The determination of iduronic acid and glucuronic acid in heparins by a new technique: Re-evaluation of the variable hexuronic acid composition of mammalian heparins

A new technique for the quantitative determination of the uronic acid components of heparin is described. Heparin was deamination with organic nitrite and methanolyzed. The monomeric derivatives of uronic acids were quantitated by gas chromotography. Depolymerization to monomeric units appeared essentially complete as judged from the yield of uronic acid derivatives. By applying the method the relative contents of iduronic acid and glucuronic acid in a number of heparin samples were estimated. In all samples examined the content of iduronic acid was larger than that of glucuronic acid. A species specific difference in the iduronic acid to glucuronic acid ration of heparin was noted. Noticeable difference of this ratio was observed also between different organs of a species of animal and among heparin fractions obtained from an organ.     

Distribution of sulphate and iduronic acid residues in heparin and heparan sulphate

1. A method was developed for determination of the uronic acid composition of heparin-like glycosaminoglycans. Polymers or oligosaccharides are degraded to monosaccharides by a combination of acid hydrolysis and deamination with HNO₂. The resulting uronic acid monosaccharides (accounting for about 70% of the uronic acid contents of the starting materials) are isolated and converted into the corresponding aldono-1,4-lactones, which are separated by g.l.c. The calculated ratios of glucuronic acid/iduronic acid are reproducible within 5%. 2. Samples of heparin from pig intestinal mucosa (molar ratio of sulphate/disaccharide unit, 2.40) and heparan sulphate from human aorta (sulphate/disaccharide ratio, 0.46) were subjected to uronic acid analysis. l-Iduronic acid constituted 77% and 19% respectively of the total uronic acid contents. 3. The correlation between the contents of sulphate and iduronic acid indicated by this finding also applied to the fractionated deamination products of the two polymers. The sulphated fragments varied in size from disaccharide to octasaccharide (or larger) and showed sulphate/disaccharide molar ratios in the range of 0.05–2.0. The proportion of iduronic acid increased with increasing ester sulphate contents of the oligosaccharides. 4. Previous studies on the biosynthesis of heparin in a cell-free system have shown that l-iduronic acid residues are formed by C-5 epimerization of d-glucuronic acid units at the polymer level; the process requires concomitant sulphation of the polymer. The results obtained in the present structural study conform to these findings, and suggest further that similar mechanisms may operate in the biosynthesis of heparan sulphate. The epimerization reaction appears to be linked to the sulphation of hydroxyl groups but does not seem to require sulphation of the target uronic acid residues. The significance of sulphamino groups in relation to the formation of iduronic acid is unknown.     

Hydrazinolysis of heparin and other glycosaminoglycans.

Heparin, carboxy-group-reduced heparin, several sulphated monosaccharides and disaccharides formed from heparin, and a tetrasaccharide prepared from chondroitin sulphate were treated at 100 degrees C with hydrazine containing 1% hydrazine sulphate for periods sufficient to cause complete N-deacetylation of the N-acetylhexosamine residues. Under these hydrazinolysis conditions both the N-sulphate and the O-sulphate substituents on these compounds were completely stable. However, the uronic acid residues were converted into their hydrazide derivatives at rates that depended on the uronic acid structures. Unsubstituted L-iduronic acid residues reacted much more slowly than did unsubstituted D-glucuronic acid or 2-O-sulphated L-iduronic acid residues. The chemical modification of the carboxy groups resulted in a low rate of C-5 epimerization of the uronic acid residues. The hydrazinolysis reaction also caused a partial depolymerization of heparin but not of carboxy-group-reduced heparin. Treatment of the hydrazinolysis products with HNO2 at either pH 4 or pH 1.5 or with HIO3 converted the uronic acid hydrazides back into uronic acid residues. The use of the hydrazinolysis reaction in studies of the structures of uronic acid-containing polymers and the implications of the uronic acid hydrazide formation are discussed.     

Isolation and partial characterization of proteoglycans from sheep lung parenchyma.

Greater than 90% of the proteoglycans of sheep lung parenchyma, as measured by uronic acid, were solubilized employing a sequential procedure with guanidine hydrochloride, dithiothreitol and Triton X-100. The amounts solubilized were 68.7%, 16.2% and 5.9%, respectively. The guanidine hydrochloride extract was chromatographed using DEAE-cellulose in urea and eluted with increasing concentrations of NaCl. A major fraction (containing a 6.5-fold enrichment of uronic acid) was obtained with 0.5 M NaCl and further purified by Sepharose Cl-6B chromatography in guanidine hydrochloride. To demonstrate the presence of protein-linked glycosaminoglycans, the void volume peak containing protein and uronic acid was digested with papain and rechromatographed. Evidence for the presence of proteoglycans was obtained by observing an almost complete loss of uronic acid in the void volume and the appearance of a uronic acid peak in the included volume, migrating in the same area as single-chain glycosaminoglycans. Electrophoretic migration and disappearance of bands in electrophoresis after digestion with specific mucopolysaccharide lyases indicated that the small amount of uronic acid remaining in the void volume was hyaluronic acid whereas the included volume contained hyaluronic acid, heparan sulfate, chondroitin sulfates and/or dermatan sulfate.     

A gas chromatographic method for the quantitative detemination of hexuronic acids in alginic acid

A method has been developed for the quantitative determination of the relative proportions of d-mannuronic and l-guluronic acids in alginic acid. To obtain homogeneous reaction conditions the viscosity of the alginic acid sample was first decreased by limited hydrolysis with mineral acid. The carboxyl groups were then esterified by reaction with 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide, and reduced with sodium borohydride. The resulting hexosans were converted by acid hydrolysis to d-mannose and an equilibrium mixture of l-gulose and 1,6-anhydro-l-gulose. These were treated with sodium borohydride; the 1,6-anhydro-l-gulose was not reduced whereas d-mannose and l-gulose were converted to d-mannitol and d-glucitol. The hexitols were estimated by gas-liquid chromatography as the n-butane boronic acid esters, and the relative proportions of the uronic acids in the alginic acid were calculated by taking into account the equilibrium ratio of l-gulose and 1,6-anhydro-l-gulose. The method can be used to analyze as little as 2 mg of alginic acid.     

Partial characterization of dermatan sulfate by proton nuclear magnetic resonance spectroscopy

The degree of galactosamine N-acetylation, iduronic acid composition, and total uronic acid/hexosamine ratios of the three dermatan sulfates of human skin, DS18, DS28, and DS35 (M. O. Longas et al. (1987) Carbohydr. Res. 159, 127-136), were determined by Fourier transform, proton nuclear magnetic resonance (FT 1H NMR) spectroscopy. Analysis of DS of varying ages was conducted at 400 MHz and 60 degrees C. Chemical shifts for H-1, H-2, H-4, and H-5 of L-IdUA were independent of those for the respective protons of D-GalNAc and D-GlcUA. The resonance intensities of H-1 and acetamido methyl protons of D-GalNac did not display the expected 1:3 ratios. Therefore, their integration values were employed to estimate the percentage N-acetylation (N-CH3/3 H-1) which was corroborated chemically. The L-IdUA content, relative to total uronic acid, was calculated from signal intensities of H-1 of L-IdUA and D-GlcUA and ascertained by quantitative chemical methods. Total uronic acid/hexosamine ratios were determined from both 1H NMR spectroscopy and chemical analyses. The data show the following N-acetylation (N-CH3/3 H-1) of galactosamine in DS:DS18, 61-72% between 17 and 60 years, unaffected by senescence; DS28, 78-86% with no age-related trend; DS35, 101% at 19 years. Furthermore, in all ages investigated, the percentage (wt/wt) L-IdUA relative to total uronic acid was 42-44% for DS18 and 37-40% for DS28. At age 19 years, DS35 had a 29% (wt/wt) L-IdUA. The total uronic acid/hexosamine ratios for DS18 and DS28 varied from 1.40:1.0 to 1.70:1.0 irrespective of age.     

Distribution of glucuronic and iduronic acid units in heparin chains

The distribution of glucuronic and iduronic acid within the chains of anticoagulantly active and inactive beef lung heparin was investigated. A fraction with an average molecular weight of 19,500 was isolated from the heterodisperse mixture and then separated into active and inactive components by affinity chromatography. Each sample was linked through its reducing terminus to tyramine, reduced with sodium borotritide, and bound covalently to Sepharose via an azo bridge. The bound reduced heparin was treated with a limited amount of HNO2 and the degraded fragments were removed. The sections of the chain contiguous with the original reducing terminus were then detached from the insoluble matrix by reaction with sodium dithionite. The recovered polysaccharide was fractionated according to size on Sephadex G-200 and the amount of each uronic acid in the individual fractions was determined. Inactive heparin showed a constant percentage of glucuronic acid in all fragments, i.e. about 8.9% of the total uronic acid. With active heparin the percentage of glucuronic acid increased with the distance from the reducing terminus of the polysaccharide chain, ranging from 9.5 to 20% of the uronic acids. These results suggest that the biosynthesis of active heparin involves unique reactions or specific processing of the macromolecule.     

Glycosaminoglycans are integral constituents of renal glomerular basement membrane

Basement membranes from canine renal glomeruli were isolated following osmotic lysis and sequential detergent treatment. Substantial amounts of uronic acid in unfractionated membranes were demonstrated with the carbazole and orcinol reactions. About 10–15% of basement membrane uronic acid was solubilized with neutral salt solutions. Denaturation in 8M urea solubilized ?70% of the uronic acid but only ?10% of basement membrane hydroxyproline; the latter was solubilized after reduction and alkylation. Uronic acid containing glycoprotein isolated by denaturation did not bind to carboxymethylcellulose and migrated as a high molecular weight band on SDS-gel electrophoresis. The ability of isolated rat glomeruli to incorporate radioactive sulfate $\text{in}\text{vitro}$ was demonstrated. These findings indicate that sulfated glycosaminoglycans are integral components of glomerular basement membrane.     

Effect of prednisolone on the glycosaminoglycan components of the regenerating articular cartilage.

Investigations were performed on the effect of prednisolone (0.5 mg/kg) on the regenerating femoral articular cartilage of the knee joint in dogs that had been subjected to semiarthroplasty. After 70 days of prednisolone treatment the dogs were killed and the regenerating articular cartilage was removed, minced, and dried with acetone. The acetone-dried material was used for the determination of galactosamine, glucosamine, uronic acid, sulphate, sialic acid and hydroxyproline. Prednisolone treatment elicited a quantitative increase in galactosamine (30.2%), uronic acid (76.2%), and sulphate (9.1%), while no difference was observed in sialic acid content between the treated and untreated groups. From the molar ratio of the measured components it appears that prednisolone produced an increase in chondroitin sulphate and hyaluronic acid, and a decrease in the keratosulphate content of cartilage. By comparing the values measured in the regenerating articular cartilage of control and prednisolone-treated dogs with the values obtained in the mature articular cartilage, we may conclude that prednisolone--at least as regards the glycosaminoglycans of the ground substance--exerts an accelerating effect on cartilage regeneration.     

Presence of heparan sulfate proteoglycan in thyroid tissue

Glycosaminoglycan (GAG) was extracted from the porcine thyroid gland with a buffer containing 5.3 M guanidine-HCl and proteolytic enzyme inhibitors and was fractionated by subsequent isodensity CsCl centrifugation. 60% of uronic acid positive materials was accumulated in the bottom one-fourth fraction with high buoyant density. More than 90% of this uronic acid positive material in the thyroid tissue was heparin or heparan sulfate (sensitive to nitrous acid treatment) and the rest was chondroitin sulfate or dermatan sulfate (sensitive to chondroitinase ABC treatment). When the accumulated high buoyant density GAG was analyzed on a Sepharose CL-6-B column, approximately 14% of the heparin sulfate were in the macromolecular portion as a form of proteoglycan because it was destroyed by the papain digestion or alkaline borohydride treatment which extensively digests protein or releases GAG from protein by the elimination reaction, respectively. This study demonstrates the existence of heparin sulfate proteoglycan in thyroid tissue for the first time.     

Studies on a novel polysaccharide gel from the fruit of Thaumatococcus daniellii (Benth)

T. daniellii gel contains residues of L-arabinose, D-xylose, D-glucuronic acid, and 4-O-methyl-D-glucuronic acid in the ratios 1.00:7.20:1.91:0.66, together with nitrogen (1?%) and ash (3.1 %). The ash-free gel contains 76% of pentose and 24% of uronic acid; 25% of the uronic acid occurs as the 4-O-methyl derivative. All of the uronic acid residues in the polysaccharide are susceptible to periodate oxidation. Methylation studies suggest that the uronic acids occur as terminal side-substituents to a xylan back-bone and that the polysaccharide is highly branched. Enzymolysis with β-D-glucuronidase liberates a substantial part of the uronic acid, but does not completely depolymerise the gel.     

A solid-phase assay for the quantitative analysis of hyaluronic acid at the nanogram level

A sensitive and accurate solid-phase assay for the quantitative determination of hyaluronic acid (HA) is described. The wells of the polystyrene microplates used were coated with glutaraldehyde followed, via a Schiff's base bond, with spermine to introduce amino groups. HA was added to the activated microwells in the presence of carbodiimide and left to bind via a peptide bond to the amino groups. Then aggrecan solution was added to the wells of the microtiter plates to interact with its G1 domain with hyaluronic acid, and the amounts of aggrecan bound were measured immunochemically. The inhibition of the binding between aggrecan and immobilized HA due to soluble HA present in reference solutions showed linearity in the range of concentrations 0.1 to 0.7 microg/ml. The reaction is specific and rapid and can be widely used for the calculation of HA in body fluids directly and in tissue samples after a brief digestion with a proteolytic enzyme.     

茶叶多糖的糖醛酸含量测定及抗氧化活性研究

采用高效液相色谱法对三个产地茶叶多糖的糖醛酸含量进行了测定,该方法具有操作简便、样品无需预处理等特点,稳定性、重现性较好,回收率高,适合于茶叶多糖糖醛酸含量的测定。结果表明不同产地茶叶多糖的糖醛酸含量有明显差异。本文同时研究了不同产地茶叶多糖对超氧自由基和DPPH自由基的清除作用,以评价其抗氧化活性。结果发现不同产地茶叶多糖的抗氧化活性与其糖醛酸含量呈正相关,糖醛酸含量越高,其抗氧化能力越强。     

Reaction of unsaturated uronic acid residues with mercuric salts. Cleavage of the hyaluronic acid disaccharide 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-glucose.

Degradation of connective-tissue polysaccharides with bacterial or fungal eliminases and subsequent characterization of the reaction products are now part of standard methodology for the analysis of these compounds. However, the scope of preparative and analytical work based on the use of eliminases has been limited by the lack of procedures for specific removal of the unsaturated uronic acid residues generated in the eliminase reactions. In the present investigation, we have shown that these residues are cleaved by mercuric salts under mild conditions that are not likely to affect other structures in an oligo- or poly-saccharide molecule. Thus the disaccharide generated from hyaluronic acid by digestion with chondroitinase AC or ABC was cleaved into a keto acid and free N-acetylglucosamine within 10 min at room temperature upon exposure to 14 mM-mercuric acetate at pH 5. The reaction of the disaccharide with mercuric salts was used for ready determination of the distribution of radioactivity between the glucuronic acid and N-acetylglucosamine moieties in radioactive hyaluronic acid that had been synthesized by IMR-90 fibroblasts from 3H-labelled monosaccharides. When the precursor was [3H]galactose, over 95% of the incorporated radioactivity was found in the glucuronic acid moiety. In contrast, cells grown in the presence of [3H]glucosamine synthesized a polysaccharide in which almost all of the label was located in the N-acetylglucosamine units. It is apparent from these experiments that the reaction of unsaturated uronic acid residues with mercuric salts provides a new tool with potential for many applications in the study of the structure and metabolism of connective-tissue polysaccharides.     

Is uronic acid oxidase involved in the hormonal regulation of abscission in explants of citrus leaves and fruits?

The role of uronic acid oxidase in abscission was studied in explants of citrus ( Citrus sinensis L. Osbeck; var. Shamouti) leaves and fruits. In leaf explants, activity of uronic acid oxidase prior to onset of abscission and the rate of abscission were markedly accelerated by ethylene and delayed by 2,4-dichlorophenoxyacetie acid. Similar results were obtained for uronic acid oxidase activity in the exocellular fraction of young fruit explants. In mature fruit explants, treated with ethylene, an immediate increase in activity was evidenty in the non-active shoot/peduncle abscission zone, whereas in the calyx abscission zone the rise in activity occurred after a prolonged exposure to ethylene, when most of the fruits had already abscised. Whenever ethylene enhanced uronic acid oxidase activity, 2,4-dichlorophenoxyacetic acid delayed it. A gradient of decreasing activity or uronic acid oxidase was recorded from both sides of the abscission zone in leaves and fruits toward the separation line, where activity was the lowest as compared with the activity found in adjacent tissues. It is suggested that uronic acid oxidase is involved in senescence and cell wall degradation. However, it is yet questionable whether this enzyme is directly related to the control mechanism of abscission.     

Auxin and gibberellin effects on cell growth and starch during abscission in cotton

An increase in starch content of cells in the abscission zone of the cotton explant appeared correlated with an increase in number of cells. A large increase in the number of cells in the abscission zone, concomitant with an increase in starch content, followed treatment with gibberellin as compared to auxin. In the final stages of abscission starch was hydrolyzed in the cells of the separation layer. Some starch remained after the petiole abscised.

A positive phloroglucinol-hydrochloric acid reaction in the cells of the petiole distal to the line of separation indicated the presence, not of lignin, but of soluble sugars and uronic acids. This reaction was especially intense following gibberellic acid treatment.

It was concluded that gibberellin in accelerating abscission leads to (1) an increase in cell number and starch content in the abscission zone, (2) the hydrolysis of starch in the separation layer just before abscission, and (3) the breakdown of polysaccharides and the release of soluble sugars and uronic acids. Auxin, an abscission retardant, either delays or prevents these events.

     

Synthesis and characterization of a biotin-alginate conjugate and its application in a biosensor construction

Biotin was covalently coupled with alginate in an aqueous-phase reaction by means of carbodiimide-mediated activation chemistry to provide a biotin-alginate conjugate for subsequent use in biosensor applications. The synthetic procedure was optimized with respect to pH of the reaction medium (pH 6.0), the degree of uronic acid activation (20%), and the order of addition of the reagents. The biotin-alginate conjugate was characterized by titration with 2-anilinonaphthalene-6-sulfonic acid (2,6-ANS), 4-hydroxyazobene-2'-carboxylic acid (HABA) and by an HPSEC-MALLS analytical method as well as by FTIR and 13C NMR spectroscopy. As a compromise between the need for a high percent of molar modification of the alginate, on one hand, and sufficient gelling capability, on the other hand, an optimal modification of 10-13% of biotin-alginate was used. The new biotin-alginate conjugate was used for the encapsulation of bioluminescent reporter cells into microspheres. A biosensor was prepared by conjugating these biotinylated alginate microspheres to the surface of a streptavidin-coated optical fiber, and the performance of the biosensor was demonstrated in the determination of the antibiotic, mitomycin C as a model toxin.     

Reaction of S-(2-amino-2-carboxyethylsulfonyl)-l-cysteine with sulfite: Synthesis of S-sulfo-l-cysteine and l-alanine 3-sulfinic acid and application to the determination of sulfite

A procedure for the simultaneous preparation of S-sulfo-l-cysteine and l-alanine 3-sulfinic acid is described. The method is based on the quantitative reaction between sulfite and S-(2-amino-2-carboxyethylsulfonyl)-l-cysteine. The yield was 95% for S-sulfo-l-cysteine and 91% for l-alanine 3-sulfinic acid. The reaction was also applied to the quantitative determination of sulfite in biological materials. In this procedure, sulfite reacts with S-(2-amino-2-carboxyethylsulfonyl)-l-cysteine. Separation of the reaction product, S-sulfo-l-cysteine, is done by ion-exchange fractionation, and it is determined with acid ninhydrin reagent 2 (M. K. Gaitonde, 1967, Biochem. J.104, 627–663). The recovery was 96.8 ± 0.3%.     

Immunopotentiator separated from hot water extract of the seed of Benincasa cerifera Savi (Tohgashi)

Summary The separation and properties of a new immunopotentiator, Benincasa cerifera mitogen (BCM) fraction, were investigated. BCM fraction was separated from hot water extract of the seed of Benincasa cerifera Savi (Tohgashi) by gel filtration using Sepharose 4B. BCM fraction is a heteropolymer consisting of uronic acid, neutral sugars, protein, and phosphorus. The proliferation and differentiation of murine B cells were markedly stimulated by BCM fraction. The in vitro development of peritoneal macrophages into antitumor macrophages was also activated by the addition of BCM fraction to cultures. BCM fraction augmented the IgM and IgG antibody responses against sheep erythrocytes (SRBC) and the induction of delayed-type footpad reaction against SRBC. The antitumor activity of BCM fraction was observed in terms of prolongation of the survival period of mice bearing Meth A fibrosarcoma. After hydrolysis with 1% acetic acid at 100° C for 4 h, marked mitogenic activity was found in a precipitate composed of 29% neutral sugars, 50% uronic acid, 1% protein, and 0.1% phosphorus. The precipitate did not contain detectable amino sugar. The possibility that the biological activities of BCM fraction may be due to contamination by bacterial lipopoly-saccharide was ruled out on the basis of the results of chemical analysis and of marked mitogenicity noted in C3H/HeJ spleen cell cultures. Abbreviations used: BCM, Benincasa cerifera mitogen; SRBC, sheep erythrocytes; PFC, plaque-forming cells; TNP-HRBC, trinitrophenylated horse erythrocytes; PBA, polyclonal B-cell activation; SI, stimulation index