Thermally induced coil-to-helix transition of sodium gellan gum with different molar masses in aqueous salt solutions

Using 5 samples of well-purified Na-gellans (Na-gellans G1-G5, weight-average molar mass M(w) = 120 x 10(3)-32 x 10(3) at 40 degrees C), the effects of molar mass on the coil-to-double-helix transition in aqueous solutions with 25 mM NaCl were studied by light scattering and circular dichroism (CD) measurements, viscometry, and differential scanning calorimetry (DSC). From the temperature dependence of M(w), molar ellipticity at 201 nm [theta]201, intrinsic viscosity [eta], and DSC exothermic curves, it was found that the coil-to-double-helix transitions for G1-G5 samples took place at almost the same temperature. The [eta] and M(w) obtained in the temperature range from 40 to 25 degrees C can be explained by a simple coil/double-helix equilibrium model using the double-helix contents determined from CD data. The van't Hoff's transition enthalpy deltaH(vH) of Na-gellans depended on M(w). It is concluded that the coil-to-double-helix transitions of Na-gellans are all-or-none type transitions, and are accelerated with increasing M(w).     

The DNA melting transition in aqueous magnesium salt solutions.

The melting transition of the magnesium salt of DNA has been systematically examined in the presence of various types of anions. The addition of ClO4- to a concentration of 3.0 N results in the biphasic optical transition, with the first phase exhibiting rapid reversibility and independence of the DNA concentration. This subtransition, which is interpreted as an intramolecular condensation to a collapsed form of DNA, is followed by a DNA concentration-dependent aggregation reaction. The aggregation can be reversed by increasing the ClO4- concentration to 6.0 N while elevating the temperature to post-transition levels. Alternatively, both the collapse and the aggregation can be prevented by melting in the presence of trichloroacetate, the most strongly chaotropic solvent for DNA which has been reported (K. Hamaguchi and E. P. Geiduschek (1962), J. Am. Chem. Soc. 84, 1329). The forces responsible for mediating both the collapse and the aggregation are superficially similar to those involved in maintaining duplex stability. The collapsed form, in particular, possibly possesses features in common with the condensed structures which can be produced in aqueous solution of certain polymers, such as polyethylene glycol (Lerman, L.S. (1971), Proc. Natl. Acad. Sci. U.S.A. 68, 1886).     

E.s.r. study of the conformational transition of spin-labelled xanthan gum in aqueous solution

The order to disorder transition of xanthan molecules in aqueous solutions has been studied using e.s.r. spectroscopy. Nitroxide spin-label was covalently attached to carboxyl groups on the xanthan side chains. The e.s.r. spectra obtained for aqueous spin-labelled xanthan solutions at varying ionic strengths contained both isotropic and anisotropic components at room temperature. The anisotropic component was attributed to the association of the side chains with the xanthan cellulosic backbone and was found to be present in greater proportions at increasing ionic strength. The spectra gradually changed with rising temperature and the proportion of anisotropic component decreased. This spectral change reflected the disruption of the side chain association with the backbone during the conformational change. Hysteresis effects were observed following sequential heating and cooling cycles suggesting that chain aggregation occurred.     

Thermally induced conformational changes in horseradish peroxidase.

Detailed differential scanning calorimetry (DSC), steady-state tryptophan fluorescence and far-UV and visible CD studies, together with enzymatic assays, were carried out to monitor the thermal denaturation of horseradish peroxidase isoenzyme c (HRPc) at pH 3.0. The spectral parameters were complementary to the highly sensitive but integral method of DSC. Thus, changes in far-UV CD corresponded to changes in the overall secondary structure of the enzyme, while that in the Soret region, as well as changes in intrinsic tryptophan fluorescence emission, corresponded to changes in the tertiary structure of the enzyme. The results, supported by data about changes in enzymatic activity with temperature, show that thermally induced transitions for peroxidase are irreversible and strongly dependent upon the scan rate, suggesting that denaturation is under kinetic control. It is shown that the process of HRPc denaturation can be interpreted with sufficient accuracy in terms of the simple kinetic scheme N -->k D where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation; N is the native state, and D is the denatured state. On the basis of this model, the parameters of the Arrhenius equation were calculated.     

A dye-binding induced conformational transition of poly-(methacrylic acid) by Auramine O in aqueous solutions. I. Experimental results

Binding of auramine O to poly-(methacrylic acid) (PMA) has been established over a large range of C⁰_p/C⁰_d values using spectroscopic methods (UV absorption and visible fluorescence emission spectra), equilibrium and sedimentation dialysis, potentiometric and viscosimetric titrations. All the results show qualitative agreement with those obtained previously with the system crystal violet-PMA although the binding seems to be less strong for auramine O than for crystal violet. From dialysis experiments binding isotherms were obtained at three different degrees of neutralization α'; at α' = 0.10 the results could be fitted to a Langmuir isotherm but at α' equal to 0.40 and 0.65 deviations with respect to such an isotherm occur. The results of potentiometric and viscosimetric titrations confirm that the conformational transition which the dye-free PMA exhibits upon ionization is affected by the dye binding. The region in which the conformational transion occurs is broadened and is less sharply defined in the presence of auramine O.     

The anomaly of oxygen diffusion in aqueous xanthan solutions

A membrane-covered polarographic oxygen electrode was used to measure oxygen diffusion coefficients in aqueous polyelectrolyte solutions of xanthan gum, sodium alginate, and sodium carboxymethylcellulose (CMC). In sodium alginate solutions, dilute xanthan solutions, and solutions containing more than 0.3 wt % CMC, oxygen diffusion coefficients decrease with increasing polymer concentrations. Interestingly, in dilute CMC solutions and concentrate xanthan solutions containing more than 0.5 wt % xanthan gum, oxygen diffusion coefficients increase with increasing polymer concentrations, and values exceeding that in pure water are generally observed.     

A rheological study of the order-disorder conformational transition of xanthan gum

The rheological properties of a moderately concentrated solution of xanthan gum in both the ordered and the disordered state have been studied. Oscillatory shear, steady shear flow, and extensional flow experiments have been performed at different temperatures, covering the order-disorder transition determined by differential scanning calorimetry (DSC). The principle of time/temperature superposition was applied to the xanthan solutions for the different types of flow. Although a master curve covering six decades of frequency could be obtained for the storage modulus over the entire investigated temperature range, less agreement was found for the other modulus. This indicates that the order-disorder transition reflects changes on the molecular scale and slight modification of the physical network structure. To the authors' knowledge, this is the first time that this transition has been observed using these different rheological techniques.     

An acid induced conformational transition of denatured cytochrome c in urea and guanidine hydrochloride solutions.

Previous work has shown that at neutral pH ferricytochrome c (horse heart) retains certain residual structures in concentrated solutions of urea or guanidine hydrochloride (Tsong, T. Y. (1974), J. Biol. Chem. 249, 1988). Present studies reveal that cooperative unfolding of these residual structures can be achieved by acidification of the protein to pH 4 in 9 M urea but can only be partially achieved in a 6 M guanidine hydrochloride solution. The evidence that the residual structures unfold in 9 M urea upon acidification is twofold. (1) Further uncoupling of the Trp-59-heme interaction occurs; this is reflected in the intensification of the tryptophan fluorescence from 55 to 90 percent relative to that of free tryptophan in the same solvent. (2) The intrinsic viscosity of the protein solution increases from 15.0 to 21 ml/g. The acidification also induces a spin-state transformation of the heme group at pH 5 both in urea and in guanidine hydrochloride. Acidic titration of the protein in urea and guanidine hydrochloride indicates that the unfolding involves the absorption of a single proton. However, the kinetics of the spin-state transformation are triphasic. These results suggest that the displacement of the ligand His-18 by a solvent molecule and the subsequent disintegration of the residual structures are complex processes and involve at least three kinetic steps. The ineffectiveness of guanidine hydrochloride as a denaturant for ferricytochrome c is shown to be due to the presence of the high concentration of Cl minus which can stabilize certain elements of the protein structure.     

Concentration-dependent conformational transition of alpha-elastin in aqueous solution

Circular dichroism measurements on aqueous solutions of alpha-elastin have given evidence of beta-bend structure at elevated concentrations. When Ca2+ was added, the concentration-dependent conformational transition was somewhat inhibited and the binding of the metal ion was shown, by means of equilibrium dialysis, to be essentially independent from alpha-elastin concentration in the range of 1 to 10 mg/ml. The results obtained are discussed in connection with the elasticity and calcification of elastin.     

Conformation of xanthan dissolved in aqueous urea and sodium chloride solutions

Quasielastic light-scattering and other physical-chemical techniques have been used to compare the conformation and intermolecular interactions of xanthan in water, aqueous sodium chloride, and urea solutions. The results showed that xanthan dissolved in 4m urea has a disordered conformation after the solution has been maintained for 3 h at 95° and then cooled to room temperature. This conformation is similar to that previously observed only in solutions having low ionic strength at higher temperatures, following disruption of the ordered, low-temperature form. “Anomalous” behavior is seen for xanthan as a function of ionic strength, in that the hydrodynamic radius increases with increase in ionic strength, whereas a decrease is typical for polyelectrolytes. These observations suggest that aggregation of rod-like chains, similar to that seen for other stiff-chain polymers, occurs for xanthan in salt solutions, where the charged groups of the polyelectrolyte are screened by the salt ions. This aggregation may explain some of the high values reported in the literature for the molecular weight.     

Physicochemical properties of aqueous xanthan solutions: Static light scattering

The secondary structure of xanthan in solutions of relatively low salt concentration and at room temperature has been investigated using static light scattering experiments. Additional evidence has been found for a dimeric structure at 25°C in 0.01M NaCl. From the experimental z-average mean square (ms) radius of gyration, a value for the persistence length p has been estimated, taking explicitly into account the polydispersity of the three samples used, which has been established by gel permeation chromatography (GPC) measurements. The experimental particle scattering functions of the three samples are consistent with theoretical estimates for polydisperse systems with the same value of p = 65 ± 10 nm and the molar mass per unit length for a dimeric structure. This secondary structure remains unaffected by the ionic strength in the 0.005–0.0lM range. Partial aggregation seems to occur at higher NaCl concentrations. Light scattering and GPC data show that heating the xanthan 0.01M NaCl solutions to about 70°C considerably reduces the M_w of the low molar mass sample (2.3 × 10⁵-g·mol^?1), contrary to what is observed for the high molar mass sample (1.8 × 10⁶-g·mol^?1). These experimental findings can be accounted for by a partial temperature-induced dissociation of the xanthan dimers according to an all-or-none mechanism. © 1994 John Wiley & Sons, Inc.     

A dye-binding induced conformational transition of poly-(methacrylic acid) by Auramine O in aqueous solutions. II. Theoretical interpretation and discussion of the experimental results

The binding of Auramine O to poly-(methacrylic acid) (PMA) is explained using a two-state model for the polyelectrolyte and preferential binding of the dye to the hypercoiled conformational state of PMA predominantly present for the dye-free polyelectrolyte at low degrees of neutralization. Bound-dye interactions were neglected leading to a binding isotherm as given by Monod et al. to which the experimental dialysis results could be fitted. It is shown that this model predicts a conformational transition from the more extended conformational state of PMA to the hyper-coiled one upon progressive binding of the dye. The experimental results obtained by potentiometric and viscosimetric titrations as well as the fluorcsence intensity measurements of the AuO—PMA system arc consistent with the conclusions based on this model.     

Modulation of the bilayer to hexagonal phase transition and solvation of phosphatidylethanolamines in aqueous salt solutions

Several salts affect the temperature of the bilayer to hexagonal phase transition of phosphatidylethanolamines. Their effects are dependent on the anion as well as the cation of the salt. Salt effects on this transition can be explained by preferential hydration and ion binding. Those salts which are excluded from the solvation sphere of the membrane promote hexagonal phase formation. For example, Na2SO4 promotes preferential hydration and is a hexagonal phase promoter while NaSCN does not do this and is a bilayer stabilizer. Unlike amphiphiles and hydrocarbons, salts can shift the bilayer to hexagonal phase transition temperature without altering the cooperativity of the transition. The effect of these salts on the gel to liquid-crystal transition is opposite to their effect on the bilayer to hexagonal phase transition. We also find that MnCl2 markedly raises the gel to liquid-crystal transition temperature. This effect is due to binding of the cation to the membrane surface. The effect is reduced with MnSO4 because of preferential hydration. Our results demonstrate that the nature of the anion as well as the cation can alter the effect of salts on lipid phase transition properties. The observed effects can be explained as resulting from preferential hydration and ion binding.     

Structural characterization of the micelle-vesicle transition in lecithin-bile salt solutions.

Small-angle neutron scattering (SANS) and dynamic light scattering (QLS) are used to characterize the aggregates found upon dilution of mixed lecithin-bile salt micelles. Molar ratios of lecithin (L) to taurocholate (TC) studied varied from 0.1 to 1, and one series contained cholesterol (Ch). Mixed aggregates of L and taurodeoxycholate (TDC) at ratios of 0.4 and 1 were also examined. In all cases the micelles are cylindrical or globular and elongate upon dilution. The radius of the mixed micelles varies only slightly with the overall composition of lecithin and bile salt which indicates that the composition of the cylindrical micelle body is nearly constant. The transition from micelles to vesicles is a smooth transformation involving a region where micelles and vesicles coexist. SANS measurements are more sensitive to the presence of two aggregate populations than QLS. Beyond the coexistence region the vesicle size and degree of polydispersity decrease with dilution. Incorporation of a small amount of cholesterol in the lipid mixture does not affect the sequence of observed aggregate structures.     

Conformation and thermostability of double-stranded nucleic acids in aqueous urea solutions

The conformation and thermostability of DNA and double-helical synthetic RNA in aqueous solutions with 0-10 M urea have been investigated. A weak dependence of DNA conformation, realized in the presence of urea, on the GC-content has been found. The increase of urea concentration leads to destabilization of DNA and synthetic RNA. The character of changes in the spectra of RNA circular dichroism at the increase of urea concentration testifies that a conformational transition (different from A----A' transition) takes place. Urea stimulates the B----Z transition in poly(dG-dC).poly(dG-dC) molecules upon NaCl addition.     

Thermally induced conformation change of succinoglycan in aqueous sodium chloride

Static and dynamic light scattering, viscosity, and optical rotation measurements have been made at eight different temperatures between 25 and 75 degrees C on two succinoglycan samples (sodium salt) with weight-average molecular weights M(w) of 7.14 x 10(5) and 3.54 x 10(5) (at 25 degrees C) in 0.01 M aqueous NaCl to investigate the thermally induced order-disorder conformation change of the polysaccharide. Additionally, viscometry and polarimetry have been performed for a sodium salt sample (M(w) = 4.55 x 10(5) at 25 degrees C) whose M(w), z-average radius of gyration (z)(1/2), and hydrodynamic radius R(H) in the aqueous salt had been determined previously. As the temperature increases, M(w), (z)(1/2), R(H), and the intrinsic viscosity for every sample sharply decrease around 55 degrees C where the specific rotation at 300 nm sigmoidally increases. In particular, M(w) at 25 degrees C (i.e., in the ordered helical state) is twice as large as that at 75 degrees C (i.e., in the disordered state). These findings substantiate that the ordered structure is composed of two chains and hence is a double helix. Data analysis shows that this helix at 25 degrees C is characterized by an unperturbed wormlike chain with a helix pitch of about 2 nm (per repeating unit) and a persistence length of about 50 nm and that upon heating, it dissociates directly (i.e., in all-or-none fashion) to disordered chains of a similar contour length but with a much smaller persistence length of about 10 nm. The temperature dependence of the light scattering second viral coefficient is discussed in relation to the association of disordered chains in the cooling process.     

Sol-gel transition temperature of PLGA-g-PEG aqueous solutions

Aqueous solutions of poly(DL-lactic acid-co-glycolic acid)-g-poly(ethylene glycol) copolymers exhibited sol-to-gel transition with increasing temperature. Further increase in temperature makes the system flow and form a sol phase again. Subcutaneous injection of a copolymer aqueous solution (0.5 mL) resulted in a formation of a hydrogel depot by temperature-sensitive sol-to-gel transition in a rat model. The reliable determination and control of sol-to-gel transition temperatures are the most important issues for this kind of sol-gel reversible hydrogel. The sol-to-gel transition temperature determined by the test tube inverting method, falling ball method, and dynamic mechanical analysis coincided within 1-2 degrees C. Fine tuning of the sol-to-gel transition temperature was achieved by varying the ionic strength of the polymer solutions and by mixing two polymer aqueous solutions with different sol-to-gel transition temperatures. The sol-to-gel transition temperature of polymer mixture aqueous solutions was well described by an empirical equation of miscible blends, indicating miscibility of the two polymer systems in water on the molecular level.     

Fluorescence of proteins in aqueous neutral salt solutions. I. Influence of anions

The effects of anions of neutral salts on the fluorescence emission of six proteins as well as on tryptophan and tyrosine were studied in relation to the structure of proteins. Most anions are good quenchers of tryptophyl and tyrosyl fluorescence, free or in proteins. The results with tryptophan and tyrosine indicate involvement of a collisional quenching mechanism due to agreement with Stern–Volmer law. The deactivation of fluorescence probably occurs because of the transition from singlet state to triplet state. Lehrer's modification of Stern–Volmer law was applied to proteins. The effective quenching constants ([K_Q]eff) and the fraction of fluorescence available ([f_a]eff) to the quencher are also calculated. In contrast to its effect on tryptophan, CH₃COO^? quenches tyrosyl fluorescence and ClO₄^? does not. The effects on fluorescence of ribonuclease and free tyrosine are similar and without any changes in emission maximum. The anions are divided into three groups based on the effect they have on tryptophan-containing proteins. (1) NO₃^?, NO₂^?, Br^?, and I^? have high [K_Q]eff values and readily quench tryptophyl fluorescence of proteins causing a shift of emission maximum to a shorter wavelength. This change is due to the specific quenching of “exposed” tryptophan residues which are accessible to quenchers and the observed residual fluorescence is from the “buried” tryptophyls. (2) ClO₄^? and SCN^? also quench fluorescence of tryptophan in proteins and have lower ([K_Q]eff) values. In their presence the fluorescence maximum is shifted to a longer wavelength, which indicates the unfolding of a protein with [(f_a)eff] = 1. (3) Cl^?, CH₃COO^?, and SO₄? do not have a direct effect on the fluorescence of tryptophan. Besides the “direct” effects, “indirect” effects on fluorophors in protein are also seen, pointing out that the neutral salts can interact in more than one manner with proteins. The effectiveness of anions in quenching fluorescence of proteins follows similar sequences which almost resemble the Hofmeister series, viz., SO₄⁼, CH₃COO^? ? Cl^? < ClO₄^? < SCN^? < Br^? < I^? < NO₃^? < NO₂^?.