A NEW METHOD FOR THE INVESTIGATION OF THE KINETICS OF THE CAPTURE REACTION IN PHOSPHATASE CYTOCHEMISTRY II. THEORETICAL AND EXPERIMENTAL STUDY OF PHOSPHATE DIFFUSION FROM THIN POLYACRYLAMIDE FILMS

Abstract

Polyacrylamide films containing [32P] inorganic phosphate are proposed as models for the investigation of diffusion of phosphate from enzymatic sites. In connection with the use of such films for the study of metal salt capture reactions in phosphatase cytochemistry, the diffusion characteristics of the system were investigated both theoretically and experimentally. Diffusion in a theoretical model was simulated with aid of a computer program based on Fick's law. For films with a thickness above a certain value it proved possible to represent the progressive decrease in concentration by a hypothetical surface layer with zero concentration, the thickness being proportional to the square root of the product of the diffusion coefficient and time. The experimentally measured decrease in concentration showed similar characteristics. A slight deviation from the theoretical model indicated a retarding effect of an unstirred layer on the diffusion process. The apparent diffusion coefficient for inorganic phosphate with a pH of 5.0 at 25°C was estimated at 3.2 x 10–6 cm2 sec–1, but in the presence of 14% sucrose (w/v) this value was reduced to 2.3 x 10–6 cm2 sec–1. In films containing 6.7% w/v cross-linked bovine serum albumin Dapp was found to be 3.1 x 10–6 cm2 sec–1. This method offers a new approach to the accurate measurement of diffusion coefficients of small molecules in macromolecular matrices and is suitable for the study of diffusion problems in cytochemistry.