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Anal. Chem. 1982, 54, 586-587
CORRESPONDENCE Cathodic Reduction of Dihydronicotinamide Adenine Dinucleotide at Mercury Electrodes Sir: There has been considerable interest in the redox chemistry of pyridine bases such as the NAD+ (nicotinamide adenine dinucleotide)/NADH couple because of their importance in biochemistry (1,Z). The electrochemical reduction of NAD* has been well characterized (3-5). Also, there have been numerous studies of the oxidation of NADH at solid electrodes (6-10). From the analytical point of view it is desirable to be able to determine NADH in aqueous solution. Because of the well-known difficulties associated with use of solid electrodes, especially in aqueous solution, it would also be desirable to be able to determine NADH voltammetrically at mercury electrodes. The oxidation of NADH occurs at +0.8 V vs. SCE in neutral aqueous media (6),which is well positive of potentials at which mercury itself is oxidized in these media. We report here on the reduction of NADH a t mercury electrodes in aqueous solution containing succinic acid. EXPERIMENTAL SECTION Solutions were prepared from analytical grade chemicals and distilled water repurified with a Millipore MilliQ system. Stock solutions of NADH (Sigma Lot No. 619-FO) were prepared in distilled water. Voltammetric data were obtained with a PARC Model 174A polarographic analyzer, an IBM Instruments Model EC225, a one-drop square wave analyzer (11),or a computer-based system of our design (12).In all cases a PARC Model 303 static mercury drop electrode was the indicator electrode. All potentials were measured and are reported with respect to a saturated calomel reference electrode. RESULTS AND DISCUSSION Aqueous succinic acid buffer solutions were employed initially in this investigation because they are widely used as the mobile phase in high-pressure liquid chromatography. Figure 1 displays square wave voltammograms for the reduction of NADH in succinic acid solution. The peak at -760 mV is characteristic of the succinic acid supporting electrolyte and varies only slightly in amplitude with addition of NADH. The peaks at -890 and -1155 mV appear on addition of NADH and are not present in solutions not containing succinic acid. The cathodic reactions responsible for these peaks may be associated with the nicotinamide moiety, for nicotinamide itself exhibits one peak at ca. -1 V which is similar to the NADH peak at -890 mV. Furthermore addition of nicotinamide to solutions of NADH in succinic acid causes an increase in the heights of the peaks associated with NADH. The value of the peak current for the most cathodic peak is somewhat greater than might be expected for a reversible one-electron reaction, For example, for the third curve of Figure 1, the peak current is about 450 nA, but the value for a reversible one-electron reaction (D= 5 x lo* cm2/s) under these conditions is about 150 nA. A similar response is observed in 0.1 F acetic acid, but no peak appears for reduction of NADH in 0.1 F oxalic acid. Cyclic voltammetry gave the same three cathodic peaks with no indication of anodic peaks on the reverse cycle. Currents again were greater than expected for a reversible one-electron reduction. Over the range of sweep rate 50-1000 mV/s, plots of log i, vs. log u were linear with slope d log i,/d log u = 0.67
Table I. Effect of Succinic Acid Concentration on Calibration Curve for NADH"
CH,A
0.02 0.04 0.06 0.08
slope, nA/ (PdmL)
intercept, nA
464 487 497 498
-73
-71
-62 -43
i ( 1 PiJ/
mL)& 393 414 436 455
a NADH 0.2-1.2 pg/mL. Current predicted by the calibration curve for 1 pg/mL NADH.
for the most cathodic peak. Furthermore, the peak potential was linear with log u with slope d E,/d log u = -41 mV up to sweep rates of 600 mV/s; a t higher sweep rates E, (=-1245 mV) is independent of sweep rate. In the normal pulse mode the limiting currents for the waves associated with NADH are almost completely depressed, and very pronounced peaks appear at -925 and -1200 mV. These data do not suffice to define the mechanism, but they are consistent with the involvement of reactant adsorption in the overall process (13-15). The adsorption of NADH has not been studied under these conditions but purines are known to be adsorbed under a variety of conditions (16) and the adsorption of adenine mononucleotide8 in acid solution is also well-known (17). It is well-known that aqueous solutions of NADH decompose on standing (6). In all cases investigated here a nearly reversible couple appeared at ca. -515 mV (&/iP,, = 0.92, E,,c - E , , = 37 mV at u = 200 mV/s) when an old stock solution was used. While no attampt was made to quantitate this phenomenon, this couple might prove highly useful analytically for the purpose of assessing the extent of decomposition of solutions of NADH. The effect of analytical concentration of succinic acid and of pH on the most negative reduction peak was determined by use of square wave voltammetry. Good linear calibration curves are obtained in the pg/mL range in all cases. The dependence on analytical concentration of succinic acid is not strong. However as the succinic acid concentration is increased, the slope of the calibration curve increases and the magnitude of the negative intercept decreases. Typical data for this dependence are given in Table I. The current predicted by the curve at concentration 1 pg/mL is given to illustrate the combined effects of changes in slope and intercept. Above the concentration of 0.1 F succinic acid the improvement in the calibration curves is insignificant, and therefore this concentration was selected for all further experiments. The effect of pH at an analytical concentration of 0.1 F succinic acid was pronounced. Succinic acid has pK values of 4.19 and 5.48. The measured value of pH in 0.1 F succinic acid was 2.35. The effect of variation of pH in the range 2.35-4.40 was investigated in solutions 0.1 F in succinic acid with added NaOH. Over this range ao,the fraction of succinic acid species present as H2A varies from 0.99 to 0.36. Data of calibration curves obtained over this range are presented in Table 11. For these data a plot of i (1pg/mL) vs. a0 is linear
0003-2700/82/0354-0566$01.25/00 1982 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 54, NO. 3, MARCH 1982
01
-550
-650
-750
-850
POTENTIRL
-350
-1050
-1150-12’
lMVJ
Figure 1. Square wave voltammograms for NADH in 0.1 M succinic acid: concentrations of NADH were 0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 pg/mL, respectively, from lowest to highest curve; initial potential, -0.550 V; step height, 5 mV; square wave amplitude, 25 mV; square wave frequency, 30 Hz; delay time at -0.550 V, 1 s;drop area, 0.0132 om2. Computer-based system.
Table 11. Effect of pH on Calibration Curve for NADHa slope, intern A/ cept, i (1 wg/ PH (IJg/mL) nA mL)b 2.35 3.24 3.65 3.92 4.17 4.40
438 418 340 263 199 96
-4 -56 -39 -39 -29 -22
434 362 301 224 170 74
a NADH 0.2-1.2 pg/mL, 0.1 F succinic acid. Current predicted by the calibration curve for 1pg/mL NADH.
(r = 0.997) with intercept -118 nA and slope 545 nA. Thus the voltammetric response appears to be associated with the succinic acid molecule itself. Because the sensitivity decreases with increasing pH, 0.1 F succinic acid appears to be the medium of choice. Under these conditions the primary hydrolysis of NADH is complete in minutes, but the peak current increases by only a few percent during the course of this reaction. It might be noted that the largest currents of Table I1 are even less than the largest currents of Table I. This is due to difference in the surface area of the electrode. The large negative intercepts of the calibration curves also deserve some comment. All currents for these curves were measured from a line tangent to the curve on each side of the peak in the customary fashion. This is the most conservative procedure, for in a practical analytical situation a true background curve in the absence of analyte is frequently impossible to obtain. The problem of subtracting the background current is further complicated in this case by two factors. First, succinic acid
587
itself (or an impurity) gives some current response in the potential range of interest (see Figure 1). Preliminary experiments with unrecrystallized succinic acid gave substantially more current in this potential region. The significant point, however, is that in the presence of NADH this background current is suppressed. Therefore the current in 0.1 F succinic acid is not the appropriate background current to subtract. The calibration curve obtained from Figure 1 (measured from the background curve) has intercept -82 nA and slope 404 nA/(pg/mL) with a correlation coefficient of 0.9998. The standard deviation about the line is 8.4 nA/ (pg/mL) and the confidence intervals (at 95% confidence) of the slope and intercept are 10 nA/(pg/mL) and 19 nA, respectively. The slope is less negative for calibration curves obtained over lower concentration ranges. We have worked routinely at 0.2 rg/mL NADH (0.26 pM). It appears that the detection limit is a t least as low as 0.1 pg/mL. We are extending these observations to applications involving enzymatic reactions of NADH and to high-pressure liquid chromatographic separation and detection of NADH. Further work is also being done to elucidate the mechanism of cathodic reduction of NADH under these conditions. ACKNOWLEDGMENT The authors wish to thank Dennis Crouse for providing samples of NADH and Andrew Webber for experimental assitance and helpful comments. LITERATURE C I T E D (1) Kaplan, N. 0. “The Enzymes”; Boyer, P. D., Lardy, H., Myrback, K., Eds.; Academic Press: New York, 1960; Vol. 3, p 105. (2) Underwood, A. L.; Burnett, J. N. I n “Electroanalytical Chemistry”; Bard, A. J., Ed.; Marcel Dekker: New York, 1972; Vol. 6, pp 1-85. (3) Eking, P. J.; O’Rellly, J. E.; Schmakel, C. 0. “Methods of Biochemical Analysis”; Wky-Interscience: New York, 1973; Vol. 21. pp 287-465. (4) Thevenot, D.; Buvet, R. J . Necwoanal. Chem. 1072, 39, 429-446. (5) Thevenot, D.; Buvet, R. J . ElecWoanal. Chem. 1072, 40. 197-207. (6) @faun. R. D.; Santhanam, K. S. V.; Elving, P. J. J . Am. Chem. SOC. 1075, 97, 2591-2598. (7) Thomas, L. C.; Christian, 0. D. Anal. Chlm. Acta 1075, 78, 271-276. (8) Blaedel, W. J.; Jenkins, R. A. Anal. Chem. 1075, 4 7 , 1337-1343. (9) Morbux. J.; Elving, P. J. Anal. Chem. 1078, 50, 1056-1062. (10) Jaegfeldt. H.; Torstensson, A.; Johansson, G. Anal. Chlm. Acta 1978, 97,221-228. (11) Yarnitzky, C.; Osteryoung, R. A,; Osteryoung, . - Janet Anal. Chem. 1080, 52, 1174-1178. Brumleve, T. R.; O’Dea, J. J.; Osteryoung, R. A,; Osteryoung. Janet Anal. Chem. 1081, 53, 702-706. Wopschall, R. H.; Shah Irving Anal. Chem. 1067, 39, 1514-1527. Wopschall, R. H.; Shah Irving Anal. Chem. 1087, 39, 1535-1542. Flanagan, J. 6.; Takahashi, K.; Anson, F. C. J. Elechoanal. Chem. 1077, 8 5 , 257-286. Klnoshtta, H.; Christian, S. D.; Kine, M. H.; Baker, J. G.; Dryhurst, Glenn I n “Electrochemical Studies of Biological Systems”; Sawyer, D. T., Ed.; American Chemical Society: Washington, DC, 1977; Chapter 8; ACS Symp. Ser., No. 38. Krznaric, D.; Valenta, P.; Niirnberg, H. W. J. &choana/. Chem. 1075, 65, 863-681.
Mumtaz S h a h J a n e t Osteryoung* Department of Chemistry State University of New York a t Buffalo Buffalo, New York 14214
REXEIVFXI for review September 8,1981. Accepted November 13, 1981. This work was supported in part by IBM Instruments, Inc.
