Purification and Characterization of Chicken Pepsinogen and Chicken Pepsin

Abstract

Abstract Chicken pepsinogen was extracted from the forestomach of chicken and purified by the consecutive application of acetone precipitation, chromatography on diethylaminoethyl cellulose, and gel filtration on Sephadex G-100. The purified pepsinogen was homogeneous on disc electrophoresis and sedimented as monodisperse material. Chicken pepsin was obtained by the activation of the pure zymogen at pH 1.8. It was separated by gel filtration from the peptides split off during activation. It was found by amino acid analyses in conjunction with molecular weight determinations that chicken pepsinogen is composed of 387 and chicken pepsin of 308 amino acid residues. The amino acid composition of the peptide fraction split off during activation accounts for the difference between zymogen and enzyme. The amino acid composition of chicken pepsin shows that it is a less acidic protein than other pepsins so far investigated. Chicken pepsin contains 39 free carboxyl groups and 16 basic groups, whereas swine pepsin (EC 3.4.4.1) contains 40 free carboxyl and only 6 basic groups. The carboxyl-terminal sequence common to chicken pepsinogen and pepsin is Leu-Ser and a proline residue apparently occupies the third or fourth position from the carboxyl terminus. Determinations of amino-terminal residues failed to reveal the amino terminus of the zymogen whereas a seryl residue was found to be amino-terminal in the enzyme. Lysine was the sole amino-terminal residue found in the peptide fraction split from the zymogen during activation. Both chicken pepsinogen and chicken pepsin contain one sulfhydryl group per molecule. The sulfhydryl group in the zymogen reacted with 5,5'-dithiobis(2-nitrobenzoic acid) at pH 7.3 in solutions containing 1% sodium dodecyl sulfate, but not in solutions in phosphate buffer or in 8 m urea. The sulfhydryl group of the enzyme reacted with 5,1'-dithiobis(2-nitrobenzoic acid) without addition of detergent. The sulfhydryl group and the two carboxyl-terminal residues are not essential for the enzymic activity of chicken pepsin. It was thus found that the chicken pepsin derivative obtained by reaction with 5,5'-dithiobis(2-nitrobenzoic acid) retained 86% of the enzymic activity. The chicken pepsin derivative obtained by the removal of the two carboxyl-terminal amino acids retained 81% of the enzymic activity. This derivative of chicken pepsin seems, however, to undergo autolysis more readily than the native enzyme. Chicken pepsinogen is stable at 24° below pH 10.5, and chicken pepsin is stable below pH 8. At pH 8.5 and above chicken pepsin is rapidly inactivated, and inactivation at 40° and pH 8.5 is accompanied by autolysis.