| Abstrakt: |
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/Cu2+ 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/Cu2+ 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 (P1) and GGGGRGGH (P2). However, the specificity constant (kcat/Km) for P2 is higher than that of P1 (6.4 x 10³ mM-1 min-1 vs. 1.3 x 10³ mM-1 min-1). This result shows that trypsin is more specific toward arginine (R) than lysine (K) in the oligopeptide sequence. [ABSTRACT FROM AUTHOR] |
