Nakata Hidetaka, Ohtsuki Takashi, Sisido Masahiko
Department of Bioscience and Biotechnology, Okayama University, Okayama 700-8530, Japan.
Anal Biochem. 2009 Jul 15;390(2):121-5. doi: 10.1016/j.ab.2009.03.049. Epub 2009 Apr 24.
We developed novel substrates for protease activity evaluation by fluorescence correlation spectroscopy (FCS). Substrates were labeled in a position-specific manner with a fluorophore near the N terminus and included a C-terminal, 30 kDa, highly soluble protein (elongation factor Ts [EF-Ts]). The C-terminal protein enhanced the substrate peptide solubility and increased the molecular weight, enabling sensitive detection by FCS. Using the labeled substrates, caspase-3 and matrix metalloproteinase-9 (MMP-9) activities were confirmed by FCS. To demonstrate the suitability of this FCS-based assay for high-throughput screening, we screened various chemical compounds for MMP-9 inhibitors. The screening results confirmed the inhibitory activity of one compound and also revealed another potential MMP-9 inhibitor. Thus, this combination of position-specific labeled protein substrates and FCS may serve as a useful tool for evaluating activities of various proteases and for protease inhibitor screening.
我们开发了用于通过荧光相关光谱法（FCS）评估蛋白酶活性的新型底物。底物在靠近N端的位置用荧光团进行特异性标记，并包含一个C端的、30 kDa的高度可溶性蛋白（延伸因子Ts [EF-Ts]）。C端蛋白提高了底物肽的溶解度并增加了分子量，从而能够通过FCS进行灵敏检测。使用标记的底物，通过FCS证实了半胱天冬酶-3和基质金属蛋白酶-9（MMP-9）的活性。为了证明这种基于FCS的检测方法适用于高通量筛选，我们筛选了各种化合物以寻找MMP-9抑制剂。筛选结果证实了一种化合物的抑制活性，还揭示了另一种潜在的MMP-9抑制剂。因此，这种特异性标记的蛋白质底物与FCS的组合可能是评估各种蛋白酶活性和筛选蛋白酶抑制剂的有用工具。
