Theoretical and experimental study of an immobilized bienzyme system. Computer calculations and electron microscopy visualization of local concentration profiles.

A cytochemical method was used in order to visualize the existence of local concentrations of metabolites within an immobilized bienzyme system. Glucose oxidase (EC 1.1.3.4) and peroxidase (EC 1.11.1.7) were immobilized together into an artificial proteic membrane obtained by a previously described co-cross-linking process using a bifunctional agent, glutaraldehyde. The cytochemical reagent 3,3'-diaminobenzidine was used as a hydrogen donor substrate and kinetic studies were performed with both free and immobilized forms of the enzymes; in the latter case, a new direct method was introduced which allowed kinetic studies on the visualization system itself. From experimental kinetic parameters, theoretical concentrations profiles were calculated by computer using numerical analysis methods. These results were discussed with special attention to the electron microscopic observations. Computer simulations were in good agreement with electron micrographs. No geometrical similarity was seen in our system between poly(3,3'-diaminobenzidine) precipitate distribution and peroxidase molecule homogeneous location.