Abstract
Linkage analysis of the carbohydrate portion of glycoproteins and glycolipids is widespread. Sequential treatment with D-galactose oxidase and tritiated borohydride is a standard method for incorporation of radioactive marker into what has been assumed to be exclusively terminal residues of D-galactose or N-acetyl-D-galactosamine. The data presented here establishes the ability of D-galactose oxidase to act upon a specific subterminal D-galactosyl residue, [----2)-D-Gal(1----], as well as upon terminal nonreducing galactosyl residues. Helix pomatia galactogen, a high molecular weight galactose homopolymer, was sequentially treated with D-galactose oxidase and tritiated borohydride. The 3H-galactogen was recovered and analyzed to determine which galactosyl units carried radioactive label. After complete methylation and then acid hydrolysis of 3H-galactogen, its partially methylated galactosyl components were reduced and acetylated for identification by gas chromatography and mass spectroscopy. Radioactivity was located by collection of effluent fractions during gas chromatography. The only subterminal residue to be labeled was the 2-linked D-galactose, although another with a free oxidizable 6-carbon was present, 3-linked D-galactose, [----3)-D-Gal(1----]. Linkage analysis of internal radiolabeled galactosyl residues could be used to detect changes in saccharide structure during cellular events.
Highlights
Linkage analysisof the carbohydrate portionof gly- polysaccharides, and glycolipids [2, 5], it has beenassumed coproteins and glycolipids is widespread
Plete methylation and acid hydrolysiosf 3H-gal- [+2)-D-Gal(l-].*Anotherresidue, [-3)-~-Gal(l+], with actogen, its partiallymethylatedgalactosyl compo- the C-6 position available for oxidatioand reduction, didnot nents were reduced and acetylated for identification become labeled. &Galactose oxidase has been reported by gas chromatography and mass spectroscopy
I1 e a. 0 tained 34pg (8.3 units) of galactose oxidase, 168 pg of horseradish peroxidase, 37 pg of #-tolidine, 0.1% Triton X-100, and 10 mM citrate buffer, pH 6.0, in a total volume of 950 ~ 1A.fter equilibrationat room temperature, 50pl of substrate were added and the change in absorbance at 425 nm recorded by a double beam Cary spectrophotometer
Summary
Materials-D-Galactose oxidase (EC 1.1.3.9), horseradish peroxidase (EC 1.11.1.7), and catalase (EC 1.11.1.6) were purchased from Sigma. 0 tained 34pg (8.3 units) of galactose oxidase, 168 pg of horseradish peroxidase, 37 pg of #-tolidine, 0.1% Triton X-100, and 10 mM citrate buffer, pH 6.0, in a total volume of 950 ~ 1A.fter equilibrationat room temperature, 50pl of substrate were added and the change in absorbance at 425 nm recorded by a double beam Cary spectrophotometer. The incubation mixture containe3d.0 mg of galactose oxidase, 0.3 mg of catalase, 7.5 mg of galactogen, 0.1 M phosphate buffer, pH 7.0, and 30 p1 of toluene in a 3.0-ml total volume. The slower moving radioactive material was eluted For reduction with tritiated potassiumborohydride the incubation from the sheet and subjected to structural characterization (see Fig. mixture containing oxidized galactogen was brought to pH10.0 with 3). Indicated two radiolabeled areas whose migration rates corresponded to 2,3,4,6-tetra-O-methyl- and 2,3,4-tri-O-methylgalactose (Fig. 1).Since we had assumed the tritium would have beenconfined tothetetra-0-methylgalactose[6],it
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