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repurification in the absence of these cations. Specific interaction (i.e., binding that is inhibited by specifc sugars) has been restored by addition of 0.5 mM Ca .-'+and 0.5 mM Mn 2+. Ideally these ions should be present in all buffers for these lectins. Similarly, Ca .-'+ and Mn 2+ were found to improve the specificity of the staining given by LTA. As a safety precaution, these cations should perhaps be included with lectins of uncertain metal ion requirement. A high background labeling of the acrylamide is a common problem with antibody staining of gels. With lectins this is less of a problem, although some, such as Con A, give higher background levels of staining than others, such as WGA, which usually gives a very clean background. A high background can usually be decreased by increasing the washing period, particularly between the first and second antibody incubations. Generally, high-titer antisera give less background than low-titer antisera. Empirically, we have found that replacing the second antibody with iodinated protein A also gives less background, z° Once again, carrier protein is important and significantly decreases the general background staining. Acknowledgment The support of an Anna Fuller Fund Postdoctoral Fellowship is gratefully acknowledged.

[6] C h a r a c t e r i z a t i o n o f Sialic A c i d s

By ROLAND SCHAUER The naturally occurring sialic acids with established structures represent a family of 15 compounds derived from neuraminic acid, systematically named 5-amino-3,5-dideoxy-o-glycero-o-galactononulosonic acid. The most common neuraminic acid derivative is N-acetylneuraminic acid, which has been found exclusively or together with other neuraminic acid derivatives in many animals and in some bacteria, as has been reviewed.'-~N-Glycolylneuraminic acid also frequently occurs in both ver' A. Gottschalk, ed., "The Chemistry and Biology of Sialic Acids and Related Substances." Cambridge Univ. Press, London and New York, 1960. 2 L. Warren, Comp. Biochem. Physiol. 10, 153 (1963). :' J. A. Cabezas, Rev. Esp. Fisiol. 29, 307 (1973). 4 H. Tuppy and A. Gottschalk, in "Glycoproteins, Their Composition, Structure and Function" (A. Gottschalk, ed.), p. 403. Elsevier, Amsterdam, 1972. S.-S. Ng and J. A. Dain, in "Biological Roles of Sialic Acid" (A. Rosenberg and C.-L. Schengrund, eds.), p. 59. Plenum, New York, 1976. R. W. Ledeen and R. K. Yu, in "Biological Roles of Sialic Acid" (A. Rosenberg and C.-L. Schengrund, eds.), p. I. Plenum, New York, 1976.

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tebrates and invertebrates. Rich sources for this compound are submandibular glands from pig, where 90% of the sialic acid fraction consists of N-glycolylneuraminic acid. 70-Acylated N-acetyl- or N-glycolylneuraminic acids are widespread in nature, too. Most frequently the O-acetyl group is found at C-9 of neuraminic acid; for instance, about 25% of the sialic acids from bovine submandibular gland glycoprotein represent N-acetyl-9-O-acetylneuraminic acid (formerly denominated N-acetyl-8O-acetylneuraminic acid, 8-'° as will be discussed below) and about J% N-glycolyl-9-O-acetylneuraminic acid. 8"°N-Acetyl-9-O-acetylneuraminic acid has also been found in man" and in gangliosides from different vertebrates. '20-Acetyl groups at C-7 are relatively rare; N-acetyl-7-Oacetylneuraminic acid and N-acetyl-7,9-di-O-acetylneuraminic acid have been isolated from bovine submandibular gland. 8-'° N-Acetylneuraminic acid and N-glycolylneuraminic acid with O-acetyl residues at C-4 are frequent components in equine tissues, especially in submandibular glands and in erythrocytes. 8-'8'13 Furthermore, N-acetyl-4,9-di-O-acetylneuraminic acid'8 and N-acetyl-4-O-glycolylneuraminic acid 8,'4 have been found in small quantities in this gland. N-Acetylneuraminic acid with an O-L-lactyl group at C-9 has recently been described in man 1' and cow. '5 N-Acetyl-8-O-methylneuraminic acid has been isolated from the starfish Distolasterias nipon TM and Asterina pectinifera, '7 and N-glycolyl-8-Omethylneuraminic acid has been purified from the starfish Asterias forbesi. ,8 N-Glycolyl-8-O-sulfoneuraminic acid was recently found in the sea urchin Echinocardium cordatum. ,8 7 H.--P. Buscher, J. Casals-Stenzel, R. Schauer, and P. Mestres-Ventura, Eur. J. Biochem., 77, 297 (1977). R. Schauer and H. Faillard, Hoppe-Seyler's Z. Physiol. Chem. 349, 961 (1968). ~' H.-P. Buscher, J. Casals-Stenzel, and R. Schauer, Eur. J. Biochem. 50, 71 (1974). ,0 j. p. Kameding, J. F. G. Vliegenthart, C. Versluis, and R. Schauer, Carbohydr. Res. 41, 7 (1975). " J. Haverkamp, R. Schauer, M. Wember, J.-P. Farriaux, J. P. Kamerling, C. Versluis, and J. IF. G. Vliegenthart, Hoppe-Seyler's Z. Physiol. Chem. 357, 1699 (1976). ,5 j. Haverkamp, R. W. Veh, M. Sander, R. Schauer, J. P. Kamerling, and J. F. G. Vliegenthart, Hoppe-Seyler' s Z. Physiol. Chem. 358, 1609 (1977). ,3 G. Blix and E. Lindberg, Acta Chem. Scand. 14, 1809 (1960). ,4 A. P. Corfield, C. Ferreira do Amaral, M. Wember, and R. Schauer, Eur. J. Biochem. 68, 597 (1976). '~ R. Schauer, J. Haverkamp, M. Wember, J. F. G. Vliegenthart, and J. P. Kamerling, Eur. J. Biochem. 62, 237 (1976). '~ N. K. Kochetkov, O. S. Chizhov, V. I. Kadentsev, G. P. Smirnova, and I. G. Zhukova, Carbohydr. Res. 27, 5 (1973). ,7 M. Sugita and T. Hori, J. Biochem. 80, 637 (1976). '8 L. Warren, Biochim. Biophys. Acta 83, 129 (1964). ,9 N. K. Kochetkov, G. P. Smimova, and N. V. Chekareva, Biochim. Biophys. Acta 424, 274 (1976).

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The unsaturated neuraminic acid derivative 2-deoxy-2,3-dehydro-Nacetylneuraminic acid, which was synthesized a few years ago by Meindl and Tuppy, 2° exists in nature. It was discovered in the urine of a sialuria patient, z~ and it has recently been detected in blood serum, saliva, and urine of normal human individuals in variable amounts. 11 With the exception of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, which has no glycosidic hydroxyl group, most of the sialic acids do not occur in a free form in biological materials. They represent glycosidic components of glycoconjugates, such as glycoproteins, glycolipids, homoand heteropolysaccharides and oligosaccharides. In these substances, the sialic acids are a-glycosidically linked to galactose, glucose, N-acetylgalactosamine or sialic acid residues. The chemistry of these compounds has been extensively reviewed. "~'2'-e~ Free sialic acids occur in minute quantities in tissues with an active sialic acid metabolism. Thus, free N-acetylneuraminic acid and N-glycolylneuraminic acid T M in addition to N-acetyl9-O-acetylneuraminic acid ~4"27 could be isolated from different tissues. These " f r e e " sialic acids partly represent CMP-glycosides. T M In the urine of a patient with sialuria, 2s however, the concentration of free N-acetylneuraminic acid was found to be about 10,000-fold higher than in the urine of normal individuals; 1T the sialuria patient excretes 7-10 g of this compound per day. A relatively high concentration of free sialic acids has been reported to occur in trout eggs and in the digestive glands of lobster and squid. 2,2:~ While sialic acids have been found in all higher animals, they do not generally occur in lower animals. 2,'~ In spite of earlier reports in literature, '~° sialic acids seem not to occur in plants. :~'32 2o p. Meindl and H. Tuppy, Monatsh. Chem. 100, 1295 (1969). 2~ j. E Kamerling, J. F. G. Vliegenthart, R. Schauer, G. Strecker, and J. Montreuil, Eur. J. Biochem. 56, 253 (1975). z2 A. Gottschalk, ed., "Glycoproteins, Their Composition, Structure and Function." Elsevier, Amsterdam, 1972. ..,:3N. Sharon, ed., "Complex Carbohydrates, Their Chemistry, Biosynthesis, and Functions." Addison-Wesley, Reading, Massachusetts, 1975. 24 j. Montreuil, Pure Appl. Chem. 42,431 (1975). 2~ A. K. Bhattacharjee, H. J. Jennings, C. P. Kenny, A. Martin, and I. C. E Smith, Can. J. Biochem. 54, 1 (1976). 2~ H. J. Schoop, R. Schauer, and H. Faillard, Hoppe-Seyler's Z. Physiol. Chem. 350, 155 (1969). 27 R. Schauer, Hoppe-Seyler's Z. Physiol. Chem. 351, 749 (1970). 2s j. Montreuil, G. Biserte, G. Strecker, G. Spik, G. Fontaine, and J.-P. Farriaux, Clin. Chim. Acta 21, 61 (1968). za L. Warren, Biochim. Biophys. Acta 44, 347 (1960). 30 K. Onodera, S. Hirano, and H. Hayashi, Agric. Biol. Chem. (Tokyo) 30, 1170 (1966). "~ J. A. Cabezas, An. Real Acad. Farm. 34, 155 0968). 32 W. Gielen, Z. Naturforsch. Text B 23, 1598 (1968).

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Isolation of Acylneuraminic Acids The acylneuraminic acids may be released from glycosidic linkage by either dilute acids or neuraminidases (EC 3.2.1.18). Acid Hydrolysis. Sialoglycoconjugates ( I - 1 0 rag) are dissolved in 1 ml of 0.1 N hydrochloric acid followed by heating at 80° for 50 min. '~'~This procedure leads to complete release of sialic acids from oligosaccharides or glycoproteins; in the case of gangliosides the heating time should be extended to at least 60 rain. About 20% of the sialic acids are d e c o m p o s e d under these hydrolytic conditions, and the O-acyl groups of the sialic acids are eliminated. For the preparation of O-acylated sialic acids the hydrolytic conditions should be milder: 0.01 N hydrochloric acid, 70 °, 1 hr.8'9 Instead of 0.01 N H C1, formic acid of about p H 2.2 can be used. Under these conditions at least 50% of the O-acyl groups are preserved:J; however, the release of the acylneuraminic acids is not quantitative. For liberation of 70--80% of the acylneuraminic acids, this hydrolysis should be repeated after intermediary dialysis of the sialic acids. Enzymic Hydrolysis. Sialoglycoconjugates ( I - 1 0 rag) are dissolved in 1 ml of 50 m M acetate buffer, pH 5.5, and 5-10 mU of neuraminidase from Vibrio cholerae or CIostridium perfringens are added. When Vibrio cholerae neuraminidase is used, the acetate buffer must contain 1 m M CaCl2. 8 Incubation is carried out at 37 ° for periods varying between 30 min and 24 hr depending on the nature of the glycoconjugate and sialic acid. The amount of sialic acid released may be measured by the colorimetric methods described below. The two bacterial neuraminidases mentioned above have wide substrate specificity including both hydrophilic (e.g., glycoproteins) and ambiphilic (e.g., gangliosides) glycoconjugates. 34''~ The glycosidic linkage of N-acetyl-4-O-acetylneuraminic acid is resistant to their action. 8 The other O-acylated sialic acids are liberated by these bacterial enzymes at slightly reduced rates. ~ Neuraminidases from other bacteria 36 or from viruses a4'a5 have also been used for liberation o f sialic acids from different glycoconjugates. Neuraminidases from brain tissues '~7,:~ or from human heart :~9 can be used for specific release of sialic acids from gangliosides. :~:~R. Schauer, A. P. Corfield, M. Wember, and D. Danon, Hoppe-Seyler's Z. Physiol. Chem. 356, 1727 0975). a4 R. Drzeniek, Histochem. J. 5, 271 (1973). :~aA. Gottschatk and R. Drzeniek, in "Gtycoproteins, Their Composition, Structure and Function" (A. Gottschaik, ed.), p. 381. Elsevier, Amsterdam, 1972. a6 A. Rosenberg and C.-L. Schengrund, in "Biological Roles of Sialic Acid" (A. Rosenberg and C.-L. Schengrund, eds.), p. 295. Plenum, New York, 1976. ~r A. Preti, A. Lombardo, B. Cestaro, S. Zambotti, and G. Tettamanti, Biochim. Biophys. Acta 350, 406 (1974). s s R. W. Veh and R. Schauer, lnt. Syrup. Carbohydr. Chem. 8th, Abstr. III 8 (1976). :~ T. L. Parker, R. W. Veh, and R. Schauer, Int. Syrup. Carbohydr. Chem., 8th Abstr. III 9 (1976).

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The acylneuraminic acids liberated by acid or neuraminidase treatment, or those already occurring in free form in tissues, are separated from macromolecular, micelle-forming, or insoluble materials by dialysis for 24 hr at 2 ° against three changes o f a 10-fold volume of water or by ultrafiltration. The diffusate or ultrafiltrate is freeze-dried, x'9'14 For large-scale preparation o f sialic acids, acid hydrolysis may be preferred. Rich sialic acid sources are colominic acid from Escherichia coli, 40 submandibular gland glycoproteins, 1,~..~collocalia mucoid,4J and the urine of the sialuria patient.'l'2~ Methanolysis. This procedure may be used for the preparation o f neuraminic acid fl-methylglycoside, which can easily be converted into radioactive N-acetylneuraminic acid, N-glycolylneuraminic acid, or other neuraminic acid derivatives? 2'*~ Submandibular gland glycoproteins (30 g) from cow or pig, or edible bird nest substance containing 7-11% sialic acids, are suspended in 500 ml of 1 N sulfuric acid in methanol and refluxed for 24 hr with stirring. 42"*~ For saponifcation o f the methyl ester of neuraminic acid //-methylglycoside, the mixture is diluted with 1 liter of water and the p H value is adjusted to 11 by the addition of solid Ba(OH)2. The mixture is refluxed for 2 hr with stirring while the pH value is maintained at 11. Thereafter, the mixture is centrifuged for 1 hr at 15,000 g, and the sediment is washed with 5 volumes of water by suspension and centrifugation under the same conditions. The combined supernatants are brought to pH 2.5 with dilute H2804. After 3 hr the mixture is centrifuged as before, and the supernatant is collected for purification of the neuraminic acid /~-methylglycoside by ion-exchange chromatography. Purification of Neuraminic Acid Derivatives A c y l n e u r a m i n i c A c i d s . Freeze-dried sialic acids from acid or neuraminidase hydrolysis are dissolved in water (10 mg/ml), and the solution is extracted twice with one volume of peroxide-flee ether to remove lipids. 33 The water phase is then passed through Dowex 50, H+-form, to remove cationic compounds, and the effluent, including two bed valumes of water washings, are slowly passed through Dowex 2-X8 (100-200 mesh, formate form). For adsorption of 1 mmol of neuraminic acid, about 400 ml o f the anion-exchange resin are used. The sialic acids are eluted with the 4-fold resin volume of a linear gradient from 0 to 2 N aqueou s formic ~oG. T. Barry and W. F. Goebei, Nature (London) 179, 206 (1957). 4~R. H. Kathan and I. D. Weeks, Arch. Bioehem. Biophys. 134, 572 (1969). 4~R. Schauer, F. Wirtz-Peitz, and H. Faillard, Hoppe-Seyler's Z. Physiol. Chem. 351,359 (1970). 4:3R. Schauer and H.-P. Buscher, Biochim. Biophys. Acta 338, 369 (1974).

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acid. 8,9,44 The gradient technique can be modified in routine analyses if sialic acid preparations contain only low quantities of other acids with similar pK values, e.g., products from intermediary metabolism. In this case, the sialic acids are eluted batchwise with three bed volumes of 1 N formic acid. All operations are performed at 2°, and the purified acylneuraminic acids are freeze-dried. O-Acyl groups of sialic acids are relatively stable with this purification method. Neuraminic Acid [3-methylglycoside. The dry neuraminic acid material resulting from methanolysis of 30 g of glycoprotein is dissolved in 50 ml of water' and slowly passed through a column (size, 4 x 70 cm) of Dowex 50, H ÷ form. The neutral effluent and two bed volumes of water washings are discarded. Neuraminic acid fl-methylglycoside is eluted with 4 liters of a linear gradient from 0 to 1 N HCI. Chloride ions are removed from the neuraminic acid-containing fractions by passage through Dowex l-X4, formate form. The eluate and two column volumes of water washings containing the neuraminic acid fl-methylglycoside are freeze-dried. 4e'43

Fractionation of Acylneuraminic Acids The sialic acid mixture obtained from ion-exchange chromatography can be fractionated into individual acylneuraminic acids by chromatography on cellulose powder (MN 2100 ff from Macherey, Nagel & Co., Diiren, FRG) using the solvent n-butanol-n-propanol-water ( 1 : 2 : 1 ; v/v/v). ~'9 Acylneuraminic acids (50-100 mg) are dissolved in this solvent and slowly passed (5-10 ml of eluate per hour) through a cellulose column (size: 2.8 × 100 cm) at 2°. Most of the known sialic acids elute as individual peaks; these, however, partly overlap depending on the relative proportions of the individual sialic acids in the mixture. Elution occurs in the following sequence: N-acetyldi-O-acetylneuraminic acids, N-acetylmono-O-acetylneuraminic acids, N-glycolylmono-Oacetylneuraminic acids, N-acetylneuraminic acid, and N-glycolylneuraminic acid. ~'9 The O-acetyl groups of the sialic acids are largely preserved during this chromatography. After elution the solvent must be removed from the individual fractions by rotary evaporation (bath temperature 35°) or freeze-drying. Otherwise, quantitative colorimetric determination of the sialic acids is not possible, and formation of the propyl and butyl esters of sialic acids occurs. A cellulose column can be used several times for fractionation of sialic acid samples. 44R. Schauer, H. J. Schoop, and H. Faillard,Hoppe-Seyler's Z. Physiol. Chem. 349, 645 (1968).

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Crystallization When the described methods are used, sialic acids can usually be obtained in a high degree of purity. Therefore, crystallization is not absolutely necessary for further purification, especially if only small quantities are available. Furthermore, crystallization o f O - a c y l a t e d sialic acids is not recommended, as these compounds are partly de-esterified during the relatively long period necessary for crystallization. H o w e v e r , the more stable N-acetylneuraminic acid, N-glycolylneuraminic acid, and 2-deoxy-2,3-dehydro-N-acetylneuraminic acid can be crystallized at 0 °, with good yield, by dissolving in a small amount of water followed by the addition of ethanol or methanol, diethyl ether and light petroleum in different ratios described elsewherC '~'2°'45or by the addition of 9 volumes of acetic acid. 42.46 Neuraminic acid fl-methylglycoside crystallizes readily from about 50% dioxane in water: the c o m p o u n d purified by ion-exchange chromatography is dissolved in water at about 40°; to this, dioxane is added dropwise until the solution becomes turbid. Crystallization begins immediately, 42 resulting in 60-80% yield when related to the neuraminic acid content o f the methanolyzate. Colorimetry Several methods for colorimetric analysis of sialic acids have been worked out and reviewed. 4'6 Some of these methods are not specific or not sensitive enough (as, for example, the direct Ehrlich reaction HT) for the exact determination of sialic acids in biological materials, which usually contain only low neuraminic acid concentrations and large amounts of substances that may interfere with the colorimetric tests. At present several principally different methods and their modifications are in widespread use. They will be discussed in the following and have partly been described in this series. Diphenol R e a c t i o n

A blue-purple color soluble in organic solvent is produced from both free and glycosidically bound neuraminic acid derivatives when heated with orcinol and Fe 3+ (Bial reagent) 48'49 or with resorcinol and Cu "+5°'51 4~L. Svennerholm, Acta Soc. Med. Upsal. 61, 75 (1956). 4, R. Kuhn and G. Baschang, Justus Liebigs Ann. Chem. 659, 156 (1962). 47G. Blix, Hoppe-Seyler's Z. Physiol. Chem. 240, 43 (1936). 48p. Brhm, S. Dauber, and L. Baumeister, Klin. Wochenschr. 32,289 (1954). 49L. Svennerholm, Ark. Kemi 10, 577 (1957). ~"This series Vol. 6 I66]. ~TThis series Vol. 8 [l].

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dissolved in concentrated aqueous HC1. The modification of the orcinol/ Fe 3+ assay worked out by B6hm et al.4S has been in continual use in our laboratory for several years. The procedure is described as follows. Reagent

Orcinol reagent: To 81.4 ml of concentrated HCI (37%) 0.2 g orcinol and 2 ml of 1% aqueous FeC13 solution are added, and the volume of the mixture is adjusted to 100 ml with water. This Bial reagent is stable at 2° for 1 wk. N-Acetylneuraminic acid or N-glycolylneuraminic acid, 1 mM, dissolved in water and adjusted to pH 5 with bicarbonate. This standard solution is stable for at least 6 months at - 1 8 °. Procedure. The whole procedure is carried out in glass centrifuge tubes with ground-glass stoppers. Of a solution containing between l0 pg and 50 /.~gof free or bound sialic acids, 1 ml is mixed with 1 ml of the Bial reagent and heated in boiling water for 15 min. The tubes are cooled in a bath of tap water, then 5 ml of amyl alcohol are added, followed by shaking to extract the blue-purple color into the organic phase. The tubes are placed into an ice bath for 5 min followed by centrifugation at 3000 g for 3 min. The clear upper organic phase is transferred to 20-mm cuvettes, and the optical density is read at 570 nm against the amyl alcohol extraction of a water blank taken through the whole procedure. A standard containing 20 /~g of crystalline N-acetylneuraminic acid or N-glycolylneuraminic acid is examined simultaneously. The amounts (in /~g) of N-acetylneuraminic acid and their O-acetylated derivatives in the samples are calculated by multiplying the extinction values by 131. The corresponding factor for N-glycolylneuraminic acid and its O-acetylated derivatives is 115. These multiplication factors are obtained from calibration curves with authentic sialic acids. Molar extinction coefficients of 5290, 6350, and 6780 mol-' cm-' have been determined for N-acetylneuraminic acid, N-glycolylneuraminic acid, and neuraminic acid fl-methylglycoside, respectively/'-' The molar extinction coefficients of O-acylated sialic acids correspond to those of the basic N-acetylneuraminic acid or N-glycolylneuraminic acid. This is due to a rapid hydrolysis of the ester groups under the strong acidic conditions. Using the method described, 5-10/~g of sialic acids can be determined accurately. The sensitivity of the test can be enhanced about 2.5-fold by a 5-fold reduction of the volumes of the sialic acid samples, Bial reagent, and amyl alcohol and by reading the absorption in a 10-mm cuvette. Correspondingly, minimum amounts of 2-3 /~g of sialic acids can be determined. The orcinol/Fe 3+ assay is accurate and reproducible and is used especially for determinations of sialic acids as components of glycoconjugates.

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For this purpose its sensitivity is sufficient. Free sialic acids are best analyzed using the more sensitive periodic acid/thiobarbituric acid assay discussed below. The specificity of the orcinol/Fe :~+assay is not absolute for sialic acids, as other monosaccharides are known to interfere with the test.4SThe same was observed with the resorcinol assay. ~°'~ In unfractionated biological materials or even in purified glycoconjugates, errors may occur in sialic acid determination, due to the presence of free or glycosidically bound pentoses, hexoses, and uronic acids. 4'4~'~°'5~ Therefore, both the orcinol and resorcinol assay are accurate and reproducible only when applied to glycoconjugates with a low content of pentoses and hexoses. In the case of a low ratio of sialic acids to the interfering sugars, the sialic acids must be released from the glycoconjugates by acids or neuraminidase and purified with the aid of ion-exchange chromatography before analysis. ~° Correspondingly, exact analytical data from free sialic acids can be obtained only after their purification. The sensitivity of the diphenol reaction is reported to be enhanced by about 50% if the orcinol/Fe 3+ reagent is replaced by the resorcinol/Cu "÷ reagent. 5°-'~'-'(In our hands, this difference is only 30%.) The specificity of the assay for sialic acids is increased if the readings are made at both 450 nm and 580 nm and the sialic acid concentration is calculated by a mathematical procedure. The sensitivity of the resorcinol/Cu '-'+ reaction is enhanced 3-6 times, and the specificity is also increased if free or glycosidically bound sialic acids are oxidized in the C-7-C-9 side chain by periodate prior to heating with the resorcinol/Cu 2+ reagent. 53 A chromophor is formed from the C-7 aldehyde of sialic acids produced by periodate oxidation,, which has an absorption maximum at 630 nm. The molar extinction coefficients for N-acetylneuraminic acid and N-glycolylneuraminic acid are 27,900 and 27,300, respectively. This test has, however, only half the sensitivity of the periodic acid/thiobarbituric acid assay for free sialic acids. Periodic Acid/Thiobarbituric Acid Reaction A sensitive and specific method for determination of free acylneuraminic acids is available using the periodic acid and thiobarbituric acid reagents. Precautions that will be discussed below are necessary to ensure the specificity of the reaction. Acylneuraminic acids as components of glycoconjugates, neuraminic acid fl-methylglycoside and 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, are negative in this test. ~2 L. Svennerholm, Biochim. Biophys. Acta 24, 604 (1957). ~3 G. W. Jourdian, L. Dean, and S. Roseman, J. Biol. Chem. 246, 430 (1971).

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Therefore, the glycosidically bound sialic acids must be released by acids or by neuraminidase before being assayed in this test. Two slightly different methods have been worked out by Warren 54 and Aminoff2~ both leading to formation of the prechromogen fl-formylpyruvic acid from acylneuraminic acids ~ and subsequently to the same red chromophor which is soluble in organic phases and has an absorbance maximum at 549 nm. The Warren periodic acid/thiobarbituric acid method has already been described in detail in this series ~1'~7, the Aminoff method, which is also in common use, is described below.

Reagents N-Acetylneuraminic acid or N-glycolylneuraminic acid, l mM, dissolved in water and adjusted to pH 5 with bicarbonate. This standard solution is kept at -18 °. Periodic acid, 25 mM, in 0.125 N H2SO4 Sodium arsenite, 2%, in 0.5 N HCI 2-Thiobarbituric acid, 0.1 M, adjusted to pH 9 with NaOH n-Butanol containing 5% (v/v) 12 N HC1

Procedure. The reactions are carried out in glass centrifuge tubes with ground-glass stoppers. To 0.5 ml of an aqueous solution containing 2-20 /zg of free sialic acids, 0.25 ml of the periodic acid reagent is added. The oxidation period is 30 rain at 37°. Then 0.2 ml of the arsenite solution is added to destroy the excess of periodate. After about 2 min the yellow color of the liberated iodine disappears, and 2 ml of the thiobarbituric acid reagent are added. The mixture is heated in a boiling water bath for 7.5 min followed by cooling in an ice bath. The chromophor is extracted by vigorous shaking with 5 ml of the acid/butanol mixture. After 3 rain of centrifugation at 3000 g, the clear organic phase is transferred to a 10-mm cuvette and the optical density is read at 549 nm against the butanol phase of a blank in which the sialic acid solution is substituted by water. Standards with 10/zg of sialic acids are run simultaneously. The amounts (/zg) of sialic acids in the assays are calculated by multiplying the extinction values by 26 in the case of N-acetylneuraminic acid and by 33 for N-glycolylneuraminic acid. The molar extinction coefficients from the Warren or the Aminoff assay given in the literature for these two sialic acids are somewhat different; this may be due mainly to differences in the purity of the sialic acid ~4 L. Warren, J. Biol. Chem. 234, 1971 (1959). ~5 D. Aminoff, Biochem. J. 81,384 (1961). ~ G. B. Paerels and J. Schut, Biochem. J. 96,787 (1965). ~7 This series Vol. 6 [67].

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preparations. For crystalline N-acetylneuraminic acid and N-glycolylneuraminic acid, we found values of 51,500 and 36,100, respectively. 42 O-Acyl groups influence the chromophor formation in the Warren or Aminoff periodic acid/thiobarbituric acid assays depending on their position in the neuraminic acid molecule. While an O-acetyl group at C-4 has no appreciable influence on color formation, ~''~an O-acetyl residue at C-7 prevents color formation, s and an O-acetyl group at C-9 reduces chromophor formation by about 60%.~'~ The latter two phenomena can be explained by prevention of the oxidation of the neuraminic acid side chain between C-7 and C-8 if an O-acetyl group is bound to C-7, and by hindrance, probably steric, of the oxidation at the same position if an O-acetyl group is linked to C-9. ~ Therefore, O-acetyl groups should be removed by dilute alkali or hydroxylamine (see below) from O-acetylated sialic acids before quantitative determination by the periodic acid/thiobarbituric acid assays. It can be calculated from the molar absorption coefficients that the periodic acid/thiobarbituric acid assay is 6-10 times more sensitive than the orcinol/Fe 3+ assay, thus enabling the determination of minimum amounts of about 2 p~g of sialic acids. The amount that can still be analyzed accurately can be decreased to about 0.5/~g of sialic acids if the quantity of the butanol phase is reduced to 1 ml to concentrate the chromophor. Even smaller sialic acid quantities may be determined by appropriate reduction of the volumes of the reagents and by using microcuvettes. The periodic acid/thiobarbituric acid assay of Aminoff or Warren is accurate and reproducible if pure acylneuraminic acids are analyzed. It becomes inaccurate, however, and may even lead to serious errors--for example, to the demonstration of sialic acids in plantsa°'32---when employed for quantitative analysis of crude sialic acid samples, especially in unprocessed biological materials. This is due to a variety of interfering substances occurring in crude biological materials, such as L-fucose, 2-deoxyribose, 2-keto-3-deoxyaldonic acids, unsaturated fatty acids, which reduce the formation of chromophor from sialic acids or lead to other red chromophors. 4'6,51,~v,SaA method to overcome these difficulties and to determine the true sialic acid concentration, in spite of the presence of chromophors derived from substances other than from sialic acids, has been worked out by Warren. ~4The chromophor formed in the sialic acid sample is measured at the wavelengths of 549 and 532 nm, and a mathematical procedure is used to calculate the sialic acid concentration. This ~ J. Haverkamp, R. Schauer, M. Wember, J. P. Kamerling,and J. F. G. Vliegenthart, Hoppe-Seyler's Z. Physiol. Chem. 356, 1575 (1975). 59This series Vol. 41 [6].

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method is valuable only as long as, first, the interfering chromophor is derived from malonaldehyde and, second, the ratio of neuraminic acid to the interfering substances is not too low. Owing to these uncertainties, purification of the sialic acid samples of biological origin by extraction of contaminating lipids with ether and by ion-exchange chromatography of the sialic acids, as was described previously/'~ before assaying them by the periodic acid/thiobarbituric acid m e t h o d is strongly recommended. Very recently a modification o f the periodic acid/thiobarbituric acid assay has. been described in which dimethyl sulfoxide is used instead of the acid/butanol mixture, s° The advantage of this procedure is a c h r o m o p h o r stable for several days, in contrast to the chromophor extracted by the butanol phase. Fluorometr& A s s a y

Sialic acids can be assayed in a much more sensitive way than is possible with the colorimetric methods described to date by using fluorometric methods. An intensive green fluorescence is produced with sialic acids in dilute HC1 in the presence of 3,5-diaminobenzoic acid. ~ H a m m o n d and Papermaster have modified the periodic acid/thiobarbituric acid assay to a fluorometric microassay that enables the accurate detection of even 10 ng of sialic acids/2 In this test the usual thiobarbituric acid chromophor extracted into the acid/butanol phase is excited at 550 nm and the emitted light is measured at 570 nm. The authors reported that the degree of contamination o f the samples with, for example, 2-deoxyribose can be determined. Enzymic Assay

A sensitive and the most specific determination of sialic acid concentration is possible by the use of acylneuraminiate pyruvate-lyase "3 isolated from Clostridium perfringens. ~4 The marketable e n z y m e cleaves acylneuraminic acids into acylmannosamines and pyruvic acid. n~'~ The concentration of the latter compound is analyzed with lactate dehydrogenase in the presence of N A D H . In spite of the equilibrium constant of 96 mM 4oL. Skoza and S. Mohos, Biochem. J. 159,457 (1976). ,i H. H. Hess and E. Rolde, J. Biol. Chem. 239, 3215 (1964). ,2 K. S. Hammond and D. S. Papermaster, Anal. Biochem. 74,292 (1976). ~3EC 4.1.3.3. ~4S. Nees, R. Schauer, F. Mayer, and K. H. Ehrlich, Hoppe-Seyler's Z. Physiol. Chem. 357, 839 (1976). "~P. Brunetti, G. W. Jourdian, and S. Roseman, J. Biol. Chem. 237, 2447 (1962). ~ D. G. Comb and S. Roseman, J. Biol. Chem. 235, -2529(1960).

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for the lyase reaction, sialic acids are quantitatively cleaved by coupling both enzyme reactions. Thus, the amount of NADH oxidized is equivalent to the amount of sialic acid present in the sample. This method and its application for the determination of N-acetylneuraminic acid and N-glycolylneuraminic acid has been described in detail in this series. 6T However, the method can be slightly modified,6S'69requiring much smaller amounts of the rather expensive lyase (0.01 U per milliliter of assay mixture). When this method is used, it should be considered that O-acyl groups at C-7 or C-9 reduce the reaction rate of the lyase by 30-50% and at C-4 by about 90%. 9'14'69Therefore, O-acetyl groups should be hydrolyzed before enzymic sialic acid determination. The presence of pyruvate in the sialic acid samples must be excluded by controls without the iyase. Owing to the possibility that analytical errors may occur in sialic acid determination it is recommended to estimate sialic acid concentrations by two different colorimetric methods. In this respect gas-liquid chromatography of sialic acids described below is of great value.

Quantitative Determination of O-Acyl Groups To determine the amount of O-acyl groups in a sialic acid preparation the hydroxamic acid reaction is carried out according to Hestrin TM with the modification of Ludowieg and Dorfman. 71Ethyl acetate, or acetyl choline serve as standards for O-acetyl determination. Esters of glycolic or lactic acid may be used as standards for the quantitative determination of the corresponding O-acyi groups found in some sialic acids, as will be described below. The method is described as follows.

Reagents Hydroxylamine hydrochloride, 0.35 M Sodium hydroxide, 1.5 M Perchloric acid, 0.75 M Perchloric acid, 0.4 M Ferric perchlorate, 0.07 M, dissolved in 0.4 M perchloric acid. The solutions are stable for 1 month at 2°. Ethyl acetate, 5 mM, dissolved in methanol-water (1 : 1; v/v). This or other standard ester solutions for calibration should be prepared ~: This series Vol. 6 [68]. ~s R. Gantt, S. Millner, and S. B. Binkley, Biochemistry 3, 1952 (1964). ~:~R. Schauer, M. Wember, F. Wirtz-Peitz, and C. Ferreira do Amaral, Hoppe-Seyler's Z. Physiol. Chem. 352, 1073 (1971). 7o S. Hestrin, J. Biol. Chem. 180, 249 (1949). 7, j. Ludowieg and A. Dorfman, Biochim. Biophys. Acta 38,212 (1960).

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immediately before use. Alkaline hydroxylamine solution, equal volumes of 0.35 M hydroxylamine hydrochloride and 1.5 M sodium hydroxide are mixed immediately before use, as the solution is unstable.

Procedure. The standard acetyl esters or the sialic acid sample (2 ml) containing 1-10 p,mol of ester are mixed with 2 ml of the alkaline hydroxylamine solution. The mixture is shaken and allowed to react for 10 min at room temperature. Then 2 ml of 0.75 M HCIO4 are added with shaking of the solution. A reddish color is developed after the addition of 1 ml of acid Fe(C104)3 solution. The optical density is determined within 10 min at 520 nm in 10-mm cuvettes against a blank in which the ester sample is replaced by water. Minimum quantities of 0.5/~mol ester groups can be assayed by this procedure. Reduction of the volumes of the reagents by a factor of 10 resulting in a total volume of 0.7 ml enables the determination of 0.05/~mol of ester in a sialic acid sample. The method is not specific with regard to the nature of the ester group. However, the concentration of total ester groups in a sample containing free or glycosidically bound O-acylated sialic acids can reproducibly and accurately be analyzed by the described procedure. Ester determination in glycoproteins requires centrifugation of the reaction mixture before reading the optical density of the chromophor, since glycoproteins are precipitated by perchloric acid. Distillation of the O-acetyl groups, for example, methyl acetate, prior to the colorimetric analysis as was described in this series ~' is not absolutely necessary for exact analysis. However, qualitative analysis of the O-acyl residues, to be described below, is necessary in addition to quantitative analysis to exclude positive ester reactions, which may be due to the presence of ester linkages between different sialic acid residues or in neuraminic acid lactones. Determination o f Glycolyl Groups The amount of glycolyl groups in a sialic acid sample can be determined according to Schoop and Faillard. 72N- and O-Glycolyl groups are converted to their ethyl esters by heating 0.01-1/zmol of sialic acid in 0.5 ml of a 20% solution of p-toluene sulfonic acid in ethanol at 95° for 8 hr. Thereafter the ethyl glycolate is distilled in vacuo and condensed in a tube containing 2 ml of ethanol and 0.1 ml of 4 N NaOH at -70 °. The distillation apparatus used is described by Schoop and Faillard. 72 After saponification of the ethyl glycolate in the condensate at 37° for 30 min and removal of the ethanol by rotary evaporation, the amount of glycolic acid 72 H. J. Schoop and H. Faillard, Hoppe-Seyler's Z. Physiol. Chem. 348, 1509 (1967).

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is determined colorimetrically by the addition of 2 ml of 0.01% 2,7dihydroxynaphthalene in concentrated sulfuric acid (Eegriwe's reagent) and heating of the mixture for 20 min in a boiling water bath. The absorbance of the reddish purple chromophor formed is monitored at 546 nm. Minimum amounts that can be measured by this method are 0.5 ~g o f glycolic acid. The distillation step is necessary because it greatly enhances the specificity of the test, as the neuraminic acid residue and other nonvolatile organic compounds which may be present in the sample interfere with glycolyl determination. Determination o f Lactyl Groups

The presence and configuration of O-lactyl groups as components o f sialic acids (N-acetyl-9-O-L-lactylneuraminic acid) isolated from cow and man'"15 can be determined with L-lactate dehydrogenases in the presence of NAD +. In the first step, the lactyl groups are released by treatment of the sialic acid sample (0.5 /~mol) with 0.05 N N a O H (0.2 ml) for 1 hr at room temperature. After neutralization of the sample L-lactate dehydrogenase, N A D + and hydrazine are added according to Hohorst, TM and the production of N A D H , which is proportional to the lactyl concentration, is followed at 366 nm. Thin-Layer Chromatography Sialic Acids. Chromatography o f sialic acids is carried out either on cellulose or silica gel thin layers of 0.2-0.5 mm thickness. The cellulose layers are prerun in 0.1 N HCI followed by drying in air to achieve a good separation of the sialic acids which are extremely sensitive to the presence o f cations on the thin-layer plates." Sialic acids (10-20/xg) are applied along a centimeter starting line. On cellulose, the following solvents are used: n-butanol-n-propanol-0.1 N HCI ( l : 2 : l ; v/v/v)74; n-butanol-pyridine-water ( 6 : 4 : 3; v/v/v)7~; and n-butanol-acetic acidwater (4 : 1 : 5; v/v/v). 7~Among these systems, which are commonly in use in our laboratory, the butanol-propanol-HCl system leads to the best and most reproducible separations. On silica gel, sialic acids are developed in n-propanol-water (7: 3; v/v). TM In all systems, development over l0 cm (solvent front) is sufficient for a good separation of sialic acids. The sialic acid spots are visualized by spraying the plates with a rea-

7:~H. J. Hohorst, in "Methoden der enzymatischen Analyse" II (H. U. Bergmeyer, ed.), p. 1425. Verlag Chemic, Weinheim, 1970. 74E. Svennerholm and L. Svennerholm,Nature (London) 181, 1154 0958). 7~M. J. Crumpton, Biochem. J. 72, 479 (1959). 76E. Granzer, Hoppe-Seyler's Z. Physiol. Chem. 328,277 0962).

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TABLE I

Rj VALUES OF ACYLNEURAMINIC ACIDS FROM THIN-LAYER CHROMATOGRAPHY ON CELLULOSE IN n-BUTANOL-n-PROPANOL-0.1 N HCI (1:2:1, v/v/va) AND ON SILICA GEL IN n-PROPANOL--WATER (7:3, V/Vb)c-e

Compound

Cellulose

Silica gel

N-Glycolylneuraminic acid N-Acetylneuraminic acid N-Acetyl-4-O-glycolylneuraminic acid N-Glycolyl-4-O-acetylneuraminic acid N-Glycolyl-9-O-acetylneuraminic acid 2-Deoxy-2,3-dehydro-N-acetylneuraminic acid N-Acetyl-9-O-L-lactylneuraminic acid N-Acetyl-4-O-acetylneuraminic acid N-Acetyl-7-O-acetylneuraminic acid N-Acetyl-9-O-acetylneuraminic acid N-Acetyl-4,9-di-O-acetylneuraminic acid N-Acetyl-7,9-di-O-acetylneuraminic acid

0.48 0.57 0.65 0.65 0.65 0.67 0.70 0.76 0.76 0.76 0.83 0.83

0.39 0.39 0.61 0.61 0.61 0.70 0.61 0.61 0.61 0.61 0.73 0.73

" E. Svennerholm and L. Svennerholm, Nature (London) 181, 1154 (1958). ~' E. Granzer, Hoppe-Seyler's Z. Physiol. Chem. 328, 277 (1962). " H.-P. Buscher, J. Casals-Stenzel, and R. Schauer, Eur. J. Biochem. 50, 71 (1974). a j. Haverkamp, R. Schauer, M. Wember, J.-P. Farriaux, J. P. Kameding, C. Versluis, and J. F. G. Vliegenthart, Hoppe-Seyler's Z. Physiol. Chem. 357, 1699 (1976). " R. Schauer, J. Haverkamp, M. Wember, J. F. G. Vliegenthart, and J. P. Kamerling, Ear. J. Biochem. 62, 237 (1976).

gent composed of 2 volumes of the orcinol/Fe 3+ solution used for colorimetric determination of sialic acids 4~ and 1 volume of water. The chromatograms are developed for 15 min at 120°, resulting in red-blue sialic acid spots. The sialic acids can also be visualized by the periodic acid/thiobarbituric acid spray reagent. 7r The reagents used for this staining technique are the same as described for the colorimetric determination of sialic acids in the modification of Aminoff. ~~ The cellulose plates are first sprayed by the periodic acid solution, covered by glass plates to prevent evaporation, and kept for 30 min at 37 °. After spraying with the arsenite solution and drying, the plates are sprayed with thiobarbituric acid and again covered by glass plates. The red sialic acid spots are developed during 15 min at 100°. A sensitive staining technique has also been worked out with the re sorcinol/Cu "+ reagent after oxidation of the sialic acids with periodate. 5:~ The Re values of sialic acids on cellulose in the butanol-propanoI-HCl system or on silica gel in the propanoi-water system are shown in Table I. It is striking that sialic acids differing only in their N-acyl group (acetyl or rr L. Warren, Nature (London) 186, 237 (1960).

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glycolyi) cannot be separated on silica gel but are well separated on cellulose. Therefore, the cellulose system is more suitable for analysis of a complex sialic acid mixture. However, none of these systems allows a separation of isomeric N-acetyl-O-acetylneuraminic acids, on the one hand, and N-glycolyl-O-acetylneuraminic acids, on the other. For further investigation of the nature of the O-acylated sialic acids, thin-layer chromatography is carried out on cellulose in two dimensions with intermediary ammonia treatment for ammoniolysis of the ester groups. 9 A sialic acid mixture or an individual N-acyl-O-acylneuraminic acid is spotted on one corner of a 10 × 10 cm plate. After chromatography in the first dimension in the butanol-propanol-HCI system, the plate is thoroughly dried under reduced pressure and placed for 2 hrs in an atmosphere of ammonia over an aqueous solution of 5 N ammonium hydroxide at room temperature. Ammonia vapor is removed from the plate under a flow of air, and the ammonia-treated sialic acids are chromatographed in the second direction again in butanol-propanol-HC1. A mixture of N-acetylneuraminic acid and N-glycolylneuraminic acid, on the one hand, and of the original sialic acid sample, on the other, are additionally chromatographed in the second dimension as references. The advantages of this method are, first, the unequivocal detection of O-acylated sialic acids, as their Rs values are characteristically reduced after ammonia treatment. Second, the nature of the parent N-glycolylneuraminic acid or N-acetylneuraminic acid derived from N-acyl-O-acetylneuraminic acids can easily be recognized by this method. Chromatography in two dimensions with intermediary ammonia treatment followed by staining with the orcinol/Fe '~+ reagent can also be applied for the detection of O-acyl residues in sialic acids bound to more complex substances. Accordingly, gangliosides show reduced migration after saponification of ester groups. 78However, the time of ammoniolysis should be extended to 12 hr for complete release of the O-acyl groups from gangliosides. The nature of the O-acyl groups can also be identified by chromatography of O-acylated sialic acids in two dimensions with intermediary treatment with alkaline hydroxylamine. Sialic acids are separated on cellulose in the butanol-propanol-HCl system in the first dimension as described above. After drying, the plate is covered by a plastic sheet with the exception of the zone where the sialic acids are suspected. This zone is sprayed with the alkaline hydroxylamine reagent described above for the colorimetric determination of ester groups, with the follow7~ R. W. Veh, J. Haverkamp, M. Sander, and R. Schauer, unpublished results, 1977.

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ing modifications: NaOH is replaced by 2.5 M ammonium hydroxide and a water-methanol mixture (1 : l; v/v) is used as solvent. After spraying, the plate is covered for 1 hr by a plastic sheet to avoid evaporation. Thereafter, the acyl hydroxamates formed are chromatographed in the second dimension using n-propanol-10% aqueous ammonium carbonate-5 N ammonium hydroxide (6: 2:1; v/v/v 9) as solvent. Acetyl hydroxamate and glycolyl hydroxamate serve as standards. The acyl hydroxamate spots are stained by spraying with a 10% aqueous FeCl3 solution leading to a blue-red color. The R s values of the hydroxamates from glycolic acid, L-lactic acid and acetic acid are 0.52, 0.65, and 0.71, respectively. ,5 The method is specific but not very sensitive. Clearly visible acyl hydroxamate spots can be obtained only from N-acyl-O-acylneuraminic acid spots containing at least 20-30 /zg of material. The method is useful to obtain a qualitative evaluation of the number and nature of O-acylated sialic acids present in a mixture. Acyl Hydroxarnates. If pure O-acylated sialic acids are available it is recommended that establishment of the nature of their O-acyl groups be carded out by derivatization in the test tube followed by unidimensional thin-layer chromatography of the resulting acyl hydroxamate. 9''~ The dried acylneuraminic acids (0.1-1 /~mol) are treated with 0.05 ml of the alkaline hydroxylamine solution used for colorimetric ester determination, in which, however, NaOH is replaced by 2.5 M ammonium hydroxide. After 1 hr at room temperature and evaporation under reduced pressure, the acyl hydroxamates are dissolved in 0.05 ml of methanol. The solutions are applied to a cellulose thin-layer plate and developed in the propanol-ammonium carbonate-ammonium hydroxide solution followed by staining with aqueous FeC13 as was described above (Rs values of the acylhydroxamates from the O-acyl groups found in natural sialic acids are as quoted earlier). Acylmannosamines. Chromatography of the acylmannosamines resulting from cleavage of acylneuraminic acids by acylneuraminate pyruvatelyase provides insight into the nature of N- and O-acyl groups of sialic acids. The incubation mixtures contain 0.2 M phosphate buffer, pH 7.2, 0.5--I /zmol of sialic acids, and 0.01 U of the lyas e in 1 ml. ~7-69 (See also the subsection "Enzymic Assay" dealing with colorimetric sialic acid analysis.) The cleavage rate of the sialic acids is checked colorimetrically in the course of 1-24 hr using the periodic acid/thiobarbituric acid assay described above. After incubation the mixtures are first passed through Dowex 50, H ÷ and then--together with 1 bed volume of water washing-through Dowex 2 - × 8, formate form. The acylmannosamines of the neutral effluent including 1 bed volume of water washing are lyophilized. The acylmannosamines are analyzed quantitatively according to Reis-

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sig et al. 7.~and qualitatively by thin-layer chromatography on cellulose in n-butanol--n-propanol-0.1 N HCi (1:2: l; v/v/v'~). They are visualized by spraying with solutions of AgNO3 and NH3 in methanol, s° Based on their R s values" (in parentheses), it was possible to distinguish the following acylmannosamines: N-glycolylmannosamine (0.45), N-acetylmannosamine (0.55), N-glycolyl-6-O-acetylmannosamine (0.63), and N-acetyl-6-O-acetylmannosamine (0.72). N-Acyl-6-O-acetylmannosamines are derived from N-acyl-9-O-acetylneuraminic acids (acyl = acetyl or glycolyl). As was mentioned before, N-acyl-4-O-acetylneuraminic acids are degraded very slowly. The reaction products are N-acylmannosamines; the 4-O-acetyl group is probably eliminated during the enzymic cleavage reaction. 69 Periodate Oxidation Determination of the oxidation rate of sialic acids by periodate may be used for structural analysis of these compounds on the one hand, s and for preparation of the C-7 and C-8 analogs of N-acetylneuraminic acid on the other, sl Periodate oxidation experiments on an analytical scale are carried out in the dark at room temperature. One milliliter of a 5 mM solution of a neuraminic acid derivative in dioxane/water, 1:1 (v/v), is mixed with an equal volume of 20 mM periodic acid or sodium metaperiodate in water. The periodate consumption is followed spectrophotometrically at 220 rim; 20-td samples of the reaction mixture are withdrawn at appropriate time intervals, diluted with 1 ml of water, and measured. ~s The C--C borids in the C-7--C-9 side chain of neuraminic acid are cleaved by periodate within 10-20 min under the conditions described if the hydroxyl groups at C-7, C-8, and C-9 are unsubstituted, s'~"'s2's'~ This high periodate consumption rate of sialic acids is appreciably reduced if the mentioned hydroxyl groups are partly substituted by ester groups. While N-acetylneuraminic acid, N-glycolyneuraminic acid, N-acetyl-4O-acetylneuraminic acid, and neuraminic acid /3-methylglycoside consume 2 mol of periodate per mole, N-acetyl-7-O-acetylneuraminic acid consumes only 1 mol ofperiodate per mole owing to a cleavage of only the C-8~C-9 diol group. ~ Remarkably, N-acetyl-9-O-acetylneuraminic acid r~ j. L. Reissig, J. L. Strominger, and L. F. Leloir, J. Biol. Chem. 217, 959 (1955). so S. M. Partridge, Biochem. J. 42, 238 (1948). ~ R. W. Veh, A. P. Corfield, M. Sander, and R. Schauer, Biochirn. Biophys. Acta 486, 145 (1977), s 2 G. Blix, E. Lindberg, L. Odin, and I. Werner, Acta Soc. Med. Upsal. 61, 1 (1956). ~s G. Dryhurst, ed., "Periodate Oxidation of Diol and Other Functional Groups," Chapter 3. Pergamon, Oxford, 1970.

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83

consumes only 0.2 mol of periodate per mole under the same conditions in spite of the presence of two vicinal free hydroxyl groups at C-8 and C-9. TM The slow reactivity of this compound with periodate is the reason for the previous assignment of its O-acetyl residue at C-8. ~ (The correct localization of the O-acetyl group at C-9 was possible by mass spectrometry as will be described below.) The low oxidation rate is explained by the trans position of the hydroxyl groups at C-7 and C-8. '~s A similar steric hindrance is believed to be the reason for the low oxidation rate of Nacetyl-9.0-L-lactylneuraminic acid, which is similar to that of Nacetyl-9-O-acetylneuraminic acid. ~ Periodate oxidation, followed by borohydride reduction of the aldehyde derivative of neuraminic acid is a valuable method for preparation of the C-7 and C-8 analogs of free or glycoconjugate-bound N-acetylneuraminic acid. ~1 (The C-7 analog is systematically called 5-acetamido-3,5-dideoxy-L-arabinoheptulosonic acid, and the C-8 analog is called 5-acetamido-3,5-dideoxy-D-galactooctulosonic acid.) Thus, glycoproteins labeled with tritium specifically in their sialic acid moiety representing mainly C-7 analogs were obtained. ~4 In addition, radioactive gangliosides were prepared with the tritium label exclusively in the modified neuraminic acid residues. ~1The ratio of the C-7 and C-8 analogs of N-acetylneuraminic acid can be influenced by variation of periodate concentration, time, and temperature of the reaction; the conditions are described in detail by Veh e t al. ~1 Under the conditions chosen, the periodate attack On the galactose residues of the gangliosides was negligible. By use of borohydride of high specific radioactivity highly labeled, modified glycoproteins, glycopeptides, and gangliosides are obtained, which may be used as substrates for measurement of the activity of glycoprotein- or glycolipid-specific neuraminidases of different origins in a very sensitive and specific manner. :~'3~'~ G a s - Liquid Chromatography

Gas-liquid chromatography has been established as a valuable tool in sialic acid analysis. Sweeley e t al. s6 originally analyzed sialic acids as their fl-methylketosides obtained by mild methanolysis, followed by trimethylsilylation (see also Laine e t a[.S7). By methanolysis, most of the O-acyl groups are, however, eliminated. ~" Therefore, the method is useful only ,4 This series Vol. 28 [15]. ~ R. Schauer, R. W. Veh, M. Wember, and H.-P. Buscher, Hoppe-Seyler's Z. Physiol. Chem. 357, 559 (1976). 8"C. C. Sweeley, R. Bentley,M. Makita, and W. W. Wells,J. Am. Chem. Soc. 85, 2497 (1963). ~7R. A. Laine, W. J. Esselman, and C. C. Sweeley,this series Vol. 28 [10]. ~ J. Casals-Stenzel,H.-P. Buscher,and R. Schauer, Anal. Biochem. 65,507 (1975).

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for determination of the total amount of neuraminic acid present in, for example, a glycoconjugate. Craven and Gehrke s9 were the first to analyze N-acetylneuraminic acid and N-glycolylneuraminic acid by direct trimethylsilylation. Based on this method, two procedures have been worked out 1°'~ for gas-liquid chromatography of the individual neuraminic acid derivatives including the O-acylated species. It was demonstrated by acyl hydroxamate formation (see above) and by mass spectrometric analysis (see next subsection) that O-acyi groups are not eliminated by the two derivatization procedures of sialic acids described below.

Preparation and Analysis o f the Trimethylsilyl Esters, Trimethylsilyl Ethers. ~ Trimethylsilyl imidazole (25-50/A) is added to decationized and lyophilized sialic acid samples (10-100/~g) under dry nitrogen. The stoppered tubes are shaken for either 15 min at room temperature or 5 min at 60° in a heating block to complete the silylation reaction, which is visible by total solubilization of the substance. Aliquots containing 0.5-10 p.g of sialic acids are chromatographed on a 1.6 m × 2 mm glass column on 3% OV-17 on Gas Chrom Q, 100-120 mesh, at 210 ° isothermally at a nitrogen flow rate of 50 ml/min and detected with a flame ionization detector. The retention times of the individual sialic acids are related either to N-acetylneuraminic acid (RNeL,NAc) as shown in Table II or to trehalose as internal standard, which is added in 1-~g quantities to the sialic acid samples. Analysis of the pertrimethylsilyl derivatives of sialic acids should be carried out immediately after preparation, as these derivatives are not very stable.

Preparation and Analysis o f the Methyl Esters, Trimethylsilyl Ethers. lo Diazomethane in ether is added dropwise to 50-100/.~g of sialic acids in 0.5 ml of methanol until a faint yellow color is obtained. The solution is immediately evaporated under reduced pressure at 30°, and the residue is dissolved in 1 ml of pyridine; 0.2 ml of hexamethyldisilazane and 0.1 ml of chlorotrimethylsilane are added. After 2 hrs at room temperature, 2 ml of chloroform and 2 ml of water are added to the turbid mixture. The chloroform layer is dried over anhydrous Na2SO4 and evaporated under reduced pressure. The residue is dissolved in 50-100 p~l of chloroform. Instead of this procedure of silylation, it is also possible to trimethylsilylate the sialic acid methyl esters directly by the addition of trimethylsilyl imidazole as described above. Gas-liquid chromatography of appropriate amounts of this solution is carried out on 2-meter × 4-mm glass columns of 3.8% SE-30 on Chromosorb W/AW-DMCS, HP, 80-100 mesh at 210 ° isothermally using a nitrogen gas flow rate of 40 ml/min.'~'2' The retention times relative to s~' D. A. Craven and G. W. G e h r k e , J. Chromatogr. 37, 414 (1968).

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TABLE II RETENTION TIMES RELATIVE TO N-ACETYLNEURAMINIC ACID (Rxeu.~Ae) OF SIALIC ACIDS ON GAS-LIQUID CHROMATOGRAPHY

Compound N-Acetylneuraminic acid N-Acetyl-4-O-acetylneuraminic acid N-Acetyl-7-O-acetylneuraminic acid N-Acetyl-9-O-acetylneuraminic acid N-Acetyl-4,9-di-O-acetylneuraminic acid N-Acetyl-7,9-di-O-acetylneuraminic acid N-Acetyl-9-O-L-lactylneuraminic acid

N-Acetyl-2-deoxy-2,3-dehydroneuraminic N-Glycolylneuraminic acid N-Glycolyl-4-O-acetylneuraminic acid N-Glycolyl-9-O-acetylneuraminic acid

acid

System A a

System B b

1.00 1.70 1.55 1.46 ND c 2.18 ND 1.81 1.78 2.79 2.56

1.00 1.18 1.04 1.13 ND 1.14 2.55 1.10 1.81 ND 2.04

" System A: Trimethylsilyl esters, trimethylsilyi ethers of acyineuraminic acids, developed on 3% OV-17 [J. Casals-Stenzel, H.-P. Buscher, and R. Schauer, Anal. Biochem. 65, 507 (1975)]. t, System B: methyl esters, trimethylsilyl ethers of acylneuraminic acids, chromatographed on 3.8% SE-30 [J. Haverkamp, R. Schauer, M. Wember, J.-P. Farriaux, J. P. Kamerling, C. Versluis, and J. F. G. Vliegenthart, Hoppe-Seyler's Z. Physiol. Chem. 357, 1699 (1976); R. Schauer, J. Haverkamp, M. Wember, J. F. G. Vliegenthart, and J. P. Kamerling, Eur. J. Biochem. 62, 237 (1976); J. P. Kamerling, J. F. G. Vliegenthart, R. Schauer, G. Strecker, and J. Montreuil, Eur. J. Biochem. 56, 253 (1975)]. " ND, not determined.

N-acetylneuraminic acid of different sialic acids analyzed by this method are shown in Table II. The different sialic acids occurring in nature and also synthetic neuraminic acid derivatives, such as N-chloroacetylneuraminic acid, N-fluoroacetylneuraminic acid, neuraminic acid /3-methylglycoside, 42 or the C-7 and C-8 analogs of N-acetylneuraminic acid, ~l can be analyzed in a reproducible way after derivatization by the two methods described. For exact analysis, minimum amounts of 0.5-1 /zg of sialic acids are required. 88 For quantitative analyses, calibration curves are necessary for each neuraminic acid derivative as differences in the detector responses, especially between the pertrimethylsilyl derivatives of O-acylated and not esterified sialic acids have been observed? ~ Analysis of sialic acids by gas-liquid chromatography is sensitive and more specific than most of the colorimetric assays, especially if the gas chromatograms obtained by the two methods described for sialic acid derivatization are compared. A further advantage is the possibility of analyzing a sialic acid mixture with greater specificity and sensitivity with regard to the different neuraminic acid derivatives than is possible with,

86

ANALYTICAL METHODS

[6]

for example, thin-layer chromatography. While the isomeric Nacyl-mono-O-acetylneuraminic acids cannot be distinguished by thinlayer chromatography, they show distinct retention times on gas-liquid chromatography (see Table II). However, the sialic acid preparations must be purified by extraction with ether :~3or n-hexane, especially if derived from gangliosides, and by ion-exchange chromatography to achieve optimal separation on gas-liquid chromatography. Development of gas-liquid chromatography of sialic acid mixtures enables combination of this method with mass spectrometry. Mass Spectrometry

While the nature of the N- and O-acyl groups of sialic acids can be determined by thin-layer chromatography and the chemical reactions described above, the position of the O-acyl groups can only tentatively and tediously be estimated by colorimetric methods and by the reaction rates of the respective sialic acids with the enzymes neuraminidase and acylneuraminate pyruvate-lyase. Even determination of the oxidation rates of sialic acids with periodate, which was frequently used in earlier studies, may lead to errors in determination of the position of the O-acyl groups, as discussed in the preceding subsection. 8.5~ In addition, relatively large quantities of pure compounds are required for structural analysis of sialic acids by classical methods. However, analysis of possible new sialic acids required the development of techniques utilizing only the small quantities of these compounds that could be isolated for study (10-100 /~g per analysis). Thus, the application of combined gas-liquid chromatography/mass spectrometry to the sialic acids shed new light on the analysis of these compounds. By this method, the structures of known sialic acids could be established unequivocally, and several new neuraminic acid derivatives were discovered in biological materials. 1°'11'15''-'~ Furthermore, structural analysis of different sialic acids as components of a mixture can be carried out in one single run. High-resolution mass spectrometric analysis of the methyl esters, trimethylsilyl ethers of acylneuraminic acids is carried out as discussed in detail in references cited in footnotes 10, 15, and 21. Seven characteristic fragment ions shown in Fig. 1 can be derived from the mass spectra of the different neuraminic acid derivatives analyzed to date. The fragmentation patterns of a variety of natural sialic acids are shown in Table III. The presence or the absence of these fragment ions, and especially their mass shifts when compared with the masses of the corresponding fragments from N-acetylneuraminic acid, enable an unequivocal analysis of sialic acids, especially with regard to the type of N-acyl groups and the type, number, and position of the O-acyl groups.

[6]

87

SIALIC ACID CHARACTERIZATION

Rsi H N ~

•

H-CH3 -I

1~1

R,O O . II CH=NH -C-R s I

OR~

\

RsCHN/,~0 \ COOCH)~I?

(g)

CH20R,

CHOR~

(o_I

/

CHOR7 I CHORa

(~}

II ~ UH=U+'()~ 0 "~,_~l/0R2~/

CH2ORg

CH2ORs L CH=ORB

-'CHOR?CHORI NH2CORs ICH2ORg-

(F__)

I[ --- R2OH R40H v

R2 0R~

(E)

(D.I

FIG. 1. Fragmentation pattern of the methyl esters, trimethylsilyl ethers of acylneuraminic acids on mass spectrometry [J. P. Kamerling, J. F. G. Vliegenthart, C. Versluis, and R. Schauer, Carbohydr. Res. 41, 7 (1975); R. Schauer, J. Haverkamp, M. Wember, J. F. G. Vliegenthart, and J. P. Kamerling, Eur. J. Biochem. 62,237 (1976)]. The mass values of the fragment ions A - G are given in Table 1II. R2, trimethylsilyl; R4 and Rr, trimethylsilyl and/or acetyl; R.~, methyl or CH~O-trimethylsilyl; R,, trimethylsilyl; R.,, trimethylsilyl or acetyl or L-lactyl.

Mass spectrometry has also been applied for analysis of the neuraminic acid concentration in biological materials. '~ Acylneuraminic acid residues are liberated by methanolysis, by which possible O-acyl groups are split off, re-N-acetylated, and trimethylsilylated. For quantitative analysis a multiple ion-detection technique was used, thus enabling a specific and extremely sensitive (minimum l ng of neuraminic acid) method for N-acetylneuraminic acid determination. Characteristic mass spectrometric fragmentation patterns have been obtained from the trimethylsilyl ethers or acetyl esters of a set of partially O-methylated methyl esters of N-acetyl-N-methyl-fl-D-neuraminic acid methyl glycosides, the preparation and analysis of which are described in references cited in footnotes 91-93. This method enables not only an ~,oI. Mononen and J. K/irkk~iinen, FEBS Lett. 59, 190 (1975). ~" J. Haverkamp, J. P. Karnerling, J. F. G. Vliegenthart, R. W. Veh, and R. Schauer, FEBS Lett. 73, 215 (1977). ~'~H. van Halbeek, J. Haverkamp, J. P. Kamerling, J. F. G. Vliegenthart, C. Versluis, and R. Schauer, Carbohydr. Res. 60 (1978) (in press). ":~A. K. Bhattacharjee and H. J. Jennings, Carbohydr. Res. 51,253 (1976).

88

[6]

ANALYTICAL METHODS T A B L E llI CHARACTERISTIC FRAGMENT IONS A - G , USED FOR IDENTIFICATION OF NATURAL SIALIC ACIDS AS THEIR METHYL ESTERS, TRIMETHYLSILYL ETHERS BY MASS SPECTROMETRYa

Fragments b Compound

A

B

C

D

E

F

G

N-Acetylneuraminic acid N-Acetyl-4-O-acetylneuraminic acid N-Acetyl-7-O-acetylneuraminic acid N-Acetyl-9-O-acetylneuraminic acid N-Acetyl-4,9-di-O-acetylneuraminic acid N-Acetyl-7,9-di-O-acetylneuraminic acid N-Acetyl-9-O-L-lactylneu raminic acid N-Glycolylneuraminic acid N-Glycolyl-4-O-acetylneuraminic acid N-Glycolyl-9-O-acetylneuraminic acid

668 638 638 638 608 608 740 756 726 726

624 594 594 594 564 564 696 712 682 682

478 448 -478 448 -478 566 536 566

298 298 -298 298 -298 386 386 386

317 -317 317 -317 317 317 -317

205 205 205 175 175 175 277 205 205 175

173 143 173 173 143 173 173 261 231 261

a j. p. Kamerling, J. F. G. Vliegenthart, C. Versluis, and R. Schauer, Carbohydr. Res. 41, 7 (1975); J. H a v e r k a m p , R. Schauer, M. W e m b e r , J.-P. Farriaux, J. P. Kamerling, C. Versluis, and J. F. G. Vliegenthart, Hoppe-Seyler's Z. Physiol. Chem. 357, 1699 (1976); R. Schauer, J. H a v e r k a m p , M. W e m b e r , J. F. G. Vliegenthart, and J. P. Kamerling, Eur. J. Biochem. 62, 237 (1976); J. P. Kamerling, J. F. G. Vliegenthart, R. Schauer, G. Strecker, and J. Montreuil, Eur. J. Biochem. 56, 253 (1975). b The nature of the fragment ions is shown in Fig. 1.

analysis of naturally occurring O-methyl derivatives of N-acylneuraminic acids, 16-is but can also successfully be applied for the determination of the type of linkage of sialic acids or other monosaccharides to the hydroxyl groups of neuraminic acid residues in polysaccharides or glycoconjugates. 91m In the latter case the glycosidically linked sialic acids are methylated with methyl sulfinyl carbanion/dimethyl sulfoxide/methyl iodide, followed by methanolysis of the glycosidic bond, re-N-acetylation of the amino group and trimethylsilylation or acetylation of the free hydroxyl groups of the neuraminic acid derivatives before analysis by mass spectrometry. The positions of the trimethylsilyl groups or acetyl groups correspond to the positions of the (nonglycosidic) hydroxyl groups to which other monosaccharide units have been linked in the intact compound. Thus, 2 - > 8 glycosidic linkages between N-acetylneuraminic acid residues in GTlb ganglioside 91 and colominic acid 91'9z could recently be verified. In addition, 2 - > 8 linkages were found in disialyl groups of rat brain glycoproteins. 94 Mass spectrometry of O-methylated neuraminic acid derivatives is 94 j. Finnel T. Krusius, and H. Rauvala, Biochem. Biophys. Res. Commun. 74,405 (1977).

[6]

SIALIC ACID CHARACTERIZATION

89

also of great value for the estimation of the position of the labile O-acyl residues in glycoconjugates. Chemical reactions have been worked out 92'~5 by which O-acyl groups in sialic acids are converted to O-methyl groups. Nuclear Magnetic Resonance Spectroscopy Application of this method to sialic acids has effectively contributed to the elucidation of the structures of both free and glycosidically bound N-acetylneuraminic acid including the position o f their O-acetyl substituents. It was shown using 1H N M R spectroscopy that the pyranose ring of both free N-acetylneuraminic acid 96"°7 and the ketosides of this compound °7'98 exists in the IC conformation. Furthermore, it can be delineated from the ~H N M R data obtained for 4,7,8,9-tetra-O-acetyl-Nacetyl-a-D-neuraminic acid benzyl glycoside methyl ester °8 and free N-acetylneuraminic acid °7 that the h y d r o x y l groups at C-7 and C-8 of the neuraminic acid side chain occupy a trans position to each other. Based mainly on 13C spin lattice relaxation (lrx) studies, Czarniecki and Thornton 99 proposed a structural model for a- or fl-methyl glycosides of N-acetylneuraminic acid in which the amido N-H is hydrogen bonded to the oxygen at C-7. Furthermore, the hydroxyl group at C-8 is hydrogenbonded to the ring oxygen, and the hydroxyl group at C-4 forms a hydrogen bond with the carbonyl group of the N-acetyl residue. No interactions of the hydroxyl at C-9 have been found. The same authors 1°° demonstrated that Ca 2÷ ions form stable complexes with the/3-anomeric form of N-acetylneuraminic acid in contrast to the biologically significant a- anomer. 13C NMR spectroscopy has also been used successfully for localization of the O-acetyl groups in the 9-O-acetyl- and 4,9-di-O-acetyl derivatives of the methyl ester o f N-acetyl-/~-D-neuraminic acid methyl glycoside synthesized by H a v e r k a m p et al.,~s or in the sialic acid residues o f different polysaccharide antigens o f N e i s s e r i a meningitidis as investigated by Bhattacharjee et al. 25,101In the latter two publications, ~ N M R spectroscopy has also been applied for study of the type of sialic acid linkages in the bacterial polysaccharides. ~ S. Hakomori and T. Saito, Biochemistry 8, 5082 (1969). ~ E. B. Brown, W. S. Brey, Jr., and W. Weltner, Jr., Biochim. Biophys. Acta 399, 124 (1975). ~7L..Dorland and J. F. G. Vliegenthart, unpublished results, 1977. '~ P. Lutz, W. Lochinger, and G. Taigel, Chem. Ber. 101, 1089(1968). ~'qM. F. Czarniecki and E. R. Thornton, J. Am. Chem. Soc. 98, 1023 (1976). ~ooM. F. Czarniecki and E. R. Thornton, Biochem. Biophys. Res. Commun. 74, 553 (1977). ~ol A. K. Bhattacharjee, H. J. Jennings, C. P. Kenny, A. Martin, and I. C. P. Smith, J. Biol. Chem. 250, 1926 (1975).