Randall E. Holt

Surface-Enhanced Resonance Raman Scattering from Cytochromes Cand P-450 on Bare and Phospholipid-Coated Silver Substrates

Surface-enhanced resonance Raman scattering (SERRS) studies of cytochrome c (cyt c) have demonstrated that the native integrity of this heme-containing protein is preserved in the presence of citrate-reduced sols. Cyt c SERRS spectra exhibit similar spin and oxidation markers as the resonance Raman scattering (RRS) signals displayed by the native protein solution. Previous SERRS investigations of cyt c showed that the protein denatures at the substrate surface. This hemoprotein denaturation was attributed to an electrostatic interaction between the porphyrin and the SERRS-active substrate. The results presented in this study suggest that the citrate ions prevent the biomolecule from directly interacting with the silver-sol surface. The native state of cyt c is also retained with the addition of phosphatidylcholine and/or phosphate buffer. Although the SERRS bandshifts correlate with the RRS spectra, the relative intensities of specific cyt c frequencies change in the presence of adsorbed citrate, phosphate buffer, and phosphatidylcholine. These compounds may influence the orientation of the protein at the substrate surface, which would account for the spectral intensity differences. In order to study the orientation effects further, additional studies of cyt c adsorbed onto silver electrodes and thin silver films were conducted. A comparison of the SERRS relative band intensities and frequency bandshifts shows that the spectra of the protein adsorbed onto phospholipid-coated substrates closely correspond to the RRS cyt c solution spectra. In contrast to the behavior exhibited by cyt c, cytochrome P-450 (cyt P-450) adsorbed onto a phosphatidylcholine silver-sol substrate leads to a low to high spin-state conversion. The shifts in the spin-state markers are ascribed to a strong interaction of the cyt P-450 with the phospholipid coating.

Low Temperature Surface-Enhanced Resonance Raman Scattering (SERRS) From Cytochromes

Surface-enhanced resonance Raman scattering (SERRS) studies of cytochrome c (cyt c) and cytochrome P450 (cyt P450) as a function of laser irradiation time have demonstrated that the proteins are extremely sensitive to photodegradation. The results suggest that previous SERRS reports of hemoprotein denaturation on Ag surfaces may reflect photosensitivity rather than an effect of the protein-surface interaction. Photodamage was eliminated by submersion of the electrode into liquid nitrogen. This procedure resulted in stable SERRS spectra, even with prolonged irradiation. The use of a diode array detector also substantially reduces the laser exposure period ( < 1 minute) required to observe SERRS spectra of the protein. The application of low temperature SERRS spectroscopy to the study of substrate binding in P450b provided evidence for spin state conversion in the presence of substrate.

Low temperature surface-enhanced resonance raman scattering (SERRS) from cytochromes

Surface-enhanced resonance Raman scattering (SERRS) studies of cytochrome c (cyt c) and cytochrome P450 (cyt P450) as a function of laser irradiation time have demonstrated that the proteins are extremely sensitive to photodegradation. The results suggest that previous SERRS reports of hemoprotein denaturation on Ag surfaces may reflect photosensitivity rather than an effect of the protein-surface interaction. Photodamage was eliminated by submersion of the electrode into liquid nitrogen. This procedure resulted in stable SERRS spectra, even with prolonged irradiation. The use of a diode array detector also substantially reduces the laser exposure period ( < 1 minute) required to observe SERRS spectra of the protein. The application of low temperature SERRS spectroscopy to the study of substrate binding in P450b provided evidence for spin state conversion in the presence of substrate.

Low Temperature surface-enhanced resonance Raman scattering (SERRS) from cytochromes

Surface-enhanced resonance Raman scattering (SERRS) studies of cytochrome c (cyt c) and cytochrome P450 (cyt P450) as a function of laser irradiation time have demonstrated that the proteins are extremely sensitive to photodegradation. The results suggest that previous SERRS reports of hemoprotein denaturation on Ag surfaces may reflect photosensitivity rather than an effect of the protein-surface interaction. Photodamage was eliminated by submersion of the electrode into liquid nitrogen. This procedure resulted in stable SERRS spectra, even with prolonged irradiation. The use of a diode array detector also substantially reduces the laser exposure period ( < 1 minute) required to observe SERRS spectra of the protein. The application of low temperature SERRS spectroscopy to the study of substrate binding in P450b provided evidence for spin state conversion in the presence of substrate.