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.
Highlights
Matrix metalloproteinases (MMPs) are a family of zinc-dependent enzymes that degrade major proteins in theHow to cite this paper: Ejupi, V., Dragusha, S., Kabashima, T., Zhu, Q.C., El-Mahdy, A.F.M., Yin, S., Shibata, T. and Kai, M. (2015) Spectrofluorometric Assays of Human Collagenase Activity Using Native Collagen and Acetyl-Peptide Substrates
We have examined DHB fluorogenic reaction [17] with N-terminal Ile-containing peptides and an acetyl peptide (Ac-GPQGIAGQ), which was reported to be a substrate [25] for human collagenase
We describe optimized assay protocols for human collagenase activity using the fluorogenic reagents dihydroxyphenylacetic acid (DHPAA) and DHB, and discuss the activity of endogenous collagenase in human cells and tissues
Summary
Matrix metalloproteinases (MMPs) are a family of zinc-dependent enzymes that degrade major proteins in theHow to cite this paper: Ejupi, V., Dragusha, S., Kabashima, T., Zhu, Q.C., El-Mahdy, A.F.M., Yin, S., Shibata, T. and Kai, M. (2015) Spectrofluorometric Assays of Human Collagenase Activity Using Native Collagen and Acetyl-Peptide Substrates. (2015) Spectrofluorometric Assays of Human Collagenase Activity Using Native Collagen and Acetyl-Peptide Substrates. Human collagenase is a MMP with the ability to cleave triple-helical native collagens at a single site, resulting in two large fragments that are approximately 1/4 and 3/4 of the initial length [2]. Elevated gene expression of this enzyme has been observed in patient tissues having malignancies such as adenocarcinoma, squamous cell carcinoma, and basal cell carcinoma [4]. This enzyme is involved in the pathogenesis of rheumatoid arthritis because it is significantly elevated in the synovial fluid and serum in such patients [5] [6]
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