Conformation of hyaluronate in neutral and alkaline solutions

Increasing the pH of a neutral salt solution of sodium hyaluronate to 12.5 produces a rapid drop in viscosity which is reversible upon restoring the pH to neutrality. Light scattering data showing a decrease in radius of gyration with no change in molecular weight and negative results with chondroitin and other acidic glycosaminoglycans suggest that the conformational change is specific for hyaluronate molecules.     

Conformation of hyaluronate in neutral and alkaline solutions.

Increasing the pH of a neutral salt solution of sodium hyaluronate to 12.5 produces a rapid drop in viscosity which is reversible upon restoring the pH to neutrality. Light scattering data showing a decrease in radius of gyration with no change in molecular weight and negative results with chondroitin and other acidic glycosaminoglycans suggest that the conformational change is specific for hyaluronate molecules.     

The molecular weight of sodium hyaluronate in amniotic fluid

The molecular weight of sodium hyaluronate in human amniotic fluid was determined by gel chromatography. Pooled samples from 16 weeks of gestation exhibited a broad molecular weight distribution (Mw = 3 X 10(5); Mn = 6 X 10(4)). Samples taken at 40 weeks showed a low molecular weight fraction (M less than 10(5) presumably of fetal origin, and a high molecular weight fraction (M greater than 10(6) which varied considerably, indicating a nonfetal origin. No hyaluronate degrading activity was detected in the fluid. In a case of renal dysplasia, only minute amounts of hyaluronate were found. These findings are in accordance with a renal excretion of a low molecular weight hyaluronate from the fetus. In 12 cases with proven neural tube defects, there was no significant difference in hyaluronate level and molecular weight compared to normal controls.     

Measurements of reducing end groups on bovine vitreous-humour hyaluronic acid by reaction with [14C]cyanide.

Bovine vitreous-humour sodium hyaluronate was purified by precipitation with cetylpyridinium chloride, CsCl-density-gradient sedimentation and gel-permeation chromatography. The number of reducing end groups in two similarly prepared hyaluronate samples was determined by reaction with K14CN, and measurements of intrinsic viscosity were performed to determine whether this reaction caused degradation of the hyaluronate. The intrinsic viscosity of one hyaluronate sample was 192ml/g, compared with a value of 187ml/g after reaction with [14C]cyanide, which indicates that the labelling reaction did not cause depolymerization of the hyaluronate. The Mr calculated from these viscosity values is approx. 60000. Fractionation of the [14C]cyanide-labelled hyaluronate by gel chromatography showed that it was composed of a polydisperse population of molecules with calculated chain lengths, based on the ratio of [14C]cyanide to uronic acid, ranging in molecular weight from 9000 to 264000, with an average Mr of 63200. On the basis of these measurements it is concluded that reaction with [14C]cyanide does not cause degradation of bovine vitreous-humour hyaluronate polysaccharide chains and that reaction with [14C]cyanide can be used to determine the molecular weight of this hyaluronate.     

Effect of molecular weight distribution on the solution properties of sodium hyaluronate in 0.2M NaCl solution

Various molecular parameters, which characterize sodium hyaluronate in 0.2M NaCl solution, were obtained at 25°C by means of the static and dynamic light scattering and low shear viscometry over the molecular weight range of 5.94–627 × 10⁴. Molecular weight distribution was obtained by using the Laplace inversion method of the autocorrelation function of the scattered light intensity and by Yamakawa theory for the wormlike chain with the stiff chain parameters for sodium hyaluronate in 0.2M NaCl (persistence length, chain diameter, molar mass per unit contour length, and the excluded‐volume strength). The molecular weight distribution thus obtained reproduced the solution properties of sodium hyaluronate well. Especially, the intrinsic viscosity showed a good agreement over four orders of molecular weight with Yamakawa theory combined with the Barrett function. Sodium hyaluronate in 0.2M NaCl solution is well expressed by the wormlike chain model affected by the excluded‐volume effect with the persistence length of 4.2 nm. © 1999 John Wiley & Sons, Inc. Biopoly 50: 87–98, 1999     

The relationship of molecular weight to electrophoretic mobility of fluorescamine-labeled proteins in polyacrylamide gels

Proteins of known molecular weights were labeled with fluorescamine and then subjected to electrophoresis through polyacrylamide gels. The electrophoretic mobilities of the fluorescamine-labeled proteins were dependent upon their respective molecular weights over a range of 17,000 to 70,000 daltons. The correlation of electrophoretic mobility of fluorescamine-labeled protein to molecular weight was similar to results obtained in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The speed with which data can be obtained with the described procedure is a definite advantage over currently employed procedures. These findings encourage the use of fluorescamine for rapid, sensitive determinations of molecular weights of proteins in polyacrylamide gels.     

Characterization and identification of the hyaluronate binding site from membranes of SV-3T3 cells

Previous research has shown that binding sites for hyaluronate are present on the surfaces of a number of different cell types. To further characterize these binding sites, membranes were prepared from SV-3T3 cells and dissolved in a solution of sodium deoxycholate. Hyaluronate binding activity was detected by mixing the sodium deoxycholate extract with [3H]hyaluronate and then adding an equal volume of saturated (NH4)2SO4, which precipitated the binding protein and any [3H]hyaluronate associated with it, but left free [3H]hyaluronate in solution. Following partial purification by hydroxylapatite chromatography, the binding site was examined by molecular sieve chromatography and by rate-zonal centrifugation, which revealed that it has a Stokes radius of 6.5 nm and a sedimentation coefficient of 4.8 S. From these values, it was possible to calculate that the sodium deoxycholate-solubilized binding site has a frictional coefficient of 1.87 and a molecular weight of 132,000. Since this latter value applies to the complex of both detergent and protein, the binding protein by itself must have a molecular weight lower than 132,000. To determine the molecular weight of the hyaluronate binding site itself, the protein was purified by the sequential application of hydroxylapatite chromatography, molecular sieve chromatography, rate-zonal centrifugation, and finally lectin-affinity chromatography on concanavalin A-agarose. Analysis of the purified material by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an 85,000 Mr protein which has been identified as the binding site. This protein was also detected on nitrocellulose blots which had been specifically stained for concanavalin A binding material, suggesting that the binding site is a glycoprotein.     

Random coil scission rates determined by time-dependent total intensity light scattering: hyaluronate depolymerization by hyaluronidase

A recently developed theory of the light scattering by random coils undergoing random scission is applied to the digestion of hyaluronate by hyaluronidase. The time dependence of the scattered light from solutions undergoing digestion was monitored. Working at a high angle with high molecular weight hyaluronate allowed the use of a powerful approximation for determining initial velocities and the Henri-Michaelis-menten coefficients, without explicit knowledge of the hyaluronate molecular weight, radius of gyration, second virial coefficient, or polydispersity. Effects due to a molecular weight dependent second virial coefficient and to non-Gaussian behavior are briefly considered. Assays were performed over nearly two orders of magnitude in substrate concentration. The initial velocities are compared with those obtained by a standard reducing sugar assay, which was performed on identical samples. The main advantages of the light scattering assay procedure over the more traditional assays are that many relatively high-precision data points can be quickly and automatically collected with simple apparatus, and that the technique is most sensitive for the initial period of digestion, where the other assays are least sensitive. The shapes of the scattering curves also provide evidence that hyaluronate in these solutions is not a stable double strand and that the hyaluronidase cleaves bond randomly. The curves also indicate that enzyme deactivation occurs, which accounts for the lower velocities yielded by the slower reductimetric assay, which is measured over longer initial periods.     

PURIFICATION OF PHOSPHATE-DEPENDENT PIG BRAIN GLUTAMINASE

Abstract— A procedure for preparing highly purified phosphate-activated glutaminase (EC 3.5.1.2, L-glutamine amidohydrolase) from pig brain is described. The main steps consist of extraction with acetone, followed by sodium sulphate fractionation of the solubilized acetone powder. Thereafter, solubilization by dialysis against a buffer containing tris-HC1, mercaptoethanol, and EDTA, followed by precipitation with phosphate-borate, is repeated twice. The final preparation contains no impurities which can be detected by polyacrylamide gel electrophoresis, isoelectric focusing, and sedimentation equilibrium centrifugation. By the latter method, molecular weight is determined to be 187,000. By polyacrylamide gel electrophoresis in sodium dodecyl sulphate, one protein band with molecular weight 64,000 is found.     

Purification of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

This paper describes a rapid purification procedure for 3-hydroxy-3-methylglutaryl coenzyme A reductase, the major regulatory enzyme in hepatic cholesterol biosynthesis. A freeze-thaw technique is used for solubilizing the enzyme from rat liver microsomal membranes. No detergents or other stringent conditions are required. The purification procedure employs Blue Dextran-Sepharose-4B affinity chromatography, and purification can be carried out from microsomal membranes to purified enzyme in 8 to 10 hours. The purified enzyme has a specific activity of 517 nmoles/min/mg protein, and it is 975-fold purified with respect to the original microsomal membrane suspension. SDS polyacrylamide gel electrophoresis of the purified enzyme shows only trace impurities; the subunit molecular weight for the enzyme measured by this technique is 47,000.     

A study of hydration of sodium hyaluronate from compressibility and high precision densitometric measurements

Sodium hyaluronate samples of various molecular weights were prepared and characterized by size exclusion chromatography and dilution viscometry. Densitometry was used for determination of the densities of sodium hyaluronate solutions and the results expressed as partial specific volumes in the respective solvents. Solution adiabatic compressibilities were measured. Hydration parameters of sodium hyaluronate were consequently determined and the values obtained discussed in relation to existing hydration models for sodium hyaluronate in water.     

Extracellular matrix metabolism by chondrocytes 5. The proteoglycans and glycosaminoglycans synthesized by chondrocytes in high density cultures

Proteoglycans were extracted from the extracellular matrix of cultures of embryonic chick chondrocytes grown at high density and were purified by CsCl density gradient centrifugation. The chemical, physical and hyaluronate binding properties of the proteoglycans were similar to those observed in proteoglycans from other hyaline cartilages. Proteoglycans in the media were also purified and on analysis showed three populations of proteoglycans to be present. One population had the physical characteristics of a typical proteoglycan subunit and bound hyaluronate, the other two populations were unable to complex with hyaluronate but one had the physical characteristics of the proteoglycan subunit and the other was of smaller molecular weight. The small molecular weight appears to be a product of the enzymatic degradation of the larger molecular weight species.     

Anti-inflammatory activity of superoxide dismutase conjugated with sodium hyaluronate

Superoxide dismutase (SOD) from bovine erythrocytes was conjugated with sodium hyaluronate (HA) with a mean molecular weight of 106 to have greater anti-inflammatory activity in vivo. Amino groups of SOD were coupled with carboxyl groups in the hyaluronate molecule using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The HA-SOD conjugate was composed of 1.5 mol of SOD molecule per 1 mol of hyaluronate on the average, and retained 70% of the activity of unmodified SOD. The conjugate was essentially non-immunogenic in mice, and exhibited much higher anti-inflammatory activities than HA or SOD in models of inflammatory diseases such as ischemic oedema of the foot-pad in mice, carrageenin-induced pleurisy and adjuvant arthritis in rats. This revised version was published online in November 2006 with corrections to the Cover Date.     

The conformation of the mucopolysaccharides. Hyaluronates

X-ray-diffraction patterns of hyaluronate fibres from a variety of sources were obtained. Sodium hyaluronate gives well-defined patterns which index on a hexagonal unit cell with dimensions a=1.17+/-nm and a fibre repeat-distance of 2.85+/-0.03nm. A further form of sodium hyaluronate is produced by annealing at 60 degrees C in 75% relative humidity. This stable state indexes on a hexagonal unit cell of unchanged fibre repeat-distance but with a=1.87nm. The chain conformation is a threefold helix. Analysis of these diffraction patterns led to two tentative structures for sodium hyaluronate, involving different packing of the polysaccharide chains. The significance of side-chain interaction is discussed. Hyaluronic acid produces an X-ray pattern different from that obtained with the sodium salt. The fibre repeat-distance is 1.96+/-0.02nm and the unit cell appears to be monoclinic. The chain conformation is a twofold helix and conformational change between free acid and monovalent salt is discussed. These findings, together with model-building experiments, are interpreted as indicating a highly ordered structure, and the physical properties of hyaluronate solutions with regard to molecular shape and polyelectrolyte behaviour are rationalized.     

Binding of hyaluronate to the surface of cultured cells

The binding of hyaluronate to SV-3T3 cells was measured by incubating a suspension of cells (released from the substratum with EDTA) with 3H-labeled hyaluronate and then applying the suspension to glass fiber filters which retained the cells and the bound hyaluronate. The extent of binding was a function of both the concentration of labeled hyaluronate and the cell number. Most of the binding took place within the first 2 min of the incubation and was not influenced by the presence or absence of divalent cations. The binding of labeled hyaluronate to SV-3T3 cells could be prevented by the addition of an excess of unlabeled hyaluronate. High molecular weight preparations of hyaluronate were more effective in preventing binding than low molecular weight preparations. The binding of [3H]hyaluronate was inhibited by high concentrations of oligosaccharide fragments of hyaluronate consisting of six sugars or more, and by chondroitin. The sulfated glycosaminoglycans (chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, heparin, and heparan sulfate) had little or no effect on the binding. The labeled hyaluronate bound to the cells could be totally removed by incubating the cells with testicular hyaluronidase, streptomyces hyaluronidase, or trypsin, indicating that the hyaluronate-binding sites are located on the cell surface.     

Flow field-flow fractionation/multiangle light scattering of sodium hyaluronate from various degradation processes

Sodium hyaluronate (NaHA) is an ultrahigh molecular weight polysaccharide that is found in body tissues, synovial fluid, the vitreous humor, and the umbilical cord, and the size characterization of NaHA is important in pharmaceutical applications. On-line field-flow fractionation/multiangle light scattering/differential refractive index (FlFFF/MALS/DRI) has been applied for the study of degradation efficiency of sodium hyaluronate (NaHA). A NaHA raw sample was degraded by different chemical or physical methods and the degraded NaHA samples were separated using field-programming FlFFF, in which separation is achieved by differences in diffusion coefficients or hydrodynamic diameters. Separation was followed by serial detection using MALS and DRI. Molecular weight distribution (MWD) and information relating to the radius of gyration of the NaHA samples were examined for the raw and degraded NaHA samples. Samples studied include: two different products of ultrasonic degradation, two products of alkaline degradation, and four different products of enzymatic degradation. While alkaline degradation showed a moderate degradation compared to ultrasonic and enzymatic methods in reducing average MW, the latter two degradation methods showed significant changes in average molecular weight and in conformation of NaHA.     

Combined alcian blue and silver staining of glycosaminoglycans in polyacrylamide gels: application to electrophoretic analysis of molecular weight distribution

Oligomeric and polymeric fragments of glycosaminoglycans may be separated for rapid analysis by electrophoresis through a 10% polyacrylamide matrix. A ladder-like series of bands is observed, in which adjacent major bands correspond to species differing in chain length by one disaccharide unit. The component species are detected by a combined alcian blue and silver staining protocol. Detection limits are less than 50 ng per band, or approximately 2-5 micrograms total load for polydisperse samples. Densitometry of the stained gel may be used to determine molecular weight averages and distribution. The applicable molecular weight ranges are approximately 4000 to 100,000 for hyaluronate, or 1500 to 40,000 for chondroitin and dermatan sulfate samples of moderate charge density heterogeneity.     

Hyaluronate-alginate gel as a novel biomaterial: mechanical properties and formation mechanism

With the aim of producing a biomaterial for surgical applications, the alginate-hyaluronate association has been investigated to combine the gel-forming properties of alginate with the healing properties of hyaluronate. Gels were prepared by diffusion of calcium into alginate-hyaluronate mixtures, with an alginate content of 20 mg/mL. The hyaluronate source was shown to have significant effect on the aspect and the properties of the gels. The gels have viscoelastic behaviour and the transient measurements carried out in creep mode could be interpreted through a Kelvin-Voigt generalised model: experimental data led to the steady state hardness and a characteristic viscosity of the gel. Gels prepared from Na rooster comb hyaluronate with weight ratio up to 0.50 have satisfactory mechanical properties, and fully stable gels are obtained after a few days; on the contrary, use of lower molecular weight hyaluronate led to loose gels for hyaluronate contents over 0.25. Gel formation was investigated by measurements of the exchange fluxes between the calcium chloride solution and the forming gel, which allowed thorough investigations of the occuring diffusion phenomena of water, calcium ion and hyaluronate. Strong interactions of water with hyaluronate reduce significantly the rate of weight loss from the gel beads and allows higher water content in steady-state gels. Calcium content in the gel samples could be correlated to the actual alginate concentration, whatever the nature and the weight ratio of hyaluronate.     

Competitive inhibition evidence for specific intermolecular interactions in hyaluronate solutions

Intermolecular associations in hyaluronate systems have been investigated by a competitive inhibition approach, monitored by low deformation oscillatory measurements of dynamic viscosity and rigidity. Solutions of sodium hyaluronate isolated by a mild procedure from rooster combs showed, under physiological conditions of pH and ionic strength, coupling behaviour typical of a transient polymer network. On addition of an equal concentration of hyaluronate segments (~60 disaccharide units), all evidence of coupling is lost and the solution rheology approximates closely to that typical of isolated chains. Such behaviour is clearly incompatible with entanglement coupling, such as occurs between synthetic polymers in solution, but parallels the behaviour of gelling polysaccharides, where co-operative interchain association is known to occur. Similar inhibition is observed in hyaluronate viscoelastic “putties” and “gels”, where further intermolecular association is promoted by suppression of interchain electrostatic repulsion and reduction of water activity. The effects are particularly pronounced for hyaluronate of lower molecular weight, where crosslinking in putties and gels may approach the minimum requirements for a cohesive network. No inhibition is observed with very short chain segments (~4 disaccharide residues) nor with longer segments (~400 disaccharides). On the basis of this evidence we suggest that hyaluronate chains interact by formation of specific co-operative junctions analogous to those characterised for plant structural polysaccharides, but having substantially shorter lifetimes. The magnitude of rheological changes on suppression of these fleeting associations by competitive inhibition suggests that they are likely to dominate the physical properties of hyaluronate in vivo.     

Hyaluronan Turnover in the Synovial Fluid in Metacarpophalangeal–and Middle Carpal Joints in Standardbred Horses

The biological turnover of hyaluronan (sodium hyaluronate) of different molecular weights (0.6×106 and 2.5×106 Daltons) was studied in the synovial fluid of the middle carpal and metacarpophalangeal joints of 6 clinically healthy Standardbred horses. The hyaluronan was radioactively labelled with 14C. The biological half-life (t1/2) was calculated from repeated synovial samples after injection of the labelled hyaluronan. The mean t1/2 in the metacarpophalangeal joints was 9.7 h for low molecular weight hyaluronan and 8.9 h for high molecular weight hya-luronan and in the middle carpal joints 16.0 h and 12.5 h respectively. There was no sig-nificant difference in turnover of the different molecular weights of hyaluronan.