Carmona Adriana K, Schwager Sylva L, Juliano Maria A, Juliano Luiz, Sturrock Edward D
Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, SP, Brazil.
Nat Protoc. 2006;1(4):1971-6. doi: 10.1038/nprot.2006.306.
Angiotensin I-converting enzyme (ACE) is involved in various physiological and physiopathological conditions; therefore, the measurement of its catalytic activity may provide essential clinical information. This protocol describes a sensitive and rapid procedure for determination of ACE activity using fluorescence resonance energy transfer (FRET) substrates containing o-aminobenzoic acid (Abz) as the fluorescent group and 2,4-dinitrophenyl (Dnp) as the quencher acceptor. Hydrolysis of a peptide bond between the donor/acceptor pair generates fluorescence that can be detected continuously, allowing quantitative measurement of the enzyme activity. The FRET substrates provide a useful tool for kinetic studies and for ACE determination in biological fluids and crude tissue extracts. An important benefit of this method is the use of substrates selective for the two active sites of the enzyme, namely Abz-SDK(Dnp)P-OH for N-domain, Abz-LFK(Dnp)-OH for C-domain and Abz-FRK(Dnp)P-OH for somatic ACE. This methodology can be adapted for determinations using a 96-well fluorescence plate reader.
血管紧张素I转换酶（ACE）参与多种生理和病理生理过程；因此，其催化活性的测定可为临床提供重要信息。本方案描述了一种灵敏且快速的程序，用于使用含有邻氨基苯甲酸（Abz）作为荧光基团和2,4-二硝基苯基（Dnp）作为淬灭受体的荧光共振能量转移（FRET）底物来测定ACE活性。供体/受体对之间的肽键水解会产生可连续检测的荧光，从而实现对酶活性的定量测定。FRET底物为动力学研究以及生物体液和粗组织提取物中ACE的测定提供了一种有用的工具。该方法的一个重要优点是使用了对该酶的两个活性位点具有选择性的底物，即用于N结构域的Abz-SDK（Dnp）P-OH、用于C结构域的Abz-LFK（Dnp）-OH以及用于体细胞ACE的Abz-FRK（Dnp）P-OH。该方法可适用于使用96孔荧光酶标仪进行测定。
