PRIMARY STRUCTURE OF A KUNITZ-TYPE TRYPSIN-INHIBITOR FROM ENTEROLOBIUM-CONTORTISILIQUUM SEEDS

A trypsin inhibitor was isolated from Enterolobium contortisiliquum se eds. Starting with a saline extract, ECTI (E. contortisiliquum trypsin inhibitor) was purified as a homogeneous protein by acetone precipita tion, ion-exchange chromatography (DEAE-Sephadex A-50), gel filtration (Sephadex G-75 and Superose 12) and reversed phase HPLC (mu-Bondapak C-18). The amino acid sequence was determined by automatic degradation and by DABITC/PITC microsequence analysis of the reduced and carboxym ethylated protein and also of purified peptides derived from the prote in by cleavage with iodosobenzoic acid and by enzymic digestion with t rypsin, chymotrypsin and Staphylococcus aureus V8 protease. ECTI conta ins 174 amino acid residues in two polypeptide chains, an cc-chain con sisting of 134 residues and a beta-chain made up of 40 residues. The i nhibitor displays a high degree of sequence identity with other Kunitz -type proteinase inhibitors isolated from the Mimosoideae subfamily. T he reactive site was identified (by homology) as the arginine-isoleuci ne peptide bond at position 64-65. ECTI inhibits trypsin and chymotryp sin in the stoichiometric ratio of 1:1 and also Factor XIIa, plasma ka llikrein and plasmin, but not thrombin and Factor Xa.