The Isolation of Porcine Ribonuclease, a Glycoprotein, from Pancreatic Juice

Porcine pancreatic RNase was isolated from the pancreatic secretion of animals with permanent fistulas by a combination of DEAE-cellulose chromatography, adsorption-elution from sulfoethyl-Sephadex, and gel filtration on Sephadex G-100. On the basis of these experiments, a practicable, large scale procedure was developed for the preparation of the enzyme in quantities of 100 to 200 mg. Porcine RNase exhibits a pronounced heterogeneity, detectable by chromatography on sulfoethyl-Sephadex C-50 and by acrylamide gel electrophoresis. At least eight major electrophoretically distinguishable components occur, and there is evidence that these themselves may be mixtures. The heterogeneity is conferred by polysaccharide associated with the enzyme. This polysaccharide is composed of fucose, mannose, galactose, N-acetylglucosamine, and N-glycolylneuraminic acid. Fractions of varying carbohydrate composition and content were obtained by chromatography on sulfoethyl-Sephadex C-50. These fractions as well as the unfractionated enzyme all had identical amino acid compositions. The amino acid analyses showed that the protein moiety in porcine RNase contains 125 residues (mol wt, approximately 14,040) in a distribution that reveals 27 differences with the bovine enzyme. The two fractions of the porcine enzyme that differed most in carbohydrate content were subjected to analytical ultracentrifugation and viscometry. The measured molecular weights (22,300 and 17,200) agreed well with the results of constituent analysis. The hydrodynamic measurements revealed the molecule to be notably asymmetric. The extent of glycosidation does not significantly influence the activity of the enzyme.