Oxidation of ABTS by Silicate-Immobilized Cytochrome c in Nonaqueous Solutions

摘要

Cytopchrome c can be readily adsorbed onto mesoporous silicates at high loadings of up to 10 mmol g~(-1) of silicate. The adsorbed protein retains its peroxidative activity, with no diffusional limitations being observed. The protein can be adsorbed onto the extrenal surface of the silicate or, provided that the pore diameter is sufficiently large, into the channels. In aqueous buffer, the catalytic activity of the adsorbed protein (for the oxidation of ABTS) decreased with increasing temperature, with the decrease being less marked for cytochrome c held within the silicate channels. Similar results were obtained in 95% methanol. Analysis of kinetic data showed that significant increases in k_cat/K_M occured in methanol, ethanol, and formamidae,with slight decreases occurring in 1-methoxy-2-propanol. The observed increases were primarily a result of substantial increasing in k_cat, while the results in 1-methoxy-2-propanol can be ascribed to increase in K_M. Resonance Raman spectroscopy indicated that the structure of the heme environment of the adsorbed protein was essentially unchanged, in aqeuous buffer and in the nonaqeous solvents, methanol, 1-methoxy-2-propanol, and ethanol. In addition, Raman spectra of the lyophilized protein indicated that there were no apparent changes in the heme structure.