SERS Investigation of NAD+ Adsorbed on a Silver Electrode

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SERS Investigation of NAD+ Adsorbed on a Silver Electrode Shi-Ping Chen,† Charles M. Hosten,*,† Alberto Vivoni,‡ Ronald L. Birke,§ and John R. Lombardi§ Department of Chemistry, Howard University, 525 College St. NW, Washington, DC, 20059; Department of Mathematics and Physical Science, Inter American University, San German, Puerto Rico 00683; and Department of Chemistry, The City College and The Graduate School and University Center of the City University of New York, and Center for Analysis of Structures and Interfaces (CASI), New York, New York 10003 Received March 8, 2002. In Final Form: July 10, 2002 The surface-enhanced Raman scattering (SERS) spectra of nicotinamide adenine dinucleotide (NAD+) at different pH values and electrode potentials were investigated at a silver electrode. For comparison purposes, the spectra of adenine, N-methylnicotinamide, and nicotinamide mononucleotide were also recorded. Good quality spectra are reported, and NAD+ adsorbs spontaneously onto the electrode at all of the pH values (9.3-3.6) studied. Normal mode calculations of the in-plane vibrations of adenine were performed to assist in band assignments and determination of adsorbate orientation. These calculations utilized Urey-Bradley force field calculations and the PM3 semiempirical molecular orbital method. From these calculations, it is concluded that at physiological pH values NAD+ adsorbs on the silver electrode in a conformer in which the adenine ring binds to the surface through the external amino group on carbon 6 and through nitrogen 7. The ribose and phosphate parts of the molecule are also involved in adsorption. In the surface conformer NAD+ is in an extended configuration with the adenine ring oriented normal (side on) with respect to the surface and the nicotinamide ring oriented flat with respect to the surface much like in an enzyme bound case.

Introduction Nicotinamide adenine dinucleotide is a ubiquitous cofactor, used by more than 100 dehydrogenase enzymes, which catalyzes reactions such as the oxidation of ethanol, lactate, and malate. In these reactions the reversible redox process occurs at the nicotinamide ring C4 position, which accepts two electrons and a proton from the substrate, to form NADH, the oxidized form of the substrate, and a proton. NAD binds to a wide range of active site shapes primarily because the molecule has a high degree of flexibility, allowing it to adopt a number of environmentally dependent conformations. Second, the molecule has a range of polarities, the hydrophobic adenine ring system, a positively charged nicotinamide ring, and a negatively charged pyrophosphate group. The conformation of NAD has been shown to be highly dependent on the environment.1-5 The molecule can adopt a folded conformation in solution, with a distance between the nicotinamide and adenine rings of 0.4-0.5 nm. When NAD is bound to enzymes, the molecule adopts an extended conformation with the adenine and nicotinamide rings separated by 1.2-1.5 nm. NAD has been shown to adopt a unique folded conformation when it is bound to flavin †

Howard University. Inter American University. § City University of New York and CASI. * To whom correspondence should be addresses. e-mail [email protected]. ‡

(1) McDonald, G.; Brown, B.; Hollis, D.; Walter, C. Biochemistry 1972, 11, 1920. (2) Riddle, R. M.; Williams, T. J.; Bryson, T. A.; Dunlap, R. B.; Fisher, R. R.; Ellis, P. D. J. Am. Chem. Soc. 1976, 98, 4286. (3) Zens, A. P.; Bryson, T. A.; Dunlap, R. B.; Fisher, R. R.; Ellis, P. D. J. Am. Chem. Soc. 1976, 98, 7559. (4) Zens, A. P.; Williams, T. J.; Wiscowaty, J. C.; R. B.; Fisher, R. R.; Dunlap, R. B.; Bryson, T. A.; Ellis, P. D. J. Am. Chem. Soc. 1975, 97, 2850. (5) Tanner, J. J.; Ty, S.-C.; Barbour, L. J.; Barnes, C. L.; Krause, K. L. Protein Sci. 1999, 83, 381.

reductase P (FRP) obtained from Vibrio harveyi.6 In this system the adenine and nicotinamide rings are stacked parallel to each other, and the distance between the rings has been shown to be 0.36 nm.6 The existence of a folded solution conformation, and an extended catalytic conformation, suggests that the unfolding of NAD might be a significant step in the process of NAD recognition, and unfolding is important for the release of the catalytically generated products. The dynamics of this folding and unfolding of the NAD molecule is not understood; however, these processes are significant in the catalytic properties of the molecule. A recent study has investigated the ability of the protein environment to facilitate the unfolding of NAD+.7 Analogues of NAD have shown potential for application as anticancer,8 antibacterial,9 and antitrypanosomal agents.10 The reduced form of NAD, NADH, functions as a substrate for NADH oxidase/flavin reductase enzymes11,12 and also provides the reducing electrons for mitochondrial ATP production. Normal Raman difference spectroscopy13,14 has been used to probe the interactions of coenzymes with apoprotein, especially the dehydrogenases. This Raman (6) Smith, P. E.; Tanner, J. J. J. Mol. Recognit. 2000, 13, 27. (7) Smith, P. E.; Tanner, J. J. J. Am. Chem. Soc. 1999, 121, 8637. (8) Franchetti, P.; Cappellacci, L.; Perlini, P.; Jayaram, H. N.; Butler, A.; Schneider, B. P.; Collart, F. R.; Huberman, E.; Grifantini, M. J. Med. Chem. 1998, 41, 1702. (9) Zhang, R.-G.; Evans, G.; Rotella, F. J.; Westbrook, E. M.; Beno, D.; Huberman, E.; Joachimiak, A.; Collart, F. R. Biochemistry 1999, 38, 4691. (10) Aronov, A. M.; Verlinde, C. L.; Hol, W. G.; Gelb, M. H. J. Med. Chem. 1998, 41, 4790. (11) Koike, H.; Sasaki, H.; Kobori, T.; Zenno, S.; Saigo, K.; Murphy, M. E.; Adman, E. T.; Tanokura, M. J. Mol. Biol. 1998, 280, 259. (12) Lei, B.; Liu, M.; Huang, S.; Tu, S.-C. J. Bacteriol. 1994, 176, 3552. (13) Chen, D.; Yue, K. T.; Martin, C.; Rhee, K. W.; Sloan, D.; Callender, R. Biochemistry 1987, 26, 4776. (14) Deng, H.; Burgner, J.; Callender, R. Biochemistry 1991, 30, 8804.

10.1021/la020235j CCC: $22.00 © 2002 American Chemical Society Published on Web 11/07/2002

NAD+ Adsorbed on a Silver Electrode

difference technique should be applicable to many enzymatic active site structure determination problems, since it is a normal Raman technique, which does not require a resonance absorption in the target molecule.15,16 However, interpretation of the spectra requires assignments of Raman bands which may not be found in the solution or the solid-state Raman spectra, due to unique configurations of the bound coenzyme. Surface-enhanced Raman scattering (SERS) can be used to investigate the vibrational spectra of biomolecules adsorbed on a metal surface where strong electric fields, electrostatic ion-pairing interactions, and metal surface binding interactions may actually provide many structural features like those found in protein-bound substrate. For this reason we decided to apply the SERS technique to the NAD+ molecule. Since its discovery by Fleischman et al.,17 one of the applications of SERS has been in the determination of the orientation of adsorbates on metal electrode or hydrosol surfaces, mainly on silver and gold sols or electrodes.18-20 Recently, we have been utilizing quantum mechanical calculations along with SERS spectroscopy to determine the orientation of molecules adsorbed on roughened silver electrode surfaces.21,22 Two calculation methods were used in performing our normal-mode analysis: frequency fit of the in-plane vibrations using a Urey-Bradley force field and Hartree-Fock ab initio methods.21 The UBFF normalmode analysis of the SERS spectra was performed by modeling the surface by means of a silver atom. With the PM3 method a zinc atom was used to model the surface because the method is parametrized for this element and not for silver. This method was used to determine the orientation of the antitumor agent 6 mercaptopurine, which is a sulfur analogue of adenine and azathioprine.21 Surface electric field selection rules for SERS23,24 have also been developed, and these aid greatly in determining the orientation of planar ring systems with respect to the metal surface. If a planar ring system is standing up on the metal surface with its molecular plane perpendicular to the surface, the intensity of in-plane vibrational modes (which transform as the polarizability component zz) should be enhanced because the electric field of the exciting light normal to the surface (z-direction) is enhanced by electromagnetic resonances in small metal surface particles. Roughened metal electrodes or metal sols yield small metal particles (nanometer range), which are one source of the surface enhancement, which provides the high sensitivities. In our SERS studies, we use Ag and Au electrodes as opposed to colloidal metal sols because frequently the reducing agent used in preparing the sol system gives extraneous vibrational bands. Furthermore, sol aggregation changes with time so that SERS spectral band frequencies and intensities of molecules adsorbed on these sols are dependent on the degree of aging of the sol. With (15) Deng, H.; Schindler, J. F.; Berst, K. B.; Plapp, B. V.; Callender, R. Biochemistry 1998, 37, 14267. (16) Wang, J. H.; Xiao, D. G.; Deng, H.; Webb, M. R.; Callender, R. Biochemistry 1998, 37, 11106. (17) Fleischman, M.; Hendra, P. J.; McQuillan, A. J. Chem. Phys. Lett. 1973, 26, 163. (18) Cotton, T. M.; Schultz, S.; Van Duyne, R. J. Am. Chem. Soc. 1982, 104, 6528. (19) Muniz-Miranda, M.; Puggelli, M.; Ricceri, R.; Gabrielli, G. Langmuir 1996, 12, 4417. (20) Xiao, Y.-J.; Chen, Y.-F.; Wang, T.; Gao, X.-X. Langmuir 1998, 14, 7420. (21) Vivoni, A.; Chen, S.-P.; Ejeh, D.; Hosten, C. M. Langmuir 2000, 16, 3310. (22) Faria, P. A.; Chen, X.; Lombardi, J. R.; Birke, R. L. Langmuir 2000, 16, 3984. (23) Creighton, J. A. Surf. Sci. 1983, 124, 209. (24) Moskovits, M.; Suh, J. S. J. Phys. Chem. 1984, 88, 5526.

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electrodes, on the other hand, one does not have these extraneous bands and time effects, and the electrode potential fixes the surface charge so that more reproducible surface conditions can be achieved. Depending on the surface charge and solvent properties, a variety of conformations of biological molecules can be “frozen out” at a roughened electrode surface, and intense surface Raman spectra were obtained for these species. The surface selection rules of SERS can be applied to elucidate the orientation of planar ring systems with respect to the surface and with respect to each other. Furthermore, variation of the electrode potential, and thus the charge on the surface, can create different environments, which affect the orientation and conformation of biomolecules adding an additional probe for structural interpretation. The majority of SERS spectra have vibrational selections rules similar to normal Raman spectra25 so that interpretation of chemical structural features in terms of normal mode assignments is straightforward. Thus, SERS vibrational studies of NAD+ were undertaken to gain additional information about the vibrational structure of NAD+ conformers and with the hope that the NAD+/protein conformation could be shown by SERS to be mimicked at the electrode solution interface. This is not to be unexpected since it has already been proposed that a dinucleoside like adenyl(3′-5′)-cytidine monophosphate has an extended configuration at a positively charged silver electrode with the adenine and phosphate binding directly to positive sites on the surface and with the cytidine repelled from the surface and extending out into the solution.26 Taniguchi et al.27 were the first to study the SERS of NAD+ adsorbed on silver and gold electrodes. In the reported spectra only two bands from adenine were observed, at 735 and 1330 cm-1, and these bands had the same wavenumber positions as the solution Raman bands of NAD+. The wavenumber position of these two bands did not change with applied voltage, suggesting that these spectra were not really surface-enhanced spectra. The authors concluded that NAD+ was adsorbed via the adenine group, with the adenine ring perpendicular to the electrode surface. A detailed SERS study, by Siiman et al., of NAD+ on silver sols28 revealed more spectral features, with spectral bands assigned to a large number of vibrations mostly in the adenine portion of the NAD+ molecule. Hester et al.29 studied the effect of excitation wavelength on the SERS of NAD adsorbed on silver sols. In a FT-near-IR SERS study of NAD adsorbed on a gold electrode surface, it was concluded that under sufficiently negative potential the NAD molecule appears to exist in a well-extended state.30 The low quality of the spectra obtained by Taniguchi et al.27 and the variation of the SERS spectra with solution conditions on colloidal silver28 led us to reexamine the SERS of NAD+ on silver electrodes. In the results reported in this study, we present wellresolved, intense SERS spectra of NAD+ adsorbed on a silver electrode. We have studied the effect of pH and electrode potential on the orientation and conformation of adsorbed NAD+. There have been several conflicting (25) Birke, R. L.; Lombardi, R. J. In Spectroelectrochemistry; Gale, R. J., Ed.; Plenum Press: New York, 1988; Chapter 6, p 312. (26) Koglin, E.; Lewinsky, H.; Sequasis, J. M. Surf. Sci. 1985, 158, 370. (27) Taniguchi, I.; Umekita, K.; Yasukouchi, K. J. Electroanal. Chem. 1986, 202, 315. (28) Siiman, O.; Rivellini, R.; Patel, R. Inorg. Chem. 1988, 27, 3940. (29) Austin, J. C.; Hester, R. E. J. Chem. Soc., Faraday Trans. 1 1989, 85, 1159. (30) Xiao, Y.-J.; Markwell, J. P. Langmuir 1997, 13, 7068.

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views (reviewed later) on the mode of interaction between adenine containing nucleotides and Ag surface sites. To assist in the interpretation of the SERS spectra of NAD+ on colloid particles and on the electrode surface, quantum mechanical frequency calculations of adenine were performed. A number of bands attributed to vibrations of adenine were observed in our NAD+ SERS spectra. The shifts in band frequencies and relative intensities between solution and SERS spectra were utilized to reveal information regarding the interaction of NAD+ with surfaces. We establish that, at pH values close to physiological pH, NAD+ has an extended configuration when it is adsorbed onto a silver electrode much like the extended configurations found in enzyme systems. The most pronounced interaction between the adsorbed NAD+ and the electrode surface is via the adenine and nicotinamide groups. The results show that for NAD+ on the surface the adenine ring always has its molecular plane oriented normal to the surface while the nicotinamide is oriented more flat with respect to the surface. Thus, the two ring systems are at more or less right angles to each other, similar to the conformation observed in holoenzymes.31 The applicability of SERS as a technique for investigating the orientation of adsorbed biomolecules possessing multiple locations for adsorbate-surface bonding is, therefore, illustrated.

Chen et al.

Figure 1. Adenine: (a) N9 tautomer, (b) N7 tautomer.

Experimental Section Nicotinamide adenine dinucleotide (NAD+), grade IIIc, and N-methylnicotinamide (1-MN+) were obtained from Sigma Chemical Co. and used without further purification. Oxidized nicotinamide mononucleotide (NMN+) was a gift from Professor R. Callender and was also used without further purification. Buffer solutions of differing pH values were prepared by combining varying volumes of 0.15 M solutions of disodium hydrogen phosphate and potassium hydrogen phosphate. Solutions of Raman-active compounds at 1 × 10-3 M concentrations were prepared on the same day of the experiment. The solutions were deaerated by bubbling prepurified grade nitrogen for 2030 min. Prior to electrochemical pretreatment, the silver electrode was polished with a slurry of 0.03 µm alumina powder and sonicated in distilled water. The oxidation-reduction cycle (ORC) to activate the electrode for SERS was carried out in the buffered solution containing the sample and consisted of a two second potential pulse from 0.0 to +0.3 V. This is refered to as in situ pretreatment as opposed to an ex situ pretreatment where the sample is added after the ORC. The advantage of in situ pretreatment for the present study is that the NAD+ target molecules will capture Ag+ surface active sites during the pretreatment. The electrochemical potential was controlled by a Princeton Applied Research model 175 universal programmer and a model 173 potentiostat/galvanostat. The sample cell consisted of a polycrystalline silver working electrode (99.999% pure) with ca. 1.6 mm2 surface area, a platinum counter electrode, and a saturated calomel electrode (SCE) reference electrode. All potentials reported in this paper are referenced to the SCE. Two Raman spectroscopic systems were used for data collection. The first consisted of a Spectra Physics 164 argon ion laser with 488 nm laser excitation. The SERS spectra were recorded on a Spex 1401 scanning double monochromator with an accumulation time of 0.6 s/cm-1. The second system consisted of an argon ion laser (Coherent, model 170C-4) tuned to the 488 nm line. The laser was focused by a 42 × 250 mm double convex lens and directed at 90° incident angle to the sample surface. Scattered radiation was collected at 90° by a Nikon f 1.2, 50 mm camera lens and collimated onto the slit of a single spectograph (SPEX, model 500M), which was configured with a 1200 grooves/mm grating (SPEX) blazed at 500 nm. The detection system was a charge coupled device detector (EG&G Princeton Applied Re(31) Deng, H.; Burgner, J.; Callender, R. Biochemistry 1991, 30, 8804.

Figure 2. Possible binding models of adenine on a silver surface. search, model 1530-AUV) containing a 256 × 1024 CCD chip in and air cooled housing. None of the spectra presented here have been smoothed. Normal mode calculations of the in-plane vibrations of adenine (Figure 1) were performed primarily because a number of adenine modes are observed in the SERS spectra of NAD and secondly because there exists some ambiguity as to the mode of interaction between adenine and a silver surface. For the normal mode calculations the initial force constants were taken from Majoube’s UreyBradley force field calculations.32 This force field was reduced using the transference approximation in order to obtain an under determined system. The experimental frequencies of adenine and the deuterated species were also taken from Majoube. The 15Nl,3 and 13C8 frequencies were obtained from Hirakawa et al.33 All observed Raman frequencies were used, to allow for a direct comparison with the SERS frequencies. IR values were used for those modes that were not observed in the Raman spectra. The geometrical parameters were obtained from Nowack et al.34 The force constants were optimized with a simplex optimization methodology. Three models describing the possible forms of attachment of adenine to the Ag surface were studied and are shown in Figure 2. Only models representing perpendicular interactions were studied since they affect the in-plane vibrations the most. Although a parallel interaction is not ruled out, the lone pair electrons of the adenine nitrogen provide the most likely binding sites. The Ag surface was represented by a silver adatom.35 The surface-molecule interactions included the Ag-N stretching and the Ag‚‚‚CR nonbonded force constants. The surface interaction force constant values were transferred directly from a previous pyridine calculation.33 The wavenumbers of the SERS spectral (32) Majoube, M. J. Raman Spectrosc. 1985, 16, 98. (33) Suzuki, S.; Orville-Thomas, W. J. J. Mol. Struct. 1997, 37, 321. (34) Nowack, M. J.; Lapinski, L.; Kwlatowski, J. S.; Lesczynski, J. Spectrochim. Acta 1991, 47A, 87.


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Figure 3. Structure of (a) NAD+, (b) NMN+, and (c) 1-NM+. bands of adenine adsorbed on silver colloid were obtained from the work of Siiman et al.28 Calculations of the N7-H tautomer and the imino conformation were also performed. The N7-H calculation was done by adding the surface interaction parameters, exchanging the N7C8 and C8-N9 stretches, and adjusting the N7-H bending and nonbonded interaction to fit the 1390 cm-1 band. While the N9 and N7 are equivalent, the hydrogens bonded to these atoms move in different environments. As a result, their bending force constants are expected to have different values. The imino calculation was done by shifting one of the amine hydrogens to N1 and shifting the π-bond from N1-C6 to C6-N1.36

Results and Discussion SERS of NAD+. Figure 3a shows the chemical structure of NAD+ with the labeling of the atoms that comprise the molecule. It should be noted that NAD+ is not a typical nucleoide since it is composed of two 5′ nucleotides connected in a head-to-head arrangement through a pyrophosphate group and thus cannot form longer oligonucleotides.37 Figure 4 shows potential-dependent SERS spectra of NAD+ adsorbed on a Ag electrode, in phosphate buffer at pH 7.4 in the potential range from 0 to -0.6 V. The potential-dependent spectra were obtained at potential steps of -0.1 V starting at 0.0 V. SERS spectra were not obtainable at potentials more negative than -0.8 V, presumably because the negatively charged molecule desorbs from the surface at these potentials. A comparison of our spectra with those reported by Taniguchi et al.27 under similar experimental conditions shows that, in addition to the two bands at 740 and 1339 cm-1 observed in both spectra, our spectra contain a large number of bands that were not observed by Taniguchi et (35) Bagus, P. S.; Hermann, K.; Buaschilcher, C. W. J. Phys. Chem. 1984, 81, 1996. (36) Vivoni, A.; Chen, S.-P.; Ejeh, D.; Hosten, C. M. Langmuir 2000, 16, 3310. (37) Otagiri, M.; Kurisu, G.; Ui, S.; Takusagawa, Y.; Ohkuma, M.; Kudo, T.; Kusunoki, M. J. Biochem. (Tokyo) 2001, 129, 205.

Figure 4. SERS of NAD+ adsorbed on a silver electrode at pH 7.4 with 488 nm laser excitation.

al.27 in their study of NAD+ adsorbed on a silver electrode. The spectra reported by Taniguchi et al.27 were obtained by signal averaging and did not show any dependence on electrode potential. Thus, their spectra appear to be very poorly enhanced. In comparison, our spectra (Figure 4) are of higher sensitivity and do show a dependence on electrode potential. On the other hand, the Raman spectra of NAD+ obtained on Ag colloids28,29 are definitely surface enhanced but show differences in band frequencies and intensities from those obtained in this study on the Ag electrode. Table 1 lists the Raman band frequencies of NAD+ obtained by a number of workers. We mainly used the normal-mode analysis of Tsuboi et al.38 for in-plane adenine vibrations and the normal Raman (NR) study of NAD+ by Yue et al.39 to assign the SERS modes. The 740 cm-1 band is assigned to the adenine ring breathing mode, ν23, and the band around 1333 cm-1 is assigned to the µ14 mode of the adenine ring containing the N7-C5 stretch and an NH2 bending mode.38 This ν14 mode of adenine couples with the ribose ring vibration and is also observed in the solution NR spectrum at 1336 cm-1. This band is a marker for the C3′ endo-anti conformation for 5′adenosine monophosphate, 5′-AMP.40 The weak band at 1235 cm-1 is close to an adenosine band vibration at 1240 cm-1, which has been assigned to the ν16 mode of adenine. This ring vibration couples the adenine ring vibration with the ribose ring vibration, again in the puckered C3′ endo(38) Tsuboi, M.; Nishimura, Y.; Hirakawa, A. Y.; Peticolas, W. L. In Biological Applications of Raman Spectroscopy; Spiro, T. G., Ed.; John Wiley & Sons: New York, 1987; Vol. 2, Chapter 3. (39) Yue, K. T.; Martin, C. M.; Chen, D.; Nelson, P.; Sloan, D.; Callender, R. Biochemistry 1986, 25, 4941. (40) Nishimura, Y.; Tsuboi, M. In Advances in Infrared and Raman Spectroscopy; Clark, R. J. H., Hester, R. E., Eds.; Heyden: London, 1986; Vol. 13, Chapter 4.

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Figure 5. SERS of 1-MN+ in 0.1 M KCl at pH 11.3 on a silver electrode at 0.0 V with 488 nm laser excitation.

anti conformation.40 The 1377 and 1655 cm-1 bands can most likely be assigned to vibrations of the adenine ring since they are close to the bands assigned by Tsuboi et al.38 as ν13 and ν7, respectively. Both of these vibrations contain in-plane C-H bending modes. The 1377 cm-1 band is weaker than the 1339 cm-1 and is close to a band at 1363 cm-1 observed on colloids by Siiman et al.28 in their SERS spectrum, which they assigned to an adenine ring C2-H deformation vibration. The 1618 cm-1 ν7 mode, which is not observed in this study, represents an external amino scissors vibration. This band may also contain a contribution from the nicotinamide carbonyl. A 1493 cm-1 band was observed in the SERS spectra of NAD+ on colloids28 and assigned to an adenine, C6-NH2 stretching vibration corresponding to ν10 of Tsuboi et al.38 However, this band is not observed in our study. The assignment of the bands to the nicotinamide nucleotide part of the NAD+ molecule is difficult since many of the nicotinamide vibrations also gain some intensity from the adenine component of the molecule.39 To develop a better understanding of the contribution of the nicotinamide part of NAD+ to the SERS spectrum, we obtained SERS spectra of a few molecules containing only this part of the dinucleotide. SERS of 1-MN+ and NMN+. We investigated the SERS of N-methylnicotinamide (1-MN+), Figure 3c, and oxidized nicotinamide mononucleotide (NMN+), Figure 3b. The SERS spectrum of 1-MN+ adsorbed on a silver electrode at pH 11.3 is presented in Figure 5, while Figure 6 shows the SERS spectrum of NMN+ also adsorbed on a silver electrode at pH 7.7. Both molecules contain the nicotinamide ring, but the NMN+ also has the ribose-phosphate linkage found in NAD+. Although the SERS spectrum of 1-MN+ has been reported in the literature,40 we have measured the spectrum with our experimental system, for purposes of

direct comparison. At pH 11.3 in 0.1 M KCl and a potential of 0.0 V, the most intense bands in the 1-MN+ spectrum (Figure 5) are found at 234 (s),1030 (vs), and 1634 cm-1 (s). The strong bands are assigned to the Ag-Cl- (234 cm-1) surface mode from adsorbed Cl-, to the nicotinamide ring breathing mode (1030 cm-1), and to a carbonyl stretching mode of the carboxy amide (1634 cm-1) which is somewhat lower in wavenumber than the CdO stretching mode found around 1700 cm-1 in solution.39 The sharpness and very strong intensity of the 1030 cm-1 band are noteworthy. In the solution Raman spectrum of 1-MN+ the 1034 cm-1 band is by far the strongest peak. The five strongest bands in the solution Raman spectrum at 530, 735, 1034, 1595, and 1632 cm-1 have their counterparts in the SERS spectrum at 528, 776, 1030, 1596, and 1634 cm-1. A major difference between the solution and SERS spectra is that although the 1030 cm-1 band is the strongest band in the SERS spectrum, it does not dominate the spectrum as it does in the solution Raman spectrum. Figure 6 shows the SERS spectrum of NMN+ at pH 7.7 and at various electrode potentials. At E values of -0.4, -0.6, and -0.8 V, intense bands are observed at 1032 cm-1 and in the 1582-1592 cm-1 region. The 1032 cm-1 band is assigned to the ring breathing mode. The band at 1634 cm-1, assigned to a carboxy amide stretching mode in the 1-MN+ SERS spectrum, is absent or shifted to lower frequency in the NMN+ SERS spectra. It is possible that the band appearing at 1616 cm-1 at 0.0 V could be this band. This shift is probably indicative of a strong interaction between the CdO group and the silver surface.39 The most striking feature of Figure 6 is the almost complete loss of intensity for the 1032 cm-1 band at 0.0 V, although other NMN+ bands are still observed in the spectrum. This is only the case for the NMN+ molecule since for 1-MN+ a strong 1032 cm-1 is observed at 0.0 V. These


Langmuir, Vol. 18, No. 25, 2002 9893 Table 1.

solutiona

pH 3.6b

pH 5.4c

pH 6.3d

pH 7.4e

pH 9.3f

Siimang

Hesterh

Markwelli

MNj

NMNk

Spirol

assignment

1687

nicotinamide

3182 3052 1700 1628 1580

1680 1584 1545

1676

1667

1655 1610 1578

1579 1512

1558 1531

1559 1530

1510 1484 1458 1422 1378

1482 1452 1423 1390

1457 1422 1381

1458 1407 1383

1460 1423 1377

1338

1329

1327

1331

1339 1320

1478

1308 1254 1224 1186

1421 1379 1354 1302

1235 1170 1157 1106

1106

1157 1109

1116 1084 1032

1108 1011

1011

1019

1026

914

920

918

924

936

842 815

844

850

817

775

785

738 707 686

743 710 695

1235 1177 1161

730

739

737

678

678

642 607

1493 1446 1416 1393 1363 1332 1313 1268 1225 1200 1180 1137

1570

1624 1573

1509

1504

1040

1090 1029 1012 980 954 929 904

1634 1596

1463 1399

1402 1378

1336 1325

1345 1322

1410

1616

1486 1450 1422 1374

1615 1577 1547 1456 1419 1377 1305

adenine NMN ribose adenine ν13 adenine ν14

1273 1244

1234

adenine ν16 1202

1182 1115

961 888 854 834 800

1618 1600 1590 1546

1030

1116 1082 1025

955 925

915

1182

1030

1114

PO2 str

1032

nicotinamide ribose

956 phosphate

858

742 719 696 633

613

837 800 754 734

820 790

818

730

729

690

700

826 776

phosphate 752 adenine ν28

682 631

654

620

a Raman bands of NAD at pH 7 with 488 nm excitation. Biochemistry 1986, 25, 4941. b-fThis study with 488 nm laser excitation. g SERS of NAD on colloidal silver at pH 8-9 with 590 nm excitation. Inorg. Chem. 1988, 27, 3940. h SERS of NAD on silver colloids at pH 4. J. Chem. Soc., Faraday Trans. 1 1989, 85, 1159. i SERS bands of NAD at -0.4 V on a gold electrode at pH 4 with 1064 nm excitation. Langmuir 1997, 13, 7068. j Raman bands of NADH at pH 7.5 with 514.5 nm excitation. J. Raman Spectrosc. 1980, 9, 369.

spectral results suggest a drastic change in orientation of NMN+ on the silver surface at 0.0 V. Our SERS spectrum of 1-MN+ shows a strong 1634 cm-1 band and medium to strong bands at 1234 and 1596 cm-1 which can be assigned to the ν9a and ν8a in-plane modes of the oxidized nicotinamide ring (benzene like vibrations) and a weak in-plane C-H stretching mode at 3052 cm-1. In-plane modes are enhanced in SERS, when the molecule is oriented perpendicular to the surface because the vibration is subjected to an enhanced EM field normal to the surface. Thus, it is quite definitive that at pH 11.3 1-MN+ is adsorbed with its molecular plane either normal or slightly tilted, but end-on with respect to the silver electrode surface. On the other hand, Siiman et al.28 concluded from their SERS spectrum that the molecule had a flat orientation on a silver colloid surface. Their conclusion was based on two arguments. First, the high relative intensity of the ring breathing mode was thought to be similar to that observed in the SERS spectrum of benzene,41,42 where the ring breathing mode is at least twice as intense as other modes in the spectrum. Benzene, however, is capable of only a flat orientation when it is adsorbed on a silver surface while nicotinamide can also (41) Bowman, W. D.; Spiro, T. G. J. Raman Spectrosc. 1980, 9, 369. (42) Cooney, R. P.; Mahoney, M. R.; McQuillan, A. J. In Advances in Infrared and Raman Spectroscopy; Clark, R. J. H., Hester, R. E., Eds.; Heyden: London, 1982.

have a side-on orientation interacting with the surface through its amide group. Second, Siiman et al.28 found only a weak CdO stretching mode at 1629 cm-1 and weak bands assignable to in-plane a1 modes of the pyridine ring such as at 1224 and 1596 cm-1.43,44 In our study, we observed the 1234 and 1596 cm-1 bands as well as a band at 3052 cm-1. This later band is observed in the SERS spectrum only when the molecule adopts the vertical type orientation on the surface.43 Furthermore, the 1-MN+ molecule is most likely adsorbed through ion pair interaction of its positive charge and an adsorbed Cl- on the Ag surface. We conclude that the strong 1032 cm-1 band is also an in-plane ring breathing mode, and it is expected to be strongly enhanced when the molecular plane is normal to the surface. Thus, the spectrum of NMN+ with a strong 1032 cm-1 band is representative of a vertical orientation of the molecule on the surface. However, in the spectrum of NMN+ at 0.0 V, the 1032 cm-1 band is greatly attenuated, most likely indicating a loss of the vertical orientation. Nicotinamide Vibrations. We now consider the bands in the SERS of NAD+ which might come from the nicotinamide part of the molecule. Nicotinamide bands are expected to be observed at 1032, 1634, 1406, and 1680 (43) Moskovits, M.; Dilella, D. D. In Surface Enhanced Raman Scattering; Chang, R. K., Furtak, T. E., Eds.; Plenum: New York, 1982. (44) Moskovits, M. Rev. Mod. Phys. 1985, 57, 78.

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Figure 6. SERS of 1-NMN+ at pH 7.7 on a silver electrode with 488 nm laser excitation: (a) 0.0, (b) -0.4, (c) -0.6, and (d) -0.8 V.

cm-1 in the SERS spectrum. The 1032 cm-1 ring breathing nicotinamide mode was observed as a weak band in the SERS spectrum of NAD as reported by Taniguchi,27 Siiman,28 and Hester.29 Xiao et al.30 have reported an intense band in the SERS of NAD adsorbed on a Au electrode. This 1025 cm-1 band at -0.4 V shifted to 1029 cm-1 at +0.4 V and was the most intense band in the SERS spectrum. Of the four nicotinamide vibrations which have been observed in the SERS spectrum of NAD, those at 1634 and 1406 cm-1 were not observed in any of the SERS spectra of NAD in this study. The bands around 1032 and 1680 cm-1 are both observed with varying degrees of intensity in the reported SERS spectra. The intensity of the SERS band around 1032 cm-1 shows a significant pH dependence. At acidic pH values, 3.6 (Figure 7) and 5.4 (Figure 8), the 1032 cm-1 band appears as a shoulder of the 1010 cm-1 band. At pH values of 6.3 (Figure 9) and 7.4 (Figure 4), the intensity of the 1032 cm-1 band increases until it becomes moderately intense relative to the other SERS bands and it is resolved from the 1010 cm-1 band. At the basic pH value of 9.3 the 1040 cm-1 band is intense, possessing a shoulder at 1067 cm-1 (Figure 10a). The 1584 cm-1 band, which derives some of its intensity from nicotinamide, is observed in the SERS spectra at pH values of 3.6, 5.4, and 6.3 but not at 7.4 and 9.3. The bands in the 1540-1590 cm-1 region of the SERS spectrum, which are assigned to the nicotinamide vibration, also show a marked pH dependence. At pH values of 3.6 and 5.4 this region of the spectrum contains a single band at 1584 cm-1. At pH values of 6.3 and 7.4 bands are observed at 1531 and 1558 cm-1 with a band at 1581 cm-1 growing into the spectrum at more negative electrode potentials (Figure 9). At pH 9.3 a 1578 cm-1 band of low to moderate intensity is observed in this region of the spectrum, and


its intensity decreases as the electrode potential is made more negative.


Langmuir, Vol. 18, No. 25, 2002 9895

Figure 8. SERS of NAD+ adsorbed on a silver electrode at pH 5.4 with 488 nm laser excitation. Figure 10. SERS of NAD+ adsorbed on a silver electrode at pH 9.3 with 488 nm laser excitation.


The strong SERS band occurring between 1570 and 1596 cm-1 for 1-MN+ and NMN+ (Figures 5 and 6) is found in NAD+ at 1580 cm-1 and has been assigned to a nicotinamide ring stretch by Siiman et al.28 The intensity

of this 1580 cm-1 band increases as the electrode potential is made more negative (Figure 4). Yue et al.39 assigned a band at 1580 cm-1 in the solution normal Raman spectrum of NAD+ to 80% ribose (on the adenine ring) and 20% nicotinamide ring vibration. Because of the presence of this band in the SERS spectrum of 1-MN+ and NMN+, it seems reasonable to assign some of the 1580 cm-1 vibrational intensity in the SERS spectrum of NAD+ to the nicotinamide portion of the molecule. A very weak band in our SERS spectrum around 1420 cm-1 also corresponds to a band that has 50% contribution from the nicotinamide ring according to Yue et al.39 Yet, another weak band at 1454 cm-1 in our SERS spectra at pH 3.6 and 5.4 corresponds to a band at 1458 cm-1 in the solution Raman spectrum, which involves vibrations of the ribose connected to the nicotinamide and a small influence from nicotinamide. The strongest and most distinct nicotinamide band in the solution Raman spectra of 1-MN+, NMN+, and NAD+, and in our SERS spectra of 1-MN+ and NMN+, comes from the 1030 cm-1 nicotinamide ring breathing vibration. This band has reduced intensity in the NAD SERS spectra at pH values of 3.6 and 5.4, is clearly discernible at pH 6.3, is of moderate intensity at pH 7.4, and is an intense band at pH 9.6. In most of the NAD+ colloidal SERS spectra this band has a low intensity.28 The absence of this band at more acidic pH values suggests either that the nicotinamide ring is oriented away from the electrode and too far from the surface (>5 Å) to be enhanced or it is in an orientation such that its in-plane ring stretch is not enhanced. The latter conclusion is consistent with the spectrum of NMN+ where at the most positive potential observed, 0.0 V, the strong 1032 cm-1 band almost completely disappears (Figure 6a). It should also be noted that other bands in this NMN+ SERS spectrum at 812,

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1420, and 1592 cm-1 have their counterpart in bands found in NAD+ SERS spectra. This suggests that when NAD+ is bound to the silver electrode at pH values of 3.6 and 5.4, the nicotinamide could be adsorbed in a position as it is in NMN+ at 0.0 V, so that its ring stretching vibration is not observed in the SERS spectrum. One such orientation could be with its molecular plane parallel to the surface. It is reasonable to assume that the positively charged nicotinamide forms an ion pair with negatively charged surface sites which may be specifically adsorbed anions, in this case hydrogen phosphate anions from the buffer solution. The weak 743 cm-1 SERS band in Figure 4 is assigned to the ring breathing mode of the adenine component of NAD+ on the surface. Nishimura and Tsuboi40 indicate that the ν23 normal mode of adenine at 724 cm-1 is strongly coupled with ribose vibrations when the hydrogen at N1 is replaced by a ribose ring. Thus, this ring breathing mode of adenine is very sensitive to the surface conformation about the adenine-ribose linkage. Two other adenine normal modes ν14 (∼1332 cm-1) and ν16 (∼1249 cm-1) also couple through N1 with ribose vibrations. These modes are sensitive to the ribose ring conformation and the anti or syn conformation of the torsional angle about the glycosidic link.45 Several conformations involving puckering of the ribose ring and the anti or syn conformation of the ribose-adenine torsional angle have been correlated with Raman normal modes. Thus, SERS bands at 1338 and 1235 cm-1 represent the normal adenine modes which can be assigned as ν14 and ν16 and which correlate with a coupling indicating the C3′ endo-ribose with an anti-configuration of the riboseadenine torsional angle orientation.40 Other bands may provide additional information on the conformation of the ribose ring, since bands at 1325 and 1343 cm-1 have been assigned as marker bands for the presence of the O-4′ endo-anti and C3′ exo-anti adenine-ribose sugar conformations, respectively.40 The plane of the sugar molecule is defined by C1-OC4 all being horizontal, with atoms lying above this plane being in the endo conformation. The C2′ and C3′ endo conformation results in a puckering of the ribose ring. This ribose arrangement of both C2′ and C3′ endo has been shown to exist when NAD+ is bound to some enzyme systems.46 Crystallographic analysis47 of the lithium salt of NAD+ confirms puckering of the ribose groups in NAD+, with the adenosine ribose displaying a C2′ endo conformation while the nicotinamide ribose is in the C3′ endo and C2′ exo twisted form. A C3′ exo-anti configuration would be represented by a band at 1255 cm-1 28 and may not exist in this study at any pH. Since the 1338 and ∼1235 cm-1 adenine ring vibrations are present in the spectra at pH 7.4 and 6.4, we can conclude that the C3′ endo-anti coupling is most likely present at these pH values for NAD on the Ag surface. On the other hand, there do not appear to be any bands in the SERS spectrum that provide information on the conformation of the nicotinamide ribose ring and its glycosidic torsional angle. Pyrophosphate Vibrations. Raman bands around 1100 and 800 cm-1 have been assigned to the νPO2 and P-O-P vibrations in NAD and NADH.28 Siiman observed bands at 1090 and 837 cm-1 while Xiao30 observed a weak shoulder at 1082 cm-1 in the SERS spectrum of NAD. These bands were assigned to the νPO2 and P-O-P (45) Smith, P. E.; Tanner, J. T. J. Am. Chem. Soc. 1999, 121, 8637. (46) Patrick, D. M.; Wilson, J. E.; Leroi, G. E. Biochemistry 1974, 13, 1813. (47) Reddy, B. S.; Saenger, W.; Muhlegger, K.; Weiman, W. J. Am. Chem. Soc. 1981, 103, 907.

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vibrations, respectively. In this study, at all the pH values investigated, a moderately intense band is observed at 1108 cm-1 while a very weak band is observed around 816 cm-1. On the basis of the work of Siiman,28 we assigned these bands to the ν(PO2) and P-O-P vibrations, respectively. The ratio of the intensities of the phosphodiester vibration to the phosphate vibration has been used to determine whether there is an ordered structure around the pyrophosphate linkage.48,49 When this ratio, IPOP/IPO2, is 6, and still have some negative charge at lower pH, it would be expected that these groups could interact with partially positive surface Ag adion sites.26 Of the strong bands observed in the SERS spectra of NAD, only the 1584 cm-1 band derives a significant proportion of its intensity from the nicotinamide group. Other bands such as those at 1420 and 1030 cm-1 owe some of their intensity to nicotinamide vibrations and are suggestive of the nicotinamide component of NAD interacting with the silver surface. Siiman28 and Barrett53 have also obtained SERS spectra of nicotinamide in which the strong nicotimamide band found in solution around 1030 cm-1 is virtually absent from the SERS spectra of NAD+. We have attributed the absence of this strong band to an orientation of the nicotinamide which is parallel to the surface so that its intense vibration at 1030 cm-1 is reduced because of the surface selection rules. These results suggest one possible structure of NAD+ on the Ag electrode surface at pH 3.6 and 5.4 which is very much like the preferred configuration of most 5′ nucleotides. At pH values more basic than 5.4, the increase in intensity of the 1032 cm-1 band along with the decrease in intensity of the 1680 cm-1 band, and its downshift, suggests a pH dependent change in the orientation of the NAD molecule. The nicotinamide component of the NAD molecule is now orientating itself perpendicular to the electrode surface, resulting in an increase in intensity of the 1032 cm-1 ring breathing mode, and the reduction in intensity and downshift of the 1680 cm-1 CdO stretch is due to strong surface interaction.

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In this case NAD+ is in an extended configuration with the nicotinamide oriented flat with respect to the surface. The adenine ring would be adsorbed in a vertical orientation relative to the surface so that the adenine and nicotinamide rings would be at nearly right angles to each other. In fact, both of the surface species predicted from the SERS spectra would have this orientation of the two ring systems no matter what configuration the adenylic part of the molecule takes. The molecule could be on the surface entirely in the extended form or the nicotinamide may be bent around but with the rings still at right angles. Since at potentials positive to the point of zero charge phosphate anions from the electrolyte may be weakly adsorbed on the surface, it is expected that the positively charged ring nitrogen of nicotinamide would be attracted to this negative surface site. This conclusion of a flat orientation of nicotinamide is consistent with the spectrum of NMN+ at 0.0 V where the spectrum shows that NMN+ is on the surface but the strong 1032 cm-1 band is lost. Conclusion We have presented a study of the effect of pH and electrode potential on the SERS spectrum of NAD+

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adsorbed on a silver electrode. From our results we conclude that, in aqueous solution at pH values close to physiological pH, NAD+ adsorbs unto the electrode with only one conformation, while NAD+ is adsorbed onto the electrode surface in an extended structure, which superficially resembles the extended structure that it adopts when it binds to the dehydrogenases and acts as a biological catalyst. The Ag adsorbed species has a configuration which is clearly more similar to the protein bound species than to the folded aqueous solution species. Acknowledgment. R.L.B. and J.R.L. are indebted to the PSC-BHE Research Award Program of the City University of New York (668274, 669267), the National Science Foundation (CHE-8711638, CHE-9100195), and the National Institutes of Health SCORE/NIGMS Grant GM08168 for financial assistance. C.M.H. is indebted to the National Cancer Institute Grant 5 RO3 CA68993 for financial assistance. LA020235J

SERS Investigation of NAD+ Adsorbed on a Silver Electrode

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