Oxidation of ABTS by Silicate-Immobilized Cytochrome c in Nonaqueous Solutions Joseph Deere, Edmond Magner,* J. Gerard Wall, and B. Kieran Hodnett Materials and Surface Science Institute and Department of Chemical and Environmental Sciences, University of Limerick, Limerick, Ireland 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. Introduction The use of silicates to immobilize proteins is well- established (1-3). Weetall (4) has reviewed the use of controlled pore glass (CPG) to immobilize proteins. These studies involved the use of CPG of pore sizes ranging from 300 to 2000 Å, with immobilization occurring via covalent linkage of the protein or enzyme to the glass. A wide range of proteins have been immobilized onto CPG. In general, it is necessary that the pore size of the glass be significantly larger than the biological molecule of interest. The amount of protein that can be immobilized depends on the pore size and surface area of the glass, with maximal activity occurring with material possessing both an optimal pore size and surface area (5). The major disadvantages in using CPG materials are their cost and more importantly their surface areas, which rapidly decrease with increasing pore size (5, 6). The use of sol-gel-derived silica glasses to encapsulate proteins has been well-documented. The synthesis of such glasses involves the hydrolysis of siloxanes, which upon polymerization and condensation yield a solid (7). The biological molecule of interest is placed in the reaction mixture and encapsulated within the solid as it is formed. A wide range of redox proteins, including cytochrome c (cyt c), hemoglobin, horseradish peroxidase, glucose oxi- dase, and nitrate reductase, have been encapsulated within sol-gels (7). The majority of these studies have focused on the development of sensors, utilizing the encapsulated biological component as the recognition element. The focus on the development of sensors is a consequence of the slow rates of diffusion of substrates through the glass to the binding sites of the protein or enzyme. Since changes in the concentration of the bio- recognition element will not adversely affect the response, diffusion-limited responses are highly advantageous in such sensors. However, such a response limits the use of these materials in biocatalysis, where slow substrate diffusion may not be desirable. A recent report has described the use of sol-gel-encapsulated horseradish peroxidase as a biocatalyst; however, the rate of reaction was still limited by mass transport (9). A range of mesoporous silicates (MPS) have been described since the first material was reported by Beck et al. (9). These materials are formed via surfactants such as cetyl-trimethylammonium bromide in solution, which act as structure directing agents. Addition of a silane results in silica polymerization around the surfactant micelle structures, forming a gel. This gel is set ther- mally, condensing the silane to form a stable material. On subsequent removal of the surfactant, a mesoporous structure remains. MPS exhibit highly ordered pore structures and very tight pore size distributions, as exemplified by the first type of these materials reported, MCM-41 (9). As a result of their mesopore structures, MPS possess large surface areas of the order of 1000 m 2 g -1 . Their silicate inorganic framework allows for a chemically and mechanically stable material (10), which is also resistant to microbial attack. In addition, it is possible to chemically modify MPS with various func- tional groups enabling electrostatic attraction or repul- sion between MPS and the protein(s) of interest to be maximized (11, 12). Unlike sol-gel immobilization, pro- tein encapsulation in MPS occurs after synthesis of the support. MPS hold promise for use as supports to immobilize enzymes (11-14) and may find applications in bio- * To whom correspondence should be addressed. Tel: +353-61- 202629. Fax: +353-61-202568. E-mail: edmond.magner@ul.ie. 1238 Biotechnol. Prog. 2003, 19, 1238-1243 10.1021/bp0340537 CCC: $25.00 © 2003 American Chemical Society and American Institute of Chemical Engineers Published on Web 05/22/2003