A malachite green colorimetric assay for protein phosphatase activity

A simple and sensitive colorimetric assay for protein phosphatase activity based on the determination of released Pi by an improved malachite green procedure (A. A. Baykov, O. A. Evtushenko, and S.M. Avaeva, 1988, Anal. Biochem. 171, 266-270) is described. Proteins must be removed or stabilized prior to Pi determination with 0.25 N sulfuric acid or 3% (w/w) perchloric acid. Alternatively, to avoid possible acid hydrolysis of phosphate groups from organic compounds during deproteinization, the protein present in the phosphatase assay mixture can be stabilized with sodium dodecyl sulfate. In this case, the excess detergent is subsequently removed by precipitation with KCl because it colors with the malachite green reagent. The above procedure was applied to the determination of phosphorylase phosphatase activity in bovine brain extracts and the results are comparable to those obtained with the radioisotopic phosphatase assay.