Oxidation of ABTS by silicate-immobilized cytochrome c in nonaqueous solutions.
Cytochrome 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 external 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) occurred in methanol, ethanol, and formamide, with slight decreases occurring in 1-methoxy-2-propanol. The observed increases were primarily a result of substantial increases in k(cat), while the results in 1-methoxy-2-propanol can be ascribed to increases in K(M). Resonance Raman spectroscopy indicated that the structure of the heme environment of the adsorbed protein was essentially unchanged, in aqueous buffer and in the nonaqueous 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.
Deere
Joseph
J
Materials and Surface Science Institute and Department of Chemical and Environmental Sciences, University of Limerick, Limerick, Ireland.
Magner
Edmond
E
Wall
J Gerard
JG
Hodnett
B Kieran
BK
D000327:Adsorption; D052160:Benzothiazoles; D045304:Cytochromes c / Q000737:chemistry*; D004789:Enzyme Activation; D004800:Enzymes, Immobilized / Q000737:chemistry; D007700:Kinetics; D008567:Membranes, Artificial; D008968:Molecular Conformation; D010084:Oxidation-Reduction; D016062:Porosity; D017640:Silicates / Q000737:chemistry*; D012996:Solutions; D012997:Solvents / Q000737:chemistry*; D013451:Sulfonic Acids / Q000737:chemistry*; D014867:Water / Q000737:chemistry
D052160:Benzothiazoles; D004800:Enzymes, Immobilized; D008567:Membranes, Artificial; D017640:Silicates; D012996:Solutions; D012997:Solvents; D013451:Sulfonic Acids; D014867:Water; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; D045304:Cytochromes c