Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland.
Anal Biochem. 2012 Aug 1;427(1):18-20. doi: 10.1016/j.ab.2012.04.025. Epub 2012 Apr 28.
The measurement of activities from individual proteases in biological samples is difficult because of the numerous proteases, their overlapping activities, and the lack of specific substrates. We applied selective protease inhibitors based on bicyclic peptides (>2000-fold selective over related proteases) to block individual proteases, allowing the quantification of their net activities. In protease mixtures, activity contributions of the serine proteases plasma kallikrein and urokinase-type plasminogen activator (uPA) were accurately quantified. In a tumor extract, we could quantify uPA activity. Because bicyclic peptide inhibitors toward virtually any protease can be generated by phage display, the approach should be applicable to any protease.
由于存在大量蛋白酶、其活性存在重叠以及缺乏特异性底物，因此很难对生物样本中个别蛋白酶的活性进行测量。我们应用基于双环肽的选择性蛋白酶抑制剂（对相关蛋白酶的选择性超过 2000 倍）来阻断个别蛋白酶，从而可以定量测定其净活性。在蛋白酶混合物中，我们可以准确地定量测定丝氨酸蛋白酶血浆激肽释放酶和尿激酶型纤溶酶原激活物(uPA)的活性。在肿瘤提取物中，我们可以定量测定 uPA 活性。由于针对几乎任何蛋白酶的双环肽抑制剂都可以通过噬菌体展示来生成，因此该方法应该适用于任何蛋白酶。
