Chromatographic purification of the plasmin-like enzyme from clamworm (Perinereis aibuhitensis Grub) and its fibrinolytic activity by metal ions.

In this study, fibrinolytic protease was isolated and purified from Perinereis aibuhitensis Grub, and the extraction process was optimized. The properties of the enzyme, such as the amino acid composition, thermal stability, optimal temperature, and pH, were investigated. After detoxification, proteins collected from fresh Clamworm (Perinereis aibuhitensis Grub) were concentrated via ammonium sulfate precipitation. The crude protease was purified using gel filtration resin (Sephadex G-100), anion exchange resin (DEAE-Sepharose FF), and hydrophobic resin (Phenyl Sepharose 6FF). The molecular weight of the protease was determined by polyacrylamide gel electrophoresis (SDS-PAGE). The optimum temperature and optimum pH of the protease were determined. The activity of crude protease in the 40-60% salt-out section was the highest, reaching 467.53 U/mg. The optimal process for purifying crude protein involved the application of DEAE-Sepharose FF and Phenyl Sepharose 6FF, which resulted in the isolation of a single protease known as Asp60-D1-P1 with the highest fibrinolytic activity; additionally, the enzyme activity was measured at 3367.76 U/mg. Analysis by Native-PAGE and SDS-PAGE revealed that the molecular weight of Asp60-D1-P1 was 44.5 kDa, which consisted of two subunits with molecular weights of 6.5 and 37.8 kDa, respectively. The optimum temperature for Asp60-D1-P1 was 40°C, and the optimal pH was 8.0.