The mechanism of the periodate-thiobarbituric acid reaction of sialic acids.
ABSTRACT: 1. The chromogen formation from N-acetylneuraminic acid in the periodate-thiobarbituric acid reaction was investigated. Measurement of periodate consumption showed an uptake of approx. 3moles/mole of substrate in neutral as well as in strongly acidic solution. Therefore the chromogen beta-formylpyruvic acid is not a direct product of the periodate oxidation; it is presumed to be formed from the true oxidation product, a hexos-5-uluronic acid, by aldol splitting during the reaction in hot acidic solution with thiobarbituric acid. 2. Methyl (methyl beta-l-threo-hexos-4-enepyranosid)uronate, an analogue of the pre-chromogen, has been shown to yield with thiobarbituric acid in acidic solution a pigment exhibiting an identical absorption spectrum and showing the same behaviour on paper chromatography as the pigment obtained from N-acetylneuraminic acid in the periodate-thiobarbituric acid assay. 3. The substitution at C-2 of methoxyneuraminic acid does not inhibit the periodate-thiobarbituric acid reaction. In neutral solution methoxyneuraminic acid is oxidized by periodate to a substance that reacts readily with thiobarbituric acid in acidic solution. When periodate oxidation is attempted in acidic solution, protonation of the amino group protects this group against oxidation, rendering methoxyneuraminic acid negative in the assay systems of Warren (1959a,b) and Aminoff (1959, 1961).
Project description:The identity of a novel form of sialyl-lactose found in milk of the echidna (Tachyglossus aculeatus) was investigated. The sialyl-lactose yielded equimolar amounts of N-acetylneuraminic acid and lactose during mild acid hydrolysis but was resistant to the action of a bacterial neuraminidase. A viral neuraminidase hydrolysed it to lactose plus a form of sialic acid that reacted positively with thiobarbituric acid reagent but whose chromatographic mobility was greater than that of N-acetylneuraminic acid. Treatment with alkali converted the sialyl-lactose into a substance with the same chromatographic mobility as N-acetylneuraminyl-(2-->3)-lactose and made it susceptible to the action of bacterial neuraminidase. The sialyl-lactose contained one mol of ester (identified as acetyl), and released one mol of formaldehyde during periodate oxidation, per mol of sialic acid. It did not contain N-glycollylneuraminic acid. These results indicate that the sialyl-lactose is N-acetyl-4-O-acetylneuraminyl-(2-->3)-lactose. Echidna milk contained, in addition, a small amount of N-acetylneuraminyl-(2-->3)-lactose.
Project description:1. The mucin of the Cowper's gland of the boar is a sialomucoprotein similar to submaxillary-gland mucin. When a solution of either mucin has been incubated for 5min or less with a particulate fraction from homogenized uterine endometriumplus-myometrium of the rabbit, 10-20% of sialyl residues (N-acetylneuraminic acid) give a positive Warren reaction for free N-acetylneuraminic acid. The particulate fraction is devoid of neuraminidase and no free (diffusible) N-acetylneuraminic acid appears during incubation. The factor that catalyses the formation of directreading non-diffusible N-acetylneuraminic acid occurs also in liver, kidney and intestinal mucosa of the rabbit. The factor is present in very small (;microsomal') particles and has not yet been solubilized. Homogenates of boar Cowper's gland contain both factor and mucin; thus direct-reading non-diffusible N-acetylneuraminic acid appears when such homogenates are stored. 2. Under optimum conditions 1mg of uterine protein catalyses the formation of 0.05-0.1mumol of direct-reading non-diffusible N-acetylneuraminic acid/min. This activity is considerably higher than the neuraminidase activities of comparable homogenates of animal tissues or of liver lysosomes. The factor is thermostable and its activity shows little variation within (i) the pH range 3-10, (ii) the temperature range 20-37 degrees C. Activity is inhibited strongly by 2,2'-bipyridyl and by ammonium pyrrolidine dithiocarbamate but is unaffected by EDTA. Its action can be simulated by low concentrations of Fe(2+). From this it may be inferred that the factor is a protein-bound from of bivalent iron. A number of pure iron-containing proteins and haemoproteins were completely inactive. The following substrates were not sources of direct-reading non-diffusible N-acetylneuraminic acid: methoxyneuraminic acid, sialyl-lactose, brain gangliosides, and sialoproteins in which N-acetylneuraminic acid is linked to galactose residues. 3. It is proposed that the factor (or Fe(2+)) reacts with the mucin in a manner that renders the C-4-C-5 bond of sialyl residues susceptible to periodate oxidation.
Project description:1. Monosaccharides, amino sugars and N-acetylneuraminic acid were determined by using an original colorimetric assay procedure, based on the detection of formaldehyde released after periodate oxidation. A range of these compounds was investigated by this method and they were all found to obey Beer's law within the concentration range 0-0.6mumole/ml. 2. A simple method for the determination of 6-deoxyhexose concentration in the presence of other monosaccharides is also described. 3. The optimum pH for the release of formaldehyde from sugars by periodate oxidation was 7.0-7.5. 4. The methods described have considerable advantages over existing assay systems and their particlar value in automatic colorimetry, where the use of concentrated acids is undesirable, is discussed.
Project description:With dimethyl sulphoxide instead of butanol in the thiobarbituric acid assay for sialic acid, a non-fading chromophore with lambdamax. = 549 nm was produced in a homogeneous solution, allowing dilution of the test mixture in case of high colour yield. This test adapted well to studies on alkaline de-O-acetylation. Bovine and rat submaxillary mucins, and rabbit Tamm-Horsfall urinary sialoproteins contain O-acetyl isomers of neuramine acid that are resistant to the thiobarbituric acid assay. Alkaline de-O-acetylation converted resistant O-acetylneuraminic acid into thiobarbituric acid-reactive sialic acid, and such conversion paralleled de-O-acetylation as measured by the ferric hydroxamate method. The colour increment was similar when the alkaline treatment of bovine submaxillary mucin either preceded or followed the acid hydrolysis. Only alkaline preptreatment was effective with rat submaxillary mucin. By selecting optimal conditions for alkaline de-O-acetylation, O-acetyl isomers can be accurately assessed by the thiobarbituric acid assay.
Project description:The selective preparation of 2-iodoxybenzenesulfonic acid (IBS, as potassium or sodium salts) by oxidation of sodium 2-iodobenzenesulfonate with Oxone or sodium periodate in water is reported. The single crystal X-ray diffraction analysis reveals a complex polymeric structure consisting of three units of IBS as potassium salt and one unit of 2-iodoxybenzenesulfonic acid linked together by relatively strong I=O···I intermolecular interactions. Furthermore, a new method for the preparation of the reduced form of IBS, 2-iodosylbenzenesulfonic acid, by using periodic acid as an oxidant, has been developed. It has been demonstrated that the oxidation of free 2-iodobenzenesulfonic acid under acidic conditions affords an iodine(III) heterocycle (2-iodosylbenzenesulfonic acid), while the oxidation of sodium 2-iodobenzenesulfonate in neutral aqueous solution gives the iodine(V) products.
Project description:1. Bovine bone sialoprotein (mol.wt. 23000) contains N-acetylneuraminic acid and N-glycollylneuraminic acid, fucose, galactose, mannose, N-acetylgalactosamine and N-acetylglucosamine residues in the form of a very small number, perhaps one, of highly branched oligosaccharide structures linked covalently to peptide. 2. Periodate oxidation of the sialoprotein results in quantitative destruction only of the sialic acid and fucose residue consistent with the earlier findings of their positions as terminal groups. 3. Terminal sialic acid residues are attached to galactopyranose residues by 2,3-linkages, and to some N-acetylgalactosamine residues (at C-6). 4. Sequential Smith degradation indicates that N-acetylgalactosamine residues may be present as points of branching (linked in C-1, C-3 and C-6) and N-acetylglucosamine residues are located in the inner part of the structure, adjacent to the carbohydrate-peptide bond(s). 5. Mannose residues appear to be linked in the 1,3-positions.
Project description:The gas-phase oxidation of methionine residues is demonstrated here using ion/ion reactions with periodate anions. Periodate anions are observed to attach in varying degrees to all polypeptide ions irrespective of amino acid composition. Direct proton transfer yielding a charge-reduced peptide ion is also observed. In the case of methionine and, to a much lesser degree, tryptophan-containing peptide ions, collisional activation of the complex ion generated by periodate attachment yields an oxidized peptide product (i.e., [M?+?H?+?O](+)), in addition to periodic acid detachment. Detachment of periodic acid takes place exclusively for peptides that do not contain either a methionine or tryptophan side chain. In the case of methionine-containing peptides, the [M?+?H?+?O](+) product is observed at a much greater abundance than the proton transfer product (viz., [M?+?H](+)). Collisional activation of oxidized Met-containing peptides yields a signature loss of 64 Da from the precursor and/or product ions. This unique loss corresponds to the ejection of methanesulfenic acid from the oxidized methionine side chain and is commonly used in solution-phase proteomics studies to determine the presence of oxidized methionine residues. The present work shows that periodate anions can be used to 'label' methionine residues in polypeptides in the gas phase. The selectivity of the periodate anion for the methionine side chain suggests several applications including identification and location of methionine residues in sequencing applications.
Project description:1. It is proposed that periodate oxidation of glycol groups in the repeating units of polysaccharide molecules can be used to probe differences in polymer shapes in solution. 2. Measurement of second-order rate constants (k2) of periodate-glycol reactions may be compared between polymers and relevant monomers, to assess perturbations due to polymer configuration. 3. Factors effecting the measurement and interpretation of k2 are discussed. Over-oxidation, free-radical side reactions, end-group effects, Donnan equilibria and polymer (or molecular-weight) effects are relevant, but their importance is either small or can be minimized in practice. 4. A small group of glycosaminoglycuronans (chondroitin 4- and 6-sulphates and hyaluronate) are oxidized 50--100 times more slowly than three other glycosaminoglycuronans of similar composition, relevant monomers or three homopolyuronides. 5. A stable configuration in solution is postulated for the periodate-resistant polymers, involving carboxylate, acetamido and hydroxy groups in hydrogen-bonded sequences on alternate sides of the molecule. The more easily oxidizable polyuronides are unable to form this configuration. 6. The effect of temperature on the postulated configuration is investigated through the Arrhenius plot of k2, measured to hyaluronate, chondroitin 6-sulphate and methyl 4-O-methyl-alpha-D-glucopyranoside. Probable transitions at high (around 90 degrees C) temperatures were observed for both polymers, with an additional transition at about 37 degrees C in the case of hyaluronate. 7. L-Iduronic acid can take up different conformations depending on the polymer environment.
Project description:Various aliphatic and aromatic amines are oxidized by sodium metaperiodate and these reactions have been studied quantitatively in acidic, unbuffered and basic media. Significant differences have been observed between the behaviour of aliphatic and aromatic amines. Certain compounds also behaved differently under acidic and basic conditions. These reactions are related to the periodate oxidation of amino acids and, from observations on a number of glycine derivatives, a reaction mechanism is proposed for this process.
Project description:The site of palmitoylation of the phosphatidylinositol moiety of the glycosyl-phosphatidylinositol membrane anchor of Trypanosoma brucei procyclic acidic repetitive protein was studied by using periodate oxidation. Analysis of the products by g.c.-m.s. allowed the assignment of 40 and 60% of the palmitate to the 2-position and the 3-position respectively of the myo-inositol ring.