Photometric microtiter assay of inorganic phosphate in the presence of acid-labile organic phosphates.

A simple and rapid colorimetric microassay for inorganic phosphate in the mild acid pH range has enabled us to perform extensive enzyme kinetic studies even in the presence of high concentrations of acid-labile substrates. The assay is performed in a 96-well microtiter plate using 30-microliters samples containing between 0.5 and 100 nmol Pi. Compared to existing microassays, color formation is linear over a much wider range of phosphate concentrations and reaction conditions. Using a computer-assisted microtiter plate reader, the data can be directly transferred to kinetic analysis software. The above assay was used to determine simultaneously up to 12 rates of the Escherichia coli maltodextrin phosphorylase-catalyzed reaction in the direction of glycogen synthesis under conditions which include screening of substrates and inhibitors at variable substrate concentrations and pH on a single 96-well microtiter plate.