Robert J. Sturgeon

Affinity chromatography of sialoglycoproteins, utilising the interaction of serotonin with N-acetylneuraminic acid and its derivatives

Serotonin, immobilised on Sepharose 4B, has been used to study the affinity chromatography of neuraminic acid and its derivatives. Free N-acetylneuraminic acid and oligosaccharides, polysaccharides, and glycoproteins containing that sugar are specifically bound to the columns. Removal of neuraminic acid from sialoglycoconjugates, or modification of the neuraminic acid residues by periodate oxidation, abolishes their ability to bind to the ligand. The presence of the N-acetyl group, but not the N-glycolyl group, and the integrity of the side chain (C-7-C-9) of the neuraminic acid are essential for binding to serotonin.

An enzymic method for the determination of the degree of polymerisation of glucans

Abstract The number-average degree of polymerisation of glucans may be determined by measurement of the sorbitol ( D -glucitol) content of an acid hydrolysate of the borohydride-reduced glucan, using sorbitol dehydrogenase. The method is applicable to both linear and branched polymers of either α- or β- D -glucose.

An enzymic assay of L-arabinose, using β-D-galactose dehydrogenase: its application in the assay of α-L-arabinofuranosidase

Abstract A new spectrophotometric method has been developed for the quantitative determination of l -arabinose, using the NAD-dependent β- d -galactose dehydrogenase from Pseudomonas fluorescens . This method has been applied to the determination of l -arabinose produced after degradation of l -arabino- d -xylan by α- l -arabinofuranosidase from Aspergillus fumigatus , and provides the basis of a sensitive, accurate, and specific method for assay of this enzyme which is technically straightforward to perform.

The oxidation of terminal D-galactofuranose residues of a galactan and a glycoprotein by a D-galactose Oxidase preparation from Dactylium dendroides

A galactan, isolated from the unicellular organism Prototheca zopfii, and a glycoprotein from a hyphal cell-wall fraction of the fungus Pithomyces chartarum have been oxidised by a D-galactose oxidase preparation from Dactylium dendroides. The oxidised polymers were subsequently reduced with sodium borotritide. The site of oxidation was identified as C-6 of non-reducing D-galactofuranosyl residues in both polymers.

The enzymic determination of d-mannitol with mannitol dehydrogenase from Agaricus bisporus

Abstract Mannitol dehydrogenase ( d -mannitol:NADP+ 2-oxidoreductase, EC 1.1.1.138), isolated from the sporocarps of Agaricus bisporus, has been purified 120-fold following fractionation with protamine sulphate, followed by hydrophobic and affinity chromatography. d -Mannitol and d -fructose appear to be the only substrate and products involved in the reaction, having Km values of 7.5 and 9.8m m , respectively. The purified enzyme has been used for the determination of d -mannitol and also the d -mannose content of glycoproteins and polysaccharides following the liberation of that hexose and reduction to d -mannitol.

An enzymic method for the determination of erythritol

Abstract An erythritol kinase has been isolated from Propionibacterium pentosaceum and purified from a contaminating glycerol kinase. A spectrophotometric method has been devised whereby erythritol can be quantitatively estimated in a series of coupled reactions with enzymes. The assay method has been applied to the analysis of the erythritol produced from a number of oligo- and poly-saccharides which have been submitted to periodate oxidation, borohydride reduction, and acid hydrolysis.

Purification of some glycoside hydrolases by affinity chromatography

Two glycoproteins have been isolated from the cell walls of bakers yeast. One is a glucan-protein complex which has been partially characterised as having a branched carbohydrate structure composed of chains of (1 leads to 3)-linked beta-D-glucosyl residues, some of which are attached by (1 leads to 6)-linkages to the main chain. Immobilization of this glycoprotein was achieved by covalent attachment to Sepharose, and the product was used to isolate a number of (1 leads to 3)-beta-D-glucan hydrolases from Helix pomatia, malted barley, and Basidiomycete QM806. The second glycoprotein, a mannan-protein complex, after immobilization, has been used in the purification of an alpha-D-mannosidase from jack-bean meal.