Журнал аналитических и биоаналитических методов

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Utilizing Enzymatic Method, Fibrin-Zymography and Liquid Chromatography Mass Spectrometry to Identify the Active Protein in Lumbrokinase Drug Substance

Hong Jiang

Lumbrokinase, extracted from the cultured earthworm of Eisenia fetida, is widely used to prevent or treat thrombosis as biochemical medicine in China. However, the molecular mechanism and protein components in the drug have not been clearly understood and the specification could not illustrate its characteristic comprehensively. In this study, we investigated the mechanism of lumbrokinase by the fibrin plate method. The results revealed that all the five manufacturers’ products contained both fibrinolytic components and kinase components. We used the method of fibrinzymography to show the existence and activity of lumbrokinase. The fibrin-zymography showed that lumbrokinase was a group of fibrinolytic proteins and there were slight differences between the products of different manufacturers. Besides, we proved the fibrin-zymogram gel could be used as the identification method to distinguish Eisenia fetida from other earthworm species. Then we identified the ingredients by mass spectrometry. The results suggested that fibrinolytic related components existed in the drug, including fifteen proteins. We further compared these proteins with other serine proteins. We observed the amino acid sequence and the catalytic triad of the identified proteins were similar with human trypsin and bovine trypsin. Besides, some also had similar characteristics with human tissue-type (tPA) plasminogen activators and urokinase (uPA) plasminogen activators. These results demonstrated lumbrokinase products contained two major groups of protein components, which suggested two different functions. One is the direct degradation of fibrin; the other is indirectly dissolving thrombus by activating the plasminogen into plasmin.