Universidad de Buenos Aires - CONICET, Laboratorio de Biofísica Molecular, Instituto de Química y Fisicoquímica Biológicas, Buenos Aires, Argentina.
Universidad de Buenos Aires - CONICET, Laboratorio de Biofísica Molecular, Instituto de Química y Fisicoquímica Biológicas, Buenos Aires, Argentina.
Anal Biochem. 2020 May 15;597:113681. doi: 10.1016/j.ab.2020.113681. Epub 2020 Mar 9.
The analytical determination of phosphate constitutes a fundamental step for the evaluation of catalytic activities of many enzymes involved in the hydrolysis of phosphate-containing biomolecules. The most sensitive colorimetric methods to quantify phosphate are based on measuring the spectral changes produced by the adsorption of malachite green to 12-molybdophosphoric acid. Malachite green methods are time-consuming because they require the preparation of color reagent on the day of use, due to its low stability. In this work we propose a modification of the malachite green method that overcomes these problems and only requires a one-step, ready-to-use stock solution including perchloric acid and Pluronic F68. The improved reaction mixture allowed the quantification of phosphate with a limit of detection of 0.22 μM, a dynamical range up to 80 μM depending on the optical path-length, and a molar absorption coefficient at 640 nm of 94000 Mcm. Color development reaches a steady level within 30 min and remains constant for at least 2 h. The high stability of the color reagent allows long-term storage for at least 1 year. The optimized procedure is especially useful to measure phosphate-containing biomolecule levels and enzyme activities when low values are critical.
磷酸盐的分析测定是评估许多参与含磷生物分子水解的酶的催化活性的基本步骤。最灵敏的比色法测定磷酸盐的方法基于测量孔雀石绿吸附到 12-钼磷酸后产生的光谱变化。孔雀石绿法耗时较长，因为由于其稳定性低，需要在使用当天制备显色剂。在这项工作中，我们提出了一种孔雀石绿法的改进方法，该方法克服了这些问题，只需一步即可使用，包括高氯酸和 Pluronic F68 的即用型储备溶液。改进后的反应混合物允许定量检测 0.22 μM 的磷酸盐，检测范围可达 80 μM，取决于光程，在 640nm 处的摩尔吸光系数为 94000 Mcm。显色反应在 30 分钟内达到稳定水平，至少 2 小时内保持稳定。显色剂的高稳定性允许至少 1 年的长期储存。当低值至关重要时，优化后的程序特别适用于测量含磷生物分子水平和酶活性。
