Hyalobiuronic Acid Research Articles

Qualitative and quantitative analysis of hyaluronan oligosaccharides with high performance thin layer chromatography using reagent-free derivatization on amino-modified silica and electrospray ionization-quadrupole time-of-flight mass spectrometry coupling on normal phase

Purified oligomers of hyalobiuronic acid are indispensable tools to elucidate the physiological and pathophysiological role of hyaluronan degradation by various hyaluronidase isoenzymes. Therefore, we established and validated a novel sensitive, convenient, rapid, and cost-effective high performance thin layer chromatography (HPTLC) method for the qualitative and quantitative analysis of small saturated hyaluronan oligosaccharides consisting of 2–4 hyalobiuronic acid moieties. The use of amino-modified silica as stationary phase allows a simple reagent-free in situ derivatization by heating, resulting in a very low limit of detection (7–19pmol per band, depending on the analyzed saturated oligosaccharide). By this derivatization procedure for the first time densitometric quantification of the analytes could be performed by HPTLC. The validated method showed a quantification limit of 37–71pmol per band and was proven to be superior in comparison to conventional detection of hyaluronan oligosaccharides. The analytes were identified by hyphenation of normal phase planar chromatography to mass spectrometry (TLC–MS) using electrospray ionization. As an alternative to sequential techniques such as high performance liquid chromatography (HPLC) and capillary electrophoresis (CE), the validated HPTLC quantification method can easily be automated and is applicable to the analysis of multiple samples in parallel.

Fast counter-electroosmotic capillary electrophoresis–time-of-flight mass spectrometry of hyaluronan oligosaccharides

Fast capillary electrophoresis-mass spectrometry measurements under counter-electroosmotic analyte migration conditions are presented. Efficient separations of a homologous series of six hyaluronan oligosaccharides (comprising 1-6 hyalobiuronic acid moieties) could be completed in 65 s. Separations were achieved in short-length fused silica capillaries under high electric field strengths of up to 1.25 kV·cm(-1). Capillary inner diameters ranging from 5 to 50 μm were investigated, resulting in an optimal value of 15 μm. The influence of capillary dimensions and buffer composition on separation efficiency and sensitivity are discussed. Optimal separations were achieved using a 28 cm × 15 μm capillary, a separation high voltage of 35 kV, a background electrolyte of 25 mM ammonium acetate adjusted to pH 8.5, and negative ionization mode. The optimized method was successfully applied to a bovine testicular hyaluronidase digest of hyaluronan. Only minimal sample pretreatment for protein-containing samples is required. The simple manual injection procedure and fast separations allow for a sample throughput of 35 samples per hour.

Syntheses of the 4-nitrophenyl glycosides of hyalobiuronic acid and chondrosine

4-Nitrophenyl [sodium β- d-glucopyranosyluronate]-(1→3)-2-acetamido-2-deoxy-β- d-glucopyranoside ( 1) and 4-nitrophenyl [sodium β- d-glucopyranosyluronate]-(1→3)-2-acetamido-2-deoxy-β- d-galactopyranoside ( 2) were prepared from the zwitterions hyalobiuronic acid [β- d-glucopyranuronic acid-(1→3)-2-amino-2-deoxy- d-glucopyranose] and chondrosine [β- d-glucopyranuronic acid-(1→3)-2-amino-2-deoxy- d-galactopyranose], respectively. Compounds 1 and 2 were not hydrolysed by bovine testicular hyaluronidase.

Group B streptococcal neuraminidase is actually a hyaluronidase.

The extracellular group B streptococcal enzyme described in numerous reports as a neuraminidase is really a hyaluronidase. Over the past 25 years, the enzyme was routinely assayed with bovine submaxillary mucin as the substrate and by the thiobarbituric acid procedure to measure released sialic acid. Characterization of the actual compound released by the enzyme revealed it to be an alpha,beta-unsaturated derivative of hyalobiuronic acid that was derived from hyaluronic acid contaminating the mucin preparation. Previous reports describing an association of elevated levels of extracellular neuraminidase with virulent strains of group B streptococci must be reevaluated with the recognition that the enzyme is really a hyaluronidase.

Mechanism of action of bovine testicular hyaluronidase. Mapping of the active site.

The reactions of purified, homogeneous bovine testicular hyaluronidase have been studied with radioactively labeled oligomers of hyalobiuronic acid, (GlcUA-GlcNAc)n, as substrates and acceptors. Transglycosylation occurs by transfer of a glycosyl residue with retention of configuration from a leaving group to an acceptor. On the basis of detailed examination of cleavage and transglycosylation patterns for the trimer; comparison of trimer, tetramer, and polymer as substrates; comparison of acceptors; equilibrium binding; and other data, it is proposed that the enzyme's active site consists of five subsites for hyalobiuronate residues. In the terminology of Schechter, I., and Berger, A. ((1966) Biochemistry 5, 3371), these are s2-s1-s' 2-s3, where the reducing terminus is to the right, and cleavage occurs between s1 and s' 1. It is proposed that subsite s'2 has a high affinity for a substrate residue, while s1 and s'1 have low substrate affinity, and s2 and s' 3 are intermediate in affinity. This proposal is seen to have mechanistic implications. The reactions of several substrates show similar bell-shaped pH dependences, with optima in the region of pH 5 to 5.5.

Preparation of tritium-labeled hyaluronic acid oligomers and their use in enzyme studies

Tritium-labeled oligosaccharides can be prepared from hyaluronic acid by the Wilzbach technique with greater ease than usual by use of the specificity of hyaluronidase in the course of purification. Labeled oligomers of hyalobiuronic acid were isolated and shown to be radiochemically homogeneous, and are used in studies of complex enzymic kinetics. The techniques used may have general use in preparing generally labeled oligosaccharides.

Digestive Activity of Lysosomes: II. THE DIGESTION OF MACROMOLECULAR CARBOHYDRATES BY EXTRACTS OF RAT LIVER LYSOSOMES

The ability of liver lysosomes to degrade hyaluronic acid and glycoproteins has been investigated. Purified liver lysosomes, prepared from rats previously treated, by injection, with Triton WR-1339, were incubated with the substrates at acid pH and 37°. Hyaluronic acid was degraded to the extent of about 85% to an equimolecular mixture of glucuronic acid and N-acetylglucosamine, with 15% of the molecule remaining in the form of hyalobiuronic acid, which proved resistant to attack by the lysosomal mixture of enzymes. With orosomucoid and fetuin as substrates, cleavage of about half the peptide bonds and removal of the larger part of the sialyl end groups of the carbohydrate chains occurred rapidly. This was followed more slowly by the release of galactose and of N-acetylglucosamine, which, however, did not exceed 30% of the theoretical yield under the conditions tried. Mannose could not be detected in the hydrolysis mixture. Apparently, considerable resistance to digestion occurs at or near the branching points in the carbohydrate portions of these glycoproteins. With submaxillary mucin as substrate, up to 80% of the disaccharide side chains were split off in the form of free N-acetylneuraminic acid and N-acetylgalactosamine. Peptide hydrolysis did not start until about half the carbohydrate portion had been removed, and then proceeded slowly, reaching 30% of theoretical yield at the end of the incubation period. Some 60% of the mannose residues were detached from ovalbumin glycopeptides. Thus, lysosomes can extensively degrade both the carbohydrate and the peptide portions of glycoproteins, but may lack the ability to bring their digestion to completion, at least with some substrates.

Synthesis of amino sugar containing disaccharides. The synthesis of hyalobiuronic acid and chondrosine.

Hyalobiuronic acid, the unit disaccharide of hyaluronic acid, and chondrosine, that of chondroitin sulfuric acid A or C, were synthesized by condensing corresponding aminosugar derivatives with acetobromo derivative of glucuronic acid by the Koenigs-Knorr reaction.

N-Acetylation of Hyalobiuronic Acid and Chondrosine

N-Acetylation of Hyalobiuronic Acid and Chondrosine

The Isolation and Synthesis of the Methyl Ester-Methyl α-Glycoside of 3-O-β-D-Glucuronosyl-N-Acetyl-D -Glucosamine (Hyalobiuronic Acid)

<b>The Isolation and Synthesis of the Methyl Ester-Methyl α-Glycoside of 3-<b><i>O</i></b>-β-D-Glucuronosyl-<b><i>N</i></b>-Acetyl-D -Glucosamine (Hyalobiuronic Acid)</b>

The Synthesis of Hyalobiuronic Acid and Chondrosine

<b>The Synthesis of Hyalobiuronic Acid and Chondrosine</b>

The Structure of Hyalobiuronic Acid and of Hyaluronic Acid from Umbilical Cord1,2

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTThe Structure of Hyalobiuronic Acid and of Hyaluronic Acid from Umbilical Cord1,2Bernard Weissmann and Karl MeyerCite this: J. Am. Chem. Soc. 1954, 76, 7, 1753–1757Publication Date (Print):April 1, 1954Publication History Published online1 May 2002Published inissue 1 April 1954https://doi.org/10.1021/ja01636a010RIGHTS & PERMISSIONSArticle Views1203Altmetric-Citations262LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (698 KB) Get e-Alerts Get e-Alerts