Evaluation of Mediator-Titrants for the Indirect Coulometric Titration of

9

Evaluation

of

Coulometric

Mediator-Titrants

Titration

for

of

the

Indirect

Biocomponents

ROBERT SZENTRIMAY, PETER YEH, and THEODORE KUWANA

Downloaded by CORNELL UNIV on October 26, 2016 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0038.ch009

Department of Chemistry, Ohio State University, Columbus, Ohio 43210

In recent years we have been interested in the development and application of the indirect coulometric titration (ICT) method 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

Sawyer; Electrochemical Studies of Biological Systems 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 e q u i l i b r i u m . Or a l t e r n a t i v e l y , a titrant may be u s e d w h o s e E ° ' v a l u e i s s u f f i c i e n t l y c l o s e to that of the 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


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 the methods of c y c l i c voltammetry, c o n t r o l l e d p o t e n t i a l c o u l o m e t r y , thin l a y e r s p e c t r o e l e c t r o c h e m i s t r y , 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


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 ' s i n ICT of biocomponents at the n a n o 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 ) . +


+

SZENTRIMAY

ι s

ET

Coulometric Titration of Biocomponents

AL.

(0

r—I

I

ε

(0

ο ο ι-Η

ο ο CO

ο ο

ο ο

ο ι—I

CD

CM ι-Η

r—»

ε (0


2

οο

(0

ο

·-!

Λ co m I

(Ο CM r** I

ϋ co »-« co I

in

σ>

(0

m

en

ο

W

CO C D* Ο •g > ^

Ο co

ι-ι

α ο £

£ ω

^ ^ >

J

ro co co m I

ϋ co en to .

5 ,

OCM

pq

5 5

·

to

(θ

(ο

0 0

- »>

l

O

·"·

l

Si O

°o

O

(0

T

°> f

U

to ^>

> - « _ ^ c o c o ^ r r r i r > w « > • • • υ ε CQ χ* ^ 1

g H

α Ο

O

1

ε

Or-» M

γ-Η

-Ό

Ό

β

(Ό ·—» Φ (0

ε

ε

*η ο

α

ε

D

*2

Λ

·2 τ*

is

g

χ>

Ό

^

Ό

8 w

a * ϋ

Ε 3 Λ

s. S Λ

Ε S Λ

à S I

Q

-

CM

f i >, 5 m

TJ
CO

Co

s

ο

3

ta ο ο

θ"

!'

Ε

ο

Ο ο

M H

a

Ν M

C/Î


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

1,500

l

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 -1.0

ι

Titration

of

I 0

Biocomponents

ι

L_ 1.0

log(Ox/Red)

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


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 g T M P D . The 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 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.



9.

SZENTRIMAY ET AL.


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, E.G., Dokl. Akad. Nauk SSSR, (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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