Surface enhanced Raman spectroscopic evidence that adsorption on silver particles can denature heme proteins

Abstract

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