AMINO-ACID-SEQUENCE DETERMINATION AND BIOLOGICAL-ACTIVITY OF THERIN, A NATURALLY-OCCURRING SPECIFIC TRYPSIN-INHIBITOR FROM THE LEECH THEROMYZON TESSULATUM

We purified a trypsin inhibitor: designated therin, from the rhynchobd ellid leech Theromyzon tessulatum. Therin was purified to apparent hom ogeneity by gel-permeation and anion-exchange chromatography followed by reverse-phase HPLC. By a combination of reduction and S-beta-pyridy lethylation, Edman degradation and electrospray mass spectrometry meas urement, 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 inhibito rs family, i.e. the first and second domains of antistasin, hirustasin , ghilanthen and guamerins (I, II). Therin is a tight-binding inhibito r of trypsin (K-i, 45 +/- 12 pM) and has no action towards elastase or cathepsin G. Furthermore, therin (10(-6) M) in conjunction with thero min, a Theromyzon thrombin inhibitor (10(-6) M) significantly diminish the level of human leucocytes activation induced by lipopolysaccharid e (10 mu g) in a manner similar to that of aprotinin. These data sugge st a leech trypsin inhibitor with possible biomedical significance.