title: Monitoring of proteolytic enzyme activity using phase transition-based peptide arrays.
abstract: 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-[γ-maleimidobutyryloxy] sulfosuccinimide ester, and a substrate peptide containing two functional groups, cysteine and tetramethyl-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 (IC(50)) 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.
authors.affiliation: Department of Molecular and Cellular Biochemistry, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do, Republic of Korea.
authors.forename: Deok-Hoon
authors.initials: DH
authors.lastname: Kong
authors.forename: Se-Hui
authors.initials: SH
authors.lastname: Jung
authors.forename: Seung-Taek
authors.initials: ST
authors.lastname: Lee
authors.forename: Young-Myeong
authors.initials: YM
authors.lastname: Kim
authors.forename: Kwon-Soo
authors.initials: KS
authors.lastname: Ha
mesh_terms: D002384:Catalysis; D020134:Catalytic Domain; D003545:Cysteine / Q000737:chemistry; D055808:Drug Discovery; D005453:Fluorescence; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D019278:Matrix Metalloproteinase 3 / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D044367:Phase Transition; D012235:Rhodamines / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013379:Substrate Specificity
chemical_list: C114557:5-carboxytetramethylrhodamine succinimidyl ester; D010455:Peptides; D012235:Rhodamines; C511768:MMP3 protein, human; D019278:Matrix Metalloproteinase 3; D003545:Cysteine