Rex Montgomery

Microheterogeneity and paucidispersity of glycoproteins : Part I. The carbohydrate of chicken ovalbumin☆

Abstract Five components of l -β-aspartamido-carbohydrate, prepared from crystalline chicken ovalbumin by digestion with pronase, have been isolated and purified by a combination of ion-exchange chromatography and zone electrophoresis. Their sequences may be represented: homology being demonstrated by enzymic hydrolysis with α- d -mannosides (E.C.3.2.1.24) or N-acetyl-β- d -glucosaminidase (E.C.3.2.1.30). The molecular dispersity of ovalbumin is discussed in the light of these results.

Alkaline degradation of glucose: effect of initial concentration of reactants

The alkaline degradation of d-glucose in aqueous Ca(OH)2 at 100°C resulted in a complex mixture of more than 50 compounds, including parasaccharinic acid, that were identified by GLC-MS. The effect of reactant concentrations on the alkaline reaction products was studied by varying the concentration of glucose (1.8–100%, w/w). Under the different reactant concentrations the same saccharinic acids were produced, which is also true when no water was added to the reaction mixture. The increase of glucose concentration favored the formation of C6 acids (2-C-methylpentonic, hexametasaccharinic, and isosaccharinic acids) and decreased the formation of <C6 acids (glycolic, lactic, glyceric, 2-C-methylglyceric, deoxytetronic, and deoxypentonic acids). The identification of parasaccharinic acid provides further support for the existence of the glucose-3,4-enediol.

The glycans of horseradish peroxidase.

Horseradish peroxidase (E.C. 1.11.1.7) isozyme c (HRPc) is a glycoprotein found to contain 21.8% carbohydrate with the average composition: 2 mol GlcNAc, 2.6 mol Man, and 0.8 mol each of Fuc and Xyl. The oligosaccharides of HRPc were investigated by a combination of High pH Anion-Exchange Chromatography with Pulsed Amperometric Detection, methylation analysis and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. The structure of the major oligosaccharide released by digestion with glycopeptidase A, accounting for between 75 and 80% of the total, was confirmed to be [sequence: see text]. Most of the remaining oligosaccharides were found to belong to the (Xyl)xManm(Fuc)fGlcNAc2 (m = 2, 4, 5, 6; f = 0 or 1; x = 0 or 1) family. Less than 5% of the oligosaccharides were of the ManmGlcNAc2 (m = 4 to 7) type. Methylation analysis of holo- and apo-HRPc and its tryptic glycopeptides support the structures proposed for the oligosaccharides. Furthermore, methylation analysis of the tryptic glycopeptides provides evidence for the heterogeneity of the oligosaccharides occurring at each of the N-linked sites.

Stereoselective ring-opening of β-D-mannopyranose 1,2-(alkyl orthoacetates)

Abstract The stability towards alkali of the 3,4,6-tri-O-acetyl-β- D -mannose 1,2-(alkyl orthoacetates) provides a route to the corresponding 3,4,6-tri-O-benzyl-β- D -mannose 1,2-(alkyl orthoacetates). Alkyl groups that have been incorporated onto the orthoacetate ring include methyl, isopropyl, and cyclohexyl. 3,4,6-Tri-O-benzyl-β- D -mannose 1,2-(methyl orthoacetate) (2) was hydrolyzed to 3,4,6-tri-O-benzyl- D -mannose; periodate oxidation converted this compound into 2,3,5-tri-O-benzyl- D -arabinose. Methanolysis of 2 led to methyl 3,4,6-tri-O-benzyl-α- D -mannoside (5) in high yield; methylation of 5, followed by debenzyiation and acetylation, afforded crystalline methyl 2-O-methyl-3,4,6-tri-O-acetyl-α- D -mannoside. An acid-catalyzed, stereoselective rearrangement of the 3,4,6-tri-O-benzyl-β- D -mannose 1,2-(alkyl orthoacetates) was observed. The resulting products were demonstrated to be the corresponding alkyl 3,4,6-tri-O-benzyl-α- D -mannosides.

Development of biobased products.

Research conducted over the past seven years by the biotechnology byproducts consortium (BBC) addresses its mission to investigate the opportunities to add value to agricultural products, byproducts and coproducts and to manage the wastewater arising from agribusinesses in an environmentally favorable way. Since a wide variety of research approaches have been taken, the results are collected in five topic groups: (1) bioremediation that includes anaerobic fermentations of wastes to produce methane and hydrogen, the genetics of methanogenesis and in situ remediation of contaminated aquifer systems, landfill leachates and industrial effluents; (2) land application of fermentation byproducts and their use in animal feeds; (3) biocatalytic studies of transformations of components of corn and soybean oils, peroxidases present in plant products, such as soybean hulls; (4) biochemical reactions for the production of de-icers from industrial water streams, biodiesel production from fats and greases, biodegradable plastics from polymerizable sugar derivatives, single cell foods derived from fungal growth on waste streams, and bacterial polysaccharides from Erwinia species; (5) separation and recovery of components by membrane technologies.

Substrate specificity of D-galactose oxidase☆

Abstract The rate of oxidation of methyl ethers of D -galactose and 2-amino-2-deoxy- D -galactose, and of oligosaccharides and polysaccharides containing D -galactosyl residues having a free hydroxyl group at C-6, has been followed by various procedures that depend either upon the hydrogen peroxide or the aldehyde groups produced, or upon the unoxidized D -galactose residues remaining in the reaction mixture. Derivatives of D -galactose having substituents on the hydroxyl group at C-4 are not oxidized. 2-Amino-2-deoxy- D -galactose residues having glycosyl substituents at C-3 are not oxidized by the enzyme, and therefore, neither are chondroitin 4-sulfate nor dermatan sulfate. No completely satisfactory procedure was found for following the oxidation reaction to termination, which, in none of the cases studied, was 100% complete.

Determination of hexosamines

Abstract A simple method for the colorimetric determination of hexosamines is presented. After deamination of the hexosamine with nitrous acid, phenol-sulfuric acid reagents are added and the absorbancy is measured at 490 mμ. Interfering substances are removed previously by ion-exchange procedures.

Acrylamide gel electrophoresis of the S-sulfo derivatives of fibrinogen☆

Abstract A polyacrylamide gel electrophoresis of sulfitolyzed fibrinogens is described, separating the S-sulfopolypeptides into three main groups, and the α(A) fraction is further resolved into a doublet.

Heterogeneity of glycans at each N-glycosylation site of horseradish peroxidase

The tryptic glycopeptides of horseradish peroxidase isozyme c (HRPc) were studied by methylation linkage analysis, exoglycosidase degradation, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOFMS). Over 90% of the predicted tryptic peptides and glycopeptides of HRPc could be identified in the unfractionated digest. Four glycans, namely (Xyl)Man3(Fuc)GlcNAc2 (major species), (Xyl)Man2(Fuc)GlcNAc2, (Xyl)Man3GlcNAc2, and Man3(Fuc)GlcNAc2 (minor species), were observed at all of the N-glycosylation sites and account for greater than 95% of the carbohydrate. Other members of this glycan family, namely (Xyl)xManm(Fuc)f GlcNAc2 (x = 0 or 1, f = 0 or 1, m = 4, 5, 6, or 7), account for the rest of the glycans. Only traces of high mannose-type glycans were detected in HRPc. Two sites, namely those at Asn-57 and Asn-267, were found to be more heterogeneous than the sites at Asn-13, Asn-158, Asn-186, 198 (doubly glycosylated peptide), Asn-214, and Asn-255. Two of the glycopeptides were observed as part of disulfide-linked species. MALDITOFMS confirmed the N-glycosylation sites previously reported [K.G. Welinder, Eur. J. Biochem., 96 (1979) 483-502] and was used to determine the heterogeneity of the glycan pool at each site.

Glycopeptides from ovalbumin: the structure of the peptide chains☆

Abstract Ovalbumin was hydrolyzed with papain and carboxypeptidase, and the carbohydrate-containing fragments were isolated as three glycopeptides. The carbohydrate composition of each glycopeptide was identical with that of ovalbumin. The studies of these glycopeptides indicate that the amino acid sequence near to the carbohydrate unit is -glu-lys-tyr-asp (carbohydrate)-.