AMINO-ACID-SEQUENCE DETERMINATION AND BIOLOGICAL-ACTIVITY OF CYTIN, ANATURALLY-OCCURRING SPECIFIC CHYMOTRYPSIN INHIBITOR FROM THE LEECH THEROMYZON TESSULATUM

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