Studies of Biological Redox Systems by Thin-Layer Electrochemical

1 Studies of Biological Redox Systems by Thin-Layer Electrochemical Techniques 1

WILLIAM R. HEINEMAN, C. WILLIAM ANDERSON, H. BRIAN HALSALL, MARILYN M. HURST, JAY M. JOHNSON, GEORGE P. KREISHMAN, BARBARA J. NORRIS, MICHAEL J. SIMONE, and CHIH-HO SU 2

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Downloaded by 80.82.77.83 on May 18, 2017 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0201.ch001

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University of Cincinnati, Department of Chemistry, Cincinnati, OH 45221 Thin-layer electrochemical techniques were developed for measuring E°' and n-values of biological redox systems. A spectropotentiostatic method combines optical measurements of the biocomponent with potential control of a thin solution layer. Coupling of the biocomponent to the electrode potential is achieved by chemical modification of the electrode or by addition of a mediator-titrant. The temperature dependence of E°' is determined easily. This condition is exemplified by cytochrome c for which measurements were sufficiently precise to resolve a nonlinear behavior in the E°' temperature dependence. Biocomponents with no observable optical properties can be studied by thin-layer pulse coulometry and thin-layer staircase coulometry. In these techniques, E°' and n are determined by measuring charge as a function of potential. The activity of galactose oxidase was measured while simultaneously controlling its oxidation state. The oxidation state is controlled by a gold electrode (coupled with ferricyanide as a mediator-titrant) in a thin-layer cell with a peroxide electrode inserted in one side. The relative activity of the enzyme is monitored by measuring peroxide production from the enzyme-catalyzed reaction of substrate. 1

Current address: Duke University, Department of Chemistry, Durham, NC 27706 Current address: University of North Carolina at Greensboro, Department of Chemistry, Greensboro, NC 27412 Current address: Yellow Springs Instrument Co., Yellow Springs, OH 45387 Current address: Abbott Laboratories, North Chicago, IL 60064 Current address: University of Houston, Department of Chemistry, Houston, TX 77004 2

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0065-2393/82/0201-0001$06.25/0 © 1982 American Chemical Society Kadish; Electrochemical and Spectrochemical Studies of Biological Redox Components Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

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BIOLOGICAL REDOX COMPONENTS

r e d o x characteristics o f b i o l o g i c a l r e d o x systems is i m p o r t a n t for u n d e r s t a n d i n g b i o l o g i c a l p r o c e s s e s s u c h as o x i d a t i v e phosphorylation a n d photosynthesis (J, 2). T h e m e a s u r e m e n t o f formal r e d u c t i o n p o t e n t i a l s ( Ε ° ' ) a n d e l e c t r o n t r a n s f e r s t o i c h i o m e t r i c s (re v a l u e s ) is a s i g n i f i c a n t a s p e c t o f t h e s e i n v e s t i g a t i o n s . M a n y b i o l o g i c a l s p e c i e s s u c h as c y t o c h r o m e s (3, 4) a n d f e r r e d o x i n s (5) u n d e r g o h e t e r o g e n e o u s e l e c t r o n t r a n s f e r v e r y s l o w l y o r i r r e v e r s i b l y . S u c h b e h a v i o r is u s u a l l y a t t r i b u t a b l e to s e v e r e a d s o r p t i o n o f t h e b i o c o m p o n e n t or to i n s u l a t i o n o f the r e d o x center from the e l e c t r o d e b y the s u r r o u n d i n g p r o t e i n structure. T h e s e b i o l o g i c a l s p e c i e s are difficult to s t u d y b y c o n v e n t i o n a l e l e c t r o c h e m i c a l m e t h o d s s u c h as c y c l i c v o l t a m m e t r y .


Investigating the

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 q u e s b a s e d o n m e d i a t o r - t i t r a n t s (6) h a v e b e e n d e v e l o p e d a n d e f f e c t i v e l y u s e d to m e a s u r e t h e E°' a n d η - v a l u e s for b i o l o g i c a l s y s t e m s t h a t e x h i b i t i r r e v e r s i b l e e l e c t r o c h e m i c a l b e h a v i o r . T h e i n d i r e c t c o u l o m e t r i c titration m e t h o d w a s a p p l i e d to t h e s t u d y o f c y t o c h r o m e c (7), c y t o c h r o m e c o x i d a s e (8-12), i n t a c t m i t o c h o n d r i a , a n d 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 (13). S o l u b l e s p i n a c h f e r r e d o x i n a l s o w a s s t u d i e d b y t h i s t e c h n i q u e (14). M e t h o d s b a s e d o n t h e t h i n - l a y e r e l e c t r o d e w e r e a p p l i e d to t h e s t u d y o f c y t o c h r o m e c (3, 15), m y o g l o b i n (15), a n d g a l a c t o s e o x i d a s e (16). T h e s e t h i n - l a y e r 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 are d e s c r i b e d a n d d i s c u s s e d i n this chapter.

Thin-Layer Cells A thin-layer e l e c t r o c h e m i c a l c e l l confines a t h i n solution layer a d j a c e n t to o n e or m o r e e l e c t r o d e s (17). T h e t h i c k n e s s o f t h i s l a y e r is t y p i c a l l y less t h a n 0.3 m m . C o m p l e t e e l e c t r o l y s i s o f e l e c t r o a c t i v e s p e c i e s o c c u r s r a p i d l y as a r e s u l t o f t h e s h o r t d i f f u s i o n a l p a t h w i t h i n the t h i n solution layer. T h i s feature enables r a p i d conversion o f redox c o u p l e o x i d a t i o n states t h r o u g h o u t t h e t h i n l a y e r b y a p p r o p r i a t e p o t e n tial control o f the w o r k i n g electrode. T h e 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 d e s c r i b e d h e r e are a l l b a s e d o n t h i s f e a t u r e o f r a p i d e l e c t r o l y s i s . A n o p t i c a l l y transparent thin-layer electrode ( O T T L E ) confines a t h i n l a y e r o f s o l u t i o n a d j a c e n t to a n e l e c t r o d e t h a t is t r a n s p a r e n t to l i g h t (18). T h i s a r r a n g e m e n t e n a b l e s s p e c t r a l a n d e l e c t r o c h e m i c a l m e a s u r e m e n t s to b e m a d e s i m u l t a n e o u s l y o n t h e t h i n s o l u t i o n l a y e r b y an optical b e a m passing through both solution a n d electrode. A n e a s i l y c o n s t r u c t e d O T T L E is t h e g o l d m i n i g r i d s a n d w i c h e d b e t w e e n t w o m i c r o s c o p e s l i d e s w i t h T e f l o n t a p e s p a c e r s , as s h o w n i n F i g u r e 1 (3). S o l u t i o n is d r a w n f r o m t h e r e s e r v o i r c u p i n t o t h e t h i n l a y e r b y s u c t i o n at t h e t o p o f t h e c e l l . T h e t h i n s o l u t i o n l a y e r is t y p i c a l l y 0 . 2 - 0 . 3 m m t h i c k , a c o m p r o m i s e v a l u e that gives m a x i m u m o p t i c a l p a t h l e n g t h w h i l e r e t a i n i n g the t h i n - l a y e r feature o f r a p i d e l e c t r o l y s i s . I n s u c h a c e l l o n l y a b o u t 4 0 / x L is e l e c t r o l y z e d i n t h e v o l u m e

Kadish; Electrochemical and Spectrochemical Studies of Biological Redox Components Advances in Chemistry; American Chemical Society: Washington, DC, 1982.


1.

HEiNEMAN ET AL.

3 Thin-Layer

A

Electrochemical

Techniques

Β

Figure 1. Front (A) and side (B) views of OTTLE (3). Key: a, point of suction application to change solution; b, Teflon tape spacers; c, microscope slides (1x3 in.); d, solution between microscopic slides; e, trans parent gold minigrid electrode; f optical path of spectrophotometer; g, reference and auxiliary electrodes; h, cup containing solution. (Re produced from Ref. 3. Copyright 1975, American Chemical Society.) d e f i n e d b y the area o f the m i n i g r i d , a l t h o u g h a f e w m i l l i l i t e r s are r e q u i r e d to use the c e l l . O x y g e n c a n b e e x c l u d e d , w h e n necessary, b y p l a c i n g t h e c e l l i n a p l a s t i c b o x fitted w i t h o p t i c a l w i n d o w s a n d filled w i t h i n e r t gas (29). S a m p l e d e o x y g e n a t i o n a n d t r a n s f e r t e c h n i q u e s w e r e d e v e l o p e d . T h e e x t e n s i v e u s e o f t h i s c e l l is a t t r i b u t a b l e t o its s i m p l i c i t y a n d ease o f fabrication. T h e t r a n s p a r e n t e l e c t r o d e u s e d m o s t o f t e n i n t h e O T T L E is g o l d m i n i g r i d (3, 18), w i t h e i t h e r 100 w i r e s / i n . (80% t r a n s p a r e n t ) o r 500 w i r e s / i n . (60% t r a n s p a r e n t ) . M e r c u r y c a n b e d e p o s i t e d o n t h e g o l d m i n i g r i d to e x t e n d t h e n e g a t i v e p o t e n t i a l r a n g e (20). O t h e r t r a n s p a r e n t e l e c t r o d e s s u c h as p l a t i n u m m e s h (21), t i n o x i d e (6), a n d r e t i c u l a t e d v i t r e o u s c a r b o n a l s o w e r e u s e d (22). S e v e r a l O T T L E c e l l s w e r e d e v e l o p e d for u s e w i t h b i o l o g i c a l s y s t e m s w h e r e a n a e r o b i c i t y a n d s m a l l t o t a l v o l u m e are i m p o r t a n t . T h e c e l l s h o w n i n F i g u r e 2 r e q u i r e s less t h a n 100 μ-L to fill (23). It is e a s i l y



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BO ILOGC IAL REDOX COMPONENTS

Figure 2. Small-volume OTTLE (23). Key: A, quartz cover plate; B, Teflon spacers; C, gold minigrid; D, quartz disk; E, plastic body; F, inlet syringe; G, overflow reservoir for reference and auxiliary electrode. (Reproduced, with permission, from Ref 23. Copyright 1979, Academic Press.)

m a c h i n e d from a L u c i t e b l o c k . T h e m a c h i n e d b l o c k facilitates the i n t r o d u c t i o n o f solution b e t w e e n the quartz c o v e r plate a n d the d i s k that defines the t h i n layer. C e l l s o f this t y p e w e r e u s e d e x t e n s i v e l y i n o u r s t u d i e s o f c y t o c h r o m e c. A c e l l o f s i m i l a r d e s i g n w a s u s e d i n c o n j u n c t i o n w i t h l i q u i d c h r o m a t o g r a p h y (24). A n O T T L E t h a t is p a r t i c u l a r l y u s e f u l for l o w t e m p e r a t u r e m e a surements was reported (25). A c i r c u l a t i n g , long-optical-path, spect r o e l e c t r o c h e m i c a l t h i n - l a y e r c e l l ( C L O S E T ) t h a t r e t a i n s t h e fast e l e c t r o l y s i s f e a t u r e o f t h e t h i n - l a y e r c e l l , b u t h a s a 1-cm o p t i c a l p a t h , w a s d e s c r i b e d (26, 27). A s e m i p e r m e a b l e thin-layer c e l l that enables c o n t r o l o f the oxida t i o n state o f a r e d o x e n z y m e w h i l e s i m u l t a n e o u s l y m o n i t o r i n g t h e p r o d u c t o f s u b s t r a t e r e a c t i o n is s h o w n i n F i g u r e 3. T h i s c e l l w a s u s e d to s t u d y g a l a c t o s e o x i d a s e [ v i d e i n f r a (16)].


Thin-Layer

1. HE N IEMAN ET AL.

Electrochemical

Techniques

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Spectropotentiostatic Technique A s p e c t r o p o t e n t i o s t a t i c t e c h n i q u e w a s d e v e l o p e d for d e t e r m i n i n g formal reduction potentials, E ° ' , a n d electron stoichiometrics, η - v a l u e s , o f r e d o x c o u p l e s (3). T h e b a s i s o f t h i s t e c h n i q u e is c o n t r o l o f the ratio [0]/[R] o f the r e d o x c o u p l e i n the t h i n solution layer b y the p o t e n t i a l a p p l i e d t o t h e c e l l . T h e r e d o x c o u p l e is i n c r e m e n t a l l y c o n v e r t e d f r o m o n e o x i d a t i o n state t o t h e o t h e r b y a series o f a p p l i e d p o t e n t i a l s (E pi) for w h i c h e a c h c o r r e s p o n d i n g v a l u e o f [0]/[R] is d e t e r m i n e d from spectra. E a c h potential is m a i n t a i n e d u n t i l electrolysis c e a s e s so t h a t t h e e q u i l i b r i u m v a l u e o f [0]/[R] i s e s t a b l i s h e d as d e fined b y t h e N e r n s t e q u a t i o n . F o r b i o l o g i c a l s y s t e m s t h a t u n d e r g o s l o w - e l e c t r o n transfer w i t h the e l e c t r o d e , a m e d i a t o r - t i t r a n t c a n b e a d d e d to c o n v e y electrons b e t w e e n e l e c t r o d e a n d b i o c o m p o n e n t , that is, c o u p l e t h e s o l u t i o n p o t e n t i a l to t h e e l e c t r o d e p o t e n t i a l . R e d u c t i o n / o x i d a t i o n o f t h e b i o c o m p o n e n t is t h u s i n d i r e c t t h r o u g h t h e m e d i a t o r t i t r a n t as s h o w n i n S c h e m e I for a r e d u c t i o n .


aP

Figure 3. Microscopic cross section of semipermeable thin-layer cell. A , 5-μ m thick porous polycarbonate support material for cellulose ace tate membrane with pores nominally 12 μ m in diameter and a pore density of 1 x 10 pores I cm . B, cellulose acetate membrane that is approximately 1 μ m thick and contains pores approximately 6 Â in diameter. C , thin layer ( ~ 4 x 10~ cm thick) containing a 500-lpi gold minigrid electrode (—2.5 μ m thick). One of the Ag/AgCl electrodes behind the thin-layer cell is used as the reference electrode for the gold minigrid electrode. The other Ag/AgCl and the Pt electrode are used to measure H 0 amperometrically. Dimensions of the electrodes and the pores are not to scale. s

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Scheme I


T h e a p p l i c a b l e N e r n s t e q u a t i o n for m e d i a t o r - t i t r a n t a n d b i o c o m p o n e n t is

£ap

P l

- £ . +

log ^

- £. +

n f t F

log

[

B

J

(1)

where m and r e f e r to t h e m e d i a t o r - t i t r a n t a n d t h e b i o c o m p o n e n t , r e s p e c t i v e l y ; a n d E°' a n d η a r e d e t e r m i n e d f r o m a N e r n s t p l o t o f t h e values f o r E a n d t h e c o r r e s p o n d i n g v a l u e s o f [ 0 ] / [ R ] , as d e t e r m i n e d f r o m t h e s p e c t r a r e c o r d e d for e a c h p o t e n t i a l . a p p I

T h e spectropotentiostatic technique was used extensively w i t h c y t o c h r o m e c (3, 2 3 , 28-30). T h e c y c l i c v o l t a m m o g r a m i n F i g u r e 4 i l l u s t r a t e s t h e e x t e n t o f i r r e v e r s i b i l i t y for c y t o c h r o m e c at a g o l d m i n i grid i n an O T T L E . T h e addition of 2,6-dichlorophenolindophenol ( D C I P ) as a m e d i a t o r - t i t r a n t e n h a n c e s t h e r a t e o f r e d u c t i o n / o x i d a t i o n o f c y t o c h r o m e c b y t h e f o l l o w i n g i n d i r e c t p r o c e s s at t h e e l e c t r o d e :

CI

a n d i n the s o l u t i o n : M

R

+ 2 c y t c(ox)

M

0

+ 2 c y t c(red)


(3)

Thin-Layer


Electrochemical

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Techniques

Q01 mA

0.2


OA

0 Ε, V

-02

-0.4

vs. SCE

Figure 4. Thin-layer cyclic voltammogram of 0.5 m M cytochrome c in 0.1 M NaCl, pH 7.0 phosphate buffer. Scan rate 1 mVls. Initial scan, positive from —0.6 V.

Spectra for cytochrome c (with the mediator-titrant added) for a series of applied potentials are shown in Figure 5. The cytochrome is fully oxidized at 300 mV and fully reduced at -600 mV vs. SCE. Spectra were recorded under conditions of equilibrium, that is, current had dropped to a negligible level and spectral changes had ceased. About 10 min is required to achieve equilibrium after a potential change, although this time is dependent on the mediator-titrant concentration and the O T T L E design. Typical Nernst plots for cytochrome c at vari ous temperatures are shown in Figure 6. Values of E° = 262±1 mV vs. SHE and η = 1.00 are obtained from the intercept and the slope, re spectively, at 25°C (3). Thus, very precise values of E°' can be obtained for a biological redox system that essentially is electrochemically ir reversible at a gold electrode. The spectropotentiostatic technique, in conjunction with the O T T L E , facilitates the acquisition of thermodynamic parameters deal ing with the temperature dependence of E°' of a biocomponent (28,29). The temperature of the O T T L E is easily controlled by clamping a cell of the type shown in Figure 2 between two water circulating thermal blocks. Nernst plots for cytochrome c at different temperatures are shown in Figure 6. The reduction potential of horse heart cytochrome c, in various sodium halide solutions in H2O and D2O, was measured over the temperature range of 25 to 50°C (28-30). All samples in D 0 and samples in H 0 not containing chloride ion give a linear tempera ture dependence. In aqueous chloride solutions, the temperature def

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» 480

ι

I 500

I

I 520

I

I 540

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1

L

560

WAVELENGTH, nm

Figure 5. Spectra of 0.5 m M cytochrome c with 0.1 m M DCIP (mediator-titrant) in 0.5 M phosphate buffer, pH 7.0, for a series of applied potentials, vs. SCE (23). (Reproduced, with permission, from Ref 23. Copyright 1979, Academic Press.) p e n d e n c e is n o n l i n e a r w i t h a n a p p a r e n t i n t e r s e c t i o n p o i n t at 4 2 ° C , s h o w n i n F i g u r e 7. T h i s n o n l i n e a r b e h a v i o r w a s i n t e r p r e t e d i n t e r m s a s t r u c t u r a l c h a n g e i n t h e b u l k w a t e r at 4 2 ° C for C 1 ~ - H 0 s o l u t i o n s . this case the g o o d p r e c i s i o n o f the s p e c t r o p o t e n t i o s t a t i c t e c h n i q u e essential i n defining the n o n l i n e a r behavior. 2


as of In is

Thin-Layer



Electrochemical

9

Techniques

LOG I0]/[R] Figure 6. Nernst plots for 0.1 m M cytochrome c, 0.1 m M DC IP (mediator-titrant), pH 7.0, 0.1 M phosphate buffer, and 0.1 M NaCl at varying temperatures, °C. [0]/[R] obtained at 550 nm from spectra recorded in OTTLE of the type in Figure 2 (15). (Reproduced, with permission, from Ref. 15. Copyright 1979, Elsevier Sequoia.) Spectropotentiostatic measurements have been made on several other b i o c o m p o n e n t s . Spectra o f m y o g l o b i n w i t h p h e n a z i n e m e t h o s u l fate as t h e m e d i a t o r - t i t r a n t g a v e a N e r n s t p l o t w i t h E = 46.4 m V vs. S H E a n d η = 0 . 9 5 (15). M e a s u r e m e n t s w e r e m a d e at v a r i o u s t e m p e r a tures on the c o p p e r b l u e proteins a z u r i n , p l a s t o c y a n i n , a n d stell a c y a n i n w i t h a p p r o p r i a t e m e d i a t o r - t i t r a n t s (31). V i t a m i n B has b e e n i n v e s t i g a t e d b y O T T L E m e t h o d s (21, 32-36). I n t h i s c a s e , n o m e d i a t o r - t i t r a n t s w e r e n e c e s s a r y , a l t h o u g h t h e e l e c t r o d e r e a c t i o n for t h e c o b a l t ( I I I ) / c o b a l t ( I I ) c o u p l e w a s v e r y s l o w (36). P h o t o s y n t h e t i c e l e c t r o n t r a n s p o r t c o m p o n e n t s p r o v e d a m e n a b l e to i n v e s t i g a t i o n b y O T T L E t e c h n i q u e s (25). or

i

2



10


TEMPERATURE, °C

Figure 7. Temperature dependence of E°' for horse heart cytochrome c in 0.10 M sodium halide solutions in H 0. Solutions contain 0.10 M sodium phosphate buffer, pH 7.0, and 0.1 mM DC1P (29). Key: O, Fl~; A, Cl~; Φ, Br~; and • , I~. (Reproduced, with permission,from Ref. 29. Copyright 1978, Elsevier Sequoia.) 2

A l t h o u g h i t w a s o r i g i n a l l y d e v e l o p e d for t h e s t u d y o f b i o l o g i c a l r e d o x c o m p o n e n t s (3), t h e O T T L E s p e c t r o p o t e n t i o s t a t i c t e c h n i q u e is e q u a l l y u s e f u l for t h e s t u d y o f i n o r g a n i c m e t a l c o m p l e x e s (37) a n d o r g a n i c c o m p o u n d s (38). T h e a d v a n t a g e o f t h e O T T L E s p e c t r o p o t e n t i o s t a t i c t e c h n i q u e is t h e e a s e w i t h w h i c h t h e r e d o x state c a n b e c o n t r o l l e d i n t h e t h i n solution layer. R e v e r s i b l e redox systems can be c y c l e d repetitively


1.

HEiNEMAN

ET AL.

Thin-Layer Electrochemical Techniques


b e t w e e n o x i d a t i o n states. A d i s a d v a n t a g e is t h e s h o r t o p t i c a l p a t h o f the O T T L E , necessitating the use o f h i g h e r concentrations o f the b i o c o m p o n e n t . S i g n a l a v e r a g i n g t e c h n i q u e s a n d m u l t i p l e passes o f t h e optical b e a m t h r o u g h the O T T L E c a n m i n i m i z e this p r o b l e m . Mediator-Titrants. Control o f the solution potential by r e d u c t i o n / o x i d a t i o n o f t h e b i o c o m p o n e n t v i a a m e d i a t o r - t i t r a n t (6) is c r u c i a l for o b t a i n i n g v a l u e s o f E°' for s o m e b i o l o g i c a l s y s t e m s ( 3 9 ) . F o r e x a m p l e , h o u r s rather t h a n m i n u t e s are r e q u i r e d to e l e c t r o l y z e c y t o c h r o m e c i n the O T T L E i n the absence o f a m e d i a t o r - t i t r a n t (3). R e q u i r e m e n t s for a n o p t i m u m m e d i a t o r - t i t r a n t for u s e i n t h e O T T L E a r e r a p i d , r e v e r s i b l e heterogeneous electron exchange w i t h the electrode a n d h o m o g e n e o u s e l e c t r o n transfer w i t h the b i o c o m p o n e n t , s t a b i l i t y i n b o t h o x i d a t i o n states, a b s e n c e o f s p e c t r a l p r o p e r t i e s t h a t i n t e r f e r e w i t h optically m o n i t o r i n g the b i o c o m p o n e n t , a n d absence o f b i n d i n g w i t h t h e b i o c o m p o n e n t c a u s i n g a s h i f t i n E° o f t h e b i o c o m p o n e n t . T h e potential range over w h i c h a mediator-titrant w i l l control solution p o t e n t i a l w a s m e a s u r e d for n u m e r o u s c o m p o u n d s (39). A t y p i c a l m e d i a t o r - t i t r a n t w i l l control the solution potential over a range o f E ± 50 m V . A large potential range can b e covered b y m i x i n g mediator-titrants w i t h £ ° ' values that s p a n the d e s i r e d range (39). f

0f

A n a l t e r n a t i v e a p p r o a c h to t h e u s e o f m e d i a t o r - t i t r a n t s for c o u p l i n g t h e e l e c t r o d e t o t h e b i o c o m p o n e n t is c h e m i c a l m o d i f i c a t i o n o f the e l e c t r o d e . A l t h o u g h r e l a t i v e l y n e w , this a p p r o a c h a l r e a d y has s h o w n p r o m i s i n g r e s u l t s for c y t o c h r o m e c (40), m y o g l o b i n (41), a n d f e r r e d o x i n (5). Spectroscopic T e c h n i q u e s . U V - v i s i b l e absorption spectroscopy was the m a i n optical t e c h n i q u e used i n conjunction w i t h O T T L E stud i e s o f b i o l o g i c a l s y s t e m s (3). L i g h t - i n d u c e d a b s o r p t i o n c h a n g e s , fluorescence y i e l d changes, a n d circular d i c h r o i s m also w e r e m e a s u r e d w i t h the O T T L E i n studies o f photosynthetic e l e c t r o n transport c o m p o n e n t s (25). The fluorescence emission spectrum o f tryptophan-59 i n cyto c h r o m e c r e c e n t l y w a s m e a s u r e d for t h e o x i d i z e d a n d r e d u c e d f o r m s ( F i g u r e 8) (42). B e c a u s e fluorescence u s u a l l y is s e n s i t i v e t o s m a l l e n v i r o n m e n t a l c h a n g e s o f t h e fluorophor, s m a l l c o n f o r m a t i o n a l c h a n g e s i n t h e p r o t e i n c a n b e d e t e c t e d . T h e fluorescence i n t e n s i t y o f t h e t r y p t o p h a n o f c y t o c h r o m e c is a t t e n u a t e d g r e a t l y b y t w o f a c t o r s : (1) effi c i e n t e n e r g y t r a n s f e r t o t h e h e m e a n d (2) q u e n c h i n g b y o t h e r c y t o c h r o m e c m o l e c u l e s i n s o l u t i o n . T a k i n g t h e s e t w o factors i n t o a c c o u n t , t h e o b s e r v e d r a t i o o f fluorescence i n t e n s i t i e s for t h e t w o f o r m s c a n b e e x p l a i n e d i f one assumes a m o v e m e n t o f the tryptophan t o w a r d the h e m e o f 0.7 ± 0.3 A o n r e d u c t i o n . B e c a u s e t h e t w o f o r m s o f t h e c y t o c h r o m e c w e r e g e n e r a t e d i n situ i n the O T T L E , this analysis c o u l d b e carried out u t i l i z i n g uncorrected spectra. I n addition, the l o w e r detec-


11


12


350

400

450

500

ληηι

Figure 8. Thin-layer fluorescence emission spectra of tryptophan-59 in cytochrome c. A, Reduced state, E = -600 mV vs. SCE; B, oxidized state, E = 200 mV vs. SCE. 288-nm excitation. Conditions: 1.0 m M cytochrome c, 0.05 m M DC1P, and 0.5 M NaCl in 0.1 M phos phate buffer, pH 7.0. (Reproduced, with permission, from Ref. 42. Copyright 1982, Academic Press.) appl

appl

tion limit o f

fluorescence

enables m e a s u r e m e n t s to b e m a d e o n m u c h

l o w e r c o n c e n t r a t i o n s o f c y t o c h r o m e c t h a n is p o s s i b l e w i t h U V - v i s i b l e absorption spectroscopy.

Thin-Layer Pulse and Staircase Coulometry T w o t h i n - l a y e r 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 w e r e d e v e l o p e d for m e a s u r i n g E°' a n d η o f b i o c o m p o n e n t s w i t h s u c h w e a k s p e c t r a l c h a n g e s t h a t t h e s p e c t r o p o t e n t i o s t a t i c t e c h n i q u e is i m p r a c t i c a l (43). T h e t w o techniques d o not require the measurement o f optical changes i n either the b i o c o m p o n e n t or the m e d i a t o r - t i t r a n t . T h e y are b a s e d o n t h e c h a r g e r e s p o n s e to p o t e n t i a l p u l s e o r p o t e n t i a l s t a i r c a s e excitation signals. B o t h techniques c a n b e u s e d w i t h the O T T L E s s h o w n i n Figures 1 a n d 2. Thin-Layer Pulse Coulometry. In thin-layer pulse coulometry, t h e e l e c t r o d e p o t e n t i a l is s t e p p e d f r o m a s i n g l e i n i t i a l p o t e n t i a l , at w h i c h t h e r e d o x c o m p o n e n t ( s ) is e n t i r e l y i n o n e o x i d a t i o n state, t o a series o f p o t e n t i a l s that c o n v e r t a n i n c r e a s i n g fraction o f the r e d o x c o m p o n e n t ( s ) i n t o a n o t h e r o x i d a t i o n state. T h e e x c i t a t i o n w a v e f o r m is s h o w n i n F i g u r e 9 A . F o r e a c h potential, the ratio o f the concentrations o f o x i d i z e d to r e d u c e d forms, [ 0 ] / [ H ] , o f t h e r e d o x s p e c i e s i n t h e t h i n


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s o l u t i o n l a y e r a d j u s t s b y e l e c t r o l y s i s to t h e v a l u e r e q u i r e d b y Nernst equation

(for a r e v e r s i b l e s y s t e m ) . T h e t h i n - l a y e r

the

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e n a b l e s t h e e q u i l i b r i u m [ 0 ] / [ R ] v a l u e c o r r e s p o n d i n g to e a c h p o t e n t i a l s t e p to b e o b t a i n e d f r o m t h e c h a r g e r e q u i r e d for e l e c t r o l y s i s o f t h e t h i n s o l u t i o n l a y e r . T h e r a n g e o f t h e s e r i e s o f p o t e n t i a l s to w h i c h t h e e l e c OR

t r o d e is s t e p p e d is s e l e c t e d to s p a n t h e E

o f t h e r e d o x c o m p o n e n t so

t h a t c h a r g e s are o b t a i n e d for t h e e x t r e m e s o f c o m p l e t e o x i d a t i o n a n d r e d u c t i o n as w e l l as for i n t e r m e d i a t e v a l u e s o f [ 0 ] / [ R ] . A p l o t o f t h e c h a r g e for e a c h p o t e n t i a l s t e p v s . t h e p o t e n t i a l to w h i c h t h e

electrode

is s t e p p e d g i v e s a Ç - Ε p l o t t h a t r e s e m b l e s t h e t r a d i t i o n a l p o l a r o g r a m . T h e E°'

a n d η - v a l u e s are o b t a i n e d f r o m t h e i n t e r c e p t a n d s l o p e , r e


s p e c t i v e l y , o f a N e r n s t p l o t i n the form o f E q u a t i o n 4.

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i n close

p r o x i m i t y to t h e r e d o x p o t e n t i a l o f t h e b i o l o g i c a l s p e c i e s . S u c h a p l o t for t h e m e d i a t o r - t i t r a n t D C I P is s h o w n b y C u r v e A i n F i g u r e 1 0 . T h e b i o c o m p o n e n t is t h e n a d d e d to t h e s o l u t i o n a n d a s e c o n d Q-E

p l o t is

o b t a i n e d . T h e r e s u l t i n g p l o t for t h e a d d i t i o n o f c y t o c h r o m e c is s h o w n b y C u r v e Β i n F i g u r e 10. I n t h i s c a s e , Q c o n t a i n s t h e c h a r g e r e q u i r e d to e l e c t r o l y z e b o t h t h e m e d i a t o r - t i t r a n t ( d i r e c t e l e c t r o n e x c h a n g e w i t h the e l e c t r o d e ) a n d the b i o c o m p o n e n t ( i n d i r e c t e l e c t r o n e x c h a n g e

Φ CL CL

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TIME

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Figure 9. Potential excitation signals for A, thin-layer pulse coulo metry and B, thin-layer staircase coulometry. (Reproduced from Ref. 43. Copyright 1981, American Chemical Society.)


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Studies of Biological Redox Systems by Thin-Layer Electrochemical

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