Electrooptical behavior of heme-like molecules adsorbed on nerve membrane

This paper discusses the electrooptical properties of iron tetrasulfonated phthalocyanine (Fe-TsPc) adsorbed on a nerve membrane. The study is based on a comparative analysis of the properties of an aqueous solution of Fe-TsPc and the properties of Fe-TsPc adsorbed on a silver electrode. The resonant Raman spectra were obtained from the adsorbed Fe-TsPc on the interfaces of a silver electrode and the nerve membrane. Considerable optical signal changes have been observed when the interfacial potential is altered. The Raman spectra are sufficiently sensitive to indicate the submolecular mechanisms of electron delocalization. The changes in Raman signals as a function of the interfacial potential provide a means for optically monitoring electrical excitability in nerve.     

Raman microprobe spectroscopy: A new tool for biofouling diagnostics

Raman vibrational spectra were obtained from crystalline amino acids and acid molecules adsorbed at the surfaces of silver electrodes in aqueous solutions. Results revealed that aromatic acids such as phenylalanine adsorbed via their aromatic ring while bases such as alanine were coordinated by their amine functional groups. Sulphur containing acids (cysteine and cystine) were found to bond through their sulphur groups. In all cases, adsorption increased towards the point of zero charge of silver, as would be expected for uncharged species. Similar experiments carried out on α‐amylase solutions showed that the enzyme molecule changes its coordination to the silver surface as a function of electrode potential, indicating that C‐S, C‐N, and aromatic ring functional groups are all present on the outer surface of the enzyme structure.     

Surface enhanced resonance Raman study of phenobarbital-induced rabbit liver cytochrome P-450 LM2

Surface enhanced resonance Raman (SERR) spectroscopy has been used to study the vibrational spectra of the heme of purified rabbit liver cytochrome P-450 LM2 which was adsorbed on colloidal silver suspensions or on a silver electrode. Bases on a comparison with the resonance Raman (RR) spectra of the 'solute' species the high sensitivity of the SERR technique is demonstrated. Two different features were chosen in order to determine the structural and functional integrity of the adsorbed P-450. Both, substrate-induced spin state changes on the oxidized P-450 and the effect of the thiolate ligand on the oxidation state marker band v4 in the reduced P-450 could be observed in the SERR spectra of the adsorbed as well as in the RR spectra of the dissolved enzyme. These findings indicate that the protein structure near the substrate binding site and the coordination by thiolate are not affected by the interaction with the metal surface. Both structural elements are crucial for the function of P-450. Thus the elementary processes of the enzymatic action of P-450 can be investigated by this highly sensitive version of RR spectroscopy.     

Surface-enhanced Raman spectroscopy of biopolymers: membrane proteins, bacteriorhodopsin and rhodopsin adsorbed on silver electrodes and silver hydrosols

Surface-enhanced Raman (SER) spectra of purple membranes of Halobacterium halobium and photoreceptor disks of the rod outer segments adsorbed on silver hydrosols were analysed. It has been shown that the intensity of SER spectra of bacterial and visual rhodopsins increases 5 X 10(4) times at adsorption. Concentration relationship of the signal intensity of SER spectra has the maximum at bacteriorhodopsin concentration about 2 X 10(-7) M. It has been shown that adsorption on silver hydrosol leads to fixation of light-induced photochemical transformations in bacterial and visual rhodopsins. Adsorption on the "smooth" electrodes at the potential of the zero charge of silver does not affect the photocycle of bacteriorhodopsin. An increase or decrease of the electrode potential relative to the zero charge point of silver leads to the accumulation of kinetic intermediate K610 and a decrease of the concentration of the form BRh570. It has been shown that on the "smooth" electrode primarily the long-range component of the SER mechanism is realized. Bands corresponding to the vibrations of the atom groups directly contacting with the metal are mainly intensified after redox cycle which increases the concentration of chemosorption centres. A conclusion is drawn that the method of SER spectroscopy of biomolecules adsorbed on "smooth" electrodes, permits obtaining information similar to that obtained from the analysis of Raman spectra of unadsorbed molecules, but at concentrations by two orders less. Adsorption on the electrodes treated with the help of redox cycle permits to obtain highly oriented preparations and to study topography of biopolymers in water solutions and suspensions.     

Active site structure and dynamics of cytochrome c3 from Desulfovibrio gigas immobilized on electrodes

Cytochrome c3 from Desulfovibrio gigas is electrostatically adsorbed on Ag electrodes coated with self-assembled monolayers (SAMs) of 11-mercaptoundecanoic acid. The redox equilibria and electron transfer dynamics of the adsorbed four-heme protein are studied by surface enhanced resonance Raman spectroscopy. Immobilization on the coated electrodes does not cause any structural changes in the redox sites. The potential-dependent stationary experiments distinguish the redox potential of heme IV (-0.19 V versus normal hydrogen electrode) from those of the other hemes for which an average value of -0.3 V is determined. Taking into account the interfacial potential drops, these values are in good agreement with the redox potentials of the protein in solution. The heterogenous electron transfer between the electrode and heme IV of the adsorbed cytochrome c3 is analyzed on the basis of time-resolved experiments, leading to a formal electron transfer rate constant of 15 s(-1), which is a factor of 3 smaller than that of the monoheme protein cytochrome c.     

Surface-enhanced resonance Raman spectroscopy of phycocyanin and allophycocyanin

High quality surface-enhanced resonance Raman (SERR) spectra were recorded from native and denatured phycocyanin and allophycocyanin on ascorbic acid treated silver hydrosols. The visible-excited SERR and resonance Raman (RR) spectra of the phycobiliproteins were very similar, indicating a predominantly electromagnetic surface enhancement mechanism. Investigation of pH-induced denaturation ofx allophycocyanin has shown that even small differences in protein/chromophore conformational are sensitively reflected by the SERR spectra. Concerning the adsorption of the protein to the metal surface, the experiments have shown that: (i) there is limited possibility for changing protein conformation during the adsorption process, (ii) there are no changes after the protein has been adsorbed onto the silver surface and (iii) for each protein an optimal activation of the silver sol has to be found for recording proper SERR spectra. The results obtained on phycobiliproteins are also discussed in connection with the interpretation of phytochrome Raman spectra.     

聚赖氨酸溴化氢结合物的表面增强拉曼光谱

银溶胶对聚赖氨酸溴化氢结合物(PLys—HBr)表现出极大表面增强拉曼(SER)效应。同PLyS—HBr的普通拉曼光谱相比,表面增强因子提高达6个数量级。实验表明,NH_3~+基是银表面增强效应的强活性基团。但是在相同条件下,聚谷氨酸钠(PGA—Na)在银溶胶中不能获得SER光谱,这可能是由于空间障碍或者COO~-基的活性不如NH_3~+基。     

Raman scattering from nucleic acids adsorbed at a silver electrode

Adsorption of nucleic acids at a silver electrode polarized to -0.6 to -0.1 V (vs. Ag/AgCl) was investigated by means of surface enhanced Raman scattering (SERS) spectroscopy. Single-stranded polyriboadenylic acid and thermally denaturated DNA adsorbed at the silver electrode yield two intense bands at 734 and 1335 cm-1 on the SERS spectra. These bands, assigned to the vibrations of adenine residue rings, were much less intense if the SERS spectra were recorded for double-helical complex polyadenylic X polyuridylic acid and native DNA. Moreover, the courses of alkaline denaturation of DNA and its digestion by deoxyribonuclease I were observed by SERS spectroscopy. The results were interpreted as support for the view that intact double-helical segments of nucleic acids are not denatured or destabilized due to their adsorption at the positively charged and roughened surface.     

Raman and surface-enhanced Raman spectroscopy investigation of vasopressin analogues containing 1-aminocyclohexane-1-carboxylic acid residue

In this work, Raman spectroscopy (RS) was employed to characterize molecular structures of [Arg8]vasopressin (AVP) and its [Acc2,D-Arg8]AVP, [Acc3]AVP, and [Cpa1, Acc3]AVP analogues. The RS band assignments have been proposed. To determine the mechanism of adsorption of the above-mentioned compounds adsorbed on a colloidal silver surface, surface-enhanced Raman spectra (SERS) were measured. The SERS spectra were used to determine relative proximity of the adsorbed functional groups of [corrected] investigated peptides and their orientation on the silver surface. The AVP and [Acc3]AVP SERS spectra (Acc: 1-aminocyclohexane-1-carboxylic acid) show that the L-tyrosine (Tyr) lies far from the metal surface, whereas the [Cpa1,Acc3]AVP spectrum (Cpa: 1-mercaptocyclohexaneacetic acid) provides evidence that Tyr interacts with the silver surface. These results suggest that [corrected] the binding of the Tyr-ionized phenolic group might be responsible for the selectivity of the analogues. We show that the aromatic ring of L-phenylalanine (Phe) of AVP and [Acc2,D-Arg8]AVP interacts with the silver surface. The strength of this interaction is considerably weaker for [Acc2,D-Arg8]AVP than for AVP. This might be due either to a longer distance between the Phe ring and the silver surface, or to the almost perpendicular orientation of the Phe ring towards the surface. The carbonyl group of the L-glutamine [corrected] (Gln) or L-asparagine [corrected](Asn) of AVP, [Acc2,D-Arg8]AVP, and [Acc3]AVP is strongly bound to the silver surface. We have also found that all peptides adsorb on the silver surface via sulfur atoms of the disulfide bridge, adopting a "GGG" conformation, except [Cpa1,Acc3]AVP, which accepts a "TGG" geometry.     

Surface-enhanced resonance Raman scattering spectroscopy of bacterial photosynthetic membranes. The carotenoid of Rhodospirillum rubrum

Resonance Raman scattering by the carotenoid, spirilloxanthin (Spx), in a suspension of chromatophores (cytoplasmic side out) isolated from the photosynthetic bacterium, Rhodospirillum rubrum, is greatly enhanced when the membranes are adsorbed onto the surface of an anodized Ag electrode. The phenomenon is the basis for surface-enhanced resonance Raman scattering (SERRS) spectroscopy. The Spx SERRS peaks observed were at 1505-1510, 1150-1155, and 1000-1005 cm-1 with laser excitation wavelengths ranging between 457.9 and 568.2 nm. Similar peaks were not observed with spheroplasts (periplasmic side out) isolated from the same species. The difference in signal detected in chromatophores and spheroplasts is not due to differences in membrane surface charge, presence of residual cell wall on the spheroplast surface, lack of adhesion of spheroplasts to metals, or large differences in pigment content per unit membrane area. Instead, the results indicate an asymmetric distribution of Spx in vivo across the membrane (i.e., it is located on the cytoplasmic side of the membrane). The results also demonstrate that the SERRS effect is extremely distance sensitive, and the thickness of a single bacterial membrane (separating the Ag electrode from the carotenoid) is sufficient to prevent detection of Spx spectra. Studies of chromatophores from the F24 strain (a reaction centerless mutant) have pin-pointed B880 antenna complex as the source of the Spx SERRS spectra, and a schematic model of the minimal structural unit of B880 is presented. This work demonstrates the potential of the SERRS technique as a probe for surface topology of pigmented membranes.     

Microperoxidase 8 adsorbed on a roughened silver electrode as a monomeric high-spin penta-coordinated species: characterization by SERR spectroscopy and electrochemistry

Microperoxidase 8 (MP8), a heme octapeptide obtained by hydrolytic digestion of cytochrome c, was adsorbed at the surface of a roughened silver electrode in order to provide a new supported biomimetic system for hemoproteins. A combination of two techniques was used to study its redox and coordination properties: electrochemistry and surface-enhanced resonance Raman (SERR) spectroscopy. This allowed us to show that MP8 could be adsorbed as a monolayer at the surface of the roughened silver electrode, where it could undergo a reversible electron transfer. Under those conditions, a redox potential of –0.4 V vs. SCE (–0.16 V vs. NHE) was measured for MP8, which was almost identical to that reported for N-acetyl-MP8 in aqueous solution. In addition, whereas MP8 appeared to aggregate in solution, and led to a mixture of high-spin penta-coordinated (5cHS) and low-spin hexa-coordinated (6cLS) iron(III) or iron(II) species, it was recovered almost exclusively as a monomeric high-spin penta-coordinated species at the surface of the electrode, both in the reduced and in the oxidized states. This then allowed a free coordination site on the iron, on the distal face of MP8 accessible to ligands. Accordingly, experiments performed in the presence of potassium cyanide demonstrated that MP8 adsorbed on a silver electrode could be ligated by a sixth CN^– ligand. Thus there is the possibility of binding several kinds of ligands such as O₂ or H₂O₂, which will open the way to biocatalysis of oxidation reactions at the surface of an electrode, or ligands such as drugs which will lead to the design of new biosensors for molecules of biological interest.     

Surface interactions of a homologous series of alpha,omega-amino acids on colloidal silver and gold

Surface enhanced Raman spectroscopy (SERS) was used to characterize a homologous series of alpha,omega-amino acids on colloidal gold and silver. Raman and SER spectra of the alpha,omega-amino acids, NH2(CH2)nCOOH (n = 3-7), are presented and analyzed, revealing the probable conformations of the molecules on the metal surfaces. The alpha,omega-amino acids interact with silver and gold through both the amine and carboxylate end groups, and modify the conformation of the molecular backbone in order to maximize these interactions. An odd-even effect is observed for backbone conformations of molecules adsorbed to the silver substrate. The anomolous SER spectrum of 5-aminopentanoic acid on gold suggests the possibility of condensation polymerization at the gold surface.     

SERRS study of the DNA binding by Ru(II) tris-(Bipyridyl) complexes bearing one carboxylic group

Surface enhanced resonance Raman scattering (SERRS) is shown to be a satisfying method to study the interaction between DNA and ruthenium complexes [Ru(bpy)(2)(Hcmbpy)][PF(6)](2), where Hcmbpy = 4-carboxy-4'-methyl-2,2'-bipyridine. Such metallic complexes are known for their fluorescence properties. To validate this spectroscopic approach we have checked that i) at a given lambda(ex), silver colloidal SERRS spectra of Ru complexes closely resemble resonance Raman spectra in aqueous solutions, intensity excepted, and ii) the DNA fragments are not altered when they are adsorbed on the Ag nanoparticles surface. This investigation shows that the intensity of the Ru complexes SERRS spectra is reduced in the presence of DNA, in particular for the specific bands assigned to the Hcmbpy ligand. This collapse demonstrates that the Ru complexes bind DNA through the Hcmbpy moiety, and intercalation is suggested as the binding mode. The DNA binding by the enantiopure Ru complexes (Delta or Lambda) is more efficient than by the racemic complexes.     

Electrogenerated chemiluminescence of luminol in neutral and alkaline aqueous solutions on a silver nanoparticle self-assembled gold electrode.

The electrochemiluminescence (ECL) behaviour of luminol on a silver nanoparticle self-assembled gold electrode in neutral and alkaline solutions was investigated using conventional cyclic voltammetry (CV). The silver nanoparticle self-assembled gold electrode exhibited excellent ECL properties for the luminol ECL system. In neutral solutions, four ECL peaks (ECL-1-ECL-4) were observed at 0.73, 1.15, -0.46 and -1.35 V (vs. SCE), respectively. The intensities of these peaks were enhanced significantly compared with those on a bulk gold electrode and a gold nanoparticle self-assembled gold electrode. It was found that ECL-1 and ECL-2 on a silver nanoparticle-modified electrode were about 1000 and 1770 times stronger than those on a bare Au electrode and were about 17 and 15 times stronger than those on a gold nanoparticle-modified electrode, respectively. In alkaline solutions, four ECL peaks were also observed that were much stronger than those in neutral solutions, and ECL-1 and ECL-2 were enhanced by about three orders and one order of magnitude compared with those on a bare Au electrode and on a gold nanoparticle self-assembled electrode, respectively. Moreover, the silver nanoparticle-modified electrode exhibited good stability and reproducibility for luminol ECL. These peaks were found to depend on a number of factors, including silver nanoparticles on the surface of the modified electrode, potential scan direction, scan rate, scan range, the presence of O2 or N2, pH values, the concentrations of NaBr and luminol, and buffer solutions. The emitter of the ECL was confirmed as 3-aminophthalate by analysing the CL spectra. The surface state of the silver nanoparticle self-assembled electrode was characterized by scanning electron microscopy (SEM) and the interface property of the electrode was studied by electrochemical impedance spectroscopy (EIS). A mechanism for the formation of these ECL peaks is proposed. The results demonstrate that luminol has excellent ECL properties, such as strong ECL intensity and good reproducibility on a silver nanoparticle-modified gold electrode, in both neutral and alkaline solutions, which is of great potential in analytical applications.     

Structural properties of bombesin-like peptides revealed by surface-enhanced Raman scattering on roughened silver electrodes

This work presents a Fourier-transform absorption infrared, Fourier-transform Raman, and surface-enhanced Raman scattering (SERS) study of the following peptides belonging to the bombesin-like family: phyllolitorin, [Leu(8)]phyllolitorin, NMB, NMC, and PG-L. The SERS study was undertaken to understand the adsorption mechanism of bombesin-like peptides on an electrochemically roughened silver electrode surface and to show changes in the adsorption mechanism with alterations in amino acids and small tertiary structures. The SERS spectra presented here shows bands mainly associated with the Trp(8) residue vibrations. The presence of mainly pyrrole coring vibrations for phyllolitorin and [Leu(8)]phyllolitorin and mainly benzene coring modes for NMB and NMC indicated that these groups interact with the roughened silver electrode surface. Furthermore, N(1)--C(8) and C(3)--C(9) bonds of the PG-L indole ring seemed to have nearly a vertical orientation on the electrode surface. In addition, distinct vibrations of the C--S fragment were observed in the SERS spectra of [Leu(8)]phyllolitorin and PG-L. The strong enhancement of the nu(C==O) vibration in the [Leu(8)]phyllolitorin SERS spectrum yielded evidence that the intact C==O bond(s) bind strongly to the silver electrode surface, whereas NMC, phyllolitorin, and NMB were located near the silver surface. This finding was supported by the presence of the nu(C--C(==O)) mode. The amide I band observed at 1642 and 1634 cm(-1) for NMB and NMC, respectively, and the Raman amide III band seen in the 1282-1249 cm(-1) range for all peptides except PG-L, indicate that the strongly hydrogen-bonded alpha-helical conformation and random-coil structure are favored for binding to the surface. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 980-992, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Physicochemical characterisation of enzymatically hydrolysed derivatives of acetylated starch

Potato starch modified to different degrees by substitution with acetyl groups was the subject of this study undertaken to determine the influence of conditions of enzymatic hydrolysis on the surface-active properties of hydrolysates of acetylated starch. The effect of acetylation of starch preparation on its susceptibility to enzymatic hydrolysis in the membrane reactor was also considered. All hydrolysates of acetylated starch samples investigated were found to bring a decrease in the surface/interfacial tension, both at the air/water and the toluene/water interfaces. For binary hydrolysate-surfactant systems, the surface mole fractions in the mixed adsorbed monolayer at the air/water interface were estimated. For mixed systems, the synergism in reducing the surface tension at the air/water interface was observed. The experimentally obtained dynamic surface tension data for the aqueous solution of acetylated starch hydrolysates were used to estimate the diffusion coefficients. Particle size distributions of the hydrolysates formed in the aqueous solutions were compared to those of commercial maltodextrin.     

Electric-field dependent decays of two spectroscopically different M-states of photosensory rhodopsin II from Natronobacterium pharaonis

Sensory rhodopsin II (NpSRII) from Natronobacterium pharaonis was studied by resonance Raman (RR) spectroscopic techniques. Using gated 413-nm excitation, time-resolved RR measurements of the solubilized photoreceptor were carried out to probe the photocycle intermediates that are formed in the submillisecond time range. For the first time, two M-like intermediates were identified on the basis of their C=C stretching bands at 1568 and 1583 cm(-1), corresponding to the early M(L)(400) state with a lifetime of 30 micro s and the subsequent M(1)(400) state with a lifetime of 2 ms, respectively. The unusually high C=C stretching frequency of M(1)(400) has been attributed to an unprotonated retinal Schiff base in a largely hydrophobic environment, implying that the M(L)(400) --> M(1)(400) transition is associated with protein structural changes in the vicinity of the chromophore binding pocket. Time-resolved surface enhanced resonance Raman experiments of NpSRII electrostatically bound onto a rotating Ag electrode reveal that the photoreceptor runs through the photocycle also in the immobilized state. Surface enhanced resonance Raman spectra are very similar to the RR spectra of the solubilized protein, ruling out adsorption-induced structural changes in the retinal binding pocket. The photocycle kinetics, however, is sensitively affected by the electrode potential such that at 0.0 V (versus Ag/AgCl) the decay times of M(L)(400) and M(1)(400) are drastically slowed down. Upon decreasing the potential to -0.4 V, that corresponds to a decrease of the interfacial potential drop and thus of the electric field strength at the protein binding site, the photocycle kinetics becomes similar to that of NpSRII in solution. The electric-field dependence of the protein structural changes associated with the M-state transitions, which in the present spectroscopic work is revealed on a molecular level, appears to be related to the electric-field control of bacteriorhodopsin's photocycle, which has been shown to be of functional relevance.     

Cytochrome c at charged interfaces. 1. Conformational and redox equilibria at the electrode/electrolyte interface probed by surface-enhanced resonance Raman spectroscopy

The structure and the electron-transfer properties of cytochrome c (cyt c) absorbed on a silver electrode were analyzed by surface-enhanced resonance Raman spectroscopy. It was found that the absorbed cyt c exists in various conformational states depending on the electrode potential. In state I the native structure of the heme protein is fully preserved and the redox potential (+0.02 V vs saturated calomel electrode) is close to the value for cyt c in solution. In state II the heme iron exists in a mixture of five-coordinated high-spin and six-coordinated low-spin configurations. It had been shown that these configurations form a thermal equilibrium [Hildebrandt, P., & Stockburger, M. (1986) J. Phys. Chem. 90,6017]. It is demonstrated that these equilibria strongly depend on the charge distribution within the electrical double layer of the silver electrode/electrolyte interface, indicating that the changes in the coordination shell are induced by electrostatic interactions. The structural alterations in state II are apparently restricted to the heme crevice, which assumes an open conformation compared to the close structure in state I. This leads to a strong decrease of the redox potentials, which were determined to be -0.31 and -0.41 V for the five-coordinated high-spin and six-coordinated low-spin configurations, respectively. On the other hand, gross distortions of the protein structure can be excluded since the reversible proton-induced conformational change of cyt c as found in solution at low pH also takes place in state II of the absorbed cyt c. The linkage of cyt c molecules to the surface is mediated by charged amino acid groups, and it depends on the potential which groups are thermodynamically favored to form such a molecular binding site. The conformational states I and II, which are in potential-dependent equilibrium, therefore refer to two different molecular binding sites. At potentials below zero charge (less than approximately -0.6 V) a rapid denaturation of the absorbed cyt c is noted, which is reflected by drastic and irreversible changes in the surface-enhanced resonance Raman spectrum. Our results are discussed on the background of previous electrochemical studies of cyt c at electrodes.     

Spectral analyses of absorption changes associated with nerve excitation in dye-stained crab nerve

Spectral characteristics of absorption changes associated with nerve excitation were studied with crab nerves stained with a homologous series of dyes, merocyanine-rhodanines and rhodanine oxonols. In these classes of dyes, the absorption changes which followed approximately the same time course as that of the action potential (fast responses) depended in a similar fashion on the wavelength and polarization of the incident light. In order to interpret those commonly observed dependencies, a mode of reorientation of the absorption oscillators of the dye molecules in the membrane matrix during nerve excitation was proposed. In addition to the fast changes mentioned above, slow responses which developed during and after the action potential were commonly observed with oxonols. The spectra of the slow changes differed from those of the fast ones, indicating a distinct mechanism on the response production. A possible mechanism of the production of fast responses was also discussed based on the proposed mode of reorientation of the absorption oscillators.     

Theoretical and pH dependent surface enhanced Raman spectroscopy study on caffeine

The surface enhanced Raman spectroscopy (SERS) spectrum of caffeine is recorded on a silver colloid at different pH values. It is discussed on the basis of the SERS "surface selection rules" in order to characterize its vibrational behavior on such a biological artificial model. To improve the previous assignments in the Raman spectrum and for a reliable, detailed analysis of SERS spectra, density functional theory calculations (structural parameters, harmonic vibrational wavenumbers, total electron density, and natural population analysis of the molecule) are performed for the anhydrous form of caffeine and the results are discussed. The predicted geometry and vibrational Raman spectra are in good agreement with the experimental data. The flat orientation of the mainly chemisorbed caffeine attached through the pi electrons and the lone pair of nonmethylated N atoms of the imidazole ring are proposed to occur at neutral and basic pH values. At acid pH values caffeine is probably adsorbed on the Ag surface through one or both oxygen atoms, more probably through the O atom of the conjugated carbonyl group with an end-on orientation. However, the changes in the overall SERS spectral pattern seem to indicate the electromagnetic mechanism as being the dominant one.