Three widely used antibiotics from the rifamycin family, rifamycin SV sodium salt, rifampicin and rifaximin, have been characterized by resonance Raman (RR) and surface-enhanced resonance Raman spectroscopy (SERRS). SERRS spectra were recorded using aqueous silver colloidal dispersions prepared with two reducing agents, sodium borohydride and hydroxylamine hydrochloride, for a range of pH values to identify the SERRS-active substrate surface most suitable for each of the three antibiotics. Rifampicin was found to give intense SERRS signals only for the borohydride-reduced colloid and only at pH < 7.7, whereas the hydroxylamine HCl-reduced colloid was the best substrate for rifaximin, giving considerably more intense SERRS spectra than the borohydride colloid. SERRS spectra of rifaximin were observed only at pH < 7.0. It is proposed that the marked pH dependence of the SERRS enhancement results from a transition from an anionic to a neutral zwitterionic state. SERRS spectra of rifamycin SV were not observed for any experimental conditions. The antibiotics display remarkably contrasting SERRS behaviour, reflecting differences in the nature of the substituent groups on the chromophore ring. A vibrational assignment of the RR spectra and detailed comparison between the RR and SERRS data have given insight into the mechanism of adsorption of the antibiotics onto the Ag surface. Rifampicin and rifaximin adsorb adopting an approximately similar vertical orientation of the chromophore ring with respect to the surface; however, rifampicin adsorbs by direct chemical interaction with the Ag whereas rifaximin does not form a direct bond with the Ag surface.

We have characterized the ferric and ferrous forms of the heme-containing (1-56 residues) N-fragment of horse heart cytochrome c (cyt c) at different pH values and low ionic strength by UV-visible absorption and resonance Raman (RR) scattering. The results are compared with native cyt c in the same experimental conditions as this may provide a deeper insight into the cyt c unfolding-folding process. Folding of cyt c leads to a state having the heme iron coordinated to a histidine (His18) and a methionine (Met80) as axial ligands. At neutral pH the N-fragment (which lacks Met80) shows absorption and RR spectra that are consistent with the presence of a bis-His low spin heme, like several non-native forms of the parental protein. In particular, the optical spectra are identical to those of cyt c in the presence of a high concentration of denaturants; this renders the N-fragment a suitable model to study the heme pocket microenvironment of the misfolded (His-His) intermediate formed during folding of cyt c. Acid pH affects the ligation state in both cyt c and the N-fragment. Data obtained as a function of pH allow a correlation between the structural properties in the heme pocket of the N-fragment and those of non-native forms of cyt c. The results underline that the (57-104 residues) segment under native-like conditions imparts structural stability to the protein by impeding solvent access into the heme pocket.