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(P2O74-). By the addition of P2O74- anion to an acidic acetonitrile–water solution containing MoO42- anion, the colorless Mo(VI) solution immediately became yellow due to the formation of 18-molybdopyrophosphate anion. The absorbance of the P2O74-–Mo(VI) mixture at, for example, 450 nm was proportional to the analytical concentration of P2O74- 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 PO43- 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, P2O74- anion can be determined spectrophotometrically in the enzymatic reaction mixture containing ATP. By the addition of ascorbic acid, the yellow P2O74-–Mo(VI) mixture turned to blue due to the reduction of the molybdopyrophosphate anion. Thus, P2O74- anion can be detected colorimetrically by the blueness. The spectrophotometric and colorimetric methods could be applied advantageously to the assay of acetyl-CoA synthetase.