A Homogeneous Caspase-3 Activity Assay Using HTRF® Technology
A Homogeneous Caspase-3 Activity Assay Using HTRF® Technology
复制标题
使用 HTRF® 技术进行均质 Caspase-3 活性测定
DOI：
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发表时间：
2002
影响因子：
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通讯作者：
H. Takemoto
中科院分区：
文献类型：
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作者：
M. Préaudat;J. Ouled;B. Alpha;G. Mathis;T. Mitsugi;Y. Aono;Koji Takahashi;H. Takemoto
半胱天冬酶是半胱氨酸蛋白酶，具有保守的活性位点，可在高度特异性的位置切割蛋白质底物。它们参与活跃细胞死亡途径的不同方面。它们中的大多数通过细胞成分的蛋白水解降解起作用。本文描述了该酶抑制剂的测定开发、测定验证和筛选，这些抑制剂可能是潜在的候选药物。该测定使用均质时间分辨荧光，基于从作为供体的铕穴状化合物到作为受体的交联别藻蓝蛋白 (XL665) 的能量转移。双标记底物生物素基-E-氨基己酰基-L-天冬氨酰基-L-谷氨酰基-L-缬氨酰基-L-天冬氨酰基-L-丙氨酰基-L-丙基-N∊-(2,4-二硝基苯基)-L-赖氨酸-酰胺(生物素-X-DEVDAPK(dnp)-NH2)与链霉亲和素穴状化合物缀合， 抗 dnp-XL665 单克隆抗体。供体和受体之间的紧密接近会产生特定的时间分辨荧光信号。在存在酶活性的情况下，底物裂解导致两个荧光探针断开连接，随后由于接近性丧失而导致特定信号消失。与荧光测定方法进行比较，讨论了优化试剂浓度、孵育时间、精度、重现性和稳健性的实验。
Caspases are cysteine proteases presenting a conserved active site that cleaves protein substrates at a highly specific position. They are involved in different aspects of the active cell death pathway. Most of them act through proteolytic degradations of cellular components. This paper describes the assay development, assay validation, and screening for inhibitors of this enzyme, which could be potential drug candidates. The assay uses homogeneous time-resolved fluorescence based on energy transfer from europium cryptate as donor to cross-linked allophyco-cyanin as acceptor (XL665). A double-tagged substrate, biotinyl-E-aminocaproyl-L-aspartyl-L-glutamyl-L-valyl-L-aspartyl-L-alanyl-L-propyl-N∊-(2,4-dinitrophenyl)-L-lysine-amide (biotin-X-DEVDAPK(dnp)-NH2), is conjugated with streptavidin cryptate and anti-dnp-XL665 monoclonal antibody. The close proximity between donor and acceptor induces a specific time-resolved fluorescence signal. In the presence of enzyme activity, the substrate cleavage induces an unlinking of the two fluorescent probes and, subsequently, the disappearance of the specific signal as a result of loss of proximity. Experiments to optimize the reagent concentration, incubation times, precision, reproducibility, and robustness are discussed in comparison with a fluorometric method.