A
serine proteinase inhibitor isolated from Leucaena leucocephala seeds (LlTI) 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) 20000 and two
polypeptide chains (alpha-chain, M(r) 15000, and beta-chain, M(r) 5000), the sequence being determined by automatic Edman degradation and by mass spectroscopy. LlTI 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. LlTI inhibits
plasmin (K(i) 3.2 x 10(-10) M), human
plasma kallikrein (K(i) 6.3 x 10(-9) M),
trypsin (K(i) 2.5 x 10(-8) M) and
chymotrypsin (K(i) 1.4 x 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 LlTI. The action of LlTI 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. LlTI 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 LlTI-treated rats.