Quantitative serine protease assays based on formation of copper(ii)–oligopeptide complexes

Abstract

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 (k_cat/K_m) for P₂ is higher than that of P₁ (6.4 × 10³ mM⁻¹ min⁻¹vs. 1.3 × 10³ mM⁻¹ min⁻¹). This result shows that trypsin is more specific toward arginine (R) than lysine (K) in the oligopeptide sequence.