176 Sialic acid Sodium

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. 

Dissolve a glycoprotein to be labeled in 50mM sodium phosphate, pH 7 (reaction buffer), at a concentration of l-10mg/ml. For sialic acid modification, place the sample in ice to cool to near 0°C. 

Flitsch and Turner reported the generation of a sialic acid DCL using an aldolase enzyme [36,37], The DCL design is shown in Scheme 2.7, and is based on the cleavage of sialic acid (58a) to A-acetylmaimosamine (56a) and sodium pyruvate (57), catalyzed by an aldolase enzyme. 

Scheme 2.7 Aldol reaction of ManNAc analogues and sodium pyruvate to produce sialic acid, catalyzed by A-acetylneuraminic acid (NANA) aldolase.

Cardo PP, Lombardo C, Gatti R (1985) A simple detection of sialic acid storage disorders by urinary free and total sialic acid determinations. Clin Chim Acta 150 129-135 Denny PC, Denny PA, Allerton SE (1983) Determination of sialic acid using 2-thiobarbituric acid in the absence of hazardous sodium arsenite. Clin Chim Acta 131 333-336... 

Bennett, E., Urcan, M., Tinkle, S., Koszowski, A., Levinson, S. Contribution of sialic acid to the voltage dependence of sodium channel gating. A possible electrostatic mechanism, J. Gen. Physiol. 

Therefore, to achieve high conversion of the substrate a tenfold excess of pyruvate is usually needed. The enzymes from Clostridium perfringens and Escherichia coli are commercially available from Toyobo the E. coli enzyme has been cloned and overexpressed, which has considerably reduced its cost [22,23], Sodium borohydride inactivates the enzyme in the presence of either sialic acid or pyruvate, indicating that the enzyme belongs to the Schiff-base-forming class 1 aldolase. This aldolase was supposed to be a... 

Eupergit 250L (Rohm Chemie, 400 mg) was added to a solution of sialic acid aldolase (8 mg, 64 U) in 1 M potassium phosphate buffer pH 7.4 (3.2 mL) containing 0.04 M sodium pyruvate and 0.02% NaN3 the suspension was stirred for 3 days at room temperature under N2. The gel was washed with 0.1 M potassium phosphate buffer pH 7 (10 mL) and stored at 4°C in this buffer in the presence of 0.04 M pyruvate and 10 3 M dithiothreitol. A unit yield of 40% was usually obtained for immobilization. 

To HEPES buffer (100 mL, 200 mM, pH 7.5) were added ManNAc 15 (1.44 g, 6 mmol), PEP sodium salt (1.88 g, 8 mmol), pyruvic acid sodium salt (1.32 g, 12 mmol), CMP (0.64 g, 2 mmol), ATP (11 mg, 0.02 mmol), pyruvate kinase (300 U), myokinase (750 U), inorganic pyrophosphatase (3 U), /V-acctylneuraminic acid aldolase (100 U), and CMP-sialic acid synthetase (1.6 U). The reaction mixture was stirred at room temperature for 2 days under argon, until CMP was consumed. The reaction mixture was concentrated by lyophilization and directly applied to a Bio-Gel P-2 column (200-400 mesh, 3 x 90 cm), and eluted with water at a flow rate of 9 mL/h at 4°C. The CMP-NeuAc fractions were pooled, applied to Dowex-1 (formate form), and eluted with an ammonium bicarbonate gradient (0.1-0.5 M). The CMP-NeuAc fractions free of the nucleotides were pooled and lyophilized. Excess ammonium bicarbonate was removed by addition of Dowex 50W-X8 (H+ form) to the stirred solution of the residual powder until pH 7.5. The resin was filtered off and the filtrate was lyophilized to yield the ammonium salt of CMP-NeuAc 17 (1.28 g, 88%). 

Figure 7 shows that the best conditions for elimination of side chains from BSM and OSM are probably 0.3M sodium borohydride in 0.12V NaOH at 45°C for about 10-12 hours. Over 90% of the total hexosamine and sialic acid were dialyzable under these conditions (36). The reduction of dehydroserine and dehydrothreonine residues was not studied,... 

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