Leucaena leucocephala serine proteinase inhibitor: primary structure and action on blood coagulation, kinin release and rat paw edema

摘要

A serine proteinase inhibitor isolated from Leucaena leucocephala seeds (L1TI) was purified to homogeneity by acetone fractionation, ion exchange chromatography, gel filtration and reverse phase chromatography (HPLC). SDS-PAGE indicated a protein with M_r 20 000 and two polypeptide chains (α-chain, M_r 15 000, and β-chain, M_r 5000), the sequence being determined by automatic Edman degradation and by mass spectroscopy. L1TI is a 174 amino acid residue protein which shows high homology to plant Kunitz inhibitors, especially those double chain proteins purified from the Mimosoideae subfamily. L1TI inhibits plasmin (K_i 3.2 * l0~(-10) M), human plasma kallikrein (K_i 6.3 * 10~(-9) M), trypsin (K_i 2.5 * 10~(-8) M) and chymotrypsin (K_i 1.4 * 10~(-8) M). Factor XIIa activity is inhibited but K_i was not determined, and factor Xa, tissue kallikrein and thrombin are not inhibited by L1TI. The action of L1TI on enzymes that participate in the blood clotting extrinsic pathway is confirmed by the prolongation of activated partial thromboplastin time, used as clotting time assay. The inhibition of the fibrinolytic activity of plasmin was confirmed on the hydrolysis of fibrin plates. L1TI inhibits kinin release from high molecular weight kininogen by human plasma kallikrein in vitro and, administered intravenously, causes a decrease in paw edema induced by carrageenin or heat in male Wistar rats. In addition, lower concentrations of bradykinin were found in limb perfusion fluids of L1TI-treated rats.