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

Valon Ejupi, Shpend Dragusha, Tsutomu Kabashima, Qinchang Zhu, Ahmed F. M. El-Mahdy, Sheng Yin, Takayuki Shibata, Masaaki Kai

Faculty of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan.

DOI: 10.4236/aer.2015.31003                

Abstract

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.

Keywords

Spectrofluorometric Assay, Human Collagenase, Metalloproteinase, Native Collagen, Acetyl Peptide

Conflicts of Interest

The authors declare no conflicts of interest.

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