Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Center for Biotechnology and Biomedicine, Leipzig University, Deutscher Platz 5, 04103 Leipzig, Germany.
Anal Chem. 2011 Oct 1;83(19):7356-63. doi: 10.1021/ac201274f. Epub 2011 Sep 14.
Proteases are widely used in analytical sciences and play a central role in several widespread diseases. Thus, there is an immense need for highly adaptable and sensitive assays for the detection and monitoring of various proteolytic enzymes. We established a simple protease fluorescence resonance energy transfer (pro-FRET) assay for the determination of protease activities, which could in principle be adapted for the detection of all proteases. As proof of principle, we demonstrated the potential of our method using trypsin and enteropeptidase in complex biological mixtures. Briefly, the assay is based on the cleavage of a FRET peptide substrate, which results in a dramatic increase of the donor fluorescence. The assay was highly sensitive and fast for both proteases. The detection limits for trypsin and enteropeptidase in Escherichia coli lysate were 100 and 10 amol, respectively. The improved sensitivity for enteropeptidase was due to the application of an enzyme cascade, which leads to signal amplification. The pro-FRET assay is highly specific as even high concentrations of other proteases did not result in significant background signals. In conclusion, this sensitive and simple assay can be performed in complex biological mixtures and can be easily adapted to act as a versatile tool for the sensitive detection of proteases.
蛋白酶广泛应用于分析科学领域，在多种常见疾病中起着核心作用。因此，人们迫切需要高度适应性和灵敏的检测方法来检测和监测各种蛋白酶。我们建立了一种简单的蛋白酶荧光共振能量转移（pro-FRET）分析方法来测定蛋白酶活性，该方法原则上可以用于检测所有蛋白酶。作为原理的证明，我们使用胰蛋白酶和肠肽酶在复杂的生物混合物中展示了我们方法的潜力。简而言之，该分析方法基于 FRET 肽底物的切割，导致供体荧光的显著增加。该分析方法对两种蛋白酶均具有高度的灵敏性和快速性。在大肠杆菌裂解物中，胰蛋白酶和肠肽酶的检测限分别为 100 和 10 飞摩尔。肠肽酶的灵敏度提高是由于应用了酶级联反应，从而导致信号放大。pro-FRET 分析方法具有高度的特异性，即使存在高浓度的其他蛋白酶，也不会产生显著的背景信号。总之，这种灵敏且简单的分析方法可在复杂的生物混合物中进行，并且可以轻松地适应作为灵敏检测蛋白酶的通用工具。
