A microcolorimetric assay of inorganic pyrophosphatase

A procedure is described for the assay of inorganic pyrophosphatase in tissues by a microcolorimetric procedure, taking advantage of the marked color intensification of phosphomolybdate by malachite green. Conditions are described for optimum enzyme activity, color stability, and sensitivity. With 1-cm cuvettes the AM660 is 100,000, allowing accurate measurement of Pi in the 1-nmol range. Reaction is conducted at 25 degrees C for 10 min in 0.5 ml of a 50 mM histidine buffer, pH 7.2, containing 0.2 mM inorganic pyrophosphate and 4 mM Mg2+, terminated by addition of 0.05 ml 2.4 M HClO4, cooled in ice, and 0.45 ml of color reagent is added. After standing 10 min at 0 degrees C, the contents are transferred to 1-cm cuvettes and the absorbance is read at 660 nm. Blanks are low, nonenzymatic hydrolysis of PPi is negligible, and color is stable without addition of detergents. The high sensitivity makes this procedure well-adapted to measurement of optimal activities in crude tissue preparations.