A MODIFIED ASSAY PROCEDURE FOR CARBOXYLIC ESTER HYDROLASES

1-Naphthol (α-naphthol) released by enzymatic hydrolysis from 1-naphthylacetate and other carboxylic esters can be determined by a condensation reaction with 4-amino-antipyrine and K3Fe(CN)6 in alkaline incubation mixtures. The condensation product has a red color which is soluble in ethyl acetate and other organic solvents. When 4-amino-antipyrine and K3Fe(CN)6 are added at the end of the incubation of the enzyme with the substrate, color formation is instantaneous but enzymatic hydrolysis continues and the color deepens. Though K3Fe(CN)6 was found to have an inhibitory effect on esteratic activity of rat brain microsomes it was insufficient to permit accurate calculation of 1-naphthol release per unit time. 4-Amino-antipyrine in low concentration does not impair enzyme activity. Extraction into ethyl acetate is recommended because the organic solvent effectively terminates enzyme activity by removing the substrate from the aqueous incubation mixture. The extraction furthermore provides clear colored solutions where the incubation mixture is turbid due to contained subcellular particles, tissue homogenates or insoluble lipids. The sensitivity of the method compares well with azo-coupling procedures for the quantitative determination of released 1-naphthol. © 1969.