Bickett D M, Green M D, Berman J, Dezube M, Howe A S, Brown P J, Roth J T, McGeehan G M
Glaxo Research Institute, Department of Biochemistry, Research Triangle Park, North Carolina 27709.
Anal Biochem. 1993 Jul;212(1):58-64. doi: 10.1006/abio.1993.1291.
Two members of the matrix metalloproteinase family of enzymes, interstitial collagenase and 92-kDa gelatinase, have been implicated in the pathogenesis of rheumatoid arthritis and tumor metastasis. In order to characterize the activities of these enzymes, we have developed a fluorogenic peptide substrate which is efficiently hydrolyzed by both enzymes. This substrate was developed based on the addition of the fluorescent tag, N-methyl-anthranilic acid (Nma), to several previously synthesized substrates that had been evaluated with respect to their turnover by interstitial collagenase. One substrate, Dnp-Pro-Cha-Gly-Cys(Me)-His-Ala-Lys-(Nma)-NH2, had favorable solubility characteristics, was > 98% quenched, and produced a single cleavage product, Dnp-Pro-Cha-Gly, with a high fluorescence yield with both interstitial collagenase and 92-kDa gelatinase. Since the assay depends on measurement of increases in fluorescence, the position of the Nma group also proved to be important for optimization of the fluorescence signal. The assay is free from interference by organomercurial compounds and the cleavage product has excitation and emission spectra compatible with filters commonly available on commercial plate readers. The assay has been adapted to a 96-well format and provides a rapid screening protocol for the evaluation of inhibitors of these enzymes.
基质金属蛋白酶家族中的两种酶，即间质胶原酶和92 kDa明胶酶，已被认为与类风湿性关节炎的发病机制和肿瘤转移有关。为了表征这些酶的活性，我们开发了一种荧光肽底物，两种酶均可有效水解该底物。该底物是在几种先前合成的底物上添加荧光标签N-甲基邻氨基苯甲酸（Nma）后开发的，这些底物已针对间质胶原酶的周转率进行了评估。一种底物Dnp-Pro-Cha-Gly-Cys（Me）-His-Ala-Lys-（Nma）-NH2具有良好的溶解性，淬灭率> 98％，并且产生单一裂解产物Dnp-Pro-Cha-Gly，对于间质胶原酶和92 kDa明胶酶均具有高荧光产率。由于该测定法依赖于荧光增加的测量，因此Nma基团的位置对于优化荧光信号也很重要。该测定法不受有机汞化合物的干扰，并且裂解产物的激发和发射光谱与商业酶标仪上常用的滤光片兼容。该测定法已适用于96孔板形式，并提供了一种快速筛选方案，用于评估这些酶的抑制剂。
