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Study of the electroreduction of NAD and NADP coenzymes in strongly acidic media
00134686/9Q53.00+0.00 b? 1990. Pergamon Press
pk.
STUDY OF THE ELECTROREDUCTION OF NAD AND NADP COENZYMES IN STRONGLY ACIDIC MEDIA Jo& Departamento
MIGUEL RoDRiGuEz MELLADO* and RAFAEL MAR~N GALV~N
de Quimica Fisica y Termodinamica Aplicada, Universidad de Grdoba, Magno s.n., 14004-Grdoba, Spain (Received
17 May 1989; in revised firm
S. Albert0
14 June 1989)
Abstract-The electroreductions of NAD and NADP coenzymes have been studied in acidic media (5 M H,SO, to pH = 2) by DP and dc polarography. Tafel slopes and reaction orders were obtained from i-E curves traced at potentials corresponding to the foot of the polarographic waves. The results show that, at very low values of coenzyme concentration (< 5 x lo-’ M) and high acidity (pH< 1), the processes are irreversible. The reduction pathways consist of the reversible transfer of an electron and an H+ ion followed by an irreversible one-electron transfer, which is the rate-determining step. At higher pH and/or concentration values this process is in competition with the electrodimerization of the coenzymes.
INTRODUCTION A great number of enzymes which are involved in redox reactions need the direct participation of the pyridine nucleotides. Two of these coenzymes, ie nicotinamide adenine dinucleotide, NAD, and nicotinamide adenine dinucleotide phosphate, NADP, are hydrogen-transferring coenzymes for the dehydrogenases. A third compound, nicotinamide adenine mononucleotide, NMN, is less well-known. Both NAD and NADP participate in the reactions which they catalyse, being simultaneously reduced. These reactions involve the transfer of an H+ ion and two electrons from the substrate to the coenzyme. The essential component of the pyridine nucleotides is the nicotinamide ring, which is the portion of the coenzymes involved in the redox reactions. Nicotinamide itself is widely distributed in plants and animal tissues. The occurrence of reversible redox reactions under physiological conditions has made advisable to study these systems electrochemically, mainly by polarographic methods[ 11. The NAD-NADH redox system has been extensively studied both potentiometrically and polarographically[2]. Most authors reported one cathodic polarographic wave in both acidic and basic solutions, its half-wave potential being pH_independent[3]. However, in buffered media containing tetraalkilammonium salts, two reduction waves have been reported[4,5]. The first of them corresponds to the irreversible one-electron diffusion-controlled reduction[5]. The second one merges in most buffered solutions with the background discharge, whereas the second reduction waves of model compounds are welldefined at the same potentials. The irreversibility of the processes was indicated by the logarithmic analyses of the waves[2]. The dimer postulated as resulting from the radical produced in the one-electron reduction of NAD has
*Author to whom correspondence should be addressed.
not been isolated and unequivocally identified. Results of electrolysis at the potential of the second wave are equivocal. Addition of cationic surfactants and vigorous ultrasonic agitation increased the rate of NADH formation[6]. The low yields of enzymatically active NADH were explained as due to the concurrent formation of dimer. A reduction mechanism involving the formation of a free radical and its subsequent dimerization was proposed for the first wave[l]. The second wave involves the reduction of the radical formed in the first step. The oxidations of NADH ahd NADPH in both aqueous media[7], acetonitrile[8] and dimethylsulfoxide[9], are bielectronic processes, but evidences of second-order reactions were reportedC9, lo]. Three pyridine carboxamides, ie nicotinamide (NA), N ,-methylnicotinamide (MNA) and isonicotinamide (INA), have been studied in strong acidic media as model compounds of the pyridinic coenzymes[ 1 l-l 33. DP and dc polarography and linear-sweep cyclic voltammetry were used. In all cases, the overall processes are irreversible but the reaction pathways are quite different. At pH < (M.5, the rate-determining step of the reduction process of NA is a protonation placed between the one-electron transfers[ 111; for MNA the process is controlled by the second electron transfer, which is irreversible[12]; finally, in the case of INA, the rds is the loss of ammonia from the unstable gem-hydroxyamine[13]. At pH > 0.5, the variations of the limiting currents with the pH and the i-t curves indicate that the processes are governed in part by chemical reactions. For NA and INA there are two competitive first-order reactions placed between the electron transfers: (a) an acid-catalysed substitution by water; (b) a non-catalysed elimination of ammonia followed by the reduction of the free aldehyde. For MNA the variation of the limiting current with the pH is due to the simultaneous occurrence of the two-electron reduction and the electrodimerization of the redical formed after the first one-electron transfer. The aim of this paper is, firstly, to carry out a detailed study on the polarographic and kinetic beha-
J. M. RoDRiGUEZ MELADO and R. M. GALV~N OH
OH A-NH, I: I:
Hz0 (a)
-IQ%
I:
I:
CH,OH
viour of NAD and NADP in an acid medium and, secondly, to propose reaction pathways consistent with both the experimental results and the previous knowiedge on the behaviour of the model compounds in those media.
EXPERIMENTAL All reagents used were Merck p. a. grade with the exception of the coenzymes, which were from Sigma of grade II and Sigma grade (NAD and NADP, respectively). The working concentration of NAD and NADP was 1 x lOA M with the exception of the experiments in which the influence of this variable was studied. Solutions were prepared immediately before each experiment by adding solid coenzyme to the background solution. The supporting electrolytes were H,SO, solutions; for pH > 1.2 a 0.1 M H,SO, solution was used, the pH being adjusted with solid NaOH. All potentials were measured against a saturated calomel electrode. Solutions were purged with purified nitrogen and the temperature was kept at 25 fO.l “C. The values of the acidity function H, were obtained from the sulfuric acid concentrations[ 141. Direct current measurements were carried out with an AMEL 465 polarograph and a dropping mercury electrode with m =0.496 mg s- ‘, t = 6.18 s (open circuit) at pH 1.94 in HClO,, h = 60 cm. DP measurements were made on an INELECSA assembly attached to a 6502-based microcomputer. A Multitech Acer 710 microcomputer was used for data treatment. All other experimental conditions are given in the literature[ll-131. The stability of the coenzymes in these media was investigated by recording DP polarograms of 1 x 10m3M reagents in 1.0 M H,SO, solutions. Results show that the solutions are stable for at least 72 h. Thus, no evidences of acid hydrolysis of the amido group were obtained.
diffusion, as is shown by both the temperature coefficients (around 2.2% “C-i) and the slopes of the log i, us log t plots (close to 0.26). The limiting currents remain constant when the pH is increased, whereas the halfwave potentials shift towards more negative values. As is shown in Fig. 1, the E,,, us pH plots are linear, having slopes close to -40 mV dec- ‘. Moreover, the half-wave potentials shift towards less negative values when the drop-time increases. Logarithmic analyses of the waves have been performed by plotting log [i/(iL-i)] us potential. Figure 2 shows the logarithmic analyses for the reduction wave of NAD at different pH values and at a NAD concentration of 1 x 10m4 M. As can be seen, the plots are linear at high acidity, having slopes close to -40 mV dec- i. When the pH increases, the graphs show deviations from linearity. The logarithmic analyses of the reduction wave of NADP show a similar pattern. The shape of the waves is affected by both the pH and the reactant concentration. At a constant pH value, the logarithmic analysis is linear at low concentrations of the coenzymes, whereas the plots show
RESULTS AND DISCUSSION The electroreductions of NAD and NADP coenzymes have been studied in acidic media, ie from 5 M H,SO, to pH = 2. In this range, both compounds show by dc polarography single well-defined waves, whose limiting currents are mainly controlled by
\ PH
HO
of half-wave (dc polarography) and peak potentials with the pH. NAD: (a) E,,,; (c) Ep. NADP: (b) E1,2; (d) E,.
Fig. 1. Variations (DP polarography)
Electroreduction
E(mV)-
Fig. 2. NAD 1 x 10e4 M. Logarithmic analyses. H,SO, concentrations: (a) 5.25; (b) 2.63; (c) 1.31; (d) 0.65 M.
of NAD and NADP
755
potentials are independent of the concentration (in the same concentration range) as can be seen in Table 1. At reactant concentration values greater than 1 x 10e4 M approximately, the ratio i,/c decreases when the concentration is increased (Table 1). Simultaneously, the half-widths of the peaks increase when the concentration is increased, as shown in Fig. 4. As can be seen, the shapes of the half-widths us log c plots are analogous to those obtained for nicotinamide (NA) and N,-methylnicotinamide (MNA) at pH values at which the corresponding reduction processes give rise to two overlapped peaks[12,13]. In the reductions of both pyridine carboxamides, the logarithmic analyses show variations with both the pH and reactant concentration similar to those obtained for the coenzymes. Moreover, the E,,, and E, potentials (at the pH values given in Fig. 4) are independent of the concentrations of reactants at low concentration values, and vary strongly at high concentrations. All these facts are due to the occurrence of two processes taking place at very close potentials, the first (less cathodic) of them corresponding to the one-electron reduction of the amide to yield a radical which suffers a dimerization, whereas the second (more cathodic) one corresponds to the reduction of the radical formed after the first electron transfer. The different depend-
Table 1. DP polarography
NAD
NADP
in 1.5 M H,SO,
c x lO’/M
- E,/mV
0.6 1.2 2.4 4.8 9.6 19.2 38.4
823 825 824 822 820 810 800
1.90 1.91 1.68 1.36 0.86
1.8 3.6 7.2 14.4 28.8 51.6
830 830 828 818 805 192
2.17 2.21 2.14 1.40 0.92 0.76
1.86
1.84
-20 I
I -660
Fig. 3. NAD. Logarithmic 10’ NAD concentrations:
-760
-em
E (mV) analyses in 0.6 M H,SO, (a) 1.25; (b) 5.03; (c) 10.7 M.
deviation from linearity when the concentration is increased, the higher the concentration, the sharper the deviation, as shown in Fig. 3 for the NAD reduction. When the acidity increases, the logarithmic analyses are linear until greater concentration values. Similar results were obtained by differential pulse polarography. When the concentrations of coenzymes are very low (ie less than 5 x lo-’ M) the DP polarograms are symmetrical having half-widths close to 73 mV, in agreement with those expected for irreversible first-order processes[15]. In addition, the peak
.I
l&N
110
z s
X
/
90
70
X
e
e
.
-x
I
I
I
-5
-4
109 t C/M I
Fig. 4. DP polarography. Half-width vs concentration: (0) NAD in 1.5 M H,SO,; (0) NADP in 1.5 M H,SO,; (x) MNA in 0.1 M H,SO,/H,PO, at pH 1.48; (0) NA in 0.2 M H,SO,.
756
J. M. RODRIGUEZ
MELAD~
and R. M. GALV~N
polarographic waves. The Tafel slopes are independent of both the pH and the concentration of reactant (at c < 1 x 10e4 M) having average values close to -40 mV dec- ’ (Fig. 5). The reaction orders with respect to both the H+ ion and the reactant concentration are close to unity. Figure 6 show the plot corresponding to the calculation of the reaction order with respect to NAD. These orders are independent of the potential in the zone where Tafel’s law is obeyed. These results, together those corresponding to the pyridine carboxamides, are shown in Table 2. From the aforementioned results, and taking into account the behaviour of the pyridine carboxamides in these media, we propose the following reduction scheme for the coenzymes at very low concentration values.
ence of these two processes on the reactant concentration causes the separation of the waves (or peaks) when this variable is increased. However, at very low concentration values, the processes are irreversible first-order ones. In the same way, the different pH-dependence of both processes causes its separation when the pH increases, this effect being more marked in the case of MNA than in that of NA. All these facts led us to think that the electroreductions of NAD and NADP in strongly acidic media are complex processes which involve electrodimerization reactions, as is known to occur at physiological pH values[ 11. Nevertheless, at very low concentration values, the processes appear as first-order ones. Tafel curves were obtained by recording the i-E curves at potentials corresponding to the foot of the
OH CONH,
+ O- +
H+
OH
W
(1)
OH i
-
NH, (2)
OH
OH
~-NH,
+ +H
(3)
OH (4)
RE
(X being H or -PO(OH), for NAD and NADP, respectively). Reaction (1) represents the transfer of an electron and a H+ ion, yielding a radical; reaction (2), which according to our data is the rate-determining step, represents the second one-electron transfer; reaction (3) is the uptake of an H+ ion, yielding a gemhydroxyamine; and reaction (4) accounts for the transformation of this species into the aldehyde.
Electroreduction of NAD and NADP d
151
Pyridinyl radicals of model compounds of the coenzymes, like that proposed in reactions (1) and (2), have been detected in homogeneous acidic media by pulse radiolysis[16,17]. Reactions (3) and (4) involve the formation of a gem-hydroxyamine and its transformation into the aldehyde, as is proposed in the literature for processes of this type[ll-13, 18, 193. Moreover, the aldehyde must be hydrated in these media[20, 211. The i-E-t relationship for the above scheme reads: RT
E=-%+~ln~,
1
where: E,,,=Eb
I
-640
-690
I
-740
E (mV) Fig. 5. NADP 1 x lo-& M. Tafel plots. H,SO, concentrations: (a) 0.4; (b) 0.2; (c) 0.05; (d) 0.025 M.
4
-
-2.5 0. I -5
I -4
I -4.5
log (C/M) Fig. 6. NADP in 1.5 M H,SO,. Plot of log i us log c at the foot of the wave. E = -680 mV.
70
2RT ctnF
K,K’k,c,c,
exp [-(l+/?)FE/RT],
-dE,,,/dpH/mV
do-’
-&S/dlog~mVdec-’ i, - i -dE,/apH/mV dec-r Half-width/mV* Tafel slope/mV dec- ’ c order H+ order * DP polarography.
(7)
where cN is the concentration of the coenzyme, K 1and k, are the potential-independent equilibrium and rate constants of reactions (1) and (2), respectively, and K’ represents the quantity: exp [ -( 1 + /I)FA$,,,/RTJ, where A& is the potential of the reference electrode. Assuming that p=OS, the theoretical values of the Tafel slope ( - 39 mV dec- ‘) and the reaction orders (unity in both cases) agree with the experimental ones. At high concentration values, the deviations from linearity of the logarithmic analyses and the anomalous half-widths values of the DP polarograms are indicative of the existence of two ill-separated waves. The deviations are easily explained assuming that the overlapped waves correspond to an electrodimerization and to the electroreduction of the radical formed after the first one-electron transfer, respectively. In this case, the difference between the half-wave (or peak) potentials should increase when the reactant concen-
Table 2. Polarographic and kinetic parameters for the coenzymes and three model compounds in strongly acidic media. Compound
(6)
The logarithmic analysis predicted by equation (5) (done in the form of E us log[i/i,-i)] plots) should yield straight lines with slopes of - 39.4 mV dec- ’ and the half-wave potential should vary towards less negative values when the drop-time is increased (19.6 mV dec- ‘). The experimental results agree with those predictions. On the basis of the above scheme, the i-E relationship for potentials corresponding to the foot of the wave (derived from the Butler-Volmer approximation) can be expressed as: i=2F
-1.5
RT 12t + -ln-+-lnc,. 2anF
i,-i
NAD
NADP
NA
INA
MNA
40
44
65
85
81
40
41
64
29
43
41 72 -41 1.1 1.0
43 15 -38 1.0 1.0
60 82 -62 1.0 1.1
90 44 -29 1.0 2.9
84 61 -40 1.1 2.0
758
J. M. RODRIGUEZMELADOand R. M.
GALVIN
7. A. J. Cunningham and A. L. Underwood, Biochemistry 6, 266 (1967). 8. H. Jaegfeldt, J. electroanaL Chem. 110, 295 (1980). 9. H. Jaegfeldt, A. Tortensson and G. Johansson, Anal. Chem. Acta 97.221 (1978).
tration is increased. In addition, the pK of the radical appearing in reactions (1) and (2) should be around 1.5[17]. Thus, above pH 1 the following reaction
should take place at the electrode. OH
CONH, +H
+
w
(8) F:
I:
The non-protonated radical has been detected in solution by pulse radiolysis being its dimerization rate constant kr6 x 10’ M-‘s-‘[22]. Due to the occurrence of the above reaction, the potential at which the second one-electron transfer takes place shifts towards negative values when the pH is increased, while that corresponding to the first electron transfer is pHindependent. This fact causes the separation of the waves when the pH increases.
REFERENCES 1. B. Janik and P. J. Elving, Chem. Rev. 68, 295 (1968). 2. J. N. Burnett and A. L. Underwood, J. org. Chem. 30, 1154 (1965). 3. C. Carruthers and J. Tech. Arch. Biochem. Biophys. 56, 441 (1955). 4. C. Carruthers and V. Suntzeff, Arch. Biochem. Biophys. 45, 140 (1953). 5. B. Ke, Biochim. Biophys. Acta 20, 547 (1956). 6. J. N. Burnett and A. L. Underwood, Biochemistry 7,3328 (1968).
10. Z. Samec, W. T. Bresnaham and P. J. Elving, J. electroanal. Chem. 133, 1 (1982). 11. R. Marin Galvin and J. M. Rodriguez Mellado, J. electroanaL Chem. 250, 399 (1988). 12. R. Marin Galvin, J. M. Rodriguez Mellado and F. Garcia Blanco, J. electroanal. Chem. 251. 163 (1988). 13. R. Marin Galvin and J. M. Rhdrigiez &fellado, J. electroanal. Chem. 260, 101 (1989). 14. R. G. Bates, Determination of pH. Theory and Practice, pp. 194-199, Wiley, New York (1973). 15. J. M. Rodriguez Mellado, M. BlBzquez, M. Dominguez and J. J. Ruiz, J. electraanal. Chem. 195. 263 (19851. 16. E. M. Kosower, A. Teuerstein and A. J. Swallow, J.’Am. them. Sot. 95,6127 (1973). 17. P. Neta and L. K. Patterson, J. phys. Chem. 78, 2211 (1974). 18. H. Lund, Acta Chem. Stand. 17,2325 (1963). 19. P. E. Iversen, Acta Chem. &and. 24,2495 (1970). 20. J. Tiroufflet and E. Laviron, C. r. Acad. Sci. 247, 217 (1958).
21. J. F. Rusling and P. Zuman, J. electroanal. Chem. 213, 245 (1986). 22. E. J. Land and J. Swallow, Biochim. Biophys. Acta 162, 2327 (1968).
pk.
STUDY OF THE ELECTROREDUCTION OF NAD AND NADP COENZYMES IN STRONGLY ACIDIC MEDIA Jo& Departamento
MIGUEL RoDRiGuEz MELLADO* and RAFAEL MAR~N GALV~N
de Quimica Fisica y Termodinamica Aplicada, Universidad de Grdoba, Magno s.n., 14004-Grdoba, Spain (Received
17 May 1989; in revised firm
S. Albert0
14 June 1989)
Abstract-The electroreductions of NAD and NADP coenzymes have been studied in acidic media (5 M H,SO, to pH = 2) by DP and dc polarography. Tafel slopes and reaction orders were obtained from i-E curves traced at potentials corresponding to the foot of the polarographic waves. The results show that, at very low values of coenzyme concentration (< 5 x lo-’ M) and high acidity (pH< 1), the processes are irreversible. The reduction pathways consist of the reversible transfer of an electron and an H+ ion followed by an irreversible one-electron transfer, which is the rate-determining step. At higher pH and/or concentration values this process is in competition with the electrodimerization of the coenzymes.
INTRODUCTION A great number of enzymes which are involved in redox reactions need the direct participation of the pyridine nucleotides. Two of these coenzymes, ie nicotinamide adenine dinucleotide, NAD, and nicotinamide adenine dinucleotide phosphate, NADP, are hydrogen-transferring coenzymes for the dehydrogenases. A third compound, nicotinamide adenine mononucleotide, NMN, is less well-known. Both NAD and NADP participate in the reactions which they catalyse, being simultaneously reduced. These reactions involve the transfer of an H+ ion and two electrons from the substrate to the coenzyme. The essential component of the pyridine nucleotides is the nicotinamide ring, which is the portion of the coenzymes involved in the redox reactions. Nicotinamide itself is widely distributed in plants and animal tissues. The occurrence of reversible redox reactions under physiological conditions has made advisable to study these systems electrochemically, mainly by polarographic methods[ 11. The NAD-NADH redox system has been extensively studied both potentiometrically and polarographically[2]. Most authors reported one cathodic polarographic wave in both acidic and basic solutions, its half-wave potential being pH_independent[3]. However, in buffered media containing tetraalkilammonium salts, two reduction waves have been reported[4,5]. The first of them corresponds to the irreversible one-electron diffusion-controlled reduction[5]. The second one merges in most buffered solutions with the background discharge, whereas the second reduction waves of model compounds are welldefined at the same potentials. The irreversibility of the processes was indicated by the logarithmic analyses of the waves[2]. The dimer postulated as resulting from the radical produced in the one-electron reduction of NAD has
*Author to whom correspondence should be addressed.
not been isolated and unequivocally identified. Results of electrolysis at the potential of the second wave are equivocal. Addition of cationic surfactants and vigorous ultrasonic agitation increased the rate of NADH formation[6]. The low yields of enzymatically active NADH were explained as due to the concurrent formation of dimer. A reduction mechanism involving the formation of a free radical and its subsequent dimerization was proposed for the first wave[l]. The second wave involves the reduction of the radical formed in the first step. The oxidations of NADH ahd NADPH in both aqueous media[7], acetonitrile[8] and dimethylsulfoxide[9], are bielectronic processes, but evidences of second-order reactions were reportedC9, lo]. Three pyridine carboxamides, ie nicotinamide (NA), N ,-methylnicotinamide (MNA) and isonicotinamide (INA), have been studied in strong acidic media as model compounds of the pyridinic coenzymes[ 1 l-l 33. DP and dc polarography and linear-sweep cyclic voltammetry were used. In all cases, the overall processes are irreversible but the reaction pathways are quite different. At pH < (M.5, the rate-determining step of the reduction process of NA is a protonation placed between the one-electron transfers[ 111; for MNA the process is controlled by the second electron transfer, which is irreversible[12]; finally, in the case of INA, the rds is the loss of ammonia from the unstable gem-hydroxyamine[13]. At pH > 0.5, the variations of the limiting currents with the pH and the i-t curves indicate that the processes are governed in part by chemical reactions. For NA and INA there are two competitive first-order reactions placed between the electron transfers: (a) an acid-catalysed substitution by water; (b) a non-catalysed elimination of ammonia followed by the reduction of the free aldehyde. For MNA the variation of the limiting current with the pH is due to the simultaneous occurrence of the two-electron reduction and the electrodimerization of the redical formed after the first one-electron transfer. The aim of this paper is, firstly, to carry out a detailed study on the polarographic and kinetic beha-
J. M. RoDRiGUEZ MELADO and R. M. GALV~N OH
OH A-NH, I: I:
Hz0 (a)
-IQ%
I:
I:
CH,OH
viour of NAD and NADP in an acid medium and, secondly, to propose reaction pathways consistent with both the experimental results and the previous knowiedge on the behaviour of the model compounds in those media.
EXPERIMENTAL All reagents used were Merck p. a. grade with the exception of the coenzymes, which were from Sigma of grade II and Sigma grade (NAD and NADP, respectively). The working concentration of NAD and NADP was 1 x lOA M with the exception of the experiments in which the influence of this variable was studied. Solutions were prepared immediately before each experiment by adding solid coenzyme to the background solution. The supporting electrolytes were H,SO, solutions; for pH > 1.2 a 0.1 M H,SO, solution was used, the pH being adjusted with solid NaOH. All potentials were measured against a saturated calomel electrode. Solutions were purged with purified nitrogen and the temperature was kept at 25 fO.l “C. The values of the acidity function H, were obtained from the sulfuric acid concentrations[ 141. Direct current measurements were carried out with an AMEL 465 polarograph and a dropping mercury electrode with m =0.496 mg s- ‘, t = 6.18 s (open circuit) at pH 1.94 in HClO,, h = 60 cm. DP measurements were made on an INELECSA assembly attached to a 6502-based microcomputer. A Multitech Acer 710 microcomputer was used for data treatment. All other experimental conditions are given in the literature[ll-131. The stability of the coenzymes in these media was investigated by recording DP polarograms of 1 x 10m3M reagents in 1.0 M H,SO, solutions. Results show that the solutions are stable for at least 72 h. Thus, no evidences of acid hydrolysis of the amido group were obtained.
diffusion, as is shown by both the temperature coefficients (around 2.2% “C-i) and the slopes of the log i, us log t plots (close to 0.26). The limiting currents remain constant when the pH is increased, whereas the halfwave potentials shift towards more negative values. As is shown in Fig. 1, the E,,, us pH plots are linear, having slopes close to -40 mV dec- ‘. Moreover, the half-wave potentials shift towards less negative values when the drop-time increases. Logarithmic analyses of the waves have been performed by plotting log [i/(iL-i)] us potential. Figure 2 shows the logarithmic analyses for the reduction wave of NAD at different pH values and at a NAD concentration of 1 x 10m4 M. As can be seen, the plots are linear at high acidity, having slopes close to -40 mV dec- i. When the pH increases, the graphs show deviations from linearity. The logarithmic analyses of the reduction wave of NADP show a similar pattern. The shape of the waves is affected by both the pH and the reactant concentration. At a constant pH value, the logarithmic analysis is linear at low concentrations of the coenzymes, whereas the plots show
RESULTS AND DISCUSSION The electroreductions of NAD and NADP coenzymes have been studied in acidic media, ie from 5 M H,SO, to pH = 2. In this range, both compounds show by dc polarography single well-defined waves, whose limiting currents are mainly controlled by
\ PH
HO
of half-wave (dc polarography) and peak potentials with the pH. NAD: (a) E,,,; (c) Ep. NADP: (b) E1,2; (d) E,.
Fig. 1. Variations (DP polarography)
Electroreduction
E(mV)-
Fig. 2. NAD 1 x 10e4 M. Logarithmic analyses. H,SO, concentrations: (a) 5.25; (b) 2.63; (c) 1.31; (d) 0.65 M.
of NAD and NADP
755
potentials are independent of the concentration (in the same concentration range) as can be seen in Table 1. At reactant concentration values greater than 1 x 10e4 M approximately, the ratio i,/c decreases when the concentration is increased (Table 1). Simultaneously, the half-widths of the peaks increase when the concentration is increased, as shown in Fig. 4. As can be seen, the shapes of the half-widths us log c plots are analogous to those obtained for nicotinamide (NA) and N,-methylnicotinamide (MNA) at pH values at which the corresponding reduction processes give rise to two overlapped peaks[12,13]. In the reductions of both pyridine carboxamides, the logarithmic analyses show variations with both the pH and reactant concentration similar to those obtained for the coenzymes. Moreover, the E,,, and E, potentials (at the pH values given in Fig. 4) are independent of the concentrations of reactants at low concentration values, and vary strongly at high concentrations. All these facts are due to the occurrence of two processes taking place at very close potentials, the first (less cathodic) of them corresponding to the one-electron reduction of the amide to yield a radical which suffers a dimerization, whereas the second (more cathodic) one corresponds to the reduction of the radical formed after the first electron transfer. The different depend-
Table 1. DP polarography
NAD
NADP
in 1.5 M H,SO,
c x lO’/M
- E,/mV
0.6 1.2 2.4 4.8 9.6 19.2 38.4
823 825 824 822 820 810 800
1.90 1.91 1.68 1.36 0.86
1.8 3.6 7.2 14.4 28.8 51.6
830 830 828 818 805 192
2.17 2.21 2.14 1.40 0.92 0.76
1.86
1.84
-20 I
I -660
Fig. 3. NAD. Logarithmic 10’ NAD concentrations:
-760
-em
E (mV) analyses in 0.6 M H,SO, (a) 1.25; (b) 5.03; (c) 10.7 M.
deviation from linearity when the concentration is increased, the higher the concentration, the sharper the deviation, as shown in Fig. 3 for the NAD reduction. When the acidity increases, the logarithmic analyses are linear until greater concentration values. Similar results were obtained by differential pulse polarography. When the concentrations of coenzymes are very low (ie less than 5 x lo-’ M) the DP polarograms are symmetrical having half-widths close to 73 mV, in agreement with those expected for irreversible first-order processes[15]. In addition, the peak
.I
l&N
110
z s
X
/
90
70
X
e
e
.
-x
I
I
I
-5
-4
109 t C/M I
Fig. 4. DP polarography. Half-width vs concentration: (0) NAD in 1.5 M H,SO,; (0) NADP in 1.5 M H,SO,; (x) MNA in 0.1 M H,SO,/H,PO, at pH 1.48; (0) NA in 0.2 M H,SO,.
756
J. M. RODRIGUEZ
MELAD~
and R. M. GALV~N
polarographic waves. The Tafel slopes are independent of both the pH and the concentration of reactant (at c < 1 x 10e4 M) having average values close to -40 mV dec- ’ (Fig. 5). The reaction orders with respect to both the H+ ion and the reactant concentration are close to unity. Figure 6 show the plot corresponding to the calculation of the reaction order with respect to NAD. These orders are independent of the potential in the zone where Tafel’s law is obeyed. These results, together those corresponding to the pyridine carboxamides, are shown in Table 2. From the aforementioned results, and taking into account the behaviour of the pyridine carboxamides in these media, we propose the following reduction scheme for the coenzymes at very low concentration values.
ence of these two processes on the reactant concentration causes the separation of the waves (or peaks) when this variable is increased. However, at very low concentration values, the processes are irreversible first-order ones. In the same way, the different pH-dependence of both processes causes its separation when the pH increases, this effect being more marked in the case of MNA than in that of NA. All these facts led us to think that the electroreductions of NAD and NADP in strongly acidic media are complex processes which involve electrodimerization reactions, as is known to occur at physiological pH values[ 11. Nevertheless, at very low concentration values, the processes appear as first-order ones. Tafel curves were obtained by recording the i-E curves at potentials corresponding to the foot of the
OH CONH,
+ O- +
H+
OH
W
(1)
OH i
-
NH, (2)
OH
OH
~-NH,
+ +H
(3)
OH (4)
RE
(X being H or -PO(OH), for NAD and NADP, respectively). Reaction (1) represents the transfer of an electron and a H+ ion, yielding a radical; reaction (2), which according to our data is the rate-determining step, represents the second one-electron transfer; reaction (3) is the uptake of an H+ ion, yielding a gemhydroxyamine; and reaction (4) accounts for the transformation of this species into the aldehyde.
Electroreduction of NAD and NADP d
151
Pyridinyl radicals of model compounds of the coenzymes, like that proposed in reactions (1) and (2), have been detected in homogeneous acidic media by pulse radiolysis[16,17]. Reactions (3) and (4) involve the formation of a gem-hydroxyamine and its transformation into the aldehyde, as is proposed in the literature for processes of this type[ll-13, 18, 193. Moreover, the aldehyde must be hydrated in these media[20, 211. The i-E-t relationship for the above scheme reads: RT
E=-%+~ln~,
1
where: E,,,=Eb
I
-640
-690
I
-740
E (mV) Fig. 5. NADP 1 x lo-& M. Tafel plots. H,SO, concentrations: (a) 0.4; (b) 0.2; (c) 0.05; (d) 0.025 M.
4
-
-2.5 0. I -5
I -4
I -4.5
log (C/M) Fig. 6. NADP in 1.5 M H,SO,. Plot of log i us log c at the foot of the wave. E = -680 mV.
70
2RT ctnF
K,K’k,c,c,
exp [-(l+/?)FE/RT],
-dE,,,/dpH/mV
do-’
-&S/dlog~mVdec-’ i, - i -dE,/apH/mV dec-r Half-width/mV* Tafel slope/mV dec- ’ c order H+ order * DP polarography.
(7)
where cN is the concentration of the coenzyme, K 1and k, are the potential-independent equilibrium and rate constants of reactions (1) and (2), respectively, and K’ represents the quantity: exp [ -( 1 + /I)FA$,,,/RTJ, where A& is the potential of the reference electrode. Assuming that p=OS, the theoretical values of the Tafel slope ( - 39 mV dec- ‘) and the reaction orders (unity in both cases) agree with the experimental ones. At high concentration values, the deviations from linearity of the logarithmic analyses and the anomalous half-widths values of the DP polarograms are indicative of the existence of two ill-separated waves. The deviations are easily explained assuming that the overlapped waves correspond to an electrodimerization and to the electroreduction of the radical formed after the first one-electron transfer, respectively. In this case, the difference between the half-wave (or peak) potentials should increase when the reactant concen-
Table 2. Polarographic and kinetic parameters for the coenzymes and three model compounds in strongly acidic media. Compound
(6)
The logarithmic analysis predicted by equation (5) (done in the form of E us log[i/i,-i)] plots) should yield straight lines with slopes of - 39.4 mV dec- ’ and the half-wave potential should vary towards less negative values when the drop-time is increased (19.6 mV dec- ‘). The experimental results agree with those predictions. On the basis of the above scheme, the i-E relationship for potentials corresponding to the foot of the wave (derived from the Butler-Volmer approximation) can be expressed as: i=2F
-1.5
RT 12t + -ln-+-lnc,. 2anF
i,-i
NAD
NADP
NA
INA
MNA
40
44
65
85
81
40
41
64
29
43
41 72 -41 1.1 1.0
43 15 -38 1.0 1.0
60 82 -62 1.0 1.1
90 44 -29 1.0 2.9
84 61 -40 1.1 2.0
758
J. M. RODRIGUEZMELADOand R. M.
GALVIN
7. A. J. Cunningham and A. L. Underwood, Biochemistry 6, 266 (1967). 8. H. Jaegfeldt, J. electroanaL Chem. 110, 295 (1980). 9. H. Jaegfeldt, A. Tortensson and G. Johansson, Anal. Chem. Acta 97.221 (1978).
tration is increased. In addition, the pK of the radical appearing in reactions (1) and (2) should be around 1.5[17]. Thus, above pH 1 the following reaction
should take place at the electrode. OH
CONH, +H
+
w
(8) F:
I:
The non-protonated radical has been detected in solution by pulse radiolysis being its dimerization rate constant kr6 x 10’ M-‘s-‘[22]. Due to the occurrence of the above reaction, the potential at which the second one-electron transfer takes place shifts towards negative values when the pH is increased, while that corresponding to the first electron transfer is pHindependent. This fact causes the separation of the waves when the pH increases.
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