[Three-step column chromatographic method for separation and purification of thrombin-like enzyme from venom of Agkistrodon halys pallas].

A three-step column chromatographic method, utilizing CM Sepharose CL-6B, DEAE Sepharose Fast Flow and Sephadex G-75 in sequence, for the separation and purification of thrombin-like enzyme from crude venom of Agkistrodon halys pallas was developed. Based on the separation results on lab-scale chromatographic column, this isolation and purification process was amplified according to the diameter of the chromatographic columns used in labscale. It was found that when the amount of the sample loaded on the large-scale columns was 25 times as that loaded on the lab-scale columns, the quality level of the thrombin-like enzyme obtained with the large-scale columns was almost the same as that obtained with lab-scale columns. The relative molecular mass of thrombin-like enzyme obtained was about 33500, and its purity was about 96%. When using this method to isolate thrombin-like enzyme from crude venom of Agkistrodon halys pallas, the total protein yield and total activity yield of thrombin-like enzyme were about 0.3% and 64% respectively, and specific activity was more than 2000 U/mg.