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Glycosaminoglycans were extracted from both young rabbit growth plate (GRP) and articular (ART) cartilage tissues and enzymatically treated to selectively eliminate chondroitin sulfates and hyaluronic acid. The procedure avoided any fractionation step that could enrich the extract with over- or under-sulfated species. Isolated heparan sulfate (HS) was characterized by mono- and bidimensional nuclear magnetic resonance (NMR) spectroscopy to quantify their specific structural features and/or by mass spectrometry to establish the disaccharide composition. Both GRP and ART HSs, despite differing in their yield (GRP at least 100 times greater than ART), exhibited a surprisingly high degree of sulfation. Quantitative two-dimensional heteronuclear single-quantum coherence-NMR analysis of GRP HS revealed unusually high N-sulfated glucosamine and 2-O-sulfated iduronic acid contents, similar to heparin. The unique pentasaccharide sequence of the binding site for antithrombin was also detected in a significant amount. High-performance liquid chromatography mass spectrometry analysis of the enzymatic digests with a cocktail of heparin lyases of both cartilaginous HSs confirmed the NMR results. As well as the discovery of an unusual HS structure in the two different types of rabbit cartilage, the feasibility of the analytical method adopted here has been demonstrated within this study. Such a method can be used to isolate and analyze HS from both normal and pathologic tissues. Characterization of healthy and pathological HS structures will contribute to improve the understanding of diseases related to malfunctions of HS biosynthesis and/or metabolism.

Determination of the pK(a) values of heparin disaccharide functional groups can provide insights into the nature of glycosaminoglycan (GAG)-protein interactions and prove useful for optimization of the charged-based separations typically used in GAG analysis. In order to gain a better understanding into the capillary electrophoresis (CE) separation process, the pK(a) values of the carboxylate and primary amine moieties of 11 heparin disaccharide standards were determined through (1)H NMR detected pH titrations. These pK(a) values were used to calculate the effective net charge of each disaccharide and compared to the electrophoretic mobilities measured by CE. Although a different migration order had been reported by other researchers, our results indicate a strong positive correlation between the two measurements, consistent with the migration order observed in our CE separations. The effect of mutarotation was also examined by (1)H NMR. Mutarotation equilibrium constants favored the alpha anomer over the beta conformation. pK(a) values determined for both anomers of the four disaccharide standards containing a GlcN primary amine indicated that the beta anomer of the GlcN residue was more acidic. Partial separation of these anomers was achieved in CE separations using either formic acid or phosphate buffer.

(1)H NMR spectroscopy has been established for the determination of uronate residues in glycosaminoglycans (GAGs) such as dermatan sulfate (DS), heparin (HP), and heparan sulfate (HS). Because of variation in the sulfonation positions in DS, HP, or HS, interpretation of spectra is difficult. Solvolysis was applied to remove O-sulfo groups from these GAG chains in dimethyl sulfoxide containing 10% methanol at 80 degrees C for 5 h. In the cases of HP and HS, N-sulfo groups on glucosamine residues were also removed under the same conditions. The resulting unsubstituted amino groups in HP and HS chains were re-N-acetylated using acetic anhydride to obtain homogeneous core structure with the exception of the variation of uronate residues. The contents of glucuronate and iduronate residues in the chemically modified DS, HP, and HS samples were analyzed by 600-MHz (1)H NMR spectroscopy. These methods were applied to compositional analysis of uronate residues in GAGs isolated from various sources., (Copyright 2001 Academic Press.)

A solution of hog intestinal heparin (average M(r) 12,000, anti-clotting activity 168 USP units/mg) in 0.2 M phosphate buffer (pH 7.2), was incubated in the presence of Fe2+ for 20 h at 50 degrees under an O2 atmosphere to yield oxidative-reductively depolymerized heparin (ORD heparin, average M(r) 3,000, anti-clotting activity 34 USP units/mg). Chemical analysis of the ORD heparin showed a 22, 26, and 14% loss of hexosamine, uronic acid, and N-acetyl group, respectively, but no remarkable loss of both total and N-sulfate groups. 1H and 13C NMR spectroscopic analysis indicated no decrease in the amount of L-iduronic acid 2-sulfate, but a marked loss of nonsulfated uronic acid (73 and 39% loss of D-glucuronic acid and L-iduronic acids, respectively, the sum of which corresponds to the chemically determined loss of total uronic acid). The results indicated that the ORD reaction of heparin proceeds essentially by destruction of monosaccharide units, except L-iduronic acid 2-sulfate residues, due to oxygen-derived free radicals, followed by secondary hydrolytic cleavage of the resulting unstable residues.

Heparan sulphate, heparin and dermatan sulphate were hydrolysed in 0.5M-H2SO4 at 100 degrees C. At intervals portions of the hydrolysate were removed and treated with HNO2 at pH 4.0 to cleave the glycosidic bonds of the N-unsubstituted hexosamine residues and to convert both free and combined hexosamines into anhydrohexoses. These hydrolysis/deamination mixtures were reduced with NaB3H4 and analysed by radiochromatography for alpha-L-iduronosylanhydrohexose, beta-D-glucuronosylanhydrohexose, and the free uronic acids and anhydrohexose. These data gave a kinetic profile of the cleavage of the alpha-L-iduronosyl and the beta-D-glucuronosyl bonds in these glycosaminoglycans. The beta-D-glucuronosyl bonds showed the expected resistance to acid hydrolysis, but the alpha-L-iduronosyl bonds were found to be as labile to acid as some neutral sugar glycosides. This unusual lability of alpha-D-iduronosyl-anhydromannitol and beta-D-glucuronosylanhydromannitol. The procedures used to follow the kinetics of glycosaminoglycan hydrolysis can also be sued to obtain quantitative analyses of L-iduronic acid, D-glucuronic acid and hexosamine in these polymers.

Bovine heparin was fractionated with respect to chain length and anticoagulant activity. Analysis of each of these fractions for iduronic and glucuronic acids demonstrated that active heparin has a greater amount of glucuronic acid than inactive heparin. The ratio of the uronic acids in the respective fractions was the same for heparin with different molecular weights. Thus, active heparin with longer chain lengths have more additional glucuronate residues than are required for the antithrombin-binding domain. The results indicate that the active and inactive heparin species differ in more than one structural characteristic and suggest a considerable divergence in their respective biosynthesis.

The disaccharides IdoA(2SO3)-anManOH(6SO3) and IdoA-anManOH (where IdoA represents alpha-L-iduronate, anManOH represents 2,5-anhydro-D-mannitol and SO3 represents sulphate ester) were prepared from bovine lung heparin using HNO2 depolymerization, borohydride reduction and desulphation, and were examined by 400 MHz 1H-n.m.r. spectroscopy. Three-bond proton-proton coupling constants around the IdoA ring were determined under a range of experimental conditions. For unsulphated IdoA all four proton-proton coupling constants varied markedly as a function of temperature, pH and solvent, providing clear evidence for a rapid conformational equilibrium. These data were analysed in terms of the three most energetically stable IdoA conformers: 1C4, 4C1, and 2S0. Predicted coupling constants for these conformers were determined using a modified Karplus-type relationship. For unsulphated IdoA in dimethyl sulphoxide the equilibrium was provoked strongly in favour of a slightly distorted 4C1 'chair' IdoA conformer for which coupling constants have not previously been reported. For sulphated IdoA in aqueous conditions and at low pH the equilibrium is strongly in favour of the alternative 1C4 chair conformer. Under many conditions, however, significant contributions from all three conformers occur for the non-reducing terminal IdoA in these disaccharides.

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.

Heparins from various sources and heparan sulfate from umbilical cords have been subjected to Smith-degradation and reaction with nitrites. These procedures were effective for providing data relating to the distribution of sulfated iduronic acid residues in the molecule. Results indicated that heparins may have, as a prominent structural feature of the molecule, non-sulfated uronic acid distributed in single sequences, much as had been shown previously for N-acetylglucosamine residues. Sulfated uronic acid, however, may occur in multiple sequences of up to 5 or 6 residues. Heparan sulfate was found to have a major proportion of its ester sulfate on iduronic acid rather than hexosamine units, thereby having sections similar to those in heparins, though in considerably lower proportion.

1H-NMR spectra of the synthetic pentasaccharide (N-sulfate-6-0-sulfate-alpha-D-glucosamine) 1----4 (beta-D-glucuronic acid) 1----4 (N-sulfate-3,6-di-0-sulfate-alpha-D-glucosamine) 1----4 (2-0-sulfate-alpha-L-iduronic acid) 1----4 (N-sulfate-6-0-sulfate-alpha-D-glucosamine), corresponding to the active site of heparin for antithrombin (AT-III), have been resolved at 500 MHz and assigned by mono- and bidimensional techniques. Vicinal proton coupling constants of the D-glucosamine residues are similar to those in the regular sequences of heparin, indicating that the 4C1 conformation of the ring, and preference for the g,g conformation of the sulfated hydroxymethyl groups of these residues are neither affected by the unique 3-0-sulfo group nor by sequence effects. By contrast, an unusually large coupling between H-2 and H-3 of the sulfated L-iduronic acid residue suggests a greater departure from the 1C4 conformation of this residue. when present in the binding sequence to AT-III than in the regular sequences. Such a departure, leading to different orientation and spacing of essential sulfate groups, may have implications for high-affinity binding to AT-III.