ANALYTICAL CHEMISTRY, VOL. 50, NO. 9, AUGUST 1978
1315
Electrocatalysis of Dihydronicotinamide Adenosine Diphosphate with Quinones and Modified Quinone Electrodes Daniel Chi-Sing Tse and Theodore Kuwana" Department of Chemistry, The Ohio State University, Columbus, Ohio 432 10
Solution rates of reaction between several quinones and dihydronicotinamide adenosine diphosphate, NADH, indicated that the LG of the reaction and the ortho structure of the quinone were important. The ortho quinones derived from dopamine and 3,4-dihydroxybenzylamine were subsequently covalently bound to carbon electrodes. The NAD' generated by the quinones was assayed and found to have high enzymatic activity. The behavior of the quinone-bound electrodes was compared to the oxidation of ascorbic acid and to the "adsorbed" 9,lO-phenanthraquinone electrode which did not oxidize NADH catalytically.
Much of the previous electrochemical investigations of the redox cofactors, nicotinamide adenosine diphosphate (NAD') and dihydronicotinamide adenosine diphosphate (NADH), has been concerned with t h e reduction of NAD' to NADH. Unfortunztely, electroreduction resulted mainly in producing an inactive dimer through a one-electron, free radical mechanism (1-3). Recently, attention has been directed t o the electrooxidation of NADH to NAD+ in attempts to develop methods to analyze substrates which can be enzymatically coupled t o NAD+/NADH ( 4 ) , or to electrochemically regenerate NAD' for enzymatically coupled substrate turnovers ( 5 , 6). T h e electrochemical oxidation of NADH on solid electrodes has been characterized by poorly defined current-potential (i-E) waves which were complicated partly by electrode fouling a t NADH concentrations above 0.1 mM. The oxidative behavior of low concentrations of NADH to eliminate such fouling has been reported by Blaedel and Jenkins (7). At platinum ( P t ) , Coughlin and co-workers (8) have suggested improved sensitivity which has been utilized for regeneration of NAD' and also for the amperometric assay of oxidoreductase enzymes. Kelly and Kirwan (9) have shown t h a t the potential dependent oxidative turnover for NADH a t a graphite electrode approached an "activity" of 90-9570. T h e NADH electrooxidation a t both Pt and carbon electrodes proceeds with considerable overpotential from the reversible potential. Blaedel and Jenkins (7) have reported t h a t t h e overpotential a t a glassy carbon electrode could be reduced some 0.2 to 0.25 V by certain "conditioning" pretreatments. These pretreatments were suggestive that perhaps oxygen containing functionalities such as hydroxyls, carbonyls, carboxyls, and quinones, which may he produced on carbon surfaces, participated in catalyzing the NADH oxidation. And our earlier observation that surface quinoidal groups could catalyze t h e oxidation of ascorbic acid ( I O ) stimulated the present work. T h e introduction of surface functionalities on electrode surfaces by a chemical reaction route is a n exciting research area of recent interest. A variety of attachment schemes have been proposed to fabricate these so-called chemically modified electrodes (CMEs) including strong surface adsorption ( I I , 12),silinization (13-16), amidization ( I O , 17-21), ether linkage (22,23),and carbon-carbon bonding ( 2 4 ) . Our approach to electrocatalysis by CMEs is t o covalently bind onto carbon 0003-2700/78/0350-1315$01 .OO/O
or metal oxide electrodes electron transfer mediators which can undergo fast electron transfer with the electrode and also with the substrate (solution species) in question. I n the present case, we have found that o-quinoidal structures, such as the catechols, appcwed to be rather specific for the fast, homogeneous oxidation of NADH t o NAD+. Thus, the determination of the homogeneous rates of t h e reaction between NADH and several quinones was undertaken to assess those quinones which might be the best candidates for covalent binding to an electrode surface in the fabrication of a CME. Such a CME would be expected to catalyze NADH oxidation through a surface EC catalytic mechanism:
33-Q
---QH, - 3 - Q + NADH
+ 2e- + 2H' + H' ---QH, + NAD' +
(1) (2)
where
2
---QH, = the surPace bound dihydroquinone
and ---Q=
the fully oxidized bound quinone
The thermodynamic requirement for the EC catalytic sequence 1 and 2 is that the reversible redox potential of NADH must be less positive in value than that of quinone/dihydroquinone. The assumption, yet to be proved, is whether those quinones (or, in general, mediators) which react rapidly in the homogeneous reaction with NADH (or in general, substrates) will also react favorably once they are bound to an electrode surface. The homogeneous rates of reaction between several quinones and NADH, and the preliminary results for electrocatalysis of NADH using catecholamines bound to graphitic electrodes will be discussed in this paper.
EXPERIMENTAL Chemicals. The 3-hydroxytyramine (commonly called dopamine, DA) hydrochloride (99% purity), 4-methylcatechol(4-MC, 99% purity), 3,4-dihydroxybenzylamirie (3.4-DHBA, 98% purity) hydrobromide, 5-hydroxy-1,4-naphthoquinone(5-OH-1,4-NPQ), and 2-anthraquinonesulfonic acid sodium salt (AQ-2-S) were obtained from Aldrich Chemical Company. Methylhydroquinone (MHQ), 9,lO-phenanthraquinorie r9,10-Pa4Q), 1,2-naphthoquinone-4-sulfonic acid sodium salt (1,2-NPQ-4-S) and 1,4naphthoquinone-2-sulfonic acid potassium salt (1,4-NPQ-2-S)were obtained from Eastnian Kodak Company. The 2,5-dihydroxyphenacetic acid (2,5-DHPA,Grade 11) and NADH (Grade 111) from yeast were obtained from Sigma Chemical Company. All chemicals were stored according to manufacturers direction or in a desiccator at 4 "C and were used without further purification. Buffer solutions were prepared by mixing 0.005 M NaH,PO, and 0.005 M Na2HP04,each containing 0 1 M NaC1, to the desired pH. All solutions were prepared freshly prior to each experiment. Demineralized water was double distilled and stored in a quartz container.
B 1978 American Chemical Society
1316
ANALYTICAL CHEMISTRY, VOL. 50, NO. 9, AUGUST 1978
Table I. Abbreviations of Compounds and Terms abbreviation compound o r term chemically modified electrode CME PG pyrolytic graphite GC glassy carbon CV cyclic voltammetry RF radio frequency 3,4-DHBA 3,4-dihydroxybenzylamine DA 3-hydroxytyramine 4-MC 4-methylcatechol 5-OH-1,4-NPQ 5-hydroxy-1,4-naphthoquinone AQ-2-S 2-anthraquinonesulfonic acid methylhydroquinone MHQ 9,lO-PAQ 9,lO-phenanthraquinone 1,2-NPQ-4-S 1,2-naphthoquinone-4-sulfonic acid 1,4-NPQ-2-S 1,4-naphthoquinone-2-sulfonic acid 2,5-DHPA 2,5-dihydroxyphenacetic acid ADH alcohol dehydrogenase NADH dihydronicotinamide adenosine diphosphate Electrochemistry. A solid state potentiostat of conventional design utilizing a positive feedback for iR compensation (25) was employed. Waveforms to the potentiostat were provided by a H P model 3300 A function generator. The cell design was similar to one previously described (26). The radio frequency (KF) plasma treatment of pyrolytic graphite (PG), (Ultracarbon Corp.. Bay City, Mich.) and glassy carbon electrodes (GC), (Tokai Co., Japan) in oxygen atmosphere was described by Evans arid Kuwana (27). The RF treatment was 5 min. Unless otherwise stated, all potentials are reported vs. a reference saturated calomel electrode (SCE). The current-time profiles for the double potential step experiments were recorded on a Tektronix Model 564B storage oscilloscope. Measurement of Rate Constants. The rate constants between various quinones and NADH were determined using three different methods depending on the initial redox state of the mediator and the magnitude of the rate. Double potential step chronocoulometry (DPSC) (28,29) and cyclic voltammetry were used for mediators in the reduced form such as DA, 4-MC, 3,4-DHBA, MHQ, and 2,5-DHPA. For the DPSC experiments, mediator concentration was 0.1 mM and NADH was 1.0 mhl. Increasing the NADH concentration to 2 mM gave the same rate constant after concentration corrections. The potential step times ranged from 5 ms to 5 s. DPSC experiments in buffer solutions alone were run to determine the background charge corrections. In the cyclic voltammetric experiments, the oxidative and reductive peak currents were monitored in the presence and absence of NADH. The quinone concentrations were 0.1 mh14.2 mM and NADH, when present, was 0.5 mM or 1.0 mM. The reaction of 1,4-NPQ-2-S, 5-OH-1,4-NPQ,or AQ-2-S with NADH was sufficiently slow that the concentration of NADH could be electrochemically monitored (oxidation) after the addition of an excess of these quinones to the NADH solution. The rate of the reaction between 1,2-NPQ-4-Sand NADH was determined using a Durrum-Gibson stop-flow instrument. Equal volumes of 1.1 X h1 1,2-NPQ-4-Sand 1.98 mM NADH were mixed and the change in the concentration of the naphthaquinone was monitored at wavelength of 400 nm. The optical absorbance vs. time data were recorded and analyzed by a Data General NOVA 1200 minicomputer. Procedure for Measurement of NAD+ Activity. The enzymatic activity of the NAD+ solution electrogenerated through the EC catalytic mechanism utilizing 3,4-DHBA was determined by spectrophotometrically monitoring the NADi to NADH conversion when ,alcohol dehydrogenase (ADH) and ethanol were added. For example, 10 mL of 1.0 X M 3,4-DHBA and 1.0 X M NADH solution was electrooxidized at a pyrolytic graphite electrode at a controlled potential of +0.35 V vs. SCE until 70-90% of NADH was oxidized. ADH (0.5 to 0.8 mg) and 100 pL aliquots of pure ethanol were added to 3.0 mL of the electrolyzed, NAD+ solution. Spectra were taken in the wavelength range of 280 to 400 nm before and after the addition of each aliquot of ethanol. The optical absorbance change at 340 nm was used to calculate the amount of NAD+ converted t o
i
06
v
U,-LJ
04
POTEYTIAL
02 VOLTS
30 vs
-02
SCE
Figure 1. Cyclic voltammograms of PG electrodes in pH 7.0 phosphate buffer (a, c) and in 0.1 mM NADH in pH 7.0 phosphate buffer (b, d). The pyrolytic graphite electrode pretreatments were: (a, b) extracted in methanol for 24 h and vacuum dried. (c, d) first extracted in methanol for 24 h, vacuum dried, then subjected to radio frequency (RF) plasma treatment. The scan rate was 50 mV/s
NADH by ADH-ethanol. Controls included unelectrolyzed solutions of NADH and 3,4-DHBA,and NAI)H electrolyzed under the same conditions but in the absence of 3,4-UHBA. The abbreviations of compounds and other terms used are summarized in Table I.
RESULTS AND DISCUSSION NADH is involved in many enzymatic reactions. However, nonenzymatically, only certain oxidants can oxidize NADH (30,31). Kubowitz (32),and Nason and co-workers (33) have indicated that the quinones and, in particular, the ortho form, reacts with NADH as follows:
R--Ph-(=0)2
+ NADH + H+
ki
R-Ph-(-OH)z
+ NAD'
(3)
where R indicates some substituent group, Ph-(=O)* is a quinone, and Ph-(-OH)2 is the reduced, dihydro form. In electrochemistry, it is convenient to oxidize the dihydroquinone to quinone which in turn can oxidize NADH in a n EC catalytic sequence as discussed earlier. The questions to be answered are: (1) Whether the ortho structure is specific to the oxidation of NADH compared to the para or meta forms; and (2) Whether the ortho or any other quinone which reacts rapidly with NADH can be appropriately attached to an electrode surface for the surface EC catalysis of NADH. Initially, the electrochemistry of NADH and several quinones (usually in the dihydro form) was examined a t P G or GC electrodes so t h a t the electrochemical behavior of each could be understood prior to the EC catalytic studies. In Figure 1, the cyclic voltammetric (CV) current-potential (i-E) curves are shown for the oxidation of NADH at a PG electrode prior to (trace b) and after (trace d ) radio frequency (RF) plasma treatment in O2 atmosphere (Po,* = 150 mTorr). As may be seen from comparison of these two curves, the peak potential, E,, for NADH oxidation is ca. 100 mV less positive at the R F treated electrode. This shift in E,. similar to that observed by Blaedel and Jenkins ( 7 ) , may be due to t h e presence of oxygen functionalities introduced on the carbon surface by the RF treatment (10). However, the decrease here may also be due to the increase in the effective surface area of the electrode. This area increase is manifested clearly by an increase in the background charging current of the treated electrode (see i--E curve, trace c) over that of the untreated one (trace a). Work is in progress t o further quantitate the effect of RF plasma treatment to NADH oxidation.
1317
ANALYTICAL CHEMISTRY, VOL. 50, NO. 9, AUGUST 1978
Table 11. Values of Redox Potentials and Kinetic Rates rate constant, M-' s-l a compound E " ' , , V vs. NHE 3,4-DHBA 0.41 t 0.02e 7.7 ( r 0.8) X lo4 DA 0.37 t 0.02e 3.6 ( i 0.4) X lo4 4-MC 0.32 z 0.04e 3 . 3 ( i 0.4) X lo' 2,5-DHPA 0.260f Ob 0.24 ?: 0.04e Ob MHQ 1,2-NPQ-4-S 0.215f 4.4 ( + 0.9) x l o z c 1,4-NPQ-Z-S 0.120f Od 5-OH-1,4-NPQ 0.033f Od AQ-2-S - 0.225f Od NADH - 0.320f Determined by DPSC. a Temperature 23 2 1 "C. N o change in NADH conDetermined by stop-flow. centration after 0.5 h when 10-fold excess of quinone was mixed with NADH. Measured in this work by means of cyclic voltammetry. f From ref. 37.
-
The CV curves for the oxidation of 3-hydroxytyramine (dopamine, DA), 3,4-dihydroxybenzylamine(3,4-DHBA), and 4-methylcatechol (4-MC) a t P G electrode in pH 7.0 solution are shown in Figure 2, traces a, c, and e, respectively. All three compounds produced well defined CV curves for the twoelectron oxidative conversion of the dihydro to the quinone form. The electrochemical data for these and several other quinones are summarized in Table 11. In the case of DA, the oxidation was characterized by a follow-up chemical reaction, a cyclization (35),which produces a new wave at ca -0.25 V vs. SCE. Such a chemical follow-up reaction was absent for 3,4-DHBA and 4-MC. The CV curves, b, d, and f i n Figure 2 are repeats of the above experiments except in the presence of 1.0 mM NADH. The electrochemical currents are now considerably enhanced by the EC coupled oxidation of NADH, and the potential for NADH oxidation is now governed by the respective quinone couples. Since the experimental conditions are similar, the shape and height of the i-E curves reflect the kinetic rates of the quinone oxidation of NADH. The NADH oxidation rate by 4-methyl-0-quinone is slower than the corresponding quinone of DA and 3,4-DHBA. One observed not only less catalytic current but also the oxidation of NADH by the electrode at 0.58 V (Figure 2f). It should be noted also that the irreversible, homogeneously coupled quinone/NADH reaction removed the reductive current on reversal of the potential scan. The values of the kinetic rates of the quinone/NADH reaction 3 are summarized in the last column of Table 11. There appears to be two factors in the rate enhancements. First, a larger free energy of reaction 3 as reflected by the more positive redox potential, E;, increased the rate. Second, there is a preference for the ortho structure. The generality of these two factors will be further tested in the future. Since reaction 3 is proton dependent, an increase in the oxidative rate between a quinone and NADH should increase with a decrease in pH. This effect of p H was examined for DA between the pHs of 6.5 to 8.5. The rate constant decreased by ca. 1.2 x lo4 M-' per pH. E n z y m a t i c Activity of NAD+. Kelly and Kirwan (9) assayed the enzymatic activity of NAD' in p H 9 pyroTable 111. Enzvmatic Activitv of NADNADH concn,
T 50kA
A
1
\-/
A
L L ._. A L I -1-._L-L L
08
06
0 4
02
P O T E U T I O ~, I / O - T S
oo
-02
-04
i s SCE
Figure 2. Cyclic voltammograms of (a) 0.1 mM DA, (b) 0.1 mM DA, 1.0 mM NADH, (c) 0.1 mM 3,4-DHBA, (d) 0.1 mM 3,4-DHBA, 1.0 mM NADH, (e) 0.1 mM 4-MC, (f) 0.1 mM 4-MC, 1.0 mM NADH, In pH 7.0 phosphate buffer with a RF plasma treated pyrolytic graphRe electrode. The scan rate was 50 mV/s
phosphate buffer by determining the acetaldehyde concentration after ADH-ethanol were added to the NADH electrolyzed solution. They reported that a maximum NAD' production of 95% occurred at an applied potential of +0.700 V vs. SCE using a carbon electrode (anode grade graphite, Union Carbide). To assess the enzymatic activity of the NAD' produced through reaction 3, a similar assay was performed except that the change in the NADH concentration was monitored by spectrophotometry. A 0.1 mM solution of 3,4-DHBA a t an applied potential of +0.35 V vs. SCE a t a PG electrode in the presence of 0.1 mM NADH (pH 7.0) was used. The coulometric charge (background corrected) was accurately measured so that the amount of NAD' produced could be calculated. In all of the runs reported in Table 111, more than 90% of the NADH was oxidized. The enzymatic activities ranged from 92 to 98%. In the control experiments where NADH was electrolyzed under the same condition as in the absence of 3,4-DHBA, only 3-4% NADH oxidation was observed. Electrochemical Activity of Bound o-Quinone CMEs. The two quinones exhibiting the highest rate of NADH oxidation in Table I1 were fortunately those containing a terminal amino group on the substituent side-chain. Thus, the reduced forms of these quinones, DA and 3,4-DHBA, were attached by arnidization t o carboxylic acid functionalities on R F / 0 2 treated P G and air oxidized GC in the presence of dicyclohexylcarbodiimide. The reaction conditions and the GC electrode pretreatments were similar to those described by Fujihira, Tamura, and Osa (36). The time of reaction was four days after which the electrodes were extracted in methanol for 24 h to remove adsorbed reactants. Since 3,4-DHBA on GC exhibited the best properties toward surface EC catalysis of NADH, the discussion will be restricted to this CME. Trace a in Figure 3 is a typical CV curve for a 3,4-DHBA CME. The oxidative E, is + 0.20 V vs. SCE which is identical to the one obtained for solution 3,4-DHBA (see trace c, Figure
NADH enzymatically generated Final ethanol 10-4 concn, M mg ADH NAD+ electrochemically generateda 1.00 2.02 0.8 0.963 0.98 1.56 0.926 0.7 0.93 2.02 0.977 0.5 0.92 2.02 0.969 0.5 a The decomposition of NADH was corrected by monitoring unelectrolyzed solution of NADH and 3,4-DHBA spectroscopically. Collections ranged from 2 to 3%of the initial NADH concentration.
ANALYTICAL CHEMISTRY, VOL. 50, NO. 9, AUGUST 1978
1318
t
..
0
ai
z
;;.;.
U
/
+ I
w LL 3 i2
L.-.
-
I
cLL-
~
____-
..
-1.
~~
PC'ENTIAL,
.
1
-;
-~
1 -. .\\.Ji 1
13
1
-/,)
, ' . . ,.,'
.'
1 ~~.. . C 6 04 ~
_ '
, ,
,-
.r
vorable as, for example, the bound 3,4-DHBA, the total unreactivity is possibly indicative of structural orientation differences between the two "bound" o-quinones. T h e "adsorbed" 9,10-phenanthraquinone may be less accessible to the 4-dihydro position of the NADH for oxidation to occur. The feasibility of fabricating a CME capable of' catalyzing the oxidation of NADH has been demonstrated. For utilizing such CME electrodes for analytical purpuses, the deleterious effect of the presence of high concentrations of NADH may be perhaps circumvented by t h e use of the pulse method described by Lane and Hubbard (37). Future studies will more fully explore the molecular/structural aspects which are necessary for the design of (:ME with redox specificity.
-,,.__
I
;J
.,. i"
!~
02 V C L T S :s
1
O C
.
-.
~~
53
~ . ~ . - 3 2
SCE
Figure 3. Cyclic vokammograms of a 3,4-DHBA modified GC electrode (a)in pH 7.0 phosphate buffer, and (b) in 0.2 mM NADH, pH 7.0 solution. The scan rate was 50 mV/s
2 ) . 'This C'V changed slightly ~ v h e nrepetitively cycled for 10 or more times. T h e surface coverage by 3,4-DHHA was calculated to be 1.3 X lo-'' niol/cm2 from the oxidative e1ect)rocheinical change, assuming TI = 2 for 3,4-DHBA. Trace b in Figure 3 is t h e repeat of the CV except in the presence of 0.2 mM NADH. The E,, of the surface EC coupled oxidation of NADH is slightly less positive than the solution E(' one (see trace d , Figure 2). This difference in E , may be due to the higher concentration of NADH in the solution case. The important aspect is the EC surface catalysis of' NADH which is being accomplished by a deliberate cheniical/structural modification of an electrode surface, Le., an o-quinone CME. Unfortunately, the activity of this electrode could not be maintained for more than a few cycles in the presence o f NADH. Because of the previous implication (7, that electrode fouling by NADH could be deleterious to an electrode, the activity of the 3,4-DHBA CME was tested by the oxidation of ascorbic acid. Indeed, a well-defined oxidative i-E characteristic of an EC catalyzed wave was obtained for the oxidation of ascorbic acid by this CME. The peak heights were reproducible from run t o run for several cycles without loss of activity. I t is reasonably safe to conclude that the decrease in the activity of the 3,4-DHBA CME with NADH is not due to loss of the bound 3,4-DHBA but rather the presence of NADH, NAD+, or the presence of small amounts of unknown side products of NADH oxidation. For ascorbic acid, the rate of oxidation by this CME was apparently quite rapid since the E , was nearly identical to that obtained in the absence of ascorbic acid. Recently, Brown et al. (12) have reported the interesting case of an electrode modification by t h e strong surface adsorption of 9,10-phenanthraquinone. This molecule is believed to be adsorbed on graphitic surfaces with the plane of the ring lying parallel to t h e surface. The E , for the oxidation was observed to be a t ca +0.20 V vs. SCE in 1 M trifluoroacetic acid which is the condition of the experiment reported. At p H 7.0 phosphate buffer, the E,, for the oxidation of 9,lOdihydroxyphenanthrene was 4 . 2 0 V vs. SCE (0.04 V vs. "E). Interestingly, this adsorbed o-quinone/dihydroquinone electrode did not "catalytically" oxidize NADH (0.2 mM). Although the free energy of this redox couple is not as fa-
LITERATURE CITED A L Underwood and Robert W. Burnett, "Electrochemistry of Biological Compounds", in "Electroanalytical Chemistry", Vol. 6, A. J. Bard, Ed., Marcel Dekker, New York. N.Y., 1973. G. Dryhurst, "Electrochemistry of Biological Molecules", Acauemic Press, New York, N.Y., 1977, Chap. 9. C. 0. Schrnakel, K. S. V. Santhanarn, and P. J. Elving, J , Am. Chem Soc., 97, 5083 (1975). See Introductory discussion in paper by F S.Cheng and G. D. Christian, Anal. Chem., 49, 1785 (1977). M. Aizawa, H W. Coughlin, and M. Charles, triochem Biophys. Acta, 385, 362 (1975). R. W. Coughlin and B. F. Alexander, Biotechnol. Bioeng., 17, 1375 (1975) W. J. Blaedel and R. A . Jenkins, Anal. Chem., 47, 1337 (1975) T. C. Wallace, M. B. Leh, and R. W. Coughlin, Biotechnol. Bioeng., 19, 901 (1977). R. M. Kelly and U. J. Kirwan, Biotechno/. Bioeng., 19. 1215 (1977). J. F. Evans, T. Kuwana, Mary T. Henne. and G. P. Royer, J . Hectroanal. Chem.. 80, 409 (1977) R. F. Lane and T. Hubbard, J Pbys Chem , 77, 1401, 1411 (1973). A. P. Brown, C. Koval, and F. C. Anson, J , Electroanal. Chem., 72, 379. (19761. P.k.-Moses and R. W. Murray, J . Am. L'hem. Soc., 98, 7435 (1976): J . Electroanal. Chem.. 77, 393 (1977). D. G. Davis and R. W. Murray, Anal. Chem., 49, 194 (1977). G. J. Leigh and C. J. Pickett, J . Chem. SOC.,Datton Trans., 1797 (1977). A. F. Diaz and K . K. Kanazawa, J . A r t . Chem. Soc.. 99, 5838 (1977). B. F. Watkins, J. R. Behling, E. Kariv, and L. L Miller, J . A m . Chem. Soc., 97, 3549 (1975). M. Fujihara, T. Matsue, and T. Osa, Chem. Len.. 875 (1976). C. A. Koval and F. C. Anson, Anal. Chem.. 50, 223 (1978). N. Oyama and F . C. Anson, J . Electroanal. Chem., 88, 289 (1978). N. Oyarna, A. P. Brown, and F. C. Anson, J , Electroanal. Chem.. 87, 435 (1978). A. W. C. Lin, P. Yeh, A. M. Yacynych, and T Kuwana, J . Electroanal. Chem., 84, 411 (1977). A. M. Yacynych and T. Kuwana, Anal. Chem., 50, 640 (1978). S. Mazur. T. Matusinovic, and K . Carnmann, J . Am. Chem. Soc., 9 9 , 3888 (1977). A. A. Pilla, J . Electrochem. Soc., 118, 702 (1971). F. Hawkridge and T. Kuwana, Anal. Chem.. 45, 482 (1973). J. F. Evans and T. Kuwana, Anal. Chem., 49, 1632 (1977). J. H. Christie, J , Nectroanal. Chem., 13, 79 (1967). P. J. Lingane and J. H. Christie, J . Elechoanal. Chem., 13, 227 (1967). L. Hellerman, F. P. Chinard, and P. A. Ramsdell, J . Am Chem. Soc., 63, 2551 (1941). W. T. Caraway and L. Hellerman. J . A m Chem. SOC , 75, 5334 (1953) F. Kubowitz, Biochem. Z . , 299, 32 (1938). W. D. Wosilait and A. Nason, J . Biol. Chem., 206, 255, 271 (1953). W. M. Clark, "Oxidation-Reduction Potentials of Organic Molecules". The Williams and Wilkins Company, Baltimore, Md., 1960 D. Tse, R. L. McCreery, and R. N. Adams, J , Med. Chem., 19, 37 (1976). M. Fujihira, A. Tarnura, and T. Osa, Chem. Len., 361 (1977). R. F. Lane and A. T. Hubbard, Anal. Chem., 48, 1287 (1976).
RECEIVEL) for review March 27, 1978. Accepted May 15, 1978. 'This work supported by funds from NSF Grant Number CHE76-81591 and US P H S Grant Number GM19181.
