Raman And Surface Enhanced Raman Spectroscopy Of Small Biological Molecules.

Abstract

Resonance Raman spectra are studied for bilirubin in chloroform, dimethyl sulfoxide, and aqueous solutions. From the known hydrogen bonding patterns in several free solutions, Raman markers for the presence or absence of internal hydrogen bonding are derived. From these measurements and time-resolved deuteration, the partial assignments of the spectra are proposed. The surface-enhanced Raman spectroscopy of bilirubin at a silver electrode is investigated in aqueous solution. Bilirubin is found to be adsorbed on the electrode as its dicarboxylate anion. Binding through a lactam carbonyl is shown. Only one pyrromethenone moiety per molecule contributes to the observed spectrum. SERS from the albumin complex is not observed, thus suggesting that bilirubin is completely enclosed by this protein. Resonance Raman spectra are investigated for bilirubin in 1:1 bilirubin/albumin complex and 1:100 bililrubin/cyclodextrin complexes, respectively. The hydrogen bond markers and bond assignments are used to interpret hydrogen bonding patterns in these complexes. The resonance Raman spectrum of the bilirubin/albumin complex demonstrates that the internal hydrogen bonds between the propionate groups and the pyrromethenone rings are ruptured. Propionate hydrogen bonding is to amino acid residues of the protein only. On the other hand, the bilirubin cyclodextrin complexes retain the usual internal hydrogen bonds. The surface enhanced Raman spectra of dopamine, norepinephrine, epinephrine, epinine, isoproterenol, 3-methoxytyramine, and catechol in pH 7.2 buffers on a silver electrode are reported. Catechol and the catecholamines are shown to be coordinated to silver through both oxygens. The methoxylated derivative is a monodentate complex. Intensities maximize near $-$0.9 V vs. S.C.E. The strongest bands in the spectra are phenolic carbon-oxygen stretches and the $\nu\sb{19b}$ modes around 1270 cm$\sp{-1}$ and 1480$\sp{-1}$ respectively. Differences in relative intensities are explained as differences in orientation of the catechol ring at the electrode surface. Ascorbate, acetylcholine, glutathione, L-Dopa, and catecholamine acetic acid metabolites are SERS-inactive under the measurement conditions. The dopamine detection limit using the intense $\nu\sb{19b}$ band is 3 $\times$ 10$\sp{-7}$ M with a 10 sec measurement time.