The mechanism of the periodate-thiobarbituric acid reaction of sialic acids

1. 4-0-Methyl-N-acetylneuraminic acid shows a strong positive periodate-thiobarbiturate reaction. The mechanism of dye formation in this test for sialic acids is discussed in view of the studies already published.2. An efficient preparation of a tritium-labelled 4-0-methyl-N-acetylneuraminic acid, with high specific radioactivity, by an oxymercuration-demercuration procedure is presented.3. Sialyltransferase activities in microsomal fractions of equine liver using desialylated fetuin are studied. The enzyme activity, assayed in a radioactive procedure, shows an apparent K , value for CMP-N-acetylneuraminic acid of 0.7 mM, whereas this value is 3.4 mM for CMP-4-0-methyl-N-acetylneuraminic acid. Differences are also observed in the maximal velocity for the two substrates.4. The equine liver system can be used to prepare substantial amounts of fetuin containing radioactive N-acetylneuraminic acid or 4-0-methyl-N-acetylneuraminic acid. The isolated reaction products show similar sialic acid release by treatments with acid or fowl-plague virus neuraminidase. In contrast, 4-0-methyl-N-acetylneuraminic acid-fetuin displays a marked resistance to desialylation by Vibrio cholerae neuraminidase.5. Free 4-0-methyl-N-acetylneuraminic acid is completely resistant to the action of acylneuraminate pyruvate-lyase. It does not inhibit the enzymic cleavage reaction of N-acetylneuraminic acid.6. The influence of a substitution at C-4 of neuraminic acid on the enzymatic reaction mechanisms is discussed.The acylneuraminic acids (sialic acids), widely distributed in animal tissues, are important components of oligosaccharides, glycolipids and glycoproteins. The occurrence and nature of various Nacylated and O-acylated neuraminic acids is now well documented [l -31, and the pattern distribution is characteristic for the animal species. Sialic acids are implicated in many cellular processes [4], and the high number of the 0-acylated derivatives points to the importance of understanding the biological role of these substances. In a preliminary approach, a precise evaluation of the behaviour of the main enzymes involved in the metabolism of sialic acids is

Section: Studies With Acylneuraminate Pyruvute-lyasementioning

confidence: 80%

Section: Studies With Acylneuraminate Pyruvute-lyasementioning

confidence: 99%

Metabolism of 4‐O‐Methyl‐N‐acetylneuraminic Acid a Synthetic Sialic Acid

Beau

Schauer

1980

“…0-Acyl groups have no significant influence on the colour yield with the orcinol reagent as was found by comparison of 0-acylated sialic acids before and after release of the 0-acyl groups. This is in contrast to the periodic acid -thiobarbituric acid method, by which the different 0-acylated sialic acids exhibit different molar absorption coefficients as was discussed by Paerels and Schut [16].…”

Section: Quantitative Determination Of Sialic Acidsmentioning

confidence: 86%

New Sialic Acids

Buscher

Casals-Stenzel

Schauer

1974

101

1. Sialic acids were released by dilute acids or by neuraminidase from glycoproteins of bovine and equine submandibular glands, from glycoproteins of equine blood plasma and from equine erythrocyte membranes. They were purified by ion-exchange chromatography and fractionated on cellulose.2. The following methods have been adapted to the structural analysis of sialic acids with regard to the nature and position of the N-acyl and 0-acyl groups. a) Thin-layer chromatography in two dimensions with intermediate ammonia treatment to remove the 0-acyl groups leading to the identification of the basic N-acylneuraniinic acids.b) Thin-layer chromatography in two dimensions with intermediate hydroxylamine treatment allowing the identification of the 0-acyl groups of sialic acids as hydroxamates. c) Gas-liquid chromatography for identification of the different N-acylated and 0-acylated sialic acids.3. The structure of the following new sialic acids have been established: a) N-glycoloyl-8-0-acetylneuraminic acid isolated from glycoproteins of bovine submandibular glands; b) N-acetyl-4-0-glycoloylneuraminic acid isolated from glycoproteins of equine submandibular glands and also occurring in equine blood plasma and equine erythrocyte membranes; c) N-acetyl-7 or 8-0-glycoloylneuraminic acid detected in glycoproteins from bovine submandibular glands.4. The 0-acetylated N-glycoloylneuraminic acid first detected in horse erythrocyte membranes by Hakomori and Saito and in horse serum by Pepper, was determined to be N-glycoloyl-4-0-acetylneuraminic acid; it was also found to occur in submandibular glands of horse.5. The sialic acid patterns from the different biological materials investigated are compared.

“…It is also known that a-keto carboxylic acids are cleaved by periodate [19] : it can therefore be expected that temperatures above ambiant, which are used in some cases [26], while ensuring more rapid glycol cleavage and hence more nearly quantitative formation of j3-formyl-pyruvate, will also provoke considerable destruction of the chromogenic a-keto acid. Indeed, it has been shown conclusively by Paerels and Shut [29], that cleavage of j3-formyl-pyruvate by periodate, and concomitant decrease of colour formation in the thiobarbiturate test takes place even at room temperature.…”

Section: Periodate Oxidation and Thiobccrbiturate Test Of 3-deoxy D-mmentioning

confidence: 99%

“…We therefore concluded that the totality of the j3-formyl-pyruvate was liberated from the aldulosonic acid when three molar equivalents of periodate had been reduced, and that cleavage of ,!I-formyl-pyruvate by periodate, though not completely arrested in the conditions of the cold acid COOH pyruvate theoretically obtainable from the deoxy octulosonate was, in fact, set free upon treatment with periodate. (The structure and chemical reactions of 3-deoxy aldulosonic acids with 5 , 6 , 7 , and 8 carbon atoms will be described elsewhere [29].) Isosaccharinic acid (3-deoxy 2-C-hydroxymethyl D-erythro-pentonic acid) (VIJ) was chosen as model substance: indeed it can be expected that besides two molar equivalents of formaldehyde, the only product formed upon periodate cleavage of this compound in the conditions of the cold acid method, would be one molar equivalent of /3-formyl-pyruvic acid.…”

Section: Periodate Oxidation and Thiobccrbiturate Test Of 3-deoxy D-mmentioning

confidence: 99%

Chemical Synthesis and Quantitative Estimation of 3‐Deoxy d‐manno‐Octulosonic Acid

Charon¹,

Sarfati²,

Strobach

et al. 1969

3-Deoxy D-manno-octulosonic acid has been synthesised starting from D-mannose via 2-deoxy D-manno heptose, cyanohydrine addition, and selective oxidation of the epimeric hydroxyl group of the resulting 3-deoxy octonic acids. When treated with periodate in 0.1 N sulphuric acid solution a t O", the deoxy octulosonic acid and other 3-deoxy aldulosonic acids all yield one molar equivalent of P-formyl pyruvate. Conditions are described in which /I-formyl pyruvate can be estimated by the thiobarbituric acid method ; hence, when treated successively with periodate and thiobarbiturate, all 3-deoxy aldulosonic acids have the same molar extinction coefficient and eight carbon atoms are known to be important intermediates of carbohydrate metabolism in various organisms, a general method was sought by which these compounds, as well as their phosphate esters, could be made readily accessible. In 1944 it was reported that o-gluconic acid and D-galactonic acid could be oxidised to aldulosonic acids by chlorate in the presence of a vanadium pentoxide catalyst [lo]. It was therefore expected that similar treatment of 3-deoxy aldonic acids would afford the corresponding aldulosonic acids. This was found to be the case : the present paper reports an unambiguous chemical synthesis of 3-deoxy D-manno-octulosonic acid starting from D-mannose. Conditions are described in which 3-deoxy D-manno-octulosonic acid and other 3-deoxy aldulosonic acids, when treated with periodate, yield one molar equivalent of ,9-formyl pyruvate; these acids can thus be estimated quantitatively in the thiobarbituric acid assay without standards. EXPERIMENTAL PROCEDURESXynthesis of 3-Deoxy D-manno-Octulosonic Acid ( V ) 1,2-Di-deoxy-l-nitro-3,4,5,6,7-penta-O-acetyl D-manno-heptene [ll, 121 (10 g) in 600 ml absolute ethanol was hydrogenated over 1 g palladium black, until 1.05 moles of hydrogen (570 ml a t 25" and 760 mm) had been consumed. The filtered solution was then concentrated in vacuo to a crystallising syrup, which was redissolved in 75 ml ethanol. Sodium hydroxide (100 ml, 2 N) was added in small portions to this solution kept below f5". The resulting deep red solution was then added rapidly, but dropwise, to a stirred solution of 25 ml concentrated sulphuric acid in 35 ml water maintained in an ice-bath. When the addition was complete, the reaction mixture was diluted with 1.5 1 water and neutralised with solid calcium carbonate. All solids were removed by filtration, the combined filtrate and washings passed through columns of ion exchange resins (Amberlite IR 120 H+ and I R 45 OH-, 250 ml each) and the effluent was concentrated in vacuo to a small volume.Sodium hydrogen carbonate (3.13 g) was added to the above solution of crude 2-deoxy D-mannoheptose and the volume adjusted to 186ml with water. This mixture was then added to 1.5 g sodium hydroxide and 3.0 g potassium cyanide in 186 ml water frozen to the walls of a 1 1 round bottomed flask. The flask was agitated until the mixture became homogeneous and then set aside a t room temperature overnig...