Assay of enzymes forming AMP+PPi by the pyrophosphate determination based on the formation of 18-molybdopyrophosphate.

The formation of 18-molybdopyrophosphate anion has been studied to develop a simple and rapid assay of the enzymatic reaction involving ATP→AMP+PPi(P(2)O(7)(4-)). By the addition of P(2)O(7)(4-) anion to an acidic acetonitrile-water solution containing MoO(4)(2-) anion, the colorless Mo(VI) solution immediately became yellow due to the formation of 18-molybdopyrophosphate anion. The absorbance of the P(2)O(7)(4-)-Mo(VI) mixture at, for example, 450nm was proportional to the analytical concentration of P(2)O(7)(4-) anion. Although the test Mo(VI) solution remained colorless by the addition of AMP, it gradually turned to yellow by ATP. The undesired color development is attributed to the formation of a yellow molybdophosphate species accompanied by the dissociation of PO(4)(3-) from the unstable ATP molecule. However, the color development became much slower when ethylenediaminetetraacetic acid was added into an assay mixture, where ATP may form a kinetically stable species. Thus, P(2)O(7)(4-) anion can be determined spectrophotometrically in the enzymatic reaction mixture containing ATP. By the addition of ascorbic acid, the yellow P(2)O(7)(4-)-Mo(VI) mixture turned to blue due to the reduction of the molybdopyrophosphate anion. Thus, P(2)O(7)(4-) anion can be detected colorimetrically by the blueness. The spectrophotometric and colorimetric methods could be applied advantageously to the assay of acetyl-CoA synthetase.