STUDIES ON TRYPSIN INHIBITORS Part IX. Synthesis and Trypsin Inhibitory Activity of the Duopentacontapeptide Corresponding to the Amino Acid Sequence of Porcine Pancreatic Secretory Trypsin Inhibitor II (Kazal)*

The synthesis of the protected duopentacontapeptide corresponding to the entire amino acid sequence 1–52 of porcine pancreatic secretory trypsin inhibitor II (Kazal type) is described. The benzyloxycarbonyltetradecapeptide tert-butyloxycarbonylhydrazide (sequence 1–14) was selectively deblocked with trifluoroacetic acid and used to acylate, by the azide procedure, the peptide free base corresponding to the sequence 15–52. The isolated material was purified by ion exchange chromatography and the protecting groups were removed by successive treatments with anhydrous hydrogen fluoride, 1M piperidine and mercuric acetate. Folding and formation of the disulfide bonds was accomplished by air oxidation in 0.02M phosphate buffer, pH8. Determination of the inhibitory capacity indicated that the synthetic material is about 50% effective, at 30:1 inhibitor:trypsin molar ratio, in inhibiting the tryptic hydrolysis of Nα-benzoyl-dl -arginine-4-nitroanilide. Full inhibition was achieved at a higher inhibitor:trypsin molar ratio. The stability constants and the standard free energy of binding of the complex between trypsin and the synthetic inhibitor have been determined.