Effect of Support Pore Size on Activity of Immobilized Sulfhydryl Oxidase

Both sulfhydryl oxidase and heat denatured milk proteins exist as highly associated, large protein complexes; therefore, the catalytic activity of immobilized forms of the enzyme should be dependent upon the pore size of the support matrix. A method was optimized for immobilization of the enzyme on succinamidopropyl-glass with water-soluble carbodiimide to activate carboxyl groups. With both heated skim milk and reduced glutathione substrates most of the activity occurred on the outer surface of the particles for enzyme immobilized on porous beads having mean pore diameters ranging from 500 A to 3000 A. Furthermore, Arrhenius plots did not indicate significant effects of pore diffusion. Also molecular sieve chromatographic experiments showed that enzyme and denatured whey proteins were excluded from the pore volume. Finally, equivalent or higher activity per unit volume of support could be obtained with nonporous materials such as glass beads.