Scorpion Anticoagulant Activity Of Parts Of The Separation And Analysis And Enzymatic Hydrolysis Kinetics Study

Objective:To study the enzymatic process of scorpion, separate and purify the anti-coagulate active components; explore the enzymatic hydrolysis reaction mechanism, calculate the thermodynamic and dynamics parameters.Methods:Taken the area of fibrinogen plate assay lysis zone and activated partial throboplstin time (APTT) as the index, combined with optical density and hydrolysis degree of protein, optimum conditions of scorpion's enzymatic process with pepsin and trypsin to obtain parameters as hydrolysis temperature,time and ratio of enzyme-substrate. Gel filtration was used to separate and purify mixture of peptide from the pepsin enzymolysis of scorpion and do preliminary research on quality standard. Based on M-M equation, investigated the dynamics during the hydrolysis process, and figure out the thermodynamic and dynamics foundation such as rate constant and activation energy and etc.Results:1) The best enzymatic process of scorpion was as follows:Scorpion power was firstly hydrolyzed for 3 hours in an artificial gastric liquid at 37℃which contained 3.0% pepsin (pepsin to substrate). Then the residue was hydrolyzed for 4 hours in artificial intestine liquid at the same temperature and contained 5.0% trypsin (trypsin to substrate).2) The hydrolysates were purified with gel filtration chromatography in Sephadex G-100, Sephadex G-50 or SephadexG-25 in proper order, eluted by deionized water, obtained the anti-coagulate active components QF50-3-③from the eluent by pharmacodynamic screening.3) Drawing the curve of hydrolysis in different conditions, deduced the dynamics dH/dt=aS 0e-bH= (19.525E0+1.2666S0) e-0.098h, and the reaction constants were determined as Km=0.00363(g·L-1), Ea=34.6577 KJ·mol-1,in accordance with the proposed kinetic model.Conclusion:established the research route of extracting scorpion, which provides a kind of research mode for animal drugs enzyme-extracting; separate and purify the anti-coagulate active components which lays the foundation for scorpion's anticoagulation pharmacodynamical research; do research on thermodynamic and dynamics during the hydrolysis process, which provides basic theoretical data for process design and condition investigation of scorpion enzymatic process.