Purification Of Earthworm Fibrinolytic Enzymes And Characteristics Of Some Components

Soybean trypsin inhibitor (SBTI) was purified partially by DEAE-Cellulose-32 chromatography. Its MW was about 20.1 kD determined by SDS-PAGE, and its inhibitory ratio was 0.9:1.The components of earthworm fibrinolytic enzymes (EFEs) were separated from Eisenia foetida by affinity chromatography using SBTI as a ligand and Sepharose-4B as a carrier. It was studied that effects on coupling rate caused by different activators such as 1,1'-carboxyl-diirnidazole (CDI) activation and chliorepoxy propcone activation. The efficiencies of affinity chromatography caused by different ligands such as SBTI and ovalbumin, different affinity time, temperature and pH of buffer were also studied. It was showed that the agarose activated with chliorepoxy propcone activation had higher coupling rate than that of 1,1'-carboxyl-diirnidazole(CDI).About twelve components could be obtained using SBTI as a ligand, however, only one main component was separated using ovalbumin as a ligand.The absorption quantity was increasing along with the increasing of affinity time, and it was saturated after 3 hours. The absorption quantity was decreasing along with the increasing affinity temperature. So the process of affinity chromatography should be undergone in lower temperature (5℃).It was also influenced by the pH of buffer markedly. The optimum pH value was between 8.5～9.0.Components 8,9 and 10 were purified through DEAE-Cellulose-32 chromatography and preparative electrophoresis from EFEs. The biochemical and enzymatic characteristics of these components were studied. They all belonged to trypsin-like serine proteinase family. They were glycoproteins, and could hydrolyze casein,fibrin and BAEE. The component 8 consisted of two subunits with MWs of 25.5 kD and 17.8 kD respectively. It acted as plasminogen activator and had trysin-like activity, no chymotrypsin-like activity. It could not resist to pepsin hydrolysis when purified. The component 9 contained one polypeptide chain only, and its MW was 33.6 kD. The component 10 consisted of two subunits with MWs of 18.7 kD and 10.9 kD respectively. Components 9 arid 10 had both plasminogen activating activity and fibrinolytic activity, both trypsin-like and chymotrypsin-like activity. But they mainly had plasminogen activating activity.