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-beta-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 (Ki, 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 microg) in a manner similar to that of aprotinin. These data suggest a leech trypsin inhibitor with possible biomedical significance.

Abstract:
We purified a chymotrypsin inhibitor, designated cytin, from the rhynchobdellid leech Theromyzon tessulatum. This 7.4-kDa peptide was purified to apparent homogeneity by gel-permeation and anion-exchange chromatographies, followed by reverse-phase HPLC. The structure of cytin was determined by reduction, S-beta-pyridilethylation, automated Edman degradation, and electrospray mass spectrometry. Cytin is formed by the association of two protein chains, which are linked by a disulfide bridge. Chain A consists of 43 and chain B of 22 amino acid residues. Chain B exhibits 40-63% sequence similarity with the N-terminal sequences of subtilisin/chymotrypsin inhibitors isolated from barley seeds. Cytin inhibited chymotrypsin (Ki 600 pM) and weakly inhibited trypsin (Ki 350 nM). This chymotrypsin inhibitor, in contrast to others isolated from leeches, does not inhibit elastase or cathepsin G. Furthermore, cytin (10 microM) significantly diminishes the level of human granulocyte and monocyte activation induced by lipopolysaccharide (1 U/ml) in a manner similar to that of aprotinin. These data indicate that this chymotrypsin inhibitor may be biomedically significant.