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THEORETICAL AND EXPERIMENTAL ASPECTS OF ENZYME DETERMINATION IN A CYTOCHEMICAL MODEL SYSTEM OF POLYACRYLAMIDE FILMS CONTAINING ALKALINE PHOSPHATASE

P. VAN DUIJN 1, E. PASCOE 1, and M. VAN DER PLOEG 1

1 Laboratory for Pathology, Biochemical Section, University of Leiden, The Netherlands

A theoretical treatment of the influence of slow substrate diffusion on the kinetics of an enzyme reaction in a cytochemical system is given. Quantitative aspects of the effect of several parameters on the diffusion of substrate were studied experimentally on a model consisting of polyacrylamide films into which alkaline phosphatase was incorporated. Spectra and staining intensity of the films were measured in a special film colorimeter. Enzyme activity in the films was determined cytochemically (with the azo dye coupling method) and biochemically. An effectiveness factor for which diffusion is not rate-limiting proved to be attainable with both methods.

Submitted on June 12, 1967


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