Carbohydrates with periodic acid

Commercially available kits (e.g., Roche Glycan Quantitation Kit) are based on the oxidation of the carbohydrates with periodic acid and subsequent coupling of the formed aldehyde with a hydrazide (e.g., digoxigenin hydrazide). The formed conjugate is estimated immunochemically by ELISA. 

The successful glycosylation of the polypeptides was monitored via SDS-PAGE with visualization via coomassie blue and periodate staining. In periodate staining, the carbohydrates were oxidized to aldehydes via treatment with periodic acid, followed by treatment with Gel-Code glycoprotein staining solution (Pierce, Rockford, IL). 

Horseradish peroxidase (HRP, EC 1.11.17) was obtained from Huka. The oxidation of carbohydrate moieties of enzyme with periodic acid (0.04 mM in 0.05 mM acetate buffer, pH 5.0) was performed according to Zaborsky and Ogletree (1974). The unreacted periodic acid was removed with 0.025-ethylene glycol. The oxidized enzymes were dialyzed against 50 mM phosphate buffer with pH 6.0 for 18 h. 

A. S. Perlin, Production of formic acid during oxidation of carbohydrates with lead tetraacetate, J. Am. Chem. Soc., 76 (1954) 5505-5508 A. S. Perlin, Oxidation of carbohydrates with periodate in the Warburg respirometer, J. Am. Chem. Soc., 76 (1954) 4101 103. 

R. spectroscopy of the products from oxidation of methyl a- and P-D-galactopyranoside with periodic acid in dimethyl sulphoxide, Carbohydr. Res., 91 (1981) 1-11. 

Sodium metaperiodate, NaI04, is a common, commercially available oxidant widely used in organic synthesis. As with periodic acid, sodium metaperiodate can be used for the glycol-cleavage oxidation reaction, which is particularly important in carbohydrate chemistry. Scheme 3.370 shows a representative example of a glycol-cleavage oxidation with NaI04 this reaction has been used in the synthesis of (25,45)-4-hydroxyproline... 

Solubilization of erythrocyte ghosts in 1 % sodium dodecyl sulfate, followed by SDS-polyacrylamine gel electrophoresis and staining with Coomassie blue reveals more than 10 well defined protein bands. When staining is performed with periodic acid-Schiff reagent (PAS), which stains carbohydrates, four bands (PAS bands) are revealed. 

Periodic acid does not oxidize mi/o-inositol according to the classical pattern. The consumption of 6 moles of oxidant with the formation of 6 moles of formic acid would be expected. Instead, a complex reaction ensues in which there is an overconsumption of oxidant and only about 4 moles of acid are produced 104). A mechanism has been advanced to account for these results 104) Similar results have been obtained with D-inositol and pinitol 106). Structural studies with periodic acid are of less application here than in other branches of carbohydrate chemistry, although useful results have been obtained Slh, 106a) by taking into account the overoxidation caused by the jS-dicarbonyl anomaly. Certain substituted derivatives, 1,3-di-O-methyl-mi/o-inositol (dambonitol) 41 o) and isopropylidene-inositols (7), for example, appear to be oxidized in the classical manner. 

Oxidative cleavage of vicinal diol functions in carbohydrates occurs with periodic acid (HIO4) or sodium metaperiodate (NaI04). The reaction proceeds through a cyclic intermediate and is similar to what we encountered with diols in Section 15.11. 

Periodic acid cleavage of vicinal diols is often used for analytical purposes as an aid m structure determination By identifying the carbonyl compounds produced the con stitution of the starting diol may be deduced This technique finds its widest application with carbohydrates and will be discussed more fully in Chapter 25... 

Periodic acid oxidation (Section 15 12) finds extensive use as an analytical method m carbohydrate chemistry Structural information is obtained by measuring the number of equivalents of periodic acid that react with a given compound and by identifying the reaction products A vicinal diol consumes one equivalent of penodate and is cleaved to two carbonyl compounds... 

Hydrazide groups can react with carbonyl groups to form stable hydrazone linkages. Derivatives of proteins formed from the reaction of their carboxylate side chains with adipic acid dihydrazide (Chapter 4, Section 8.1) and the water-soluble carbodiimide EDC (Chapter 3, Section 1.1) create activated proteins that can covalently bind to formyl residues. Hydrazide-modified enzymes prepared in this manner can bind specifically to aldehyde groups formed by mild periodate oxidation of carbohydrates (Chapter 1, Section 4.4). These reagents can be used in assay systems to detect or measure glycoproteins in cells, tissue sections, or blots (Gershoni et al., 1985). 

Certain solubility factors are to be considered in work with periodate at different pH values. Above pH 5, disodium paraperiodate (Na2HJ06) tends to crystallize from aqueous solutions.246 Below pH 5, the sodium salt of periodic acid is more soluble, whereas above pH 5, the potassium salt is best employed. Phosphate-buffer solutions cause erroneous results,23 69a 60 61 and should be avoided in oxidations of carbohydrates. 

Actually, on oxidation of the carbohydrate with sodium periodate, three moles of periodate are consumed and one mole of formic acid is formed. These data agree with the assumption that the disaccharide contains a pyranose and a furanose ring. The possibility that the disaccharide is made up of glucofuranose and sorbopyranose can also be eliminated on the basis of the periodate oxidation data. Glucofuranose would contain two pairs of adjacent hydroxyls, on carbon atoms 2 and 3 and on 5 and 6, and the sorbopyranose would have three adjacent hydroxyls, on carbon atoms 3, 4 and 5. In oxidizing such a disaccharide, a total of four moles of periodate would thus be used, giving rise to one mole of formic acid. This is inconsistent with the experimental data. 

A more general method for carbohydrates that can be used equally well on handsections and on sections of plastic embedded materials uses the Periodic acid SchifFs (PAS) reaction or one of its variants [29] for vicinal hydroxyl groups [22,23]. Sections are treated with 1%W/V Periodic acid at room temperature for 10 minutes and rinsed. The aldehydes created by this treatment are detected with SchifFs reagent (decolorized para-rosaniline) by immersion for 30 minutes. A red color (or fluorescence with excitation at 540 nm) indicates vicinal hydroxyl groups in starch and many other carbohydrates. If the specimen has been fixed with aldehydes then an aldehyde-blocking step must precede this reaction [22]. The best treatment is immersion for 24 hr in a saturated solution of dimedone. 

Protein concentrations were determined according to the method of Lowry et al. (30). Electrophoresis of proteins in polyacrylamide gels was carried out at 4°C, using the discontinuous buffer system No. 1 described by Maurer (31) and modified by Emert et al. (1). Protein was stained with 0.1% Coomassie Brilliant Blue R250 in a water-acetic acid-methanol (45 10 45) solution. Carbohydrates were stained with the periodic acid-Schiff (PAS) reagent using the method described by Lang (32). 

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