Swelling behavior of hyaluronic acid/polyallylamine hydrochloride multilayer films

The reversible swelling behavior of multilayer films containing hyaluronic acid and polyallylamine hydrochloride was investigated using in situ ellipsometry, since many of the natural functions and applied uses of hyaluronic acid are related to the extraordinary ability of this biopolymer to swell, and to respond conformationally to the local solution environment. This swelling was observed to be substantial, and depended strongly on the film thickness, the pH conditions used to prepare the films, and the swelling solution pH and ionic strength. The swelling results were also rationalized in terms of the dissociation behavior of the polyelectrolytes in the multilayer assemblies, measured by the zeta potential, on colloidal particles. The films were found to swell by as much as 8 times their dry thickness, and the extent of film hydration was observed to depend on the thickness of the films in a nonlinear fashion. This was related to the internal structure of the films, which is dictated by the assembly pH conditions. In addition, the swelling solution pH and ionic strength influence the electrostatic environment in the films and, in turn, have a substantial effect on the overall swelling behavior.     

Interfacial behaviour of bovine testis hyaluronidase

The interfacial properties of bovine testicular hyaluronidase were investigated by demonstrating the association of hyaluronidase activity with membranes prepared from bovine testis. Protein adsorption to the air/water interface was investigated using surface pressure-area isotherms. In whichever way the interfacial films were obtained (protein injection or deposition), the hyaluronidase exhibited a significant affinity for the air/water interface. The isotherm obtained 180 min after protein injection into a pH 5.3 subphase was similar to the isotherm obtained after spreading the same amount of protein onto the same subphase, indicating that bovine testicular hyaluronidase molecules adopted a similar arrangement and/or conformation at the interface. Increasing the subphase pH from 5.3 to 8 resulted in changes of the protein isotherms. These modifications, which could correspond to the small pH-induced conformational changes observed by Fourier-transform IR spectroscopy, were discussed in relation to the pH influence on the hyaluronidase activity. Adding hyaluronic acid, the enzyme substrate, to the subphase tested the stability of the interfacial properties of hyaluronidase. The presence of hyaluronic acid in the subphase did not modify the protein adsorption and allowed substrate binding to a preformed film of hyaluronidase at pH 5.3, the optimal pH for the enzyme activity. Such effects of hyaluronic acid were not observed when the subphase was constituted of pure water, a medium where the enzyme activity was negligible. These influences of hyaluronic acid were discussed in relation to the modelled structure of bovine testis hyaluronidase where a hydrophobic region was proposed to be opposite of the catalytic site.     

pH-responsive properties of multilayered poly(L-lysine)/hyaluronic acid surfaces

Multilayer films have been prepared by the sequential electrostatic adsorption of poly(L-lysine) and hyaluronic acid onto charged silicon surfaces from dilute aqueous solutions under various pH conditions. Microelectrophoresis was used to examine the local acid-base equilibria of the polyelectrolytes within the films as a function of the total number of layers in the film and the assembly solution pH. The acid-base dissociation constants were observed to deviate significantly from dilute solution values upon adsorption, to be layer dependent only within the first 3-4 layers, and to show sensitivity to the assembly solution pH. As a result, some of the physicochemical properties of the films have also been found to exhibit pH-responsive behavior. For example, the thickest films result when at least one of the polyelectrolytes is only partially dissociated in solution. As well, the pH-dependent degree of dissociation of the surface functional groups can be used to vary the contact angle of a water droplet by as much as 25 degrees and the coefficient of friction by up to an order of magnitude. In addition, the extent to which PLL/HA films can be made to swell in solution can be varied by a factor of 7 depending on the assembly solution and swelling solution pH. The anomalies found in the dissociation constants account for the trends in these pH-dependent properties. Here, we demonstrate that knowledge of the acid-base dissociation behavior in multilayer films is key to understanding and controlling the physical properties of the films, particularly surface friction and wettability, which are fundamentally important factors for many biomaterials applications. For example, we outline a mechanism whereby biopolymer thin films can be electrostatically adsorbed under highly charged "sticky" conditions and then quickly transformed into stable low-friction films simply by altering the pH environment.     

Optical properties and viscosity of hyaluronic acid in mixed solvents: Evidence of conformational transition

Circular dichroism, optical rotatory dispersion, and viscosity of hyaluronic acid at various solvents compositions, concentrations, and pH values have been studied. The data show a large change in the molecular properties in organic/water solvents such as ethanol, p-dioxane, or acetonitrile/water at pH ? pK_a. At this pH range of aqueous solution, hyaluronic acid shows a CD minimum near 210 nm whereas in the presence of organic solvent it exhibits a strong negative dichroism (below 200 nm) and a positive band near 226 nm. It undergoes a sharp, cooperative transition with respect to pH and solvent. The observed CD features are assigned to the π-π* and n-π* transitions of the amide and carboxyl chromophores. The ORD results show a gradual blue shift of trough at 220 nm with increasing magnitude of rotation when the organic solvents and hydrogen ion concentrations are increased. A one-term Drude's equation was used to analyze the ORD data, and the result show a variation of dispersion parameters with different solvents in accordance with the observed CD changes. The intrinsic viscosity of hyaluronic acid in mixed solvent at pH 2.6 is lower than that of aqueous solution. All the observed property changes of hyaluronic acid are reversed on addition of foramide in mixed solvents indicating that the hydrogen bonds are involved in this transition. The observed spectroscopic and hydrodynamic features are attributed to a conformational change of hyaluronic acid in a mixed solvent involving intramolecular hydrogen bonding between the acetamido and carboxyl groups. The possible conformational state of hyaluronic acid in solution under various conditions is discussed in terms of the reported helical structure of hyaluronic acid from x-ray diffraction studies.     

The histochemical specificity of Streptomyces hyaluronidase and chondroitinase ABC.

Synopsis Treatment of tissue sections with enzymes which degrade specific types of glycosaminoglycans should provide a means for localizing glycosaminoglycans in tissue sections. The feasibility of this technique was examined by utilizing endogenously labelled glycosaminoglycans in chick and quail embryos. Less than 8% of the total glycosaminoglycans appear to be lost non-specifically during fixation and dehydration. BothStreptomyces hyaluronidase and chondroitinase ABC degraded more than 90% of their respective substrates and demonstrated minimal non-specific extraction of other glycosaminoglycans. The selectivity of chondroitinase ABC for sulphated glycosaminoglycans was substantially increased by raising the pH of the incubation buffer to 8.6. At this pH, chondroitinase ABC degraded negligible amounts of hyaluronic acid. Use of bothStreptomyces hyaluronidase and chondroitinase ABC confirmed that embryonic hyaluronic acid binds Alcian Blue under conditions that were previously believed specific for sulphated glycosaminoglycans. We suggest that this may be due to the increased molecular weight of embryonic hyaluronic acid compared to the hyaluronic acid in adult tissues. The results presented suggest that treatment of adjacent sections with buffer, chondroitinase ABC at pH 8.6, andStreptomyces hyaluronidase and subsequent staining with Alcian Blue provides a method for localizing and quantitating glycosaminoglycans in tissue sections.     

Biosynthesis of hyaluronic acid by Streptococcus.

Synthesis of hyaluronic acid was investigated in a cell-free system derived from a strain of Group A streptococci. Preparative procedures were improved so that an enzyme system 70 times more active than that previously reported was obtained. The hyaluronic acid synthesized could be separated into trichloroacetic acid-soluble and -insoluble fractions. On the basis of pulse-chase experiments, it was shown that the trichloroacetic acid-insoluble fraction is a precursor of the soluble fraction. The release of the trichloroacetic acid-insoluble hyaluronic acid is specifically blocked with p-chloromercuribenzoate, without inhibition of chain elongation. The addition of butanol to trichloroacetic acid resulted in solubilization of all of the hyaluronic acid. No detectable difference in molecular size was observed between the two hyaluronic acid fractions, both of which were estimated to be more than one million daltons in size. Testicular hyaluronidase digestion of either one of the two types of hyaluronic acid yielded no high molecular weight fragments, indicating that hyaluronic acid is not bound covalently to protein. However, following incubation of enzyme assay mixtures with UDP-[14C]GlcUA, even in the absence of UDP-GlcNAc, radioactive high molecular weight hyaluronic acid was obtained which suggests that the enzyme system elongates rather than initiates hyaluronic acid chains. Tunicamycin did not inhibit hyaluronic acid synthesis, indicating lack of participation of an intermediate of pyrophosphorylpolyisoprenol type. The results obtained are consistent with the hypothesis that chain elongation of hyaluronic acid proceeds by alternate addition of monosaccharides from UDP-sugars by a membrane-bound synthesizing system followed by release of completed hyaluronic acid chains.     

Possible requirement of collagen gel substratum for production of mucin-like glycoproteins by primary rabbit tracheal epithelial cells in culture

Summary Primary rabbit tracheal epithelial cells growing on either plastic surface or collagen gel produce high molecular weight glycoconjugates. Biochemical characterization of these materials show they are exclusively hyaluronic acid when cells are grown on plastic surface, but a mixture of hyaluronic acid and mucin-like glycoproteins when grown on collagen gel. This research suggests that the substratum plays an important role in the maintenance or differentiation or both of mucous cells in culture.     

Effects of Streptomyces hyaluronidase digestion on the histochemical reactions of proteoglycans in cartilage compared with its effects on certain non-cartilaginous tissues

Summary To test the value ofStreptomyces hyaluronidase in carbohydrate histochemistry, the effects of digestion with the enzyme on the staining of cartilage and non-cartilaginous tissues by Alcian Blue (AB) pH 1.0, AB pH 2.5, high iron diamine, low iron diamine, aldehyde fuchsin, dialysed iron-ferrocyanide and AB pH 2.5-periodic acid-Schiff were studied by light microscopy. The results obtained lead to the conclusion that theStreptomyces enzyme releases not only hyaluronic acid but also chondroitin sulphates and keratan sulphates in cartilage. Since hyaluronic acid is known to be linked to chondroitin sulphate proteoglycans, the enzyme is of limited value in localizing hyaluronic acid in cartilage. However, it is useful in localizing hyaluronic acid in most non-cartilaginous tissues.     

Laser light scattering measurements on vitreous and rooster comb hyaluronic acids

Molecular weights and translational diffusion coefficients have been measured for rooster comb and vitreous hyaluronic acid (HA) at pH 7.2 and 11. The results indicate that the molecular weight, second virial coefficient and translational diffusion coefficient for vitreous HA can be reversibly decreased by increasing the solution pH from 7.2 to 11, whereas the physical properties of rooster comb HA are independent of pH studied. In addition, it is reported that the second virial coefficient for vitreous HA is negative, suggesting intermolecular interactions exist in solution at both neutral and alkaline pH as opposed to rooster comb HA which exhibits a positive second vitrial coefficient associated with decreasing molecular weights may be related to the accessibility and number of hydrogen bond forming groups. Differences in the dependence of molecular weight on pH between vitreous and rooster comb HA may be due to differences in the number of intramolecular interactions per molecule. These studies indicate that molecules of low molecular weight HA are able to form higher molecular weight complexes and differences in the organization of the polysaccharide chains may contribute to the differences in molecular weight of HAs isolated from various tissues.     

Conformational transition of hyaluronic acid: carboxylic group participation and thermal effect

The chiroptical, viscosity and titration studies of hyaluronic acid in mixed organic/water solvent show a reversible conformational transition of the molecule depending upon pH, solvent composition, temperature, and molecular weight. Neither methylhyaluronate nor chondroitin undergoes conformational transition of this type. These results indicate that hydrogen bonding between the protonated carboxylic group and carbonyl oxygen of the acetamido group is directly involved in the conformational change. Results with chondroitin provide further support for the 4-fold helical structure that we have proposed for hyaluronic acid in mixed organic/water solvent. The thermal stability of the conformation has been studied under various pH values and solvent compositions.     

Snake venom hyaluronidase: An evidence for isoforms and extracellular matrix degradation

The present study attempts to establish the isoforms of hyaluronidase enzyme and their possible role in the spreading of toxins during envenomation. Screening of venoms of 15 snakes belonging to three different families revealed varied hyaluronidase activity in ELISA-like assay, but with relatively similar pH and temperature optima. The zymograms of individual venoms showed varied activity banding patterns and indicated the presence of at least two molecular forms of the enzyme. During envenomation, activity of hyaluronidase is considered crucial for the spreading of toxins and is presumed to distort the integrity of extracellular matrix through the degradation of hyaluronic acid in it. This property has been addressed through localization of hyaluronic acid in human skin and muscle tissue sections using the probe, biotinylated hyaluronic acid binding protein. Faint and discontinuous staining pattern of hyaluronidase treated tissue sections over intense staining of untreated tissue sections confirm the selective degradation of hyaluronic acid in extracellular matrix and thus provide an evidence for the spreading property of the enzyme.     

金黄色葡萄球菌透明质酸裂解酶HylS的异源表达与特性研究

透明质酸酶能够将透明质酸聚糖降解成具有抗氧化等生物活性的低分子量寡糖.微生物来源透明质酸酶具有酶学性质多样和易于重组表达等特点,是开发透明质酸酶的研究热点.通过基因组测序获得一个潜在的金黄色葡萄球菌来源透明质酸裂解酶基因hylS,将其进行了大肠杆菌BL21(DE3)异源重组表达,并对重组酶进行了酶学特性和酶解产物抗氧化...     

Rheology of hyaluronic acid

The dynamic viscoelastic properties of hyaluronic acid solutions have been measured over the frequency range 0.02–1.67 cps. The effects of varying temperature, hyaluronic acid concentration, pH, and ionic strength on the dynamic shear moduli were studied. The solutions exhibited a sharp transition from viscous to elastic behavior as the strain frequency increased. No entanglement coupling of the hyaluronic acid molecules was evident over the concentration range 2.0–4.0 mg./ml. Solutions at pH 2.5 showed a pronounced elastic behavior relative to both higher and lower pH's. This effect was attributed to a stiffening of the hyaluronic acid molecule at this pH.     

Hyaluronic acid in cartilage and proteoglycan aggregation

1. Dissociation of purified proteoglycan aggregates was shown to release an interacting component of buoyant density higher than that of the glycoprotein-link fraction of Hascall & Sajdera (1969). 2. This component, which produced an increase in hydrodynamic size of proteoglycans on gel chromatography, was isolated by ECTEOLA-cellulose ion-exchange chromatography and identified as hyaluronic acid. 3. The effect of pH of extraction showed that the proportion of proteoglycan aggregates isolated from cartilage was greatest at pH4.5. 4. The proportion of proteoglycans able to interact with hyaluronic acid decreased when extracted above or below pH4.5, whereas the amount of hyaluronic acid extracted appeared constant from pH3.0 to 8.5. 5. Sequential extraction of cartilage with 0.15m-NaCl at neutral pH followed by 4m-guanidinium chloride at pH4.5 was shown to yield predominantly non-aggregated and aggregated proteoglycans respectively. 6. Most of the hyaluronic acid in cartilage, representing about 0.7% of the total uronic acid, was associated with proteoglycan aggregates. 7. The non-aggregated proteoglycans were unable to interact with hyaluronic acid and were of smaller size, lower protein content and lower keratan sulphate content than the disaggregated proteoglycans. Together with differences in amino acid composition this suggested that each type of proteoglycan contained different protein cores.     

Light and electron microscopic studies on the localization of hyaluronic acid in developing rat cerebellum

The hyaluronic acid-binding region was prepared by trypsin digestion of chondroitin sulfate proteoglycan aggregate from the Swarm rat chondrosarcoma, and biotinylated in the presence of hyaluronic acid and link protein. After isolation by gel filtration and HPLC in 4 M guanidine HCl, the biotinylated hyaluronic acid-binding region was used, in conjunction with avidin-peroxidase, as a specific probe for the light and electron microscopic localization of hyaluronic acid in developing and mature rat cerebellum. At 1 w postnatal, there is strong staining of extracellular hyaluronic acid in the presumptive white matter, in the internal granule cell layer, and as a dense band at the base of the molecular layer, surrounding the parallel fibers. This staining moves progressively towards the pial surface during the second postnatal week, and extracellular staining remains predominant through postnatal week three. In adult brain, there is no significant extracellular staining of hyaluronic acid, which is most apparent in the granule cell cytoplasm, and intra-axonally in parallel fibers and some myelinated axons. The white matter is also unstained in adult brain, and no staining was seen in Purkinje cell bodies or dendrites at any age. The localization of hyaluronic acid and its developmental changes are very similar to that previously found in immunocytochemical studies of the chondroitin sulfate proteoglycan in nervous tissue (Aquino, D. A., R. U. Margolis, and R. K. Margolis. 1984. J. Cell Biol. 99:1117-1129; Aquino, D. A., R. U. Margolis, and R. K. Margolis. J. Cell Biol. 99:1130-1139), and to recent results from studies using monoclonal antibodies to the hyaluronic acid-binding region and link protein. The presence of brain hyaluronic acid in the form of aggregates with chondroitin sulfate proteoglycans would be consistent with their similar localizations and coordinate developmental changes.     

Streptococcal hyaluronic acid: proposed mechanisms of degradation and loss of synthesis during stationary phase.

Streptococcal hyaluronic acid was found to distribute into two discrete sizes. Cellular hyaluronic acid from strain D181 had an average molecular weight of 10 X 10(6), whereas the average molecular weight of extracellular hyaluronic acid from the same strain was 2 X 10(6). Cellular streptococcal hyaluronic acid was purified to homogeneity. Proteases were unable to cleave the purified cellular polymer, indicating that a peptide was not involved in cross-linking five extracellular hyaluronate polymers to form a cell-bound complex. Lipids apparently are not part of the cellular hyaluronic acid because phosphorus and glycerol were not detected by radioisotopic techniques, and denaturing conditions did not change the size of the polymer. Membranes obtained from various strains of group A and C streptococci cleaved the cellular form of the hyaluronate polymer demonstrating the presence of a membrane-bound hyaluronidase-like activity. By contrast, this activity was not found in the extracellular products of the strains studied. Furthermore, membranes derived from streptococci at the stationary phase of growth no longer had the capacity to synthesize hyaluronic acid. The loss of this property appeared to be due to changes in the structure of the membrane.     

A reversed-phase ion-pair high-performance liquid chromatography method for bovine testicular hyaluronidase digests using postcolumn derivatization with 2-cyanoacetamide and ultraviolet detection

A reversed-phase ion-pair HPLC method for separating hyaluronic acid oligomers, using a polymeric C18 column at alkaline pH, is described. As the concentration of the ion-pairing agent tetrabutylammonium hydroxide increased, over the range of 0.01 to 0.06M, the capacity factors (k') of tetra- to dodecasaccharide decreased. The change in k', for each increment in pairing agent, increased with oligomer molecular weight. When changing mobile phase pH from 7 to 8, k' dramatically decreased and remained unchanged from pH 8 to 11. The isocratic separation was optimized to resolve tetrato dodecasaccharide at pH 9.0 in under 19 min. The postcolumn derivatizing agent 2-cyanoacetamide reacted with the reducing N-acetylglucosamine end groups of hyaluronic acid oligomers to yield reaction products that were monitored at 27 nm. In a series of control experiments using decasaccharide and N-acetylglucosamine, it was found that maximum product formation took place at pH 9 and was greatly influenced by borate buffer concentration. The optimum concentration for 2-cyanoacetamide was 0.33% and a temperature of 100 degrees C gave the best signal to noise ratio for the postcolumn reaction. The method is linear and reproducible, and has a lower limit of detection for tetrasaccharide of 20 ng (25 pmol). This system is suitable for studying the degradation kinetics of purified hyaluronic acid oligomers by bovine testicular hyaluronidase. Extension of the method to fluorescent and electrochemical detection and its applicability to other glycosaminoglycans is discussed.     

Hyaluronic Acid in Synovial Fluid

Twelve horses with traumatic arthritis were treated with intraarticular injection of hyaluronic acid mixed with cortisone and the results compared with 6 horses treated only with cortisone. There was a significantly better improvement in the group injected with a mixture of hyaluronic acid and cortisone. Further studies have given the same results in traumatic arthritis in horses if hyaluronic acid alone is injected. After injection of hyaluronic acid a large number of granulated monocytes appeared in the synovial fluid, but no inflammatory signs were observed. It is possible that this macrophage invasion is instrumental in producing improvement in the condition of the joint. The injected hyaluronic acid may also adhere to the surface of articular cartilage producing an “clastic cushion” protecting the cartilage surface. Experimental mechanical damage was also inflicted on the surface of articular cartilage in dogs and monkeys, and smoother healing was achieved if hyaluronic acid was injected into the joints after the damage. Injections of hyaluronic acid seem to be of value in treating traumatic arthritis or similar conditions.     

Synthesis of 1-(2,6,6-Trimethyl-4-hydroxy-1-cyclohexen-1-yl)-1,3-butanedione

Streptococcus dysgalactiae IID 678, belonging to group C of the streptococci, secreted a large amount of hyaluronidase (hyaluronate lyase, EC 4.2.2.1) into a culture medium containing hyaluronic acid. The purification procedures of hyaluronidase were 70% ammonium sulfate precipitation, ECTEOLA-cellulose chromatography, phospho-cellulose chromatography, and gel filtration on Sephacryl S-300. The hyaluronidase was purified approximately 27,000-fold from the culture filtrate. The purified enzyme was homogeneous by SDS-poIyacrylamide gel electrophoresis. The enzyme degradated only hyaluronic acid and chondroitin to zl 4,5-unsaturated disaccharides and did not act on other glycosaminoglycans containing sulfate groups, while the degradation rate of chondroitin was about 1/60 of that of hyaluronic acid. The optimum pH was wide, from pH 5.8 to pH 6.6, and the optimum temperature was 37°C. Fe^{2 +}, Cu^{2 +}, Pb^{2 +}, and Hg^{2 +} ions inhibited the activity strongly and Zn²⁺ inhibited it by half. The molecular weight of the enzyme was estimated to be 125,000 by gel filtration and 117,000 by SDS-polyacrylamide gel electrophoresis. The enzyme was different immunochemically from the hyaluronidase from Streptococcus pyogenes belonging to group A.     

Kinetic studies on activation of peptide bond formation by hyaluronic acid.

1. In this study, a cell-free system derived from Escherichia coli has been used in order to examine in detail the effect of hyaluronic acid on peptide bond formation with the aid of puromycin reaction. 2. This reaction is activated by hyaluronic acid. 3. The degree of activation of peptide bond formation depends on the molecular size of hyaluronic acid. 4. The kinetic analysis revealed that the hyaluronic acid acts as a mixed-type nonessential activator. 5. The presence of hyaluronic acid improves about 9-fold the activity status of ternary complex as it can be calculated by k3/k5 ratio.