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 column, DEAE Sepharose Fast Flow column and Sephadex G-75 column 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 three-step column chromatographic isolation and purification process was scaled-up according to the diameter of the used chromatographic columns. Then, it was found that, when the amount of the crude venom sample loaded on large-scale chromatographic column was 25 times as that loaded on the lab-scale chromatographic column, the quality level of the thrombin-like enzyme obtained with the large-scale chromatographic column was almost the same as that obtained with the lab-scale chromatographic column. When using this three-step column chromatographic method to isolate and purify thrombin-like enzyme from crude venom of Agkistrodon halys pallas, the total mass yield, the total activity yield and the purity of the obtained thrombin-like enzyme were about 0.3%, 64% and 96%, respectively, and the specific activity of thrombin-like enzyme was not lower than 2000 U/mg.