Inorganic phosphate determination in the presence of a labile organic phosphate: assay for carbamyl phosphate phosphatase activity

Abstract

An assay was developed for carbamyl phosphate phosphatase (CAP phosphatase) activity. This enzyme activity is difficult to assay because of the chemical instability of carbamyl phosphate (CAP). We chose to measure CAP phosphatase activity by monitoring release of inorganic phosphate (Pi). This entailed preparing CAP with low endogeneous Pi, constructing enzyme incubation and deproteination conditions for minimal chemical hydrolysis of CAP and employing a Pi assay designed for use with labile organic phosphates. With Ehrlich ascites tumor cell homogenate, CAP phosphatase assay was linear with time and increments of protein. In developing this procedure, the most critical aspect was the definition of reliable conditions for measuring Pi. The Pi assay used is a modification of the method of Baginski, Foa, and Zak [Clin. Chim. Acta 15, 155-158 (1967)] which is insensitive to hydrolysis of organophosphates after the last reagent is added. Color development in the Pi assay is significantly inhibited by cold and by greater than 0.3 mmol of trichloroacetic acid or greater than 1.5 mumol of CAP. However, under conditions utilized for measuring CAP phosphatase activity there is no interference. Absorbance in the Pi assay is stable and linear from 5 to 300 nmol of Pi. The assay is also suitable for measurement of ATPase activity and Pi determinations in general.