A continuous fluorescence resonance energy transfer angiotensin I-converting enzyme assay
A continuous fluorescence resonance energy transfer angiotensin I-converting enzyme assay
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DOI：
10.1038/nprot.2006.306
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Sturrock, Edward D.
中科院分区：
文献类型：
--
作者：
Carmona, Adriana K.;Schwager, Sylva L.;Sturrock, Edward D.
血管紧张素I转换酶（ACE）参与多种生理和病理生理状况；因此，对其催化活性的测量可以提供重要的临床信息。该协议描述了使用荧光共振能量转移 (FRET) 底物测定 ACE 活性的灵敏且快速的程序，其中邻氨基苯甲酸 (Abz) 作为荧光基团，2,4-二硝基苯基 (Dnp) 作为淬灭受体。供体/受体对之间的肽键水解产生可以连续检测的荧光，从而可以定量测量酶活性。 FRET 底物为动力学研究和生物体液和粗组织提取物中的 ACE 测定提供了有用的工具。该方法的一个重要优点是使用对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 和用于体细胞 ACE 的 Abz-FRK(Dnp) P-OH。该方法可适用于使用 96 孔荧光板读数器进行测定。
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.