Edyta Podstawka

Part I: Surface-Enhanced Raman Spectroscopy Investigation of Amino Acids and Their Homodipeptides Adsorbed on Colloidal Silver

Surface-enhanced Raman scattering spectra (SERS) were measured for various amino acids: L-methionine (Met), L-cysteine (Cys), L-glycine (Gly), L-leucine (Leu), L-phenylalanine (Phe), and L-proline (Pro) and their homodipeptides (Met-Met, Cys-Cys, Gly-Gly, Leu-Leu, Phe-Phe, and Pro-Pro) in silver colloidal solutions. The geometry and orientation of the amino acids or dipeptides on the silver surface, and their specific interaction with the surface, were deducted by detailed spectral analysis of the SERS spectra. This analysis has allowed us to propose the particular surface geometry of amino acids or dipeptides and also implied that C–C bonds were almost parallel to the surface, as evidenced by the absence of marker bands in the skeletal C–C stretching region of the spectra. Additionally, using “time-dependent” SERS measurements we solved an existing controversy regarding the binding specificity of Gly-Gly on the silver surface.

Adsorption of S–S Containing Proteins on a Colloidal Silver Surface Studied by Surface-Enhanced Raman Spectroscopy

We present a Raman and surface-enhanced Raman scattering (SERS) study of the following proteins containing S–S group(s): α-chymotrypsin (α-CHT), insulin, lysozyme, oxytocin (OXT), Streptomyces subtilisin inhibitor (SSI), and trypsin inhibitor (STI). The SERS study is performed in order to understand the adsorption mechanism of the above-mentioned proteins on a colloidal silver surface. The SERS spectra presented here show bands associated mainly with aromatic amino acid vibrations. In addition, two distinct vibrations of the –C–S–S–C– fragment are observed in the Raman and SERS spectra, i.e., v(SS) and v(CS). The enhancement of the v(SS) vibration in the SERS spectra yields evidence that the intact disulfide bridge(s) is (are) located near the silver surface. This finding is supported by the presence of the v(CS) mode(s). The presence of vs(COO−) and v(C–COO−) in the SERS spectra in the 1384–1399 cm−1 and 909–939 cm−1 regions, respectively, indicate that the negatively charged COO− groups (aspartic and glutamic acids) assist in the binding on the positively charged silver surface. The Raman amide I and III bands observed in the 1621–1633 and 1261–1289 cm−1 ranges, respectively, indicate that the α-helical conformation is favored for binding to the surface over the random coil or β-sheet conformations. In addition, the presence of the imino group of Trp and/or His indicates that these amino acid residues may also bind to the silver sol.

Part III: Surface-Enhanced Raman Scattering of Amino Acids and Their Homodipeptide Monolayers Deposited onto Colloidal Gold Surface

Surface-enhanced Raman scattering (SERS) spectra were measured for monolayers of various amino acids: L-methionine (Met), L-cysteine (Cys), L-glycine (Gly), L-leucine (Leu), L-phenylalanine (Phe), and L-proline (Pro) and their homodipeptides (Met-Met, Cys-Cys, Gly-Gly, Leu-Leu, Phe-Phe, and Pro-Pro) deposited onto a colloidal gold surface. Orientation of amino acids and their homodipeptides, as well as specific-competitive interactions of their functional groups with the gold surface, were predicted by detailed spectral analysis of the obtained SERS spectra. The analysis performed allowed us to propose a particular surface geometry for each amino acid and homodipeptide on the gold surface. In addition, we compared the structures of these molecules adsorbed on colloidal gold and silver surfaces.

Part II: Surface-Enhanced Raman Spectroscopy Investigation of Methionine Containing Heterodipeptides Adsorbed on Colloidal Silver

Surface-enhanced Raman scattering (SERS) spectra of methionine (Met) containing dipeptides: Met-X and X-Met, where X is: L-glycine (Gly), L-leucine (Leu), L-proline (Pro), and L-phenylalanine (Phe) are reported. Using pre-aggregated Ag colloid we obtained high-quality SERS spectra of these compounds spontaneously adsorbed on colloidal silver. Additionally, we measured Raman spectra (RS) of these heterodipeptides in a solid state as well as in acidic and basic solutions. The RS and SERS spectra of Met-X and X-Met presented in this work appear to be different. One of the most prominent and common features in the SERS spectra of all these dipeptides is a band in the 660–690 cm−1 range that is due to the C–S stretching, v(CS), vibration of Met. This suggests that all the abovementioned compounds adsorb on the silver surface through a thioether atom. On the other hand, the SERS spectra of X-Met show clearly that not only the S atom but also the carboxylate group interact with the colloid surface as manifested by the enhancement of bands in the 920–930 and 1380–1396 cm−1 regions. These bands are ascribed to the v(C–COO−) and vsym(COO−) vibrations, respectively. Additionally, a SERS spectrum of Phe-Met indicates that the interaction of the thioether atom, amine group, and aromatic side chain with the silver surface is favorable and may dictate the orientation and conformation of adsorbed peptide.

Potential-dependent studies on the interaction between phenylalanine-substituted bombesin fragments and roughened Ag, Au, and Cu electrode surfaces.

In this work, we report systematic surface-enhanced Raman spectroscopy (SERS) and generalized two-dimensional correlation analysis (G2DCA) studies of the structures of five specifically modified phenylalanine-substituted C-terminal bombesin 6-14 fragments (BN(6-14)). The fragments studied have all been tested as chemotherapeutic agents in cancer therapy, and they form amino acid sequences in bombesin: cyclo[d-Phe(6),His(7),Leu(14)]BN(6-14), [D-Phe(6),Leu-NHEt(13),des-Met(14)]BN(6-14), [D-Phe(6),Leu(13)-((R))-p-Cl-Phe(14)]BN(6-14), [D-Phe(6),beta-Ala(11),Phe(13),Nle(14)]BN(6-14), and [D-Tyr(6),beta-Ala(11),Phe(13),Nle(14)]BN(6-14). We adsorbed these fragments onto roughened Ag, Au, and Cu electrode surfaces, using a potential range from -1.200 to 0.400 V, at physiological pH. We compared the adsorption mechanism of each fragment on these substrates, as well any changes observed with varying electrode potential, to determine the relationship between adsorption strength and geometry of each of the peptides wherever it was possible. For example, we showed that none of these fragments directly interact with the Ag, Au, and Cu surfaces via residues of Phe (phenylalanine) and Trp(8) (L-tryptophane at position 8 of the BN amino acid sequence) or by an amide bond, due to a very small shift in wavenumber of their characteristic vibrations. Specific interactions were recognized from the broadening, wavenumber shift, and increase in intensity of the W18 Trp(8) mode near 759 cm(-1) and decrease in nu(12) vibration frequency of the Phe residue. In general, more intense SERS bands were observed due to the Phe ring, compared with the Trp(8) ring, which suggested a preferential adsorption of phenylalanine over tryptophane. For [D-Tyr(6),beta-Ala(11),Phe(13),Nle(14)]BN(6-14), the data also suggest some interaction of a D-Tyr(6) residue (D-tyrosine at position 6). Finally, only slight rearrangements of these moieties on the substrates are observed with changes in electrode potential.

Potential-dependent characterization of bombesin adsorbed states on roughened Ag, Au, and Cu electrode surfaces at physiological pH.

This paper reports the direct surface-enhanced Raman spectroscopic (SERS) and generalized two-dimensional correlation analysis observations of the different orientations of the neurotransmitter bombesin (BN) chemisorbed on electrochemically roughened Ag, Au, and Cu electrode surfaces at different applied electrode potentials and at physiological pH. The presence of the indole ring of Trp(8) and the amide bond between Gln(7) and Trp(8) of BN on these surfaces generates a specific SERS profile of BN adsorbed on the roughened Ag and Au electrodes that is affected by the electrode potential. Furthermore, for BN on Au, slight changes are observed in the band enhancement in comparison to what is observed for this neurotransmitter immobilized on Ag. In addition, there are larger changes in the spectra triggered by the substitution of Ag with Au electrodes and Ag with Cu electrodes than by substitution of Au with Cu electrodes.

Adsorbed States of Phosphonate Derivatives of N-Heterocyclic Aromatic Compounds, Imidazole, Thiazole, and Pyridine on Colloidal Silver: Comparison with a Silver Electrode

Here, we report a systematic surface-enhanced Raman spectroscopy (SERS) study of the structures of phosphonate derivatives of the N-heterocyclic aromatic compounds imidazole (ImMeP ([hydroxy(1H-imidazol-5-yl)methyl]phosphonic acid) and (ImMe)(2)P (bis[hydroxy-(1H-imidazol-4-yl)-methyl]phosphinic acid)), thiazole (BAThMeP (butylaminothiazol-2-yl-methyl)phosphonic acid) and BzAThMeP (benzylaminothiazol-2-yl-methyl)phosphonic acid)), and pyridine ((PyMe)(2)P (bis[(hydroxypyridin-3-yl-methyl)]phosphinic acid)) adsorbed on nanometer-sized colloidal particles. We compared these structures to those on a roughened silver electrode surface to determine the relationship between the adsorption strength and the geometry. For example, we showed that all of these biomolecules interact with the colloidal surface through aromatic rings. However, for BzAThMeP, a preferential interaction between the benzene ring and the colloidal silver surface is observed more so than that between the thiazole ring and this substrate. The PC(OH)C fragment does not take part in the adsorption process, and the phosphonate moiety of ImMeP and (ImMe)(2)P, being removed from the surface, only assists in this process.

Resonance raman studies of heme structural differences in subunits of deoxy hemoglobin.

Low frequency resonance Raman (RR) spectra are reported for deoxy hemoglobin (Hb), its isolated subunits, its analogue bearing methine-deuterated hemes in all four subunits (Hb-d(4)), and the hybrids bearing the deuterated heme in only one type of subunit, which are [alpha(d4)beta(h4)](2) and [alpha(h4)beta(d4)](2). Analyzed collectively, the spectra reveal subunit-specific modes that conclusively document subtle differences in structure for the heme prosthetic groups in the two types of subunits within the intact tetramer. Not surprisingly, the most significant spectral differences are observed in the gamma(7) mode that has a major contribution from out of plane bending of the methine carbons, a distortion that is believed to relieve strain in the high-spin heme prosthetic groups. The results provide convincing evidence for the utility of selectively labeled hemoglobin hybrids in unraveling the separate subunit contributions to the RR spectra of Hb and its various derivatives and for thereby detecting slight structural differences in the subunits.

The orientation of BN-related peptides adsorbed on SERS-active silver nanoparticles: comparison with a silver electrode surface.

We used surface-enhanced Raman scattering (SERS) to characterize the adsorption behavior of bombesin (BN) and five BN-related peptides, including phyllolitorin, [Leu(8)]phyllolitorin, neuromedin C (NMC), neuromedin B (NMB), and PG-L (Pseudophryne guntheri), in a silver colloidal solution. Our experiments show that the pyrrole coring of the Trp and aromatic ring of Phe of these peptides are preferentially adsorbed on silver nanoparticles. However, the geometry of the rings and the strength of the interactions with this surface vary among peptides. Additionally, these peptides are weakly coordinated to the colloidal silver surface through the CO fragment of a peptide bond, between Gln/Leu/His and Trp residues, and CNC and SC fragments. Also, using the recently reported SERS spectra of these peptides immobilized onto an electrochemically roughened silver electrode surface, we demonstrate substrate-induced changes in the adsorption behavior of these peptides. Comparative analysis indicates that the interactions between peptides and the enhancing surfaces depend strongly on the geometry of the Trp, CONH, and SC fragments of these biomolecules etched on the surfaces.

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.