An improved malachite green assay of phosphate: Mechanism and application
An improved malachite green assay of phosphate: Mechanism and application
复制标题
改进的孔雀石绿测定磷酸盐：机制和应用
DOI：
10.1016/j.ab.2010.10.025
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发表时间：
2011-02-01
影响因子：
2.9
通讯作者：
Liao, Fei
中科院分区：
文献类型：
--
作者：
Feng, Juan;Chen, Yuan;Liao, Fei
磷酸盐的经典孔雀石绿色（MLG）测定法（在酸化磷酸盐反应溶液后加入MLG）可耐受罂粟碱、西地那非和一些类似疏水胺的干扰。共振瑞利散射信号，由聚（乙烯醇）的干扰的缓解，和沉淀的一些黄色的配合物支持的疏水胺的干扰和磷钼酸盐的配合物的不可逆聚集减少的磷钼酸盐的量可访问MLG和造成的干扰。通过在磷酸盐的酸化反应溶液中加入MLG，在磷钼酸盐和这种疏水胺的复合物有效聚集之前优先形成磷钼酸盐和MLG的复合物，从而开发了具有对常见疏水胺抗性的磷酸盐的改进的MLG测定。采用改进的MLG法测定磷酸盐，并利用一种磷酸酶从AMP中释放磷酸盐，成功地测定了罂粟碱对重组人环核苷酸磷酸二酯酶（PDE）同工酶4和兔脑PDE同工酶混合物的半抑制浓度。因此，改进的MLG测定磷酸盐是一个有利的和通用的技术，用于开发光谱方法来表征和筛选酶的抑制剂，释放磷酸盐在其行动。(C)2010年爱思唯尔公司All rights reserved.
The classical malachite green (MLG) assay of phosphate, which added MLG after molybdate to the acidified reaction solutions of phosphate, tolerated interference from papaverine, sildenafil, and some similar hydrophobic amines. Resonance Rayleigh scattering signals, the alleviation of interference by poly(vinyl alcohol), and the precipitation of some yellow complexes supported that the irreversible aggregation of the complexes of a hydrophobic amine of interference and phosphomolybdate reduced the amounts of phosphomolybdate accessible to MLG and caused the interference. By adding MLG before molybdate to the acidified reaction solutions of phosphate, the complexes of phosphomolybdate and MLG were preferentially formed before the complexes of phosphomolybdate and such a hydrophobic amine effectively aggregated; thereby, an improved MLG assay of phosphate with the resistance to common hydrophobic amines was developed. Using the improved MLG assay of phosphate and a phosphatase to release phosphate from AMP, a spectrometric method successfully estimated the half-inhibition concentrations of papaverine on the recombinant human cyclic nucleotide phosphodiesterase (PDE) isozyme 4 and the mixture of PDE isozymes from rabbit brain. Therefore, the improved MLG assay of phosphate was a favorable and universal technique for developing spectrometric methods for characterizing and screening inhibitors of enzymes that release phosphate during their actions. (C) 2010 Elsevier Inc. All rights reserved.