Two Trypsin Inhibitors from Porcine Pancreatic Juice

Abstract

Abstract Two trypsin inhibitors of the Kazal type have been isolated from porcine pancreatic juice. They have been prepared in 65% yield by gel filtration on Sephadex G-75 at pH 8.1 in the presence of 10-4 m diisopropyl phosphofluoridate, filtration through diethylaminoethyl cellulose at pH 9, and equilibrium chromatography on sulfoethyl Sephadex at pH 5.4. The major component, Porcine Inhibitor I, has a chemical molecular weight of 6024 and contains 56 residues per molecule: Asp4, Thr6, Ser6, Glu7, Pro5, Gly4, Ala1, Cys6, Val4, Ile3, Leu2, Tyr2, Lys4, Arg2. The minor component, Porcine Inhibitor II, contains 1 residue each fewer of glutamic acid, threonine, serine, and proline. The amino-terminal amino acid sequences, Thr-Ser-Pro-Gln-Arg-Glx-Ala and Arg-Glx-Ala (where Glx denotes glutamyl or glutaninyl), have been determined for Porcine Inhibitors I and II. It is likely that Inhibitor II is equivalent to residues 5 through 56 of Inhibitor I. The inhibitor isolated from bovine pancreatic juice (Greene, L. J., Rigbi, M., and Fackre, D. S., J. Biol. Chem., 241, 5610 (1966)) contains 56 amino acid residues and has the amino-terminal sequence Asn-Ile-Leu-Gly-Arg-Glu-Ala. There are a minimum of eight differences in amino acid sequence when Porcine Inhibitor I is compared with bovine inhibitor. The polypeptides are homogeneous by equilibrium chromatography, electrophoresis, and amino acid analysis, and also on the basis of the stoichiometry of their interaction with trypsin. The preparations appear to contain small amounts of dimer not in rapid equilibrium with monomer. The monomer molecular weights, determined by equilibrium centrifugation, and the minimum molecular weights, determined from the stoichiometry of the interactions with trypsin, are consistent with the chemical molecular weights calculated from amino acid composition. The inhibitors are secreted in the pancreatic juice in the free form (not combined with trypsin) and together are equivalent to 0.1% of the total protein in pancreatic juice. The relative concentrations are 4:1 for Inhibitors I and II, respectively. Burck, Cerwinsky, and Grinnan (Abstracts of the American Chemical Society Meeting, Chicago, September 1967, p. C-22) have prepared a fraction containing both inhibitors from acid extracts of porcine pancreas, and Fritz et al. (Hoppe-Seyler's Z. Physiol. Chem., 348, 405 (1967)) have isolated a porcine inhibitor containing 53 residues of similar composition and properties. A partial purification of prekallikrein was achieved when porcine pancreatic juice was filtered on Sephadex G-75.