Dielectric changes in hyaluronate solutions at microwave frequencies as a function of concentration,system pH,and temperature

The dielectric response of human umbilical cord hyaluronic acid in various environments has been studied at microwave frquencies using a resonant microwave cavity as a probe. Both the real and imaginary parts of complex dielectric constant and the loss tangent for hyaluronate solutions are obtained by utilizing equations for perturbation of a resonant cavity. Dielectric changes at room temperature have been observed in aqueous solutions of hyaluronic acid as a function of concentration ranging from 0 to 350 mg/ml. The data indicate the existence of ordered phases in hyaluronate solutions at selective concentrations, that is, exhibiting lyotropic-type transitions. Hyaluronate solutions at 1.5 and 3 mg/ml concentrations have been studied at various pH in the range of 6–8 and at constant ionic strength 0.1. A temperature-dependent transition in hyaluronate solution of 120 mg/ml concentration has been observed at physiological temperature. It is shown that this temperature-dependent behavior can be related to the orientational polarizability term in the Debye theory of polar molecules in liquids.     

Changes in the refractive index ellipsoid isotropies,symmetric anisotropies,and depolarization ratios of potassium hyaluronate solutions as a function of pH

A new method is presented for studying the tertiary and possibly the secondary structure of a biopolymer, which is applied to the pH-dependent structural transition of potassium hyaluronate solutions. The method involves a new application of Dirac dispersion theory polarization dependence measures to isotropic and anisotropic components of the refractive index ellipsoid. Changes in the dipole moment may also be observed by this method. The pH-induced changes in viscosity and optical properties of hyaluronic acid previously reported appear to be dependent on changes in secondary and tertiary structure.     

The effects of pH on hyaluronate as observed by light scattering

Hyaluronate was investigated over a wide pH range, and at near zero and intermediate ionic strength, using dynamic and total intensity light scattering. Commercially obtained rooster comb hyaluronate was purified, and solutions were prepared in pure water by low-power bath ultrasonication and subsequent filtering. These solutions were of low polydispersity and appeared to contain single molecules of hyaluronate. Despite the absence of added electrolyte, these solutions yielded well-behaved Zimm plots. Increasing ionic strength and changing pH decreased radii of gyration and increased diffusion constants. Except for what appeared to be slow hydrolysis at either extreme of pH, molecular weights remained constant under all pH and ionic strength conditions. Under all solvent conditions investigated, diffusion coefficients increased with decreasing hyaluronate concentration. Unsonicated, lightly centrifuged solutions without added electrolyte were polydisperse, and their light scattering intensity was dominated by what appeared to be stable hyaluronate aggregates. The results are interpreted in terms of the polyelectrolyte properties of hyaluronate and its tendency to form stable entanglements, especially at low ionic strength. Previous light scattering studies in the literature on hyaluronate have shown widely varying results. The present article briefly reviews this literature and attempts to explain the variation among the previous results, emphasizing the Kuhn statistical segment length as an indicator of whether results are influenced by polydispersity or contaminants causing hyaluronate aggregation.     

Metabolic regulation of carbon and electron flow as a function of pH during growth of Sarcina ventriculi

The physiology and biochemistry of Sarcina ventriculi was studied in order to determine adaptations made by the organism to changes in environmental pH. The organism altered carbon and electron flow from acetate, formate and ethanol production at neutral pH, to predominantly ethanol production at pH 3.0. Increased levels of pyruvate dehydrogenase (relative to pyruvate decarboxylase) and acetaldehyde dehydrogenase occurred when the organism was grown at neutral pH, indicating the predominance of carbon flux through the oxidative branch of the pathway for pyruvate metabolism. When the organism was grown at acid pH, there was a significant increase in pyruvate decarboxylase levels and a decrease in acetaldehyde dehydrogenase, causing flux through the non-oxidative branch of the pathway. CO₂ reductase and formate dehydrogenase were not regulated as a function of growth pH. Pyruvate dehydrogenase possessed Michaelis-Menten kinetics for pyruvate with an apparent K _m of 2.5 mM, whereas pyruvate decarboxylase exhibited sigmoidal kinetics, with a S_0.5 of 12.0 mM. Differences in total protein banding patterns from cells grown at pH extremes suggested that synthesis of pyruvate decarboxylase and other enzymes was in part responsible for metabolic regulation of the fermentation products formed.     

Vacuum-ultraviolet circular dichroism of sodium hyaluronate oligosaccharides and polymer segments

The CD spectrum of an enzymatically derived sodium hyaluronate (NaHA) segment preparation with chain length 18 ± 3 disaccharide units [NaHAseg, ( NaGlcUA GlcNAc)_15–20°. NaGlcUA, sodium D -glucuronate; GlcNAc, 2-acetamido-2-deoxy-D -glucose] in H₂O was recorded to 180 nm using a computer-controlled vacuum-uv CD instrument. Near 190 nm the spectrum is of low intensity, similar to the sum of the free monosaccharide contributios, attributed to the π–π* transitions of the acetamido and carboxylate substituents. In contrast, much smaller oligosaccharides, also derived from high-molecular-weight NaHA by enzymatic digestions, show CD spectra in H₂O with prominent bands centered near 190 nm. The oligosaccharide spectra can be matched as linear combinations of interior sugar residue (? NaHAseg) and end sugar residue CD contributions. End residues from oligosaccharides of the type (NaGlcUA-GlcNAc)_n show a negative CD band near 190 nm. End residues from oligosaccharides of the reverse sequence (GlcNAc-NaGlcUA)_n show a positive CD band near 190 nm. Averaging of the two end-residue spectral contributions yields an approximate match for the spectrum of NAHAseg below 200 nm. It is proposed that the low intensity CD of NaHA in the π–π* region is the result of large-magnitude, oppositely signed contributions, which can be visulized by studying oligosaccharides.     

Circular dichroism of polynucleotides: dimers as a function of conformation

Working within the restrictions of a model, we have calculated the circular dichroism of the dinucleoside phosphates ApA, CpC, and CpA for various conformations. Comparing the calculated curves with those measured in aqueous solution we find agreement for (1) ApA as a right-handed helix with both bases either as in B-form DNA, or else rotated 180° around the glycosidic bond, (2) CpC as the right-handed conformation with both bases as in DNA, (3) ApC as either the right-handed conformation with both bases as in DNA, or else as a left-handed helix with both bases rotated 180°, and (4) CpA as either a left-handed helix with both bases in a left-handed DNA, or else in the right-handed conformation with both bases rotated 180°. In addition, we have investigated circular dichroism as a measure of unstacking. We find that opening the bases to a 90° total angle (base planes perpendicular) reduces the intensity of the calculated bands to 20% of their original value. Further, we find that allowing the sliding of one base past the other does not lead to a temperature dependence consistent with experiment.     

Flow dichroism of T7 DNA as a function of salt concentration

Measurements have been made on the flow dichroism of T7 DNA as a function of NaCl concentration. In high salt, our results are compatible with an optical factor of ?1.4 and a persistence length of 470 Å. The former is in agreement with expectations from the x-ray diffraction structure of fibrous B–DNA, and the latter is in the midrange of recent determinations. As salt concentration is decreased, the persistence length increases. The relation of our study to other recent investigations and with current theories of the electrostatic contribution to persistence length is discussed. We note that the separation of the electrostatic expansion factor into long- and short-range effects is somewhat arbitrary and might affect the interpretation of different experimental results in different ways. Finally, our hydrodynamic factors are consistent with a chain which is partially free-draining. This goes against the traditional interpretation but is in agreement with two recent observations.     

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.     

Flow birefringence of xanthan and other polysaccharide solutions

A xanthan sample with molecular weight M = 2.2 x 10(6) was investigated in three solvents: bidistilled water, 0.2 M aqueous NaCl and cadoxen by flow birefringence and viscometry methods in dilute solutions. It was shown that the optical shear rate coefficients of xanthan in aqueous and cadoxen media differ by two orders of magnitude. An estimation of xanthan optical anisotropy in different conformational states has been made and compared with values for other polysaccharides: dextran, pullulan, cellulose and chitosan. The process of denaturation and the flow birefringence of renaturated xanthan in aqueous solutions (after heat treatment at 121 degrees C) have also been studied.     

Electric birefringence and dichroism of acridine orange and methylene blue complexes with polynucleotides

Electro-optic measurements are reported for the polynucleotides poly A, poly U, poly G, and NaDNA, and their complexes with acridine orange (AO). Measurements were also made on the methylene blue (MB) complex with NaDNA. Poly U, poly G, and NaDNA complexes with AO as well as the NaDNA–MB complex were found to exhibit positive birefringence and perpendicular dichroism indicating the dye molecules are oriented with their long axes perpendicular to the applied electric field. The opposite was found for the AO complex with poly A, which showed positive birefringence and parallel dichroism, indicating that in this case the dye molecules are oriented with their long axes parallel to the field.     

Noncovalent intercalative complex formation and kinetic flow linear dichroism of racemic syn- and anti-benzo[a]pyrenediol epoxide-DNA solutions

In order to gain a better understanding of the molecular basis underlying the differences in biological activities of the diastereomeric syn and anti diol epoxides of benzo[a]pyrene (trans-7,8-dihydroxy-syn-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, respectively), their interactions with DNA in aqueous solutions were studied and compared. Kinetic flow linear dichroism experiments indicate that both diastereomers (racemic mixtures) form intercalation complexes immediately after mixing; the association constant (23 degrees C, ionic strength approximately 0.005) is significantly smaller (5200 M-1) in the case of the syn than in the case of the anti diastereomer (12 200 M-1). This difference is attributed to the greater bulkiness of the 7,8,9,10 ring of the syn stereoisomer, which is in the quasi-diaxial conformation as compared to the less bulky quasi-diequatorial conformation of the anti diastereomer. In contrast, the intercalative association constants of the tetraols derived from the hydrolysis of the two diol epoxides are similar in value. Upon formation of noncovalent syn-BPDE-DNA intercalation complexes, the reaction rate constant for the formation of tetraols (approximately 98%) and covalent adducts (approximately 2%) increases from 0.009 to 0.05 s-1 at pH 9.5 in 5 mM tris(hydroxymethyl)aminomethane buffer. The conformations of the aromatic chromophores of BPDE were followed by the kinetic flow dichroism technique as a function of reaction time; while the anti diastereomer changes conformation from an intercalative to an apparently external binding site, the syn diol epoxide molecules do not appear to undergo any measurable reorientation during or after the covalent binding reaction.(ABSTRACT TRUNCATED AT 250 WORDS)