EFFECT OF WATER-MISCIBLE ORGANIC-SOLVENTS ON THE CATALYTIC ACTIVITY OF CYTOCHROME-C

The effect of five water-miscible organic solvents (tetrahydrofuran, N ,N-dimethylformamide, acetonitrile, 2-propanol, and methanol) on the o xidation of pinacyanol chloride (Quinaldine Blue) by horse heart cytoc hrome c was determined. Hydrogen peroxide was used as the oxidant, and a change in catalytic property of the dissolved protein was observed after a certain threshold concentration of the organic solvent had bee n reached. The maximum specific activity was correlated with the Dimro th-Reichardt parameter for the solvents, which is directly related to the free energy of the solvation process. The kinetic constants for th e oxidation of pinacyanol chloride were determined in systems containi ng different proportions of tetrahydrofuran. The best catalytic effici ency (k(cat)/K(M,app)) was obtained in a system containing 50% tetrahy drofuran in phosphate buffer. In a mixture containing 90% tetrahydrofu ran, cytochrome c showed 18% of its maximum activity. The inactivation of cytochrome c was mainly due to the presence of hydrogen peroxide, and a direct correlation was found between the inactivation constant a nd the concentration of hydrogen peroxide in the system. The chemical modifications and immobilization of cytochrome c were able to change i ts biocatalytic activity and stability in the organic solvent system. The kinetic constants and the inactivation of three other type c cytoc hromes, from Saccharomyces cerevisiae, Pseudomonas aeruginosa, and Des ulfovibrio vulgaris Hildenborough in a system containing 90% tetrahydr ofuran were compared with those of cytochrome c from horse heart. Cyto chrome C551 from P. aeruginosa showed the best stability against hydro gen peroxide and a higher catalytic efficiency than that of horse hear t cytochrome c.