Purification of Sialic Acids

The aeylneuraminic acids can be released from their glycosidic linkages either by dilute (aqueous or methanolie) acids or sialidases. Special care must be taken in the isolation of the rather labile, O-acv-lated sialic acids, which arc partially non-susceptible to the action of sialidases. Furthennore, in gangliosides, non-O-acetylated sialic acid residues occur, and these are also more or less resistant towards these enzymes. 

Almost total release of sialic acids from glycoproteins or oligosaccharides can be achieved108,107 by heating the material in 0.05 M sul- 

After the mild-hydrolysis step at 70°, the sialic acids liberated are removed from the sample by dialysis or ultrafiltration at 2°, and the macromolecular material is rehydrolyzed, using, however, the stronger acidic conditions of 0.1 M acid. The dialysis time ranges between 6 and 24 h, depending on the volume and viscosity of the hydrolysis mixture. Therefore, the optimum dialysis time should be evaluated by determinations of sialic acid in the eluate, or by addition of a trace of radioactive Nen5Ac. The dialyzates, or filtrates, are combined, and processed as will be described. By using this procedure, the overall yield of purified sialic acids is 70-80%, and the loss of O-acetyl groups107 is 40%. 

Only a few bacterial and viral sialidases have been purified to high purity or even to protein homogeneity. 0 Complete purification of sia-lidase on a preparative scale from the culture filtrate ofC. perfringens was achieved111 by using poly(acrylamide) gel-electrophoresis as the final purification step (see Section VI,1). It is necessary that such purified sialidases be available, as the presence of proteases or other gly-cosidases in the enzyme preparations would lead to severe errors, not only in studies of substrate specificity, but also in cell biological and medical studies (see Sections VI and VII). 

For routine, enzymic hydrolysis of sialic acids on a preparative or an analytical scale in our laboratory, the substrates are incubated with bacterial sialidases in 50 mM acetate buffer at pH 5.5 and 37° for times ranging between a few minutes and several hours, depending on the substrate.107 After incubation, the sialic acids liberated are separated by dialysis, ultrafiltration, or protein precipitation at 0-2°, and purified as will be described in the following Section. 

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Varki, A., and Diaz, S., 1984, The release and purification of sialic acids from glycoconjugates Methods to minimize the loss and migration of O-acetyl groups. Anal. Biochem. 137 236-247. 

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... 

Several alternative methods for the determination of sialic acid in body fluids and tissues have been described. Most of these methods make use of the classic periodate-TBA assay in combination with purification using HPLC [13]. Another method makes use of fluorometric HPLC of sialic acids after derivatization with a fluorogenic compound [9]. The most promising new method for the determination of free sialic acid in urine (and probably also other body fluids and tissues) is the HPLC-tandem mass spectrometry method [19]. This method is rapid, accurate, and sensitive, and is more robust than earlier methods. The only disadvantage is the expensive equipment that is required, which makes it only economical for specialized metabolic laboratories. Since this equipment is used for many different metabolic assays, the investment is certainly warranted, and nowadays almost essential for any metabolic laboratory. 

Halcomb and Chappell developed a route to CMP-NeuAc 88 that promises to be general for the synthesis of virtually any derivative thereof [44,45]. The route (Scheme 36) utilizes a condensation of sialic acid derivative 99 with the phosphoramidite 112 to afford the phosphite 113 in 62% yield. Oxidation of the phosphite provided the phosphotriester 114 [46], which was taken directly to the next transformation without purification (owing to its instability to chromatography). Deal-lylation of the phosphate gave compound 115 (61% for two steps), which was stable to silica gel chromatography. Compound 115 was deacylated with methoxide, and its methyl ester was subsequently saponified with NaOH to provide CMP-NeuAc 88. The derivatives shown in Scheme 37 were synthesized according to this protocol and were investigated as substrates for sialyltransferases (see below). 

The content of sialic acid was also more reduced in our extract (5.5%), pleading for a lower degree of purification, although a 3.8% content of sialic acid was found in purified kidney erythropoietin by Kuratow-ska (7). 

The role of sialic acid residues in determining the life-time of circulating cells and glycoproteins and the importance of desialylation have been discussed, Chromatography on Blue Dextran 2000 coupled to agarose has been used in the rapid separation of factor X from citrated human plasma a 2(XX)-fold purification was achieved, Inhibition by the antithrombin-heparin cofactor of the conversion of factor IX into its active form by factor IXa has been examined. The process is time-dependent and requires a 1 1 combination... 

Combination of ion-exchange purification with ether or hexane extraction has proved to be necessary in quantitation of sialic acids released by mild acid hydrolysis from membrane preparations (Schauer er al 1975, Schauer 1978 see also chapter C). The extraction removes lipid material which would otherwise yield similar pigments in the assay, and which is not always removed after ion-exchange chromatography. 

Interference due to protein, especially haemoglobin, results in a reduction of sensitivity, and proteins should therefore be removed prior to analysis of sialic acid. A method using ethanol/chloroform precipitation has been put forward (Rivetz et al. 1980), but such deproteination methods still require additional correction for lipid, and the ion-exchange purification and lipid extraction should be part of the procedure to achieve reproducible and accurate quantitation (LEDEENandYu 1976, Schauer 1978, 1982). 

An automated assay based on the Aminoff method, including acidic butan-l-ol and combining the sensitivity of the assay with ion-exchange purification, has been put forward by Krantz and Lee (1975) and remains the most sensitive of all automated methods. It can detect 1.5 nmoles of sialic acid ( 0.5 xg), eliminates major interference due to 2-deoxyribose and precludes any corrections for the presence of such interference. 

Both methods, NMR and FAB-MS, require preparation and purification of individual sialylated oligosaccharides, glycopeptides, or gangliosides before analysis, making this approach less attractive if only low amounts of the corresponding materials are available. Therefore, sensitive methods for isolation and analysis of sialic acids and sialoglycoconjugates are required. Several HPLC methods have been developed that fulfill this need. 

Higa, H. H., Manzi, A., and Varki, A., 1989b, O-Acetylation and de-O-acetylation of sialic acids. Purification, characterization, and properties of a glycosylated rat liver esterase specific for 9-0-acetylated sialic acids, J. Biol. Chem. 264 19435-19442. 

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