The formation of a dimeric product of raffinose by the action of galactose oxidase

• 1.1. Galactose oxidase is known to catalyze the oxidation of the C-6 hydroxymethyl group of galactose to an aldehyde group.
• 2.2. When the products of a galactose oxidase-catalase treatment of raffinose were examined by gel filtration and ion exchange chromatography, we found that in addition to the expected 6'-aldehydoraffinose, two other components were present.
• 3.3. One component was neutral and had an elution volume close to that of maltopentaose and on treatment with sodium borohydride or hypoiodite, this component was converted to raffinose or 6'-carboxyraffinose, respectively.
• 4.4. Fast atom bombardment mass spectroscopy in the negative mode indicated that the major molecular ion had an M/Z of 1003.
• 5.5. These data are consistent with this component being a dimer of 6'-aldehydoraffmose.

     

Synthesis and rapid purification of UDP-[6-3H]galactose

UDP[6-3H]galactose of high specificity can be obtained by oxidation of the C-6 hydroxymethyl group of UDP-galactose by galactose oxidase and subsequent reduction by sodium borotritide. One-step purification of the nucleotide sugar involves anion-exchange chromatography on a Pharmacia Mono Q column. Radiolabeled UDP-N-acetylgalactosamine can also be synthesized and purified by this procedure. Both nucleotide sugars can be used for sugar incorporation studies using the appropriate glycosyltransferase.     

Oxidation of methyl α-d-galactopyranoside by galactose oxidase: products formed and optimization of reaction conditions for production of aldehyde

The reaction conditions of galactose oxidase-catalyzed, targeted C-6 oxidation of galactose derivatives were optimized for aldehyde production and to minimize the formation of secondary products. Galactose oxidase, produced in transgenic Pichia pastoris carrying the galactose oxidase gene from Fusarium spp., was used as catalyst, methyl α-d-galactopyranoside as substrate, and reaction medium, temperature, concentration, and combinations of galactose oxidase, catalase, and horseradish peroxidase were used as variables. The reactions were followed by ¹H NMR spectroscopy and the main products isolated, characterized, and identified. An optimal combination of all the three enzymes gave aldehyde (methyl α-d-galacto-hexodialdo-1,5-pyranoside) in approximately 90% yield with a substrate concentration of 70 mM in water at 4 °C using air as oxygen source. Oxygen flushing of the reaction mixture was not necessary. The aldehyde existed as a hydrate in water. The main secondary products, a uronic acid (methyl α-d-galactopyranosiduronic acid) and an α,β-unsaturated aldehyde (methyl 4-deoxy-α-d-threo-hex-4-enodialdo-1,5-pyranoside), were observed for the first time to form in parallel. Formation of uronic acid seemed to be the result of impurities in the galactose oxidase preparation. ¹H and ¹³C NMR data of the products are reported for the α,β-unsaturated aldehyde for the first time, and chemical shifts in DMSO-d₆ for all the products for the first time. Oxidation of d-raffinose (α-d-galactopyranosyl-(1-6)-α-d-glucopyranosyl-(1-2)-β-d-fructofuranoside) in the same optimum conditions also proceeded well, resulting in approximately 90% yield of the corresponding aldehyde.     

Purification of galactose oxidase from Dactylium dendroides by affinity chromatography on melibiose-polyacrylamide

Galactose oxidase is a fungal enzyme which is known to oxidize the C-6 hydroxymethyl of galactose and galactosamine to an aldehyde group. It has been widely used in glycoconjugate research, for example in the labeling of asialoglycoproteins. We have developed a simple affinity purification for galactose oxidase using melibiose-polyacrylamide. This affinity procedure was used to purify the enzyme from ammonium sulfate precipitates of culture filtrates of Dactylium dendroides. The material containing proteases and other contaminants is eluted in the buffer wash. The galactose oxidase is then specifically eluted from the column with buffer containing 0.1 M D-fucose or D-galactose. Using this procedure, the enzyme was also purified from commercial samples of galactose oxidase which contain high proteolytic activity.     

Chemical modifications of carboxylated chitosan

C-6-carboxylated chitosan obtained by oxidation of chitosan was selectively modified in order to obtain derivatives similar to bacterial antigens. Selective O-acetylation of 6-carboxyl chitosan afforded a modified polysaccharide with the 2-amino group available for further modifications to create carbonyl groups. A deaminative degradation reaction allowed the formation of oligosaccharides with terminal aldehyde groups. Reductive alkylation with lactose introduced lactityl branches which were oxidized with galactose oxidase to give aldehyde groups in its -galactose residues.     

Preparation of radiolabeled GM2 and GA2 gangliosides.

GM2 and GA2 gangliosides from the brain of a patient who died of Sandhoff's disease were purified by solvent partition, silicic acid and silica gel column chromatography, and silica gel preparative thin-layer chromatography. They were tritiated in the terminal N-acetylgalactosamine residue using galactose oxidase and sodium [3H]borohydride with the inclusion of catalase and peroxidase into the oxidation reaction. The specific activities were 4.62 X 10(8) dpm/mumol of GM2 ganglioside and 5.54 X 40(7) dpm/mumol of GA2 ganglioside. The addition of catalase and peroxidase to the tritiation procedure is recommended.     

The synthesis of high-specific-activity UDP-[6-3H]galactose, UDP-N-[6-3H]acetylgalactosamine, and their corresponding monosaccharides.

Tritiated uridine-5'-diphosphogalactose (UDP-[3H]Gal) has been widely used to study oligosaccharide biosynthesis and structure. It can be synthesized either chemically or enzymatically using galactose oxidase to oxidize the hydroxyl moiety at C-6 to an aldehyde (6-aldo-UDP-Gal), which is then reduced back to the alcohol with tritiated sodium borohydride. Although the enzymatic approach is simple and efficient, there are several problems associated with it. First, incomplete oxidation to the aldehyde reduces the final specific activity. Second, if the galactose oxidase is not removed from the 6-aldo-UDP-Gal prior to reduction, the resulting UDP-[6-3H]Gal can be reoxidized to 6-aldo-UDP-[6-3H]Gal. We present evidence for the occurrence of this compound in one commercially obtained preparation of UDP-[6-3H]Gal. Finally, if an excess of 6-aldo-UDP-Gal is used for good yield, it is necessary to quench the reduction with nonradioactive borohydride, again reducing the final specific activity. We have devised a rapid, inexpensive, and efficient synthesis of UDP-[6-3H]Gal that circumvents all of these problems. Galactose oxidase is used to produce 6-aldo-UDP-Gal and the completeness of this reaction is confirmed on polyethyleneimine (PEI) cellulose TLC plates. The 6-aldo-UDP-Gal is purified on silica gel 60 TLC plates. This purified compound is then reduced with tritiated sodium borohydride, with the aldehyde present in excess. Unreacted 6-aldo-UDP-Gal is then purified away from the product UDP-[6-3H]Gal by chromatography on PEI cellulose. Radiochemically pure UDP-[6-3H]Gal with a specific activity of 10 Ci/mmol was obtained using the above scheme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Light microscopic histochemical detection of terminal galactose and N-acetylgalactosamine residues in rodent complex carbohydrates using a galactose oxidase--Schiff sequence and peanut lectin--horseradish peroxidase conjugate

A technique was investigated for the direct visualization on paraffin sections of galactose and N-acetylgalactosamine residues terminating saccharide chains in complex carbohydrates. Sections were incubated with the enzyme galactose oxidase (GO), which oxidizes the C-6 hydroxyl of galactose or N-acetylgalactosamine (GalNAc) residues, and the resulting aldehyde was visualized by its reaction with Schiff's reagent. Submaxillary and sublingual glands, pancreas, stomach, duodenum, and ileum from mice and rats were stained with the GO-Schiff sequence and results were compared with staining by a peanut lectin-horseradish peroxidase (PL-HRP) conjugate that binds selectively to terminal galactose and preferentially to the terminal dimer beta-D-Gal-(1 leads to 3)-D-GalNAc. Three classes of reactive sites were revealed: 1) those reactive with both GO-Schiff and PL-HRP, 2) those stained with the GO-Schiff sequence but unreactive with PL-HRP, and 3) those GO-Schiff unreactive but PL-HRP positive. Based on the carbohydrate binding specificity of GO and PL, it is suggested that tissue complex carbohydrates in group one contain terminal beta-galactose residues with unmodified hydroxyls at C-2, C-4, and C-6, whereas those in group two contain terminal GalNAc residues. The structure of oligosaccharides in group 3 sites remains enigmatic.     

Galactose oxidase action on galactose containing glycolipids--a fluorescence method

Features that alter the glycolipid sugar headgroup accessibility at the membrane interface have been studied in bilayer lipid model vesicles using a fluorescence technique with the enzyme galactose oxidase. The effects on oxidation caused by variation in the hydrophobic moiety of galactosylceramide or the membrane environment for galactosylceramide, monogalactosyldiacylglycerol and digalactosyldiacylglycerol were studied. For this study we combined the galactose oxidase method for determining the oxidizability of galactose containing glycolipids, and the fluorescence method for determining enzymatic hydrogen peroxide production. Exposed galactose residues with a free hydroxymethyl group at position 6 in the headgroup of glycolipids were oxidized with galactose oxidase and subsequently the resultant hydrogen peroxide was determined by a combination of horseradish peroxidase and 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red). Amplex Red reacts with hydrogen peroxide in the presence of horseradish peroxidase with a 1:1 stoichiometry to form resorufin. With this coupled enzyme approach it is also possible to determine the galactolipid transbilayer membrane distribution (inside-outside) in bilayer vesicles.     

Use of dye affinity chromatography for the purification of Aerococcus viridans lactate oxidase

Lactate oxidase was purified from Aerococcus viridans (A. viridans) by dye affinity chromatography and FPLC ion exchange chromatography. The lactate oxidase could be purified by comparatively simple procedures, the purification achieved from a crude extract of A. viridans was 41-fold with a specific activity of 143 units/(mg of protein). The purified enzyme was a L-lactate oxidase, which catalyses the conversion of L-lactate in the presence of molecular oxygen to pyruvate and H(2)O(2). This purified lactate oxidase showed an apparent molecular mass of 48,200 in SDS-PAGE and the native molecular weight, as estimated by FPLC gel filtration, was 187,300. This molecular weight indicates that lactate oxidase exists in tetrameric form after gel filtration. To differing degrees, all the triazine dyes tested were inhibitors of lactate oxidase, solutions of free triazine dyes showing an inhibition mechanism which was both time- and pH-dependent.     

Purification and characterisation of a minor low-sulphated dermatan sulphate-proteoglycan from ray skin.

A minor low-sulphated dermatan sulphate proteoglycan was isolated from ray skin by extraction with 2% sodium dodecyl sulphate, followed with ion-exchange chromatography, gel chromatography and density gradient centrifugation. The proteoglycan with a relative molecular mass (Mr) ranging from 70 to 120 kDa is composed of about two dermatan sulphate chains (Mr 33 kDa) bound on a protein core of Mr 27 kDa, and oligosaccharides consisting of uronic acids, hexosamines and neutral sugars. The major amino acids of the protein core were glycine (corresponding to about one-fourth of the total amino acids), serine, threonine, glutamic acid/glutamine, leucine and cysteine, together amounting to 56% of the total. The isolated proteoglycan does not interact with hyaluronic acid and does not form self-aggregates. Dermatan sulphate was rich in iduronic acid (62% of total uronic acid) and composed of non-sulphated (44%), and mono-sulphated disaccharides bearing esterified sulphate groups at positions C-4 (53%) or C-6 (3%) of the N-acetyl galactosamine. HPLC analysis of a pure preparation of dermatan sulphate, showed the presence of galactose and glucose possibly as branches on the dermatan sulphate chain.     

Polysaccharide and oligosaccharide changes in germinating lupin cotyledons

In germinating lupin cotyledons, there was a rapid depletion of raffinose series oligosaccharides, a temporary increase in sucrose and constant low levels of reducing monosaccharides. The major polysaccharide fraction was extracted with hot NH₄ oxalate—EDTA solution and had the constitution of intercellular/cell wall polysaccharide. GLC examination of component sugars showed that as cotyledons expanded this fraction was depleted and that there was selective hydrolysis of arabinose and galactose, so that the uronic acid proportion increased. Gel and DEAE-cellulose chromatography showed that this fraction became more heterogeneous. The neutral and acidic fractions were separated and the component sugars, viscosities, gel chromatographic behaviour and sedimentation constants of these determined. The results indicated that in the later phase of plant cell wall expansion in germinating lupin cotyledons the arabinogalactan side chains of the pectic polysaccharide fraction are selectively hydrolysed leaving a primary wall with a high uronic acid content.     

Structure of the heparan sulfate-protein linkage region. Demonstration of the sequence galactosyl-galactosyl-xylose-2-phosphate

We have treated bovine lung heparan sulfate with alkaline [3H]borohydride to end label the chains with [3H]xylitol. After subsequent periodate oxidation-alkaline elimination products were separated by gel permeation and ion exchange chromatography. The linkage region fragment expected to have 2 galactoses and 1 [3H]xylitol residue appeared in the tetra-/trisaccharide region after gel filtration and was bound to the anion exchange resin. A similar negatively charged fragment, expected to have 2 galactoses, 1 xylose and 1 serine, was isolated after periodate oxidation-alkaline elimination of unlabeled heparan sulfate. The negative charge was due to the presence of alkaline phosphatase-labile phosphate ester. The molar ratio of galactose:phosphate:xylose was 2.17:1.19:1.00. The phosphate ester was associated with the xylose/[3H] xylitol moiety as indicated by the formation of phosphoxylose/-xylitol by beta-galactosidase digestion of the phosphorylated trisaccharide. Furthermore, orcinol reactivity disappeared after periodate oxidation of the dephosphorylated trisaccharide. The phosphate ester must be located to C-2 of xylose/xylitol as the 1-3H radioactivity could be released by periodate oxidation when it was preceded by alkaline phosphatase treatment. It is estimated that almost every chain of heparan sulfate carries 2-phosphoxylose. It would be of interest to know if glycosaminoglycan chains that are artificially initiated onto exogeneous beta-D-xylosides also acquire the 2-phosphoxylose moiety.     

N-acetyl-D-galactosaminyl-β-(1→4)-D-galactose: A terminal non-reducing structure in human blood group Sda-active Tamm-Horsfall urinary glycoprotein

Human Sd^a-active Tamm-Horsfall urinary glycoprotein labelled with galactose oxidase and tritiated sodium borohydride was found to contain both galactose and N-acetylgalactosamine as [³H]-labelled terminal non-reducing sugars. Fragmentation of the macromolecule achieved by hydrazinolysis and acid hydrolysis was followed by fractionation of the degradation products by gel filtration, ion exchange and paper chromatography. A major product was a disaccharide which contained unlabelled galactose and [³H]-labelled N-acetylgalactosamine. Sugar analysis, sodium borohydride reduction, methylation analysis and enzymic degradation enabled the structure N-acetyl-D-galactosaminyl-β-(1→4)-D-galactose to be assigned to the disaccharide.     

以聚乙二醇为层析伴侣同时制备SOD、过氧化氢酶和血红蛋白

发展了一条从红细胞裂解液中同时制备超氧化物歧化酶(SOD)、过氧化氢酶和血红蛋白的新工艺。采用0 75 %的聚乙二醇600作为层析伴侣,使血红蛋白直接流过阴离子交换层析柱,同时吸附SOD和过氧化氢酶。经过梯度洗脱获得SOD和过氧化氢酶组分,再经过疏水性相互作用层析与凝胶过滤层析相串联,使SOD和过氧化氢酶得到纯化。纯化后的SOD和过氧化氢酶的比活力分别达到15932u/mg和65918u/mg ,血红蛋白的纯度达到99.9%以上。总回收率为:SOD ,47.4% ;过氧化氢酶,29.6% ;血红蛋白,88.7%。     

In vitro labeling of the sialic acid moiety of glycoconjugates with carbon-14

Labeling of sialoglycoproteins with carbon-14 in vitro was performed by reacting the aldehyde groups, generated by mild periodate oxidation of the terminal sialyl groups, with 14C-labeled sodium cyanide to produce the labeled cyanohydrin derivatives (Kiliani reaction). Labeling with tritium was carried out by reduction of the aldehyde groups generated on the sialyl residues with 3H-labeled sodium borohydride following standard procedures. The behavior of both types of labeled specimens of fetuin and ovine submaxillary mucin, individually and in mixtures, was investigated by gel-filtration chromatography, gel electrophoresis, and cesium bromide gradient ultracentrifugation. The labeled sialyl residues were subjected to partial characterization: color yield with the resorcinol and thiobarbituric acid reagents, behavior on ion-exchange chromatography, and susceptibility to mild acid and enzymatic hydrolyses. In addition to these model glycoproteins, this procedure was also utilized to label the sialoglycoproteins present in human tracheobronchial secretions collected from normal subjects and patients with chronic bronchitis. The potential uses of this approach for comparative studies of normal and pathological sialoglycoconjugates available in minute amounts is described. The extension of this approach to the labeling of the galactosyl and N-acetylgalactosaminyl moieties of glycoconjugates following treatment with galactose oxidase is outlined.     

Chondroitin proteoglycans from squid skin. Isolation, characterization and immunological studies

Two populations of proteochondroitins were isolated from 4 M guanidine hydrochloride extracts of squid skin by a combination of ion exchange, gel chromatography and density gradient centrifugation. The proteoglycans, Mr 4.8 x 10(5) and 2.8 x 10(5), contained four and two chondroitin chains respectively and unusual oligosaccharides with uronic acid and sulphate groups, and had different amino acid and neutral sugar composition. The chondroitin chains isolated after alkaline borohydride treatment contained varying amounts of glucose, galactose, mannose, fucose and xylose, most likely as branches. Both proteoglycans were antigenic to the rabbit and showed considerable cross-reactivity as assessed by competition experiments using the ELISA technique. The proteoglycans reacted neither with exogenous hyaluronic acid nor with each other to form aggregates.     

Carbohydrate specificity of the galactose-recognizing receptor of rat peritoneal macrophages

The galactose-recognizing system of rat peritoneal macrophages mediates the binding and uptake of desialylated blood cells and glycoproteins. To characterize the specificity of this receptor, binding studies were performed with various galactose derivatives as competitive inhibitors and sialidase-treated erythrocytes or asialoorosomucoid as ligands for receptors, which were either membrane-bound or isolated after solubilization. From the results obtained it can be concluded that galactose is recognized via its hydrophobic and/or hydrophilic regions, formed by the accumulation of OH-functions on one side and of H-atoms on the other ("side effect"), whereas the binding partner or the anomeric configuration of galactose has no significant influence. Although it became apparent that not a single hydroxyl group of the sugar is responsible for binding, the hydroxyl at C-4 seems to be most important, followed by the OH-group at C-3. Those at C-1, C-2 and C-6 do not play a great role. This order of importance ("position effect") was found with galactose, derivatized by methylation or otherwise, and with diastereomers of galactose. Whereas the recognition of a single galactose residue leads to weak binding only, an appropriate arrangement of several of these ligands in one molecule results in an enormous increase in the binding strength of each galactose residue. This "cluster effect" was observed not only with membrane-bound but also with solubilized receptor. However, the binding of asialoorosomucoid by the latter was better inhibited with free galactose, when compared with the membrane-bound receptor.     

Composition of Root Mucilage Polysaccharides from Lepidium sativum

Root mucilage polysaccharides were recovered from roots of 3-d-oldcress seedlings by washing with water, followed by ethanol precipitationof the high molecular weight material. The redissolved polysaccharidewas fractionated by combined gel filtration chromatography onBiogel A50 and ion exchange chromatography on DEAE-Sepharose/DEAE-Trisacrylinto four heterogeneous fractions. The fractions could be assignedto two groups based on monosaccharide composition and behaviourduring ion-exchange chromatography. Group One polysaccharidescontained fucose as the major 6-deoxyhexose and were low inuronic acid, not binding to the ion-exchange column. Group Twopolysaccharides contained rhamnose as the major 6-deoxyhexoseand were uronic acid rich. It is suggested that these representroot cap and root epidermal mucilage components respectively. Key words: Root mucilage, recognition, polysaccharides     

Analysis of guanase by agarose gel electrophoresis and activity staining

A method was developed to separate guanase by agarose gel electrophoresis and to detect its activity by staining of the bands with a mixture of the enzymes xanthine oxidase, catalase, and aldehyde dehydrogenase, the coenzyme NADP+, and a substrate of guanine, ethanol, phenazine methosulfate, nitrotetrazolium blue, and KCN in Tris-(hydroxymethyl)methylamine buffer (pH 8.0). Serum samples showed bands 1 (faster moving) and 2 corresponding to the positions of albumin and alpha 2-globulin, respectively, found by serum protein staining. The same bands were detected with guanase from human liver and kidney, although band 2 from the latter samples was not as distinct as that from the liver samples.