Evaluation of fluorometric and zymographic methods as activity assays for stromelysins and gelatinases.
To measure matrix metalloproteinase (MMP) activity in a large number of samples it is advisable to use easily automated methods. We have evaluated 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) by zymogram analysis and fluorescent substrate degradation assays. FITC-casein and the fluorogenic peptide Dnp-Pro-beta-cyclo-hexyl-Ala-Gly-Cys(Me)-His-Ala-Lys-(N-Me-Abz)-NH 2 were used as fluorescent substrates. FITC-casein was more efficiently degraded than the fluorogenic peptide by all MMPs tested except MMP-9. MMP-2 was not significantly able to degrade the fluorogenic peptide. Gelatin zymography was the most sensitive method to detect the activity of both gelatinases but quantitation problems compromise 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. Fluorometric methods represent a good alternative for MMP activity measurement, especially when a large number of samples must be processed.
Quesada
A R
AR
Pharmacia & Upjohn Research Department, Madrid, Spain.
Barbacid
M M
MM
Mira
E
E
Fernández-Resa
P
P
Márquez
G
G
Aracil
M
M
D002118:Calcium / Q000502:physiology; D002364:Caseins / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D017364:Collagenases / Q000032:analysis* / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D005470:Fluorometry; D018093:Gelatinases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D019278:Matrix Metalloproteinase 3 / Q000032:analysis* / Q000378:metabolism; D020783:Matrix Metalloproteinase 7; D020780:Matrix Metalloproteinase 9; D061965:Matrix Metalloproteinase Inhibitors; D008666:Metalloendopeptidases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D011506:Proteins / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000037:antagonists & inhibitors; D019716:Tissue Inhibitor of Metalloproteinase-2
D002364:Caseins; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D061965:Matrix Metalloproteinase Inhibitors; D011506:Proteins; D011993:Recombinant Fusion Proteins; D019716:Tissue Inhibitor of Metalloproteinase-2; D004492:Edetic Acid; D017364:Collagenases; D018093:Gelatinases; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3; D020783:Matrix Metalloproteinase 7; D020780:Matrix Metalloproteinase 9; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium