ENZYME-CATALYZED CONVERSION OF PYROPHOSPHATE TO ORTHOPHOSPHATE: KINETIC CHARACTERIZATION BY FLOW INJECTION ANALYSIS AND P-31 NMR

Abstract

Flow injection method for the selective detection of orthophosphate in the presence of pyrophosphate (diphosphate) was designed to characterize the inorganic pyrophosphatase (EC3. 6. 1. 1)-catalyzed hydrolysis of pyrophosphate. Fast bluecoloration of molybdophosphate by a mixed reductant of Sn (II) and hydrazine sulfate was employed. The proposed method provided kinetic data that were enough to determine a catalytic rate constant and a Michaelis constant. P-31 NMR spectroscopy was also employed to confirm the conversion of pyrophosphate to orthophosphate.