State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, PR China.
Anal Chim Acta. 2013 Jun 19;784:72-6. doi: 10.1016/j.aca.2013.04.066. Epub 2013 May 13.
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.
一种新的用于蛋白酶活性和抑制剂筛选的连续荧光开启分析方法已经建立。使用了荧光标记的单链 DNA（FAM-DNA）和细胞色素 c（cyt c）。当 FAM 荧光团与 cyt c 的血红素辅基之间发生电子转移时，FAM-DNA 与 cyt c 结合会有效地猝灭其荧光。在存在蛋白酶（如胰蛋白酶）的情况下，cyt c 被消化成小肽片段。FAM-DNA 被释放，从而恢复 FAM 荧光。通过添加抑制剂可以降低 cyt c 的消化速率。因此，获得的荧光恢复程度降低。我们的分析方法的检测限为 1 nM 胰蛋白酶，对于来自蛋清的抑制剂和来自大豆的抑制剂，IC50 值分别为 3.23 μg mL(-1) 和 0.303 μg mL(-1)。我们的方法可用于各种生化应用中蛋白酶活性的传感，以及用于筛选蛋白酶抑制剂作为治疗各种相关疾病的药物。
