Vilozny Boaz, Schiller Alexander, Wessling Ritchie A, Singaram Bakthan
Department of Chemistry and Biochemistry, University of California, 1156 High Street, Santa Cruz, CA 95064, USA.
Anal Chim Acta. 2009 Sep 7;649(2):246-51. doi: 10.1016/j.aca.2009.07.032. Epub 2009 Jul 22.
In-vitro fluorescent enzyme assays have been developed for sucrose phosphorylase (SPO) and phosphoglucomutase (PGM). These assays make use of a selective carbohydrate sensing system that detects the unlabeled enzymatic products fructose and glucose-6-phosphate. The system comprises 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt as the reporter unit and boronic acid appended viologens as selective receptors with working ranges from 70 microM to 1.0 mM for fructose (SPO) and 190 microM to 2.0 mM for glucose-6-phosphate (PGM). The change in fluorescence can be converted into product concentration, allowing initial reaction velocities and Michaelis-Menten kinetics to be calculated. The assays are also carried out in multiwell plate formats, making them suitable for high-throughput screening of enzyme inhibitors. Rapid PGM inhibition screening is demonstrated with EDTA and LiCl. The PGM assay can also be used for enzyme quantification with a detection limit of 50 ng mL(-1).
已开发出用于蔗糖磷酸化酶（SPO）和磷酸葡萄糖变位酶（PGM）的体外荧光酶测定法。这些测定法利用了一种选择性碳水化合物传感系统，该系统可检测未标记的酶促产物果糖和6-磷酸葡萄糖。该系统包含8-羟基芘-1,3,6-三磺酸三钠盐作为报告单元，以及硼酸连接的紫精作为选择性受体，对果糖（SPO）的工作范围为70微摩尔至1.0毫摩尔，对6-磷酸葡萄糖（PGM）的工作范围为190微摩尔至2.0毫摩尔。荧光变化可转化为产物浓度，从而能够计算初始反应速度和米氏动力学。这些测定法也以多孔板形式进行，使其适用于酶抑制剂的高通量筛选。用乙二胺四乙酸（EDTA）和氯化锂展示了快速的PGM抑制筛选。PGM测定法还可用于酶定量，检测限为50纳克/毫升（-1）。
