Preparative High-voltage Paper Electrophoresis Research Articles

The structure of the polysaccharide moiety of Rhodopseudomonas sphaeroides ATCC 17023 lipopolysaccharide.

The chemical structure of the polysaccharide moiety of the lipopolysaccharide Rhodopseudomonas sphaeroides ATCC 17023 was established. Mild acetic acid hydrolysis of isolated lipopolysaccharide, followed by preparative high-voltage paper electrophoresis afforded three oligosaccharides. They were characterized by chemical and physicochemical studies to be: GlcA(alpha 1----4)dOclA8P, Thr(6') GlcA(alpha 1----4)GlcA and GlcA(alpha 1----4)dOclA, where GlcA is D-glucuronic acid and dOc1A is 3-deoxy-D-manno-octulosonic acid. Carboxyl-reduction of the lipopolysaccharide followed by acid hydrolysis gave a trisaccharide: GlcA(alpha 1----4)Glc(alpha 1----4)Glc, showing the presence of three residues of glucuronic acids in the O-specific chain and indicating that only two of them are reducible by NaBH4. The linkage between the polysaccharide and lipid A was shown to be through a single 1,4-linked residue of dOc1A attached by a 2,6'-linkage to the lipid A moiety.

Control of glycoprotein synthesis. The purification by preparative high voltage paper electrophoresis in borate of glycopeptides containing high mannose and complex oligosaccharide chains linked to asparagine.

The heterogeneity of glycopeptides prepared from pronase digests of various glycoproteins has previously been demonstrated primarily by lengthy paper chromatography of oligosaccharides released from the glycopeptides by enzymatic or chemical means. We describe the use of high voltage paper electrophoresis in borate for the resolution of seven glycopeptides from ovalbumin and seven glycopeptides from human immunoglobulin G. This technique has been used in the preparation of glycopeptide substrates required for the study of certain glycosyltransferases and glycosidases.

Isolation and characterization of [formula omitted]- and [formula omitted]- glycosidic carbohydrate units of human chorionic gonadotropin

The detailed structures of the carbohydrate moiety of hCG have been determined. The structural analyses were carried out on all four asparagine-linked glycopeptides as well as serine-linked carbohydrate chains. The glycopeptides were prepared from the tryptic hydrolysates of the reduced-S-carboxamidomethyl hCG-α and hCG-β and were purified by chromatography on Sephadex G-50 and preparative high voltage paper electrophoresis at pH 1.8. The serine-linked carbohydrate chains were cleaved by β-elimination with alkali in the presence of sodium borohydride and were purified by chromatography on Sephadex G-25. All glycopeptides and the oligosaccharide were examined for homogeneity by high voltage paper electrophoresis, paper chromatography, and chemical composition. The structural studies involved the determination of intersugar and anomeric linkages and monosaccharide sequences and were carried out by a combination of several techniques such as periodate oxidation, methylation and sequential enzymatic degradation.

Glycopeptides isolated from ovine submaxillary mucin.

Desialized ovine submaxillary major mucin was digested extensively with pronase. The digestion product was gel-filtered through Sephadex G-25 then fractionated by ion-exchange column chromatography on Hitachi custom resin No. 2614. Of the resulting fractions, the fractions containing relatively simple glycopeptides were purified by rechromatography on the same ion-exchange column, followed by preparative high-voltage paper electrophoresis. The isolated glycopeptides, A12d, A14a, A17c, and B12, were shown to be homogeneous by high-voltage paper electrophoresis at pH 1.9, 3.7, and 6.5 and by paper chromatography. The results of chemical analysis, alkali treatment, determination of the amino acid sequence and digestion with alpha-N-acetylgalactosaminidase of these glycopeptides indicated their structures to be as follows: glycopeptide A12d, O-alpha-N-acetylgalactosaminyl-Ser-Glx-Pro-Gly; glycopeptide A14a, O-alpha-N-acetylgalactosaminyl-Ser; glycopeptide A17c, O-alpha-N-acetylgalactosaminyl-Ser-Gly-Gly-(O-alpha-N-acetylgalactosaminyl-)Thr-Glx; glycopeptide B12, Gly-(O-alpha-N-acetylgalactosaminyl-)-Ser-Ala.

Fractionation of sialyl oligosaccharides of human milk by ion-exchange chromatography

The sialyl oligosaccharides of human milk are partially separated by ion-exchange chromatography on DEAE-cellulose in acetic acid-pyridine buffers, pH 5.4. One neutral and seven sialyl oligosaccharide fractions are obtained in essentially quantitative yields. The method permits the large-scale preparation of sialyl oligosaccharide fractions from which individual sialyl oligosaccharides may be purified without the necessity of preparative high-voltage paper electrophoresis.

Isolation of Glycopeptides from the Lectin-stimulated Human Peripheral Lymphocyte Cell Surface<xref ref-type="fn" rid="fn1"><sup>1</sup></xref>

Three radioactive glycopeptides were isolated from human peripheral lymphocytes stimulated with Wistaria floribunda mitogen in the presence of D-[14C]glucosamine hydrochloride by mild trypsin digestion followed by gel filtration and preparative high-voltage paper electrophoresis. The carbohydrate compositions of these glycopeptides suggest that one has a sugar chain of the type found in serum glycoproteins, consisting of sialic acid, galactose, N-acetylglucosamine, mannose, and fucose in a molar ratio of 2:2:4:2:1, and the other two have sugar chains like those of mucins, consisting of sialic acid, galactose, and N-acetylgalactosamine in a molar ratio of 1 or 2:1:1. The results of enzymic degradation with purified glycosidases indicate that these sugar chains are similar in structure to their counterparts in human erythrocyte membranes.

The amino acid sequence of pike-whale (lesser-rorqual) pancreatic ribonuclease

Pancreatic RNAase (ribonuclease) from the pike whale (lesser rorqual, Balaenoptera acutorostrata) was isolated by affinity chromatography. The protein was digested with different proteolytic enzymes. Peptides were isolated by gel filtration, preparative high-voltage paper electrophoresis and paper chromatography. The amino acid sequence of peptides was determined by the dansyl-Edman method. Although we do not have an amino acid composition for the whole protein, all peptide bonds were overlapped by one or more peptides. Residues 85-96 are bridged by a peptide of unstaisfactory composition and the sequence here depends, at least in part, on homology for its confirmation. Another region in which a similar situation obtains is residues 39-40. This pancreatic RNAase differs at 24-33% of the positions from all other mammalian pancreatic RNAases sequenced to date, except for pig RNAase, from which it differs by 19%. This indicates that whale RNAase has evolved independently during the larger part of the evolution of the mammals. Lesser-rorqual pancreatic RNAase is partially glycosidated (30%) at asparagine-76 in an Asn-Ser-Thr sequence (residues 76-78). Pig RNAase also has carbohydrate attached to asparagine-76 and is identical with lesser-rorqual RNAase in residues 76-98. Detailed evidence for the sequence has been deposited as Supplementary Publication SUP 50066 (11 pages) at the British Library Lending Division, Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms ginen in Biochem. J. (1976) 135, 5.

The amino acid sequence of dromedary pancreatic ribonuclease.

Dromedary (Camelus dromedarius) RNAase (ribonuclease) was isolated from pancreatic tissue by affinity chromatography. Peptides obtained by digestion with different proteolytic enzymes and CNBr were isolated by gel filtration, preparative high-voltage paper electrophoresis and paper chromatography. Peptides were sequenced by the dansyl-Edman method. All peptide bonds were overlapped by one or more peptides. The polypeptide chain consists of 123 amino acids. A deletion (position 39) was observed in an external loop of the polypeptide chain (residues 35-40), as was found earlier to horse RNAase (Scheffer & Beintema, 1974). A heterogeneity was found at position 103 (glutamine and lysine). Dromedary RNAase differs at 23-32% of the positions from all other pancreatic RNAases sequenced to date. In evolutionary terms this indicates that dromedary RNAase has evolved independently during the larger part of the evolution of the mammals. Detailed evidence for the sequence has been deposited as Supplementary Publication SUP 50046 (14 pages) at the British Library (Lending Division), Boston Spa, Wetherby, W. Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1975) 145, 5.

Release of ethanolamine pyrophosphate during mild acid hydrolysis of the lipopolysaccharide of Pseudomonas aeruginosa.

Low molecular weight solutes released during mild acid hydrolysis of the lipopolysaccharide of Pseudomonas aeruginosa were isolated from the fraction containing the partially degraded polysaccharide, by successive chromatography on columns of Sephadex G-75 and G-10, followed (as necessary) by preparative high voltage paper electrophoresis. The major components identified were 2-keto-3-deoxyoctonic acid, basic amino acids (free and bound), inorganic orthophosphate, ethanolamine phosphate and ethanolamine pyrophosphate. Ethanolamine pyrophosphate has not previously been found in acid hydrolysates of lipopolysaccharides. Ethanolamine phosphate and some of the orthophosphate were apparently produced by breakdown of ethanolamine pyrophosphate.

Glycopeptides isolated from bovine submaxillary mucin

Extensive pronase digestion of the desialized bovine submaxillary major mucin (BSM) resulted in a mixture of glycopeptides. Separation of the glycopeptides into nine fractions, fractions a to i, was achieved by linear gradient elution at constant pH (2.6) of 0.05–0.2 m pyridine-formate buffer from Dowex 50-X2 (pyridinium form). Glycopeptide b 6 was isolated from fraction b by preparative high-voltage paper electrophoresis, and glycopeptides g 5and e 7 from fractions g and e, respectively, by preparative high-voltage paper electrophoresis and preparative paper chromatography, in succession. Homogeneity of these glycopeptides was shown by high-voltage paper electrophoresis at pH 1.9, 3.7, and 6.5 and by paper chromatography. The results of the N-terminal analysis by dansylation method with or without sequential Edman degradation and the analytical data before and after a β-elimination reaction of glycopeptides b 6, g 5, and e 7 indicated the structures of these glycopeptides as follows: Glycopeptide b 6, seryl-( O-α- N-acetylgalactosaminyl-)-serine; glycopeptide g 5, glycyl-( O-α- N-acetylgalactosaminyl-)-serine; glycopeptide e 7, seryl-( O-α- N-acetylgalactosaminyl-)-threonyl-glycyl-( O-α- N-acetylgalactosaminyl-)-serine.