Department of Functional Molecular Science, Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima 734-8553, Japan.
Sensors (Basel). 2017 Aug 15;17(8):1877. doi: 10.3390/s17081877.
We introduce two types of fluorescence-quenching assay for alkaline phosphatases (APs) by using a carboxytetramethyl-rhodamine (TAMRA)-labeled phosphate-binding tag molecule (TAMRA-Phos-tag). In the first assay, TAMRA-labeled -phosphorylethanolamine (TAMRA-PEA) was used as an artificial AP-substrate. TAMRA-Phos-tag specifically captured TAMRA-PEA to form a 1:1 complex at pH 7.4; the intensity of the fluorescence peak of the complex at 580 nm (λ = 523 nm) was significantly reduced to 32% of the average value for the two individual components as a result of the mutual approach of the TAMRA moieties. As TAMRA-PEA was dephosphorylated by AP, the resulting TAMRA-labeled ethanolamine dissociated and the fluorescence increased in a manner dependent on the AP dose and the time. In the second assay, pyrophosphate (PP), a natural AP-substrate, was used as a bridging ligand to form a dimeric TAMRA-Phos-tag complex. The dimerization reduced the fluorescence intensity to 49% of that in the absence of PP. As pyrophosphate was hydrolyzed to two orthophosphate moieties by AP, the 580-nm fluorescence recovered in a time-dependent manner. By examining the initial slope of this time-dependent fluorescence recovery, we succeeded in evaluating the 50% inhibitory concentrations of orthovanadate toward two AP isozymes under near-physiological conditions.
我们介绍了两种利用羧基四甲基若丹明（TAMRA）标记的磷酸结合标签分子（TAMRA-Phos-tag）检测碱性磷酸酶（APs）的荧光猝灭法。在第一种测定方法中，我们使用 TAMRA 标记的 -磷酸乙醇胺（TAMRA-PEA）作为人工 AP 底物。TAMRA-Phos-tag 特异性地捕获 TAMRA-PEA，在 pH7.4 下形成 1:1 复合物；由于 TAMRA 部分的相互接近，该复合物在 580nm（λ=523nm）处的荧光峰强度显著降低至两个单独组分平均值的 32%。由于 AP 将 TAMRA-PEA 去磷酸化，产生的 TAMRA 标记乙醇胺解离，荧光强度随 AP 剂量和时间的变化而增加。在第二种测定方法中，焦磷酸（PP），一种天然的 AP 底物，用作桥联配体以形成二聚体 TAMRA-Phos-tag 复合物。二聚化将荧光强度降低至无 PP 时的 49%。由于 AP 将焦磷酸水解为两个正磷酸部分，580nm 处的荧光恢复呈时间依赖性。通过检查该时间依赖性荧光恢复的初始斜率，我们成功地在接近生理条件下评估了两种 AP 同工酶的钒酸盐对其 50%抑制浓度。
