ELECTRON MICROSCOPIC AND KINETIC STUDIES DEALING WITH AN ARTIFICIAL ENZYME MEMBRANE APPLICATION TO A CYTOCHEMICAL MODEL WITH THE HORSERADISH PEROXIDASE-3,3'-DIAMINOBENZIDINE SYSTEM

Abstract

A new colorimetric method for measuring horseradish peroxidase (HRP) activity, in solution, with 3,3'-diaminobenzidine (DAB) as the hydrogen donor is presented. The same method was also used to test HRP activity when immobilized in an artificial proteic membrane. The reaction was shown to be of first order in both cases. Direct spectrophotometric measurements dealing with HRP membrane show that the total amount of insoluble polymer produced does not increase linearly with the quantity of enzyme introduced in the membrane. Moreover, a study of the DAB permeability through membranes with or without peroxidase activity was performed to allow the study of diffusion limitations. Membranes have also been studied by electron microscopy. We have especially visualized the existence of product concentration profiles inside the membrane. The results show that in the membrane there is no geometrical similarity between the distribution profiles of the enzyme and of the insoluble product.