413

Bioelectrochemistry and Bioenergetics, 19 (1988) 413-423

A section of J. Electroanal. Chem., and constituting Vol. 253 (1988)
Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands

F A D used as a mediator in the electron transfer b e t w e e n

piatinmn and several b i o m o l e c u l e s
H. Durliat, M.B. Barrau and M. Comtat
Loboratoire de G~nie Chimique, UA C N R S 192, Laboratoire de Chimie Physique et Electrochimie,
UniversitJ Paul Sabatier, i18 Route de Narbonne, 31062 Toulouse C~dex (France)
(Received 30 July 1987, in revised form I February 1988)

ABSTRACT
Flavin adenine dinucle~tide is used as a mediator to reduce cytochrome c, glucose oxidase and
methemoglobin and to oxidize ferredoxin on the platinum electrode. Thin layer spectroelectrochemistry
allows for precise and simple comparison of these biomacromolecules in the presence or absence of F A D .
Their biological activity is not modified by the electrochemical treatment, and electrochemical preparation of reduced or oxidized biomolecule solutions on a preparative scale can be considered.

INTRODUCTION

Heterogeneous electron transfer between metals or carbon a nd biomolecules is

known to be difficult, if not impossible. However, when carried out in a fast way, it
can be applied in analytical or preparative chemistry. M a n y papers are therefore
being published on the acceleration of these transfers.
Research has been done on the use of surface activators [1-9]. These are
electroinactive organic molecules used in order to avoid the denaturing adsorption
of biomolecules and facilitate their fixation on the electrode. Other studies deal with
the modulation of electrochemical activity by means of electrolysis, using charged
ions or complexes in order to modify the local electrostatic environment at the
electrode/solution interface [10-14]. Electrochemical treatments such as repetitive
potentiodynamic perturbations have been considered [15,16].
The development of the use of mediators fixed in polymer films must also be
noted [17-24].
Finally, m a n y mediators in solution are still being used either with analytical
alms in m i n d or in order to determine thermodynamic da ta [25-29].
03024598/88/$03.50

1988 Elsevier Sequoia S.A.

414

The drawbacks in using these mediators fie in their ability to form complexes
with the biomolecules. They can also lead to biological products deprived of part of
their activity.
The study presented here deals with the use of flavin adenine dinucleotide ( F A D )
as a mediator in the electron transfer between platinum and various biomolecules.
This molecule has several of the qualities necessary for a mediator. Previous
electrochemical-type studies showed that electron exchanges between F A D and
various metals are f a s t . Besides, homogeneous reactions between F A D and the
molecules u n d e r s t u d y are fast too. Finally, since F A D is naturally associated ~,,ith
some molecules studied in electron transfer processes, a better biocompadbility than
that observed with most organic or inorganic mediators can be expected.
EXPERIMENTAL

Apparatus
Thin layer spectroelectrochemistry is the method used. The working electrode
was a platinum grid slotted between two glass slides separated by a 0.05 cm gap.
The auxiliary electrode was made of platinum and has a big surface area. The
reference electrode was a saturated calomel one, to wb~c.h. all potentials will be
referred in the following. The general setup has already been described [15]. It
allows many electrochemical methods to be carried out, particular!y linear sweep
voltammetry and controlled potential electrolysis, which are more specifically used
in this study.
The volume of the solutions treated" was about 40 mm 3. The circuits included a
T;lcussel potentiostat (type PRT 20 x 2), a Tacussel signal generator (type Servovit)
arid a Sefram XY recorder (type Luxy-trace).
Optical measurements were carried out by means of a 845iA Hewlett Packard
spectrophotometer linked to a computerize~l apparatus for the data to be stored and
read out on a plotter.

Reagents
The variou~ solutions were made from twice distilled water plus high purity salts
of various origins.
The buffered solution, pH 7.2, was a mixture of 0.2 M monopotassium and
disodium phosphate. The buffered solT~tion, pH 5.3, was a mixture of 0.13 M
monopotassium phosphate and 0.005 M citric acid.
Horse heart cytochrome c was provided either by Sigma (type VI) or Boehringer.
The protein fraction in reduced fo~,i, was oxidized by potassium ferricyanide; excess
ferricyanide and ferrocyanide formed were eliminated through a Sephadex (325
column.
Methemoglobin was of placental origin; the fraction of oxyhemoglobin present
was oxidized by potassium ferricyanide. The products are separated when applied
on a Sephadex (325 column.

415

Glucose oxidase (GO) from Aspergillus niger, ferredoxin from Clostridium

pasteurianum and flavin adenine dinucleotide were provided by Sigma.
In the case of some experiments with controlled potential, a bigger volume cell
was also used. This parallelepipedic cell was waterproof; ~ e working electrode was
set parallel to the larger surfaces sides. The beam could thus be introduced in order
to follow the reactions by in situ absorbance measurements. The cell was about 1 cm
thick; its volume was about 25 cm 3.

Procedure
The experiments consisted of a comparison between current-potential curves
obtained with protein alone in solution and those observed with F A D + protein
mixtures.
Before any experiment, the platinum grid was oxidized with a flame for several
minutes.
The experiments began with an electrolysis at a constant potential of --0.1 V for
about 15 min, in order to reduce and eliminate the dissolved oxygen present in the
thin layer. The cells were designed so that the experiments could be carried out in
strictly anaerobic conditions. Unless indicated otherwise, the tracings of the current-potential curves begin with a cathodic sweep.
in order "~o comply with the hypothesis concerning thin layer electrochemistry,
the sweep rate of the potentials was adapted to the thickness of the cell: 3.3 m V / s ,
unless specifically mentioned.
Glucose oxidase biological activity before and after electrochemical treatmen,.s
was measured by the following two reactions:
glucose + O2 c'--c'P--~gluconicacid + H202
H202 + reduced o-dianisidine --, oxidized o-dianisidine + H 2 0
The enzyme activity was calculated from the rate of disappearance of reduced
o-dianisidine as followed by spectrophotometry at 436 nm.
For hemoglobin, the equifibrium curves corresponding to the reaction:
deoxyhemoglobin + 02 --* oxyhemoglobin
were compared before and after electrolysis. The experiments consisted of lowering
the pressure in the tonometer sufficiently for all the oxyhemoglobin to be transformed into deoxyhemoglobin. Known volumes of air were then introduced in the
apparatus and the oxy- eald deoxyhemoglobin concentration ratio was determined
by spectrophotometry. Experiments were carried out at 37 C after dilution of the
samples in a pH 7.0 phosphate buffer. Comparison concerned:
(a) the P5o value which corresponds to the oxygen pressure in equilibrium with a
solution containing the two forms in equimolecular quantitie~
(b) the Hill coefficient, i.e. the slope of the straight line [30]:
[oxyhemoglobin]
og-[deoxyhemoglobin] - f (Po2)

416

RESULTS A N D DISCUSSION

FAD

T h e electrochemical b e h a v i o u r of this molecule has been studied previously

[16,31,32]. The a p p a r e n t s t a n d a r d potentials which c o r r e s p o n d to two-electron
transfer are close to --0.34 V a t p H 5.3 a n d to - 0 . 4 5 V a t p H 7.2. T h e conversion
f r o m the oxidized to the reduced form takes place t h r o u g h a semi-quinonic
intermediate ( F A D s ) which allows for a n o x i d e - r e d u c t i o n reaction b e t w e e n F A D
a n d an enzyme whose prosthetic g r o u p only allows for the e x c h a n g e o f one electron.
W h e n the a d s o r b e d molecules are t r a n s f e r r e d on graphite, the film electrodes are
very stable, a n d the transfer c o n s t a n t s higher t h a n 1 s - 1 [33].
O n e m u s t r e m e m b e r that this molecule does not a b s o r b light a b o v e 500 nm.
Cytochrome c

M a n y studies o n this biological molecule can be found in the literature. H e t e r o geneou~ electron transfer rate c o n s t a n t s observed are very varied: 3.5 x 10 -5 c m / s
with methylviologen as a fixed m e d i a t o r [34], a b o u t 1 0 - 2 c m / s with b i p y r i d i n e used
as a surface activator [2,3,35]. A t the m e r c u r y electrode the r a t e c o n s t a n t is f r o m
10 - l i to 10 - 2 c m / s [36]. O n solid metallic o r semi-conducting electrodes, the s a m e
disparity c a n be observed [10,11,35-40].
F o r repetitive p o t e n t i o d y n a m i c p e r t u r b a t i o n s with a p l a t i n u m electrode in contact with the c y t o c h r o m e c solution, a t a potential sweep rate o f 8.3 m V / s , r a t e
constants of the o r d e r of 2 x 10 -5 c m / s are o b t a i n e d [15].
-0.5
=

-0i5

01.5

-4 .-

4 -

UIV' vs, SI~E

0
I

. .

0.5
0.8

"-%,,\

Off

I~.2

~.

l i

'

,~" ~

..J

-2

-'
" .....

[
480

~/n~.,,
6O0

Fig. 1. I - U curve and absorption spectra for a cytochrom c solution; concentration--0.48 r a M ;

p H = 7.2; sweep rate = 0.33 m V / s . (1) Cytochrome entirely oxidized (U ;~ 0); (2) during the electrochemical reduction; (3) cytochrom in the reduced form (U---- - 0 . 6 V).
Fig. 2. I - U curve (
- ) , and absorbance at 550 ntn ( . . . .
) for a 0.50 m M cytochrome +0.10 m M
F A D solution, pH = 7.2; sweep r~lte = 0.33 m V / s . At 550 nm only cytochrome absorbs (top).

0o6

417

The standard potential of the oxidized cytochrome c / r e d u c e d eytochrome c

system is --0.012 V. "l-he experiments were performed at p H 7.2 in a phosphate
buffer.
Spectral modifications due to preliminary reduction of dissolved oxygen in the
presence of cytochrome c in the thin layer were never observed. A reaction between
superoxide ion and oxidized cytochrome can therefore never be obtained.
Sweep voltammetry modifies the rate constant of the electron transfer reaction
between platinum and cytochrome in the absence of potentiodynamic perturbations.
W h e n cytochrome c is the only species in solution, the current-potential curve
corresponding to the first sweep presents a relatively ill-defined reduction and
oxidation peak separated by about 0.3 V, due to the irreversibility of the system
(Fig. 1). However, the absorption spectra show the total transfo,~zation of the
molecules during the tracing of the curve for potentials in the range of the solvent
electroactivity. D uring the second sweep the peaks are considerable lower, the
system becomes more irreversible (the heteroEcncc=s re.~ction rate constant is below
10 -1 c m / s ) and only a small fraction o f - t h e molecules is reduced before the
potential of - 0 . 6 V is reached. This beha_viour ma y be due to strong adsorption of
the molecule on platinum. U nder these conditions, the potential at which the
cytochrome reduction rate becomes relatively high is lower than the F A D reduction
potential, Although comparison of the apparent standard potentials shows a thermodynamically unfavourable oxi ~dized cytochrome c - F A D reaction, the irreversibility of the cytochrome system means that the role of flavin as a mediator can be
considered.

9t

9O

701"-~
o
501-~

30"

p"

10
0

-'< 50

C~

10

2O

ft,o

Fig. 3. Constant potential electrolysis for a cylochrome solution performed in a waterproof ceil.
t.~.ytochrom" 0.07 m ~ r ; p H = 7.2; applied potential - --0.54 V; v o l u m e - - 6 cn~. ( | ) Without F A D ; (2)
with 0.01 m ~ / FAD.

418

A l o n g the electron transfer respiratory chain, the s p o n t a n e o u s reaction is b e tween F A D H 2 a n d the oxidized f o r m of c y t o c h r o m e c. T h u s the s t a n d a r d a p p a r e n t
potential o f the F A D H , / F A D system is lower than that o f cyt cox/cyt C,,d.
If the transfer between oxidized c y t o c h r o m e c a n d p l a t i n u m w e r e fast, two
i n d e p e n d e n t reductions with two specific reduction p e a k s would be observed b y
thin layer electrochemistry.
The first sweep traced in a solution c o n t a i n i n g a mixture of c y t o c h r o m e a n d
flavin corresponds to the sum o f the curves o b t a i n e d with these species a l o n e in
solution; the a b s o r b a n c e m e a s u r e m e n t s show t h a t reduction of c y t o c h r o m e takes
place before the complete reduction of flavin. Similarly, F A D oxidation takes place
at a potential lower than that of cytochrome. T h e flavin system is still a fast system.
In the second sweep, a single p e a k a p p e a r s in reduction, b u t the a b s o r b a n c e
s p e c t r u m (Fig. 2) shows tile t r a n s f o r m a t i o n of all the c y t o c h r o m e molecules at the
potential at which F A D reduction takes place. T h ~ is still true for a c y t o c h r o m e to
F A D concentration ratio equal to 5. The a m o u n t ~f electricity e x c h a n g e d confirms
this result. Because of the F A D disproportionation, the following catalytic m e c h a nism can be proposed, where F A D s is the semi-quinonic form:
F A D + e - -* F A D s
F A D s + Cytox "-~ F A D + cyt~d
D u r i n g oxidation the only change is the decrease in the heterogeneous reaction
rate of cytochrome, which corresponds to a higher irreversibility o f the systera. This
behaviour is m a i n t a i n e d t h r o u g h o u t the following sweeps.
F A D was used as a catalyst to reduce c y t o c h r o m e solutions with larger volume~
than that of the thin layer cell. T h e results are s h o w n in Fig. 3 for a solution
electrolyzed at --0.54 V, which corresponds to the reduction of F A D .
F o r short times the c y t o c h r o m e reduction rate is low because electric energy is
used first to reduce dissolved oxygen. T h e r a t e increases later, a n d the c y t o c h r o m e
reduction rate can be observed to b e multiplied by a factor close to 3 in the presence
of flavin. F A D begins to a p p e a r in its reduced form w h e n 90% o f the c y t o c h r o m e
has already been reduced.
Glucose oxidase
Electron transfer between glucose oxidase a n d a n electrode w a s observed in the
presence of methylviologen included in a polymeric film fixed o n a gold electrode
[41], without a m e d i a t o r on a m e r c u r y electrode [41] a n d with c y a n u r i c chloride used
to fix G O on a graphite electrode [42]. O n platinum, direct transfer w i t h o u t loss of
enzymatic activity after electrochemical reduction a n d reoxidation h a s also b e e n
d e m o n s t r a t e d [16].
Glucose oxidase is studied here at p H 5.3. The a b s o r p t i o n s p e c t r u m o f this
molecule is very close to that of F A D . T h e a p p a r e n t s t a n d a r d potential at p H 5.3 is
close to - 0 . 2 9 V [26], b u t the system is irreversible; therefore the reduction a n d the
reoxidation reactions take place at very different potentials. D u r i n g the tracing of
the c u r r e n t - p o t e n t i a l curves in a thin layer cell, reduction begins at a b o u t --0.3 V

419

4~
"~

-o'.4

-0.1

uiv ~,~scE' - o ~ T ~ ~

-4

.'r:
Ii

.,'__\

Is

-12

3EO

~./n~

---~-

600

Fig. 4. I - U curve and absorption spectra at various potentials for a solution containing 0.32 r n M O 0
a n d 0.32 m M F A D . p H = 5.3; sweep r a t e ----0.33 m V / s . Spectra I t o 4 w e r e o b t a i n e d duriltg r e d u c t i o n at
- 0 . 1 0 , - 0 . 2 8 , --0.32 a n d - 0 . 4 6 V respectively.

b u t the reaction is slow a n d only a fraction o f the molecules is reduced at a potential

higher t h a n that for solvent reduction ( - 0 . 5 V at this p H ) . A t this potential,
electrolysis must last a b o u t 1 h for all the molecules to b e transformed. T h e color of
the solution goes f r o m yellow to colorless a n d the a m o u n t of electricity is in
agreement with the molecules t r a n s f o r m e d (oxygen w a s reduced preliminarily).
Reoxidation is difficult too, a n d 1 h electrolysis at a potential of + 0 . 5 V is
necessary. The high difference between the oxidation a n d the reduction potentials is
characteristic of a n irreversible system.
Figure 4 shows the results which c o r r e s p o n d to a solution in which glucose
oxidase a n d F A D are present in equimolecular a m o u n t s . The c u r r e n t - p o t e n t i a l
curve shows a m a r k e d reduction p e a k a n d the a m o u n t of electricity indicatc~ the
reduction of all the glucose oxidase a n d F A D molecules. However, the o x i d a t i o n
p e a k concerns only the flavin molecules and protein reoxidation remains difficult.
T h e reduction o f glucose oxidase is similar w h e n the F A D c o n c e n t r a t i o n is five
times lower, indicating t h a t F A D ac~s as a mediator. O n the other h a n d , n o decrease
in the enzymatic activity induced b y the electrochemical reduction is observed.

Methemoglobin
A few studies have been carried o u t on the m e r c u r y electrode in which this
molecule shows a half-wave potential of the o r d e r o f --0.6 V a n d in which
electrolysis a t - 0 . 1 V gives a solution whose s p e c t r u m is identical to t h a t of the
oxyhemoglobin to be o b t a i n e d [43]. T h e heterogeneous transfer c o n s t a n t on tin
oxide in the presence of C l - ions is 5.2 X 10 -6 c m / s [44]. ( A c o n s t a n t o f 3.8 x 10 - u
c m / s on a gold electrode modified b y methylviologen [45] is o b t a i n e d w i t h spe~/~
whale myoglobin.)

420
0

24 I
C

8-"~
8
0

UIV vs. $~E

-8

-24

460

650

Fig. 5. I - U curve a n d a b s o r p t i o n spectra for a solution c o n t a i n i n g initially 0.7 m M M e t H b , 1 m M

F A D , 1.5 m M f e r r i c y a n i d e + f e r r o c y a n i d e ; p H = 7.2; sweep r a t e - - 0 . 3 3 m V / s . Spectra ?. a n d 3 are
characteristic o f M e t H b a n d d e o x y h e m o g l o b i n respectively.

Methemoglobin is studied at p H 7.2. This molecule shows n o electrochemical

reaction on the c u r r e n t - p o t e n t i a l curve. Electron e x c h a n g e with p l a t i n u m is very
difficult a n d a b o u t 10 h are necessary to reduce a diluted solution (10 / t M ) b y
electrolysis at - 0 . 7 V. Electrochemical reoxidation could not be carried out. W h e n
the solution is m o r e c o n c e n t r a t e d (0.7 r a M ) reduction becomes negligible.
U n d e r o u r conditions, the electrochemical reoxidation o f d e o x y h e m o g l o b i n o n
p l a t i n u m electrode is not possible. To regenerate methemoglobin, a second m e d i a t o r
is necessary which will react with d e o x y h e m o g l o b i n in a fast way.
Figure 5 shows the c u r r e n t - p o t e n t i a l curve which c o r r e s p o n d s to a solution
containing methemoglobin, F A D a n d f e r r i - f e r r o c y a n i d e mixture.
A t p e a k A, the evolution of spectra 1 to 3, with two isobestic points, shows the
reduction of all the methemoglobin molecules into deoxyhemoglobin.
F A D is a m e d i a t o r in the reduction reaction with the following m e c h a n i s m :
FAD + e- ~ FAD s
4 F A D s + M e t H b --~ 4 F A D + H b
F A D is reduced to F A D H 2 w h e n the m e t h e m o g l o b i n has d i s a p p e a r e d .
Peak B-corresponds to the oxidation of the a d s o r b e d h y d r o g e n f o r m e d at the e n d
of the previous sweep. Reoxidation of F A D takes place later ( p e a k C). W h e n the

42~
ferrocyanide oxidation potential is reached, a new mttalytic p h e n o m e n o n appea~'s
for the reoxidation of deoxyhemoglobin (pea k D):
[ F e ( C N ) 4 ] 4 - =. [ F e ( C N ) 6 ] ' - + e -

4[ F e ( C N ) 6 ] 3 - + H b =-) 4[ F e ( C N ) 6 ] 4 - + M e t H b
T h e o r d e r of the spectra is the opposite of the o n e presented for the reduction of
methemoglobin b y F A D , a n d the s p e c t r u m o b t a i n e d at a poteutial of 0.5 V is
identical to the initial one. D u r i n g the negative sweep, ferricyarfidv is reduced in the
thin layer ( p e a k E).
W h e n the f e r r i - f e r r o c y a n i d e mixture is eliminated b e f o r e the e x p e r i m e n t t h r o u g h
a Sephadex G25 column, the p h e n o m e n a o b s e r v e d between --0.1 a n d - 0 . 5 5 V are
n o t changed.
D u e to the catalytic properties o f F A D , 25 cm 3 of 0.7 m M m e t h e m o g l o b i n in the
presence o f 0.15 m M F A D were reduced in a n o n - o p t i m i z e d parallelepipedic cell in
a few hours [46]. The equilibrium curve for the oxidation o f d e o x y h e m o g l o b i n is
identical in each case to the one observed previous to the electrochemical experiment.

Ferredoxin
Papers o n different types of ferredoxins are fairly n u m e r o u s in the literature a n d
various electrochemical methods have been used o n c a r b o n or gel d electrodes in the
presence of methylviologen [20,47-52] or on modified m e r c u r y electrodes [53-55].
A ferredoxin solution at p H 7.2 does not show a n y electrochemical reaction o n
the c u r r e n t - p o t e n t i a l curve at a p l a t i n u m electrode. Electrolysis at a controlled
potential close to the potential of the reduction of the solvent leads to a very slow
reduction of tile molecules. F o r tile reoxidation, a change in the a b s o r b a n c e
spect.mm a p p e a r s only when the electrode is at a potential close to 0.0 V a n d
indic~ates a d e n a t u r a t i o n of the molecules (the s p e c t r u m o f the reoxidized molecule
is n o t similar to the initial one).
Figure 6 shows the results o b t a i n e d with a solution containing F A D a n d
ferredoxin.
In a c c o r d a n c e with the values of the a p p a r e n t s t a n d a r d potentials, the reduction
o f F A D occurs before the reduction of ferredoxin. Moreover, F A D m a k e s the
reduction of ,~erredoxin easier as the reaction begins at a b o u t - 0 . 1 5 V ( s p e c t r u m 4)
a n d a fee., minutes o f electrolysis at a potential of - 0 . 7 V is. sufficient to t r a n s f o r m
all the molecules. N o explanation for this p h e n o m e n o n ce~a be given so far.
T h e reoxidation o f ferredoxin co).xesponds to the shoulder prior to the p e a k o n
the c u r r e n t - p o t e n t i a l curve. The spe~tral c h a n g e in inverse o r d e r can be observed to
occur as for the reduction. S p e c t r u m 4 corresponds to a potential of --0.48 V at
which oxidized F A D appears.
The catalytic p h e n o m e n o n c a n now be observed for the oxidation of the protein.
T h e p e a k centred at 0.2 V is p r o b a b l y due to a n a d s o r p t i o n p h e n o m e n o n . S p e c t r u m
a n d c u r r e n t - p o t e n t i a l curve d o n o t change t h r o u g h o u t the following sweeps.

422

"

-0~

U / V vs. SCE

-0.7

-S

o.s

380

x/
500

Fig. 6. l - U c u r v and absorption spectra for a 0.13 m M ferredoxin-.~0.46 m M F A D solution, p H = 7.2;

swe:p rat--0.33 m V / s . B o ~ molecules absorb light in the rang~: of wavelengths used. During the
reduction, F A D is reduced first up to a potential of --0.51 V (s.ne0trum 4); then the ferredoxin is reduced
at - 0 . 6 V (spectrum 6). During the rcoxidafion, ferredoxin is roox;dizcd first up to --0.48 V (spectrum
4), then F A D is oxidized up to --0.3 V (spectrum 1). Spectra 2 and 3: the fertedoxin is in the oxidized
form while F A D is partly in the oxidized and partly in ',_he t ~ l u c e d form. Spectrum 5: F A D is in the
reduced form while ferredoxin is partly in the oxidized and partly in the reduced form.

The catalytic role of F A D for the electron transfer between platinum aTtd some
biological molecules is clca~ly demonstrated.
This property may be used in the analytical field, where various sensor.~ can be
proposed, and in preparative chemistry. The elimination of methemogtobin in
hemoglobin solutions by electrochemical reduction is a first example of its application.
Due to their very different molar masses, oxyhemoglobin and flavin are easy to
separate. Ultrafiltration could be carried out, and an apparatus which wouht
associate an electrolyser and an ultrafiltration system for the recyc!ing of flavh~
could be elaborated.
REFERENCES

1
2
3
4
5
6
7

F.A. Armstrong, H.A.O. Hill and N J , Walton, Q. Rev. Biophys., 18 (1986) 261.
M J . Eddowes and H.A.O. Hi!l, J. Am. Chem. Soc., 101 0 9 7 9 ) 4461.
W 3 . Albery, M.J. Eddowes, H.A.O. Hill and A.IL PAIlman, J. Am. Chem. Sot., 103 (1981) 3904.
K. Uosaki and H.A.O. Hill, J. ElectroanaL Chem., 122 (1981) 321.
P.M. Allen, H.A.O. Hill and NJ. Walton, J. Electroanal. Chem., 178 (1984) 69.
A.E.G. Cass, G. Davis, H.A.O. Hill and D J . Nancarrow, Biochim. Biophys. Acta, 828 (1985) 51.
H.A.O. Hill and N.J. Walton, J. Am. Chem. Soc., 104 (1982) 6515.

423

8
9
10
11
12
13
14
15
16
17
18
19

J.O.D. Coleman, H.A.O. Hill, N.J. Walton and F.R. Whatley, FEBS Lett., 154 (1983) 319.
H.A.O. Hill, N J . Walton and DJ. Whitford, J. Eleetroanal. Chem., 187 (1985) 109.
P. Yeh and T. Kuwana, Chem. Lett., (1979) 1145.
N J . Lewis and M.S. Wrighton, Science, 211 (1981) 9,',4.
C.F. Bowden, F.M. Hawk,ridge and H.N, Blount, J. Electroanal. Chem., 161 (1984) 355.
M.A. Harmer and H.A.O. Hill, J. Electroanal. Chem., 189 (1985) 229.
F.A. Armstrong, H.A.O. Hill, B.N. Oliver and D. Whitford, J. Am. Chem. Soc., 107 (1985) 1473.
H. Durliat and M. Comtat, Anal. Chem., 54 (1982) 856.
H. Durliat and M. Comtat, Anal. Chem., 56 (1984) 148.
P.R. Moses, L. Weir and R.W. Murray, Anal. Chem., 47 (1975) 1882.
R.W. Murray, Ace. Chem. Res., 13 (1980) 135.
W.J. Albery and A.R. Hillman, Annual Reports on the Progress of Chemistry, Vol. 78C, The Royal
Society of Chemistry, London, 1981, p. 377.
20 H.L. Landrum, R.T. Salmon and F.M. Hawkridge, J. Am. Chem. Sou., 99 (1977) 3154.
21 N.K. Cenas, A.K. Pocius and J.J. Kulys, Bioelectrc~hem. Bioenerg., 11 {1983) 61.
22 S. Chao, J.L. Robbins and M.S. Wrishton, J. Am. Chem. Sc~., 10S (1983) 181.
23 N.K. Cenas and J.J. Kulys, Bioelectrochem. Bioenerg., 8 (1981) 103.
24 J.J. Kulys and A.S. Samalius, Bioelectrochem. Bioener8., 10 (1983) 385.
25 G.S. Wilson, Methods in Enzymology, Vol. 54, Academic Press, New York, 1978.
26 M.L. Meckstroth, B.J. Norris and W.R. Heineman, Bioeleetrochem. Bioenerg., 8 (1981) 63.
27 S. Kwee and H. Lund, Bioeleetrochem. Bioenerg', 2 (1975) 231.
28 A.E.G. Cass, G. Davis, M.J. Green and H.A.O. Hill, J. Electroanal. Chem., 190 (1985) 117:
29 M.L. Fullz and R.A. Durst, Anal. Chim. Acta, 140 (1982) 1.
30 D. Labie and V. Byckova, N. Rev. Hematol., 11 (1971) 57.
31 O. Dryhurst, Electrochemistry of Biological Molecules, Academic Press, New York, 1977, Ch. 7.
32 O.S. Ksenzhek and S.A. Petrova, Bioelectrochem. Bioenerg., 11 (1983) 105.
33 L. Gorton and G. Johansson, J. Electroanai. Chem., 113 (1980) 151.
34 J.F. Castner and F.M. Hawkridge, J. Electroanal. Chem., 143 (1983) 217.
35 J. Haladjian, R. Pilard, P. Bianco and P.A. Serre, Bioelectrochem. Bioener8~, 9 (1982) 91.
36 J. Haladjian, P. Bianco and P.A. Serre, Bioelectrochem. Bioenerg., 1 (1974) 478.
37 F. Scheller and H. Priimke, Stud. Biophys., 60 (1976) 137.
38 M.R. Tarasevich and V.A. Bogdanovskaya, Bioelectrochem. Bioenerg', 3 (1976) 589.
39 S.R. Betso, M.H. Klapper and L.B. Anderson, J. Am. Chem. Soc., 94 (1972) 8197.
40 T.M. Cotton, S.G. Schultz and R.P. Van Duyne, J. Am. Chem. Sot., 102 (1980) 7960.
41 F. Scheller mLt2G. Strnad, Adv. Chem. Set., 201 (1982) 219.
42 R.M. lanniello, T J . Lindsay and A.M. Yacynych, Anal. Chem., 54 (1982) 1098.
43 F. Scheller, M. Janchen, J. Lampe, J. Blank, R.J. Prtimke and E. Palecek, Biochim. Biophys. Acta, 4T2
(1975) 157.
44 V.J. Razumas, A.A. Zapalskyte and J J . Kulys, Stud. Biophys., 103 (1984) 57.
45 H.F. Bowden, F.M. Hawkridse and H.N. Braun, J. Eleetroanal. Chem., 116 (1980) 477.
46 H. Durliat and M. Comtat, J. Biol. Chem., 262 (1987) 11497.
47 L.H. Rickard, H.L. Landrum and F.M. Hawkridge, Bioelectrochem. Bioenerg., S (1978) 686.
48 C.D. Crawley and F.M. Hawkridge, Biochem. Biophys. Res. Commun., 99 (1981) 516.
49 F.A. Armstrong, H.A.O. Hill and N.$. Walton, FEBS Left., 145 (1982) 241.
50 P. Bianco, J. Haladjian and L. Asso, J. Chim. Phys., 80 (1983) 763.
51 C.D. Crawley and F.M. Hawkridge, J. El~-ctroan~J. Chem., 159 (1983) 313.
52 C. Van Dijk, 3". Van Eijs, J.W. Van Leeuwen and C. Veeger, FEBS Left., 166.(1984) 76.
.53 P, Bianco and J. Haladjian, Biochem. Biophys. Res. Commun., 78 (1977) 323.
54 P,D.J. Weitzman, i.R. Kennedy and R.A. Caldweii, FEBS Lett., 17 (1971) 241.
55 I.R. Miller and M.M. Werber, J. Electroanal. Chem., 100 (1979) 103.