Purification And Partial Characterization Of The Trypsin Inhibitor From Cicer Arietinum L.

Trypsin Inhibitor (TI) is a kind of Serine proteinase inhibitors. It exists in a wide range oforganisms including animals, plants and microorganisms. TI takes part in many physiologicalprocesses through adjusting a series of proteinase activities. TI, one of the most importantprotease inhibitors, has great medicinal value for its effectiveness in treatments of acutepancreatitis, emphysema, multifunctional failure, anti-shock, cardiac surgery to reduce bloodloss, preventing cerebral edema and cerebral ischemia, prevention of premature and unilateralpulmonary ischemia-reperfusion injury, and other related diseases treatments. Therefore,seeking new and highly effective TI will provide theoretical meaning as well as practicalvalue.A new trypsin inhibitor, designated CPTI, was isolated and purified from the seeds of Cicerarietinum L. by ammonium sulfate grade precipitation, anion exchange chromatography, andgel filtration on Sephadex G-100, and subsequently partial characterization of CPTI was alsostudied.The crude extract was obtained by decorticating, grinding, as well as defatting of Cicerarietinum seeds with petroleum ether. We employed the orthogonal design method to test theeffects of four factors, including temperature of extraction solution (A), volume of extractionsolution (B), pH value of extraction solution(C) and time of extraction (D), on the extractionof trypsin inhibitor, then after practised the optimized procedure. For the sake of highinhibition rate, Cicer arietinum L. was selected to extract TI for further characterization. Theoptimized criteria of TI extraction procedures were pH4.0,40times of solvent volume,50℃and1.5h for extraction per time. The trypsin inhibitor was purified further from the crudeextract by precipitating with35%~75%(NH4)2SO4, ion-exchange chromatography on DEAEcellulose (DEAE-52) and gel filtration on Sephadex G-100. The molecular weight of CPTI is25.7KDa by SDS-PAGE. CPTI was of competitive type inhibtor with a Ki value of3.88×10-7mol/L (BAPNA as substrate). When heated up to100℃for60min,it still had78%residualactivities, so CPTI was thought to be heat-tolerating. The studies on enzyme-catalyzedreaction showed that the inhibitor from chickpea seeds had a strong inhibition on trypsin, slight inhibition on chymotrypsin, no inhibition on pepsin, papain and subtilisin.