Surface Enhanced Raman Spectroscopy evidence that absorption on silver particles can denaturate heme proteins.

Raman spectra are reported for micromolar solutions of the heme proteins hemoglobin (Hb), cytochrome b5 (cyt b), and cytochrome c (cyt c), absorbed on aqueous silver sols, using 413.1-nm excitation, in resonance with the heme Soret band. The surface enhanced Raman scattering (SERS) spectra of HbOz and of cyt b show distinct changes relative to the solution resonance Raman spectra of these molecules. These changes clearly indicate formation of surface bound hemin pox0 dimers, implying that the heme prosthetic groups have been extracted from their binding pockets in at least some of the protein molecules. The extent of dimer formation is preparation-dependent and may depend on silver particle aggregation, as well as the surface potential, which are difficult to control. Dimer formation is less pronounced for cytochrome c but is still readily observed; it is suggested that Ag' ions at the surface catalyze the cleavage of the heme-protein thioether bonds. The reduced forms of these proteins showed SERS spectra which were similar to the solution RR spectra, although some conversion to Fe"' was generally observed; no w-oxo dimer formation was detected. These results indicate that heme extraction is facilitated under oxidizing conditions, perhaps via increased surface charge on the Ag surface. The heme environment is unperturbed for reduced cyt c, as judged by the rich low-frequency SERS spectrum. For deoxy hemoglobin, however, the Fe-imidazole stretching band appears to shift from 215 to 200 cm-' in the SERS spectrum, suggesting some perturbation of the hemeprotein linkage.