Koller E, Wolfbeis O S
Anal Biochem. 1984 Nov 15;143(1):146-51. doi: 10.1016/0003-2697(84)90569-4.
A direct and continuous kinetic method for the photometric and fluorometric determination of various acid phosphatases is described. It is based on new coumarin-derived phosphates, which after enzymatic hydrolysis undergo dissociation to form intensely colored and strongly fluorescent phenolate anions. The latter have absorption maxima ranging from 385 to 505 nm, and fluorescence maxima between 470 and 595 nm. The new substrates were compared with respect to their rate of enzymatic hydrolysis, optimum pH, and detection limits of acid phosphatase from potato and wheat germ. Detection limits of 0.001 unit/ml were found by photometry, and as low as 0.00006 unit/ml by fluorometry. The principal advantages of the new substrates over existing ones are longwave absorptions and emissions, large Stokes shifts, and the low pKa values of the corresponding phenols, thus allowing a direct and continuous assay of acid phosphatase even in weakly acidic solutions.
描述了一种用于光度法和荧光法直接连续测定各种酸性磷酸酶的动力学方法。该方法基于新的香豆素衍生磷酸盐，其经酶促水解后发生解离，形成颜色强烈且荧光强烈的酚盐阴离子。后者的吸收最大值在385至505nm之间，荧光最大值在470至595nm之间。比较了这些新底物在酶促水解速率、最佳pH值以及马铃薯和小麦胚芽酸性磷酸酶检测限方面的情况。通过光度法发现检测限为0.001单位/毫升，通过荧光法低至0.00006单位/毫升。新底物相对于现有底物的主要优点是长波吸收和发射、大斯托克斯位移以及相应酚类的低pKa值，因此即使在弱酸性溶液中也能直接连续测定酸性磷酸酶。
