Monitoring of proteolytic enzyme activity using phase transition-based peptide arrays

초록

We have developed an assay using peptide arrays based on phase transition from the glass substrate to the liquid for monitoring quantitative protease activity in real-time. Peptide arrays were fabricated using a bifunctional cross-linker, N-[gamma-maleimidobutyryloxy] sulfosuccinimide ester, and a substrate peptide containing two functional groups, cysteine and tetramethy1-6-carboxyrhodamine (TAMRA) on the C- and N-terminus, respectively. The phase transition-based peptide arrays were characterized by analyzing the substrate peptide cleaved from the solid substrate by matrix metalloproteinase-3 (MMP-3). We successfully used this assay to determine the quantitative proteolytic activity of MMP-3 in a dose-dependent manner. In addition, parameters including Michaelis constant (K-m), maximum rate of enzymatic reaction (V-max), and half maximal inhibitory concentration (IC50) were determined by analyzing the concentrations of substrate peptide cleaved by MMP-3. Therefore, this new assay has potential for the quantitative analysis of enzyme kinetics of protease and informs research developments in drug discovery utilizing kinetic studies. (C) 2012 Elsevier B.V. All rights reserved.

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