Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
Anal Biochem. 2011 Nov 15;418(2):247-52. doi: 10.1016/j.ab.2011.07.021. Epub 2011 Jul 24.
Alkaline phosphatase serves both as a model enzyme for studies on the mechanism and kinetics of phosphomonoesterases and as a reporter in enzyme-linked immunosorbent assays (ELISAs) and other biochemical methods. The tight binding of the enzyme to its inorganic phosphate product leads to strong inhibition of catalysis and confounds measurements of alkaline phosphatase activity. We have developed an alkaline phosphatase substrate in which the fluorescence of rhodamine is triggered on P-O bond cleavage in a process mediated by a "trimethyl lock." Although this substrate requires a nonenzymatic second step to manifest fluorescence, we demonstrated that the enzymatic first step limits the rate of fluorogenesis. The substrate enables the catalytic activity of alkaline phosphatase to be measured with high sensitivity and accuracy. Its attributes are ideal for enzymatic assays of alkaline phosphatase for both basic research and biotechnological applications.
碱性磷酸酶既是研究磷酸单酯酶的机制和动力学的模型酶，也是酶联免疫吸附测定（ELISA）和其他生化方法中的报告酶。酶与无机磷酸盐产物的紧密结合导致强烈的催化抑制，并使碱性磷酸酶活性的测量复杂化。我们开发了一种碱性磷酸酶底物，其中在“三甲基锁”介导的过程中，当 P-O 键断裂时，罗丹明的荧光被触发。尽管该底物需要非酶的第二步才能表现出荧光，但我们证明了酶的第一步限制了荧光发生的速率。该底物能够以高灵敏度和准确性测量碱性磷酸酶的催化活性。其特性非常适合用于基础研究和生物技术应用的碱性磷酸酶酶促测定。
