Periodate oxidation studies in the elucidation of the structures of sialic acid containing oligosaccharides

The structures of sialo-oligosaccharide alditols as determined by ¹H-NMR spectrometry together with methylation analysis did not correspond with those derived previously from quantitative periodate oxidation data alone. Possible causes of the discrepancy were explored in the periodate oxidation methodology. No free sialic acid was released by the acidity of the periodate in the course of oxidation at pH 4.5. The anionic properties of the sialic acid residues were therefore utilized to separate the periodate oxidation products and thereby establish the position of the sialic acid in the oligosaccharide chain.     

The kinetics of suppression of proliferation of oxidized murine lymphoid cells by sodium borohydride reduction.

Murine splenic lymphoid cells are stimulated to proliferate following mild oxidation with sodium periodate. To assess the class of cells responding, we used periodate treatment alone or in association with concanavalin A, a T-cell mitogen, or lipopolysaccharide (LPS), primarily a B-cell mitogen. Brief periodate treatment followed by culturing with concanavalin A gave no additive proliferative response to that seen using concanavalin A alone, while culturing periodate-treated cells with LPS gave approximately an additive response. Furthermore, periodate failed to stimulate spleen cells from neonatally thymectomized mice while LPS produced significant stimulation of proliferation, suggesting that periodate is stimulating a class of T lymphoid cells or a subpopulation of T cells. Studies were performed to determine an optimal concentration of borohydride which would suppress proliferation in lymphoid cells initially oxidized with periodate. It was observed that 2 mM borohydride would suppress proliferation of oxidized cells yet permit a normal response of these cells to another T-cell mitogen, concanavalin A. Higher concentrations of borohydride, from 3 to 5 mM, would also suppress proliferation of oxidized cells but would interfere with the ability of these cells to respond to concanavalin A, perhaps due to cell damage. Studies were performed to determine when it was possible to suppress periodate-induced mitogenesis by reducing with borohydride at various times after the initial oxidation. It was observed that 2 mM borohydride treatment could suppress stimulation through 8 hr after the original periodate oxidation and that from 12 hr through 20 hr after the initial periodate oxidation, borohydride was incapable of inhibiting proliferation. Additional studies demonstrate that optimal mitogenesis induced by periodate or concanavalin A is contingent upon a serum factor.     

Inhibition by sodium periodate of the in vitro antibody response of mouse spleen cells and its reversal by borohydride or aldehyde blocking reagents

Mild oxidation of mouse spleen cells by sodium periodate induces blastogenesis with enhancement of thymidine incorporation. Concomitantly, the specific in vitro response of these cells to sheep red blood cells and trinitrophenyl-polyacrylamide and the nonspecific polyclonal B-cell response to lipopolysaccharide are markedly inhibited. Exposure of these cells to sodium borohydride and hydroxylamine following periodate treatment reduces blastogenesis and prevents periodate-induced suppression. Data suggest that the integrity of aldehyde moieties generated by periodate oxidation is necessary for blastogenesis and induction of suppressor cells in mouse spleen cell culture.     

Estimation of in vivo aminoacylation by periodate oxidation: tRNA alterations and iodate inhibition.

Two problems associated with periodate oxidation in determining the extent of aminoacylation of tRNA are discussed. One of the products of this reaction, sodium iodate, was found to inhibit tRNA charging. In addition, periodate oxidation also appears to alter sites other than the 3′-end on at least two isoacceptor species of tRNA^Leu.     

Periodate oxidation of chitosans with different chemical compositions

Periodate oxidation of chitosans with different chemical compositions were investigated by determining the consumption of periodate consumed, and the amount of ammonia and formaldehyde liberated during the reaction. Oxidised chitosans were further characterised by size-exclusion chromatography with online multi-angle light scattering (SEC-MALLS) to obtain the molecular weight distributions, and by elemental analysis to obtain the N/C ratio. Chitosans became only partially oxidised by periodate, reaching degrees of oxidation around 0.5, when oxidising with excess periodate. Overconsumption of periodate is attributed to the extensive depolymerisation, which occurs concomitantly with the oxidation, thereby exposing novel reducing and non-reducing ends which consume additional periodate. Both the rate and extent of overoxidation, and the rate of depolymerisation decreased with increasing F(A). A chitosan-specific degradation mechanism is probably involved in the depolymerisation in addition to the general free-radical-mediated degradation.     

The manual and automated determination of aldonic acids and their O-glycosyl derivatives

Manual and automated spectrophotometric methods are described for the specific determination of aldonic acids by periodate oxidation and reaction with 2,3,4-trihydroxybenzoic acid. In combination with analyses for formaldehyde released on periodate oxidation, and for total aldose, the measurement of glyoxylic acid is employed for the determination of the substitution pattern of _O-glycosylaldoic acids.     

Loss of basophilic (sulphated) material from sections of cartilage treated with periodate solution

Synopsis Fresh-frozen sections of cartilage quickly lost much basophilic material (presumably chondroitin sulphate) when placed in a 2% aqueous solution of sodium periodate. The loss is due specifically to the periodate anion. Hydrogen peroxide was as active as sodium periodate in this respect, but little loss occurred in the presence of various thiols.Proteoglycan and connective tissue polysaccharide showed rapid and marked decreases in viscosity on oxidation in low concentrations of aqueous periodate, although only limited oxidation of glycols had taken place.Pretreatment of sections with cetylpyridinium chloride solution greatly diminished the loss of basophilia as did the incorporation of high concentrations (>15%) ofn-propanol in the periodate solution. Acid solutions (pH 3.0) during oxidation, or as a pretreatment, also reduced the loss. This fixation could not be reversed by exposure to neutral buffer or concentrated salt solutions.It is suggested that the loss of basophilia from sections treated with sodium periodate solutions is due to the degradation of proteoglycan, possibly by a free radical mechanism. The relevance of the loss to the structure of cartilage is considered.     

Periodate oxidation and alkaline degradation of heparin-related glycans

Heparin, heparan sulphate, and various derivatives thereof have been oxidised with periodate at pH 3.0 and 4° and at pH 7.0 and 37°. Whereas oxidation under the latter conditions destroys all of the nonsulphated uronic acids, treatment with periodate at low pH and temperature causes selective oxidation of uronic acid residues. The reactivity of uronic acid residues depends on the nature of neighbouring 2-amino-2-deoxyglucose residues. d-Glucuronic acid residues are susceptible to oxidation when flanked by N-acetylated amino sugars, but resistant when adjacent residues are either unsubstituted or N-sulphated. L-Iduronic acid residues in their natural environment (2-deoxy-2-sulphoamino-d-glucose) are resistant to oxidation, whereas removal of N-sulphate groups renders a portion of these residues periodate-sensitive. Oxidised uronic acid residues in heparin-related glycans may be cleaved by alkali, producing a series of oligosaccharide fragments. Thus, periodate oxidation-alkaline elimination provides an additional method for the controlled degradation of heparin.     

The periodate oxidation of amino acids with reference to studies on glycoproteins

1. All α-amino acids are oxidized by periodate, but at different rates. 2. The rates of oxidation of individual α-amino acids vary with pH. In general, oxidation proceeds more rapidly at alkaline pH. 3. Serine, threonine, cysteine, cystine, methionine, proline, hydroxyproline, tryptophan, tyrosine and histidine are rapidly and extensively oxidized by periodate. 4. Cysteine, cystine, methionine, tryptophan, tyrosine and histidine are oxidized by periodate when they are substituted in the carboxyl and amino groups, as in a polypeptide chain.     

Isotachophoresis for periodate oxidation analysis of a small amount of carbohydrate.

The present study demonstrates that formate, periodate, and iodate, in a reaction mixture for periodate oxidation of carbohydrates, are simultaneously and conveniently determined by isotachophoretic analysis. The operating condition of the electrophoretic method involves the addition of 0.3 vol of dioxane and 0.2% Triton X-100 to a deoxygenated leading electrolyte of 10 mm HCl buffered with 20 mm imidazole, pH 7.0. These additives are essential for complete resolution of formate and periodate in respective zones. It has been shown that the analyzable amounts of these products in the oxidation reaction are less than 5 nmol which corresponds to less than μg of carbohydrates. This value is about one-thousandth of that required for the conventional methods of periodate oxidation analysis.     

Mechanism of lymphocyte activation by periodic acids. Blastogenesis and induction of immunomodulator populations]

Mild periodate treatment is mitogenic for T lymphocytes. With murine spleen cells periodate oxidation is effective between C8 and C9 on sialyl acid residues. With human blood lymphocytes this oxidation occurs between C7 and C8 of these residues. In vitro immune response is inhibited by periodate treatment. Activation of an immunosuppressive T lymphocyte population is obtained. Similar results are performed with human blood lymphocytes.     

A micromethod for the quantitation of sialoglycoconjugate immobilization on insoluble supports: Its use in the preparation of supports with varying ligand concentration

A micromethod is described for the evaluation of immobilization of sialoglycoconjugates on insoluble supports. Ligands were radioactively labeled in their sialic acid moieties after mild periodate oxidation and borotritide reduction, or in the glycosylamino residue after borotritide reduction of the Schiff's base formed between reducing sialooligosaccharides and β-(p-aminophenyl)-ethylamine. Sephadex G-25, Sepharose 4B, and Cellulose MN 2100 were activated by CNBr or periodate oxidation. The hydrazido derivatives of these supports were prepared using both activation methods, and activated to azido-supports using nitrous acid. Controlled Pore Glass-glycophase activated by periodate oxidation was also studied. The investigation of conditions for the binding of the radioactive ligands was carried out in the microassay using 0.5-ml aliquots of the activated supports. The stability of the bound ligands in dependence on various parameters was investigated using the immobilized radioactive ligands. Multivalent linkages formed between ligand and support gave increased stability to release compared to monovalent attachment, for cyanogen bromide activation. The use of periodate activation yielded ligands with much greater stability even for monovalent linkages.The microassay was used successfully to predict conditions for the batchwise preparation of immobilized ligands.     

Mild and efficient oxidation of Hantzsch 1,4-dihydropyridines with sodium periodate catalyzed by a new polystyrene-bound Mn(TPP)Cl

Mild and efficient oxidation of Hantzsch 1,4-dihydropyridines with sodium periodate catalyzed by Mn(TTP)Cl supported on polystyrene-bound imidazole is reported. This heterogeneous catalyst is of great stability and reusability in the oxidation of 1,4-dihydropyridines with sodium periodate without significant loss of its catalytic activity.     

The effect of periodate oxidation and α-mannosidase treatment of Dolichos biflorus lectin

The effects of periodate and α-mannosidase treatment of the Dolichos biflorus lectin were determined. Destruction by periodate of 16% of the mannose residues of the lactin had no effect on its ability to agglutinate type A erythrocytes, precipitate blood group A + H substance or to be precipitated by concanavalin A. Removal of up to 40% of the mannose by either periodate or α-mannosidase rendered the lecton nonprecipitable by concanavalin A. The lectrin treated by α-mannosidase retained its ability to agglutinate erythrocytes and precipitate blood group A + H substance, but the lectin treated with periodate lost most of its activity.The results suggest that the complete integrity of the carbohydrate unit of the lectin is not necessary for its activity and that the periodate may be affecting the protein portion of the molecule as well as its carbohydrate residues. No conversion of form A to form B of the lectin was observed with either periodate oxidation or α-mannosidase treatment.     

Effects of chemical disruption on the biological activities of sea urchin egg jelly

In an attempt to relate the biological activities of sea urchin egg jelly to the structural characteristics of the acid glycoprotein molecule, the jelly was oxidized with H₂O₂ and sodium periodate, and digested with trypsin and pronase. The non-dialyzable products of H₂O₂ and periodate oxidation, and a fucose-rich fraction isolated from enzyme-digested jelly by column chromatography, were tested for their capacity to induce sperm agglutination and acrosome reaction in Hemicentrotus pulcherrimus. It was found that a degree of enzyme digestion sufficient to remove about 80% of the amino acids reduced, but failed to eliminate, the capacity of the jelly to elicit agglutination and acrosomal reaction. Mild oxidation with H₂O₂ suppressed sperm agglutination, but more drastic treatment was required to destroy the capacity of the jelly to induce the acrosome reaction. The loss of both these biological activities after periodate oxidation was found to parallel the release of sialic acid.     

Oxidation of tissue polysaccharides by periodic acid in dimethyl sulfoxide and its anhydrous and aqueous mixtures.

Periodic acid (1% w/v) solvated by anhydrous dimethyl sulfoxide (DMSO) readily induced a strong Schiff reaction in a variety of structures containing polysaccharides, but not glycogen. With the increasing amounts of water added to DMSO, glycogen was also oxidized, while the selective localization of other polysaccharides remained unimpaired. Periodate, solvated in the anhydrous acetic acid-DMSO mixture, rapidly induced concomitant oxidation of nucin and glycogen-containing structures. Sodium bisulfite addition derivatives of carbonyls, induced by periodate oxidation in DMSO, were stained meta- and orthochromatically with toluidine blue at controlled pH. Certain metachromatic tissue components were strongly birefringent in polarized light in contrast to the identical structures oxidized by aqueous periodate. Marked differences in staining reactions elicited in identical structures by periodate in DMSO as compared with aqueous periodate suggest that DMSO-periodate method considerably enhances the range of histochemical oxidations by periodate.     

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

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).     

Dihydroxypropylation of amino groups of proteins: use of glyceraldehyde as a reversible agent for reductive alkylation

The mode of derivatization of amino groups of proteins by glyceraldehyde, an aldotriose, depends on the presence or absence of reducing agent. In the presence of sodium cyanoborohydride, the Schiff base adducts of the aldehyde with the amino groups are reduced, and dihydroxypropylation of amino groups takes place (reductive mode). The reductively glycated lysine residue, N epsilon-(2,3-dihydroxypropyl)lysine, is a substituted alpha-amino alcohol. This alpha-amino alcoholic function of the derivatized lysine should be susceptible to periodate oxidation, and this oxidation is anticipated to result in the regeneration of the lysine residue. This aspect has been now investigated. Indeed, on mild periodate oxidation (15 mM periodate, 15 min at room temperature) of dihydroxypropylated ribonuclease A, nearly 95% of its N epsilon-(2,3-dihydroxypropyl)lysine residues were regenerated to lysine residues. The removal of the dihydroxypropyl groups by periodate oxidation could be accomplished within a wide pH range with little variation in the recovery of lysines. The possible usefulness of this reversible chemical modification procedure in the primary structural studies of proteins was investigated with a tryptic peptide of dihydroxypropylated streptococcal M5 protein, namely, DHP-T4. This 12-residue tryptic peptide contains one internal N epsilon-(dihydroxypropyl)lysine. The dihydroxypropylated peptide released most of its dihydroxypropyl groups on mild periodate oxidation. Redigestion of the periodate-treated peptide with trypsin generated the two expected peptides, demonstrating the generation of a trypsin-susceptible site. Reductive dihydroxypropylation of amino groups of RNase A resulted in the loss of its enzyme activity, the extent of inactivation increasing with the concentration of the glyceraldehyde used.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced chemiluminescence of lucigenin with epinephrine in cationic surfactant micelles containing periodate

Epinephrine (EP) species involved in the lucigenin chemiluminescence (CL) were identified in alkaline solution by comparing the time course of the CL response and the formation of EP oxidation products. EP quinone and adrenolutine (AL) were found to be responsible for the lucigenin-CL reaction. The mechanism of the lucigenin-CL enhancement was investigated using cationic micellar hexadecyltrimethylammonium hydroxide (CTAOH), periodate, and a mixture of micellar CTAOH and periodate. The CL enhancement in the presence of micellar CTAOH and periodate could be explained in terms of increases in the oxidation rate of EP to EP quinone and the intramolecular oxidation rate of adrenochrome to AL.     

Covalent attachment of immunoglobulins to liposomes via glycosphingolipids

We describe a method for covalent binding of proteins to large unilamellar liposomes which involves the periodate oxidation of glycosphingolipids in the vesicle membrane. Proteins such as IgG and F(ab′)₂ may then be attached to the aldehyde groups on the glycolipid by Schiff-base formation at pH 9.5 and reduction with NaBH₄, or by reductive amination with NaBH₃CN at pH 8.4. Exposure of the vesicles to periodate, protein coupling and separation from unbound protein by a novel method of flotation in discontinuous dextran gradients does not release the vesicle contents when performed at pH 8.4. Studies on the oxidation of neutral glycolipid-containing vesicles, and on the oxidation of encapsulated glycerol 1-phosphate show that periodate influx into neutral vesicles during a 4 h exposure is appreciable at pH 5.5 but not at pH 8.4. Under optimal conditions, approx. 20% of the protein may be coupled to vesicles, and a ratio of 100–200 μg of protein/μmol of lipid is readily achieved. This method will be of great importance for the antibody-mediated targeting of vesicles to cells.