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Measurement of activity of collagenolytic MMP and inhibitors of MMPs using radiolabeled collagen substrate

Lookup NU author(s): Emeritus Professor Tim Cawston

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Abstract

This protocol describes how to purify and radiolabel collagen for use as a substrate to assay collagenolytic members of the matrix metalloproteinases (MMPs). This assay measures enzymes that specifically cleave native triple helical collagen. After incubation of the MMP enzyme with the collagen substrate at 37 degrees C, undigested collagen is removed by centrifugation. Radiolabeled cleaved fragments remain in the supernatant, which is then counted in a scintillation counter; a linear increase in the release of radiolabeled collagen fragments occurs with enzyme level and time. Methods are included for the activation of the proenzyme forms of these MMPs and the assay can also be adapted to measure inhibitors of the collagenolytic MMPs. This assay can be completed in 18 h.

Publication metadata

Author(s): Cawston TE

Publication type: Article

Publication status: Published

Journal: Nature Protocols

Year: 2009

Volume: 4

Issue: 3

Pages: 286-290

ISSN (print): 1754-2189

ISSN (electronic): 1750-2799

Publisher: Nature Publishing Group

URL: http://dx.doi.org/10.1038/nprot.2008.244

DOI: 10.1038/nprot.2008.244

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