The partition of glycosaminoglycan-quaternary ammonium complexes: I. The effect of phase composition

The complexes formed between quaterny ammonium cations and polyanionic glycosaminoglycans can be partitioned between partially miscible aqueous inorganic salt and alcohol phases. Small changes in salt concentration can completely shift the complex from one phase to the other. The effect of the phase composition variables: the type of inorganic salt, the type of quaternary ammonium salt, and the alcohol used, were systematically investigated. The sharp transition from solubility in the upper non-aqueous phase to solubility in the lower, aqueous phase was found to be strongly affected by the type of inorganic salt. This transition occurred at higher salt concentrations when NaCl, KCl, or LiCl were used than when CaCl₂ or MgCl₂ were used. Differences in behavior among glycosaminoglycans were larger for NaCl than for CaCl₂. The complex is stabilized to dissociation by salt by increasing hydrophobicity of the non-aqueous phase. However, aggregation of the complex into an insoluble form is also favored by an increasingly hydrophobic environment. The most consistent partition was observed with 1- and 2-butanol. The partition isotherm of chondroitin 4-sulfate was investigated at constant salt concentration. It was found that the partition coefficient varies with the concentration of chondroitin 4-sulfate, although the magnitude of this effect could be diminished by increasing the quaternary ammonium salt concentration.     

The partition behavior of complexes of glycosaminoglycans and quaternary ammonium salts

Complexes of purified glycosaminoglycans and hexadecylpyridinium chloride are shown to be capable of partition between aqueous and butanol phases. The partition coefficient of these complexes is dependent upon the concentration of the supporting electrolyte as well as the concentration of the quaternary ammonium salt. A sharp transition, during which the complex changes from complete solubility in the butanol phase to complete solubility in the aqueous phase, occurs over a very narrow range of salt concentrations. The various glycosaminoglycans show differences sufficient to permit fractionation at least into nonsulfated, monosulfate, and polysulfated classes by simple partition.     

Chromatography of glycosaminoglycans on hydrophobic gel. Correlation between chromatographic behavior of glycosaminoglycans on phenyl-sepharose CL-4B and their solubility in the presence of high concentrations of ammonium sulfate

The effect of bound sulfate groups and uronic acid residues of glycosaminoglycans on their behavior in chromatography on hydrophobic gel was examined by the use of several pairs of depolymerized chondroitin, chondroitin 4- or 6-sulfate, and dermatan sulfate having comparable degree of polymerization. Chromatography on Phenyl-Sepharose CL-4B in 4.0-2.0 ammonium sulfate containing 10m hydrochloric acid showed that: (a) The retention of depolymerized chondroitin 4- or 6-sulfate on the gel varies with the temperature, whereas the depolymerized samples of chondroitin and dermatan sulfate does not show a temperature dependence (this is not the case for hyaluronic acid or dextrans). (b) Among depolymerized samples of chondroitin and chondroitin 4- and 6-sulfate that have a similar degree of polymerization, chondroitin 4- and 6-sulfate showed the highest retention. (c) The retention on the gel of chondroitin 6-sulfate, chondroitin 4-sulfate, and dermatan sulfate decreased in this order. The solubility in ammonium sulfate solution of the polysaccharides agreed well with the chromatographic behavior, suggesting that the fractionation by the hydrophobic gel largely depends on the ability to precipitate on the gel rather than on the hydrophobic interaction between gel and polysaccharide.     

Isolation and characterization of glycosaminoglycans from the Furth murine mastocytoma.

New methods for isolation and fractionation by partition are described and compared with existing techniques. Substantially purer products were isolated by partition as compared to precipitation with hexadecylpyridinium chloride. The glycosaminoglycans isolated fron Furth murine mastocytoma tumor were found to consist of 78-80% heparin, 12-13% chondroitin sulfate, and 8-9% hyaluronate. Dermatan sulfate was not detected. Two heparin-like glycosaminoglycans could be isolated by partition fractionation in the phase system 1-butanol/aqueous NaCl containing hexadecylpyridinium chloride. The composition of one was typical of heparins. However, the other glycosaminoglycan contained only 0.47 moles N-sulfate/mole uronate, but had electrophoretic and partition properties characteristic of heparin.     

Thermodynamics of the partition of chondroitin sulfate–hexadecylpyridinium complexes in butanol/aqueous salt biphasic solutions

The thermodynamics of the partition of chondroitin sulfate–hexadecylpyridinium complexes wee studied in order to gain further insight into the mechanisms responsible for the sensitivity of the relative solubility of these complexes in aqueous slat and butanol phases to small changes in slat concentration. The dependence of the partition coefficient was measured as a function of temperature at three different salt concentrations. Increasing the temperature was found to favor the form of the complex which was soluble n the aqueous phase. Although the transition could be induced by temperature changes, he transition occurred over a 20°C range in temperature. The transition from the aqueous phase to the butanol phase was strongly exothermic, with ΔH = ?22.3 kcal/mol polymer. The value of ΔS was found to be dependent on the salt concentration, ranging from ?72.7 e.u./mol polymer in 0.05125M NaCl to ?77.1 e.u./mol polymer in 0.05375M MaCl. When placed on a disaccharide basis, the corresponding values are ΔH = ?402 cal/mol and ΔS = ?1.31 to ?1.3 e.u./mol. The sharpness of he transition was found to be due t the similarity in magnitude of ΔH and TΔS, and on the dependence of the later upon the salt concentration.     

The effect of changes in salt concentration and pH on the interaction between glycosaminoglycans and cationic polypeptides.

Circular dichroism (CD) spectroscopy has been used to investigate the effects of changes in salt concentration and pH on the interactions between basic polypeptides and connective tissue glycosaminoglycans in dilute aqueous solution. The polypeptides undergo conformation-directing interactions in the presence of glycosaminoglycans, which are subject to transitions as the ionic strength and pH are varied. For poly(L -lysine), the conformational change due to interaction breaks down as the ionic strength (monovalent ions) is increased. Based on the ionic strength at which disruption occurs, the glycosaminoglycans can be placed in order of increasing strength of interaction: chondroitin 6-sulfate, hyaluronic acid, chondroitin 4-sulfate, heparin, and dermatan sulfate. Prior to the conformational transition, scattering effects are observed, indicating the development of larger aggregates. Each glycosaminoglycan induces α-helicity for poly(L -arginine), which does not break down as the ionic strength is increased, indicating a stronger interaction for this polypeptide. The pH-induced transitions are in the pH range 2.5–3.8 and are probably related to deionization of carboxyl groups. For poly(L -lysine) the conformational effect is disrupted at low pH. For poly(L -arginine), the transitions are not complete, but appear to correspond to an increase in scattering.     

Modulation of cAMP-dependent protein kinase by derivatives of chondroitin sulfate

Here, we report investigations about the direct effect of glycosaminoglycans, such as dermatan sulfate, chondroitin 4- and 6-sulfate upon cAMP-dependent protein kinase activity. The results indicate that glycosaminoglycans strongly influence the phosphorylation activity of this enzyme against histone type IIa and [Val⁶,Ala⁷]-kemptide. While chondroitin 4-sulfate and dermatan sulfate exhibit inhibitory effects, chondroitin 6-sulfate shows a stimulating effect. In addition, the chondroitin 6-sulfate is also able to reduce the chondroitin 4-sulfate and dermatan sulfate specific inhibition.     

Correlation for the partition behavior of proteins in aqueous two-phase systems: effect of surface hydrophobicity and charge

Correlations to describe the effect of surface hydrophobicity and charge of proteins with their partition coefficient in aqueous two-phase systems were investigated. Polyethylene glycol (PEG) 4000/phosphate, sulfate, citrate, and dextran systems in the presence of low (0.6% w/w) and high (8.8% w/w) levels of NaCl were selected for a systematic study of 12 proteins. The surface hydrophobicity of the proteins was measured by ammonium sulfate precipitation as the inverse of their solubility. The hydrophobicity values measured correlated well with the partition coefficients, K, obtained in the PEG/salt systems at high concentration of NaCl (r = 0.92-0.93). In PEG/citrate systems the partition coefficient correlated well with protein hydrophobicity at low and high concentrations of NaCl (r = 0.81 and 0.93, respectively). The PEG/citrate system also had a higher hydrophobic resolution than other systems to exploit differences in the protein's hydrophobicity. The surface charge and charge density of the proteins was determined over a range of pH (3-9) by electrophoretic titration curves; PEG/salt systems did not discriminate well between proteins of different charge or charge density. In the absence of NaCl, K decreased slightly with increased positive charge. At high NaCl concentration, K increased as a function of positive charge. This suggested that the PEG-rich top phase became more negative as the concentration of NaCl in the systems increased and, therefore, attracted the positively charged proteins. The effect of charge was more important in PEG/dextran systems at low concentrations of NaCl. In the PEG/dextran systems at lower concentration of NaCl, molecular weight appeared to be the prime determinant of partition, whereas no clear effect of molecular weight could be found in PEG/salt systems.     

The isolation, identification and characterization of sulfated glycosaminoglycans synthesized in vitro by human eosinophils

Human eosinophils were purified to greater than 92% using 16-30% metrizamide gradients, and these cells cultured for up to 72 h in vitro to label sulfated glycosaminoglycans. Over 90% of the sulfated glycosaminoglycan-containing material was extracted in 4 M guanidine HCl and had a hydrodynamic size similar to a glycosaminoglycan marker with an approximate average molecular weight of 60,000. Treatment of this salt-extracted 35S-labeled glycosaminoglycan-containing material with 0.5 M NaOH resulted in a change in mass to approx. 20,000 daltons, suggesting that the larger molecules were proteoglycans with side chains with an approximate molecular weight of 20,000. These salt extracted presumptive 35S-labeled proteoglycans were protease insensitive and behaved in a highly charged fashion on DEAE-cellulose. The composition of 35S-labeled glycosaminoglycans from human eosinophils as identified using selected polysaccharides was 70-81% chondroitin 4-sulfate, 9-12% chondroitin 6-sulfate, and 5-12% dermatan sulfate. The predominance of chondroitin 4-sulfate in human eosinophils is similar to the predominance of chondroitin 4-sulfate in human neutrophils and human platelets.     

The binding of chondroitin 6-sulfate to plasma low density lipoprotein

The interaction in vitro of several sulfated glycosaminoglycans with low density lipoproteins (LDL) has been studied. Chondroitin 6-sulfate and heparin were the only ones to produce turbidity when added to LDL in presence of Ca2+. However, when these two glycosaminoglycans were applied to LDL-affinity columns in presence of Ca2+, only chondroitin 6-sulfate was retained. Partially desulfated chondroitin 6-sulfate was not retained on LDL-affinity column, indicating the relevance of sulfate groups in the binding of LDL. Since chondroitin 4-sulfate and heparin, with a sulfate content respectively equal to and greater than that of chondroitin 6-sulfate, are not retained on LDL-affinity columns, the factors relevant to the binding of LDL are probably the conformation of the glycan in solution and the orientation of its sulfate groups.     

Countercurrent chromatography--a new method for the fractionation of glycosaminoglycans

A new method is described for continuous flow fractionation of glycosaminoglycans (GAG) by partition in two-phase systems. In the 1-butanol/aqueous NaCl two-phase system containing hexadecylpyridinium chloride fractionation is dependent primarily upon chemical composition, or charge density, of the GAG and is relatively independent of molecular weight. Chondroitin 4-sulfate was fractionated according to degree of sulfation and could be completely resolved from heparin. A heparin sample was shown to contain three discrete components differing with respect to sulfaminohexose and sulfate substitution.     

Novel approach towards recovery of glycosaminoglycans from tannery wastewater

Poly ethylene glycol (PEG)-poly acrylic acid (PAA) based aqueous two-phase system (ATPS) was selected as a practical model to recover glycosaminoglycans (GAGs) from tannery wastewater. The influence of PEG molecular weight, tie line length (TLL), pH, temperature and NaCl concentration on the partition coefficient of glycosaminoglycans from tannery wastewater was studied. Partition coefficient of glycosaminoglycan decreases on increase of PEG molecular weight, NaCl concentration and temperature, whereas it increases with increase of pH. In the PEG-rich phase, increased partitioning of GAGs was observed with increase in TLL. The partitioning of GAGs was better in PEG 4000 at pH 8.0, 20 °C with a yield of 91.50%. This study demonstrates the potential application of ATPS processes for the recovery of GAGs from complex biological suspensions.     

Isolation and Purification of Chondroitin 6-Sulfate Protein From Umbilical Cord

Intact chondroitin 6-sulfate protein can be extracted from umbilical cord with dilute saline. Hyaluronic acid which is also extracted, is removed by precipitation with cetylpyridinium chloride followed by washing of the precipitate with aqueous sodium chloride. Subsequent purification is effected by passage through cation and anion exchange resins. Elution from the latter with salt solutions of increasing concentration yields chondroitin 6-sulfate proteoglycan in two fractions. The product is isolated from each of the fractions as the calcium salt by fractional precipitation with ethanol. The protein moiety can be cleaved from the mucopolysaccharide either by proteolytic digestion or treatment with alkali. The results obtaired on reaction with alkali and with sodium borohydride indicate that the polysaccharide is covalently linked to the protein through a serine unit.     

Highly sulfated glycosaminoglycans inhibit aggrecanase degradation of aggrecan by bovine articular cartilage explant cultures.

The catabolism of 35S-labeled aggrecan and loss of tissue glycosaminoglycans was investigated using bovine articular cartilage explant cultures maintained in medium containing 10(-6) M retinoic acid or 40 ng/ml recombinant human interleukin-1alpha (rHuIL-1alpha) and varying concentrations (1-1000 microg/ml) of sulfated glycosaminoglycans (heparin, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate and keratan sulfate) and calcium pentosan polysulfate (10 microg/ml). In addition, the effect of the sulfated glycosaminoglycans and calcium pentosan polysulfate on the degradation of aggrecan by soluble aggrecanase activity present in conditioned medium was investigated. The degradation of 35S-labeled aggrecan and reduction in tissue levels of aggrecan by articular cartilage explant cultures stimulated with retinoic acid or rHuIL-1alpha was inhibited by heparin and heparan sulfate in a dose-dependent manner and by calcium pentosan polysulfate. In contrast, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate and keratan sulfate did not inhibit the degradation of 35S-labeled aggrecan nor suppress the reduction in tissue levels of aggrecan by explant cultures of articular cartilage. Heparin, heparan sulfate and calcium pentosan polysulfate did not adversely affect chondrocyte metabolism as measured by lactate production, incorporation of [35S]-sulfate or [3H]-serine into macromolecules by articular cartilage explant cultures. Furthermore, heparin, heparan sulfate and calcium pentosan polysulfate inhibited the proteolytic degradation of aggrecan by soluble aggrecanase activity. These results suggest that highly sulfated glycosaminoglycans have the potential to influence aggrecan catabolism in articular cartilage and this effect occurs in part through direct inhibition of aggrecanase activity.     

Impact of various glycosaminoglycans upon cAMP-dependent protein kinase

The physiological effects of the second messenger cAMP are displayed by cAMP-dependent protein kinase-medicated phosphorylation of specific target proteins which in turn control diverse cellular functions. We have determined this enzyme substrate phosphorylation in the presence of various glycosaminoglycans using a cAMP-dependent protein kinase isolated from rat liver. The results indicate that sulfated and unsulfated polysaccharides are able to inhibit phosphorylation of histone type IIa catalysed by cAMP-dependent protein kinase. Based on their impact upon substrate phosphorylation, glycosaminoglycans can be divided into three groups: group I with the highest inhibitory effect: dermatan sulfate and heparan sulfate; group II: chondroitin 4-sulfate and group III with the lowest inhibitory effect: chondroitin 6-sulfate, keratan sulfate and hyaluronic acid.     

Inhibition by 5-bromo-2'-deoxyuridine of differentiation-dependent changes in glycosaminoglycans of the retina.

Embryonic chick neural retinas incorporated radio-labeled precursors into glycosaminoglycans in the same relative amounts whether cultured as intact tissues, cell aggregates, or monolayers. Incubation with 5-bromo-2′-deoxyuridine inhibited histogenesis and caused the pattern of synthesis to remain more like that in undifferentiated tissue, when compared with controls without this nucleoside analog. This was determined by the level of incorporation and the ratios of chondroitin sulfate to heparan sulfate and chondroitin-4-sulfate to chondroitin-6-sulfate incorporation. Incubation with 4-methylumbelliferyl-β-D-xylopyranoside stimulated synthesis and release of chondroitin sulfate and heparan sulfate into the medium. The results taken together imply that the production of specific glycosaminoglycans during the course of differentiation in the retina is regulated at the gene level in parallel with histogenesis in this tissue.     

Decreased ganglioside neuraminidase activity in fibroblasts from mucopolysaccharidosis patients. Inhibition of the activity in vitro by sulfated glycosaminoglycans and other compounds

The neuraminidase activities towards the ganglioside substrates GD1a, GD3 and GM3 were found to be markedly diminished in homogenates of fibroblasts cultured from patients with various genetic mucopolysaccharidoses. Mixing normal and patients' fibroblast homogenates revealed this effect to be due to the presence of diffusible inhibitors. The neuraminidase acting on the trisaccharide sialyllactose, on the other hand, showed normal activity in all the cell lines tested. Experiments in vitro revealed the sulfated glycosaminoglycans chondroitin 4-sulfate and heparin, the polysaccharide dextran sulfate, and the trypanocidal drug suramin to be strongly inhibitory on the ganglioside GD1a neuraminidase activity of normal fibroblast homogenates. Regarding chondroitin 4-sulfate, this inhibition was of the non-competitive type. A disulfated tetrasaccharide prepared from chondroitin 4-sulfate, on the other hand, was not at all inhibitory. These and additional findings led us to propose a model for the interaction between enzyme and inhibitor, involving a 'clamping' mechanism by the polysulfated compounds. We conclude that the decreased ganglioside neuraminidase activities of mucopolysaccharidosis fibroblasts are due to an inhibition by the accumulated sulfated glycosaminoglycans and that such inhibition is responsible for the storage of certain gangliosides in the tissues of the patients.     

Spectroscopic properties of complexes of acridine orange with glycosaminoglycans II. Aggregated complexes-evidence for long-range order.

Aggregated complexes of acridine orange with dermatan and chondroitin sulfates have been studied in aqueous solution by absorption and circular dichroism spectroscopy. Aggregation was found to be favored at high-dye and glycosaminoglycan concentrations, and in solutions where anionic sites of the glycosaminoglycan are effectively complexed with dye. The aggregates can be removed from solution by centrifugation at 27,000 × g for 1 hr or by filtration through a membrane containing pores of 0.1 μm diameter. The aggregated complexes exhibit large-magnitude-ellipticity circular dichroism bands. In addition, the circular dichroism spectrum observed for a solution containing aggregated acridine orange/chondroitin 4-sulfate complexes is nearly a mirror image of that obtained for aggregated acridine orange/dermatan sulfate complexes. Cooperative alterations (sharp transitions) in the circular dichroism ellipticities of the aggregates occur at elevated temperatures, and result in spectroscopically distinct aggregates upon cooling. The circular dichroism properties and temperature effects are attributed to a supramolecular ordering of acridine orange/glycosaminoglycan complexes within the aggregates, which can be reorganized to a more stable form at high temperatures. Mixed aggregates, containing two different glycosaminoglycans, can be formed. The circular dichroism properties of the mixed aggregates also indicate the existence of long-range order in the arrangement of the complexes. Mixed aggregates containing dermatan sulfate and either chondroitin 4-sulfate or chondroitin 6-sulfate resemble pure dermatan sulfate aggregates in circular dichroism characteristics.     

Human venous and arterial glycosaminoglycans have similar affinity for plasma low-density lipoproteins

We compared the glycosaminoglycan content of human venous and arterial walls. The most abundant glycosaminoglycan in human veins is dermatan sulfate whereas chondroitin 4/6-sulfate is preponderant in arteries. The concentrations of chondroitin 4/6-sulfate and heparan sulfate are approximately 4.8- and approximately 2.5-fold higher in arteries than in veins whereas dermatan sulfate contents are similar in the two types of blood vessels. Normal and varicose saphenous veins do not differ in their glycosaminoglycan contents. It is known that certain glycosaminoglycan species from the arterial wall, mainly high-molecular-weight fractions of dermatan sulfate+chondroitin 4/6-sulfate have greater affinity for plasma LDL. These types of glycosaminoglycans can be identified on a LDL-affinity column. We now demonstrated that a similar population of glycosaminoglycan also occurs in veins, although with a lower concentration than in the arteries due to less chondroitin 4/6-sulfate with affinity for LDL. The concentrations of dermatan sulfate species, which interact with LDL, are similar in arteries and veins. The presence of these glycosaminoglycans with affinity to plasma LDL in veins raises interesting questions concerning the role of these molecules in the pathogenesis of atherosclerosis. Possibly, the presence of these glycosaminoglycans in the vessel wall are not sufficient to cause retention of LDL and consequently endothelial dysfunction, but may require additional intrinsic factors and/or the hydrodynamic of the blood under the arterial pressure.