Isolation,Purification And Properties Of A Thrombin-Like Enzyme From Agkistrodon Acutus Venom

Agkistrodon acutus is a monotypic venomous snake of the serpentes viperidae crotalinae,a second-class endangered terrestrial wildlife,which is widely distributed in East China.The main active constituents of Agkistrodon acutus venom(AAV)are proteins and peptides,which have pharmacological effects such as anti-cardiovascular disease,anti-thrombosis,anti-tumor,analgesia,anti-inflammatory and anti-bacteria.As a class of anti-thrombosis drugs,snake venom thrombin-like enzyme preparations can reduce the content of fibrinogen by hydrolyzing fibrinogen in the body,thereby reducing blood viscosity to improve blood flow,and exert anti-thrombosis effects.In this study,Agkistrodon acutus venom was used as the study object,and a thrombin-like enzyme was purified from it by a variety of chromatographic techniques and its properties were studied.The main research work is as follows.A thrombin-like enzyme named TLE-Ⅰ with an exact molecular weight of 30391 Da was isolated and purified from Agkistrodon acutus venom by three-step chromatography method with DEAE-Sepharose Fast Flow anion exchange chromatography,Heparin-Sepharose 6 Fast Flow affinity chromatography,and Sephacryl S-200 HR gel chromatography.(1)TLE-Ⅰ was identified as a single band by SDS-PAGE electrophoresis.(2)The purity of TLE-Ⅰ was 100% and 92.888% by Molecular Sieve High Performance Liquid Chromatography and C18 Reverse High Performance Liquid Chromatography,respectively.(3)The protein concentration of TLE-Ⅰ was 0.616 mg/m L and the total protein content was 7.392 mg as measured by the Folin-phenol agent method,and the total purification multiplicity was about 618 times after the three-step column chromatography.(4)The enzyme activity of TLE-Ⅰwas measured to be 25056 U,and the specific activity was 3389 U/mg.(5)The experimental results showed that the optimum reaction p H of TLE-Ⅰ was 7.5 and the optimum reaction temperature was 45°C.(6)The results of fibrinogen hydrolysis experiments showed that TLE-Ⅰ was able to hydrolyze fibrinogen with strong hydrolytic activity.(7)Through LC-MS protein identification and amino acid sequence determination,the amino acid sequences of TLE-Ⅰ were VIGGVECDINEHRFLV,and its peptide coverage and amino acid sequence were consistent with the Agkistrodon acutus venom thrombin-like enzyme numbered“Q9I8X2” included in the protein database Uniprot,and TLE-Ⅰ is identified as Agkistrodon acutus venom thrombin-like enzyme.In conclusion,a method for the isolation and purification of thrombin-like enzymes from snake venom of Agkistrodon acutus was investigated in this experiment,the isolation and purification of target components were realized by using the different charges of proteins,biological activity based on heparin anticoagulant effect,and the difference in molecular weight between the proteins.The purification method is simple and the purification efficiency is high,which provides an effective way to further develop and utilize snake venom thrombin-like enzymes.