Department of Chemical and Biomolecular Engineering, 4 Engineering Drive 4, National University of Singapore, Singapore 117576, Singapore.
Anal Chem. 2010 Oct 15;82(20):8604-10. doi: 10.1021/ac101695x.
A fluorescence "turn-on" assay for monitoring protease activity is developed on the basis of a water-soluble carboxylated polyfluorene derivative, PFP-CO₂Na, and its different fluorescence response toward cytochrome c (cyt c) and its fragments. PFP-CO₂Na is synthesized via Suzuki coupling polymerization between 2,7-dibromo-9,9-bis(3'-tert-butyl propanoate)fluorene and 1,4-bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-benzene, followed by treatment with trifluoroacetic acid and Na₂CO₃. The fluorescence of PFP-CO₂Na can be significantly quenched by cyt c due to complexation-mediated electron transfer between the polymer and protein. Using the complex of PFP-CO₂Na/cyt c as a substrate, a real-time fluorescence turn-on assay for trypsin activity study has been developed. Addition of trypsin to the substrate solution induces gradual recovery of the fluorescence intensity for PFP-CO₂Na due to trypsin-catalyzed hydrolysis of cyt c, which dissociates the heme moiety from the polymer vicinity. The time-dependent fluorescence intensity increase of PFP-CO₂Na in the presence of trypsin allows us to derive the initial reaction rates and k(cat)/K(m) (5350 M⁻¹ s⁻¹) for trypsin-catalyzed hydrolysis. Addition of trypsin inhibitor efficiently inhibits trypsin-catalyzed hydrolysis reaction of cyt c, which leads to a decreased fluorescence turn-on response of PFP-CO₂Na.
一种基于水溶性羧基化聚芴衍生物 PFP-CO₂Na 的荧光“开启”分析方法被开发出来，用于监测蛋白酶活性，其对细胞色素 c（cyt c）及其片段具有不同的荧光响应。PFP-CO₂Na 通过 2,7-二溴-9,9-双（3'-叔丁基丙酰基）芴与 1,4-双（4,4,5,5-四甲基-1,3,2-二恶唑烷-2-基）-苯之间的 Suzuki 偶联聚合合成，然后用三氟乙酸和 Na₂CO₃处理。由于聚合物和蛋白质之间的复合物介导的电子转移，PFP-CO₂Na 的荧光可以被 cyt c 显著猝灭。使用 PFP-CO₂Na/cyt c 复合物作为底物，开发了用于研究胰蛋白酶活性的实时荧光开启分析。向底物溶液中加入胰蛋白酶会导致 PFP-CO₂Na 的荧光强度逐渐恢复，这是由于胰蛋白酶催化 cyt c 的水解，使血红素部分从聚合物附近解离。在存在胰蛋白酶的情况下，PFP-CO₂Na 的荧光强度随时间的增加允许我们得出胰蛋白酶催化水解的初始反应速率和 k(cat)/K(m)（5350 M⁻¹ s⁻¹）。胰蛋白酶抑制剂的加入可有效抑制胰蛋白酶催化的 cyt c 水解反应，从而降低 PFP-CO₂Na 的荧光开启响应。
