Heparin-enhanced zymographic detection of matrilysin and collagenases.
Unlike the gelatinases (MMP-2 and -9), matrilysin (MMP-7) and collagenases (MMP-1 and -13) are difficult to detect at low levels in conventional casein or gelatin zymography. In this report, heparin was used to enhance the zymographic assays for MMP-7, -1, and -13. With the addition of heparin to the enzyme sample, MMP-7 can be detected at a level of 30 pg in transferrin zymography and MMP-1 and -13 can be detected at a level of 0.2 ng in gelatin zymography. Carboxymethylated transferrin is used instead of casein as a substrate for assaying rat MMP-7. This substrate does not require a prerun step or substrate cross-linking to give uniform staining and clear band formation. It is necessary for heparin to run to the same region of the gel as the enzyme to produce its enhancing effect. For MMP-7 movement of heparin and enzyme is almost equal; for the collagenases it is necessary to add heparin to each well after the electrophoretic run is underway. Possible mechanisms of activity enhancement are discussed.
Yu
W H
WH
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, FL 33101, USA. sean-y@helix.mgh.harvard.edu
Woessner
J F
JF
D000818:Animals; D002417:Cattle; D017364:Collagenases / Q000032:analysis* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology; D018093:Gelatinases / Q000032:analysis / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology*; D006801:Humans; D020783:Matrix Metalloproteinase 7 / Q000032:analysis* / Q000378:metabolism; D051381:Rats; D014168:Transferrin / Q000378:metabolism
D014168:Transferrin; D006493:Heparin; D017364:Collagenases; D018093:Gelatinases; D020783:Matrix Metalloproteinase 7