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The Reactive Site of Trypsin Inhibitors

From the ¹ From the Department of Chemistry, Purdue University, Lafayette, Indiana 47907

Virgin soybean trypsin inhibitor has 1 NH₂-terminal aspartic acid or asparagine residue and no COOH-terminal residues that can be released by carboxypeptidase B. Upon incubation with 1 mole % of trypsin for 24 hours at pH 3.75, virgin inhibitor is converted to a modified inhibitor which has 2 NH₂ terminals aspartic acid or asparagine and isoleucine. Approximately 1 mole of arginine is released from it by the action of carboxypeptidase B. Reduction and carboxymethylation of virgin inhibitor leads to formation of one major fragment as judged by Sephadex chromatography; the same treatment of modified inhibitor leads to formation of two fragments. The sum of the amino acid compositions of these two fragments accurately corresponds to the amino acid composition of soybean trypsin inhibitor. The smaller fragment is composed of approximately 64 amino acids and has NH₂-terminal aspartic acid or asparagine residue and a COOH-terminal arginine residue. It is concluded that the modification reaction consists of conversion of single chain inhibitor to two chains held by a single disulfide bond by tryptic cleavage of a single Arg-Ile bond located approximately between residues 64 and 65.

Evidence is also presented that modification of chicken ovomucoid consists of conversion of single chain to two chain protein by cleavage of a single Arg-Ala bond. The fragments may be held by a disulfide bond or bonds.

Compilation of considerable amount of evidence obtained by others suggests that part of the reactive site of many, or possibly all, naturally occurring trypsin inhibitors consists of a trypsin-accessible Arg-X or Lys-X bond contained within a disulfide loop.

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