Studies On Separation And Purification Of Lumbrokinase

Lumbrokinase is a common medicine used in the treatment of cardiovascular and cerebrovascular diseases. It is a antithrombotic active substance which found in a traditional Chinese herbal medicine-earthworms, has role of promoting blood, lowering blood pressure and lowering blood viscosity. It has a special affinity with thrombosis, can track thrombolysis, effectively dissolve microsphere and repair damaged endothelial cells. Lumbrokinase has significant advantages of fewer side effects, safety, more significant effect and broad market prospects.At present, there are many researches on extraction and separation of lumbrokinase in China. Ammonium sulfate precipitation is always used in purification process, which is Time-consuming, complicated separation and purification, not suitable for industrial production. Lumbrokinase can be applied to the treatment and prevention of thrombosis.How to effectively extracted plasmin-lumbrokinase from earthworms is a significant application. In this study, crude extracts of lumbrokinase using the thermal stability characteristics, which use method of adopting appropriate temperature to remove part of hybrid protein. This can facilitate post-purification; It can obtain more than95%purity of the lumbrokinase using only a two-step chromatographic separation, which greatly simpli fies purification steps to improve the purity of the lumbrokinase.In this paper, we centrifuge earthworm homogenate twice, use300K ultrafiltration hollow fiber column and5K ultrafiltration hollow fiber column to purify and concentration, reducing the burden of post-purification. We obtain the best conditions by optimizing the heat treatment, It add before the second centrifugation step to remove heat-labile protein impurities.300K hollow fiber column and5K hollow fiber column can further purify and concentrate the crude extract, which reduce the crude extract and volume of the hybrid protein, simplify the post-processing step.After optimization of a series of conditions, we determine using the cyanogen bromide-activated Sepharose4B affinity gel to capture protein,10K ultrafiltration column to remove ammonia and concentrate protein, DEAE anion exchange chromatography to obtain more than95%purity of the lumbrokinase.Through a series of purification, we obtain lumbrokinase which specific activity is210000U/mg, relative molecular weight is28000by SDS-PAGE analysis, purity of over95%by HPLC.