Surface enhanced resonance Raman scattering as a probe of the spin state of structurally related cytochromes P‐450 from rat liver

Abstract

Surface enhanced resonance Raman scattering (SERRS) was observed from structurally related drug‐induced rat liver cytochromes P‐450 adsorbed on a silver colloid. Careful control of pH and the sequence of addition of components to the sol is required to prevent protein denaturation at the surface due to conversion to P‐450's biologically inactive form P‐420 or haem loss. A low‐spin P‐450 (PB_3a), a mixed low‐and high‐spin P‐450 (PB_3b) and a predominantly high‐spin P‐450 (MC_1a) were investigated. Spectra recorded in the 1300–1700 cm⁻¹ frequency region, containing the oxidation state marker ν₄ at 1375 cm⁻¹ (Fe³⁺) and spin state markers ν₁₀ (1625 cm⁻¹, high‐spin; 1633 cm⁻¹, low‐spin) and ν₁₉ (1575 cm⁻¹, high‐spin; 1585 cm⁻¹, low‐spin) were used to differentiate between the spin states of the various forms of cytochrome P‐450. As well as the established spin state marker bands, the intensity of a band at 1400 cm⁻¹ appeared to depend on the high‐spin content. Thus, with this method SERRS from silver colloids can be used to determine spin states of related cytochromes P‐450 in dilute solution (10⁻⁸ M) and may be of value in studies of protein‐substrate interactions.