Kinetic microphotometric activity determination in enzyme containing gels and model studies with tissue sections

Abstract

The dependence of microphotometrically recorded reaction rate on local enzyme concentration was studied as a basic prerequisite of comparative microphotometric enzyme activity determinations at initial rate conditions in tissue sections. Polyacrylamide gels containing defined concentrations of glucose-6-phosphate dehydrogenase served as a model. Optimal conditions of preparing enzyme containing gels are reported. Measurements in which either thickness of gel sections or enzyme concentration was varied proved the linear relationship between local enzyme concentration and microphotometrically recorded reaction rate. Sections of enzyme containing gels as well as cross-sections of rat muscles were used as models for studying possible influences of heterogeneous chromophore distribution (distributional error). No such influences could be detected during the initial phase of the staining reaction which suggests that distributional error is of no significance for kinetic microphotometric enzyme activity determination at initial rate conditions.