A new trypsin inhibitor, bdellin for Korean leech (bdellin-KL), was isolated from blood sucking leech, Hirudo nipponia. It has a molecular mass of 16156.4 Da and pI value of 4.0. The inhibitor was stable at range of pH 1.3 through 10.0 and temperatures ranging from 20 C to 90 C. The bdellin-KL showed inhibition constants of $1.67±0.73×10^{-9}$ M for trypsin and $1.76±1.29×10^{-9}$ M for plasmin but a poor activity against chymotrypsin. Over 60 amino acid residues of N-terminal region showed a high similarity with those of the high-molecular mass bdellin B-3 (HMB), a European leech-derived trypsin-plasmin inhibitor. The first 10 residues that contained P1 reactive site of Lys and the spacing of six cysteines in the peptide are well conserved for the two phylogenetically different species. A N-terminal fragment of 48 amino acids generated by endoproteinase Asp-N with limited proteolysis showed an equivalent inhibitory activity to the intact bdellin-KL. The active fragment was as stable at extreme pH and high temperature as the natural bdellin isolated from the leech.
