Search

Ten oligomannose-type sugar chains (ManGlcNAc2-Man5GlcNAc2) were prepared from various glycoproteins and fluorescence labeled with 2-aminopyridine. The fluorescent pyridylamino (PA)-sugar chains were first separated into five fractions according to their molecular sizes by HPLC on a TSK gel Amide-80 column. Each fraction was then separated into the component PA-sugar chains by reversed-phase HPLC on a Capcell Pak C18 column according to their chemical structures. The method is useful for studying the substrate specificities of alpha-mannosidases with Man5GlcNAc2-PA as a substrate.

Fluorescence labeling was used in the analysis of partial acetolysis products of oligomannose-type sugar chains with five to nine mannose residues. The principle of the method was the pyridylamination of fragments obtained by the partial acetolysis of pyridylamino sugar chains and the identification of the fragments with an HPLC apparatus equipped with a fluorescence spectrophotometer. The method was tested by analysis of eight oligomannose-type sugar chains with known chemical structures and was found to be effective for analysis of branching structures with samples of 0.5 nmol.

Amino acid analyzer and gas chromatographic analyses of methanolysates of model compounds of N-glycosidic and O-glycosidic N-acetylglucosamine shows that the main product of N-glycosidic N-acetylglucosamine is glucosamine and that of O-glycosidic N-acetylglucosamine is methyl α-glucosaminide. The amounts of N-acetylglucosamine in glycoproteins determined by methanolysis followed by gas-liquid chromatography represent the amount of O-glycosidic N-acetylglucosamine. A new method was developed to determine O-glycosidic and N-glycosidic N-acetylglucosamine in glycoproteins. Glucosamine and methyl α-glucosaminide in the methanolysates of glycoproteins are determined in an amino acid analyzer using the buffer system that is usually used for analysis of basic amino acids in the two-column system. The contents of O- and N-glycosidic N-acetylglucosamine of Taka-amylase A, ovalbumin, and egg white and yolk flavoproteins determined by the method agree reasonably well with the values calculated from the carbohydrate structures or compositions of the compounds. Therefore, this method can be used to determine the number of N-glycosidically linked carbohydrate chains together with the amount of O-glycosidic N-acetylglucosamine in glycoproteins.

A mixture of p-nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranoside (FG5P) and p-nitrophenyl alpha-D-glucoside (GP) was incubated with cyclomaltodextrin glucanotransferase (CGTase) [EC 2.4.1.19]. Analysis of the digest by HPLC showed that the products were p-nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-alpha-D-glucopyranoside (FG4P) and p-nitrophenyl alpha-D-maltoside (G2P), and no other product could be detected. Based on the reaction, a sensitive method to assay for CGTase was developed.

Human salivary alpha-amylase isozymes were rapidly separated from each other by high-performance liquid chromatography with a postcolumn assay. The eluate from the HPLC column was mixed continuously with an intramolecularly quenched fluorescent substrate, p-nitro-phenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-alpha-D-glucopyranoside delivered by a pump. The mixture was incubated in a reaction coil, and the fluorescence intensity was continuously measured by a fluorescence detector. The assay was based on the marked increase in fluorescence with the enzymatic cleavage of the glycosidic bond of the substrate that links the fluorogenic and quenching moieties.

A new method of the analysis of neutral sugars was developed based on the separation of their corresponding glycamines. Sugars and ammonia were combined by means of the reduction with sodlum cyanoborohydride. The glycamines thus obtained were quantitatively analyzed by an automatic amino acid analyzer. Satisfactery results were obtained in the analyses of the constituent sugars of several polysaccharides and glycoconjugates.

A method for the quantitation of picomole amounts of neutral and amino sugars in glycoconjugates was developed. Glycoconjugates were hydrolyzed with a mixture of equal amounts of 4 M trifluoroacetic acid and 4 M hydrochloric acid, and the free amino groups were acetylated. Sugars were coupled with 2-aminopyridine. After the excess reagents were removed by gel-permeation high-performance liquid chromatography, the fluorescent pyridylamino derivatives of sugars were separated and quantified by high-performance liquid chromatography on a reversed-phase column. This method allowed the determination of 0.01-10 nmol of sugars. About 100 pmol of several glycoconjugates were analyzed by the present method, with satisfactory results.

A mixture of the pyridylamino (PA) derivatives of 12 oligomannose-type sugar chains was fractionated into five fractions (mannose5N-acetylglucosamine2-PA approximately mannose9N-acetylglucosamine2-PA) by size-fractionation HPLC with a MicroPak AX-5 column. Each fraction thus obtained was then analyzed by reversed-phase HPLC with a Cosmosil 5C18-P column. In this way, the 12 PA-oligomannose-type sugar chains were completely separated from each other. The method was used to identify the structures of oligomannose-type sugar chains of human C3, the third component of human complement.