Quasi-elastic light scattering studies of hyaluronic acid solutions

Autocorrelation functions for the intensity of laser light scattered from solutions of hyaluronic acid have been measured under a variety of experimental conditions. The form of these functions is consistent with the large amount of long-range intra- and intermolecular interactions characteristic of polyelectrolyte solutions. Further experiments have been performed in order to study the effect of hyaluronic acid on the Brownian diffusion of polystyrene spheres. Using several different sphere sizes as solution probes, the importance of the ionic environment in determining hyaluronic confirmation has been demonstrated.     

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.     

Electro-optic scattering studies on deoxyribonucleic acid

Measurements have been made of the intensity of light scattered from aqueous solutions of calf thymus DNA with and without the application of electric fields. For fields approaching 150 V/cm and frequencies below 2.5 KHz, changes (ΔI) of up to 10% in the residual scattered intensity were observed. In agreement with previous dielectric and electric birefringence measurements, a low frequency dispersion of ΔI was observed, from which a rotary diffusion constant (D) of 1200 s^?1 was determined. Interpreting the electric field data in terms of the classical dipolar orientation theory led to values of 2.4 × 10^?25 cm (7.4 × 10^?14 esu) and 4.3 × 10^?25 cm (13 × 10^?14 esu) for the permanent dipole moment and the anisotropy of the electric polarisabilities respectively. Furthermore the permanent dipole moment was along the major molecular axis and the particles orientated in the field as rigid entities. The zero field data indicated a molecular shape which was not rodlike but corresponded to the Kratky-Porod “stiffness” parameter of x = 24 for the wormlike coil model. Although curved, the molecules appeared to orientate in low-intensity electric fields as rigid, but not rodlike molecules. The implications of this on recent discrepancies in D determined by two or more dynamic relaxation methods is briefly discussed.     

Light scattering studies of tetramethyl ammonium gellan

Tetramethyl ammonium (TMA) gellan does not gel. Light scattering studies suggest that in solutions of TMA gellan, in tetramethyl ammonium chloride (TMACI), the gellan molecules assemble end to end to produce elongated fibrous structures. Such fibrils are envisaged as resulting from double-helix formation between the ends of neighbouring gellan molecules. Fibrils with molecular weights ranging from (1.06 ± 0.06) × 10⁵ to (4.5 ± 0.1) × 10⁶ have been observed. The molecular weights obtained depended upon the pore size of the filters used to clarify the solutions. The formation of strong gels, in the presence of gel promoting cations, is attributed to a localized ordered lateral association, or crystallization of regions of these fibrils. It is suggested that such a model for gelation may be of general applicability to a number of polysaccharide systems.     

Light scattering studies of supercoiled and nicked DNA

Static and dynamic light scattering measurements were made of solutions of pGem1a plasmids (3730 base pairs) in the relaxed circular (nicked) and supercoiled forms. The static structure factor and the spectrum of decay modes in the autocorrelation function were examined in order to determine the salient differences between the behaviors of nicked DNA and supercoiled DNA. The concentrations studied are within the dilute regime, which is to say that the structure and dynamics of an isolated DNA molecule were probed. Static light scattering measurements yielded estimates for the molecular weight M, second virial coefficient A₂, and radius of gyration R_G. For the nicked DNA, M = (2.8 ± 0.4) × 10⁶g/mol, A₂ = (0.9 ± 0.2) × 10⁻³ mol cm³/g², and R_G = 90 ± 3 nm were obtained. For the supercoiled DNA, M = (2.5 ± 0.4) × 10⁶ g/mol, A₂ = (1.2 ± 0.2) × 10⁻³ mol cm³/g², and R_G = 82 ± 2.5 nm were obtained. The static structure factors for the nicked and supercoiled DNA were found to superpose when they were scaled by the radius of gyration. The intrinsic stiffness of DNA was evident in the static light scattering data. Homodyne intensity autocorrelation functions were collected for both DNAs at several angles, or scattering vectors. At the smallest scattering vectors the probe size was comparable to the longest intramolecular distance, while at the largest scattering vectors the probe size was smaller than the persistence length of the DNA. Values of the self-diffusion coefficients D were obtained from the low-angle data. For the nicked DNA, D = (2.9 ± 0.3) × 10⁻⁸ cm²/s, and for the supercoiled DNA, D = (4.11 ± 0.21) × 10⁻⁸ cm²/s. The contribution to the correlation function from the internal dynamics of the DNA was seen to result in a strictly bimodal decay function. The rates of the faster mode Γ_int, reached plateau values at low angles. For the nicked DNA, Γ_int = 2500 ± 500 s⁻¹, and for the supercoiled DNA, Γ_int = 5000 ± 500 s⁻¹. These rates correspond to the slowest internal relaxation modes of the DNAs. The dependence of the relaxation rates on scattering vector was monitored with the aid of cumulants analysis and compared with theoretical predictions for the semiflexible ring molecule. The internal mode rates and the dependence of the cumulants moments reflected the difference between the nicked DNA and the supercoiled DNA dynamical behavior. The supercoiled DNA behavior seen here indicates that conformational dynamics might play a larger role in DNA behavior than is suggested by the notion of a branched interwound structure. © 1996 John Wiley & Sons, Inc.     

Light scattering studies of bovine skin proteodermatan sulfate

The proteodermatan sulfate (PDS) of bovine skin is a low molecular weight proteoglycan with a molecular structure consisting of a protein chain and a sulfated polysaccharide chain covalently linked at the 4-serine of the protein. Static and dynamic laser light scattering methods have been used to determine the weight-average molecular weight, Mw, zeta-average radius of gyration, Rg zeta, and zeta-average translational diffusion coefficient, Dto, zeta, of bovine skin PDS. We have also characterized the two components of PDS, i.e., the protein core and the dermatan sulfate (DS) chain. (The latter contained an N-terminal-linked penta- or tetrapeptide.) Interpretation of the PDS data is complicated by the block copolymer nature of its structure. When appropriate corrections are made, our results indicate that Mw for PDS monomer is 62,000 when dissolved in 4M guanidine hydrochloride (GdnHCl), and increases to 610,000 in 0.15M NaCl. Mw for the core protein in 4M GdnHCl is 39,000, and this also increases substantially to 650,000 in 0.15M NaCl. In contrast, Mw for the DS chain is 24,000 in 0.15M NaCl, indicating that there is minimal self-association of DS in 0.15M NaCl. Thus we conclude that the self-association of PDS involves the protein core. Comparison of Rg zeta and Rh, the average hydrodynamic radius, suggests that trace amounts of aggregation persist for the PDS and its core protein even in 4M GdnHCl. This conclusion is supported by evaluation of the second moments of the dynamic light scattering correlation function. Comparisons of the observed Dto, zeta for PDS with predicted values using hydrodynamic theory are consistent with a "lollipop" conformation for the molecule.     

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.     

The hydrodynamic resistance of hyaluronic acid: estimates from sedimentation studies

The problem of estimating the permeability of hyaluronic acid as a function of concentration has been examined. A previously known method of obtaining permeability from sedimentation studies has been employed, and the results have been compared to those obtained from convection studies, in which solvent is forced through the hyaluronic acid. The two sets of results were seen to be at variance, which could be explained by a flow-induced polarization of the hyaluronic acid in the convection studies. The polarization phenomenon was described in terms of a convection-associated compaction balancing the hyaluronic acid's intrinsic resistance to compression. Based on these results and other arguments, it was suggested that data from sedimentation studies provide a more accurate estimate of hyaluronic acid permeability than do convection experiments. The implications of this finding in a physiological context were briefly discussed.     

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.