EVALUATION OF FLUOROMETRIC AND ZYMOGRAPHIC METHODS AS ACTIVITY ASSAYSFOR STROMELYSINS AND GELATINASES

To measure matrix metalloproteinase (MMP) activity in a large number o f samples it is advisable to use easily automated methods, We have eva luated and compared the activity of stromelysin-1 (MMP-3), matrilysin (MMP-7), 72 kDa gelatinase A (MMP-2) and 92 kDa gelatinase B (MMP-9) b y zymogram analysis and fluorescent substrate degradation assays, FITC -casein and the fluorogenic peptide lohexyl-Ala-Gly-Cys(Me)-His-Ala-Ly s-(N-Me-Abz)-NH2 were used as fluorescent substrates. FITC-casein was more efficiently degraded than the fluorogenic peptide by all MMPs tes ted except MMP-9, MMP-2 was not significantly able to degrade the fluo rogenic peptide, Gelatin zymography was the most sensitive method to d etect the activity of both gelatinases but quantitation problems compr omise its use, The degradation of fluorogenic substrates by MMPs could be inhibited by the chelating agent EDTA and by the tissue inhibitor of metalloproteinases 2 (TIMP-2), an MMP-specific inhibitor, Fluoromet ric methods represent a good alternative for MMP activity measurement, especially when a large number of samples must be processed.