Continuous assay for acid phosphatase using phenyl phosphate.

A continuous spectrophotometric assay for the determination of the initial rate of an acid phosphatase-catalyzed reaction in an acidic environment, using phenyl phosphate as a substrate, is presented. The method is based on the continuous determination of phenol, a product of the enzymatic hydrolysis, by the kinetic measurement of its absorbance. The method allows for the direct estimation of acid phosphatase activity in an acidic solution. This is possible without interrupting the reaction by alkalization or precipitation required for commonly used end-point colorimetric detection procedures for phosphate or phenol, and without the use of any coupled assays. The method has been developed for acid phosphatase activity determination in an aqueous solution and in sodium bis(2-ethylhexyl)sulfosuccinate (AOT)-isooctane-water reverse micelles in a broad pH range (pH 3.8 to 8.8). The proposed procedure has been used for the determination of kinetic constants (K(m) and kcat) for human prostatic acid phosphatase in aqueous solutions, and in AOT-isooctane-water reverse micelles, at pH 3.8, 4.5, and 5.7.