A sensitive, specific assay for tissue collagenase using telopeptide-free [3H]acetylated collagen.
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 10X more sensitive than the Cawston and Barrett method and can readily detect 1-8 mU collagenase (1 unit equals 1 microgram collagen cleaved/min at 30 degrees 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.
D000107:Acetylation; D000818:Animals; D002918:Chymotrypsin / Q000378:metabolism; D003094:Collagen / Q000378:metabolism; D005260:Female; D003012:Microbial Collagenase / Q000032:analysis* / Q000378:metabolism; D010196:Pancreatic Elastase / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D012867:Skin / Q000032:analysis; D013696:Temperature; D014357:Trypsin / Q000378:metabolism
D003094:Collagen; D002918:Chymotrypsin; D010196:Pancreatic Elastase; D014357:Trypsin; D003012:Microbial Collagenase