THE PROTON ACTIVITY AT CRYOGENIC TEMPERATURES - A POSSIBLE INFLUENCE ON THE SPIN-STATE OF THE HEME IRON OF CYTOCHROME P-450(CAM) IN SUPERCOOLED BUFFERED SOLUTIONS

The electronic absorption spectra for camphor-bound cytochrome P-450(c am) have been analysed in the temperature range between 78 K and 298 K . The well-known high-spin/low-spin equilibrium has been detected betw een 298 K and 220 K. Depending on the cooling rate, below 220 K a new species was found in the absorption spectra. In contrast, the electron ic absorption spectra for camphor-free cytochrome P-450(cam) between 7 8 K and 295 K show no significant spectral changes. The conversion bet ween the spin states of camphor-bound cytochrome P-450(cam) and the ap pearance of the new species do not correspond to the temperature-induc ed change in the p alpha(H) value of the aqueous glycerol mixture cont aining phosphate or cacodylate buffer (p alpha(H) 7.0). For this study a spectroscopic procedure for the determination of the temperature de pendence of the pa, value of the solvent for the range 78-295 K is pre sented using dyes as pH-indicators. It is shown that the state of the acid-base equilibrium frozen in is strongly dependent on the cooling r ate of the mixture.