Assay of enzymes forming AMP plus PPi by the pyrophosphate determination based on the formation of 18-molybdopyrophosphate
Assay of enzymes forming AMP plus PPi by the pyrophosphate determination based on the formation of 18-molybdopyrophosphate
复制标题
DOI：
10.1016/j.ab.2011.10.031
复制
发表时间：
2012-02-01
影响因子：
2.9
通讯作者：
Hamano, Yoshimitsu
中科院分区：
文献类型：
--
作者：
Katano, Hajime;Tanaka, Rina;Hamano, Yoshimitsu
本文研究了18-β-焦磷酸阴离子的形成，建立了一种简单、快速测定ATP -> AMP + PPi（P_2O_（74-））酶促反应的方法。在含有MoO_（42）-阴离子的酸性乙腈-水溶液中加入P_2O_（74）-阴离子后，无色的Mo（VI）溶液立即变成黄色，生成了18-羟基焦磷酸根阴离子。P2 O 74-Mo（VI）混合物在例如450 nm处的吸光度与P2 O 74-阴离子的分析浓度成比例。虽然测试Mo（VI）溶液通过添加AMP保持无色，但其通过ATP逐渐变为黄色。不希望的显色归因于伴随着PO 43-从不稳定的ATP分子中解离而形成的黄色二磷酸盐物质。然而，显色变得慢得多时，乙二胺四乙酸被添加到测定混合物中，其中ATP可以形成动力学稳定的物种。因此，P2 O 74-阴离子可在含有ATP的酶反应混合物中通过电化学测定。通过添加抗坏血酸，黄色的P2 O 74-Mo（VI）混合物变成蓝色，由于还原的P2 O 74-Mo（VI）阴离子。因此，P2 O 74-阴离子可以通过蓝色比色检测。分光光度法和比色法可用于乙酰辅酶A合成酶的测定。(C)2011 Elsevier Inc. All rights reserved.
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. (C) 2011 Elsevier Inc. All rights reserved.