Microdetermination of Individual Neutral and Amino Sugars and N-Acetylneuraminic Acid in Complex Saccharides*
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J. Biochem., 76, 783-789 (1974) Microdetermination of Individual Neutral and Amino Sugars and N-Acetylneuraminic Acid in Complex Saccharides* Seiichi TAKASAKI and Akira KOBATA Department of Biochemistry, Kobe University School of Medicine, Kusunoki-cho, Ikuta-ku, Kobe 650 Received for publication, April 4, 1974 A very sensitive method for the quantitative determination of individual neutral and amino sugars and N-acetylneuraminic acid in complex saccharides has been developed. The usefulness of this method was confirmed by applying it to the analysis of several milk oligosaccharides of known structure. Heterosaccharide complexes on the cell surface commonly found in glycoproteins and glyco are important as immunodeterminants, func lipids. tional structures of viruses and hormone re This method is based on quantitative radio ceptors, and may also play a key role in cell isotope labelling of sugars with [3H]-sodium to-cell interactions. However, in the charac borohydride, as first applied by Howell et al. terization of heterosaccharides, the determina for the analysis of total hexose and hexosamine tion of the sugar components has always been contents of IgG (4), and separation of sugar a problem. alcohols by borate paper electrophoresis. The sugar composition in heterosaccharide complexes is usually determined by an appro MATERIALS AND METHODS priate method after hydrolysis in acidic con ditions. Sugars and Reagents-Sorbitol and man After the development of the gas chromato nitol were purchased from Seikagaku Kogyo graphic (1, 2) and ion exchange chromato Co., Ltd. N-Acetylneuraminic acid and galac graphic methods (3), the required sample size titol were purchased from Sigma Chemical Co., was reduced down to scores of micrograms, Ltd. Fucitol and N-acetylneuraminic acid al but this is still too large to be applicable for cohol** were synthesized from fucose and N- the study of cell surface components. acetylneuraminic acid by sodium borohydride This paper deals with a sensitive ana reduction (5). N-Acetyglucosaminitol and N- simple method for the determination of sugars acetylgalactosaminitol were synthesized by the method of Crimmin (6). Glucosaminitol hydro chloride and galactosaminitol hydrochloride * This work has been supported in part by Research Grants from the Jane Coffin Childs Memorial Fund ** The reduction product of N-acetylneuraminic acid for Medical Research, the Naito Foundation, and the Scientific Research Fund (1973-1974) of the Ministry in which the ketone group is converted to a secondary of Education of Japan. alcohol group. Vol. 76, No. 4, 1974 783 784 S. TAKASAKI and A. KOBATA were obtained from their N-acetyl derivatives again evaporated to dryness. The evaporation by hydrolysis in 2 N HC1 for 2 hr at 100•Ž. with 1 M acetic acid was repeated five times After decolorization with a small amount of to ensure the removal of any exchangable active charcoal, the mixtures were filtered. tritium as [3H]-H2O. The filtrates were lyophilized and crystallized For the simple measurement of radioac from aqueous ethanol. Milk oligosaccharides tivity incorporation, the residue was dissolved were isolated from human milk as reported in 1.0 ml of water and a 0.1-0.5 ml aliquot previously (7). [3H]-Sodium borohydride, spe was counted with 7 ml of Bray's solution (10) cific activity 346 mCi/mMole, was purchased using a Packard liquid scintillation counter from Amersham Radiochemical Centre. It was model 3320. Since comMercial [3H]-sodium dissolved in freshly distilled anhydrous di borohydride contains several percent of non methylformamide at 32 ƒÊmoles/ml and stored volatile radioactive material, a blank experi in a tube with an airtight screw cap at -10•Ž. ment without sugar was always done with this Under these conditions no detectable change simple determination method. The blank was of the reductant was observed even after 5 unnecessary in the case of paper electrophore months. Sialidase [EC 3.2.1.18] purified from tic determination of radioactive sugar alcohol Clostridium perfringens was purchased from mixtures (vide infra), as the nonvolatile con Worthington Biochemical Co., Ltd. taminant remains at the origin as a single Buffers-Borate buffer (8), 0.2 M in borate, peak in the electrophoretogram and does not was prepared by dissolving 19.1 gm of borax interfere with the determination of any of the in 500 ml of water, adjusting the pH to 9.50 sugar alcohols. with 1.0 M NaOH, and adding water to make Determination of the Monosaccharide Com a final volume of one liter. Borate buffers of position of Complex Sugars-i) Measurements 0.06 and 0.04 M were prepared by simply dilut of reducing values: For the correct determina ing the 0.2 M solution with distilled water. tion of the monosaccharide composition of Paper Electrophoresis-High voltage paper complex sugars, the total reducing value of electrophoresis of sugar alcohols was performed the acid hydrolysate must be known. For this in various concentrations of borate buffer, pH purpose, complex sugars containing approxi 9.50 on Whatman No. 1 paper. mately 1-2 nanomoles of monosaccharides Sugar alcohols (less than 10 ƒÊg) were were heated in 1 N HCl for 2 hr at 100•Ž. spotted and were located after electrophoresis The hydrolysate was freed from acid by re with periodate-benzidine reagent (9). peated evaporation with water (five times). Paper electrophoresis in pyridine-acetate The residue was dissolved in 5 pl of 0.05 N buffer, pH 5.4 (13 ), was used to separate the NaOH and mixed with 5, 10, and 20 ƒÊl of reduction product of the acid hydrolysate of [3H]-sodium borohydride solution, as in the 3•Œ-sialyllactose. standard reduction procedure. The radioac Standard Reduction Procedure for Mono tivity incorporated was measured by the simple saccharides with [3H]-Sodium Borohydride method described in the standard reduction Five nanomoles or less of monosaccharides in procedure. This preliminary experiment gives 5 ƒÊ1 of water was mixed with 20 pl of alkaline an approximate reducing value by simple cal [3H]-sodium borohydride solution. The solu culation from the specific activity of the re tion was prepared by mixing 40 pl of dimethyl agent. The specific activity can be obtained formamide solution of [3H]-sodium borohydride by determining the radioactivity incorporated (vide supra) with 1 ml of 0.05 N NaOH. The into 5 nmoles of glucose by the standard re pH of the mixture was 12.0. The reaction duction procedure. mixture was incubated at 30•Ž for 4 hr, and The optimal conditions for the hydrolysis the reaction was stopped by adding 100 ƒÊ1 of of various complex heterosaccharides into glacial acetic acid. The acidic mixture was monosaccharides could vary according to the then evaporated to dryness and the residue sample, and a time course study for each sam was dissolved in 0.5 ml of 1 M acetic acid and ple may be necessary for the accurate deter- J. Biochem. MICRODETERMINATION OF SUGAR COMPOSITION 785 mination of monosaccharide composition by this neutral oligosaccharide fraction, the number method. The following procedures were con of N-acetylneuraminic acid moieties in the firmed to be useful for the analysis of sugar original sample could be obtained. A blank composition in milk oligosaccharides, experiment without sugar was necessary for úA) Procedure I: The complex sugar sam estimation of the radioactivity incorporated into ple, 0.5-1.0 jug, was heated in 0.4 ml of 1.0 N the neutral oligosaccharide fraction, since the HCI for 2 hr at 100•Ž. The hydrolysate was nonvolatile radioactive contaminant in [3H]- evaporated to dryness and the residue was sodium borohydride was included in this frac freed from acid by evaporation with 0.5 ml of tion. The neutral oligosaccharide fraction can water five times. The residue was dissolved further be analyzed for its monosaccharide in 20 pl of 0.05 N NaOH and subjected to the composition by procedure I. standard reduction procedure. The labelled sample was spotted on Whatman No. 1 paper, RESULTS AND DISCUSSION and subjected to paper electrophoresis in borate Separation of Sugar Alcohols by Paper buffer. The electrophoretogram was monitored Electrophoresis-Nine sugar alcohols derived with a Packard radiochromatoscanner model from monosaccharides usually found in glyco 7201 and the radioactive region was cut out, proteins and glycolipids were subjected to paper extracted with 1 ml of water in a counting electrophoresis in various concentrations of vial and the radioactivity determined with a borate buffer, pH 9.5. As shown in Fig. 1-B, Packard liquid scintillation spectrometer model N-acetylneuraminic acid alcohol, galactitol, 3320 with 7 ml of Bray's solution (10 ). fucitol, mannitol, sorbitol, N-acetylgalacto- As will be discussed later, N-acetyl- saminitol, and N-acetylglucosaminitol were suc neuraminic acid was completely destroyed un- cessfully separated from each other when elec der these conditions and did not give any false trophoresis was carried out with 0.06 M borate radioactive peaks. buffer, 40 V/cm for 2.5 hr, Glucosaminitol and úB) Procedure II: This procedure is ap galactosaminitol could not be separated, but plicable for the determination of N-acetyl- were separated from seven other sugar alco neuraminic acid in oligosaccharides. O1igo hols under these conditions. saccharides, 0.5-1.0 ƒÊg, were dissolved in 0.2 These two amino sugar alcohols, however, ml of 0.01 N HC1 and heated for 10 min at were clearly separated when the electrophore- 100•Ž. The reaction mixture was lyophilized and subjected to the standard reduction pro cedure. The labelled sample was freed from boric acid by repeated evaporation with 2% acetic acid in methanol, and then separated into N-acetylneuraminic acid alcohol and a neutral oligosaccharide either by paper electro phoresis in pyridine-acetate buffer, or on an ion exchange column as follows. The residue was dissolved in 0.5 ml of H20 and passed through a column (0.5 cm di ameter) containing 3 cm of Amberlite AG-50 Fig.