Amino acid sequence determination and biological activity of therin, a naturally occuring specific trypsin inhibitor from the leech Theromyzon tessulatum

Abstract

We purified a trypsin inhibitor, designated therin, from the rhynchobdellid leech Theromyzon tessulatum. Therin was purified to apparent homogeneity by gel-permeation and anion-exchange chromatography followed by reverse-phase HPLC. By a combination of reduction and S-β-pyridylethylation, Edman degradation and electrospray mass spectrometry measurement, the complete sequence of therin (48 amino acid residues; m/z, 5376.35 ± 0.22 Da) was determined. Therin exhibits an approximately 30 % sequence similarity with peptides of the antistasin-type inhibitors family, i.e. the first and second domains of antistasin, hirustasin, ghilanthen and guamerins (I, II). Therin is a tight-binding inhibitor of trypsin (K_i, 45 ± 12 pM) and has no action towards elastase or cathepsin G. Furthermore, therin (10⁻⁶ M) in conjunction with theromin, a Theromyzon thrombin inhibitor (10⁻⁶ M) significantly diminish the level of human leucocytes activation induced by lipopolysaccharide (10 μg) in a manner similar to that of aprotinin. These data suggest a leech trypsin inhibitor with possible biomedical signifance.