化学
荧光团
荧光
蛋白酶
生物化学
胰蛋白酶
检出限
血红素
寡核苷酸
细胞色素c
DNA
酶
色谱法
物理
量子力学
线粒体
作者
Dongli Liao,Yongxin Li,Jian Chen,Cong Yu
标识
DOI:10.1016/j.aca.2013.04.066
摘要
A new continuous fluorescence turn-on assay for protease activity and inhibitor screening has been developed. A fluorophore labeled single stranded DNA (FAM-DNA) and cytochrome c (cyt c) were employed. The fluorescence of the FAM-DNA was efficiently quenched when binding to cyt c, through the electron transfer between the FAM fluorophore and the heme cofactor of cyt c. In the presence of a protease, such as trypsin, cyt c was digested into small peptide fragments. The FAM-DNA was released, which resulted in the recovery of the FAM fluorescence. The rate of the cyt c digestion could be reduced via the addition of an inhibitor. As a result, reduced degree of the fluorescence recovery was obtained. The limit of detection of our assay is 1 nM trypsin and the IC50 values are 3.23 μg mL(-1) and 0.303 μg mL(-1) for the inhibitor from egg white and the inhibitor from soybean, respectively. Our method could be used for the sensing of protease activity for various biochemical applications, and for the screening of protease inhibitors as drugs for the treatment of various related diseases.
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