Quantitative serine protease assays based on formation of copper(II)-oligopeptide complexes.
A quantitative protease assay based on the formation of a copper-oligopeptide complex is developed. In this assay, when a tripeptide GGH fragment is cleaved from an oligopeptide chain by serine proteases, the tripeptide quickly forms a pink GGH/Cu(2+) complex whose concentration can be determined quantitatively by using UV-Vis spectroscopy. Therefore, activities of serine proteases can be determined from the formation rate of the GGH/Cu(2+) complex. This principle can be used to detect the presence of serine protease in a real-time manner, or measure proteolytic activities of serine protease cleaving different oligopeptide substrates. For example, by using this assay, we demonstrate that trypsin, a model serine protease, is able to cleave two oligopeptides GGGGKGGH () and GGGGRGGH (). However, the specificity constant (kcat/Km) for is higher than that of (6.4 × 10(3) mM(-1) min(-1)vs. 1.3 × 10(3) mM(-1) min(-1)). This result shows that trypsin is more specific toward arginine (R) than lysine (K) in the oligopeptide sequence.
Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585, Singapore. cheyk@nus.edu.sg.
Xiaokang
X
Ding
Kun-Lin
KL
Yang
D003300:Copper / Q000737:chemistry* / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism; D009942:Organometallic Compounds / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D057057:Serine Proteases / Q000032:analysis* / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
D009842:Oligopeptides; D009942:Organometallic Compounds; D003300:Copper; D057057:Serine Proteases