TY - JOUR
T1 - A sensitive, specific assay for tissue collagenase using telopeptide-free [3H]acetylated collagen
AU - Dean, David D.
AU - Woessner, J. Frederick
N1 - Funding Information:
ACKNOWLEDGMENTS This research was supported by National Institutes of Health Grants AM-16940 and HD-06773. D.D.D. is a Fellow of the Arthritis Foundation. We thank Ms. Marie Seizer and Ms. Carolyn Taplin for their excellent technical assistance, Dr. Z. Gunja-Smith for performance of the hydroxyproline assays, and Dr. Thomas Curry for his comments on the manuscript.
PY - 1985/7
Y1 - 1985/7
N2 - 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.
AB - 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.
KW - collagen, telopeptide-free
KW - collagenase assay
KW - collagenase, tissue
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U2 - 10.1016/0003-2697(85)90642-6
DO - 10.1016/0003-2697(85)90642-6
M3 - Article
C2 - 2994518
AN - SCOPUS:0021881764
SN - 0003-2697
VL - 148
SP - 174
EP - 181
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 1
ER -