Salvi A, Mayer J M, Carrupt P A, Testa B
Institut de Chimie Thérapeutique, Université de Lausanne, Switzerland.
J Pharm Biomed Anal. 1996 Nov;15(2):149-55. doi: 10.1016/0731-7085(96)01837-7.
This paper describes a new spectrofluorimetric assay for continuously monitoring the enzymatic hydrolysis of medicinal esters. The procedure is based on the stoichiometric quantity of protons generated by the hydrolysis of the substrate, which produces changes in the fluorescence of a pH-sensitive dye. The pH indicator, 2', 7'-bis(car-boxyethyl)-5(6)-carboxyfluorescein, was selected due to its favourable pKa for studies under physiological conditions. Moreover, the presence of a domain in the spectra (< 442 nm) where fluorescence intensities are independent of pH allows measurements of wavelength ratios that cancel artifacts and lower sample-to-sample variability. The indicator did not affect the catalytic activity of purified hog liver carboxylesterase or human serum albumin. This assay is easy to perform and appears to be especially useful for studying enzymatic reactions with half-lives of the order of minutes or hours.
本文描述了一种用于连续监测药用酯酶促水解的新型荧光分光光度法。该方法基于底物水解产生的化学计量的质子，这会导致对pH敏感的染料荧光发生变化。选择pH指示剂2',7'-双（羧乙基）-5（6）-羧基荧光素是因为其在生理条件下研究的有利pKa。此外，光谱中存在一个荧光强度与pH无关的区域（<442nm），这使得可以测量消除伪影并降低样品间变异性的波长比。该指示剂不影响纯化的猪肝羧酸酯酶或人血清白蛋白的催化活性。该测定易于进行，对于研究半衰期在几分钟或几小时量级的酶促反应似乎特别有用。
