Sialic acid periodate oxidation

Oxidation of polysaccharides using 10 mM or greater concentrations of sodium periodate at room temperature results in the cleavage of adjacent hydroxyl-containing carbon-carbon bonds on other sugars besides just sialic acid residues (Lotan et al., 1975). High concentrations... 

Add 106-108cells/ml in a PBS solution (lOmM sodium phosphate, 0.15M NaCl, pFI 7.4) containing ImM sodium periodate and incubate on ice for 30 minutes in the dark. This level of periodate addition will target the oxidation only to sialic acid residues (Chapter 1, Section 4.4). If additional sites of glycosylation also are to be labeled, increase the periodate concentration to 10 mM and do the reaction at room temperature in the dark. 

Dissolve the glycoprotein(s) to be labeled in ice-cold ImM sodium periodate, lOmM sodium phosphate, 0.15 M NaCl, pH 7.4, for the exclusive oxidation of sialic acid residues. For general carbohydrate oxidation, increase the periodate concentration to 10 mM in PBS at room temperature. 

The following protocol describes the use of biotin-hydrazide to label glycosylated proteins at their carbohydrate residues. Control of the periodate oxidation level can result in specific labeling of sialic acid groups or general sugar residues (Chapter 1, Section 4.4). 

Such studies, and others on an O-phosphonomannan155 and a tei-choic acid,168 relied on judicious comparisons (of shift) with signals of model compounds, and these are simpler than conventional, analytical procedures. For example, it is difficult to methylate alkali-labile O-phosphonomannans, and sialic acid and KDO-containing polymers would require difficultly available, O-methylated standards. In addition, periodate-oxidation analyses are restricted to polymers having fortuitously amenable, chemical structures. 

Exact analysis of sialic acid is required in biologieal experiments where the biological role of sialic acid is frequently studied with the aid of sialidases, and the amount of sialic acids released is determined. This is also important for periodate oxidation studies on biological systems, where modification of sialic acids by periodate is only assumed, but chemical analysis of this effect by isolation and analysis of the modified sialic acids is seldom performed. These uncertainties in determinations of sialic acid can be overcome by the purification procedures already described. Furthermore, it must be stressed that unequivocal determination of the structure of a sialic acid, especially... 

An increase in sensitivity of 30-50% can be achieved if resorcinol and Cu2+ are used.30 This increase may be by a factor of 3 to 6 if the sialic acid side-chains are oxidized by periodate prior to application of the resorcinol-Cu2+ reaction.127... 

A further method for estimation of sialic acid using periodic acid has been reported by Massamiri and coworkers.121 Fonnaldehyde produced by mild, oxidative cleavage of the D-en/f/iro-glycerol-l-yl side-chain of Neu can be detected by 3-methyl-2-benzothiazolinone hydrazone, which yields a green-blue color (absorbance maximum 625 nm). This test, which is slightly more sensitive than the thiobarbi-... 

Periodate oxidation of sialic acids had earlier been used for structural determination of O-substituted sialic acids.89 Whereas, for example, one mole of a 4-O-acetylated sialic acid consumes 2 moles of periodate within 10-20 min at 0°, the same amount of the 7-O-acetyl isomer is oxidized by only one mole. As already discussed in connection with the periodic acid-thiobarbituric acid assay, 9-O-substituted sialic acids exhibit a very low rate of oxidation as compared with the unsubstituted sialic acids13 this observation originally led to the erroneous assignment89 of the 9-O-acetyl group to 0-8. All other sialic acids having O-acetyl groups on the side chain are expected to be unaffected by periodate. 

A prerequisite for the specificity of the staining procedures based on periodate oxidation is exclusive oxidation of the sialic acid residues by low concentrations of periodate during short reaction-times. The... 

Histochemical demonstration of most of the O-acetylated sialic acids is possible because substituents on the side chain of Neu hinder periodate oxidation of this part of the molecule to an extent dependent on the number and position of the O-acetyl groups already mentioned. Correspondingly, removal of these ester groups by alkaline treatment (0.5% KOH in 70% ethanol91) may increase the staining reaction of a sialic acid. For example, the presence of O-acylated sialic acids has been demonstrated in colonic, epithelial mucin of man and various mammals (summarized in Ref. 91), in healthy and diseased, human small-intestine,188-190 in bovine submandibular gland,182 in mouse and rat erythrocyte-membranes,191 and in human lymphocytes.192... 

The group of 7- and 8-mono-O-acetylated, 7,9- or 8,9-di-O-acetylated, and 7,8,9-tri-O-acetylated sialic acids (the existence of which in bovine, submandibular glands is well established82) is specifically stained in the following way. Tissue slices are treated with 0.5 M periodate for 120 min at 25°, causing complete oxidation of the side chain to the C7 aldehyde of the unsubstitutecl and 9-mono-O-acetylated sialic acids. 

After reduction of the aldehyde with borohydride, the ester groups of the remaining mono-, di-, and tri-O-acetylated sialic acids that are unaffected by these vigorous conditions of oxidation are hydrolyzed by alkali, the products are subjected to mild oxidation with periodate,... 

A method for selective, radioactive labelling of sialic acids, especially in cell membranes, by mild oxidation with periodate followed by reduction with borotritide, has heen described by Gahmberg and Andersson.200 This procedure can be used either for isolation and characterization of the labelled, cell-surface glycoconjugates (see, for example, Ref. 201) or for autoradiography of tissues and cells (for example, erythrocytes).142... 

In the spermatozoa of the sea urchin Anthocidaris crassipina, nine sialoglycolipids were found, two of which were isolated and their structures established from the results of acid hydrolysis, methylation, and periodate oxidation.139 The major component of the glycolipid mixture is a disialo-glycolipid, NeuAc-(2- 8)-NeuAc-(2- 6)-Glc-Cer. The least-polar, minor component has the structure of a monosialoglycolipid, NeuAc-(2 - 6)-Glc-Cer. In these compounds, glucose is again the only neutral monosaccharide, and the sialic acid is attached to it at 0-6. 

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