c t i o n from a f i l m - f r e e surface. The m a g n i t u d e o f t h e c h a n g e s i n t h e p o l a r i z a t i o n s t a t e i n d u c e d by t h e s u r f a c e f i l m depends on t h e t h i c k n e s s and the o p t i c a l c o n s t a n t s o f the adsorbed f i l m m a t e r ial. Some p r e l i m i n a r y e l l i p s o m e t r i c m e a s u r e m e n t s h a v e b e e n made o n a q u e o u s s o l u t i o n s o f a d e n i n e a n d d e o x y adenosine i n contact with a plane (or n e a r l y p l a n e ) mercury e l e c t r o d e s u r f a c e . A t y p i c a l experimental arr a n g e m e n t u s e d i n t h e s e s t u d i e s i s shown i n F i g u r e 2 . Only q u a l i t a t i v e or s e m i - q u a n t i t a t i v e s t u d i e s are p o s s i b l e with the arrangement s i n c e d i f f i c u l t i e s are e n c o u n t e r e d w i t h m a i n t a i n i n g an a b s o l u t e l y f l a t m e r c u r y s u r f a c e and a v o i d i n g b u b b l e f o r m a t i o n a t v e r y n e g a t i v e potentials. H o w e v e r , some o f t h e p o t e n t i a l c a p a b i l i t i e s o f e l e c t r o e l l i p s o m e t r y c a n be i l l u s t r a t e d b y g i v i n g the r e s u l t s of a simple experiment with deoxyadenosine. F i g u r e 12 shows t h e c a p a c i t a n c e v s . p o t e n t i a l c u r v e f o r a 35mM s o l u t i o n o f d e o x y a d e n o s i n e at pH 7 a t t h e DME. I t w i l l be r e c a l l e d t h a t e l e c t r o c a p i l l a r y and c a p a c i t a n c e r e s u l t s s u g g e s t t h a t between about - 0 . 2 to c a . - 1 . 1 V deoxyadenosine i s adsorbed f l a t on the e l e c t r o d e , w h i l e i n the c a p a c i t a n c e p i t
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
4.
a
1 and
e
text.
-5695
measurements.
capacitance
C
e
-4091^ -4941
cal.
AG°/
measurements.
see! T a b l e
14±0.5
D e t e r m i n e d by
terms
e
1.0±0.2^ 4.2±0.5
3
xl0" / '
electrocapillary
d e f i n i t i o n of
ο
il/mole
Β
9
-0.40
5
SCE
/
e
-- 0° .- 5 4 9c° 4
W
V vs.
Ε
C
66±5 C
C
e
molecule
2.52±0.2
1 Λ
10 χ10
43±3^ 46±2
"2
Area/S^per
3.83±0.24^ 3.63±0.16
cm
Γ /mole
g e n e r a l i z e d Frumkin e q u a t i o n f o r 6 - m e t h y l a d e n i n e and a t pH 9 d e t e r m i n e d b y c a p a c i t a n c e a n d e l e c t r o c a p i l l a r y
D e t e r m i n e d by
For
C
0. 5 ± 0 . 6
6-Dimethyl-
C
0. 85±^0.3 0. 4 0 ± 0 . 2
α
Parameters o f the 6-dimethyladenine measurements.
6-Methyladenine
Compound
Table
8.
KiNosmTA E T A L .
Interfacial
Behavior
-0.80
of Purines
1.00
137
1.20
POTENTIAL (VOLTS vs SCE )
Figure 9. Capacitance vs. potential curves for 6-dimethyladenine in borate pH 9. Upper trace is background electrolyte, lowest trace is 19.7mM 6-dimethyladenine.
Adenine
Figure
10. Hydrogen bonding between thymine and adenine in DNA
Figure 11. Possible modes of binding of adenine to the mercury electrode surface when in perpendicular orientation in region of anomalous capacitance pits
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
138
ELECTROCHEMICAL
STUDIES O F B I O L O G I C A L S Y S T E M S
39.20h
-1.15V -1.15V
-1.15V
ω 39.10H
-1.05 V 39.00h
-1.05V
-1.05V
-1.05V
Figure 13. Variation in ellipsometric polarizer angle at a plane mercury electrode in 35mM deoxyadenosine in Mcllvaine buffer pH 7.0 as the potential is switched from -1.05V to -1.15V vs. SCE
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
8.
KINOSHITA
E T AL.
Interfacial
Behavior
of Purines
139
region (-1.1V t o -1.5V) i t i s adsorbed p e r p e n d i c u l a r to the electrode surface. The v a r i a t i o n i n t h e e l l i p someter p o l a r i z e r angle (P) f o r t h e same s o l u t i o n a t a p l a n e mercury e l e c t r o d e as t h e e l e c t r o d e p o t e n t i a l i s v a r i e d between - 1 . 0 5 V ( o u t s i d e t h e c a p a c i t a n c e p i t ) a n d - 1 . 1 5 V ( i n s i d e t h e c a p a c i t a n c e p i t ) i s shown i n F i g u r e 1 3 . Ρ v a r i e s c o n s i s t e n t l y by about 0 . 1 5 ° ( a b s o l u t e ) as t h e p o t e n t i a l i s s w i t c h e d back a n d f o r t h . A s s u m i n g a r e a s o n a b l e r e f r a c t i v e i n d e x o f 1.4 8 t o 1.50 f o r d e o x y a d e n o s i n e , t h i s change i n Ρ c o r r e s p o n d s t o a change i n t h i c k n e s s o f about + 5 § f o r t h e p o t e n t i a l change from - 1 . 0 5 V t o - 1 . 1 5 V ( u s i n g t h e c a l c u l a t i o n a l m e t h o d s o f M c C r a c k i n [63]. Such an i n c r e m e n t i n f i l m t h i c k n e s s agrees w e l l w i t h t h a t i n f e r r e d from c a p a c i t a n c e and e l e c t r o c a p i l l a r y measurements, i . e . , e x p e c t e d on t h e b a s i to p e r p e n d i c u l a r (-1.15V Acknowledgements : The a u t h o r s would l i k e t o a c k n o w l edge f i n a n c i a l s u p p o r t o f t h i s work t h r o u g h N a t i o n a l I n s t i t u t e s o f H e a l t h G r a n t N o . GM 2 1 0 3 4 . We w o u l d a l s o l i k e t o t h a n k D r . R o b e r t S . H a n s e n o f t h e Iowa S t a t e U n i v e r s i t y f o r h i s v a l u a b l e comments a n d s u g g e s t i o n s r e g a r d i n g some a s p e c t s o f t h i s w o r k . The v a l u a b l e a s s i s t a n c e o f D r . E r i c E n w a l l d u r i n g some o f t h e i n i t i a l ellipsometric studies i s also gratefully acknowledged. Literature Cited 1. Hill, T. L., J. Amer Chem. S o c . (1958), 80, 2142. 2. O'Konski, C. T. and K a t c h a l s k y , Α., Biophysical J. (1965), 5, 667. 3. Neumann, E . , and K a t c h a l s k y , Α . , P r o c . N a t . Acad. Sci., U.S.A. (1972), 69, 993. 4. R o b e r t s , R. B. and F l e x n e r , L. Β., Q u a r t Rev. B i o p h y s . (1969), 2, 135. 5. K a t c h a l s k y , Α . , and Neumann, E . , I n t e r n . J. N e u r o s c i . (1972), 3, 175. 6. K a t c h a l s k y , A. and O p l a t k a Α., J. Med. Sci. (1966) 2, 4. 7. K a t z , B. in " B i o p h y s i c a l Science-Α Study Program," p. 466, J. L . O n c l e y ( e d . ) , 1959. 8. Ganesan, A. T. and L e d e r b e r g , L., Biochem. B i o p h y s . Res. Commun. (1965), 18, 824. 9. R y t e r , Α . , B a c t . Rev. (1968), 32, 39. 10. Comings, D. E . , Ann. J. Human G e n e t i c s (1968), 20, 440. 11. O ' B r i a n , R. L., S a n y a l , A. B., and S t a n t o n , R. H., Exp. Cell. Res. (1972), 76, 106.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
140
ELECTROCHEMICAL STUDIES OF BIOLOGICAL SYSTEMS
12. Jacob, F., Brenner, S., and C u z i n , F., C o l d S p r . Harb. Symp. Quant. Biol. (1963), 28, 329. 13. Becker, R. O., J. Bone Joint Surg. (1961), 43A, 643. 14. Becker, R. O. and Murray, D. G., Clin. Ortho. (1970), 75, 1969. 15. Becker, R. O., Nature (1973), 235, 109. 16. Becker, R. O. and Spadaro, J. Α., Bull. N.Y. Acad. Med. (1972), 48, 627. 17. Cope, F. W., Bull. Math. B i o p h y s . (1963), 25, 165. 18. Cope, F. W., A r c h . Biochem. B i o p h y s . (1963), 103, 352. 19. Cope, F. W., J. Chem. Phys. (1964), 40, 2653. 20. Cope, F. W., Bull. Math. B i o p h y s . (1965), 27, 237. 21. Cope, F. W., E x p e r i e n t i a 22. Cope, F. W., i Systems," T. E . K i n g , H. S. Mason and M. M o r r i s o n ( E d s . ) , W i l e y , New York, 1965. 23. Cope, F. W., and S t r a u b , K. D., Bull. Math. B i o p h y s . (1969), 31, 761. 24. Cope, F. W., Adv. Biol. Med. Phys. (1970), 13, 1. 25. Mohilner, D. M. in "Electroanalytical Chemistry," A. J. B a r d (Ed.), V o l . 1, p. 241, Dekker, New York, 1966. 26. Pilla, Α. Α . , B i o e l e c t r o c h e m . B i o e n e r g . (1974), 1, 227. 27. E i s e n b e r g , S., Ben-or, S., and D o l j a n s k i , F., Exptl. Cell. Res. (1962), 26, 451. 28. Mayhew, E . and Weiss, L., Exptl. Cell Res. (1968), 50, 441. 29. Mayhew, E., J. Gen. P h y s i o l . (1966), 49, 717. 30. B r e n t , T. P. and F o r r e s t e r , J. Α . , Nature (1967), 215, 92. 31. Purdon, L., Ambrose, E. J., and Klein, G., Nature (1958), 181, 1586. 32. Ambrose, E. J., P r o c . 7th Canadian Cancer Res. Cont., p. 247, Pergamon, T o r o n t o , 1967. 33. Weiss, L. and Hauscha, T. S., I n t . J. Cancer (1970), 6, 270. 34. Field, Α . V., Tytell, Α . Α., Lampson, P. G., and H i l l e m a n , M. R., P r o c . Nat. Acad. Sci. U.S.A. (1967), 58, 1004. 35. Schell, P. L., Biochem. B i o p h y s . A c t a (1971), 240, 472. 36. J a n i k , B. and Sommer, R. G., B i o p o l y m e r s (1973) 12, 2803. 37. J a n i k , B. and Elving, P. J., J. Amer. Chem. Soc. (1970), 92, 235.
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8. KINOSHITA ET AL.
Interfacial Behavior of Purines
141
38. J a n i k . B. and Elving, P. J., Chem. Rev. (1968), 68, 295. 39. D r y h u r s t , G. and Elving, P. J., T a l a n t a (1969), 16, 855. 40. Vetterl, V., Coll Czech. Chem Commun. (1966), 31, 2105. 41. Vetterl, V., Coll. Czech. Chem Commun. (1969), 34, 673. 42. Vetterl, V., J. Electroanal. Chem. (1968), 19, 169. 43. Vetterl, V., B i o p h y s i k (1968), 5, 255. 44. L o r e n z , W., Z. E l e k t r o c h e m (1958), 62, 192. 45. Webb, J. W., J a n i k , Β., and Elving, P. J., J. Amer. Chem. Soc. (1973), 95, 991. 46. K r z n a r i k , D., V a l e n t a P. and Nürnberg H W. J. E l e c t r o a n a l 47. K i n o s h i t a , Η. J. E l e c t r o a n a l . Chem., s u b m i t t e d f o r publication (1976). 48. Damaskin, B. B., Petrii, Α . Α., and B a t r a k o v , V., " A d s o r p t i o n o f O r g a n i c Compounds on E l e c t r o d e s , " p. 16, Plenum, New York, 1971. 49. Grahame, D. C., Coffin, Ε. M., Cummings, J. P., and P o t h , Μ. Α., J. Amer. Chem. Soc. (1952), 74, 1207. 50. Frumkin, Α. Ν., Z. Phys. Chem. (1925), 116, 466. 51. Broadhead, D. E., B a i k e r i k a r , K. G., and Hansen, R. S., J. Phys. Chem. (1976), 80, 370. 52. R e f e r e n c e 48, p. 113. 53. Reference 48, p. 112. 54. Our c o m p u t a t i o n a l program employs an o p t i m i z i n g program w r i t t e n by Dr. E. E n w a l l and i n c o r p o r a t i n g an a l g o r i t h m g i v e n by Marquardt, D. W., J. Soc. I n d u s t . A p p l . Math. (1963), II, 431. 55. Hansen, R. S., P e r s o n a l communication. 56. B a i k e r i k a r , K. G., and Hansen, R. S., J. Coll. I n t e r f a c e Sci. (1975), 52, 277. 57. Reference 51, p. 70. 58. A l b e r t y , R. Α., Smith, R. Μ., and Bock, R. Μ., J. Biol. Chem. (1951), 193, 425. 59. Donohue, J., A r c h . Biochem. B i o p h y s . (1968), 128, 591. 60. T s ' o , P. O. P., in " B a s i c Principles in N u c l e i c A c i d C h e m i s t r y , " V o l . 1, pp. 537-543, Academic, New York, 1974. 61. S c h w e i z e r , M. P., Broom, A. D., Hollis, D. P. and Ts'o, P. O. P., J. Amer. Chem. Soc. (1968), 90, 1042. 62. R o s s e t t i , G. P. and van Holde, Κ. E., Biochem. B i o p h y s . Res. Commun. (1967), 26, 717.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
142
63.
ELECTROCHEMICAL STUDIES O F BIOLOGICAL SYSTEMS
M c C r a c k i n , F. L., " A Fortram P r o g r a m of Ellipsometric M e a s u r e m e n t s , " NBS No. 479, 1969.
for Analysis Technical Note
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
9
Evaluation
of
Coulometric
Mediator-Titrants
Titration
for
of
the
Indirect
Biocomponents
ROBERT SZENTRIMAY, PETER YEH, and THEODORE KUWANA Department of Chemistry, Ohio State University, Columbus, Ohio 43210
In recent years we hav bee interested i th development and application of the indirec for the accurate assessment of stoichiometry (n value) and energetics (Ε value) of bioredox components such as cytochrome c oxidase and "blue" copper laccases. The initial reason for developing ICT was the advantage of being able to work with fairly small volumes under anaerobic conditions and to conven iently add electrochemical charge accurately and incrementally on nanoequivalent levels (1). The optically transparent electrode also provided a means of easily acquiring spectral information during the titration (2). Other advantages of ICT over c l a s s i c a l potentiometric methods became obvious during our progress and these w i l l be discussed subsequently. The fundamental problem in the accurate assessment of n and E values of biocomponents is the slow heterogeneous electron transfer between the biocomponent and an indicator electrode such as platinum. This is particularly severe with large macromolecules where the redox site may be surrounded by some periphery structure such as a protein. Thus, one or more "mediators" are usually added to the solution so that redox coupling is enhanced between the biocomponent(s) and the electrode. In the ICT method, a titrant (either a reductant or oxidant) is electrochemically generated to transfer charge to the biocompo nent. For example, the reaction sequence for a reduction i s : o'
o'
Electrode reaction: M Q Solution reaction:
M
R
x
+ ne" +
E n z
Q
= M M
X
= Qx
E R
+
E n z
°'M R
^ ^
where reaction (1) occurs at the electrode to generate the titrant, M R which in turn reduces the biocomponent, EnzQx/ to Enz^. The equilibrium of reaction (2) is given by: 143
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
144
ELECTROCHEMICAL
STUDIES O F B I O L O G I C A L S Y S T E M S
ΔΕ°' = E?' - E ? ! = R T In Κ Enz M eq
(3)
where E ^ z i s the formal e l e c t r o d e p o t e n t i a l for the h a l f - r e a c t i o n :
Enz
Q x
+ ne" = Enz
R
E j ^
(4)
The η and E g ^ v a l u e s are determined from the " b e s t " fit between the e x p e r i m e n t a l and computer s i m u l a t e d plots of the e l e c t r o c h e m i c a l c h a r g e , q , and the change in the o p t i c a l a b s o r b a n c e , £A, of a b i o c o m p o n e n t ( s ) . As i n potentiometry, one or more mediators may be added i n s m a l l but known q u a n t i t i e s to a c c e l erate the attainment of may be u s e d w h o s e E ° biocomponent (usually w i t h i n 1 8 0 mv) so that it a c t s as a m e d i a tor. T h u s , we have c h o s e n to c a l l redox titrants employed i n the I C T method as m e d i a t o r - t i t r a n t s ( M - T ' s ) . z
The e a r l i e r c h o i c e s of M - T ' s were those e m p l o y e d in p o t e n tiometric titrations of b i o c o m p o n e n t s , n o t a b l y those reported for s t u d i e s of components in the r e s p i r a t o r y s y s t e m , or those w h o s e redox properties seemed s u i t a b l e for M - T ' s as known from our e x p e r i e n c e or from the e l e c t r o c h e m i c a l l i t e r a t u r e . H o w e v e r , no s y s t e m a t i c study has been p r e v i o u s l y reported to e x p e r i m e n t a l l y e v a l u a t e and c o m p i l e a l i s t of redox compounds w h i c h c o u l d serve as M - T ' s . Such a l i s t w o u l d be p a r t i c u l a r l y v a l u a b l e to those e x a m i n i n g bioredox components at p o t e n t i a l s where s u i t a b l e M - T ' s are p r e s e n t l y u n a v a i l a b l e or where w e l l known mediators or M - T ' s have f a i l e d to give r e p r o d u c i b l e r e s u l t s for a p a r t i c u l a r b i o c o m p o n e n t . T h u s , a l o n g - r a n g e o b j e c t i v e of our laboratory has been to c o m p i l e a l i s t of p o s s i b l e M - T ' s w h o s e p o t e n t i a l s are graded i n increments of some 2 0 to 4 0 mV's over a total p o t e n t i a l range of ca_. + 1 . 0 0 to - 1 . 0 0 v o l t v e r s u s N H E . Such a c o m p i l a tion i s s t i l l i n c o m p l e t e . In t h i s paper, the e x p e r i m e n t a l a s s e s s ment of s e v e r a l p o s s i b l e redox compounds as M - T ' s along w i t h their e f f e c t i v e n e s s i n the ICT of test biocomponents w i l l be r e ported . The " i d e a l " properties sought for M - T ' s are l i s t e d in Table I. The c o n s t r a i n t s imposed by these properties are so r e s t r i c t i v e that very few compounds f u l f i l l a l l of t h e m . There a r e , fortunately, s i t u a t i o n s where some properties are l e s s important than o t h e r s . For e x a m p l e , let us a s s u m e that M i n r e a c t i o n (1) and (2) i s c h e m i c a l l y u n s t a b l e and d e c o m p o s e s w i t h a h a l f - l i f e of ca_. 1 hour to another p r o d u c t . If Ε°^ i s much l e s s than E g ^ ( K of r e a c t i o n (2) i s large) and the forward rate of r e a c t i o n (2) i s f a s t , R
z
e q
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
9.
szENTRiMAY E T A L .
Coulometric
Titration
of
Biocomponents
145
then the c o n c e n t r a t i o n of M w i l l be s m a l l s u c h that any l o s s through d e c o m p o s i t i o n w i l l be m i n i m a l . The s i t u a t i o n i s s o m e what different, h o w e v e r , w h e n a compound i s u s e d as a mediator in a potentiometric t i t r a t i o n . The mediator i n this c a s e a c t s as a redox buffer and i s most e f f e c t i v e near a 1:1 ratio of o x i d i z e d to r e d u c e d s p e c i e s . I n s t a b i l i t y of one s p e c i e s may then be detrimental d e p e n d i n g on the rate of d e c o m p o s i t i o n , properties of the d e c o m p o s i t i o n p r o d u c t , and whether the product i n t e r a c t s w i t h the b i o c o m p o n e n t . As a matter of f a c t , the most d i f f i c u l t property to a s s e s s i n Table I i s whether any of the redox s p e c i e s i n h i b i t or i n t e r a c t with any p a r t i c u l a r b i o c o m p o n e n t . A priori judgement i s d i f f i c u l t . A l s o , i n the ICT method, two M - T ' s may or may not be c o m p a t i b l e w i t h e a c h other. To a s s e s s the above properties and problems w i t h M - T ' s th trolled potential coulometry c o u l o m e t r i c titrations of M - T v e r s u s M - T , and M - T titrations of b i o c o m p o n e n t s , p a r t i c u l a r l y the test s y s t e m of cytochrome £ , have been e m p l o y e d . R
Primary attention w i l l be devoted to d i s c u s s i o n of v a r i o u s ferrocene and b i p y r i d y l i u m compounds as M - T ' s . Although e v a l u a t i o n of a l l the properties of v a r i o u s M - T ' s i s s t i l l i n p r o g r e s s , a l i s t i n g of M - T ' s h a s been c o m p i l e d and i s h e r e i n i n c l u d e d s i n c e others may find one or more of these M - T ' s u s e f u l . TABLE I Properties of " I d e a l " M e d i a t o r - t i t r a n t s 1.
w e l l - d e f i n e d η v a l u e (n = 1 preferrable for most c a s e s )
2.
known E ° ' v a l u e under e x p e r i m e n t a l c o n d i t i o n s
3.
fast heterogeneous and homogeneous e l e c t r o n transfer
4.
s o l u b l e i n aqueous media at or near p H 7.0
5.
stable redox
6.
good o p t i c a l window
7.
does not i n h i b i t or interact w i t h biocomponents
species
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
146
ELECTROCHEMICAL
STUDIES O F B I O L O G I C A L S Y S T E M S
R e s u l t s And D i s c u s s i o n B i p y r i d y l i u m Salts ( V i o l o g e n s ) . The negative v a l u e of the e l e c t r o d e p o t e n t i a l for the b i p y r i d y l i u m s a l t s (viologens) makes them a t t r a c t i v e as r e d u c t a n t s . They have been p r e v i o u s l y e m p l o y e d as one e l e c t r o n r e d u c i n g m e d i a t o r - t i t r a n t s i n the I C T of biocomponents (1-4). Others have u t i l i z e d these s a l t s for c o u l o metric and k i n e t i c s s t u d i e s (5-7) as w e l l . S t e c k h a n (8) h a s e v a l u a t e d the s p e c t r o e l e c t r o c h e m i c a l c h a r a c t e r i s t i c s of s e v e r a l v i o l o g e n s , the h a l i d e s a l t s of 1, l ' - d i b e n z y l - 4 , 4 ' - b i p y r i d y l i u m , 1, l ' - e t h y l e n e - 2 , 2 ' - b i p y r i d y l i u m , 1, l ' - d i m e t h y l - 4 ^ ' - b i p y r i d y l i u m , and 1 , 1 ' - p r o p y l e n e - 2 , 2 ' - b i p y r i d y l i u m d i c a t i o n s u s i n g o p t i c a l l y transparent e l e c t r o d e s ( O T E ' s ) . The d i s c u s s i o n to follow extends and i l l u s t r a t e s the further a p p l i c a t i o n s of these v i o l o g e n s for u s e as M - T ' e q u i v a l e n t (10-100) l e v e l s . The properties of v i o l o g e n s examined to date are l i s t e d i n Table II. The E ° ' v a l u e s (pH = 7 . 0 , p h o s phate buffer with i o n i c strength of 0.15) range from -358 mV to - 5 5 6 mV for the first e l e c t r o n r e d u c t i o n (reaction (5)). A second r e d u c t i o n step (reaction (6)) r e d u c e s the r a d i c a l c a t i o n to the neutral s p e c i e s . In most of these v i o l o g e n s , the neutral s p e c i e s , V
+ +
Vt
+ e~ = V+
E°'
(5)
+ e" = V°
E°'
(6)
V ° , i s i n s o l u b l e and i s strongly adsorbed on the electrode s u r f a c e . The r a d i c a l c a t i o n , v t , a l s o may form s p a r i n g l y s o l u b l e s a l t s , d e p e n d i n g on the p a r t i c u l a r v i o l o g e n and the counter i o n present i n s o l u t i o n . A t y p i c a l c u r r e n t - p o t e n t i a l (i-E) pattern for c y c l i c voltammetry at a tin oxide O T E at p H 7.0 is shown i n Figure 1. The r e v e r s e o x i d a t i v e w a v e for the neutral s p e c i e s v a r i e s and often shows t y p i c a l c h a r a c t e r i s t i c s for the e l e c t r o l y s i s of adsorbed r e a c t a n t . The 1st w a v e for the r e d u c t i o n of V to v t and the o x i d a t i o n of the r a d i c a l back to the d i c a t i o n is u s u a l l y r e v e r s i b l e . For the quantitative generation of the r a d i c a l c a t i o n as a reductant for a s o l u t i o n r e a c t i o n during c o u l o m e t r i c t i t r a t i o n s , it i s imperative that the s o l u t i o n c o n d i t i o n s and the p o t e n t i a l be s e l e c t e d so that i n s o l u b i l i t y or a d s o r p t i o n does not o c c u r . The s e p a r a t i o n between E 9 ' and E°/' i s u s u a l l y s u f f i c i e n t for most v i o l o g e n s that the r a d i c a l c a t i o n c a n be q u a n t i t a t i v e l y generated without interference from the neutral s p e c i e s . This has been demonstrated p r e v i o u s l y for methyl v i o l o g e n by s e t t i n g the p o t e n t i a l of the chromoamperometric experiment at no more than 20 mv n e g a t i v e of Ε ° ' ( 8 , 9 ) . +
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
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148
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STUDIES O F B I O L O G I C A L
SYSTEMS
The i n i t i a l steps i n the preparation for ICT of a biocomponent are to quantitate the c o u l o m e t r i c generation of the M - T ( s ) and to c h e c k on the a n a e r o b i c i t y of the e l e c t r o l y s i s c e l l . Figure 2 shows the plot of the change i n the o p t i c a l a b s o r b a n c e , ΔΑ, at 595 nm v e r s u s the e l e c t r o c h e m i c a l c h a r g e , q , for the generation of BVt at a tin oxide Ο T E . The p o t e n t i a l a p p l i e d to the Ο TE w a s - 0 . 6 0 V v s A g / A g C l reference e l e c t r o d e . The subsequent r e m o v a l of b e n z y l v i o l o g e n r a d i c a l c a t i o n , B V t , as i n d i c a t e d by the d e c r e a s e of M i n Figure 2 w a s a c c o m p l i s h e d through the e l e c t r o l y t i c generation of m o l e c u l a r oxygen (3) at a platinum microelectrode (applied p o t e n t i a l of +1.40 V v s A g / A g C l r e f e r e n c e ) . In the particular c e l l u s e d , two to three minutes of s o l u t i o n s t i r r i n g w a s required between e a c h charge i n j e c t i o n so that redox e q u i l i b r i u m c o u l d be attained throughout the s o l u t i o n . A spectrum was obtained after e a c h e q u i l i b r a t i o n and befor d u c t i o n s , a charge c o r r e c t i o n of 15 + 3% was required to correct for background charge w h i c h w a s e v a l u a t e d from c o u l o m e t r i c experiments i n the a b s e n c e of the M - T . W i t h some tin oxide O T E ' s , this c o r r e c t i o n w o u l d be as low as 2% w h i c h depended on the s o l u t i o n c o n d i t i o n s for the e x p e r i m e n t . It i s therefore n e c e s s a r y to c a r e f u l l y e v a l u a t e t h i s background c o r r e c t i o n for e a c h r u n . W i t h the 15% c o r r e c t i o n , the average η v a l u e s c a l c u l a t e d from the s l o p e s (3,4) of the ΔΑ-q plots were 1.08 + 0.03 and 1.08 + 0 . 0 6 for o x i d a t i o n and r e d u c t i o n of BV, r e s p e c t i v e l y . The background c o r r e c t i o n for the Pt m i c r o e l e c t r o d e w a s l e s s than 1% of the total charge for O 2 g e n e r a t i o n . It i s a l s o a d v i s a b l e to titrate a M - T a g a i n s t another o n e . Such a c o u l o m e t r i c titration i s i l l u s t r a t e d i n Figure 3 where the o p t i c a l a b s o r b a n c e of the 1, Γ - b i s (hydroxymethyl) f e r r i c i n i u m ion (BHMF+) at w a v e l e n g t h of 640 nm i s being f o l l o w e d . The B H M F + i s q u a n t i t a t i v e l y generated at a t i n oxide O T E by a p p l y i n g +0.60 V v s A g / A g C l reference e l e c t r o d e . The r e d u c t i o n of the ion w a s through the e l e c t r o g e n e r a t i o n of BVt S i n c e larger increments of charge were employed for t h i s titration as compared to the amount for most b i o c o m p o n e n t s , the background c o r r e c t i o n s are c o n s i d e r a b l y l e s s (2 + 1%). The redox c y c l i n g of B H M F + / B H M F c a n be r e p r o d u c i b l y repeated s e v e r a l times without any n o t i c e a b l e d e v i a t i o n i n the ΔΑ-q c u r v e s . It s h o u l d be noted that the s e quence of the BHMF+ r e a c t i o n w i t h BVt i s t y p i c a l of the ec c a t a l y t i c regeneration m e c h a n i s m : BV++ + e " = BVt BHMF
+
+ BVt = BV " " + B H M F 4
1
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
(7)
9.
Coulometric
SZENTRIMAY E T A L .
I
Titration
of
ι
ι
ι
I
!
I
-0.2
-0.4
-0.6
-0.8
-1.0
-1.2
Ε
volt
vs
149
Biocomponents
Ag/AgCl
Figure 1. Typical cyclic voltammetric i - E curve for viologens. Reduction of .96mM concentration of 1,1'dihydroxyethyl-4,4'-bipyridylium chloride in phos phate buffer pH 7.0 (ionic strength 0.15) at tin oxide OTE; scan rate 96 mv/S. (A) i - E of 1st wave only; (B) i - E of 1st and 2nd reduction wave.
0
10
20
30
40
mCoulombs/ml Figure 2. Change in optical absorbance vs. charge plot for the generation and removal of benzylviohgen radical cation. Concentration of benzylviologen chlo ride l.OOmM in phosphate buffer at pH 7.0 (ionic strength 0.15); monitoring wave length of 595 nm using a cell with an optical path length of 1.25 cm, cell volume 1.33 ml. Increase in absorbance corresponds to the generation of the benzyl viologen radical cation.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
150
ELECTROCHEMICAL
STUDIES O F B I O L O G I C A L
SYSTEMS
P o t e n s o m e t r i c measurements c a n be made c o n c u r r e n t l y during coulometry of BVt or B H M F generation by monitoring the e l e c t r o d e p o t e n t i a l of a s e c o n d s m a l l Pt e l e c t r o d e i n the c e l l . The E ° ' v a l u e of the B H M F + / B H M F c o u p l e determined i n this manner w i l l be d i s cussed shortly. In Figure 4 , t y p i c a l r e s u l t s are presented for the I C T titration of the test b i o c o m p o n e n t , cytochrome £ (cyto c ) . The c y t o c_ c o n c e n t r a t i o n was 2 1 . 2 μΜ and the c e l l volume w a s 1.33 ml (4). The redox c o n c e n t r a t i o n of c y t o c w a s monitored by f o l l o w i n g the o p t i c a l a b s o r b a n c e at a w a v e l e n g t h of 550 nm after e a c h i n c r e mental a d d i t i o n of c h a r g e . The two M - T ' s d i s c u s s e d above were employed for this ICT redox c y c l i n g . S e q u e n t i a l r e d u c t i v e and o x i d a t i v e c y c l i n g c o u l d be repeated as many as eight times w i t h out any o b v i o u s change to the shape of the M - q c u r v e s . E a c h charge increment i n thi The average η v a l u e s for four c o n s e c u t i v e c y c l e s as shown i n Figure 4 were 1.01 + 0 . 0 1 (6% c o r r e c t e d for background charge) and 1.06 + 0 . 0 4 (3% c o r r e c t e d for background charge) for r e d u c tions and o x i d a t i o n s , r e s p e c t i v e l y . The quantitation for the ICT of c y t o c _ u s i n g these two M - T ' s i s e x c e l l e n t and i s i n good a g r e e ment w i t h previous r e s u l t s (1_). The abrupt change i n the s l o p e of the M - q plot during r e d u c t i o n i n d i c a t e s that c y t o c_ is f u l l y r e d u c e d and that an e x c e s s of BVt had been g e n e r a t e d . T h i s change c o n v e n i e n t l y marks the end point for the t i t r a t i o n . T h u s , i n the o x i d a t i o n s , the e x c e s s must be removed prior to the c o m m e n c e ment of the o x i d a t i o n of r e d u c e d c y t o c_. W h e n an e x c e s s of BVt w a s present i n the s o l u t i o n , a slow l o s s of the r a d i c a l was found as e v i d e n c e d by the d e c r e a s e of the optical absorbance. The rate of l o s s w a s about 0.002 to 0.004 absorbance unit per minute or c o r r e s p o n d e d to about 0.2 to 0 . 4 nanomoles per m i n u t e . T h i s l o s s m a y , i n part, e x p l a i n the 8% error i n the coulometry of BVt found e a r l i e r (results as shown i n Figure 2). H o w e v e r , to date we have found the p r e c i s i o n i n the I C T of biocomponents to be w i t h i n 5% u s i n g this M - T , as e x pected s i n c e BVt i s b e i n g c o n s u m e d as g e n e r a t e d . The r e a s o n for the l o s s of this r a d i c a l when it i s i n e x c e s s i s p r e s e n t l y u n e x plained . Both methyl and b e n z y l v i o l o g e n have been e x t e n s i v e l y u s e d as r e d u c t i v e M - T ' s i n the titration of the heme p r o t e i n s , c y t o c_ and cytochrome c o x i d a s e , w i t h good r e s u l t s . Preliminary r e s u l t s from the I C T of tree l a c c a s e (10) and of heme proteins in s u b mitochrondrial p a r t i c l e s have produced w e l l defined M - q c u r v e s w h i c h are interpetable and a s s i g n a b l e to the e x p e c t e d c o m p o n e n t s . At p r e s e n t , there i s no a - p r i o r i r e a s o n w h y the other v i o l o g e n s l i s t e d i n Table II w i l l not a c t as s a t i s f a c t o r y M - T ' s for +
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
szENTRiMAY E T A L .
Ο
Coulometric
40
Titration
80
of
Biocomponents
120
160
mCoulombs Figure 3. Change in optical absorbance vs. charge plot for the generation and removal of lJ'-bis(hydroxymethyl)ferricinium ion. Concentration of l,r-bis(hydroxymethyl)ferrocene 1.02mM and 1.22mM benzylviologen chloride in phosphate buffer at pH 7.0; cell parameters same as those used in experiment shown in Figure 2. Increase in absorbance corresponds to the generation of the ferricinium ion.
mCou'-)mbs / ml Figure 4. Change in optical absorbance vs. charge plot for the ICT of 21.2 /xM cytochrome c. The M-T s are those used for the experiment shown in Figure 3 using same experimental conditions. Increase in absorbance at 550 nm corresponds to the reduction of cytochrome c.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
422+6
465+5°
ferrocene (parent)
1, Γ - b i s
644+12
1, Γ - d i c a r b o x y l i c a c i d
T h e symbol S i n d i c a t e s water s o l u b i l i t y ; Tween-20.
627+11
ferrocenylmethyl t r i methyl ammonium s a l t
a
589+8
chloro
e
530+10°
monocarboxylic
acid
480+5
hydroxy-2 -phenylethyl
(hydroxymethyl)
402+10
hydroxyethyl d
365+10°
acetic acid
E ° ' ( m V v s . Ν HE)
341+9
derivatives
1, Γ - d i m e t h y l
Ferrocene
I,D
57
S
3
625
630
638
615
625
630
655
d
^(nm) 1
3
1
420
385
335
370
etcm"
e
l b
M~ )
(hr)
0.50
;> 24
14
d
4.3
32
1 / 2
>25
t
I , D means i n s o l u b l e and detergent s o l u b i l i z e d u s i n g 3%
40
S
S
64
57
I,D
61
I,D S
d
S
S
I,D
Solubility
57
62
58
57
51
p
AE (mV)
E l e c t r o c h e m i c a l and O p t i c a l Properties of Ferrocenes
TABLE III
1
>
1
ο r ο ο ο
1
M g > r
η
Ο
M f W
or to
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
T a k e n from Ref.
T h e counter a n i o n was
d
e
perchlorate.
O T T L E = O p t i c a l l y Transparent Thin L a y e r E l e c t r o d e .
c
(17).
T h e w a v e l e n g t h maximum for the o x i d i z e d form, the f e r r i c i n i u m s .
D
154
E L E C T R O C H E M I C A L STUDIES O F B I O L O G I C A L S Y S T E M S
reductive titrations. Ferrocene and Ferrocene D e r i v a t i v e s The unique structure and properties of ferrocene and its d e r i v a t i v e s have r e s u l t e d i n a great d e a l of t h e o r e t i c a l and e x p e r i mental s t u d i e s during the l a s t two d e c a d e s . Of p a r t i c u l a r i n t e r e s t to us was the w i d e range of p o s i t i v e redox p o t e n t i a l s a c c e s s i b l e through the v a r i e t y of s u b s t i t u e n t s of f e r r o c e n e , the f a c i l e e l e c t r o n - t r a n s f e r p r o p e r t i e s , the c l e a r o p t i c a l window i n the v i s i b l e r e g i o n for ferrocenes i n their r e d u c e d form, and the w e l l d e f i n e d η v a l u e of u n i t y . Their formal redox p o t e n t i a l s and polarographic v a l u e s have been reported i n v a r i o u s literature c o m p i l a t i o n s (Π_, 12_, 13_ problems w i t h the u s e o l i m i t e d s o l u b i l i t y of some ferrocenes i n their r e d u c e d form and the i n s t a b i l i t y of the o x i d i z e d form, the f e r r i c i n i u m i o n s , i n aqueous s o l u t i o n s , p a r t i c u l a r l y near p h y s i o l o g i c a l p H ' s . The number of oxidants for b i o l o g i c a l a p p l i c a t i o n s h a v e been quite l i m i t e d w i t h the most familiar ones b e i n g the metal c y a n i d e s (Fe, M o , W) w h i c h c a n be d e l i t e r i o u s w i t h c e r t a i n biocomponents at higher c o n c e n t r a t i o n s (15,16). There have been few r e c e n t s t u d i e s w h i c h encouraged the further e x a m i n a t i o n of ferrocenes as M - T ' s . The l i m i t e d s o l u b i l i t y c o u l d be c i r c u m v e n t e d by s o l u b i l i z a t i o n i n m i c e l l e s as formed by n o n - i o n i c detergent s u c h as T w e e n - 2 0 (17). U s i n g such s o l u b i l i z a t i o n , F u j i h i r a , et a l . , demonstrated the I C T of r e d u c e d c y t o c_ o x i d a s e by electrogenerated f e r r i c i n i u m i o n (16). The E ° ' v a l u e s for v a r i o u s ferrocenes i n phosphate buffer at p H of 7.0 are g i v e n i n Table III. The ferrocenes i n the Table span a range of p o t e n t i a l s from +340 to +644 mV and were s e l e c t e d from a l i s t of 24 ferrocenes w h i c h have been e x a m i n e d . C y c l i c voltammetry at a Pt O T E w a s u s e d for determination of the E ° ' v a l u e s e x c e p t as noted otherwise i n the t a b l e . The trend i n the E ° ' v a l u e s i s i n agreement with that e x p e c t e d on the b a s i s of substituent effects (11, 12 , 13 , 14). The lower r e l a t i v e p o t e n t i a l s for ferrocene m o n o c a r b o x y l i c a c i d (FMCA) and ferrocene a c e t i c a c i d (FAA) may be due to the a c i d - b a s e e q u i l i b r i u m i n w h i c h the b a s i c form predominates at p H 7 . 0 . T h u s , these two compounds are more e a s i l y o x i d i z e d than e x p e c t e d from substituent effect c o n s i d e r a t i o n s alone and are i n agreement with c a l c u l a t i o n s of Penden, et a l . (18). The i n s o l u b l e compounds (labelled I, D) i n Table III were s o l u b i l i z e d u s i n g 3% Tween 20 a c c o r d i n g to the procedure of Yeh and Kuwana (17). The c y c l i c voltammetric i - E c u r v e s of these detergent s o l u b ï ï i z e d ferrocenes e x h i b i t e d r e v e r -
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
9.
SZENTRIMAY E T AL.
Coulometric
Titration
of
155
Biocomponents
s i b l e to n e a r l y r e v e r s i b l e b e h a v i o r . The difference between the o x i d a t i v e and r e d u c t i v e peak p o t e n t i a l s (AE ) was l e s s than 70 mV for these f e r r o c e n e s . Three f e r r o c e n e s , F M C A , FAA and B H M F (1, l ' - b i s - ( h y d r o x y methy'Jferrocene) w i t h E ° ' v a l u e s of +530, 365 and 4 6 5 , r e s p e c t i v e l y were examined quite thoroughly, p r i m a r i l y b e c a u s e of their s o l u b i l i t y (up to about 10 m M i n phosphate buffer at p H 7.0) and their attractive redox p o t e n t i a l s . T y p i c a l c y c l i c i - E c u r v e s for B H M F at both t i n oxide Ο TE and Pt e l e c t r o d e s are shown for c o m p a r i s o n purposes i n Figure 5. The " i r r e v e r s i b l e " i - E for the tin oxide e l e c t r o d e i s quite t y p i c a l for most ferrocenes at this e l e c t r o d e . The r e v e r s i b i l i t y v a r i e s w i t h e a c h e l e c t r o d e and the p H (19). T h u s , the p o t e n t i a l required for the d i f f u s i o n c o n t r o l l e termined for e a c h c o u l o m e t r i The s t a b i l i t y of these three f e r r i c i n i u m i o n s , F M C A + , F A A and B H M F + was determined by monitoring their o p t i c a l a b s o r b a n c e after they were generated by c o u l o m e t r y . In Figure 6 the plots of a b s o r b a n c e , A , v e r s u s time are shown for these ions i n aqueous s o l u t i o n at p H 7 . 0 . The w a v e l e n g t h was set at 630 or 640 nm w h i c h is the long w a v e l e n g t h maximum c h a r a c t e r i s t i c of the ferriciniums (see Figure 5 for spectra of B H M F and B H M F ) . These plots are c h a r a c t e r i s t i c of first order k i n e t i c s and the h a l f - l i v e s are 0 . 5 0 , 4 . 3 and ^ 24 hours for F M C A + , F A A and B H M F + , r e s p e c t i v e l y . It i s i n t e r e s t i n g t h a t , w h e n a l l of the i o n s have been c o m p l e t e l y l o s t , 5 0 - 7 5 % of the i n i t i a l c o n c e n t r a t i o n c a n be r e g e n erated a g a i n by o x i d a t i v e e l e c t r o l y s i s . These r e s u l t s tend to support the interpretation by Penden, et a l . (20) that the f e r r i c i n i u m i o n undergoes a h y d r o l y s i s r e a c t i o n i n v o l v i n g a d i s p r o p o r t i o n a t i o n m e c h a n i s m . T h i s d i s p r o p o r t i o n a t i o n produces o n e - t h i r d ferric hydroxide and the r e m a i n d e r , the parent f e r r o c e n e . The h a l f - l i v e s for other ferriciniums are l i s t e d in Table III. Irrespec tive of the i n s t a b i l i t y of f e r r i c i n i u m i o n s , they c a n be employed as o x i d a t i v e M - T ' s if their E ° ' v a l u e s are s u f f i c i e n t l y p o s i t i v e of the biocomponent so that their e q u i l i b r i u m c o n c e n t r a t i o n r e mains r e l a t i v e l y s m a l l during the c o u l o m e t r i c t i t r a t i o n s . P o t e n t i o m e t r i c , voltammetric and spectra data for B H M F , F M C A and FAA (concentrations ca_. 10 mM) were a l s o obtained u s i n g the s p e c t r o e l e c t r o c h e m i c a l method at the transparent thin l a y e r c e l l u s i n g a g o l d minigrid e l e c t r o d e . The data are summarized i n Table III. The thin l a y e r e x p e r i m e n t a l procedures as d e s c r i b e d by H e i n e m a n (21) were a d o p t e d . The effect of the differing E ° ' v a l u e s of these three ferrocenes i s c l e a r l y i l l u s t r a t e d i n Figure 7 w h i c h shows the ΔΑ-q plots for the I C T of c y t o c (ca_. 20 μ Μ ) . The ΔΑ-q curve for e a c h titration p
+
+
+
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
156
ELECTROCHEMICAL
STUDIES O F B I O L O G I C A L S Y S T E M S
Ε , Volts vs. Ag/AgCl .6
400
500
600
700
.6
.4
.2
0
-^2
800
Wavelength (nm) Figure 5. (A) (left) Absorption spectra of the 1,Γ-bis(hydroxymethyl)ferrocene and its oxidized form, the ferricinium ion. (B) (right) Cyclic voltammetric i - E curves for l.OmM l,V-bis(hydroxymethyl)ferrocene at tin oxide OTE (top of figure). Area of electrode 0.8 cm (bottom of figure is for same compounds at Ft electrode, area of electrode ca. 2 cm ). Solution contains phosphate buffer at pH 7.0. 2
2
Figure 6. First order kinetic plots for the loss of the ferricinium ions. FMCA* = O ; FAA* = Q; BHMF* = ·. Concentrations were l-2mM in phosphate buffer at pH 7.0.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
9.
SZENTRIMAY E T AL.
Coulometric
Titration
of
157
Biocomponents
has b e e n n o r m a l i z e d to a common e n d - p o i n t (100% ùh) and charge scale (equivalents/mole). The data points at M greater than 100% represent the generation and subsequent r e m o v a l of e x c e s s BVt ( Ε ° ' = - 3 5 8 mV vs_ NHE) w h i c h was the e l e c t r o g e n e r a t e d reductant i n a l l three c a s e s . W i t h F M C A + as the oxidant ( Ε ° ' = +530 mV v s N H E ) , the /$-q plot i s n e a r l y l i n e a r s i n c e i t s E ° ' i s 275 mV more p o s i t i v e than that of c y t o c . W i t h B H M F , t h e ^ _ - q p l o t shows s l i g h t curvature as c y t o c_ becomes n e a r l y f u l l y o x i d i z e d . T h i s curvature r e f l e c t s the e q u i l i b r i u m between B H M F / B H M F ( Ε ° ' = 465 mV ys_ NHE) and c y t o C j Q / c y t o c ^ s i n c e the K (K = 1Q3.56) i r e l a t i v e l y s m a l l . The s i t u a t i o n becomes more a c c e n tuated w i t h FAA+/FAA ( Ε ° ' = 365 mV v s NHE) s i n c e the K is only ] Q1.86 There i s pronounced curvature a l o n g the entire ΔΑ-q p l o t . The s o l i d l i n e s through c a l c u l a t e d a s s u m i n g th good agreement between e x p e r i m e n t a l and c a l c u l a t e d ^A-q p l o t s g i v e c o n f i d e n c e of the attainment of redox e q u i l i b r i u m and that these ferrocenes do not i n h i b i t or interact c h e m i c a l l y w i t h c y t o c . +
+
X
e q
e q
S
e q
>
#
Similar data are presented i n Figure 8 for the q u a n t i t a t i v e ICT of cytochrome c o x i d a s e u s i n g BVt as reductant and B H M F as o x i d a n t . The s o l i d l i n e s i n t h i s figure are the computer s i m u l a t e d ΔΑ-q c u r v e s w h i c h were c a l c u l a t e d by a s s u m i n g the E ° ' v a l u e s of 35 0 mV ys_ N H E (high p o t e n t i a l c o p p e r - h e m e pair) and 210 mV v s N H E (low p o t e n t i a l c o p p e r - h e m e pair) (3). The e q u a l c o n t r i b u t i o n of e a c h heme to the t o t a l a b s o r b a n c e change at the monitoring w a v e l e n g t h of 604 nm w a s a s s u m e d . C o m p a r i s o n between the r e d u c t i v e and o x i d a t i v e ΔΑ-q c u r v e s show a s m a l l degree of h y s t e r e s i s w h i c h i s i n d i c a t i v e of some i r r e v e r s i b i l i t y . R e v e r s i b i l i t y had been p r e v i o u s l y reported for this enzyme u s i n g detergent s o l u b i l i z e d ferrocene w h i c h was e l e c t r o l y z e d to f e r r i c i n i u m i o n as an oxidant (16). H o w e v e r , both F M C A + and B H M F o x i d a t i o n s of c y t o c o x i d a s e have shown behavior v a r y i n g between near r e v e r s i b i l i t y to the type of a n i s o tropy shown i n Figure 8. The e x a c t nature of this a n i s o t r o p y i s not k n o w n . Numerous interpretations of the redox b e h a v i o r of c y t o c_ o x i d a s e have been proposed i n the literature i n c l u d i n g v a r i o u s states of o x i d i z e d and oxygenated o x i d a s e (22). Schroedel and H a r t z e l l (23) have r e c e n t l y interpreted these types of t i t r a tion c u r v e s to a redox m e c h a n i s m w h i c h r a t i o n a l i z e s difference between the r e d u c t i v e and o x i d a t i v e c u r v e s . +
+
The e l e c t r o g e n e r a t i o n of f e r r i c i n i u m ions or the c h e m i c a l o x i d a t i o n of ferrocenes to f e r r i c i n i u m s that are f a i r l y stable p r o v i d e s o x i d a n t s w h i c h p o s s e s s many of the properties of i d e a l M - T ' s . They w i l l greatly expand the a r s e n a l of o x i d a n t s w h i c h were p r e v i o u s l y l i m i t e d to few metal c y a n i d e s and metal c o m p l e x e s
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
158
ELECTROCHEMICAL
STUDIES O F B I O L O G I C A L
SYSTEMS
Figure 7. "Normalized change in absorbance vs. charge plots for the ICT of cytochrome c using benzylviologen and differ ent ferriciniums. Q, 25μΜ cytochrome c and 0.60 mM FAA; Δ , 20 Μ cytochrome c and 1.02 mM BHMF; ·, 21 Μ cyto chrome c and 0.75 mM FMCA. 1-2 mM benzylviologen using phosphate buffer at pH 7.0 (ionic strength 0.15). Solid lines draum through the experimental points are computer simulated curves for the experiment. μ
• 0
μ
I
I 4
I
I β
Equivalents / Oxidase
I
I 12
L
Figure 8. Normalized change in optical absorbance vs. charge plot for the ICT of cytochrome c oxidase. 15.6μΜ cytochrome c oxidase (100% ΔΑ = .375 a.u./cm); 0.33mM l,Y-bis(hydroxymethyl)ferrocene and l.OmM benzylviolo gen; phosphate buffer at pH 7.0 (ionic strength 0.15); cell parameters same as those in expenment shown in Figure 2. Solid lines are computer simulated curves assuming the oxidase to be η = 4 (see text for E ° ' values).
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
9.
Coulometric
szENTRiMAY E T A L .
Titration
of
159
Biocomponents
s u c h as those d e r i v e d from b i p y r i d y l l i g a n d s . Preliminary e x p e r i ments w i t h the o x i d a t i o n of tree l a c c a s e and the heme proteins i n the s u b m i t o c h o n d r i a l p a r t i c l e s u s i n g ferriciniums have produced r e s u l t s interprétable i n v i e w of previous works (10). Molybdenum Octacyanide In search of M - T ' s w i t h E ° ' v a l u e s much more p o s i t i v e than the commonly u s e d f e r r i c y a n i d e , molybdenum o c t a c y a n i d e ( M O ( C N ) Q / M O ( C N ) Q ) w a s e v a l u a t e d . It had been u t i l i z e d p r e v i o u s l y for potentiometric and k i n e t i c s t u d i e s of the l a c c a s e s (24, 25). The generation of M o f C N ) ^ as a c a t a l y t i c oxidant at m i c r o molar l e v e l s had a l r e a d y been reported u s i n g v a r i o u s e l e c t r o d e s (26 2 7 , 28). The commonly u s e d t i n oxide O T E i n our laboratory was thought to be superio these p o t e n t i a l s s i n c e tin oxide has a h i g h o v e r p o t e n t i a l and has a surface that i s a l r e a d y o x i d i z e d . L a i t i n e n and C o n l e y (29) have reported the quantitative generation of Ag(II) w i t h the current e f f i c i e n c i e s being higher at this e l e c t r o d e compared to either Pt or A u . Experimental care must be taken i n h a n d l i n g molybdenum o c t a c y a n i d e due to its p h o t o s e n s i t i v i t y (30). 4
3
3
f
A t y p i c a l c y c l i c i - E curve for 10.6 m M M O ( C N ) Q u s i n g a t i n oxide thin layer s p e c t r o e l e c t r o c h e m i c a l c e l l i s shown i n Figure 9 . The shape of the i - E curve i s c h a r a c t e r i s t i c of a r e v e r s i b l e thin l a y e r e l e c t r o c h e m i c a l s y s t e m with u n c o m p e n s a t e d iR d r o p . * Spectra obtained c o n c u r r e n t l y during p o t e n t i a l - s t e p e l e c t r o l y s i s of the M O ( C N ) Q 4 , a c c o r d i n g to the method of H e i n e m a n , et a l . (21) are a l s o presented i n Figure 9. If a n e r n s t i a n plot of the a p p l i e d p o t e n t i a l ( E p i ) v e r s u s the logarithmic ratio of the o x i d i z e d to r e d u c e d forms of the molybdenum o c t a c y a n i d e as determined by the change i n the o p t i c a l a b s o r b a n c e at 388 n m , are p l o t t e d , a l i n e a r l i n e r e s u l t s . The average E ° ' determined from four s u c h experiments gave a v a l u e of +798 + 3 mV v s - N H E . T h i s v a l u e compares w e l l w i t h those p r e v i o u s l y reported at the same i o n i c strength (30, 31). The η v a l u e c a l c u l a t e d from the s l o p e of the plots i s 1 . 0 0 + . 0 2 . T h u s , the molybdenum o c t a c y a n i d e appeared as a good o x i d i z i n g M - T (See Figure 10). 4
a p
* T h i s i s confirmed by the fact that E ° ' , c a l c u l a t e d as E ° ' = E + E p / 2 , w a s found to be independent of s c a n r a t e . Here E and E p are the a n o d i c and c a t h o d i c peak p o t e n t i a l s for the c y c l i c voltammograms. p
C
p
C
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
a
a
ELECTROCHEMICAL
STUDIES O F B I O L O G I C A L S Y S T E M S
Wavelength (nm) Figure 9. Thin layer spectroelectrochemical data for molybde num octacyanide. (upper right) Cyclic voltammetric i—Ε curve for Mo(CN) ~ ' in a tin oxide thin hyer spectroelectrochemical cell, (left) Spectra obtained during incremental addition of charge for oxidation of Mo(CN) ~*; 10.63mM molybdenum octa cyanide in 0.50M NaCl, phosphate buffer at pH 7.0. The wavy baseline on the spectrum is from the tin oxide electrode (inter ference pattern). 8
3/ 4
8
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
9.
SZENTRIMAY E T AL.
Coulometric
Titration
of
Biocomponents
161
Our i n i t i a l test of molybdenum o c t a c y a n i d e i n a M - T v e r s u s M - T c o u l o m e t r i c titration f a i l e d when BV++ was u s e d as the other M - T b e c a u s e p r e c i p i t a t i o n occurred as BVt was e l e c t r o g e n e r a t e d . After e x a m i n i n g v a r i o u s other p o s s i b l e r e d u c t a n t s , the e l e c t r o r e d u c t i o n of a n t h r a q u i n o n e - 2 - s u l f o n a t e at tin oxide O T E proved to be c o m p a t i b l e w i t h the molybdenum o c t a c y a n i d e . U s i n g the above M - T ' s , the I C T q u a n t i t a t i o n w a s a c c o m p l i s h e d for c y t o c_ (Figure 11). The r e s u l t s from four r e d u c t i v e - o x i d a t i v e c y c l e s gave an average η v a l u e of 1.07 + 0 . 0 4 (uncorrected, about 4% background c o n t r i b u t i o n ) . The s e c o n d ΔΑ-q curve shown i n Figure 11 i s for the r e d u c t i v e and o x i d a t i v e titration of c y t o c_ o x i d a s e . The shape of this ΔΑ-q curve i s c o n s i d e r a b l y different from that obtained for o x i d a s e titrated by BVt and B H M F (see Figure 8). It i s e v i d e n c e of either finite c o m p l e x a t i o n by d i s s o c i a t e d c y a n i d e i o n or by M O ( C N ) Q ^ of c y t o c_ o x i d a s e o n l y p a r t i a l l y a c t i v e toward o x y g e n . T h u s , there appears to be s e r i o u s i n h i b i t i o n produced by the p r e s e n c e of the molybdenum o c t a c y a n i d e to cytochrome c_ o x i d a s e . Irrespective of the previous r e s u l t s for the u s e of molybdenum o c t a c y a n i d e as a n oxidant for the c h e m i c a l titration of l a c c a s e s (24), the a b s e n c e of i n h i b i t i o n or i n t e r a c t i o n by this compound to the redox states of l a c c a s e s t i l l remains to be p r o v e n . Part of the s u c c e s s i n u s i n g the molybdenum o c t a c y a n i d e i n the l a c c a s e titrations may be due to the lower c o n c e n t r a t i o n s (less than 40 μ Μ ) e m p l o y e d . However, these ICT r e s u l t s should be i n d i c a t i v e of the p r e c a u t i o n s w h i c h must be taken i n the u s e of M - T ' s for b i o c o m p o n e n t s , p a r t i c u l a r l y those that have c o m p l e x i n g l i g a n d s w h i c h c a n be s l o w l y and i r r e v e r s i b l y d i s s o c i a t e d and then taken up by the biocomponent(s). +
The molybdenum o c t a c y a n i d e titration of c y t o c_ o x i d a s e s e r v e s to e m p h a s i z e the importance of performing multiple I C T ' s u s i n g s e v e r a l M - T ' s for c o n f i r m a t i o n of η and E ° ' r e s u l t s . Other M - T ' s Our s e a r c h and c h a r a c t e r i z a t i o n of p o s s i b l e M - T ' s are s t i l l far from being c o m p l e t e d . Some 60 redox compounds have been i d e n t i f i e d now as p o s s i b l e M - T ' s and c h a r a c t e r i z a t i o n of these and many more i s a n t i c i p a t e d for future w o r k . Table IV l i s t s s e v e r a l compounds w h i c h h a v e been reported and u s e d as m e d i a tors or those w h i c h have been g i v e n p r e l i m i n a r y s c r e e n i n g i n our laboratory and may prove to be u s e f u l . For e x a m p l e , the 2 , 2 ' b i p y r i d y l c o m p l e x e s of r u t h e n i u m , iron and osmium were examined b e c a u s e of their v e r y p o s i t i v e formal p o t e n t i a l s . H o w e v e r , the r e d u c e d forms of these metal c o m p l e x e s are h i g h l y c o l o r e d i n the v i s i b l e r e g i o n of the spectrum (see Table IV for Xmax Çdata) a
n
d
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
0 ,
371 (366)
418 (415)
798 (774)
844 (877)
1,074 (1,096)
#
3 6
45
44
43
41
3 6
1,107 (1 1 2 0 )
3 4
a
NHE) '
+ 1,272 (1,274)
E (mV,
b
/
N,N-Dimethyl-pphenylenediamine 2
1
1
Molybdenum o c t a cyanide
Iron h e x a c y a n i d e
1
1
1
1
p
63
55
61
58
55
60
η 4E (mV)
Osmium t r i s - ( 2 , 2 ' bipyridine)
Iron t r i s - ( 2 , 2 ' bipyridine)
Iron t r i s - ( l , 1 0 phenanthroline)
,
Ruthenium t r i s (2 2 -bipyridine)
Compound b
3 7
475 (44 7 ) 4 1
518 (522)39
507 (510)
450 (445)35
_1
1
M" )
3 7
13,800 (13,700)
4 2
8,800 (8,650)39
11,000 (ΙΙ,ΙΟΟ)
16,000 (14,600)35
€(cm
Reduced Form
Redox Compounds U s e f u l as M e d i a t o r - t i t r a n t s
TABLE IV
9
550 (550)
418
388
(610)
5 4
4 0
(590)38
(418)35
_1
M
350
1,140
1,365
(330)
(600)
)
4 0
3 8
3 5
_ 1
(δ,ΙΟΟ)
€(cm
O x i d i z e d Form a
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
-10 (-53)
49
5 0
-3 (+33)
Pyocyanine
5-Hydroxy-l , 4 naphthoquinone
Phenazine m e t h o sulphate
92 (80)
2
2 ,6-Dichlorophenolindophenol
2
2
2
2
2
2,3,4,5-Tetramethylp-phenylenediamine 40
60
70
85
80
100
p
η 4E (mV)
Ν , Ν , Ν ' , Ν'-Tetramethylp-phenylenediamine 2
Compound
1,2 - N a p h t h o q u i n o n e
b
157
(227)
2 1
4 7
4 5
4 8
270 (2 7 0 )
a
NHE) '
257 (240)
E°'(mV, b
Reduced Form
Redox C o m p o u n d s U s e f u l a s M e d i a t o r - t i t r a n t s
TABLE IV ( C O N T I N U E D )
682
420
430
405
(600)
473 (480)
560 (565)
2 1
5 4
4 6
1
4,090
2,400
6,860
2,700
(2,060)
370
2 1
4 6
1
M" )
12,000 (12,470)
Çfcm'
O x i d i z e d Form 9
CD
4
I—« Œ> CO
Co
s
ο
3
ta ο ο
θ"
!'
Ε
ο
Ο ο
M H
a
Ν M
C/Î
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
5 1
'
'
b
c
c
2
2 2
2-Amino-1,4naphthoquinone
Anthraquinone - 2 sulphonate
2 -Amino-4 -pteridone
s are i n p a r e n t h e s e s ,
2
b
m a x
450
X
(nm) a
l
1,500
e(cm~
O x i d i z e d Form
c
V
Q
u
e
s
o
f
1
M^ )
r e f . (51) gave Ε 1/2 l - 0 . 3 0 and - 0 . 90 v o l t w ith r e s p e c t to S C E for 2 -• a m i n o - 6 , 7 - d i h y d r o - 4 pteridone and 2 - a m i n o - 4 - p t e r i d o n e , respe c t r / e l y , i n \ DH 9 borate buffer.
(pH 7. 0 , i o n i c strength 0.15)
135
p
η AE (mV)
2-Amino-6,7-dihydro4-pteridone
Compound
^supporting e l e c trolyte u s e d i s phosphate buiEfer
^literature v a l u e
5 1
5 8
5 2
(-660)
(-225)
-133 (-137)
(-60)
a
E°'(mV,NHE) '
Reduced Form
Redox Compounds U s e f u l a s M e d i a t o r - t i t r a n t s
TABLE IV ( C O N T I N U E D )
9
C/3 KJ C/5
>
Ω
ο ο
W
ο
Μ
α
Η ci
> f
Μ
g
ο η
w
H—»
SZENTRIMAY E T A L .
Coulometric
_J
ι
Titration
of
I
Biocomponents
ι
L_
Figure 10. Plot of the applied potential (E ned) vs. the optically determined logarithm of concentration ratio of oxidized to reduced molybdenum octacyanide. Experimental conditions same as those in Figure 9. apv
Cytochrome c
Oxidase
Equivalents/Mole
Enzyme
Figure 11. Normalized plots of optical absorbance vs. charge for the ICT of cytochrome c and cytochrome c oxidase with molybdenum octacyanide and anthraquinone 2-sulfonic acid, (left) ICT of cytochrome c (22μΜ); (right) ICT of cytochrome c oxidase (12μΜ). M-T's of LOmM Mo(CN) ~ and 1.3mM anthra quinone 2-sulfonic acid. 13μΜ phenazine methosulfate added to the oxidase solution to insure equilibrium. 8
3
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
166
ELECTROCHEMICAL
STUDIES O F B I O L O G I C A L
SYSTEMS
and may c a u s e problems for o p t i c a l monitoring of b i o c o m p o n e n t s . The o x i d i z e d forms of these c o m p l e x e s may be q u a n t i t a t i v e l y generated at the t i n oxide Ο TE and M - T y s ^ M - T I C T s have been performed u s i n g b e n z y l v i o l o g e n as the other M - T w i t h s a t i s f a c tory r e s u l t s . H o w e v e r , o x i d a t i v e I C T ' s of r e d u c e d c y t o c w i t h these M - T ' s e x h i b i t e d d r a w n - o u t ΔΑ-q c u r v e s i n d i c a t i v e of o x i d a t i o n of more than the iron heme m o i e t y . It i s i n t e r e s t i n g to note that the p r e v i o u s l y d i s c u s s e d molybdenum c y a n i d e gave q u a n t i tative η = 1 titration of c y t o c w h i c h s u g g e s t s that o x i d a t i o n of other parts of c y t o c_ o c c u r s at p o t e n t i a l s above ca_. +800 mV. The s u b s t i t u t e d p - p h e n y l e n e d i a m i n e s have been p r e v i o u s l y e m p l o y e d as mediators and M a c k e y (4) demonstrated the q u a n t i t a tive e l e c t r o g e n e r a t i o n of the d i i m i n e i n the c a s e of t e t r a - m e t h y l p - p h e n y l e n e d i a m i n e ( T M P D ) . He a l s o obtained good r e s u l t s for the I C T of c y t o c_ o x i d a s e u s i n p h e n o l h a s been examined thoroughly i n the O T T L E c e l l and u s e d for potentiometry of c y t o c_ by Heineman (21). Although o n l y a few naphthaquinones appear i n the t a b l e , s e v e r a l other n a p h t h a q u i n o n e s , p a r t i c u l a r l y those s u b s t i t u t e d a p p r o p r i a t e l y for greater water s o l u b i l i t y , are b e i n g c h a r a c t e r i z e d . These naphthaquinones w i l l serve as M - T ' s i n the p o t e n t i a l range of +100 to +200 mV. Phenazine m e t h o - and e t h o - s u l f a t e are w e l l known mediators w h i c h have been w i d e l y u s e d i n potentiometric titrations of b i o c o m p o n e n t s . The pteridones h a v e been s u g g e s t e d for c o u p l i n g to N A D r e d u c t i o n by Kwee and Lund (33). W e hope that further work w i l l be forthcoming from their laboratory u t i l i z i n g these p t e r i d o n e s . 1
+
Acknowledgement The f i n a n c i a l support provided by NSF Grant MPS 73-04882 and N I H - P H S Grant N o . G M 19181 i s gratefully a c k n o w l e d g e d .
Literature Cited 1. Hawkridge, Fred and Kuwana, Theodore, Anal. Chem., (1973) 45, 1021. 2. Heineman, William and Kuwana, Theodore, Acc. Chem. Res., (1976) 9, 241. 3. Heineman, William and Kuwana, Theodore, Biochem. Biophys. Res. Commun., (1973) 50, 892. 4. Mackey, L.N., Kuwana, T., and Hartzell, C.R., FEBS Lett., (1973) 36, 326. 5. Rodkey, F.L. and Donovan, J.A. Jr., J. Biol. Chem., (1959) 234, 677.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
9.
SZENTRIMAY ET AL.
Coulometric Titration of Biocomponents
167
6. Thevenot D. and Leduc, P., 3rd International Symposium on Bioelectrochemistry, Juelich (1975). 7. Ke, B. and Hawkridge, F.M., unpublished results. 8. Steckan, Eberhard and Kuwana, Theodore, Ber. Bunsenges. Phys. Chem., (1974) 78, 253. 9. Mackey, L.N. and Kuwana, Theodore, 3rd International Symposium on Bioelectrochemistry, Juelich (1975). 10. Szentrimay, Robert, (1976) Ph.D. Thesis, Ohio State University. 11. Hennig, Horst and Gürtler, Oswald, J. Organometal. Chem., (1968) 11, 307. 12. Mason, J.G. and Rosenblum, Myron, J. Am. Chem. Soc. (1960) 82, 4206. 13. Gubin, S.P. and Perevalova (1962) 143, 1351 14. Perevalova, E.G., Gubin, S.P., Smirnova, S.A. and Nesmeyanov, A.N., Dokl. Akad. Nauk. SSSR (1964) 155, 857. 15. Yu, C.A. and Yu, Linda, Biochem. Biophys. Res. Commun., (1976) 70, 1115. 16. Fujihira, Υ., Kuwana, T. and Hartzell, C.R., Biochem. Biophys. Res. Commun., (1974) 61, 488. 17. Yeh, P. and Kuwana, T., J. Electrochem. Soc., (1976) 123, 1334. 18. Penden, Α.Α., Leont'evskaya, P.K., L'vova, T.I. and Nikolskii, B.P., Dokl. Akad. Nauk. SSSR, (1969) 189, 115. 19. Strojek, J.W. and Kuwana, T., Electroanalytical Chemistry and Interfacial Electrochemistry, (1968) 16, 471. 20. Penden, Α.Α., Zakharevskii, M.S. and Leont'evskaya, P.K., Kinetika: Kataliz, (1966) 7, 1074. 21. Heineman, W.R., Norris, B.J. and Goelz, J.F., Anal. Chem., (1975) 47, 79. 22. Caughey, W.S., Wallace, W.J., Volpe, J.A. and Yoshikawa, S., in "The Enzymes" (P.D. Boyer ed.) Volume XIII Part C, p. 299, Academic Press, New York, 1976. 23. Schroedel, Nancy, (1976) Ph.D. Thesis, The Pennsylvania State University. 24. Reinhammar, Bengt R. M., Biochimica et Biophysica Acta, (1972) 275, 245. 25. Pecht, Israel, Israel Journal of Chemistry, (1974) 12, 351. 26. Mendez, Hernandez and Lucenta, F., An. Quim, (1968) 64, 71. 27. Mendz, Hernandez, J. Acta Salmanticensia, Cienc (19671968) 33, 41.
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28. Cordova-Orellana, Lucena-Conde, F., Talanta (1971) 18 505. 29. Laitinen, H.A. and Conley, J.M., Anal. Chem., (1976) 48, 1224. 30. Kolthoff, I.M. and Tomsicek, Wm. J., J. Phys. Chem., (1936 40, 247. 31. Malik, W. andAli,S.I., Indian J. Chem., (1963) 1, 374. 32. Nickolls, P. and Chance, Β., in "Molecular Mechanisms of Oxygen Activation", (O. Hayaishi, ed.) p. 479 Academic Press, New York 1974. 33. Kwee, S. and Lund, Η., Bioelectrochemistry and Bioenerget i c s , (1975) 1, 137. 34. Schilt, Α.Α., Anal. Chem., (1963) 35, 1599. 35. Miller, R.R., Brandt Soc., (1955) 77, 3178 36. Dwyer, F.P. and McKenzie, H.A., J. Proc. Roy. Soc. N.S. Wales, (1947) 81, 93. 37. Fortune, W.B. and Mellon, M.G., Ind. Eng. Chem., Anal. Ed., (1938) 10, 60. 38. Harvey, A.E. and Manning, D.L., J. Am. Chem. Soc., (1952) 74, 4744. 39. Moss, M.L. and Mellon, M.G., Ind. Eng. Chem., Anal. Ed., (1942) 14, 862. 40. Schilt, Α.Α., "Analytical Applications of 1,10-Phenanthroline and Related Compounds", Pergamon Press, New York, (1969). 41. Dywer, F.P., Gibson, N.A. and Gyarfas, E.C., J. Proc. Roy. Soc. N.S. Wales, (1942) 84, 80. 42. Burstall, F.H., Dwyer, F.P. and Gyarfas, E.C., J. Chem. Soc., (1950), 953. 43. (a) Volke, J., Collect. Czechoslov. Chem. Commun., (1968) 33, 3044. (b) Volke, J. and Volkova, V., Collect. Czechoslov. Chem. Commun., (1969) 34, 2037. 44. Kolthoff, I.M. and Tomsicek, W.J., J. Phy. Chem., (1935) 39, 945. 45. Michaelis, L. and H i l l , E.S., J. Am. Chem. Soc., (1933) 55, 1481. 46. Albrecht, A.C. and Simpson, W.T., J. Am. Chem. Soc., (1955) 77, 4455. 47. Dutton, P.L., Wilson, D.F. and Lee, C.P., Biochem., (1970) 9, 5077. 48. Dickens, F. and McIlwain, H., Biochem. J., (1938) 32, 1615.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
9. SZENTRIMAY ET AL.
Coulometric Titration of Biocomponents
169
49. Friedheim, E.A.H., Biochem. J., (1934) 28, 180. 50. Friedheim, Ε.A.H. and Michaelis, L., J. Biol. Chem. (1931) 91, 355. 51. Kwee, S. and Lund, H., Biochim. Biophys. Acta, (1973) 297, 285. 52. Fieser, L.F. and Fieser, M., J. Am. Chem. Soc., (1934) 56, 1565. 53. Conaut, J.B., Kahn, H.M., Fieser, L.F. and Kurtz, S.S., J. Am. Chem. Soc., (1922) 44, 1382. 54. Michaelis, L., Schubert, M.P. and Granick, S., J. Am. Chem. Soc., (1939) 61, 1981.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
10 Rotating Ring Disk Enzyme Electrode for Biocatalysis Studies 1
RALPH A. KAMIN, FRANK R. SHU, and GEORGE S. WILSON Department of Chemistry, University of Arizona, Tucson, Ariz. 85721
In recent years there has been considerable interest i n catalytic surface reaction interest. This has bee development of immobilized enzyme technology (1,2) and electrochemical sensors based on electroactive product formation within an enzyme layer (3,4). In comparing the kinetic behavior of an immobilized enzyme with i t s soluble counterpart, it is necessary to establish that the overall reaction rate i s catalysis rather than mass transport limited. It has been shown, for example, that immobilized enzymes i n flowing streams give apparent Michaelis constants K ', that are flow rate dependent (5). Under conditions where the overall reaction i s limited by mass-transport supply of substrate to the catalytic surface, K ' i s larger than expected. One i s then tempted to conclude that the properties of the enzyme have been modified by immobilization. On the contrary, increasing flow (mass transport) rates may lead to a limiting value for K ' essentially identical to that of the soluble enzyme (6). The rotating disk electrode as described by Levich (7) appears to offer an experimentally facile means for varying the rate of substrate mass transport. The addition of a concentric ring (rotating ring disk electrode) (8) permits independent monitoring of the reaction at the disk surface. We have recently (9) derived the theory describing the response of the rotating disk enzyme electrode. In the present work we report further experimental studies in support of this theoretical model. The system selected for study i s the glucose/glucose oxidase reaction: M
M
M
Glucose + 0 2 o
l u c
s e
g ° ) oxidase
Gluconic Acid + H 0 22 o
o
(1)
The peroxide produced i s either monitored directly or coupled 1
Present address: Smith-Kline Instruments, 880 W. Maude Ave., Sunnyvale, CA 94086 170
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
10.
KAMIN
ET
Rotating
AL.
Ring
Disk Enzyme
171
Electrode
with the i n d i c a t o r r e a c t i o n : H 0 2
2
+ 2H
+
+ 2Γ
m
o
l
r
b
d
a
t
e
>
I
2
+ 2H 0 2
(2)
Experimental Instrumentation, The f our^ e l e c t r o d e p o t e n t i o s t a t used i n these s t u d i e s was s i m i l a r to that described by Shabrang and Bruckenstein (10). The r o t a t i n g d i s k e l e c t r o d e , Model DT-6, was purchased from the Pine Instrument Co., Grove C i t y , PA. The d i s k was a 0.5 cm deep c a v i t y with a radius of 0.382 cm ac cording to the manufacturer's s p e c i f i c a t i o n s . When f i l l e d with carbon paste the c a l c u l a t e d d i s k area was 0.46 cm^. The width of the platinum r i n g e l e c t r o d e was 0.024 cm and was separated from the d i s k by a 0.01 e f f i c i e n c y measured experimentall paste e l e c t r o d e (8) was 0.18 and was i n good agreement with experimental r e s u l t s . A platinum wire counter e l e c t r o d e and a Ag/AgCl reference e l e c t r o d e ( E = 0.200 v) were employed. A Pine Instruments Model PIR r o t a t o r was used to c o n t r o l e l e c trode r o t a t i o n speed. o f
Preparation of Glucose Oxidase E l e c t r o d e . The carbon paste was prepared i n the usual manner from 5 g of graphite powder #38 ( F i s h e r S c i e n t i f i c Co.) and 3 ml of Nujol except that 10 mg (except where otherwise s p e c i f i e d ) of n-octadecylamine ( t e c h n i c a l grade, A l d r i c h ) was a l s o added. The carbon paste was packed f i r m l y i n t o the d i s k c a v i t y of the DT-6 e l e c t r o d e which was then p o l i s h e d with a piece of weighing paper. A f t e r the r i n g and gap were c a r e f u l l y cleaned, the e l e c t r o d e was allowed to r o t a t e i n a 12.5% glutaraldehyde s o l u t i o n f o r 10 - 15 min. followed by a 1 minute washing with c o l d 0.2M phosphate b u f f e r pH 6.5. (Glutaraldehyde must be f r e s h l y p u r i f i e d and s t o r e d below 0°C as i t r e a d i l y polymerizes (11))· The r o t a t i n g e l e c t r o d e was dipped i n t o a bovine serum albumin s o l u t i o n (0.1 g/ml) (BSA F r a c t i o n V 96-99%, Sigma Co.). After 2 - 3 minutes the e l e c trode was washed f o r 1 minute i n c o l d phosphate b u f f e r . The e l e c t r o d e was then removed from the r o t a t o r and p o s i t i o n e d with the e l e c t r o d e surface f a c i n g up. A glucose oxidase s o l u t i o n prepared by d i s s o l v i n g 0.3 g of the enzyme (Glucose Oxidase E.C. 1.1.3.4 Sigma Type II 15,000 units/g) i n 1 ml. of 5% g l u t a r a l dehyde s o l u t i o n (buffered with phosphate at pH 6.5) was a p p l i e d to the d i s k s u r f a c e . A f t e r standing at room temperature f o r 5 min., the excess enzyme s o l u t i o n was discarded and the gap and r i n g were c a r e f u l l y cleaned. Rotating the e l e c t r o d e i n c o l d phosphate b u f f e r at 2500 rpm f o r 5 min. aids i n removing phys i c a l l y entrapped or weakly bonded enzyme. When not i n use the e l e c t r o d e was stored i n phosphate b u f f e r at 5°C.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
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STUDIES O F
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S o l u t i o n s and Reagents Unless otherwise mentioned, a l l chemicals used were reagent grade. The stock s o l u t i o n of 0.1 M glucose was allowed to mutar o t a t e at room temperature f o r at l e a s t 24 hr. before using. When the course of the r e a c t i o n was measured by f o l l o w i n g I2 formation (Reaction 2) a K I - b u f f e r c a t a l y s t described p r e v i o u s l y (12) was employed. Where d i r e c t monitoring of peroxide format i o n (Reaction 1) i s p o s s i b l e the glucose i s d i s s o l v e d i n a 0.05 M phosphate b u f f e r pH 6.5. Procedure. The enzyme e l e c t r o d e was allowed to r o t a t e f o r about 30 sec. i n the glucose s o l u t i o n at which time a p o t e n t i a l was a p p l i e d to the r e s p e c t i v e i n d i c a t i n g e l e c t r o d e . The i o d i n e formed i n Reaction 2 was monitored at the d i s k by applying a p o t e n t i a l of -0.2 V v mation (no i o d i d e present by h o l d i n g the p o t e n t i a l at -0.2 V followed by a step to 0.75 V at which p o i n t the current t r a n s i e n t was measured. The p o t e n t i a l was then returned to -0.2 V u n t i l the next measurement. Enzyme E l e c t r o d e T h e o r e t i c a l Model. The d e t a i l s of the d i g i t a l s i m u l a t i o n c a l c u l a t i o n s f o r t h i s e l e c t r o d e have been presented elsewhere (9). Our model assumes the existence of an enzyme l a y e r extending i n t o s o l u t i o n from the e l e c t r o d e surface (X=0). This uniformly d i s t r i b u t e d t h i n enzyme l a y e r i s assumed not to i n t e r f e r e with d i f f u s i o n of species to or from the e l e c t r o d e s u r f a c e . The enzyme l a y e r l i e s w i t h i n the minimum hydrodynamic l a y e r j u s t i f y i n g the assumption that s o l u t i o n flow i n the e l e c t r o d e v i c i n i t y i s a l s o unaffected by the immobilization process. Michaelis-Menten k i n e t i c theory i s assumed to describe the enzymatic r e a c t i o n . Figure 1 i l l u s t r a t e s the nature of the concentration gradi e n t s at the e l e c t r o d e surface f o r a p a r t i c u l a r set of condit i o n s . The steady s t a t e product (or coupled product) concentrat i o n gradient i s f i r s t simulated f o r the r o t a t i n g e l e c t r o d e at open c i r c u i t . Product concentration increases as substrate pene t r a t e s the enzyme l a y e r from the s o l u t i o n s i d e . I f a p o t e n t i a l i s a p p l i e d to the d i s k i n a region where the product i s e l e c t r o a c t i v e , i t s concentration at the e l e c t r o d e surface drops to zero. E v e n t u a l l y the steady-state c o n d i t i o n shown i n Figure 1 i s a t t a i n e d . I t w i l l be noted that the concentrations i n the outer p o r t i o n of the enzyme l a y e r are r e l a t i v e l y unaffected by the pot e n t i a l perturbation. The r a t e of product formation i s given by Michaelis-Menten theory d
[P] dt
k C /(K /[S]+l) 3
E
M
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
(3)
10.
Rotating
KAMiN E T A L .
Ring
Disk Enzyme
173
Electrode
where i s the rate constant f o r the i r r e v e r s i b l e conversion of the enzyme-substrate complex i n t o products and K^. the M i c h a e l i s constant. C^, i n t h i s case i s the a n a l y t i c a l concentration of a c t i v e enzyme i n the immobilized l a y e r and S the substrate con c e n t r a t i o n i n the enzyme l a y e r . In order to evaluate the r e l a t i v e e f f e c t s of c a t a l y s i s and convective mass transport a r e a c t i o n v e l o c i t y parameter, V, i s defined: = W k ^ I
v
(
4
)
The convection time constant, t ^ , has been derived p r e v i o u s l y by P r a t e r and Bard (13) and i s given by t
k
= (0.51)-
2 where ν i s the kinematic v i s c o s i t y (cm /sec) and ω the r o t a t i o n speed i n rad/sec. For a given enzyme e l e c t r o d e , V r e f l e c t s the amount of product formed i n a given time and i s dependent only on ω, to which i t i s i n v e r s e l y p r o p o r t i o n a l . For l a r g e values of V e.g. V > 10 the c a t a l y s i s rate i s extremely f a s t and the o v e r a l l r e a c t i o n becomes convection mass transport l i m i t e d . For V < 0.1 the enzymatic r e a c t i o n i s c a t a l y s i s r a t e l i m i t e d . Thus, by v a r y i n g the e l e c t r o d e r o t a t i o n speed, the f l u x of sub s t r a t e can be modulated to change the nature of the r a t e l i m i t i n g process. The r a t i o C/K^ where C i s the bulk substrate con c e n t r a t i o n a l s o serves to define the current response. We have a l s o shown (9) that an optimal r o t a t i o n speed f o r current mea surement w i l l r e s u l t from increased substrate mass transport on one hand and decreased product production due to short contact time with the c a t a l y t i c l a y e r on the other. The steady s t a t e current r e l a t i o n s h i p s are presented below: Case I - Mass Transport Limited Rate (V > 10) From s i m u l a t i o n i t can be shown (by analogy to a L i n e weave r-Burk p l o t (14)):
nFAdk C 3
1/2 E
k
2
" d ~
1.22
D t
- k_
C
t
3 E k C
(6)
where i i s the steady s t a t e current at the d i s k , d i s the en zyme l a y e r thickness; b i s a f u n c t i o n only of ω and D. A l l other parameters have the usual e l e c t r o c h e m i c a l s i g n i f i c a n c e . At low substrate concentrations the f i r s t term of Equation 6 i s much greater than b and the steady-state current becomes 0.65nFAD V 2 /
1 / 6
U)
1 / 2
C
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
(7)
174
ELECTROCHEMICAL
STUDIES O F
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which i s p r a c t i c a l l y i d e n t i c a l to the L e v i c h equation (7) f o r a r o t a t i n g d i s k e l e c t r o d e as expected. Case I I - C a t a l y s i s Limited Rate (V<0.1) An expression analogous to Equation 6 can be derived f o r t h i s case:
s For high substrate concentrations written i
max
( 0 » Κ ^ ) Equation
8 can
= i nFAdk^ b 3 Ε
be
(9)
I t can be seen that a the r e l a t i o n s h i p "Snax i
*Sl C
+ 1
w i l l y i e l d the M i c h a e l i s
(10)
constant.
C h a r a c t e r i s t i c s of the Immobilized Enzyme Layer Figure 2 gives a rough schematic r e p r e s e n t a t i o n of the im m o b i l i z e d l a y e r . The presence of the amine d i s s o l v e d i n the N u j o l and/or adsorbed on the graphite p a r t i c l e s appears to be e s s e n t i a l to the formation of a s t a b l e enzyme l a y e r of high biological activity. I f product formation i s to be monitored at the d i s k then an optimal amount of amine must be used. I f the concentration i s too low, then i n s u f f i c i e n t coupling s i t e s are a v a i l a b l e whereas too much amine causes current suppression. At the amine l e v e l suggested above the e l e c t r o c h e m i s t r y of I ( c y c l i c voltammetry and RDE l i m i t i n g currents) are v i r t u a l l y i d e n t i c a l i n the presence or absence of the amine i n the c a r bon paste. Based on the L e v i c h Equation (7) the e f f e c t i v e e l e c t r o d e area i s approximately 50% of the p r o j e c t e d area sug g e s t i n g that about h a l f of the graphite i s i n contact with the s o l u t i o n at the e l e c t r o d e s u r f a c e . The BSA added i n the next step a l s o appears to be e s s e n t i a l to enzyme l a y e r s t a b i l i t y . It probably functions along with the glutaraldehyde as a m u l t i f u n c t i o n a l c r o s s - l i n k i n g reagent. L i m i t i n g d i s k currents f o r I3 r e d u c t i o n are i d e n t i c a l f o r a pure carbon paste e l e c t r o d e and an enzyme e l e c t r o d e (no substrate present) suggesting that the immobilization process does not i n t e r f e r e with d i f f u s i o n c o n t r o l l e d reduction of the e l e c t r o a c t i v e s p e c i e s . L i t t l e i s known at present about the micro environment of the immobilized enzyme a c t i v e s i t e . The pH optimum i s s h i f t e d
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
10.
Rotating Ring Disk Enzyme Electrode
ΚΑ M I N E T A L .
ENZ. LAYER
RELATIVE
R -
NH
Figure 1. Simuhtion of steady state product concentration gradients conditions CKM — 3.334, V = 0.0113, d = 0.007 cm, αηήω = 1600 rpm. ( ), disk profile at open circuit; ( j, profile for disk electrode polanzed at product potential.
THICKNESS
H H R -|— Ν = C - ( C H ) C=0 2
2
3
NH NH 9
H H Ν = C-(CH ) C=0 2
BSA
3
t NH
Η Η Ν = C(CH ) C = Ν 2
0
3
9
I H H R 4 - Ν = C-(CH ) C = Ν
I
2
h NH
H H 0=C-(CH ) C=0
3
ON —1
3
H H • N=C(CH ) C=N — j Z 2
Figure 2.
3
0
2
2
0
0
| — N=C(CH ) Ν = C(CH ) C=
- NH
3
0
2
NH
R «
3
H H 0= C-(CH ) C=0
NH
R -
HYOROO LAYER
0
+
R -
175
3
Scheme for enzyme layer formation
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
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ELECTROCHEMICAL
STUDIES O F
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SYSTEMS
to about 6.5 which i s around one pH u n i t higher than the s o l uble enzyme (6). Such a l k a l i n e s h i f t s may be caused by a r e duction of p o s i t i v e charge on the enzyme due to the coupling process. This would occur as p o s i t i v e l y charged ε-amino l y s y l or guanido groups react with the glutaraldehyde (15). The M i c h a e l i s constant might be a f f e c t e d i n an analogous manner by the e l e c t r o s t a t i c p o t e n t i a l of the a c t i v e s i t e microenvironment f o r cases of charged s u b s t r a t e s . The p o s s i b i l i t y of vary ing t h i s p o t e n t i a l by changing the p o t e n t i a l a p p l i e d to the d i s k i s an a t t r a c t i v e one, e s p e c i a l l y i n view of present e l e c trochemical methodology. When p r o p e r l y immobilized, a s t a b l e c a t a l y t i c l a y e r i s ob t a i n e d . A f t e r an i n i t i a l decay i n a c t i v i t y , probably due to non-bonded enzyme, the a c t i v i t y decays slowly. We have ob served only 15% decay a f t e r 30 days and have used e l e c t r o d e s f o r glucose determination not known at present whethe l a y e r "washout", enzyme denaturation, or p o s s i b l y both. Use of the Ring f o r Reaction
Monitoring
There are some important advantages i n using the r i n g f o r monitoring the course of the enzymatic r e a c t i o n . F i r s t , the e l e c t r o d e to which the enzyme i s attached may not be s u i t a b l e or optimal f o r monitoring product formation. We have not been able to o b t a i n r e p r o d u c i b l e r e s u l t s f o r peroxide when monitored d i r e c t l y at the r o t a t i n g d i s k whereas the platinum r i n g i s q u i t e suitable. Second, the course of the r e a c t i o n can be measured without p o t e n t i a l p e r t u r b a t i o n of the c a t a l y t i c surface or a l t e r n a t i v e l y at a p o t e n t i a l at which n e i t h e r products nor reactants are e l e c t r o a c t i v e . F i n a l l y , chemical and e l e c t r o c h e m i c a l c h a r a c t e r i s t i c s of r e a c t i o n products can be determined i n a manner analogous to a conventional r o t a t i n g r i n g d i s k e l e c t r o d e . Figure 3 demonstrates the manner i n which the r i n g " t r a c k s " the d i s k as the rate of the enzymatic r e a c t i o n i s v a r i e d by changing substrate concentration. The slope of Figure 3 (equal to 0.4) i s independent of ω over a wide range. In t h i s case the r i n g current i s measured with the d i s k at open c i r c u i t . If a p o t e n t i a l c h a r a c t e r i s t i c of 1^ reduction i s a p p l i e d simul taneously to both r i n g and d i s k an "apparent c o l l e c t i o n e f f i c i e n c y " of 0.30 i s obtained. This i s only a 25% reduction over the c o n d i t i o n s of Figure 3 suggesting that a l a r g e p o r t i o n of the product which i s produced i n the enzyme l a y e r can be c o l l e c t e d a f t e r i t d i f f u s e s back i n t o the s o l u t i o n . The d i r e c t p r o p o r t i o n a l i t y between the d i s k and r i n g currents over a wide range of c o n d i t i o n s makes p o s s i b l e the e v a l u a t i o n of enzyme k i n e t i c parameters at the r i n g . Using Equation 10 and the steady-state r i n g current, a K of 9.59 mM was obtained. This i s i n good agreement with values obtained at the d i s k using Reaction 2 (9) as w e l l as with p r e v i o u s l y reported values f o r M
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
KAMIN
E T AL.
Rotating
Ring
Disk Enzyme
Electrode
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both the s o l u b l e and immobilized enzyme (1). In Figure 4 the r e l a t i o n s h i p between substrate concentration and steady-state r i n g current i s demonstrated. As i n the case of the disk,the current i s observed to decrease as the r o t a t i o n speed i s increased. At 1600 rpm, however, the g r e a t l y enhanced substrate mass t r a n s p o r t r e s u l t s i n higher s e n s i t i v i t y at the expense of decreased l i n e a r range and absolute current. When a p o t e n t i a l i s a p p l i e d to the r i n g a current t r a n s i e n t r e s u l t s which decays to steady-state i n about 60 sec. I t i s p o s s i b l e to shorten the measurement time and improve s e n s i t i v i t y by measuring the current before steady-state i s reached. As expected, the current at time t i s l i n e a r l y p r o p o r t i o n a l to g l u cose concentration and a s e n s i t i v i t y improvement o f at l e a s t 10% over steady-state i s observed without s i g n i f i c a n t d e t e r i o r a t i o n of p r e c i s i o n ( b e t t e r than 2%). Conclusion The possibility of producing a s t a b l e c a t a l y t i c surface a t tached to an e l e c t r o d e has been demonstrated. Using a r o t a t i n g r i n g d i s k enzyme e l e c t r o d e , the e f f e c t s of substrate mass t r a n s port and kinetic c o n t r o l of surface c a t a l y z e d r e a c t i o n s can be s t u d i e d . The use o f the r i n g makes p o s s i b l e the independent monitoring of the r e a c t i o n s o c c u r r i n g a t the d i s k . The evalua t i o n of kinetic parameters has been demonstrated paving the way f o r d e t a i l e d e l e c t r o c h e m i c a l c h a r a c t e r i z a t i o n of immobilized biosurfaces. Acknowledgemen t This work was supported i n part by N a t i o n a l Science Founda t i o n Grant CHE 73-08683-A03. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8.
W e e t a l l , Η. Η., Anal. Chem., (1974), 46, 602-615A. Wiseman, Α., ed., "Handbook o f Enzyme Biotechnology," Halsted Press, New York (1975). M e l l , L. D. and J . T. Maloy, Anal. Chem., (1975), 47, 299307. G u i l b a u l t , G. G. and G. J . Lubrano, Anal Chim. Acta, (1973) 64, 439-455. Hornby, W. E., M. D. Lilly, E. Crook and P. D u n n i l l , Biochem J., (1968) 107, 669-674. Smith, G. L., M. S. T h e s i s , U n i v e r s i t y of Arizona (1975). L e v i c h , V. J . , "Physicochemical Hydrodynamics," P r e n t i c e H a l l , Englewood Cliffs, N. J . (1962). Albery, W. J . and M. L. Hitchman, "Ring-Disk E l e c t r o d e s " , Oxford U n i v e r s i t y Press, London (1971).
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
10.
9. 10. 11. 12. 13. 14. 15.
KAMIN
ET
AL.
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Ring
Disk
Enzyme
Electrode
179
Shu, F. R. and G. S. Wilson, Anal Chem., (1976), 48, 16791686. Shabrang, M. and S. Bruckenstein, J. Electrochem Soc., (1975), 122, 1305-1311. Hadju, J . and P. F r i e d r i c h , Anal. Biochem, (1975), 65, 273-280. Malmstadt, H. V. and H. L. Pardue, Anal Chem, (1961), 33, 1040-1047. P r a t e r , Κ. B., and A. J . Bard, J . Electrochem Soc., (1970), 117, 207-213. Mahler, H. R. and E. H. Cordes, " B i o l o g i c a l Chemistry," Harper and Row, New York (1966). G o l d s t e i n , L. in "Methods in Enzymology" V o l . 19, G. E. Perlmann and L. Lorand, Eds. Academic Press, New York, (1970), pp. 935-978.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
11 Electrokinetic Potentials i n a Left Ventricle/Aorta Simulator EUGENE FINDL and ROBERT J. KURTZ ARK Research, Farmingdale, N.Y. 11735
There a r e two term in e l e c t r o c h e m i s t r that sound alike but c o v e field, electrode kinetics first term is used t o d e s c r i b e e l e c t r o d e r e a c t i o n rates. The second term (our s u b j e c t h e r e i n ) i s used to d e s c r i b e interfacial electrochemical phenomena o b s e r v e d when an electrolyte and a solid s u r f a c e move w i t h r e s p e c t t o each o t h e r . Electrokinetic phenomena a r e classically d i v i d e d i n t o f o u r c a t e g o r i e s , i.e., e l e c t r o p h o r e s i s , e l e c t r o - o s m o s i s , s e d i m e n t a t i o n p o t e n t i a l s and streaming potentials. There are however, s e v e r a l lesser known electrokinetic effects, namely motoelectric effects, Ueda effects and a c o u s t o - e l e c t r i c effects. Of t h e s e electrokinetic phenomena, s t r e a m i n g p o t e n t i a l s , under t u r b u l e n t f l o w c o n d i t i o n s , are t h e o r i z e d to c o n t r i b u t e significantly t o the electrical signals attributed t o mammalian hearts. I t is w e l l known t h a t b l o o d f l o w s t h r o u g h the h e a r t and c e r t a i n b l o o d v e s s e l s under t u r b u l e n t flow c o n d i t i o n s ( 1 ) . T h e r e f o r e , based upon the premise t h a t s t r e a m i n g p o t e n t i a l s o f significant magnitude can be g e n e r a t e d by such f l o w c o n d i t i o n s , we d e c i d e d t o i n v e s t i g a t e the possibility that streaming p o t e n t i a l s c o n t r i b u t e a s u b s t a n t i a l part o f the p o t e n t i a l s seen in an electro-cardiogram (EKG). The work of Miller and Dent(2) is o f particular interest in this regard. They p r e s e n t e d e x p e r i m e n t a l e v i d e n c e , b o t h i n v i t r o and i n v i v o ( w i t h d o g s ) , t h a t s t r e a m i n g p o t e n t i a l s a r e the cause o f a t l e a s t the Τ wave p o r t i o n o f the EKG. 0 t h e r s ( 2 - 6 ) have r e p o r t e d the e x i s t e n c e of s t r e a m i n g p o t e n t i a l s i n c a r d i o - v a s c u l a r components. 180
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
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Our a p p r o a c h t o t h e p r o b l e m o f d e m o n s t r a t i n g t h a t t h e EKG i s a t l e a s t p a r t i a l l y d u e t o s t r e a m i n g p o t e n t i a l s has been t o f i r s t demonstrate i n v i t r o , w i t h m e c h a n i c a l l e f t v e n t r i c l e s i m u l a t o r s , t h a t an EKG l i k e s i g n a l c a n b e g e n e r a t e d . Second, to d e m o n s t r a t e t h a t EKG l i k e s i g n a l s c a n be g e n e r a t e d i n v i v o u s i n g a m e c h a n i c a l , p u l s a t i l e , h e a r t pump. Some o f t h e e x p e r i m e n t a l r e s u l t s o f o u r f i r s t step are presented h e r e i n . Experimental
Apparatus
Our l a t e s t l e f t v e n t r i c l e s i m u l a t o r (LVS III) i s shown o n F i g u r e 1. £Two o t h e r m o d e l s , L V S I and LVS II, were o f s i m i l a r d e s i g n . ] A dc m o t o r d r i v e n cam m o v e s a s h a f diaphragm. As t h e d i a g r a f l u i d i n t h e " l e f t v e n t r i c l e " chamber i s f o r c e d o u t , t h r o u g h a o n e way r u b b e r v a l v e a n d i n t o a distensible balloon (aorta). During the v e n t r i c u l a r c y c l e , when c o m p r e s s i o n o c c u r s , t h e " a o r t a " expands due t o t h e o u t w a r d f l o w o f f l u i d . When t h e diaphragm i s brought back t o i t s s t a r t i n g p o s i t i o n , f l u i d from the " a o r t a " flows back i n t o the "left v e n t r i c l e " c h a m b e r t h r o u g h a s e c o n d one way v a l v e . S t r o k e v o l u m e s w e r e v a r i e d b e t w e e n 20 a n d 50 m l b y v a r y i n g t h e f l o w r e s t r i c t i o n c a u s e d by t h e c h e c k valves. E l e c t r o d e s ( A g / A g C l ) were p l a c e d a t v a r i o u s l o c a t i o n s on t h e " l e f t v e n t r i c l e . " The e l e c t r o d e s were p l a c e d o u t o f t h e f l o w i n g s t r e a m s t o m i n i m i z e moto-electric effect artifacts. [ M o r e w i l l be s a i d about t h i s problem area l a t e r . ] Coaxial c a b l e l e a d s were a d a p t e d t o c o m p r e s s i o n f i t t i n g s to connect the e l e c t r o d e s to the readout instrumentation. S i m u l a t o r e l e c t r o d e s w e r e n u m b e r e d a s shown o n F i g u r e 2. The L V S I I v e r s i o n h a d a n a d d i t i o n a l e l e c t r o d e (#6) p l a c e d i n t h e body o f t h e L V S . W a t e r b a t h e l e c t r o d e s ( F i g u r e 3) w e r e l a b e l e d A , B , C a n d D. T h e s e were l o c a t e d a s f o l l o w s : A, n e a r one v a l v e ; B, 2-6 cm f r o m A ; C , 2-6 cm f r o m D; D, near the other v a l v e . The s i m u l a t o r s w e r e made i o n i c a l l y c o n d u c t i v e by d r i l l i n g many s m a l l h o l e s i n t o t h e l e f t v e n t r i c l e chamber and t h e v a l v e h o l d e r s e c t i o n s . Bulk t r a n s f e r of f l u i d from the L V S * s and the w a t e r b a t h i n t o w h i c h t h e y w e r e s u b m e r g e d was p r e v e n t e d by a c o a t i n g o v e r the h o l e s . Several d i f f e r e n t c o a t i n g s were e v a l u a t e d . Among t h e
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
Figure I. Left ventricle simulator
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
11.
FiNDL AND KURTZ "*5
-®
"
Left Ventricle/Aorta Simulator ^
1
^
I
^
183
*4
&
\
\
/
Detail of electrodes inside tank Figure 3.
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m a t e r i a l s were a t h i n l a t e x , a c e l l u l o s e acetate r e v e r s e o s m o s i s membrane, c o l l a g e n , c o l l o d i o n and a cellulose battery separator material. Of t h e s e , c o l l o d i o n a n d t h e c e l l u l o s e membranes a p p e a r e d t o be t h e m o s t s a t i s f a c t o r y . The o t h e r s w e r e e i t h e r too r e s i s t i v e or too d i f f i c u l t to mechanically a t t a c h to the L V S ' s . T h e l e f t v e n t r i c l e was i m m e r s e d i n a w a t e r bath to simulate the "aqueous environment" o f a mammalian b o d y . F i g u r e 3 i l l u s t r a t e s how f o u r A g / A g C l e l e c t r o d e s were a t t a c h e d t o t h e b a t h . A T e k t r o n i x 751^ dual channel, storage o s c i l l o s c o p e w i t h 7A22 d i f f e r e n t i a l a m p l i f i e r s was used to monitor the e l e c t r o k i n e t i c potentials. I n p u t i m p e d a n c e was i n c r e a s e d t o l O * ^ ohms b y means o f s o l i d s t a t tests, i n addition we a l s o m o n i t o r e d t h e " l e f t v e n t r i c u l a r " pressure, u s i n g a s t r a i n gauge t r a n s d u c e r . Test
Results
Examples o f the e l e c t r o k i n e t i c p o t e n t i a l s g e n e r a t e d b y t h e s i m u l a t o r s i s shown o n F i g u r e s 4, 5i 6 a n d 7. A l l t e s t s were r u n a t a m b i e n t temperature, u s i n g v a r i o u s c o n c e n t r a t i o n s of NaCl as the e l e c t r o l y t e . F i g u r e k i l l u s t r a t e s the waveforms o b t a i n e d from v a r i o u s c o m b i n a t i o n s of e l e c t r o d e s u s i n g the LVS I I . The u p p e r t r a c e s a r e t h e e l e c t r o k i n e t i c p o t e n t i a l s while the lower t r a c e i l l u s t r a t e s the l e f t ventricular pressure. Electrokinetic p o t e n t i a l s were m e a s u r e d h a v i n g t h e following v e r t i c a l deflection scale factors, Electrode Pair 1-.2+
l-,3 l-,5 1-.6+
2-,5 2-,6+
+
+
+
Scale (mv/div)
50 200 200 20 20 200 200 20 50
Electrode Pair 3 - A
+
3-.5 3-.6 ^-,5
+
+
k~,6 5-,
+
+
6
+
Scale (mv/div)
20 200 200 200 200 20
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
FiNDL A N D KURTZ
Left
VentricleIAorta
Simulator
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T h e L V S was n o t i m m e r s e d i n t h e w a t e r b a t h . Pulse r a t e was 66 p u l s e s p e r m i n u t e ( P / M ) . The e l e c t r o l y t e was s a l i n e h a v i n g a r e s i s t i v i t y o f 3 x l C P ohm cm. Peak v e n t r i c u l a r p r e s s u r e was 320 mm H g . F i g u r e 5 i l l u s t r a t e s the waveforms o b t a i n e d from t h e LVS I I I u s i n g i t s v a r i o u s electrode combinations. I n t h i s c a s e , t h e p u l s e r a t e was 76 P/M a n d t h e s a l i n e h a d a r e s i s t i v i t y o f 1,040 ohm cm, w h i c h i s a b o u t t h a t o f m a m m a l i a n t i s s u e . S t r o k e v o l u m e was « 50 m l . Figure 6 i l l u s t r a t e s the t e s t r e s u l t s obtained w i t h the LVS III immersed i n t h e water b a t h . The t o p t r a c e shows t h e p o t e n t i a l s a s m e a s u r e d b y e l e c t r o d e s ( A , B , C , D ) immersed i n the b a t h . The b o t t o m t r a c e show e l e c t r o d e s 1 and 2 Figure 7 i l l u s t r a t e s the effect of stroke volume on t h e a m p l i t u d e o f t h e s i g n a l . Pulse rate was 72 P/M f o r a l l p h o t o s . S t r o k e v o l u m e was v a r i e d by c h a n g i n g t h e l e n g t h o f t h e p i s t o n p u s h i n g the LVS III diaphragm. A s e r i e s o f t e s t s was made u s i n g t h e L V S III to determine the e f f e c t of e l e c t r o l y t e c o n d u c t i v i t y on s i g n a l v o l t a g e l e v e l . The r e s u l t s a r e shown o n T a b l e 1. Table
1
Effect of Electrolyte Conductivity Signal Voltage Level Electrolyte Conductivity (ohm-i.cm !) 3
1.1·10" 2.0.10-3 9.1-10-Jf 2
2.9ΊΟ-7 1.5·10"7 I.O.IO-7
3.0-10-f 2.2·10"5 1.4-10-5
Signal Peak
7mvT 0.5 3 5
on
Voltage Level Average (mV) 0.12 0.60 0.87
12 18 20
2.40 2.95 3.82
40 44 47
7.88 9.24 10.86
T h e o r e t i c a l l y , streaming p o t e n t i a l i s inversely proportional to conductivity. Figure 8 i l l u s t r a t e s that our experimental r e s u l t s approximate the t h e o r e t i c a l v a l u e s o v e r the range 10~2 t o 10"5 o h i r r ^ c n T . 1
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
FiNDL A N D KURTZ
Left
Ventricle/Aorta
Simulator
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Figure 7. Effect of stroke volume on signal amplitude. Electrodes 4+,5 of the LVS HI. Average stroke umes are given below photos.
10
-3
Ί
\ c 10" Experiment al
The o r e t i c a Y
10"
2 Average
4
6
Tulsatile
Potential
S
10
12
(Millivolts)
Figure 8. Effect of electrolyte conductivity on pulsatile electrokinetic potential (electrode 4 and 5; LVS III)
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
11.
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Ventricle/Aorta
Simulator
Discussion H i s t o r i c a l l y , streaming p o t e n t i a l s were f i r s t described by Quincke i n 1859.(2.) Other 1 9 century i n v e s t i g a t o r s included Z o l l n e r ( 8 ) , Edlund(£) Haga(lO) and Clark(11). Helmholtz(12) added a t h e o r e t i c a l basis f o r streaming potential> incorporating h i s famous double l a y e r theories into the explanation. Smoluchowski r e f i n e d Helmholtz's theories i n her t r e a t i s e published i n 1921. Most recent e f f o r t i n the f i e l d has been concerned with the determination of zeta p o t e n t i a l s by using streaming p o t e n t i a l measurements. With but few exceptions( 11^,1^, 1Λ) the experi mental e f f o r t described i th streamin potential literatur through c a p i l l a r y tubes or porous plugs. The region of turbulent flow has been l a r g e l y ignored u n t i l recently. Kurtz, F i n d l , Kurtz and Stormo(15) extended the region of streaming p o t e n t i a l measurements well i n t o the turbulent region, u t i l i z i n g tubing up to 3.8 cm i n diameter. Further they extended the basic streaming p o t e n t i a l r e l a t i o n s h i p s well into the turbulent flow region. t h
f
Laminar region 2
Ε = 8[DcL/nd k]u Turbulent
region
Ε - [0.0^(pA)°- ][DCL/nd - k]u 7 5
1
2 5
1
7 5
where d = tubing diameter u = f l u i d velocity D = d i e l e c t r i c constant ζ = zeta p o t e n t i a l Ε = streaming p o t e n t i a l μ = f l u i d v i s c o s i t y k = f l u i d conductivity ρ = f l u i d density L = electrode spacing The major d i f f e r e n c e between the two r e l a t i o n ships i s that i n the turbulent region, as compared to the laminar region, the streaming p o t e n t i a l increases as a function of v e l o c i t y to the 1.75 power rather than l i n e a r l y . Further, the p o t e n t i a l diminishing e f f e c t of enlarging tubing diameter i s much l e s s i n the turbulent region, i . e . , d ~ i n laminar versus d " * * i turbulent. Overall, the streaming p o t e n t i a l increases much more r a p i d l y i n the turbulent region than would have been expected using extrapolations from the laminar flow region. 2
1
2
n
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
190
E L E C T R O C H E M I C A L STUDIES O F BIOLOGICAL SYSTEMS
There are two other e l e c t r o k i n e t i c e f f e c t s that might be capable of generating p o t e n t i a l s such as were i l l u s t r a t e d . These are moto-electric effects(l6,12,18) and Ueda effects(1£,20). Unlike streaming p o t e n t i a l s , these two e f f e c t s are due to the i n t e r a c t i o n between an electrode and an e l e c t r o l y t e moving r e l a t i v e to i t . [Streaming p o t e n t i a l s on the other hand, are due to the r e l a t i v e motion between an e l e c t r o l y t e and a s o l i d surface, generally an i n s u l a t o r . The p o t e n t i a l i s measured by a p a i r of electrodes which are not located i n the moving e l e c t r o l y t e . ] Moto-electric e f f e c t s are r e a d i l y d i s t i n g u i s h able by the slow response of t h i s p o t e n t i a l to changes i n f l u i d v e l o c i t y Further l f f l u i d d i r e c t i o n doe of p o l a r i t y of the p o t e n t i a l as i s the case with streaming p o t e n t i a l s . In general, moto-electric e f f e c t s simply cause a s h i f t i n baseline and do not contribute to p u l s a t i l e p o t e n t i a l s . Ueda e f f e c t s are e s s e n t i a l l y due to rapid vibratory movement of an electrode-electrolyte i n t e r f a c e . This v i b r a t i n g motion r e s u l t s i n a sinusoidal, a l t e r n a t i n g p o t e n t i a l being developed. It i s t y p i c a l l y caused by a mechanical v i b r a t i o n of the e l e c t r o l y t e , such as caused by banging a water bath with a hard object, or by r a p i d l y c l o s i n g a valve causing a water hammer i n a pipe. [ U l t r a sonic acousto-electric e f f e c t s such as those described by Yeager et al.(20,21,22) are of too high a frequency to be factors i n our experimental results.] We have investigated the p o s s i b i l i t y that Ueda e f f e c t s were contributing to the p o t e n t i a l s generated by the LVS*s. When e l e c t r o l y t e s of high r e s i s t i v i t y (> lO^Qcm) were used, h i t t i n g the LVS with a metal object d i d produce measurable sinusoidal voltages. With e l e c t r o l y t e s of lower r e s i s t i v i t y , the e f f e c t was l e s s s i g n i f i c a n t . The Ueda e f f e c t does not account f o r the p u l s a t i l e p o t e n t i a l s measured i n the water bath, but i t d i d contribute a "sinusoidal noise" signal due to wave motions i n the bath. A factor that indicates that i t was indeed streaming p o t e n t i a l s that we measured was that the wave shape was dependent upon the rubber check valves. These valves were i n d i v i d u a l l y cast i n our laboratory by hand, using a s u r g i c a l latex. Each had a character of i t s own. As a r e s u l t , the opening and closing c h a r a c t e r i s t i c s of each
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
11.
FiNDL A N D KURTZ
Left Ventricle/Aorta
Figure
9.
Wave form parison
Simulator
com-
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
191
192
ELECTROCHEMICAL
STUDIES O F B I O L O G I C A L S Y S T E M S
differed. This resulted i n a noticeable variation i n s i g n a l waveform. I t i s much more r e a s o n a b l e t o assume t h e s e v a r i a t i o n s i n s i g n a l waveforms a r e due t o f l o w v a r i a t i o n s a n d t h u s s t r e a m i n g p o t e n t i a l s r a t h e r t h a n due t o a c o u s t i c e f f e c t s c a u s i n g Ueda potentials. As a f i n a l i n d i c a t i o n t h a t t h e p o t e n t i a l s m e a s u r e d were i n d e e d s t r e a m i n g p o t e n t i a l s , F i g u r e 9 i l l u s t r a t e s a c o m p a r i s o n b e t w e e n t h e wave s h a p e o f e l e c t r o d e s 4 and 5 and the flow o f blood i n t o the human a o r t a ( i ) . N o t e t h e c l o s e s i m i l a r i t y o f wave form. The p o t e n t i a l s m e a s u r e d u s i n g t h e L V S * s w i t h n o r m a l s a l i n e , were a n o r d e r o f m a g n i t u d e l o w e r than those t y p i c a l l y obtained i n v i v o , using mammals. I t was n o t o u r o b j e c t i v e t o d u p l i c a t e in vivo surface-electrolyt potential levels. However, i t i s f e l t t h a t such l e v e l s c a n be a t t a i n e d i n v i t r o , u s i n g b l o o d a s an e l e c t r o l y t e , c o l l a g e n l i n e d plumbing and p u l s a t i l e blood flow conditions as occur i n v i v o . I n summary, i t h a s b e e n shown t h a t p u l s a t i l e flow of s a l i n e e l e c t r o l y t e s generates electrok i n e t i c p o t e n t i a l s remarkably s i m i l a r to i n vivo EKG's. This fact, i n conjunction with p r i o r research^ 2), i n d i c a t e s that the present assumption t h a t EKG p o t e n t i a l s a r e d u e s o l e l y t o m u s c l e a c t i o n p o t e n t i a l s n e e d s t o be r e - e x a m i n e d . Acknowledgments The a s s i s t a n c e o f L i n d a Stormo a n d S i d n e y Golden o f our research s t a f f i n the conduct o f t e s t s and p r e p a r a t i o n o f t h i s paper i s g r a t e f u l l y acknowledged. Abstract Several left ventricle/aorta mechanical s i m u l a t o r s were f a b r i c a t e d t o e v a l u a t e t h e p o s sibility o f g e n e r a t i n g EKG like electrical signals by electrokinetic methodology. The s i m u l a t o r s produced p u l s e d t u r b u l e n t flows, s i m u l a t i n g mammalian h e a r t pumping c o n d i t i o n s . EKG like s i g n a l s were g e n e r a t e d by t h e m o t i o n o f t h e electrolyte through the simulators.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
11. FINDL AND KURTZ Literature
Left Ventricle/Aorta Simulator
193
Cited
1.
I b e r a l l , Α., Cardon, S., Young, Ε., "On Pulsatile and Steady Arterial Flow, t h e GTS C o n t r i b u t i o n , " General T e c h n i c a l S e r v i c e s Inc., Upper Darby, Pa. (1973) LC 72-96894. 2. M i l l e r , J . R., Dent, R. F., Lab and Clin. Med., (1943), 28, 168. 3. Sawyer, P. Ν., Himmelfarb, Ε., L u s t r e n , I . , Z i s k i n d , Η., B i o p h y s . J . , (1966), 6, 641. 4. C i g n e t t e , Μ., "Streaming P o t e n t i a l s , Theory and Examples in Biological Systems," in P r o c . 1 I n t e r . Symp. Biol. A s p e c t s o f E l e c t r o c h e m . , S. M i l l a z o , P. E. Jones, L. Rampazzo eds., Birkhauser Verlag Basel (1971) 5. K u p f e r , Ε., J. 53, 16. 6. S r i n i v a s a n , S., Sawyer, P. Ν., J . Coll. I n t e r f a c . Sci., (1970), 32, 456. 7. Quinke, G., Ann. P h y s i k , (1859), 2, (107), 1. 8. Z o l l n e r , F., Ann. P h y s i k , (1873), 2, (148), 640. 9. Edlund, Ε., Ann. P h y s i k , (1875), 2, (156), 251. 10. Haga, Η., Ann. P h y s i k , (1877), 3, ( 2 ) , 326. 11. C l a r k , J . W., Ann. P h y s i k , (1877), 3, ( 2 ) , 335. 12. Helmholtz, H. L. F., Ann. P h y s i k , (1879), 3, (7), 337. 13. Dorn, E., Ann. P h y s i k , (1880), 3, ( 9 ) , 513. 14. Boumans, Α. Α., P h y s i c a , (1957), 23, 1038. 15. K u r t z , F., Findl, Ε., K u r t z , Α., Stormo, L., J . Coll. I n t e r f a c . S c i e n c e , ( i n p r e s s ) . 16. P r o c o p i u , S., Ann. P h y s i k , (1913), 37, 229. 17. Zucker, E. R., "A Critical Evaluation of Streaming P o t e n t i a l Measurements," Ph.D. T h e s i s , Columbia Univ., (1959). 18. Newberry, Α., T r a n s . E l e c t r o c h e m . Soc., (1934), 67, 25. 19. Ueda, T., e t al., J. E l e c t r o c h e m . Soc. Japan, (1951), 19, 142. 20. Yeager, Ε., Hovorka, F., J. A c o u s t i c a l Soc. America, (1953), 25, 445. 21. W i l l i a m s , Μ., Rev. Sci. Instr., (1948), 19, 640. 22. Packard, R. G., J . Chem. Phys., (1953), 21, 303. st
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
12 Differential Pulse Polarographic Analysis for Ethylenediaminetetraacetate ( E D T A ) and Nitrilotriacetate ( N T A ) i n Phytoplankton Media RICHARD J. STOLZBERG Harold Edgerton Research Laboratory of the New England Aquarium, Central Wharf, Boston, Mass. 02110 In t h e c o u r s e o f investigating t h e effects o f t r a c e metal s p e c i a t i o we have found it desirable tration of small q u a n t i t i e s o f strong organic l i g a n d s . These i n c l u d e artificial l i g a n d s such as e t h y l e n e d i a m i n e t e t r a a c e t a t e (EDTA), nitrilotriacetate (NTA), and t r i s ( h y d r o x y m e t h y l ) a m i n o m e t h a n e ( t r i s ) added by the e x p e r i m e n t e r and some l e s s w e l l - d e f i n e d e x t r a cellular m e t a l b i n d i n g o r g a n i c s (EMBO) added by t h e phytoplankton. Artificial l i g a n d s have traditionally been added by algal p h y s i o l o g i s t s because a wide variety o f a l g a e c a n be grown in media c o n t a i n i n g complexed t r a c e m e t a l s . In a d d i t i o n , precipitation of t h e medium is reduced, e n a b l i n g t h e e x p e r i m e n t e r t o p r e p a r e a more r e p r o d u c i b l e medium ( 1 ) . The h y p o t h e s i s has been made t h a t p l a n k t o n might a c t i v e l y produce EMBO f o r much t h e same r e a s o n - t o improve the medium f o r growth e i t h e r by d e t o x i f y i n g potentially t o x i c m e t a l s such as copper (2) o r by making i r o n a v a i l a b l e as a s o l u b l e c h e l a t e d s p e c i e s (3,4). Our work c u r r e n t l y i n v o l v e s correlating t h e c o n c e n t r a t i o n o f complexed and uncomplexed s p e c i e s o f copper w i t h p h y t o p l a n k t o n p r o d u c t i v i t y and w i t h t h e p r o d u c t i o n o f EMBO. W e l l d e f i n e d artificial media t h a t l e n d themselves t o c o n v e n i e n t c h e m i c a l manipu lation and which s u p p o r t a good growth o f a l g a e a r e used. Table I g i v e s the composition o f both the s y n t h e t i c seawater (SSW) used in t h e analytical development work and t h e artificial medium d e s i g nated Cu-IV. In media c o n t a i n i n g l e s s than 5 χ 10 M EDTA o r NTA, a c c u r a t e measurement o f specific l i g a n d concen t r a t i o n i s q u i t e important. Small v a r i a t i o n s i n l i g a n d c o n c e n t r a t i o n may i n c e r t a i n c a s e s produce -6
194
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
12.
EDTA
STOLZBERG
and NTA
in Phytoplankton
TABLE Composition
of
I
Synthetic
Component
195
Media
Seawater
and
Cu-IV
SSW
Cu-IV
NaCl
4.3
χ
ΙΟ"
1
M
4.3
χ
ΙΟ"
1
M
KC1
9.4
χ
1(Γ
3
M
9.4
χ
ΙΟ"
3
Μ
χ
ΙΟ"
3
Μ
7.5
χ
ΙΟ"
3
Μ
1.2
χ
10~
3
Μ
5
Μ
MgS0
4
2.
CaCl
2
9.5
NaN0
3
NaH P0
4
4.8
χ
10~
Na Si0
3
2.6
χ
ΙΟ"
4.4
χ
10~
to
5 χ
ΙΟ"
6
Μ
to
5 χ
1θ"
β
Μ
to
8 χ
10~
2
2
Boron EDTA
5 χ
ΙΟ"
ΝΤΑ
5 χ
ΙΟ""
Tris
8 χ
ΙΟ"
7
7
5
Μ
4
5
3
Μ
Μ
Μη
2.2
χ 1θ"
Ζη
2.2
χ
1θ""
7
Μ
5.4
χ
10~
8
Μ
3.7
χ
10~
9
Μ
Co
(inorganic)
Vitamin
Β
1 2
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
6
Μ
196
ELECTROCHEMICAL
STUDIES O F B I O L O G I C A L
SYSTEMS
i m p o r t a n t changes i n copper s p e c i a t i o n . Loss of l i g a n d due t o p h o t o d e g r a d a t i o n i s a d i s t i n c t p o s s i b i l i t y when c u l t u r i n g a l g a e , p a r t i c u l a r l y i n l o n g term experiments under h i g h l i g h t i n t e n s i t y . The s e n s i t i v i t y of ferric-EDTA to photodegradation has b e e n known f o r o v e r two d e c a d e s (5) . The s u s c e p t i b i l i t y o f b o t h f e r r i c - and c u p r i c - N T A t o p h o t o d e g r a d a t i o n has been documented r e c e n t l y {6,]_,S). Sorption of ligands can a l s o present experimental d i f f i c u l t i e s i n dense a l g a l c u l t u r e s . Changes i n m e t a l s p e c i a t i o n c a n be e x p e c t e d f r o m any o f t h e s e mechanisms t h a t m i g h t r e d u c e l i g a n d o r m e t a l c o n c e n trations. F i n a l l y , t h e p r o d u c t i o n o f EMBO b y t h e c e l l s s h o u l d be t a k e n i n t o c o n s i d e r a t i o n when calculating speciation. T h e q u a n t i t y o f EMBO p r o d u c e d by t h e c e l l s c o u l d b t o t a l complexing capacit EDTA o r NTA p r e s e n t . The a n a l y t i c a l methodology f o r s p e c i f i c and s e n s i t i v e d e t e r m i n a t i o n o f EDTA a n d NTA i n s a l i n e waters i s not w e l l developed i n s p i t e o f the wide r a n g e o f t e c h n i q u e s d e v e l o p e d f o r NTA i n f r e s h w a t e r and sewage s l u d g e (100 . The most w i d e l y used s p e c i f i c t e c h n i q u e s i n n o n - s a l i n e w a t e r a r e gas chromatography (11-13) and e l e c t r o c h e m i s t r y (14-18). The p r e s e n c e o f l a r g e q u a n t i t i e s o f d i s s o l v e d salts i n seawater c l e a r l y favors e l e c t r o c h e m i c a l techniques. E l e c t r o c h e m i c a l r e d u c t i o n o f t h e CdNTA c o m p l e x , f i r s t u s e d a n a l y t i c a l l y f o r t h e d e t e r m i n a t i o n o f NTA i n EDTA (14^) a n d t h e n l a t e r a d a p t e d f o r NTA d e t e r m i n a t i o n i n l a k e and r i v e r w a t e r s ( 1 6 , 1 7 ) , i s t h e system of c h o i c e . R e d u c t i o n o f NTA c o m p l e x e s o f l e a d , b i s m u t h , a n d i n d i u m (_15,1_8) h a s b e e n u s e d a n a l y t i c a l l y , b u t t h e CdNTA {19_,2C0 a n d C d E D T A ( 2 1 , ,22,23) e l e c t r o c h e m i s t r y h a s b e e n c h a r a c t e r i z e d i n d e t a i l and appears r e l a t i v e l y w e l l behaved. C l a s s i c a l DC p o l a r o g r a p h y h a s b e e n u s e d t o m e a s u r e 1 t o 10 ppm NTA i n l a k e w a t e r (16^) , b u t t h i s t e c h n i q u e c o u l d n o t be u s e d i n s e a w a t e r . The r e d u c t i o n c u r r e n t due t o t h e l a r g e q u a n t i t y o f cadmium a d d e d t o d i s p l a c e c a l c i u m f r o m t h e NTA w o u l d swamp t h e s m a l l c u r r e n t i n c r e m e n t s d u e t o CdNTA r e d u c t i o n . D i f f e r e n t i a l pulse polarography (DPP) i s more s e n s i t i v e t h a n DC p o l a r o g r a p h y , and i t c a n be u s e d t o measure s m a l l c u r r e n t s a t a p o t e n t i a l c a t h o d i c o f an e l e c t r o c h e m i c a l l y a c t i v e s p e c i e s p r e s e n t a t much g r e a t e r c o n c e n t r a t i o n .
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
12.
EDTA
STOLZBERG
Theoretical
and NTA
in Phytoplankton
197
Media
Considerations
T h e r e d u c t i o n o f t h e CdNTA c o m p l e x a t - 0 . 9 V (vs SCE) i s i r r e v e r s i b l e a n d d i f f u s i o n controlled (20). A t pH 8 t h e r e d u c t i o n o f t h e CdEDTA c o m p l e x a t - 1 . 2 t o - 1 . 3 V i s more i r r e v e r s i b l e t h a n t h a t for CdNTA. I t i s d i f f u s i o n c o n t r o l l e d o n l y when t h e c o n c e n t r a t i o n o f t h e s u p p o r t e l e c t r o l y t e i s h i g h (2_1) . S e p a r a t i o n o f t h e uncomplexed Cd (at - 0 . 6 V ) , the CdNTA, and t h e CdEDTA waves w i l l p r e s e n t no p r o b l e m s u s i n g DPP. I n s e a w a t e r , t h e CdEDTA c o m p l e x r e d u c t i o n c u r r e n t i s e x p e c t e d t o be d i f f u s i o n c o n t r o l l e d due to the presence of a high c o n c e n t r a t i o n of salts, and t h e s e n s i t i v i t y s h o u l d be s u f f i c i e n t f o r t h e determination of micromolar q u a n t i t i e s of ligand. The a d d i t i o n o f a l a r g a l a r g e f r a c t i o n o f b o t h l i g a n d s t o be p r e s e n t i n t h e b u l k o f t h e s o l u t i o n as t h e cadmium complex (see below). E q u a t i o n 1 d e s c r i b e s the g e n e r a l i z e d competition r e a c t i o n b e t w e e n c a d m i u m a n d c o m p e t i n g m e t a l (M) for EDTA o r NTA ( L ) . Charges have been o m i t t e d f o r clarity. C o n c e n t r a t i o n s t a b i l i t y c o n s t a n t s and m o l a r c o n c e n t r a t i o n s are used throughout. CdL + Μ ^
Cd + ML,
K„ = C
w
h
a
n
e
d
r
=
e
.
TmTTTlT
=
CdL
[ [ C
C
d
L
( l a )
(lb)
]
d]
(D
CdL
[L]
Using Ringbom's concept o f c o n d i t i o n a l s t a b i l i t y c o n s t a n t s a n d h i s n o t a t i o n (24^) , t h e c o m p e t i t i o n s t a b i l i t y c o n s t a n t , K , c a n be r e w r i t t e n as a conditional competition constant c
K'
=
'ML K '
K
c
d
[Cd'] [M<]
=
L
[ML] [CdL]
(2)
where M' r e f e r s t o a l l o f M n o t a s s o c i a t e d w i t h L . I n t h e c a s e where t h e cadmium c o n c e n t r a t i o n , C ç £ , i s much g r e a t e r t h a n t h e EDTA o r NTA c o n c e n t r a t i o n , C , the f o l l o w i n g approximation i s v a l i d : L
[ML]
^
C -[CdL] L
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
(3)
198
ELECTROCHEMICAL
Substituting equation arranging results i n
[
c
d
L
1
•
κ» C
STUDIES O F B I O L O G I C A L
3 into equation
tM'f'l
[cd'l
2 and
SYSTEMS
re
^
(
4
)
Under the c o n d i t i o n s o f the a n a l y s i s i n seawater (where M = C a ) , t h e f o l l o w i n g a p p r o x i m a t i o n s a r e a l s o valid: [M» ] ^ [Cd']
C
~,
M C
(5)
Cd
(6)
Substituting int following relationship
[CdL]
=
t
^ (K»C ) x
C
c
d
+
M
^
C
c
c
L
(7)
d
Equation 7 p r e d i c t s that the c o n c e n t r a t i o n of e l e c t r o a c t i v e CdL s p e c i e s i n t h e b u l k o f s o l u t i o n i s a l i n e a r f u n c t i o n of the a n a l y t i c a l c o n c e n t r a t i o n of L when and Cç; a r e c o n s t a n t . Under the c o n d i t i o n s o f a n a l y s i s i n s y n t h e t i c s e a w a t e r (2.4 χ Ι Ο " M Cd), a p p r o x i m a t e l y 80% o f t h e NTA a n d 100% o f t h e EDTA a r e a s s o c i a t e d w i t h cadmium. Figure 1 p l o t s the f r a c t i o n o f NTA a n d EDTA a s s o c i a t e d w i t h c a d m i u m a s a f u n c t i o n o f cadmium c o n c e n t r a t i o n . A c o m p l i c a t i o n can e x i s t i f the method o f standard additions i s used. I f enough l i g a n d i s added s u c h t h a t t h e c o n d i t i o n C c d ^ C j j no l o n g e r h o l d s , e q u a t i o n 6 i s no l o n g e r v a l i d . However, the r e l a t i o n ship d
4
[Cd ] 1
-
C
c
d
c a n be s u b s t i t u t e d rearrangement, the results: [CdL]
2
-
(K^C
M
-
[CdL]
(8)
i n t o equation 4 and, a f t e r following quadratic equation
+ C
c
d
+ C ) L
[CdL]
+ C
c
d
C
L
=
0
(9)
T h e s o l u t i o n o f t h i s e q u a t i o n o f [CdL] p r e d i c t s a non-linear p l o t of current vs. concentration of ligand, the c u r v a t u r e of which i s p a r t i c u l a r l y pronounced at high concentrations of ligand. T h i s e f f e c t w i l l be d i s c u s s e d i n a l a t e r s e c t i o n , s p e c i f i c a l l y f o r NTA.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
12.
EDTA
STOLZBERG
and NTA
in Phytoplankton
199
Media
Experimental Apparatus. T h e e l e c t r o c h e m i c a l s y s t e m u s e d was a P r i n c e t o n A p p l i e d R e s e a r c h M o d e l 174 P o l a r o g r a p h i c A n a l y z e r a n d a M o d e l 172A m e r c u r y d r o p - t i m e r . Output was t o a H o u s t o n I n s t r u m e n t s O m n i g r a p h i c 2000 X - Y recorder. T h e e l e c t r o c h e m i c a l c e l l was s i m i l a r t o t h e o n e d e s c r i b e d b y G i l b e r t a n d Hume (25), but a dropping mercury e l e c t r o d e (DME) was u s e d r a t h e r t h a n a wax i m p r e g n a t e d g r a p h i t e e l e c t r o d e . Synthetic s e a w a t e r was u s e d i n t h e s e a w a t e r - s i l v e r reference electrode. M i x i n g o f t h e a n a l y t e was d o n e w i t h a m a g n e t i c s t i r r e r and T e f l o n c o a t e d s t i r r i n g b a r . D a t a a n a l y s i s was p e r f o r m e d o n a Wang 600 p r o g r a m mable c a l c u l a t o r . Reagents. Glass d i s t i l l e d , deionized water; r e a g e n t g r a d e c h e m i c a l s ; and t r i p l e d i s t i l l e d m e r c u r y were used t h r o u g h o u t t h i s s t u d y . Prepurified n i t r o g e n g a s p r e s a t u r a t e d w i t h w a t e r was u s e d t o deoxygenate samples. Procedure. Optimum c o n d i t i o n s f o r t h e d e t e r m i n a t i o n o f NTA a n d EDTA d i f f e r e d a s shown i n T a b l e II. W i t h t h e s m a l l e r q u a n t i t y o f cadmium a d d e d , baseline n o i s e i n t h e r e g i o n o f t h e C d E D T A wave was r e d u c e d . H o w e v e r , t h e l i n e a r r a n g e was e x t e n d e d f r o m 4 χ 1 0 " ^ M to 2.4 χ 1 0 " M EDTA i f t h e c a d m i u m s p i k e was i n c r e a s e d from 0.1 ml t o 0.6 m l . 4
TABLE Analytical 500 ppm c a d m i u m
Peak (a) (b)
EDTA 0.10 or 0.60
No
Yes, i f <10-5 M
c a l c i u m by
0.1 M
No
Yes
10 m i n 0.7
scan
potential 10.00 pulse
(a)
spike
Deoxygenate Voltage
Procedure NTA 0.60 ml
cadmium
Equilibrate
Increase
II
(b)
ml sample amplitude
to
-0.90V
C
E
D
ml T
A
10 m i n -1.1V
-1.0
to
01.6V
-1.25V
+ 1.00 m l t r i s pH 7 . 9 25 o r 50 mV, s c a n r a t e 5 mV
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
sec
200
ELECTROCHEMICAL
STUDIES O F B I O L O G I C A L
SYSTEMS
T h e m e t h o d o f s t a n d a r d a d d i t i o n s was generally used i n e v a l u a t i n g the t e c h n i q u e . However, l i n e a r c a l i b r a t i o n c u r v e s c o u l d b e made f o r NTA i f t h e sum o f s t r o n g l i g a n d s p r e s e n t was < 1 0 " M a n d f o r EDTA i f EDTA c o n c e n t r a t i o n was l e s s t h a n t h e c a d m i u m concentration. Thus, f o r a n a l y s i s of a wide v a r i e t y o f m e d i a , s t a n d a r d a d d i t i o n s were n o t n e c e s s a r y . It was t h e r e f o r e p o s s i b l e t o d o s i m u l t a n e o u s d e t e r m i n a t i o n s o f NTA a n d EDTA i f t h e t o t a l s t r o n g l i g a n d c o n c e n t r a t i o n was <10~^ m. standard additions could b e made ( k e e p i n g t h e t o t a l f i n a l l i g a n d c o n c e n t r a t i o n b e l o w 1 0 " 4 m) o r c a l i b r a t i o n c u r v e s c o u l d b e u s e d . I n n e i t h e r c a s e was c a l c i u m a d d e d b e c a u s e t h e s e n s i t i v i t y o f t h e NTA d e t e r m i n a t i o n d r o p p e d p r e c i p i t o u s l y . T h e q u a n t i t y o f c a d m i u m a d d e d was 0 . 6 m l t o e n s u r e adequate s e n s i t i v i t 4
Results
and
Discussion
General Evaluation. F i g u r e 2 shows a d i f f e r e n t i a l p u l s e p o l a r o g r a m o f t h e cadmium c o m p l e x e s o f NTA a n d EDTA i n SSW. W i t h two m i n o r e x c e p t i o n s , the t h e o r e t i c a l c o n s i d e r a t i o n s have been p r o v e n v a l i d . One e x c e p t i o n t o t h e b e h a v i o r p r e d i c t e d b y t h e o r y i s t h a t t h e q u a n t i t y o f c a l c i u m p r e s e n t i n SSW was a c t u a l l y n o t s u f f i c i e n t t o p r o d u c e a wave t h a t i s diffusion controlled. T h i s d e v i a t i o n from t h e o r y does not s e r i o u s l y l i m i t the t e c h n i q u e because the wave h e i g h t i s n o n e t h e l e s s p r o p o r t i o n a l t o C E D T A The s e c o n d e x c e p t i o n i s t h a t s i m p l e c o m p e t i t i v e t h e o r y d o e s n o t p r e d i c t e l e c t r o d e r e s p o n s e when c o p p e r competes w i t h cadmium f o r t h e l i g a n d . H y d r o g e n i o n c o n t r o l i s n e c e s s a r y t o k e e p pH i n t h e r a n g e o f 7 t o 8. A b o v e pH 8 t h e a d d e d c a d m i u m tends to p r e c i p i t a t e . B e l o w pH 6 o r 7 , t h e r a p i d d i s s o c i a t i o n r e a c t i o n o f t h e p r o t o n a t e d CdHNTA s p e c i e s b e c o m e s i m p o r t a n t (20) . CdNTA" ^
Cd
2 +
+ NTA "
CdHNTA ^
Cd
2 +
+ HNTA "
3
2
Slow
(10)
Fast
(11)
The s e n s i t i v i t y o f t h e t e c h n i q u e i s r e d u c e d i n even s l i g h t l y a c i d s o l u t i o n because the c o n c e n t r a t i o n of the species reduced at - 0 . 9 V i s diminished. Form a t i o n o f t h e C d H E D T A " s p e c i e s a t pH 5 p l a y s a s i m i l a r r o l e i n d e c r e a s i n g the r e d u c t i o n c u r r e n t at -1.25 V.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
EDTA
STOLZBERG
and NTA in Phytoplankton
Media
201
1.0
/Β
[CdL] c L
/
0.5-
/ 0.0 l—ι
Figure 1. Fraction of ligand associated with Cd in SSW as function of C - (A) NTA, K 'C = 5.99 X JO' ; (B) EDTA, 0.1 M Ca added, K/C = 5.06 X 10~ ; (C) EDTA, K 'CM = 4.76 Χ 10 . ca
C
2+
5
M
M
6
1
c
Figure 2. Differential pulse pohrogram of 1 X 10~ M NTA and 2.5 X 10 M EDTA in SSW. 25-mV pulse height, 5-mV/sec scan rate, 1-sec drop time. 5
5
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
202
ELECTROCHEMICAL
STUDIES O F B I O L O G I C A L
SYSTEMS
Linearity. W i t h i n the l i m i t s of the accuracy of the t e c h n i q u e , the peak c u r r e n t a t - 0 . 9 V i s a l i n e a r f u n c t i o n o f NTA c o n c e n t r a t i o n f r o m z e r o t o 1 χ 10 M i n SSW c o n t a i n i n g 2 . 6 7 χ 10 M Cd. S o l u t i o n of e q u a t i o n 9 p r e d i c t s t h a t t h e f r a c t i o n o f NTA a s s o c i a t e d w i t h cadmium d e c r e a s e s from 0.816 t o 0 . 7 6 1 i n t h a t r a n g e due t o d e p l e t i o n o f u n c o m p l e x e d cadmium, b u t t h i s d e c r e a s e i s n o t a p p a r e n t from the d a t a . A d e f i n i t e decrease i n r e l a t i v e response i s observed e x p e r i m e n t a l l y above 1 0 " M NTA. F i g u r e 3 compares experimentally observed behavior with that p r e d i c t e d w i t h n o c o m p e t i t i o n (K£ = 0 ) , a n d w i t h two n o n - z e r o v a l u e s o f K£. The v a l u e o f calculated for calcium c o m p e t i t i o n u s i n g c o n s t a n t s i n r e f e r e n c e 24 i s 1 0 " " · The good f i t o f t h e e x p e r i m e n t a l l y o b s e r v e d p o i n t s to the l i n e c a l c u l a t e the system behaves a ligand. A p l o t o f EDTA c o n c e n t r a t i o n i n SSW v s . p e a k c u r r e n t a t a p p r o x i m a t e l y - 1 . 2 5 V i s l i n e a r from . ΙΟ" M t o 2 . 5 χ ΙΟ"" M i n t h e p r e s e n c e o f 2 . 6 7 χ 10~ M Cd. Response a t 5 χ Ι Ο " M i s generally less than t h a t e x p e c t e d by e x t r a p o l a t i n g t h e l i n e a r p o r t i o n o f the c a l i b r a t i o n curve back toward the o r i g i n . This d e c r e a s e i n r e l a t i v e r e s p o n s e i s due t o t h e s l o w r e a c t i o n b e t w e e n m i c r o m o l a r q u a n t i t i e s o f EDTA a n d cadmium. M a l j k o v i c a n d B r a n i c a (26) o b s e r v e d that the r e a c t i o n between 2 χ 1 0 " M Cd and 2 t o 7 χ 1 0 " M EDTA i n n a t u r a l s e a w a t e r p r o c e e d e d o n l y 3 1 - 6 9 % o f t h e way t o t h e e q u i l i b r i u m v a l u e i n 10 m i n . In the presence o f 2.5 χ 1 0 M EDTA t h e r e a c t i o n i s r a p i d , however. In our u s u a l a n a l y t i c a l t e c h n i q u e , t h e EDTA p r e s e n t i n t h e s a m p l e r e a c t s w i t h t h e c a d m i u m f o r o n l y t h e 10 m i n u t e d e o x y g e n a t i o n p e r i o d . We a l s o h a v e o b s e r v e d e x p e r i m e n t a l l y t h a t t h e r e a c t i o n i s i n c o m p l e t e i n 10 m i n u t e s i n s p i t e o f t h e r e l a t i v e l y h i g h c o n c e n t r a t i o n o f cadmium p r e s e n t . When t h e s a m p l e i s e q u i l i b r a t e d w i t h t h e c a d m i u m s p i k e f o r an h o u r , the d e c r e a s e d r e l a t i v e r e s p o n s e is n o t o b s e r v e d w i t h 5 μΜ EDTA p r e s e n t a n d l i n e a r i t y h a s been o b s e r v e d t o e x t e n d t o t h i s l e v e l and b e l o w . t
4
2
5
2
4
6
6
6
- 5
U s i n g c l a s s i c a l DC p o l a r o g r a p h y , Raspor and B r a n i c a (21) o b s e r v e d t h a t t h e l i m i t i n g c u r r e n t o f t h e CdEDTA wave i s s t r o n g l y d e p e n d e n t o n t h e c o n c e n t r a t i o n , c h a r g e , and n a t u r e o f t h e s u p p o r t i n g electrolyte cations. A d d i t i o n o f 10~*- M c a l c i u m t o d i l u t e N a C l s o l u t i o n s c o n t a i n i n g EDTA i n c r e a s e s s e n s i t i v i t y b y i n c r e a s i n g r e l a t i v e r e s p o n s e , a s shown i n Table III. The a d d i t i o n o f c a l c i u m t o s e a w a t e r
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
12.
EDTA
STOLZBERG
and NTA
in Phytoplankton
l i k e w i s e i n c r e a s e s the s e n s i t i v i t y of f o r EDTA i n t h a t m e d i u m . TABLE Effect
of
Calcium
Ca
2
+
and M g
the
technique
III
on CdEDTA Peak
2 +
203
Media
Current
or
Magnesium Concentration (molar)
CdEDTA C u r r e n t Ca Additions Φ) (nA) 2
+
None
CdEDTA C u r r e n t Mg Additions ( ) (nA) 2 +
b
0.0
0.0
8.0
2.7
1
χ 1 0 ~
3
5
χ
3
1
χ 1 0 ~
2
3 4 . 1
1 5 . 1
5
χ
2
46.9
2 6 . 3
1
χ
51.0
3 0 . 0 0
(a)
1 0 "
1 0 "
ΙΟ"
1
5 χ 1 0 " M EDTA, 2 . 4 χ Ι Ο " M Cd, pH 7 . 9 Mean o f d u p l i c a t e v o l t a g e scans 4
5
(b)
( a )
1 0 "
3
M NaCl,
P r e c i s i o n and A c c u r a c y . The p r e c i s i o n o f t h e a n a l y s i s f o r NTA i n SSW h a s b e e n d e t e r m i n e d b y r e p l i c a t e a n a l y s i s o f s a m p l e s o f NTA a l o n e a n d i n t h e p r e s e n c e o f EDTA. T a b l e IV p r e s e n t s r e s u l t s f o r five such experiments. The s t a n d a r d d e v i a t i o n o f the a n a l y s i s i n t h e 1 . 5 t o 5 yM NTA r a n g e i s a p p r o x i m a t e l y 6 χ 1 0 ~ 7 M . A c c u r a c y i s e x c e l l e n t e x c e p t when s i m u l t a n e o u s s t a n d a r d a d d i t i o n s o f NTA a n d EDTA a r e made to greater than 1 0 " M t o t a l l i g a n d . T a b l e V p r e s e n t s a n a l o g o u s r e s u l t s f o r EDTA d e t e r m i n a t i o n s i n SSW a n d i n t h e m e d i u m A q u i l (27), which resembles Cu-IV. The a c c u r a c y i s e x c e l l e n t f o r 5 χ 1 0 ~ 6 t o 5 χ 1 0 ~ M EDTA. The p r e c i s i o n i n c r e a s e s s i g n i f i c a n t l y with the a d d i t i o n of 1 0 " M C a , although accuracy i s not affected. If t h e cadmium s p i k e i s a l l o w e d t o e q u i l i b r a t e w i t h t h e s a m p l e , s a t i s f a c t o r y a n a l y s e s c a n b e made o f s a m p l e s c o n t a i n i n g l e s s t h a n 1 χ 1 0 ~ M EDTA. The p r e c i s i o n o f the r e p l i c a t e d d a t a would suggest that the l i m i t of d e t e c t i o n i n seawater i s a p p r o x i m a t e l y 1 yM NTA a n d 1 yM E D T A . The d e t e c t i o n l i m i t f o r NTA i n d e e d i s a p p r o x i m a t e l y 1 y M . 4
5
1
2
+
5
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
204
ELECTROCHEMICAL
TABLE Precision
and A c c u r a c y
of
NTA
Determination^
. NTA F o u n d * '
Standard Deviation
Replicates
1 . 5 7 μΜ NTA i n SSW
1.63
μΜ
0.68
μΜ
6
5 . 2 4 μΜ NTA i n SSW
5.40
μΜ
0.68
μΜ
5
5 . 2 4 μΜ NTA i n Cu-IV
5.5
μΜ
0.69
μΜ
5
μΜ
0.48
μΜ
6
5 . 2 4 μΜ NTA + 50 μΜ EDTA i n SSW(c) 5 . 0 0 μΜ NTA + 50 μ Μ Ε ϋ Τ Α i n SSW (d)
10.6
4.77
SYSTEMS
IV
(
Sample
STUDIES O F B I O L O G I C A L
ν
(a) (b) (c) (d)
25 mV p u l s e a m p l i t u d e , 5 m V / s e c s c a n r a t e , 0 . 6 m l 500 ppm C d p e r 10 m l s a m p l e Standard a d d i t i o n analysis S i m u l t a n e o u s s t a n d a r d a d d i t i o n s o f NTA a n d _ E D T A . F i n a l s t r o n g l i g a n d c o n c e n t r a t i o n 2 . 2 χ 10 M A d d i t i o n s o f NTA o n l y
As s e e n i n F i g u r e 4, t h e l i m i t i n g f a c t o r s are the s m a l l r e d u c t i o n c u r r e n t , the presence of baseline n o i s e , and t h e r a p i d l y d e c r e a s i n g c u r r e n t from t h e u n c o m p l e x e d cadmium r e d u c t i o n . F o r EDTA, the d e t e c t i o n l i m i t i s m o r e n e a r l y 2 o r 3 μΜ. Figure 5 p r e s e n t s a p o l a r o g r a m o f 5 μΜ EDTA i n A q u i l , a n d o n c e a g a i n t h e s m a l l c u r r e n t and n o i s y b a s e l i n e are important factors. Response does drop o f f sharply n e a r 2 t o 3 μΜ E D T A , a n d t h i s p h e n o m e n o n a p p e a r s t o set the lower l i m i t of d e t e c t i o n at t h i s time. Competitive Reactions. F o r t h i s t e c h n i q u e t o be s u c c e s s f u l , a l a r g e a n d c o n s t a n t f r a c t i o n o f NTA a n d EDTA m u s t b e a s s o c i a t e d w i t h c a d m i u m n e a r t h e e l e c trode surface. In most p o l a r o g r a p h i c a n a l y s e s i t i s
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
STOLZBERG
EDTA
and NTA
in Phytoplankton
Media
Figure S. Concentration of CdNTA in bulk of solu tion as function of total NTA concentration. Cad mium concentration = 2.67 X 10~ M; χ = experi mental points calculated from wave height; — = calculated lines for various values of K/. 4
Figure 4. Polarogram of 1.6 μΜ NTA and standard addi tions. 25-mV pulse, 5-mV/ sec scan rate, 1-sec drop time. (1.) 1.6 Μ NTA, du plicated; (2.-4.) spiked with successive 1.6-μΜ NTA in crements, duplicated. μ
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
ELECTROCHEMICAL
STUDIES O F B I O L O G I C A L
Figure 5. Polarogram of 5 μΜ EDTA and standard additions. Same conditions as Figure 4. (1.) 5 Μ EDTA, in triplicate; (2., 3.) spiked with successive 5μΜ EDTA increments, dupli cated. μ
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
SYSTEMS
12.
EDTA
STOLZBERG
and NTA
in Phytoplankton
207
Media
TABLE V Precision
and A c c u r a c y
Sample
o f EDTA
Standard Deviation
EDTA F o u n d
5.0 χ Ι Ο " M EDTA i n A q u i l , 0.1 M C a * added
Determination^
Replicates
6
2
3.0 χ 1 0 ~
6
M
0.8 χ Ι Ο "
M
0.42 χ 1 0 "
6
M
5
M
4
M
6
5.0 χ Ι Ο " M EDTA i n A q u i l , 0.1 M C a 6
2
added< )
4.48 χ 1 θ "
b
1.00 EDTA 5.00 EDTA 5.24 NTA i
χ 10" M i n SSW χ 10" M + χ 10" M n SSw(c)
6
6
0.93 χ 10"° M
3 . 5 χ 10
5 . 0 9 χ 10
5
M
5.0x
10"
M
6
5.00 χ 1 0 " M EDTA i n SSW
5 . 5 5 χ 10
b
M
3.1x
r 10" M
5
5.00 χ 1 0 " M EDTA i n SSW, 0.1 M C a added
5.13 χ 10"^ M
5
b
5
6
5
6
b
5
2
v
;
+
1.1 χ 1 0 ~
b
M
5
2 5 mV p u i s e a m p l i t u d e , 5 m V / s e c s c a n r a t e , 0 . 6 m l 500 ppm C d a d d e d p e r 10 m l s a m p l e e x c e p t w h e r e noted, standard addition analysis.
( k ^ O . l m l 500 ppm C d a d d e d p e r 10 m l s a m p l e , b r a t e cadmium s p i k e 1 h r b e f o r e a n a l y s i s . Simultaneous
additions
equili
o f NTA a n d E D T A .
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
208
ELECTROCHEMICAL
STUDIES O F B I O L O G I C A L
SYSTEMS
d e s i r a b l e t o know i f t h e s o l u t i o n n e a r t h e e l e c t r o d e s u r f a c e r e f l e c t s t h e s p e c i a t i o n and c o n c e n t r a t i o n o f e l e c t r o c h e m i c a l l y a c t i v e components i n the b u l k o f solution. We h a v e s t u d i e d t h e c o m p e t i t i o n b e t w e e n c a d m i u m a n d a number o f o t h e r m e t a l s f o r b o t h ligands. When t h e c o m p e t i n g m e t a l i s c a l c i u m , f e ' r r i c i r o n , z i n c , n i c k e l , or c o b a l t , the observed r e d u c t i o n o f t h e h e i g h t o f t h e C d L wave i s p r o p o r t i o n a l t o t h e r e d u c t i o n i n the c a l c u l a t e d f r a c t i o n of L a s s o c i a t e d with Cd. When t h e c o m p e t i n g m e t a l i s c o p p e r , the o b s e r v e d wave i s much l a r g e r t h a n i s e x p e c t e d f r o m a c a l c u l a t i o n of l i g a n d s p e c i a t i o n i n the bulk o f solution. The c o p p e r c o m p e t i t i o n e x p e r i m e n t s have demon s t r a t e d two p o i n t s . A) T h e a n a l y t i c a l t e c h n i q u e w o r k s w e l l i n the presenc although thermodynamic won't. B) M e t a l s p e c i a t i o n m e a s u r e m e n t s made i n w a t e r u s i n g e l e c t r o c h e m i c a l t e c h n i q u e s c a n be m i s l e a d i n g i f care i s not taken i n i n t e r p r e t i n g r e s u l t s . In the system s t u d i e d here the e q u i l i b r i u m e s t a b l i s h e d near the e l e c t r o d e i s q u i t e d i f f e r e n t than t h a t i n the b u l k o f s o l u t i o n due t o an a c c u m u l a t i o n o f ligand near the e l e c t r o d e surface. Analysis of Media. The p r e p a r a t i o n o f p h y t o p l a n k t o n g r o w t h medium r e q u i r e s t h e a d d i t i o n o f n u t r i e n t s (Ν, P, S i ) and m i c r o n u t r i e n t s ( t r a c e m e t a l s , l i g a n d s , v i t a m i n s ) t o SSW. Results presented i n T a b l e s IV and V d e m o n s t r a t e t h e a p p l i c a b i l i t y o f t h e t e c h n i q u e t o s a m p l e s o f medium as w e l l as s y n t h e t i c seawater. No d e c r e a s e i n p r e c i s i o n o r a c c u r a c y was observed i n e i t h e r Cu-IV or A q u i l . We h a v e f o l l o w e d p h o t o d e g r a d a t i o n o f t h e a r t i f i c i a l medium A S P - 7 ( 8 . 1 χ 1 0 " M EDTA, 3.66 χ ΙΟ"" M NTA) u n d e r r a t h e r e x t r e m e c o n d i t i o n s ( d i r e c t s u n l i g h t , m i d - s u m m e r , no t e m p e r a t u r e c o n t r o l s ) and o b s e r v e d r a p i d , b u t sometimes e r r a t i c disappearances o f b o t h NTA a n d E D T A . No s u c h d e c r e a s e s w e r e o b s e r v e d under normal c u l t u r i n g c o n d i t i o n s over a p e r i o d of two w e e k s . The r e l a t i v e s t a n d a r d d e v i a t i o n o f r e p l i c a t e a n a l y s e s was £ 5 % . The p r e s e n c e o f even moderate d e n s i t i e s o f S k e l e t o n e m a c o s t a t u m , a m a r i n e d i a t o m , i n t h e medium decreases the r e l i a b i l i t y o f measurements. Centrif u g a t i o n o f the sample i s thus n e c e s s a r y f o r a n a l y z i n g growing c u l t u r e s . T h i s t e c h n i q u e i s u s e f u l f o r m e a s u r e m e n t o f NTA a n d EDTA c o n c e n t r a t i o n s i n a w i d e v a r i e t y o f m e d i a . I f degradation or s o r p t i o n processes are o c c u r r i n g 5
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
4
12.
EDTA
STOLZBERG
and NTA
in Phytoplankton
Media
209
and m e t a l s p e c i a t i o n i s o f i n t e r e s t , a s p e c i f i c t e c h n i q u e s u c h a s t h i s one i s n e c e s s a r y . This is p a r t i c u l a r l y so i f b o t h l i g a n d s a r e p r e s e n t , as i n ASP 7 . The p r e c i s i o n o f the t e c h n i q u e l i m i t s i t s a b i l i t y t o measure submicromolar changes i n l i g a n d concentration. If degradation i s not o c c u r r i n g , a n o n - s p e c i f i c t e c h n i q u e c o u l d b e u s e d (9^) . However, t h i s t e c h n i q u e i s the method o f c h o i c e i f specific m e a s u r e m e n t s o f NTA a n d EDTA i n s e a w a t e r a r e needed at the micromolar level.
Abstract A t e c h n i q u e f o r the a n a l y s i s o f s y n t h e t i c s e a water and p h y t o p l a n k t o n media f o r e t h y l e n e d i a m i n e t e t r a a c e t a t e (EDTA differential pulse polarograph The a d d i t i o n o f a p p r o x i m a t e l y 2.4 x 10 M cadmium t o the a n a l y t e c o n v e r t s a l a r g e fraction of either l i g a n d t o the r e d u c i b l e cadmium complex. With 5 x 10 M l i g a n d p r e s e n t , the s t a n d a r d d e v i a t i o n o f the t e c h n i q u e is a p p r o x i m a t e l y 6 x 10 M f o r NTA and 4 x 10 M f o r EDTA. An e x a m i n a t i o n o f how a c c u r a t e l y the e l e c t r o d e r e a c t i o n reflects the s p e c i a t i o n o f t h e s e l i g a n d s in the b u l k s o l u t i o n has a l s o been made. With c a l c i u m , nickel, c o b a l t , and z i n c added as competing c a t i o n s , the e l e c t r o d e response is as e x p e c t e d . In the p r e s e n c e o f copper, the p r o p o r t i o n o f l i g a n d a s s o c i a t e d w i t h cadmium, as i n d i c a t e d by the r e d u c t i o n c u r r e n t , was much g r e a t e r than p r e d i c t e d by thermodynamic c o n s i d e r a t i o n s a l o n e . -4
-6
-7
-7
Credit T h i s r e s e a r c h was s u p p o r t e d by the Oceanography S e c t i o n , N a t i o n a l S c i e n c e F o u n d a t i o n , G r a n t DES7421642. Literature 1) 2) 3) 4) 5)
Cited
P r o v a s o l i , L., M c L a u g h l i n , J . J . Α., and Droop, M. R., A r c h i v . für M i k r o b i o l o g i e (1957), 25, 392. Steemann N i e l s e n , E. and Wium-Andersen, S., P h y s i o l . P l a n t . (1971), 24, 480. Murphy, T. P., Lean, D. R. S., and Nalawajko, C., S c i e n c e (1976), 192, 900. Levandowsky, M. and Hutner, S. Η., Ann. N.Y. Acad. Sci. (1974), 245, 16. Jones, S. S. and Long, F., J . Phys. Chem. (1952), 56, 25.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
210
6) 7) 8) 9)
10) 11) 12)
13) 14) 15) 16) 17) 18) 19) 20) 21) 22) 23) 24) 25) 26) 27)
E L E C T R O C H E M I C A L STUDIES
O F BIOLOGICAL
SYSTEMS
S t o l z b e r g , R. J. and Hume, D. Ν . , E n v i r o n . Sci. T e c h n o l . (1975), 9, 654. L a n g f o r d , C., Wingham, Μ., and Sastri, V., ibid. (1973), 7, 820. T r o t t , T., Henwood, R., and L a n g f o r d , C., ibid. (1972), 6, 367. S t o l z b e r g , R. J. and R o s i n , D., "Complexing c a p a c i t y measurements u s i n g t h e sodium form o f C h e l e x 100: G e n e r a l t h e o r y and application t o s e a w a t e r - l i k e m a t r i c e s " , p r e s e n t e d a t 172nd N a t i o n a l ACS M e e t i n g , San F r a n c i s c o , CA (1976). M o t t o l a , Η. Α., Toxicol. E n v i r o n . Chem. Rev. (1974), 2, 99. S t o l z b e r g , R. J. and Hume, D. Ν . , A n a l . L e t t . (1973), 6, 829. Aue, W., H a s t i n g s Hill, H., and Moseman 72, 259. Warren, C. and Malec, Ε . , ibid. (1972), 64, 219. D a n i e l , R. and L e B l a n c , R., A n a l . Chem. (1959), 31, 1221. Afghan, B. and Goulden, P., E n v i r o n . Sci. T e c h n o l . (1971), 5, 601. A s p l u n d , J. and Wanninen, E., A n a l . Lett. (1971), 4, 267. Wernet, J. and Wahl, Κ., F r e s e n i u s ' Ζ. A n a l . Chem. (1970), 251, 373. Haberman, J. P., A n a l . Chem. (1971), 43, 63. K o r y t a , J. and K o s s l e r , I . , Collect. Czech. Chem. Commun. (1950), 15, 241. Raspor, B. and B r a n i c a , Μ., J. Electroanal. Chem. (1975), 59, 99. Raspor, B. and B r a n i c a , Μ., ibid. (1973), 45, 79. Raspor, B. and B r a n i c a , Μ., ibid. (1975), 60, 35. Schmid, R. W. and Reilley, C. Ν., J. Amer. Chem. Soc. (1958), 80, 2101. Ringbom, Α., "Complexation in Analytical Chem istry", p. 35, I n t e r s c i e n c e , Ν . Y . (1963). Gilbert, T. R. and Hume, D. Ν., A n a l . Chim. A c t a (1973), 65, 451. M a l j k o v i c , D. and B r a n i c a , M., L i m n o l . Oceanogr. (1971), 16, 779. M o r e l , F. M. M., W e s t a l l , J. C., R e u t e r , J. G. and C h a p l i c k , J. P., T e c h n i c a l Note #16, Ralph M. Parsons Lab., M.I.T., Cambridge, MA, Sept. 1974.
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
INDEX A Absorbance 158 iron tetraphenylporphyrin complexes 74 optical ( Δ Α ) 148,149,151 values, peak 8 Accuracy 203 Acetylene 84 electrocatalytic reduction of 86,87,91 reduction rate 90 Adenine 12 binding to mercury electrod differential capacitance curves for.. 126 Frumkin equation for 129,134 Adenosine mononucleotides 116 Adsorption of adenosine mononucleotides 116 free energy of 132 on potential, dependence 132 Ag/Ag N o 33 Amine level 174 π Anion radical reactions 51, 55 Anthraquinone 2-sulfonic acid 165 Antiferromagnetic coupling 65 Aorta simulator, left ventricle/ 180 Aprotic solvents 51 Aquocobalamin(Bi2a) 1 Associated proximate structure (APS) 47 Attraction coefficient <*) 122 Autooxidation of B 1
Bioredox components, stoichiometry and energetics of 143 Bipyridylium salts (viologens) 146 l,l'-Bis(hydroxymethyl) ferri cinium ion ( B H M F ) 148,151 1, l'-Bis- ( hydroxymethyl ) ferrocene (BHMF) 155,156 Bis pyridine complexes 51 Bis ( 8-quinolinolato ) magnesium ( II ) 99 dihydrate 99 "Blue" copper laccases 143 +
Bridging atoms, sulfur Bridging groups Buffer effects 1,3-Butadiene Butyronitrile
88 66 82 85,92 54
C
3
i 2
Β Bi2
autooxidation of 1 cyanocobalamin 1 reduction of 9 - C N ( dicyanocobalamin ) 1 Bi2a ( aquocobalamin ) 1 Back integration method 121 Benzylviologen 158 radical cation 148,149 Binuclear center, dissociation of the .. 94 Biocatalysis studies, rotating ring disk enzyme electrode for 170 Biocomponents, indirect coulometric titration of 143 Biological processes, electrical activity associated with 114
Cadmium 197 ligand associated with 201 C d E D T A peak current 203 C d E T A complex reduction 197 C d N T A complex, electrochemical reduction of the 196 Capacitance 120,125,137 measurements, differential 117 Catalysis limited rate 174 Catalytic current 90 7Γ Cation radicals 51, 55 Cell, surface potential of 115 Cellulose membranes 184 Charge plots 158 Charge surface 131 Chelates, metal 32 Chemical reaction coupled to the electron transfer 67 Cobalamin(s) 1 systems, spectroelectrochemistry of the 7 Cob(I)alamin 3,5 Cob(II)alamin 3,5 Cob(III)alamin 5,6 Cobalt (II) tetraphenylporphyrin 57 Co 7Co couple 41 C o , complexes of 39 CoTPP 56 Co(II)TPP 51,57,58,61 2
1 +
2+
211
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
ELECTROCHEMICAL
212
Coenzyme Bi (5'-deoxyadenosylcobalamin ) 21 Collodion 184 Competition stability constant ( K ) .. 197 Competitive reactions 204 Complex (es) with anionic tetraazamacrocyclic ligands 41 binuclear molybdenum(V) 78,83 binuclear oxo- and sulfido-bridged molybdenum ( V ) -cysteine and EDTA 80 cobalt 39 of C o ( I I ) T P P with pyridine 58 coordination number of 52 of dianionic ligands, N i 42, 43 di-/x-oxo-bridged Mo(V)-systeine .. 78 dry cave 49 iron (II) 39,40,46 tetraphenylporphyrin 7 manganese(II) and-(III) 8-quinolinol 97,100,105 manganese 98,100 metal 32 mono and bis pyridine 51 of M o ( V ) 79 nickle 39,45,48 oxidized and reduced 52 porphyrin 51 solution, structure of the 103 with unsaturated neutral ligands ... 34, 37 Conductivity, electrolyte 188 Controlled potential electrolysis 71 Convection time constant ( i ) 173 Coordination number of the complex 52 Copper laccases, "blue" 143 Coulometric titration ( C T ) , indirect 143 Coulometry 27,71 Coupled electron—proton transfer 94 Cu-IV 195 Cyanocobalamin ( B i ) 1 Cyanocob ( III ) alamin 3 Cyclic voltammetry 27, 67 Cytochrome c 26,150,151 oxidase 143,158 2
c
2+
2 +
k
2
D D . C . polarography 26 d-d Transition energies 48 dADP 127 dAMP 127,131 dATP 127 Deoxyadenosine 126,127,138 Frumkin equation for adenine . 1 2 9 , 1 3 4 Deoxyadenosine-5'-phosphate 129 5'-Deoxyadenosyl-cobalamin (coenzyme B i ) 21 Dianionic ligands, N i complexes of .42, 43 2,6-Dichlorophenolindophenol 7,166 2
2 +
STUDIES O F B I O L O G I C A L
SYSTEMS
Dicyanocobalamin B i - C N 1 Dicyanocob( III ) alamin 5 Differential capacitance measure ments 117,126,138 Differential pulse polarography 67,194 Dimer 65 6-Dimethyladenine 135-137 Di-/x-oxo-bridged Mo ( V )-cystein complex 78 Dioxygen carrying ability of the M (II) metalloporphyrin 51 Disk current 177 Dissociation of the binuclear center 94 of the binuclear Mo (III) products 82 of manganese complexes 100 D M S O solution 58,100 DNA 113 Dropping mercury electrode (dme) .. 26 2
Ε Ε (half wave potential) 51 E* (linear free energy correlation ) ...33,44 E ° ' value(s) (energetics) 143,155 Ec catalytic regeneration mechanism 148 E D T A ( ethylenediaminetetraacetate) 194,201 complexes 80 determination 207 Electrical activity associated with biological processes 114 Electrical fields on natural polynucleo tides, structure effects of 113 Electrically charged membrane 113 Electroanalytical chemistry of heme c 28 Electroanalytical chemistry of porphyrin c 27 Electrocapillary data 120 maximum potential ( E C M ) 121 measurements 119 studies 125 Electrocardiogram ( E K G ) 180 Electrocatalytic hydrogénation 91 Electrochemical charge (q) 148 measurements 66 phenomena, interfacial 180 reaction mechanisms 30 reduction of adduct 91 reduction of binuclear molybdenum ( V ) 83 Electrode(s) enzyme 172 glucose oxidase 171 mercury 116,137 optically transparent ( O T E ) 146 platinum 68
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
213
INDEX
F Electrode ( s ) ( continued ) positioning 183 Ferricinium ions 156 potential s) 34,90 Ferrocene(s) 152,154 processes 32 acetic acid (FAA) 154 reaction(s) 143 derivatives 154 of CoTPP 56 monocarboxylic acid (FMCA) 154 of FeTPPCl 58 Formation constants of the oxidized products of each 71 and reduced complex 52 scan rate dependence of half Free energy of adsorption 132 wave potential and peak Free energy correlation, linear 44,46 current for three70 Frumkin equations 123,129,134,136 redox coupling between the biocomponent(s) and 143 G rotating ring disk enzyme 170 Glucose/glucose oxidase reaction .170,171 standard calomel (SCE) 53 Glucose oxidase electrode 171 system, three53 Growth medium, phytoplankton 208 suspended in tank 183 Electroellipsometry system 119 H Electrokinetic potential s ) 180,185,187,18 51,67 Electrolysis, controlled potential 71 Half wave potential (E) as function of temperature 61 Electrolyte conductivity 186,188 for three-electrode reactions, scan Electrolytes, supporting 27 rate dependence of 70 Electron-proton transfer, coupled .... 94 Electron transfer reactions 51, 53,67,75 Hammet substituent constants (σ ) .. 43 Electronic spectral bonds, low energy 46 Hanging drop mercury electrode (hdme) 27 Electroosmosis 180 Heme c 26,28-30 Εlectrooxidation—reduction of heme c 30 Heme-proteins 32, 46, 47 Electrooxidation-reduction of Hemin dimers, isoelectronic 66 porphyrins 67 Homogeneous hydrogénation 91 Electrophoresis 180 Homogeneous redox reaction 91 Electroreduction of Hydrogen bonding between thymine cyanocob(III)alamin 3 and adenine 137 Ellipsometric polarized angle (P) .... 138 Hydrogen evolution 91 Ellipsometric studies 135 Hydrogénation, electrocatalytic and Energetic (Ε°' value) of bioredox homogeneous 91 components 143 Entropy I changes 51 I-E curve for viologen 149 data Indirect coulometric titration (ICT) 143 for metal redox reaction of 114 Co(II)TPP 57 Injury potential Instrumentation 27 for metal redox reaction of Interface, mercury solution 113 Fe(II)TPP 59 Interfacial electrochemical for oxidation or reduction of a phenomena 180 neutral porphyrin ring 55 Interfacial tension 122 of electron transfer reactions at Iron porphyrin ring 53 oxidation state complexes 65 Enzyme(s) porphyrin redox properties 65 electrode, rotating ring disk 170,172 tetraphenylporphyrin 59,74 immobilized 170 ( II ) complexes 40,46 layer 172,174,175 (II) tetraphenylporphyrin 60 Ethane 90,92 Fe , complexes of 40 Ethylene 85,90 Fe /Fe couple(s) 32,48 Ethyenediaminetetraacetate FeTPPCl 58,68 (See EDTA) ( FeTPP ) N ( /x-nitro-bis-[α,β,γ,δtetraphenylporExtracellular metal binding organics phinatoiron] ) ... 65,67-69,70,71,73 (EMBO) 194 ρ
2+
2+
3+
2
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
ELECTROCHEMICAL
214
Iron (continued) ( F e T P P ) 0 ( /x-oxo-bis-tetraphenylporphinatoiron( III ) ) 65, 68, 73 F e ( I I ) T P P in D M F , pyridine binding by 51, 59, 62 Isoelectronic hemin dimers 66 Isoelectronic iron (IV) dimers 75 2
Κ K (competition stability constant) .. K ' (Michaelis constants) c
M
197 170
L Laminar region 189 Lewis acid Ill Lewis base 51 Ligand(s) Ill anionic tetraaza macrocyclic 41 artificial 19 associated with C d 201 binding of metalloporphyrins 51 complexation, axial 52 iron (II) complexes of 46 macrocyclic 32 N i complexes of dianionic 42, 43 saturated neutral tetraaza 34, 35 structural modifications 32 unsaturated neutral 37 unsaturated tetraaza 14-membered neutral macrocyclic 45 Linear free energy correlation 44, 46 Linearity 202 2 +
STUDIES O F B I O L O G I C A L
Mercury (continued) dropping (dme) hanging drop (hdme) solution interface Metal chelates complexes ion, central ion couples redox reaction of C o ( I I ) T P P redox reactions of F e ( I I ) T P P Metalloporphyrins 6-Methyladenine Methylene chloride Michaelis constants (Κ ') Michaelis-Menten theory Mitochondrial, S O D M - N distance, ideal Molar absorptivities for iron tetraΜ
biological function of catalysts, reduced octacyanide oxidation state solution chemistry (III) products, binuclear ( V ) complexes Monitoring, ring for reaction Mono pyridine complexes Mononucleotides, adenosine Moto-electric effects
SYSTEMS
26 27 113 32 32 32 32 57 59 26, 51 136 66, 74 170 172 97 34
95 84 159,160,165 93 93 82 78,80,83 176 51 116 190
Ν M Macrocyclic ligands, complexes of .... 32 Magnetic moment 103 Magnetic susceptibilities of manganese complexes 100 Manganese complexes dissociation and mag netic susceptibilities of 100 complexes, redox behavior of 98 SOD 97 -(II) and -(III) 8-quinolinol complexes 97,100,105 Mass transport limited rate 173 Mass transport, rate of substrate 170 M cil vaine buffer 138 Mechanisms, electrochemical reaction 30 Mediator-titrant ( M - T ) .143,145,161,162 Membranes cellulose 184 electrically charged 113 potentials biological 114 Mercury electrode 80 adenine 137 adsorption of adenosine mono nucleotides at a 116
N-bridged dimer 65 n-value 14,143 N i complexes 39,42,43,45,48 Ni /Ni 33 N i ( M A C ) \ oxidation of 37 ^-Nitrido-bis-[a,/?,%8-tetraphenylporphinatoiron] ( F e T P P ) N 65 μ-Nitrido-bis- [ α,β,γ,δ-tetraphenylporphyriniron] 65 Nitrilotriacetate ( N T A ) .194,201,204,205 Nitrogenase substrates 78,84 Nucleic acids 113 2 +
2 +
3 +
2
2
Ο One-electron reduction 18 Optical adsorbance(AA) 148,149,151 Optical spectroscopy 66, 72 Optically transparent electrodes (OTE) 146 Ottle cell, H g - N i 1 Oxidase, cytochrome c 158 Oxidation of the central metal ion 32 of ( F e T P P ) N 69,75 2
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
215
INDEX
Oxidation (continued) of a neutral porphyrin ring 55 potential for isoelectronic iron (IV) dimers 75 properties of metal chelates 32 state complexes, iron 65 state, molybdenum 93 ju-Oxo-bis ( 8-quinolinolato-8-quinolinol ) manganese ( III ) dimethanol 99 /x-Oxo-bistetraphenylporphinatoiron(III) " ( F e T P P ) ° 65 Oxo-bridged complexes of M o ( V ) , electrochemistry of 79, 80 Oxygen binding site, heme protein .... 47 Oxygen reaction of the complexes with 108
Purines, biologically important Pyridine binding by Co ( II ) T P P in D M S O .. binding by F e ( I I ) T P P in D M F .... to cobalt ( II ) tetraphenylporphyrin, addition of Co (II) T P P complexation with to iron (II) tetraphenylporphyrin, addition of
2
Ρ Peak absorbance values 8 Peak current, C d E D T A 203 Peak current for three-electrode reactions 70 Peroxide 176 p-Phenylenediamines, substituted .... 166 Phytoplankton growth medium 194,208 Phytoplankton productivity 194 Platinum electrode 68,104 Polarizer angle, ellipsometric 138 Polynucleotides, electrical fields on natural 113 Porphomethene moiety 30 Porphyrin(s) 26 c 26,27,29 complexes, π cation and π anion radical reactions of 51 electrooxidation-reduction of 67 redox properties, iron 65 ring, reactions at 53, 55 Potential s ) 133 biological membranes 114 of a cell, surface 115 curves 137 dependence of adsorption on 132 electrocapillary maximum ( E C M ) 121 injury 114 for isoelectronic iron ( IV ) dimers, oxidation and reduction 75 of metal ion couples 32 sedimentation 180 step 15 streaming 180 voltammetric peak 106 Precision 203 Product concentration gradient, steady state 175 Product formation 172 Proteins, heme 32,46,47 Pulsatile electrokinetic potential 188
113 61 62 57 58 60
Q q (electrochemical charge)
148
R Radical cation, benzylviologen Radicals, anion and cation
149 55
behavior of manganese complexes .. 98 compounds as mediator-titrants 162 coupling between biocomponent(s) and electrode 143 mechanism 105 model for mitochondrial superoxide dismutase 97 properties, iron porphyrin 65 properties of Μη( II ) - and Mi( III ) 8-quinolinol complexes 105 properties of a N-bridged dimer ... 65 Reaction(s) of Co (II) T P P 57 of F e ( I I ) T P P 59 homogeneous 91 Reduction of acetylene, electrocatalytic ...86,87,91 of adduct, electrochemical 91 of B 9 of binuclear molybdenum ( V ) complexes, electrochemical 83 C d E T A complex 197 of the C d N T A complex, electrochemical 196 of the central metal ion 32 of ( F e T P P ) N , electrochemical ... 67 of a neutral porphyrin ring 55 of nitrogenase substrates 78, 84 one-electron 18 potentials for isoelectronic iron (IV) dimer 75 rate, acetylene 90 reaction sequence for a 143 Regeneration mechanism ec catalytic 148 Reoxidation of cob(I)alamin 3,5 Reoxidation of cob(II)alamin 5 Ring current, steady state 177,178 Ring for reaction monitoring 176 Rotating ring disk enzyme electrode .. 170 1 2
2
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
216
ELECTROCHEMICAL
S Scan rate dependence Seawater, synthetic (SSW) Sedimentation potentials Signal amplitude Signal voltage level Sinusoidal voltages
70 194,195 180 188 186 190
Skeletonema costatum
208
Solution reaction 143 Solvents 27 Spectroelectrochemistry of the cobalamin systems 7 Spreading pressure values, surface .... 121 Stability constant, competition (K ) .. 197 Standard addition 198, 205, 206 Standard calomel electrode (SCE) .53,117 Steady state product concentration gradient 175 Steady state ring current 177,17 Stoichiometry, of bioredox components 143 Streaming potential s ) 180,191 Stroke volume 188 Structural effects of electrical fields .. 113 Structural modifications, ligand 32 Substituent constants, Hammet (σ ) 43 Substituent effect 43 Substrate mass transport, rate of 170 Sulfido-bridge complexes of Mo( V) .79, 80 Sulfur bridging atoms 88 Superoxide dismutase (SOD) 97 Superoxide ion 97 Surface charge 131 coverage 128,133 potential of a cell 115 reactions, catalytic 170 spreading pressure 121,131 c
STUDIES O F B I O L O G I C A L
SYSTEMS
Tetramethyl-p-phenylenediamine (TMPD) Tetraphenylporphyrin complexes Thermodynamic ( s ) of Co(II)TPP complexation with pyridine data for addition of pyridine to cobalt ( II ) tetraphenyl porphyrin for addition of pyridine to iron ( II ) tetraphenyl porphyrin of electron transfer and ligand binding of metalloporphyrins .. Time constant, convection (f ) Titration of biocomponents, indirect coulometric k
166 74 58
57 60 51 173 143
+
Trans-Co (13-16 ane-N )Cl 36 Transition energies (d-d) 48 Tris ( hydroxymethyl ) aminomethane (tris) 194 Turbulent region 189 4
2
ρ
Τ t (convection time constant) 173 TBAP 68 TEAP-DMSO 104 Temperature, half wave potential as function of 61 Tetraaza macrocyclic ligands 34, 35, 41, 45 Tetraaza tetradentate macrocycles .... 32 Tetrabutylammonium perchlorate (TBAP) 66
U Ueda effects
190 V
Van't Hoff plot 61, 62 Velocity parameter, reaction (V) 173 Ventricle/aorta simulator, left 180 Viologens (bipyridylium salts) 146,147,149 Vitamin B i 1 Voltage level, signal 186 Voltages, sinusoidal 190 Voltammetric peak potentials 106 2
k
W
Waveform(s)
187,191 Ζ
Zinc etioporphyrin Zinc octaethylporphyrin ZnTPP
In Electrochemical Studies of Biological Systems; Sawyer, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.
55 55 54
