title: Assay of enzymes forming AMP+PPi by the pyrophosphate determination based on the formation of 18-molybdopyrophosphate.
abstract: 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.
authors.affiliation: Fukui Prefectural University, Eiheiji-cho, Fukui 910-1195, Japan. hajime@fpu.ac.jp
authors.forename: Hajime
authors.initials: H
authors.lastname: Katano
authors.forename: Rina
authors.initials: R
authors.lastname: Tanaka
authors.forename: Chitose
authors.initials: C
authors.lastname: Maruyama
authors.forename: Yoshimitsu
authors.initials: Y
authors.lastname: Hamano
mesh_terms: D000106:Acetate-CoA Ligase / Q000032:analysis / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D003124:Colorimetry; D011756:Diphosphates / Q000378:metabolism*; D008982:Molybdenum / Q000378:metabolism; D013053:Spectrophotometry
chemical_list: D011756:Diphosphates; D000249:Adenosine Monophosphate; D008982:Molybdenum; D000255:Adenosine Triphosphate; D000106:Acetate-CoA Ligase