The primary structure of porcine pancreatic ribonuclease. I. The distribution and sites of carbohydrate attachment.

Reduced, S-aminoethylated porcine ribonuclease was subjected to tryptic hydrolysis. A sequence of fractionation steps that included gel filtration and chromatography over SE-Sephadex C-50, Dowex 50-X2, and Dowex l-X2 afforded a series of glycopeptide fractions which were recognized to derive from three separate regions of the molecule. Heterogeneity of the attached polysaccharide chains precluded the isolation of glycopeptides homogeneous with respect to polysaccharide and hampered the preparation of fractions suitable for amino acid sequence determination. However, fractions representative of individual segments of the primary structure of the protein moiety were obtained. Their amino acid sequences were determined by conventional procedures. These fractions represent 35 of the 124 amino acid residues in the molecule. By reference to the amino acid sequence of porcine ribonuclease, determined in concurrent experiments, the three sites of polysaccharide attachment were recognized to be at positions 21, 34, and 76, designated as Sites I, II, and HI, respectively. Aspartic acid occupies each of these positions and it is presumed that the carbohydrate-peptide attachment in each is represented by a flN-aspartamido 2 acetamido 1,2 dideoxyglucopyranoside unit. By reference to the structure of bovine ribonuclease, the three attachment sites are recognized to be at residues which have external side chains in regions of the molecule remote from the active site. The polysaccharides at the three sites have notably different compositions. The side chain at Site II contains 2 N-acetylglucosamine and 6 mannose residues. With respect to composition and site of attachment it is homologous with the single polysaccharide side chain in bovine ribonuclease B. The side chains at Sites I and III are about twice as large as that at Site II and display a more complex composition. In addition to N-acetylglucosamine and mannose, they contain galactose, fucose, and N-glycolylneuraminic