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

• 作者: Ahmed F. M. El-Mahdy Masaaki Kai Qinchang Zhu Sheng Yin Shpend Dragusha Takayuki Shibata Tsutomu Kabashima Valon Ejupi
• 来源期刊: 酶研究进展(英文)
• 学科: 医学
• 关键词: Spectrofluorometric Assay HUMAN COLLAGENASE Metalloproteinase NATIVE COLLAGEN ACETYL Peptide
• 年，卷（期）: myjjzyw_2015,（1）
• 页码范围: 19-29
• 页数: 11页
• 分类号: R73

摘要

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.