Resonant Raman scattering from cytochrome c: Frequency dependence of the depolarization ratio

The depolarization ratio has been measured for several peaks in the resonant Raman spectrum of horse heart ferrocytochrome c (reduced) using six argon and krypton ion laser lines near resonance with the β absorption band. The depolarization ratio is observed to be a strong function of laser frequency so that previous mode symmetry assignments for heme proteins based on data at a single frequency are unreliable. For the anomalously polarized peaks (p₁ > 3/4), the depolarization ratio increases to a maximum when the laser frequency is midway between the a and βabsorption bands. These data are compared with a theoretical expression for the dispersion derived using perturbation theory to treat mixing of the D_4h basis states. Quantitative estimates of the relative contributions of the different symmetry representatives are obtained.