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Langmuir 1997, 13, 7068-7074
Potential Dependence of the Conformations of Nicotinamide Adenine Dinucleotide on Gold Electrode Determined by FT-Near-IR-SERS Yi-Jin Xiao*,† and J. P. Markwell‡ Department of Chemistry, Peking University, Beijing 100871, China, and Department of Biochemistry, University of NebraskasLincoln, Lincoln, Nebraska 68588 Received January 13, 1997. In Final Form: August 7, 1997X High-quality Fourier transform surface-enhanced Raman scattering (FT-SERS) of a dilute solution of nicotinamide adenine dinucleotide (NAD) was recorded on a roughened Au electrode. It was found that the detection limit could be lower as much as 3 orders of magnitude by using a near-infrared laser as the exciting source and an in-situ roughening technique. The SERS of NAD shows a strong potential dependence in the non-Faradaic regions. In regions of positive electrode potential, only the bands responsible for the adenine and nicotinamide moieties can be observed. In contrast, with a negative shift of the potential, several additional strong bands representing the ribose and phosphate moieties are also evident. Based upon the SERS behaviors of NAD in the regions of different potential, an adsorption mechanism for dilute NAD on the gold electrode was proposed. With this mechanism, the stacked NAD molecule is considered to be opened to some extent on an electric charged electrode. Specifically, under sufficiently negative potential, the NAD molecule appears to exist in a well-extended state on the gold electrode, leading to the tight adsorption of the entire NAD molecule on the electrode.
1. Introduction It is well-known that the coenzyme couple nicotinamide adenine dinucleotide (NAD)/NADH plays an important role in many biological redox processes.1-3 The reaction properties and conformations of this coenzyme have been extensively studied using a wide range of disciplines and technologies. A number of studies have shown that this coenzyme mainly assumes a stacked conformation in solution.4-6 In contrast, X-ray diffraction analysis has indicated an extended NAD conformation when bound in biochemical systems.1,7,8 Knowledge of the factors influencing the folding process would be desirable and could lead to an increased understanding of the role of this cofactor in enzymatic redox reactions. Various techniques have been used to identify the conformations assumed by NAD in different environments such as aqueous solution,4-6 binding to enzymes or metal ions,1,7,8 crystallization,9,10 or adsorption on metal * To whom correspondence should be addressed. † Peking University. ‡ University of NebraskasLincoln. X Abstract published in Advance ACS Abstracts, November 15, 1997. (1) Boyer, P. D. The Enzymer, 3rd ed.; Academic: New York, 1975; Vol. 2. (2) Sund, H. Pyridine Nucleotide-dependent Dehydrogenases; de Gruyter: Berlin, 1977. (3) Lehninger, A. L. Principles of Biochemistry, 2nd ed.; Worth: New York, 1993; Chapter 13, p 364. (4) Sarma, R. H.; Mynott, R. J. J. Am. Chem. Soc. 1973, 95, 7470. (5) Hamill, W. D.; Pugmire, R. J.; Grant, D. M. J. Am. Chem. Soc. 1974, 96, 2885. (6) Perahia, D.; Pullman, B.; Saran, A. In Structure and Conformation of Nucleic Acids and Protein-Nucleic Acid Interactions; Sundaralingam, M., Rao, S. T., Eds.; University Park Press: Baltimore, 1975; p 685. (7) Saenger, W. Principles of Nucleic Acid Structure; SpringerVerlag: New York, 1984; p 404. (8) Lee, C.-Y.; Eichner, R. D.; Kaplan, N. O. Proc. Natl. Acad. Sci. (USA) 1973, 70, 1593. (9) Reddy, B. S.; Saenger, W.; Muhlegger, K.; Weimann, G. J. Am. Chem. Soc. 1981, 103, 907. (10) Saenger, W.; Reddy, B. S.; Muhlegger, K.; Weimann, G. In Pyridine Nucleotide-Dependent Dehydrogenases; Sund, H., Ed. ; de Gruyter: Berlin, 1977, p 222.
S0743-7463(97)00042-5 CCC: $14.00
surface.11-19 Because a charged metal surface may serve to mimic a biological interface20 and provide informatioin about the electrochemistry of the bound NAD, there is considerable interest in studies of such models. However, most of such measurements reported previously were performed under ex-situ conditions, and the proposed conformation models were usually based upon indirect evidence. Raman spectroscopic techniques, especially surfaceenhanced Raman scattering (SERS), were recently developed and widely used to investigate the states of biological molecules.21,22 However, normal Raman spectroscopy is unable to identify the conformation of NAD since a totally identical spectrum would be given by a folded or extended molecule.23 Most conventional SERS measurements are performed using a visible laser as the exciting source. The problem of photodecomposition of the adsorbed compounds has made the conclusions from visible SERS somewhat questionable. For example, the marked photooxidation of adenine, a constituent moiety of NAD, adsorbed on a roughened silver electrode has (11) Schmakel, C. O.; Santhanam, K. S. V.; Elving, P. J. J. Am. Chem. Soc. 1975, 97, 5083. (12) Bresnahan, W. T.; Elving, P. J. J. Am. Chem. Soc. 1981, 103, 2379. (13) Samec, Z.; Bresnahan, W. T.; Elving, P. J. J. Electroanal. Chem. 1982, 133, 1. (14) Bresnahan, W. T.; Moiroux, J.; Samec, Z.; Elving, P. J. Bioelectrochem. Bioenerg. 1980, 125, 7. (15) Takamura, K.; Mori, A.; Kusu, F. Bioelectrochem. Bioenerg. 1981, 8, 229. (16) Takamura, K.; Mori, A.; Kusu, F. Bioelectrochem. Bioenerg. 1982, 9, 499. (17) Moiroux, J.; Deycard, S.; Malinski, T. J. Electroanal. Chem. 1985, 194, 99. (18) Siiman, O.; Rivellini, R.; Patel, R. Inorg. Chem. 1988, 27, 3940. (19) Austin, J. C.; Hester, R. E. J. Chem. Soc., Faraday Trans. 1 1989, 85 (5), 1159. (20) Drhurst, G. Electrochemistry of Biological Molecules; Academic Press: New York, 1977, p 473. (21) Paisley, R. F.; Morris, M. D. Prog. Anal. Spectrosc. 1988, 11, 111. (22) Cotton, T. M.; Kim, J.-H.; Chumanov, G. D. J. Raman Spectrosc. 1991, 22, 729. (23) Yue, K. T.; Martin, C. L.; Chen, D.; Nelson, P.; Sloan, D. L.; Callender, R. Biochemistry 1986, 25, 4941.
© 1997 American Chemical Society
Conformations of NAD on Gold Electrode
been observed by Otto et al.24 Some of the SERS spectra reported previously were confirmed to have resulted from the presence of products of photodecomposition. An additional complication is that the poor S/N ratio of visible SERS can lead to ambiguous conclusions. Fortunately, near-infrared Fourier transform Raman scattering techniques (near-IR-FT-Raman) have recently been developed. The prediction of significant advantages for this technology such as elimination of the effects of photolysis and fluorescence has been confirmed by several reports.25-27 Previous studies on the SERS of NAD28,29 have shown that a gold electrode may give satisfactory SERS signals. However, these SERS experiments were performed in concentrated solutions. The dilution of NAD would likely result in a lower S/N ratio or the complete disappearance of SERS spectra. In the present work, a very defined SERS was obtained in as low as 10-6 mol‚L-1 NAD solutions on an in-situ roughened Au electrode. Almost all of the characteristic NAD bands can be clearly recognized in the spectra. The most interesting phenomena are that a significant shifting of the SERS bands did occur during the potential scanning process and that some weak bands became quite strong. From the observed SERS behavior of NAD, an adsorption model is proposed. 2. Experimental Section Nicotinamide adenine dinucleotide (NAD) was from Sigma Chemical Co. (grade 1, 100%). All of the other agents are of analytical grade. Fourier transform surface-enhanced Raman scattering measurements were carried out on a Bruker IFS 66/FRA 106FT Raman spectrometer equipped with a diode-pumped Nd:YAG laser exciting at 1064 nm. A 180° backscattering geometry was used. Laser power at the sample was ca. 100 mW. All of the spectra were recorded by 200 scans at a resolution of 2 cm-1, and none of the presented spectra 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 plate (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 used. The roughening of the electrode was achieved by 30 electrochemical oxidation-reduction cycles (ORC) between +1.1 and -0.3 V vs SCE at a speed of 0.5 V s-1. The solutions for the ORC process and SERS measurements consisted of 0.1 mol‚L-1 KCl, 10 mmol‚L-1 Na2HPO4, and a 1.2 µmol‚L-1 NAD solution, the pHs of which were adjusted by adding aliquots of HCl or KOH solutions into the bulk. The voltages for the roughening and the polarization of the electrode were controlled using a M173 potentiostat and a M175 program generator (EG&G, San Diego, CA).
3. Results and Discussion The SERS of an Au electrode in dilute NAD solutions shows a marked potential dependence. As shown in Figure 1, the bands near 730, 1030, and 1320 cm-1 are predominantly enhanced in all of the potential regions. The other bands are very weak or absent under 0 V or with an open (24) Otto, C.; Hoeben, F. P.; Greve, J. J. Raman Spectrosc. 1991, 22, 791. (25) Fleischmann, M.; Sockalingum, D. Spectrochim. Acta 1990, 46A (2), 285. (26) Liang, E. J.; Engert, C.; Kiefer, W. J. Raman Spectrosc. 1993, 24, 775. (27) Goral, J.; Zichy, V. Spectrochim. Acta 1990, 46A (2), 253. (28) Xiao, Y.-J.; Wang, T.; Wang, X.-Q.; Gao, X.-X. J. Electroanal. Chem. 1997, in press. (29) Taniguchi, I.; Umekita, K.; Yasukouchi, K. J. Electroanal. Chem. 1986, 202, 315.
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ciruit and became more pronounced as the potential was moved in positive or negative directions. All peaks decreased sharply and tended to disappear when the potential was more negative than -1.0 V. Since the bulk concentration of NAD was maintained at no more than 2 µM and the SERS measurements were performed in nonFaradaic regions, no Raman signal from the solution was detected and all the spectral information appears to be informative about the states of the adsorbates. All of the SERS bands are assigned and listed in Table 1, based on the suggestion23 that the Raman spectra bands of NAD can be simply attributed to the four constituent molecular moieties labeled in the schematic structure of NAD: 4 3
5
–
O O P O CH2
6
+
N1
O CNH2 nicotinamide (N)
2
ribose (R) phosphate (P)
OH OH NH2
O
6 1
N
2 3N –O P O CH 2 O
OH
5
N7
4
N9
8
adenine (A)
ribose (R) OH
3.1. SERS Behavior of NAD on a Positively Charged Electrode. Under positive polarization, the main characteristic of SERS for NAD is the appearance of peaks at 1598, 1553, 1470, and 1380 cm-1 and a pair of overlapping strong peaks near 1343 and 1320 cm-1. In addition, the band near 730 cm-1 shifted upward by 7 cm-1. For the convenience of discussion, these bands are defined as Group 1. All the bands in Group 1 can be assigned to vibrations of the adenine moiety of NAD as listed in Table 1. Based on the results of ab initio SCF MO calculation, Watanabe et al.30 assigned a 1476 cm-1 band to a combination of C5-N7-C8 stretching and C6NH2 stretching. The pronounced enhancement of the 1470 cm-1 band may provide evidence to support the suggestion that, in positive potential regions, the adenine moiety tends to be adsorbed onto the electrode surface through the -NH2 group and N7 atom. With both solution Raman and SERS spectra of adenine, there are strong characteristic bands near 1338 cm-1 21,23,31,32 that are usually assigned to skeleton vibration of the adenine ring. This band is clearly dependent on the potential. Under positive potentials, it forms a strong doublet peak near 1318 and 1342 cm-1, while under negative potentials, the shoulder band (1342 cm-1) becomes quite weak or completely disappears. The 1318 cm-1 band seemed to be rather stable since its position was unchanged and its amplitude is strong under positive electrode potential; near 0 V or with an open circuit it is low. Suh et al.33 assigned the 1317 cm-1 band to C6-NH2 of the amino group of adenine. They observed that this band is weak among the SERS spectra of adenine adsorbed on silver colloid. This was interpreted to mean adenine was lying flat on the surface in negative potential regions. (30) Watanabe, T.; Kawanami, O.; Katoh, H.; Honda, K. Surf. Sci. 1985, 158, 341. (31) Ervin, K. M.; Koglin, E.; Sequaris, J. M.; Valenta, P.; Nurnberg, H. W. J. Electroanal. Chem. 1980, 114, 179. (32) Koglin, E.; Sequaris, J. M.; Valenta, P. J. Mol. Struct. 1980, 60, 421. (33) Suh, J. S.; Moskovits, M. J. Am. Soc. 1986, 108, 4711.
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Xiao and Markwell Table 1. SERS Bands (cm-1) of NAD on Gold Electrode Compared with Normal Raman Bands of NAD in Solutionsa SERS on gold electrode -0.4 V
+0.4 V (vs SCE)
1624 s
normal Raman in solnb 1628 w, br
1598 s 1573 s 1504 m 1402 br 1378 sh, w 1345 sh, w 1322 s 1273 w 1182 s 1116 s 1082 sh, w 1025 vs 915 w, br 818 s
1580 s 1553 s 1509 w, sh 1470 vs 1386 m 1343 s 1319 vs
1029 vs
1510 m 1422 m 1378 s 1338 vs 1254 m 1186 w, br 1116 m 1084 w 1032 vs 914 w 800 vw
736 s 729 s 631 br 320 m
320 m
730 s 642 w, br 324 w, br
assigntb N amide I, ν(CdO) N ring 0.8A + 0.2N A ring A ring, ν(C6-NH2) 0.5A + 0.5N A A A ring, NH2 rock A R/N R P N P R A ring, NH2 bend A 0.5A + 0.5N
a
Abbreviations: vs, very strong; s, strong; m, medium; w, weak; vw, very weak; sh, shoulder; br, broad; N, nicotinamide; A, adenine; P, phosphate; R, ribose. b From refs 18 and 23.
Figure 1. SERS spectra of NAD adsorbed on a roughened gold electrode, 1.2 µmol‚L-1 NAD + 10 mmol‚L-1 H2PO4- + 100 mmol‚L-1 KCl in distilled water, pH 4.0. Applied voltages (V vs SCE): a, open; b, +0.4; c, +0.2; d, 0.0; e, -0.2; f, -0.4; g, -0.6; h, -0.8. About 5 min of waiting time in each voltage.
In addition, the upward shift by several wavenumbers of the band near 730 cm-1, which was assigned to a totally symmetric ring-breathing vibration of the adenine moiety,23 implied that this band may mix with the in-plane NH2 bend27 and that an adenine-metal complex via -NH2 may form on the positively charged electrode.34 The shifting of the bands, compared to the normal spectra in solution, implies the formation of some metaladenine complexes. Especially evident, the strong band near 1340 cm-1, which has been assigned to the N7-C5 stretching mode,18,30,35 suggests the formation of a close linkage between the N7 atom of the adenine moiety and an electrode. Indeed, it has been confirmed that, for purine nucleotides, the metal-heterocyclic nitrogen at N7 is preferred over other atoms for metal binding.18,36 Furthermore, almost all of the bands discussed above are concerned with vibration modes of NH2 of the adenine moiety as shown in Table 1, which substantiates the direct contact of NH2 to the electrode. It seems likely that, with positive potentials, the NH2 group and the N7 atom of the adenine moiety are adsorbed in a vertical orientation with respect to the surface of the electrode. This conformation is consistent with Taniguchi’s model29 in which the vertical adsorption through NH2 and N7 takes place under negative electrode potentials. (34) Speca, A. N.; Mikulski, C. M.; Iaconianni, F. J.; Pytlewski, L.; Karayannis, N. M. J. Inorg. Nucl. Chem. 1981, 43, 2771. (35) Cotton, T. M.; Schultz, S. V.; Van Duyne, R. P. J. Am. Chem. Soc. 1980, 102, 7960. (36) Saenger, W. Principles of Nucleic Acid Structure; SpringerVerlag: New York, 1984; Chapter 8, pp 201-219.
3.2. SERS Behavior of NAD on a Negatively Charged Electrode. As mentioned above, as the electrode potential becomes more negative, the bands of Group 1 may progressively decrease and then completely disappear, while several other new bands at 1624, 1573, 1182, 1116, 818, and 631 cm-1, defined as Group 2, would appear and increase in intensity up to the limit of -0.4 V. It is interesting to note that, in contrast to those of Group 1, the bands in Group 2 are mainly concerned with the vibrations of nicotinamide, ribose, and phosphate moieties, as shown in Table 1. Compared to the adenine moiety, the nicotinamide group has been less studied, because few signals ascribable to the nicotinamide can be recorded by conventional methods. The higher resolution of spectra obtained from the present study may be able to provide more information about the molecular orientation of NAD on the electrode. The Raman spectra of NAD in solutions23 show a strong band near 1030 cm-1, which was assigned to the ringbreathing vibration of the nicotinamide moiety; this band is quite weak or undefined in conventional SERS.18,19,29 Unlike the case with visible SERS, the FT-SERS of NAD on a gold electrode gives a strong band near 1029 cm-1 with all applied potentials. At more negative potentials, this band is more enhanced and the frequency slightly downshifted by 4 cm-1. In addition, a strong band near 1624 cm-1, which is assignable to the carbonyl stretching mode of the carboxamide group,18,37 also appears with negative potentials. In contrast, this band is quite weak in the aqueous solution23 and in the SERS of Ag colloids.18 Obviously, for a molecule on a negatively charged electrode, a reorientation may occur that produces a suitable orientation for enhancement from the carbonyl vibration. Another important signal of the NAD SERS in Figure 1 is that two strong bands near 1116 and 1182 cm-1, which are assignable to the ribose moiety, appeared and increased in intensity as the potential was made more (37) Parlick, D. M.; Wilson, J. E.; Leroi, G. E. Biochemistry 1974, 13, 2813.
Conformations of NAD on Gold Electrode
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Figure 2. SERS spectra of NAD in the solutions of pH 6.4. The other conditions are the same as those in Figure 1.
Figure 3. SERS spectra of NAD in the solutions of pH 9.0. The other conditions are the same as those in Figure 1.
negative up to the limit of -0.4 V. Obviously, the appearance of these bands mean that, under negative electrode potentials, not only adenine and nicotinamide moieties but also the ribose moiety may be in intimate contact with the electrode. 3.3. pH dependence of SERS. SERS of dilute NAD shows a significant pH dependence. A satisfactory spectra could only be obtained in a pH region of 4-10. Higher or lower pH values decrease the band intensity and weaken the spectrum. This behavior has also been observed in the Raman spectra of NAD in solution.23 The decrease or disappearance of the bands at higher and lower pH were attributed to the degradation and protonation of NAD, respectively. It is interesting to note that the bands of Group 1 are more pH sensitive than other bands. Figures 1-3 show the SERS behavior of NAD at various pH values. Obviously, higher pH intensifies the bands of Group 1. For example, at pH 9 the bands of Group 1 are evident until the applied potential is decreased to -0.6 V. Conversely, at pH 4 these bands completely disappeared when the applied potential was decreased to 0.0 V. 3.4. Discussions on the SERS Mechanism of NAD. Based on current understanding,22,38 the surface enhancement phenomenon can originate from either of two major mechanisms, i.e., the electromagnetic mechanism (EM) or the charge-transfer mechanism (CT). Generally, the EM mechanism is adopted in the interpretation of longrange enhancement, which is not sensitive to the molecular orientation of the adsorbates and consequently SERS would be similar to the normal Raman spectra of the
molecules in solution. On the other hand, SERS from a CT mechanism would be different from the solution phase spectra. The changes may occur in the intensities and positions of the bands, and some new bands may be observed. In terms of these two essential SERS mechanisms, various theoretical explanations for the SERS behavior of NAD or some relative molecules adsorbed on metal surfaces have been proposed. On the basis of EM mechanism,33,39 Moskovits and Suh have derived surface selection rules, which are similar to the surface selection rules for infrared spectroscopy40,41 and unenhanced surface Raman spectroscopy.42,43 It has been demonstrated that these selection rules are valid in the discussion of the SERS mechanism and adsorption models of phthalazine molecules and some DNA bases adsorbed on the silver surfaces.33,39 However, the straightforward interpretation of the surface selection rules to predict the adsorption orientation of an adenine molecule or its analogues have led to some ambiguous conclusions. For example, based on the fact that no remarkable signals attributable to the out-of-plane vibration of the adenine ring, which would be enhanced if the ring was lying flat on the surface, were observed, the adenine moiety was deduced to be adsorbed vertically on the electrode surface.29 In contrast, using the same selection rules in the explanation of a similar spectrum, Suh and Moskovits proposed a flat orientation for adenine
(38) Chang, R. K.; Furtak, T. E., Eds. Surface Enhanced Raman Scattering; Plenum Press: New York, 1982.
(39) Moskovits, M.; Suh, J. S. J. Phys. Chem. 1984, 88, 5526. (40) Greenler, R. G. J. Chem. Phys. 1966, 44, 310. (41) Allara, D. L.; Baca, A.; Pryde, C. A. Macromolecules 1978, 11, 1215. (42) Hallmark, V. M.; Campion, A. J. Chem. Phys. 1986, 84, 2933. (43) Hallmark, V. M.; Campion, A. J. Chem. Phys. 1986, 84, 2942.
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adsorbed on a silver surface. The main evidence was the lack of enhancement for the C-H mode at ca. 3080 cm-1, which would be expected to be strongly enhanced in a vertical orientation,33 because it also made strong in-plane contributions to the Raman polarizability tensor. To harmonize the theoretical explanation of SERS data observed in different conditions, Austin et al.19 proposed that SERS from either the adenine molecule or the adenine moiety of NAD mainly resulted from the CT mechanism. It was believed that the CT contributions to SERS of adenine or NAD are crucial elements. It is the contribution of the CT mechanism that makes the determination of the orientation of adenine by surface selection rules somewhat uncertain. By the CT enhancement mechanism, the phenomena could reasonably explain that only the ring skeleton vibrations could be enhanced while the exocyclic vibrations (e.g., C-H) could not. This explanation was supported by the observation of the SERS band intensity variations with electrode potential. On the other hand, some of the researchers explained their SERS data in terms of the assumption that the most intense lines in the SERS spectra are due to the atoms in closest proximity to the surface30,32,44 or the groups of the molecule closely situated near the surface.45 Specifically, with this assumption, the SERS behavior of the adenine molecule and some derivatives of adenine adsorbed on a silver electrode has been carefully analyzed by Otto et al.46 and the external amino group of adenine was suggested to be the adsorption site on the positively charged electrode. Taking into account the SERS behavior of NAD in different conditions and the theoretical explanations reported before, we believe that SERS of NAD adsorbed on a gold surface is mainly caused by the CT mechanism. The significant differences between the SERS spectra and the normal Raman spectra of NAD in solutions supported the CT mechanism. Furthermore, the good quality SERS recorded in very low NAD concentrations (∼10-6 mol‚L-1) is in itself evidence for a significant contribution from a CT enhancement mechanism.19 The significant changes observed with SERS of NAD in the present work are interpreted to imply that the conformation of NAD on the electrode is quite sensitive to the applied voltage. The constituent NAD moieties, especially the adenine and nicotinamide groups, may change their conformations and orientations independently during scanning of the electrode potential. 3.4.1. Adsorption Model of NAD in the Positive Potential Region. The new SERS bands (Group 1) indicate that an extra adsorption model arises from positive polarization. The shifting of the bands, compared to the normal spectra in solution, indicates the formation of some metal-adenine complexes. Almost all of the bands in Group 1 arising from the vibration modes of NH2, as shown in Table 1, implied a direct contact of NH2 with the electrode. A likely explanation is that, under positive applied potentials, the adenine moiety of NAD is adsorbed in a vertical orientation with respect to the electrode, with contact mediated by the NH2 group. This orientation was supported by the following reasons: (1) The bands related to the vibration of C6-NH2 got extra enhancements; some of the bands were even much (44) Brabec, V.; Niki, K. Collect. Czech. Chem. Commun. 1986, 51, 167. (45) Otto, C.; de Mul, F. F. M.; Huizinga, A.; Greve, J. J. Phys. Chem. 1988, 92, 1239. (46) Otto, C.; van den Tweel, T. J. J.; de Mul, F. F. M.; Greve, J. J. Raman Spectrosc. 1986, 17, 289.
Xiao and Markwell
more intense than the totally symmetric ring vibration band near 730 cm-1. (2) The intensities of the bands in Group 1 showed a significant decrease with the movement of the potential in the negative direction, which implied the formation of the complex with lone electron pair of the NH2 and the positively charged electrode. Otherwise, in the case of parallel orientation, i.e., the ring π-electron binding with the electrode, the intensities of the bands would increase with lowering of the electrode potential.19 (3) The shifting of the band 730 cm-1 to higher wavenumber can be taken as evidence of the formation of the complex of the NH2 group with the metal surface, because a similar band shifting had also been found in the spectra of the adenine-metal complex.34 In addition, the strong band near 1340 cm-1, which has been assigned to the N7-C5 stretching mode,18,30,35 suggests the formation of a close linkage between the N7 atom of the adenine moiety and the electrode. Indeed, it has been confirmed for purine nucleotide that the metalheterocyclic nitrogen binding at N7 is preferred over other atom.18,36 This adsorption orientation is consistent with the pH dependence of SERS. A moderately high or neutral pH will enhance the bands of Group 1, whereas a low pH will significantly decrease or eliminate these signals. This is because in acidic media the contact site of NAD should be protonated and tends to be rejected by the positively charged electrode; consequently, the relevant bands (Group 1) lose the surface enhancement effects. In contrast, a moderate or higher pH may deprotonate the NAD contact site, promote binding to the electrode, and consequently facilitate the Raman enhancement. It has long been confirmed47 that, depending on the environment, NAD may exist in two main conformations. One is a folded (or stacked) conformation in which the adenine and nicotinamide rings are stacked and parallel to each other at a distance of 3.5 Å. The other one is an extended (or opened) conformation with two rings separated by about 13 Å and perpendicular to each other. In the positive potential regions, in addition to the bands ascribable to adenine, another strongest band also appeared near 1029 cm-1. We interpret this peak as the nicotinamide moiety approaching quite close to the Au surface. Furthermore, a strong band at 1598 cm-1, which seems assignable to ν(ring) of the nicotinamide, appeared in positive potential regions. However, the band near 1630 cm-1, assignable to the amide I of the carboximide of nicotinamide, was less enhanced under positive electrode potentials. This type of SERS behavior has also been observed on a colloidal silver by Siiman et al.,18 who proposed a flat or side-on orientation for the nicotinamide to explain the phenomenon. However, due to the restriction of the steric orientation,47 it is impossible for a stacked NAD to be adsorbed vertically on the surface with the NH2 group of the adenine moiety and the nicotinamide ring in a side-on orientation. So, the SERS behavior mentioned above implied a flat orientation for the nicotinamide moiety adsorbed on a positively charged electrode. According to the distance dependence of SERS intensity, it has been predicted18 that if both adenine and nicotinamide are coordinated to the surface, the SERS band intensity ratio I1030/I730 would remain about the same as the 2:1 ratio observed in solution Raman spectra of NAD. On the other hand, if only the adenine moiety was linked to the electrode surface with nicotinamide exposed to the (47) Dolphin, D.; Poulson, R. Pyridine Nucleotide Coenzymes; John Wiley & Sons: New York, 1987; Part A.
Conformations of NAD on Gold Electrode
Figure 4. (A) SERS spectrum of NAD at +0.4 V vs SCE (extended from Figure 1b). (B) Possible conformation of NAD adsorbed on a positively charged gold electrode (modified from ref 28): P, P; b, N; L, O; O, C.
solution, the ratio would be much lower or even no SERS band relative to the nicotinamide could be observed. This may be the reason that very weak bands or no SERS band assignable to the nicotinamide was detected on a silver electrode.29 Obviously, this is not the case for the gold electrode. The SERS bands assignable to adenine and nicotinamide moieties underwent strenghened enhancement, which indicates that both adenine and nicotinamide are directly adsorbed on the surface, while the other moieties (ribose and phosphate) may be exposed to the solution for very weak SERS signals from these moieties observed at positive electrode potentials. On the basis of the arguments mentioned above, we suggest a partly expended adsorption model for a dilute NAD on the positively charged gold electrode. As shown in Figure 4, the adenine and the nicotinamide moieties are directly adsorbed on the surface with a vertical and a flat orientation, respectively. 3.4.2. Adsorption Model of NAD in the Negative Potential Region. The generally accepted structure for NAD adsorbed on a negatively charged electrode was firstly proposed by Moiroux and Elving.48 In their model, the adenine moiety of NAD is adsorbed in a flat orientation on the electrode and the nicotinamide moiety is stacked and parallel with the adenine. However, this model was based on indirect evidence. Obviously, this stacked model is completely incompatible with the SERS behavior of NAD in the present work. As mentioned previously, the bands assignable to all of the constituent moieties of NAD would be enhanced at negative electrode potentials. This suggests that all the moieties must be close to the electrode. It is difficult to imagine that the adenine and nicotinamide both touch the surface and yet still maintain a stacked configuration. So, we believed that the NAD molecules are opened and unstacked by a negatively charged electrode. However, SERS spectra of NAD in the negative potential region are substantially different from those of NAD in (48) Moiroux, J.; Elving, P. J. J. Electroanal. Chem. 1979, 102, 93.
Langmuir, Vol. 13, No. 26, 1997 7073
Figure 5. (A) SERS spectrum of NAD at -0.4 V vs SCE (extended from Figure 1f). (B) Possible conformation of NAD adsorbed on a negatively charged gold electrode (modified from ref 28): P, P; b, N; L, O; O, C.
the positive potential region. This means a potential induced reorientation taking place in the potential scanning. The good quality SERS (as in Figures 1-3) recorded in very low NAD concentrations indicates that the NAD molecule may form a charge-transfer complex with a gold surface. However, the NAD-metal complex formed on a negatively charged electrode would be quite different from that formed on a positively charged electrode. The sharp decrease and then disappearance of bands in Group 1 and the increase of bands in Group 2 with potential scanning in the negative direction indicate the changes of the conformations and orientations of NAD. Specifically, the adenine moiety binds to the negatively charged electrode with its ring π-electrons and takes a flat geometry on the surface, instead of that connected to the surface mainly with the external amino group as in the case of the positively charged electrode. Here, the charge transfer would be between the Feimi level of the metal and the lowest unoccupied level of the adenine moiety.19,21 The increase in band intensities of group 2 as the potential is lowered from 0.0 to 0.6 V is in agreement with this chargetransfer complex model. Under the lower-energy excitation (red or near-infrared), the SERS bands resulting from the CT mechanism would get more enhancements as the electrode potential moved to the negative region, because the negative electrode potential may lead to a raising of the metal-donor level closer to the ligand-acceptor level. This CT mechanism can also be used to explain the SERS behavior that only ring modes (not exocyclic C-H modes) of the adenine moiety got enhancement.19 Furthermore, the 1624 cm-1 band has been used to determine whether the nicotinamide takes a end-on or a flat orientation.18 The end-on orientation should give a high SERS intensity to a band near 1630 cm-1, assignable to the vibrational modes ν(CdO), while the flat orientation may give no band near 1630 cm-. So, we believe that, on an electrode with negative potential, the nicotinamide moiety may take an end-on orientation. Actually, it has been found17 that the electronic structure of the carbonyl of the carboxamide group in the NAD molecule is considerably different from that of a normal
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carbonyl, with important contribution from the resonance form, -(O-)CdN+H2. This resonance form leads to appreciably greater polar character on the carboxamide group of NAD and consequently makes the group more readily adsorbed by the negatively charged electrode. In contrast to the adenine or nicotinamide moieties, the ribose and phosphate moieties generally give weak or very weak bands to normal Raman or SERS, unless they are adsorbed quite close to the electrode to obtain enhancement. As shown in Table 1, several SERS bands assignable to ribose appeared in the negative potential region and implied the proximity of ribose to the electrode. From steric considerations, the end-on orientation of nicotinamide may result in the β-riboside being displaced from the electrode surface. Therefore, these SERS bands may be due to the ribose moiety attached to the adenine moiety which lies flat on the surface. Figure 5 shows a typical SERS spectrum and a possible conformation for the NAD molecule adsorbed on a negatively charged gold electrode.
Xiao and Markwell
SERS made it possible to produce a more reliable pattern for NAD’s adsorption in a ultradilute solution. (2) Under positive or negative polarization conditions, NAD was adsorbed on the gold electrode with two conformations, in which the flexibility of bound phosphate provides a degree of freedom for the adenine and nicotinamide moieties to independently change their orientations. Under negative electrode potential, the adenine moiety of NAD adsorbed onto the gold electrode mainly in a flat orientation, whereas the nicotinamide moiety was in an end-on orientation. In contrast, 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. While, the nicotinamide moiety was adsorbed in a flat orientation with respect to the electrode surface. (3) The quite different spectral behaviors of NAD observed with near-IR-SERS and visible SERS suggest the necessity for further approaches on the current SERS models.
4. Conclusion (1) SERS of NAD on a gold electrode can be largely improved by an in-situ roughening method and by use of a near-IR laser as the exciting source. A high quality of
Acknowledgment. Research was supported by the National Science Foundation of China. LA970042+
