Catalytic and spectroscopic properties of cytochrome-c, horseradish peroxidase, and ascorbate oxidase embedded in a sol-gel silica matrix as a function of gelation time

In this study, we investigated the optical features of the redox metal-depe ndent proteins cytochrome-c, horseradish peroxidase (HRP), and ascorbate ox idase embedded in a sol-gel-processed silica matrix as a function of gelati on time. Circular dichroism, absorbance, and fluorescence spectroscopies re vealed that the sol-gel process affects the complex structure of the dimeri c ascorbate oxidase (although the prosthetic coppers still remain bound to the enzyme) but not that of monomeric cytochrome-e and HRP. Any modificatio ns in ascorbate oxidase occurred in the initial gelation phase; the drying process induced no further alterations and the enzyme remained stable for m onths. Unfolding-refolding experiments on cytochrome-e revealed severely re stricted motility in the protein moiety in the xerogel, the concentrated ma trix that forms after drying. The diffusion time of the solvent within the matrix, which regulated the enzyme-substrate reaction rate, depended on the thickness of the monolith, not on the dryness of the specimen.