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Spectrofluorometric Assays of Human Collagenase Activity Using Native Collagen and Acetyl-Peptide Substrates

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DOI: 10.4236/aer.2015.31003    3,862 Downloads   4,576 Views  


A selective, sensitive, and convenient assay for human collagenase is required because of its implication in diseases such as rheumatoid arthritis, osteoarthritis, and tumors. Here, a novel assay for human collagenase activity is described in which enzymatic degradation of native collagen or acetyl peptide is determined by using a fluorogenic reaction for oligopeptides. The oligopeptides are quantified spectrofluorometrically with either 3,4-dihydroxyphenylacetic acid or 1,2-dihydroxybenzen reaction in the presence of sodium periodate and sodium borate (pH 7 - 8). These reactions can selectively convert N-terminal Gly-containing oligopeptides and N-terminal Ile-containing oligopeptides to fluorescence (FL) compounds, respectively, but not proteins, acetyl peptides or amino acids. Under optimized conditions using 1.65 μM collagen IV or 1.5 mM Ac-GPQGI- AGQ as substrates, this assay exhibits a proportional relationship between FL intensities and human collagenase-3 (MMP-13) concentrations. It can assay endogenous collagenase activities in several biological samples, such as cultured human cells and cheek tissue.

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The authors declare no conflicts of interest.

Cite this paper

Ejupi, V. , Dragusha, S. , Kabashima, T. , Zhu, Q. , El-Mahdy, A. , Yin, S. , Shibata, T. and Kai, M. (2015) Spectrofluorometric Assays of Human Collagenase Activity Using Native Collagen and Acetyl-Peptide Substrates. Advances in Enzyme Research, 3, 19-29. doi: 10.4236/aer.2015.31003.


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