7420
Langmuir 1998, 14, 7420-7426
Effects of Glutamate Dehydrogenase Enzyme on the SERS Spectra of Nicotinamide Adenine Dinucleotide on a Gold Electrode Yi-Jin Xiao,* Yan-Feng Chen, Ting Wang, and Xiao-Xia Gao Department of Chemistry, Peking University, Beijing, 100871 China Received April 15, 1998. In Final Form: September 29, 1998 Near-infrared Fourier transform Raman spectroscopy was used to study the SERS behaviors of nicotinamide adenine dinucleotide (NAD) in a solution containing Glutamate dehydrogenase enzymes. It was found that the SERS spectra of NAD adsorbed on a gold electrode should be largely changed by addition of glutamate dehydrogenase (GDH) in the electrolyte solutions. The potential dependence of the SERS revealed that, in an adequate negative potential region, NAD adsorbed on a gold electrode can be combined with the enzyme to a certain extent. The SERS spectra of NAD/GDH exhibit some different properties from those shown for normal SERS spectra of NAD alone in two aspects: (1) The existence of the enzymes led to decrease or disappearance of several strong bands under open circuit conditions. The reductions of the SERS bands are quite consistent with the changes of normal Raman spectra of NAD in combination with some NAD-dependent dehydrogenase in solutions. The protonation of the adenine moiety was proposed to explain this SERS behavior. (2) Several new bands, some of which can be attributed to the backbone peptide of the enzyme, appeared and increased in intensity with a negative shift of the electrode potential. The appearance of the new bands was attributed to the close approach of the active site of the enzyme induced by the coenzyme preadsorbed on the surface.
Introduction Electrochemistry of redox enzymes is of current interest because of the potential applications in bioelectronics and biosensors.1-5 A variety of electrochemical methods have been used to investigate the enzymes contacting an electrode, directly or through a mediator.6-20 However, ordinary electrochemical techniques are insufficient in identifying the reactive sites or species on the electrode interface. Detailed information, that is, conformations, orientations, as well as active sites for * To whom correspondence should be addressed. (1) Bardea, A.; Katz, E.; Buckmann, A. J.; Willner, I. J. Am. Chem. Soc. 1997, 119, 9114-9119. (2) Heller, A. Acc. Chem. Res. 1990, 23, 128-134. (3) Heller, A. J. Phys. Chem. 1992, 96, 3579-3587. (4) Gopel, W.; Heiduschka, P. Biosens. Bioelectron. 1994, 9, iii. (5) Yacynych, A. M. In Advances in Biosensors; Turner, A., Ed.; JAI Press Ltd.: London, 1992; Vol. 2. (6) Gros, P.; Zaborosch, C.; Schlegel, H. G.; Bergel, A. J. Electroanal. Chem. 1996, 405, 189-195. (7) Degani, Y.; Heller, A. J. Am. Chem. Soc. 1988, 110, 2615-2620. (8) Schuhmonn, W.; Ohara, T.; Heller, A.; Schmidt, H. L. J. Am. Chem. Soc. 1991, 113, 1394-1397. (9) Willner, I.; Katz, E.; Riklin, A.; Kasher, R. J. Am. Chem. Soc. 1992, 114, 10965-10966. (10) Willner, I.; Lapidot, N.; Riklin, A.; Kasher, R.; Zahavy, E.; Katz, E. J. Am. Chem. Soc. 1994, 116, 1428-1441. (11) Willner, I.; Riklin, A.; Shoham, B.; Rivenzon, D.; Katz, E. Adv. Mater. 1993, 5, 912-915. (12) Ohara, T. J.; Rajagopalan, R.; Heller, A. Anal. Chem. 1994, 66, 2451-2457. (13) Ohara, T. J.; Rajagopalan, R.; Heller, A. Anal. Chem. 1993, 65, 3512-3517. (14) Gregg. B. A.; Heller, A. J. Phys. Chem. 1991, 95, 5976-5980. (15) Gregg. B. A.; Heller, A. Anal. Chem. 1990, 62, 258-263. (16) Willner, I.; Mandler, D. Enzyme Microb. Technol. 1989, 11, 467483. (17) Willner, I.; Katz, E.; Lapedot, N.; Baiierle, P. Bioelectrochem. Bioenerg. 1992, 29, 29-45. (18) Yokoyama, K.; Sode, K.; Tamiya, E.; Karube, I. Anal. Chim. Acta 1989, 218, 137-142. (19) D’Costa, E. J.; Higgins, I. J.; Turner, A. P. F. Biosensors 1986, 2, 71-87. (20) Schmidt, H. L.; Schuhmann, W. Biosens. Bioelectron. 1996, 11, 127-135.
adsorbates, is beyond the resolution of electrochemistry, while the technique of surface-enhanced Raman spectroscopy (SERS) could provide molecular level information for the molecules at the interface,21,22 especially suitable for the in-situ investigation of the gold-electrolyte interface. As important oxidoreductase enzymes, nicotinamide adenine dinucleotide (NAD)-dependent dehydrogenases have been extensively studied by various techniques in recent years. The colloidal SERS technique has been proposed to observe the structural changes of NAD induced by active-site binding to a dehydrogenase.23,24 Unfortunately, no such enzyme-cofactor SERS signal could be obtained on a colloid silver.23 The lack of significant surface enhancement was attributed to NAD being deeply buried in the dehydrogenase protein and implied that use of this technique to track the combination of enzymes, coenzyme, and substrate in solution was problematic. Recently, we have designed an original experimental procedure which obtained high-quality spectra attributable to NAD when bound to a dehydrogenase enzyme using the near-infrared Fourier transform surface-enhanced Raman scattering (NIR-FT-SERS) technique. This technique permits monitoring of the process of combination between an NAD-dependent dehydrogenase and its coenzyme. The SERS behavior of NAD under the perturbation of the enzymes showed that NAD molecules adsorbed on the electrode can be progressively complexed by the enzymes, and this process can be governed by adjusting the applied voltages. In the present work, we (21) Fleischmann, M.; Hill, I. R. In Comprehensive Treatise of Electrochemistry; White, R. E., Bockris, J. O’M., Conway, B. E., Yeager, E., Eds.; Plenum Press: New York, 1984; Vol. 8, p 373. (22) Chang, R. K., Furtak, T. E., Eds. Surface Enhanced Raman Scattering; Plenum Press: New York, 1982. (23) Austin, J. C.; Hester, R. E. J. Chem. Soc., Faraday Trans. 1 1989, 85, 1159-1168. (24) Siiman, O.; Rivellini, R.; Patel, R. Inorg. Chem. 1988, 27, 39403949.
10.1021/la980432b CCC: $15.00 © 1998 American Chemical Society Published on Web 11/25/1998
NAD on a Gold Electrode
Langmuir, Vol. 14, No. 26, 1998 7421
Figure 1. SERS spectra of NAD adsorbed on a gold electrode. 1 × 10-5 mol‚L-1 NAD, 10 m mol‚L-1 H2PO4, and 100 mmol‚L-1 KCl (pH 7.0). Applied voltages (V vs SCE): a, + 0.4; b, 0.0; c, -0.2; d, -0.4; e, -0.6; f, -0.8. A waiting time of about 5 min at each voltage was used before recording the spectra.
will report the results of the studies on the interaction of NAD and glutamate dehydrogenase (GDH) on a gold electrode. Experimental Section Nicotinamide adenine dinucleotide (NAD) (grade 1, 100%) and beef liver glutamate dehydrogenase were purchased from Sigma Co. All of the other reagents were of analytical grade. Fourier transform surface-enhanced Raman scattering measurements were carried out on a Bruker IFS 66/FRA 106FTRaman spectrometer equipped with a diode-pumped Nd:YAG laser exciting at 1064 nm. A 180° backscattering geometry was used. The laser power at the sample was approximately 100mW. All of the spectra were acquired by 200 scans at the resolution 2 cm-1; none of the spectra presented have been smoothed. A specifically designed 4 mL spectroelectrochemical cell with a CaF2 window was used in the studies. The cell was equipped with a three-electrode system consisting of a Pt counter electrode, a polycrystalline gold disk (φ ) 5 mm) embedded in a Teflon holder as a working electrode, and a saturated calomel reference electrode. The working electrode was carefully polished with emery paper (No. 1500) and ultrasonically cleaned in distilled
water before use. The roughening of the electrode was achieved electrochemically by 30 oxidation-reduction cycles (ORCs) from +1.1 to -0.3V versus SCE at 0.5 V s-1. The solutions for the ORC performance consisted of 0.1 M KCl, 10 mM Na2HPO4, and 1 × 10-5 M NAD. The voltages for the roughing and the polarization of the electrode were controlled using an M173 potentiostat and an M175 program generator (EG&G, USA). After the roughening, an aliquot of GDH was injected into the electrolyte solution.
Results and Discussion 1. SERS Spectra of NAD on a Gold Electrode. As shown in Figure 1, a very defined SERS pattern was obtained on a roughened Au electrode with NAD solutions of 1 × 10-5 M. All of the SERS bands are assigned as in Table 1, on the basis of the suggestion25 that the Raman spectrum bands of NAD can be simply attributed to the (25) Yue, K. T.; Martin, C. L.; Chen, D.; Nelson, P.; Sloan, D. L.; Callender, R. Biochemistry 1986, 25, 4941-4947. (26) Xiao, Y.-J.; Markwell, J. P. Langmuir 1997, 13, 7068-7074.
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Table 1. SERS Bands (cm-1) of NAD and NAD + GDH on a Gold Electrodea -0.4 V
open circuit NAD
NAD + GDH
NAD
NAD + GDH
1628m
1665m 1627m
1571s
1584s
1600m 1554m
assignmentb amide I, GDH N N N A
1501w 1468s
1470m 1418m
1375w 1338s 1317s
1320s
1321s
1114w 1025vs 825w 729m
1113w 1028s 828w 732w
1180m 1116m 1025vs 818m 731s
1323m 1230m 1186m 1115m 1027vs 820w 732m
A N A A A amide III, GDH R2/N R2/P N, ring R A, ring
a Abbreviations: A, adenine; N, nicotinamide; P, phosphate; R, ribose; vs, very strong; s, strong; m, medium; w, weak. b References 25, 26, and 28 and the references therein.
adenine, nicotinamide, ribose, and phosphate constituent molecular moieties in the structure of NAD:
To explain the SERS behavior of NAD on a gold electrode, we previously proposed an adsorption mechanism.26,27 The SERS bands assignable to adenine and nicotinamide moieties both underwent strengthened enhancement, indicating that both are directly adsorbed onto the electrode surface. The ribose and phosphate moieties may be exposed to the solution, since very weak SERS signals were observed from these moieties at a positive electrode potential. Under a positive electrode potential, the adenine moiety adopted a vertical configuration, in which the amino group and N7 are coordinated to the electrode surface, whereas the nicotinamide moiety is apparently adsorbed in a flat orientation with respect to the electrode surface. The substantial changes of the SERS spectra with increasingly negative potential indicate a potentialinduced molecular reorientation. Specifically, the adenine moiety changes from an end-on surface interaction with the positively charged electrode dominated by interaction of the extraring amino group to a face-on interaction involving the ring electrons. In contrast, the nicotinamide moiety is in an end-on orientation with respect to the surface. Additionally, the changes observed with SERS during potential scanning imply that the adsorption (27) Xiao, Y.-J.; Wang, T.; Xang, X.-Q.; Gao, X.-X. J. Electroanal. Chem. 1997, 433, 49-56.
intensity of NAD on the electrode is quite sensitive to the applied voltage. A tight chemical adsorption should occur between the NAD molecule and the positively charged electrode surface. However, under negative potentials, the NAD molecule mainly undertakes a physical adsorption and loosely contacts the electrode surface. This difference in the interaction of NAD with the electrode is the basis of our experiments below to attempt observation and monitoring of the interactions of NAD with dehydrogenase enzymes on the electrode by the SERS method. 2. GDH-Induced Changes in NAD SERS. As mentioned above, NAD molecules are adsorbed onto the electrode surface and give strong surface-enhanced Raman scattering under positive potentials or nearby 0 V. However, as shown in Figure 2, significant changes in the SERS of NAD are produced by the addition of GDH. The affects of GDH on the SERS of NAD depend on the polarization state of the electrode surface. With significantly positive potentials (>0.2 V vs SCE), the addition of GDH produced minimal changes in the NAD SERS signals. This implies a tight chemical adsorption of NAD on the positively charged electrode. In contrast, at electrode potentials of 0 V or less, the addition of GDH results in significant changes in the SERS of NAD. 2.1. Effects of GDH on the SERS of NAD under Open Circuit Conditions. Time-dependent SERS spectra under open circuit after the addition of GDH are shown in Figure 3. Substantial decreases in the intensities of the SERS bands of the adenine moiety upon the addition of GDH are found in the 1554, 1470, 1341, and 741 cm-1 bands. In addition, the spectra display a slight decrease in intensity for the 1027 cm-1 band, which is assignable to the breathing vibration of the nicotinamide moiety. The partial disappearance of the adenine bands could represent a characteristic interaction between NAD and GDH. These SERS behaviors, especially the decrease of the 1341 and 1470 cm-1 bands, are in agreement with the changes of normal Raman (NR) and resonance Raman (RR) spectra of NAD or NADH bound to dehydrogenases in aqueous solutions.29-32 It has been observed that the 1335 and 1483 cm-1 bands of NAD were greatly lowered in intensity or completely vanished upon binding to dehydrogenases in solution.29,30,32,33 These data were tentatively interpreted as an indication of protonation at N7 or N1 of the adenine ring.34 Indeed, this proposition was supported by our previous experimental results26,27 that the lowering of the pH value should always decrease the intensities of the corespondent bands of NAD under open circuit or nearby 0 V. It is interesting to note that a negative movement of the electrode potential may also lead to a similar change of the SERS spectra of NAD in the case of no enzyme in the solution. This is probably because the negatively charged surface should also result in a pH decrease near the electrode-electrolyte interface. Therefore, we believe that the changes in the SERS spectra of NAD upon combination with GDH are due to the protonation of the adenine moiety. This SERS (28) Carey, P. R. Biochemical Applications of Raman and Resonance Raman Spectroscopy; Academic Press: New York, 1982. (29) Austin, J. C.; Wharton, C. W.; Hester, R. E. Biochemistry 1989, 28, 1533-1538. (30) Deng, H.; Zheng, J.; Burgner, J.; Sloan, D.; Callender, R. Biochemistry 1989, 28, 1525-1533. (31) Deng, H.; Yue, K. T.; Martin, C.; Rhee, K. W.; Sloan, D.; Callender, R. Biochemistry 1987, 26, 4776-4784. (32) Deng, H.; Burgner, J.; Callender, R. Biochemistry 1991, 30, 88048811. (33) Chen, D.; Yue, K. T.; Martin, C.; Rhee, K. W.; Sloan, D.; Callender, R. Biochemistry 1987, 26, 4776-4784. (34) Nadolny, C.; Zundel, G. J. Mol. Struct. 1996, 385, 81-87.
NAD on a Gold Electrode
Langmuir, Vol. 14, No. 26, 1998 7423
Figure 2. SERS spectra of NAD adsorbed on a gold electrode in the solution containing 10 µmol‚L-1 GDH. Applied voltages (V): a, 0.0; b, -0.2; c, -0.4; d, -0.6; e, -0.8; f, -1.0. The other conditions are the same as those in Figure 1.
behavior supports the suggestion that the entrance of NAD into the binding site of the enzyme should result in a protonation of the adenine moiety.29,30,32,33 As can be seen in Figure 3b, the SERS band near 1320 cm-1 remains unchanged in intensity with the addition of the enzymes. This band has been assigned to the C6NH2 vibration of the amino group of the adenine moiety.26 The strong band at 1320 cm-1 is interpreted as showing the direct contact of the NH2 group to the electrode surface. This phenomenon is very consistent with the structural features of NAD in all enzymatic environments.29 The edge of the adenine ring is generally oriented toward the enzyme whereas C6-NH2 is oriented toward the solution. This binding feature is the structural basis for the general application of affinity chromatography utilizing immobilized NAD linked to a solid support through the N6 atom.29 2.2. Potential Dependence of NAD SERS on the Presence of GDH. The potential dependence of the spectra on the presence of the enzyme reveals an extremely interesting and surprising effect. As shown in Figure 2, upon scanning
the potential in a negative direction, several new bands appear and increase in intensity up to the limit -0.6 V. It is worth noting that some of these bands cannot be attributed to any known vibration mode of the NAD molecule. A comparison of the SERS bands in Figure 2c with that in Figure 1d illustrates significant GDH-induced changes in the SERS spectra of NAD on the negatively charged electrode. Several new bands near 1663, 1418, and 1230 cm-1 appear. In addition, the 1571 cm-1 band, which has been assigned to the mix of CdC and CdO vibrations of the nicotinamide moiety, shifts from 1571 to 1584 cm-1 in the SERS spectrum compared with a shift from 1570 to 1580 cm-1 in the NR spectrum. The shift of the SERS band in Figure 2c is evidence for formation of a NAD-GDH complex near the electrode surface. Obviously, the possible presence of multiple species (NAD, NADH, NAD-GDH, GDH, etc.) in solution and on the surface complicates the interpretation. However, as shown in Figure 4, the enzyme alone (in the case of no NAD adsorbed on the electrode) does not give any recognizable SERS signal in the resolution. Even if the
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Figure 3. Time-dependent SERS spectra of NAD preadsorbed on a gold electrode after addition of GDH into the electrolyte solution under the open circuit condition: (a) before addition of GDH; (b) 10 min after addition of GDH; (c) 30 min after addition of GDH. The other conditions are the same as those in Figure 1.
enzyme and coenzyme were mixed stoichiometrically before the SERS measurement, we also failed to obtain any SERS signal. In additon, the NADH molecules, which, if any, appear at very nagative potentials, cannot be recognized by SERS measurement.26 Furthermore, SERS may give information about only the molecules adsorbed on the surface. Therefore, it is reasonable to attribute the SERS bands in Figure 2 to NAD adsorbed on the surface and the active site of the enzyme introduced to the surface by NAD. The most important change in the SERS spectra of NAD with the addition of the enzyme is the appearance of 1663 cm-1 band, which is located in the region of amide CdO stretching motions, while this band cannot be simply assigned to any group in the system, because not only the NAD coenzyme but also the polypeptide backbone of the GDH enzyme has some amide groups. However, no band near 1663 cm-1 has ever been detected in normal Raman spectra of NAD in solution or in an enzymatic environment or in SERS of NAD on a metal surface. The nicotinamide
moiety of NAD gives an amide I band at 1630 cm-1. Furthermore, if it were accurate to assign the 1663 cm-1 band to the nicotinamide moiety of NAD, the upward shift of the band from 1630 to 1663 cm-1 should be caused by the hydrogen bonding existing between the carbonyl group of the nicotinamide moiety of the coenzyme and the active site of the enzyme. However, the hydrogen bonding may principally result in a downward shift of the carbonyl stretching vibration,35 rather than an upward shift. So, we intend to assign this band to the polypeptide backbone amide CdO stretching motions. On the basis of both theoretical calculations and empirical observation of bands in the normal vibrational spectra (normal Raman and IR) of polypeptides in solutions,28,30,36,37 the bands centered at approximately 1665 cm-1 are believed to reflect the amide I vibration of turn (35) Surewicz, W. K.; Mantsch, H. H. Biochim. Biophys. Acta 1988, 952, 115-130. (36) Ryttersgaard, J.; Larsen, B. D.; Holm, A.; Christensen, D. H.; Nielsen, O. F. Spectrochim. Acta, Part A 1997, 53, 91-98.
NAD on a Gold Electrode
Langmuir, Vol. 14, No. 26, 1998 7425
Figure 4. SERS spectra of GDH in the case of no NAD adsorbed on the surface. Applied voltages (V vs SCE): a, 0.0; b, -0.2; c, -0.4; d, -0.6; e, -0.8; f, -1.0. The other conditions are the same as those in Figure 1.
segments, while the 1230 cm-1 band is attributed to the amide III vibration of GDH. Generally, the amide I band represents the CdO stretching vibrations of the amide groups.35 The exact frequency of the band depends on the nature of hydrogen bonding involving the amide CdO and NH moieties. The bands in this frequency region usually reflect the particular secondary structure adopted by the polypeptide chains. However, because an enzyme generally contains a variety of tertiary structure domains, a conventional vibration spectrum usually gives complex, overlapping amide bond contours representing R-helices, β-sheets, turns, and nonordered structures. Due to the inherently large widths of these overlapping component bands and the poor signal-to-noise ratio in aqueous solutions, individual features cannot normally be resolved, making any interpretation questionable, even at a qualitative level.35 However, the appearance of very resolved SERS bands assignable to the vibration modes of the (37) Krimm, S.; Bandekar, J. Adv. Protein Chem. 1986, 38, 181364.
polypeptide backbone suggests a potential use of the SERS method in the study of the secondary structures of enzymes. When the potential was negatively moved, the other characteristic bands changed in the same manner as that for those in the normal SERS spectra of NAD in the system without enzyme. This is because some of the NAD molecules are still adsorbed on the electrode in the normal model, without being affected by the enzyme. In light of the suggestion38 that SERS signals of macromolecules are only detected from components in direct contact with the electrode surface, we propose that specific signals can be ascribed to the formation of the enzyme-NAD binary complex upon the electrode. The 1663 cm-1 band strongly implies turn35 segments close to the electrode surface. Because the enzyme itself does not give any SERS band in this region, it may reflect the NADdependent approach of the enzyme to the electrode surface. (38) Koglin, E.; Sequaris, J. M. Top. Curr. Chem. 1986, 134, 1-57.
7426 Langmuir, Vol. 14, No. 26, 1998
The increase of the amide I signal at 1663 cm-1 with more negative electrode potentials may implicate a surface potential-induced approach of the enzyme to the electrode and interaction with the NAD cofactor to produce the Raman enhancement. When the electrode potential is made more negative than -0.6 V, all of the SERS bands decrease and then disappear completely at potentials more negative than -1.0 V, indicating the desorption of the adsorbates. 3. Further Discussions on the SERS Behaviors of the NAD-GDH System. The potential-dependent SERS behaviors of NAD in the presence of GDH demonstrate that, under certain potentials, at least a portion of the NAD molecules on the electrode can interact with the enzyme. When this occurs, the adenine moiety function appears to partition into a hydrophobic pocket of enzyme whereas the nicotinamide moiety is partly left exposed near the surface of the electrode. From an understanding of the general spectroscopic properties of proteins,28,35,36 the frequency of an amide I mode, arising from polypeptide backbone amide CdO stretching motions, is dependent on its hydrogen-bonding environment and hence its secondary structure. Specifically, a frequency near 1663 cm-1 is typically found in turn motifs.35,39-44 The amide III regions, due to polypeptide backbone N-H in-plane bending and C-N stretching motions, are also sensitive to secondary structures. Because turns are often found near the surface of a protein,45 the SERS bands may indicate a greater interaction of these structural elements with the surface of the enzyme. (39) Susi, H.; Byler, M. Methods Enzymol. 1986, 130, 290-311. (40) Byler, M.; Susi, H. Biopolymers 1986, 25, 469-487. (41) Harris, P. I.; Lee, D. C.; Chapman, D. Biochim. Biophys. Acta 1986, 874, 255-265. (42) Yang, P. W.; Mantsch, H. H.; Arrondo, J. L. R.; Saint-Girons, I.; Guillou, Y.; Cohen, G. N.; Barzu, O. Biochemistry 1987, 26, 2706-2711. (43) Surewicz, W. K.; Moscarello, M. A.; Mantsch, H. H. Biochemistry 1987, 26, 3881-3886. (44) Arrondo, J. L. R.; Mantsch, H. H.; Mullner, N.; Pikula, S.; Martonosi, A. J. Biol. Chem. 1987, 262, 9037-9043. (45) Lehninger, A. L.; Nelson, D. L.; Cox, M. M. Principles of Biochemistry, 2nd ed.; Worth Publishers: New York, 1993; pp 390392.
Xiao et al.
However, we cannot explain the development of a strong band at 1418 cm-1 with increasing negative potential. This band has not been previously reported in either normal Raman spectroscopy or SERS of NAD in solution or bound to a enzyme. It is interesting to note that the reduced coenzyme NADH shows a strong band near 1422 cm-1 in solution and 1416 cm-1 when bound to dehydrogenases.29-32 It therefore seems possible that the NAD molecules are reduced, catalyzed by the enzyme, to NADH on the negatively charged electrode. The electrochemical reduction of NAD catalyzed by dehydrogenase or hydrogenase enzymes has been investigated,1,6 and it is likely that the binding to the dehydrogenase greatly reduced the overpotential for the electroreduction of the NAD on the electrode. However, this suggestion is inconsistent with two other SERS behaviors of the system. First, the relative intensity of the 1027 cm-1 band was almost unchanged by interaction with the dehydrogenase under conditions of negative potential. It has been known that only the oxided nicotinamide moiety of NAD shows aromatic nature and gives a ring-breathing motion near 1030 cm-1. In contrast, reduced nicotinamide is not aromatic and produces no band near 1030 cm-1.30 Second, the 1680 cm-1 band, which is normally apparent in spectra of NADH in solution, did not appear at all. We interpret this SERS behavior as an unfavorable orientation of the reduced nicotinamide moiety with respect to the electrode surface. Obviously, further systematic studies will be required to validate the ability of SERS spectra to provide structural information about the structure and reaction mechanisms of NAD-dependent dehydrogenases and other enzymes, which will complement the existing methods currently employed in this active area of research. Acknowledgment. The research was supported by the National Science Foundation of China (Grant No. 29675001). LA980432B
