Structure of potato carboxypeptidase inhibitor: disulfide pairing and exposure of aromatic residues

Abstract

The determination of the covalent structure of a carboxypeptidase inhibitor from potatoes containing 39 amino acid residues was completed by analysis of the pairing of the 6 half-cystine residues. Since the native inhibitor is resistant to fragmentation by proteases, the protein was first subjected to cleavage at aspartic acid residues by exposure to 0.03 N HCl at 110.degree. C for 10 h to yield a fragment containing 2 chains (residues 6-15 and residues 18-39) held together by 3 disulfide bonds. Digestion with subtilisin and Pronase [Streptomyces griseus], respectively, yielded sets of peptides from which, by diagonal electrophoresis and amino acid analysis, the paired cystinyl residues were identified as Cys-8 to Cys-24, Cys-12 to Cys-27 and Cys-18 to Cys-24. Charge-transfer titration of the native inhibitor with N-methylnicotinamide chloride suggests that 1 of the 2 tryptophan residues and the single tyrosine residue are exposed to the solvent.