Electrochemical regeneration of nicotinamide adenine dinucleotide

362

Biochimica et Biophysica Acta, 385 (1975) 362--370

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 27615 ELECTROCHEMICAL REGENERATION OF NICOTINAMIDE ADENINE DINUCLEOTIDE

MASUO AIZAWA, ROBERT W. COUGHLIN and MARVIN CHARLES Department of Chemical Engineering, Lehigh University, Bethlehem, Pa. 18015 (U.S.A.)

(Received October 21st, 1974 )

Summary Reduced nicotinamide adenine dinucleotide (NADH) has been characterized electrochemically by solid electrode voltammetry and controlled potential electrolysis. Photometric and enzymatic assay showed that enzymatically active nicotinamide adenine dinucleotide (NAD ÷) could be regenerated electrolytically from its reduced form without the use of so-called electron mediators. Complete regeneration of enzymatically active NAD can be expected in pyrophosphate buffers and phosphate buffers during the electrolysis. Advantages of electrochemical regeneration of coenzymes are discussed, especially with regard to immobilization of enzymes.

Introduction Many potential applications of enzymes hold great promise for future expansion particularly by immobilization on various supports [1,2] which permits easy handling and reuse. Coenzymes such as nicotinamide adenine dinucleotide (NAD*), however, are not automatically regenerated for further use, whether immobilized or not. Enzymatic and chemical regeneration of NAD ÷ from NADH requires additional chemical reactions which consume reagents and can change pH. Electrolytic oxidation of NADH to produce an enzymatically active NAD + is an example of an electrochemical system for regenerating coenzymes, which proceeds without any addition of chemicals. The electrolytic reduction of NAD ÷ has been thoroughly investigated [3--5]. These studies have been primarily concerned with reaction mechanisms and employed polarography [6--12], voltammetry [13,14] and coulometry. Little attention has been paid to the oxidation of NADH, however, although it has been mentioned in passing during discussion of reduction [11,13]. This paper concerns the electrolytic oxidation of NADH. Voltammetry

363 was employed for the electrochemical characterization of NADH. Furthermore, controlled potential electrolysis of NADH was carried out to confirm that the regenerated NAD ÷ was enzymatically active. Experimental

Materials. The NADH (reduced form) was obtained from Sigma Chemical Co. This material was found to be 92% pure at the time of the tests by its absorption at 340 nm with a molar extinction coefficient of 6.20 • 103. The alcohol dehydrogenase (EC 1.1.1.1) derived from yeast was obtained from Sigma Chemical Co. All other chemicals were reagent grade and deionized water was used throughout. Voltammetry. An H type cell (E.H. Sargent Co., No. S-29392) with a fritted glass separator was used for voltammetric studies. The working electrode was a platinum wire (1.0 mm outer diameter X 10 mm) and the counter electrode a platinum gause (30 X 40 mm). The electrode potential, which was referred to saturated calomel electrode at 25 ° C, was controlled and scanned continuously with a Heath Model EUA-19-2 Polarography Module and Model EUW-19B Operational Amplifier. Controlled potential electrolysis. A two c o m p a r t m e n t type of cell was used for the controlled potential electrolysis. Both anode and cathode were platinum gauze (30 X 40 mm). The anolyte was stirred magnetically during the electrolysis. The anode potential was controlled with reference to saturated calomel electrode and the anolyte was assayed for its absorbance at 340 nm every 5 min during the electrolysis. Enzymatic assay. Alcohol dehydrogenase and ethanol were used for determining the enzymatic activity of the electrolytically regenerated NAD. The assay was carried out in 0.1 M pyrophosphate buffer (pH 9.0) at 25°C. Photometric measurements. A Bausch and Lomb Spectronic 700 was used for determining the absorbence at 340 nm and the spectrum in the wavelength range from 240 to 340 nm. Results and Discussion

Voltammetric studies on the oxidation o f N A D H One of the difficulties to be expected in electrochemical characterization of nicotinamide adenine dinucleotide (NAD ÷) and its reduced form {NADH) may be the adsorption of these on the surface of electrodes. Multi-sweep voltam m e t r y suggested t h a t such adsorption was indeed significant in the case of the platinum electrodes used in these measurements. The working electrode which was carefully cteaned before measurement was immersed in a sample solution and the electrode potential was held 10 s at 0.3 V vs saturated calomel electrode and then scanned up to 0.7 V with a constant sweep rate of 0.2 V/min. The potential was immediately switched back to 0,3 V after it reached 0.7 V and the potential sweep was repeated. The electrolyte contained 0.5 mM NADH in 0.1 M pyrophosphate buffer (pH 7.6). The multi-sweep voltammogram obtained in the above m e t h o d is shown in Fig. 1. Each subsequent sweep displays a positive shift of the peak current

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Fig. 1. M u l t i - s w e e p v o l t a m m o g r a m . T h e e l e c t r o d e p o t e n t i a l w a s h e l d 10 s at 0.3 V vs s a t u r a t e d c a l o m e l e l e c t r o d e a n d t h e n s c a n n e d up to 0.7 V w i t h a c o n s t a n t s w e e p r a t e o f 0.2 V / r a i n . T h e p o t e n t i a l w a s i m m e d i a t e l y s w i t c h e d b a c k to 0.3 V a f t e r it r e a c h e d 0.7 V a n d t h e p o t e n t i a l s w e e p w a s r e p e a t e d : (a) 1st, (b) 2 n d , (c) 3rd, a n d ( d ) 4 t h a n d 5 t h s c a n n i n g s . T h e e l e c t r o l y t e c o n t a i n e d 0 . 5 m M N A D H in 0.1 M p h o s p h a t e b u f f e r ( p H 7.6).

m a x i m u m toward more positive potentials and a corresponding decrease in the peak current; this behavior may be attributed to slow approach toward equilibrium adsorption of the cofactor on the electrode during the multi-sweep experiments. A steady-state and reproducible voltammogram was obtained by the fifth sweep. Because such large changes in both the peak potential and the current may be caused by adsorption of NADH and the oxidized species on the surface of the working electrode, it appears reasonable to consider only reproducible current vs potential curves obtained with each initial sweep, when the working electrode is first cleaned by electrolysis before each measurement. Therefore only the voltammograms for the initial sweep with a cleaned electrode were used t h r o u g h o u t the investigation. In a cyclic voltammogram for a platinum electrode in an acidic solution, two peaks of hydrogen pre-wave may be obtained [15]. These pre-waves serve as a highly sensitive probe to detect adsorbed species on the electrode surface. Most adsorbed species depress the current of these pre-waves and extreme suppression by both NAD and NADH has been reported [16]. The hydrogen pre-waves were checked for the working electrode before each measurement of the present work. The cyclic voltammetry was carried out for the working electrode in 0.5 M H2 SO4 solution in the range o f - - 0 . 3 0 to +0.9 V vs saturated calomel electrode. The potential sweep rate was 2.0 V/min. One of the typical current vs potential curves for the oxidation of NADH is presented in Fig. 2. The anolyte contained 0.5 mM NADH in 0.1 M pyro-

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Fig. 2. V o l t a m m o g r a m for oxidation of N A D H . The anolyte contained 0.5 m M N A D H in 0.I M pyrophosp h a t e b u f f e r ( p H 9 . 0 ) , t h e c a t h o i y t e was 0.1 M p y r o p h o s p h a t e b u f f e r ( p H 9 . 0 ) , a n d t h e p o t e n t i a l s w e e p rate was 0.2 V / r a i n . T h e c u r r e n t is p l o t t e d as t h e r a t i o to t h e p e a k c u r r e n t ip.

phosphate buffer (pH 9.0), and the potential sweep rate was 0.2 V/min. The current is plotted as the ratio to the peak current ip, and the potential as the difference from the initial potential, vt, where v and t are the constant potential sweep rate and time respectively. Only one anodic wave appears for the oxidation of NADH, in contrast to the cathodic polarogram of NAD+ wherein two steps can be detected in the wave. This suggests that the oxidation of NADH proceeds in one step by a two-electron reaction. It is recognized that the electrode process of NAD/NADH is irreversible. For numerous irreversible processes the effect of the backward reaction can be neglected when the over-voltage is larger than about 0.12/n V, n being the total electrons involved in the electrode process; in this case n = 2. If the effect of the backward process is neglected, Delahay [17] has shown that the current vs potential relationship is: i = ~'/'nFA~3'/'D'/'c°×([Jt)

(1)

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(2)

where F , A , D , c ° , R , T , and v are Faraday constant, surface area of the electrode, diffusion constant, concentration, gas constant, temperature (°K), and poten-

366

tial sweep rate, respectively; na is the n u m b e r of electrons in the rate-determining step o f the electrode process; ~ is the transfer coefficient for the electrode process. X(fit) is the function defined by Delahay [ 1 7 ] . From the definition of the function, = 0.2827rV2nFA(anaF/RT)V2vV2Dl/~c '' and

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The d ep en d e nce of the peak current on potential sweep rate is presented in Fig. 3 for a c o n c e n t r a t i o n of NADH = 0.5 mM. The peak potential is proportional to the square r o o t of the potential sweep rate in accord with Eqn 3. In Fig. 4 the relationship between the c o n c e n t r a t i o n of NADH and the peak current is shown for two different potential sweep rates of 0.2 and 1.0 V/min. The peak current changes linearly with the c o n c e n t r a t i o n of NADH up to at least 1.0 mM. The p r o d u c t ana and the diffusion constant D were calculated using Eqns 2, 3 and 4 with the following results: an = 0.57, D = 2.4 • 10 -6 cm 2 • s-~, at 25°C. The polarographic diffusion constant of NAD previously reported to be 3.40 • 10 -6 cm 2 • s -1 [ 1 8 ] , agrees satisfactorily with the present work. The peak potential was observed t o be d e p e n d e n t on pH and this is shown in Fig. 5 for a potential sweep rate o f 0.2 V/min. The buffers used were 0.1 M p h o s p h ate and 0.1 M p y r o p h o s p h a t e . The slope AE/ApH is approx. 35 m V / p H in substantial agreement with the Nernst equation.

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R e l a t i o n s h i p b e t w e e n the c o n c e n t r a t i o n of NADH and the peak current. Fig. 4. R e l a t i o n s h i p b e t w e e n t h e c o n c e n t r a t i o n of N A D H a n d t h e p e a k c u r r e n t . T h e s u p p o r t i n g e l e c t r o l y t e was 0.1 M p y r o p h o s p h a t e b u f f e r ( p H 9.0). T h e p e a k c u r r e n t is s h o w n for t w o d i f f e r e n t s w e e p rates o f 0 . 2 ( - - e - - ) a n d 1.0 V / m i n ( ~ - - ) .

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Fig. 6. E f f e c t of p H o n t h e p e a k p o t e n t i a l E p . T h e s u p p o r t i n g e l e c t r o l y t e s used w e r e 0.1 M p h o s p h a t e b u f f e r ( p H 6 - - 8 . 5 ) a n d 0.1 M p y r o p h o s P h a t e b u f f e r (DH 8 . 0 - - 9 . 5 ) . T h e a n o l y t e c o n t a i n e d 0.5 m M N A D H , a n d t h e p o t e n t i a l was s c a n n e d at a c o n s t a n t r a t e o f 0.2 V / r a i n .

368

Electrolytic oxidation at controlled electrode potential The voltammetric behavior of NADH suggests that the o p t i m u m electrode potential for the electrolytic oxidation o f NADH at pH 9.0 should be around 0.7 V vs saturated calomel electrode. The controlled potential electrolysis of NADH was carried out in pH 9.0 p y r o p h o s p h a t e buffer, that particular buffer baying been chosen because alcohol dehydrogenase, one of the c o m m o n enzymes coupled with NAD ~ shows the m a x i m u m activity under these conditions. A stirred 10-ml volume of anol yt e containing 0.5 mM NADH in pyrophosphate buffer (pH 9.0) and an unstirred cat hol yt e containing pyrophosphate but no NADH were electrolyzed, during which the anode potential was controlled at 0.7 V vs saturated calomel electrode. The time behavior of current and absorbance (at 340 nm a characteristic absorption band of NADH) of the anolyte are shown in Fig. 6. The electrolysis was continued for 100 min. Ultraviolet spectra o f the anolyte were de t e r m i ne d before and after the electrolysis as shown in Fig. 7. The spectrum after the electrolysis is positively identified as essentially that of NAD + in pH 9.0 p y r o p h o s p h a t e buffer (absorption band at 260 nm). P h o t o m e t r i c det er m i nat i on of absorption at 340 and 260 nm of the p r o d u c t solution indicated that 92% of NADH was oxidized by the electrolysis carried o u t for 100 min. The n u m b e r of equivalents electrolyzed in this time was a p p r o x i m a t e l y 1 • 10 -~ F, with 0.5 • 10 -s mol of NADH contained in the

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Fig. 7. U l t r a v i o l e t s p e c t r a e f t h e a n o l y t e . T h e s p e c t r a w e r e d e t e r m i n e d f o r t h e a n o l y t e b e f o r e ( . . . . . . and after( )thecontrolled potentialelectrolysisofNADH t o N A D +.

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initial anolyte. Thus it appears that the two-electron oxidation proceeded to 92% conversion during the controlled potential electrolysis for this time. Enzymatic assay was also made of the product. Alcohol dehydrogenase and ethanol were used as enzyme and substrate added in 3-ml aliquots. The final concentration of alcohol dehydrogenase and ethanol were 0.07 mg/ml and 0.4 M, respectively. Change in absorption at 340 nm was recorded continuously. Absorption at 340 nm increased with the addition of alcohol dehydrogenase and ethanol. It indicates that the oxidized product was reduced again in the enzyme reaction. The photometric determination showed that the product was fully enzymatically active. Conclusion Reduced nicotinamide adenine dinucleotide (NADH) was oxidized directly by an electrolytic m e t h o d w i t h o u t the use of so-called electron mediators. The electrolytic oxidation of NADH proceeds in one step by a two-electron reaction and the electrolytic product is fully enzymatically active. It is possible therefore to regenerate enzymatically active nicotinamide adenine dinucleotide from its reduced form by a direct electrolytic method. A recovery of more than 90% of the original NADH can be expected in pyrophosphate and phosphate buffers during electrolysis. The electrolytic regeneration of enzymatically active NAD is possible, therefore, under o p t i m u m conditions for enzyme reactions coupled with NAD. Neither any addition of chemicals nor changes in pH, ionic strength and temperature are involved. A subsequent paper will report on simultaneous reaction

370

and regeneration in a flow system in which continuous operation is possible. It is believed that the electrolytic regeneration of NAD should offer significant advantages, especially in practical use. Acknowledgment The authors acknowledge with gratitude support for this research under N.S.F. Grant No. GI35997.

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