Quantitative zymography of matrix metalloproteinases by measuring hydroxyproline: application to gelatinases A and B.
Gelatinases A and B are metalloproteinases involved in the degradation of the extracellular matrix. Detection and quantification of these enzymes in physiological and pathological conditions such as rheumatoid arthritis, tumor invasion and metastasis may be clinically useful. Gelatin zymography is an electrophoretic technique specific for gelatinases. It can be used to detect the activity of both the active and latent forms. We have standardized this technique for the active and latent forms of gelatinase A and for the latent form of gelatinase B. We measured the extent of gelatin degradation with an EDC scanning densitometer (Helena). The value recorded was directly proportional to the amount of enzyme. Gelatinase activity was quantified from the gel by assaying hydroxyproline as an index of gelatin breakdown. Gelatin zymography was found to be useful in characterizing gelatinases A and B by their molecular weights and measuring their specific activity by a standardized analysis of the degraded gelatin substrate.
Lê
J
J
Laboratoire de Biochimie, Faculté de Médecine, Saint-Etienne, France.
Dauchot
P
P
Perrot
J L
JL
Cambazard
F
F
Frey
J
J
Chamson
A
A
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D006909:Hydroxyproline / Q000032:analysis*; D020778:Matrix Metalloproteinase 2 / Q000032:analysis* / Q000737:chemistry; D020780:Matrix Metalloproteinase 9 / Q000032:analysis* / Q000737:chemistry
D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9; D006909:Hydroxyproline