Purification And Characterization Of Autoproteolytical Protein From Agkistrodon Acutus

This article researched for the reason of the proteolytic phenomenon of the proteins of Agkistrodon acutus snake venom adsorbed in thiophilic adsorption chromatography and purify and characterize the proteolytic proteins.The proteins in the eluant of the thiophilic adsorption chromatography of MW 44kDa were converted to a new protein of MW29kDa when they were put under room temperature over night.To reveal the reason of above phenomenon, we purified the eluant by POROS 20HQ ion-exchange chromatography and got five proteins. One of them(HQ-S) was proved to can be proteolytic. When it was verified to be pure by SDS-PAGE, PAGE and CLC, it could be concluded that HQ-5's autoproteolysis just was the cause of the proteolytic phenomenon of eluant of the thiophilic adsorption chromatographyHQ-5 was identified as a glycoprotein containing 3.5% neutral carbohydrates. Its molecular weight was determined to be 47.6kDa by SDS-PAGE and about SO.SkDa under reduced conditions. It is a metalloprotease, having a basic proteolytic activity with the optimum pH of 9.5 and being inhibited by EDTA . Its anti-coagulation activity was characterized. HQ-5 could be converted to a new protein autoproteolytically when it was incubated hi the basic solution or higher temperature.The new protein (TSK-2) was isolated and purified to homogeneity by TSK G3000SW chromatography from the elutriant of thiophilic adsorption, which was incubated in the condition of 40℃ for 2 hours. Its molecular weight was determined to be 26.8kDa by SDS-PAGE and the same under reduced conditions. Its anti-coagulation activity was characterized. The N-terminal sequence of TSK-2 is RTEFQRYMEIW.It is shown that the obtained proteins(HQ-5 & TSK-2) can be identified as novel components of anti-coagulation enzyme family in the venom of Agkistrodon acutus by the analysis of the N-terminal sequence of TSK-2.