A sensitive, specific assay for tissue collagenase using telopeptide-free [3H]acetylated collagen

David D. Dean, J. Frederick Woessner

Research output: Contribution to journalArticlepeer-review

69 Scopus citations

Abstract

Collagenase is assayed by incubation with soluble, telopeptide-free collagen extracted from rat skin and labeled with [2-3H]acetic anhydride. Collagen is cleaved by collagenase and the resulting fragments are digested with trypsin and chymotrypsin. Undigested collagen is recovered by precipitation with trichloroacetic acid, collected on glass-fiber filters, and quantitated by liquid scintillation spectrometry. This procedure combines features of the Cawston and Barrett (T. E. Cawston and A. J. Barrett, 1979, Anal. Biochem. 99, 340-345) and the Ryhänen et al. (L. Ryhänen et al., 1982, Collagen Rel. Res. 2, 117-130) methods. The first method provides a simple way to prepare large quantities of uniform substrate, while the second increases the specificity of the assay by removal of the labeled telopeptides. The assay is reproducible and linear with time and enzyme concentration. It is approximately 10× more sensitive than the Cawston and Barrett method and can readily detect 1-8 mU collagenase (1 unit equals 1 μg collagen cleaved/min at 30°C). The substrate is resistant to elastase, trypsin, and chymotrypsin and is completely degraded by bacterial collagenase. Collagenase is the only tissue metalloprotease found, to date, that cleaves the substrate.

Original languageEnglish (US)
Pages (from-to)174-181
Number of pages8
JournalAnalytical Biochemistry
Volume148
Issue number1
DOIs
StatePublished - Jul 1985
Externally publishedYes

Keywords

  • collagen, telopeptide-free
  • collagenase assay
  • collagenase, tissue

ASJC Scopus subject areas

  • Molecular Biology
  • Biophysics
  • Biochemistry
  • Cell Biology

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