Determination of Intrinsic Properties of Immobilized Enzymes 2. Kinetic Studies on Sepharose-Staphylococcal Nuclease in the Presence of Diffusional Limitations

Using a small substrate (thymidine 5'-(p-nitrophenyl phosphate) 3'-phosphate), the kinetics of staphylococcal nuclease insolubilized on CNBr-activated Sepharoses 4B and 6B are affected by internal diffusional limitations. Since we demonstrate that we are working under conditions in which external mass-transfer resistances do not influence the reaction rate, we propose a simple theoretical model that considers only the case of mixed enzymic reaction-internal diffusion kinetics. In the Eadie-Hofstee plots we find very good agreement between theory and experiment. The model accounts very well for the results obtained by changing support texture, reaction conditions, and/or enzyme concentration in the insoluble derivatives, variables that modify the diffusional restrictions of the system.