Significant immunological cross-reactivity of plant glycoproteins

Plant glycoproteins generally cross-react because of the presence of identical or related complex glycans which are highly immunogenic. The use of mild periodate oxidation of glycans after glycoprotein transfer from sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels to nitrocellulose membranes prior to immunodetection is a way of identifying the carbohydrate antigenic determinants of a glycoprotein as the basis for antigenic cross-reaction. Periodate oxidation can distinguish between antibodies directed against carbohydrate and against peptide antigenic determinants, the latter being unaffected by oxidation. Immunoblotting performed after periodate treatment allows the detection of common protein epitopes.     

Periodate oxidation analysis of carbohydrates : Part I. Spectrophotometr1c determination of glyoxal in dialdehyde fragments formed from glycosides with 2,4-dinitrophenylhydrazine

A simple procedure for the determination of glyoxal in dialdehyde fragments, formed from glycosides by periodate oxidation, is proposed. By heating sample solutions prepared by dilution of reaction mixtures for periodate oxidation, with an aqueous dimethylsulfoxide solution of 2,4-dinitrophenylhydrazine hydrochloride. followed by addition of an aqueous ethanolic solution of potassium hydroxide, intense color with an absorption maximum at 576 nm developed. The spectrophotoinetric method based on this color reaction makes it possible to determine selectively 1·10^?2–2·10^?1 μmole amounts of conjugated glyoxal without a prior liberation process. Data for glyoxal content obtained by this procedure are discussed in relation to overoxidation.     

Acetals of Dehydro-L-Ascorbic Acid 2-Hydrazones. Access to the Chiral Building Block (R)-Glycerol Acetonide

5,6-O-Isopropylidene, cyclohexylidene and benzylidene derivatives of L-threo-2,3-hexodiulosono-1,4-lactone 2-phenylhydrazone were prepared. Reduction of the isopropylidcne derivative was followed by treatment with base, and then periodate oxidation and reduction to give (R)-glycerol acetonide. Hydrogen bonding in the hydrazones has a role in forming geometrical isomers.     

An Easy and Efficient Method for Cleavage of Carbon-Nitrogen Double Bonds Under Non-Aqueous and Neutral Conditions

1-Benzyl-4-aza-1-azoniabicyclo[2.2.2]octane periodate (BAABCP) (1), readily prepared as an orange solid from commercially available DABCO (1,4-diazabicyclo [2.2.2]octane, performs oxidation in anhydrous conditions. Under these conditions, the reagent converts phenylhydrazones, p-phenylhydrazones, semicarbazones, and β-keto sulfoxide hydrazones to the corresponding carbonyl compounds and β-keto sulfoxides respectively, the yields and enantioselectivity are excellent.     

Preparation of the stabilized glycoenzymes by cross-linking their carbohydrate chains

Each of the three high-mannose type glycoproteins studied, acid phosphatase, invertase, and glucose oxidase, could be specifically cross-linked through its carbohydrate chains. The procedure involves periodate oxidation of carbohydrate residues followed by reaction of the generated aldehyde groups with adipic acid dihydrazide as a cross-linker. The amount and size as well as solubility of the formed polymers could be efficiently controlled by varying the reaction conditions, i.e., the oxidation degree and the concentrations of glycoproteins, cross-linker, and hydrogen ions during the cross-linking reaction. It was found that the quantity and size of polymers increased with oxidation degree and protein concentration and by lowering the pH. When the protein concentration was above and pH below certain values, depending on the glycoenzyme, insoluble polymers formed. The soluble cross-linked polymers retained a high level of original activity, and the minor decrease in specific activity noticed was shown to occur during the periodate oxidation step. The cross-linked glycoenzymes are much more resistant to denaturation by high temperature and by changes in pH, demonstrating the usefulness of this method in preparation of the stabilized glycoprotein derivatives.     

Detection of cerebrospinal fluid leakage by specific measurement of transferrin glycoforms

A simple and rapid detection of cerebrospinal fluid (CSF) leakage would benefit spine surgeons making critical postoperative decisions on patient care. We have assessed novel approaches to selectively determine CSF β2‐transferrin (β2TF), an asialo‐transferrin (aTF) biomarker, without interference from serum sialo‐transferrin (sTF) in test samples. First, we performed mild periodate oxidation to selectively generate aldehyde groups in sTF for capture with magnetic hydrazide microparticles, and selective removal with a magnetic separator. Using this protocol sTF was selectively removed from mixtures of CSF and serum containing CSF aTF (β2TF) and serum sTF, respectively. Second, a two‐step enzymatic method was developed with neuraminidase and galactose oxidase for generating aldehyde groups in sTF present in CSF and serum mixtures for magnetic hydrazide microparticle capture. After selectively removing sTF from mixtures of CSF and serum, ELISA could detect significant TF signal only in CSF, while the TF signal in serum was negligible. The new approach for selective removal of only sTF in test samples will be promising for the required intervention by a spine surgeon.     

Synthesis of high-capacity immunoaffinity sorbents with oriented immobilized immunoglobulins or their Fab' fragments for isolation of proteins

Two methods for synthesizing high-capacity immunoaffinity sorbents on Sepharose and Separon HEMA E-1000 are described. The first is the oriented immobilization of monovalent immunoglobulin Fab fragments on a maleimide derivative of Sepharose via the formation of a covalent bond between the SH group of the Fab fragment at the C-terminus of the molecule and the maleimide covalently coupled to Sepharose. The second method is based on the oxidation of the immunoglobulin carbohydrate component, located in the Fc fragment, by periodate with subsequent immobilization of the derivatives on hydrazide derivatives of Sepharose or Separon. Sorbents for the isolation of monoclonal antibodies from the culture supernatants and the elongation factor EF-G from a crude extract of Escherichia coli cells were obtained. These sorbents are characterized by a high capacity, minimal non-specific sorption and high stability.     

Viscometric determination of dialdehyde content in periodate oxycellulose Part II. Topochemistry of oxidation

The kinetics of periodate oxidation of cellulose was followed through the alkaline degradation of the dialdehyde groups by measuring the viscometric degree of polymerisation and the alkali consumption. The obtained results show that a fast but limited attack of periodate occurs in the amorphous region of cellulose, causing the decrease of degree of polymerisation to its levelling-off value. The alkali consumption indicates at least two further slower reactions, that lead to the asymptotic complete oxidation of cellulose units. With the pseudo first-order approximation, the oxidation half-time of these three reactions can be calculated, corresponding to 1.2, 20 and 854 h respectively. In spite of the high oxidation of the analysed samples (up to about 46%), the residue after alkaline degradation shows a relatively high value of degree of polymerisation rather than the narrow molecular weight distribution of oligomers expected from a random oxidation, thus indicating that periodate oxidises cellulose in isolated domains. The sequence of analyses over the same sample utilised in this work (titrimetry, weight loss and viscometry), performed at room temperature in mild conditions, makes it possible to investigate the topochemistry of oxidation of paper and textiles of historic and artistic value with microdistructive techniques on a single, very small fragment of material.     

Determination of sucrose in sugar-cane juice and molasses by flow-injection spectrophotometry

An automated procedure is proposed for the spectrophotometric determination of sucrose in sugar-cane juice and nonlasses. The previously diluted and filtered sample is introduced into a flow-injection analyzer designed with two merging streams, producing two samples zones. One zone is transported directly towards the merging-stream confluence; the other zone reaches this site after flowing through a heated coil in which partial and reproducible sucrose inversion is attained under controlled conditions of acidity and temperature. At the confluence point, a buffered periodate stream is added to oxidize the sugar. The consumption of periodate, which mainly reflects the fructose content, is measured spectrophotometrically as a transient lowering of the iodine concentration, produced by reaction of periodate with iodide. The two processed zones proceed sequentially to the flow cell and two peaks are recorded. The sucrose content in the sample is proportional to the difference in peak heights. System dimensioning and the effects of temperature, pH, reagent concentrations, flow rates and the presence of other reducing sugars in the sample are discussed. With the proposed system, about 30 sugar-cane juice samples can be analyzed per hour, and sample clarification is not required. Precise results (r.s.d. <1%), in agreement with those obtained by h.p.l.c., are achieved for sucrose contents of 11–14% (w/v) in cane juice. Modifications of the system for analysis of molasses (16–52% w/w sucrose) are described.     

Quantitative determination of paralytic shellfish poisoning toxins in shellfish using prechromatographic oxidation and liquid chromatography with fluorescence detection: interlaboratory study

An interlaboratory study was conducted for the determination of paralytic shellfish poisoning (PSP) toxins in shellfish. The method used liquid chromatography with fluorescence detection after prechromatographic oxidation of the toxins with hydrogen peroxide and periodate. The PSP toxins studied were saxitoxin (STX), neosaxitoxin (NEO), gonyautoxins 2 and 3 (GTX2,3 together), gonyautoxins 1 and 4 (GTX1,4 together), decarbamoyl saxitoxin (dcSTX), B-1 (GTX5), C-1 and C-2 (C1,2 together), and C-3 and C-4 (C3,4 together). B-2 (GTX6) toxin was also included, but for qualitative identification only. Samples of mussels, both blank and naturally contaminated, were mixed and homogenized to provide a variety of PSP toxin mixtures and concentration levels. The same procedure was followed with samples of clams, oysters, and scallops. Twenty-one samples in total were sent to 21 collaborators who agreed to participate in the study. Results were obtained from 18 laboratories representing 14 different countries.     

Electrogenerated chemiluminescence: Part XXVII. E.C.L. and electrochemical studies of selected laser dyes

The electrogenerated chemiluminescence (e.c.l.) and electrochemistry of the laser dyes, coumarin-2, coumarin-30, rhodamine-6G (perchlorate), rhodamine-B (perchlorate), oxazine-1 (perchlorate), and Nile Blue (perchlorate) were studied in acetonitrile using 0.1 M tetra-n-butylammonium perchlorate (TBAP) as a supporting electrolyte. Rather low intensity e.c.l. was obtained for all dyes except Nile Blue. A study of the electrochemical oxidation and reduction of coumarin-30, oxazine-1 and rhodamine-6G using cyclic voltammetry and controlled potential coulometry demonstrated that chemical side reactions of the electrogenerated reactants are responsible for the low e.c.l. efficiency. In several cases the one-electron transfer reaction at the electrode is followed by a dimerization reaction. The neutral free radical formed on reduction of oxazine-1 was investigated by electron spin resonance spectroscopy and coupling constants for it are reported. Some experiments in which the e.c.l. of mixtures of the dyes with rubrene or 9,10-diphenylanthracene were determined are also described.     

Micro and submicro iodometric determination of arsenite and sulphite ions by amplification reactions

Two methods are described for the micro and submicro iodometric determination of arsenite and sulphite ions involving 3- and 6-fold amplification reactions, respectively. The arsenite method is based on oxidation with an excess of periodate, masking of the unreacted periodate with molybdate, and final iodometric titration of the iodate released. The sulphite method depends upon oxidation with iodine and removal of its excess by extraction with chloroform, and oxidation of the iodide formed to iodate, which is determined iodometrically as usual. The two methods are simple, rapid, and accurate. The average recoveries obtained are 99.9 and 99.3% for arsenite and sulphite, respectively.     

Phase Transfer Promoted Ruthenium Oxide Oxidations of Carbohydrates II

ABSTRACT

The use of a phase transfer catalyst, benzyltriethylammonium chloride (BTEAC), is described in conjunction with the ruthenium dioxide/periodate : water/chloroform system for the oxidation of carbohydrate alcohols to the corresponding ketone, aldehyde, or carboxylic acid. The method was found to be applicable to carbohydrates appropriately protected as acetals, ethers, or containing a benzoyloxy group not positioned to readily undergo β-elimination. While the method was very suitable for the oxidation of carbohydrate secondary alcohols to ketones, it was found to be less suitable for the oxidation of a carbohydrate primary alcohol to the corresponding aldehyde or carboxylic acid. Evidence presented suggests that under the mildly basic conditions of the reaction, ruthenium tetraoxide is converted to ruthenate and perruthenate ions in the aqueous solution and then the perruthenate ion is carried by the phase transfer catalyst into the organic layer where oxidation of the substrate occurs. A number of examples illustrating the scope of the method are presented.     

Catalytic cycloaddition of diazoalkanes to fullerene C60

Cycloaddition to fullerene C₆₀ of monosubstituted diazomethanes generated in situ by oxidation of aldehyde hydrazones in the presence of Pd(acac)₂-2 PPh₃-4 Et₃Al as catalytic system resulted in selective formation of homofullerenes in which the alkyl substituent is located above the plane of the five-membered ring in C₆₀. Under analogous conditions, unsymmetrical disubstituted diazomethanes generated from the corresponding ketone hydrazones gave rise to mixtures of stereoisomeric 5,6-open adducts.     

SEPARATION OF SOME AROMATIC CARBONYL COMPOUNDS BY THIN LAYER CHROMATOGRAPHY OF THEIR ISONICOTINOYL HYDRAZONE DERIVATIVES

Abstract

The separation of 13 isonicotinoyl hydrazones derived from some isomeric aromatic carbonyl compounds by thin layer chromatography is described. Methods for separating and identifying the components of complex mixtures of these hydrazones are discussed.     

Effect of pH on the oxidation of paralytic shellfish poisoning toxins for analysis by liquid chromatography

The effect of pH on the oxidation of individual PSP toxins using both periodate and peroxide oxidations was studied. It was found that the optimum pH for individual toxins varied considerably. For periodate oxidations, pH 8.2 produced the maximum yield of fluorescent products for neosaxitoxin and GTX1/GTX4 while the non-hydroxylated toxins (saxitoxin, GTX2/GTX3, decarbamoyl saxitoxin, GTX5) showed optimum pHs from about pH 10-11.5. Neosaxitoxin and GTX1/GTX4 did not produce significant fluorescent oxidation products with peroxide oxidation at any of the pHs studied (pH 8.2-12.8). The non-hydroxylated toxins all showed optimum pHs above pH 12 with peroxide oxidation. Yields of fluorescent products of these toxins decreased substantially at pHs below pH 12. Neosaxitoxin and GTX1/GTX4 each produced three product peaks at pH 8.2 with periodate oxidation. There was no pH where these toxins produced predominantly a single oxidation product. Decarbamoyl saxitoxin always produced two oxidation products with both oxidation reactions at the pHs studied. However, the relative yields of the products changed with pH. At low pH the second eluting product predominated, while at higher pH values the first eluting product predominated. This pattern was observed for both oxidation reactions. The other non-hydroxylated toxins produced mainly single unique products with both oxidation reactions over the pH range studied. No single pH was found optimum for the oxidation of both hydroxylated and non-hydroxylated toxins without a significant compromise in yield of oxidation products. This has implications for the post column oxidation liquid chromatographic methods, since small changes in pH of the post column oxidant can both positively and negatively affect the yields of oxidation products of toxin mixtures leading to increased error in the subsequent quantitation of these compounds.     

Volumetric determination of periodate ion after the oxidation of carbohydrates

Summary On the basis of a method previously known for inorganic mixtures, an accurate, rapid, and universal volumetric method for determining periodate ion after the oxidation of carbohydrates, including oligosaccharides and deoxy sugars, has been developed.     

Hydrophosphorylation of monosaccharides oximes and hydrazones

The hydrophosphorylation of monosaccharides oximes and hydrazones was studied; similarity and difference of this process compared to the reaction of secondary glycosylamines with the hydrophosphoryl compounds are shown. A number of the original α-aminophosphoryl compounds is obtained including those containing carbohydrate fragment in their compositions.     

Simultaneous determination of iodate and periodate by kinetic spectrophotometric method using principal component artificial neural network

A kinetic spectrophotometric method for the simultaneous determination of iodate and periodate in mixtures was proposed. The method was based on the reaction of periodate and iodate with pyrogallol red in sulfuric acid media. The reaction was monitored spectrophotometrically by measuring the decrease in absorbance of pyrogallol red at 470 nm. Kinetic data collected at 470 nm were processed by principle component artificial neural network (PC-ANN) method. The constructed model was able to predict the concentration of two species in the range of 0.1?C15.0 and 0.1?C17.0 ??g/mL for iodate and periodate, respectively. The proposed method was applied to the simultaneous determination of iodate and periodate in several real samples with satisfactory results.     

Oriented immobilization of periodateoxidized monoclonal antibodies on amino and hydrazide derivatives of eupergit C

Amino and hydrazyno derivatives of Eupergit C were prepared by reaction of the beads with hexamethylene diamine (HMD) and adipic acid dihydrazide (ADH), respectively. Monoclonal antibodies (mAbs) against carboxypeptidase A (CPA) and horse radish peroxidase (HRP) were prepared, and those that did not inhibit the respective enzymatic activities were selected. The carbohydrate moieties of these antibodies were oxidized by reaction with sodium periodate and then coupled onto the modified beads. The oxidation and coupling reactions were optimized to achieve highly active matrix-conjugated antibodies. Thus, antibody-matrix conjugates that possessed antigen-binding activities close to the theoretical value of 2 mol antigen bound/mol immobilized antibody were obtained.