Solution properties of an exopolysaccharide from a Pseudomonas strain obtained using glycerol as sole carbon source

We report the solution properties of a new exopolysaccharide (EPS) obtained from a Pseudomonas strain fed with glycerol as the sole source of carbon. This high molecular mass (3 × 10⁶ g mol⁻¹) biopolymer is essentially made of galactose monomers with pyruvate and succinate groups imparting a polyelectrolyte character. The Smidsrod parameter B computed from the ionic strength dependence of the intrinsic viscosity indicates that the EPS backbone is rather flexible. In salt free aqueous solutions, the zero shear viscosity scaling with concentration follows a typical polyelectrolyte behavior in bad solvent, whereas at high ionic strength the rheological response is reminiscent from neutral polymers. Light scattering data indicate that the EPS adopts a globular conformation as a result of hydrophobic interactions. EPS solutions are stable within 4 days as particle sizing does not indicate EPS aggregation. Both globular conformation and stability against precipitation from solution are attributed to the low charge density of the polyelectrolyte.     

Solution properties of sodium alginate

Sodium alginate fractions derived from three different sources—Laminaria hyperboria (75% guluronate), Fucus vesicularus (95% mannuronate), and Azotobacter vinelandii (85% mannuronate)—were investigated in aqueous solution over a wide range of ionic strength and pH using the techniques of light scattering, viscometry, and osmometry. Light-scattering data extrapolated to infinite ionic strength yielded b₀ = 4.7 ± 0.3 and 3.0 ± 0.2 nm for the unperturbed effective bond lengths of the guluronate- and mannuronate-rich samples, respectively. These values are in the same ratio as predicted by conformational analysis, although lower by a factor of 0.7, probably due, in part at least, to the fact that measurements cannot be made on pure homopolymers. A comparison of the light-scattering and the viscosity data indicated that Φ in the Flory-Fox equation is lower than for more flexible polymers and increases with molecular weight, probably due to decreasing hydrodynamic permeability. Mark-Houwink exponents obtained from data extrapolated to infinite ionic strength were found to be considerably greater than 0.5, and we attribute this entirely to a variation in Φ. Comparison of the results obtained for the two mannuronate-rich samples indicated that the value of Φ and its variation with molecular weight can, in the case of alginates, differ markedly for chains, which, although having chemical differences, have similar chain statistics.     

Radii of gyration of sodium carboxymethylcellulose in aqueous and mixed solvent media from viscosity measurement

The viscosities of three sodium carboxymethylcellulose samples with molecular weights of 90,000 [degree of substitution (DS): 0.7], 250,000 (DS: 0.9), and 700,000 (DS: 0.9) have been reported in water and methanol–water mixtures in salt-free and salt-containing solutions at 35 °C. The results were analyzed in terms of a phenomenological approach for the viscosity of polymer solutions to determine the intrinsic viscosities [η] of the polyelectrolyte samples. This contribution presents a new and convenient method for the determination of the root-mean-square radii of gyration of the polyion chains using the [η] values obtained as a function of the added salt concentration. The polyion coils are found to expand at low ionic strength and these collapse drastically with increasing ionic strength. Addition of methanol to the medium in which these samples are dissolved causes a contraction of the polyion chains, although this influence is less pronounced than that of the added salt.     

Viscosity study of DNA. II. The effect of simple salt concentration on the viscosity of high molecular weight DNA and application of viscometry to the study of DNA isolated from T4 and T5 bacteriophage mutants

Polyelectrolyte expansion effects on high molecular weight bacteriophage DNA have been studied by examining the influence of simple salt concentration upon the intrinsic viscosity, [η]. The viscosity–molecular weight exponent a in the expression [η] = KM^a diminishes from 0.8 in 0.005M simple salt to a limiting value of 0.6 for salt concentrations greater than 0.6M at 25°C. The ε parameter of the N^1+ε hydrodynamic representation thus varies from approximately 0.2–0.07 over this range of salt concentration. The intrinsic, viscosity of DNA decreases slightly with increasing temperature at low and moderate salt concentrations but becomes independent of temperature at high salt concentrations. The expansion of the DNA molecular domain is linear in the reciprocal square root of the simple salt concentration. Viscosity differences among DNA's isolated from several bacteriophage T5 mutants reflect small differences in molecular weight which are in agreement, with sine determination by other techniques. The DNA's isolated from various rII mutants of T4 bacteriophage including some very large deletion mutations were found to be identically the same size in accord with current genetic ideas. Details of the representation and extrapolation of viscosity data are discussed and the sensitivity of the technique is evaluated.     

Interaction of aminoacridines with deoxyribonucleic acid: viscosity of the complexes

The intrinsic, viscosities at zero shear rate of defined complexes of proflavine, 9-aminoacridine, and 9-amino-l,2,3,4-tetrahydroaeridine with calf thymus DNA have been determined at, various ionic strengths by means of rotating cylinder viscometers. By controlled adjustment, of the composition of the mixtures, the amount of bound acridine (r moles/g.-atom DNA phosphorus) was maintained constant at different dilutions. The intrinsic viscosities of the complexes increased with r up to r values (ca. 0.16–0.20) corresponding to the end of the process of strong binding of the acridinium cations. However, complex formation between the acridines and thermally denatured DNA caused either a marked decrease in viscosity (at the low ionic strengths of 0.0015 and 0.005) or no change at all (ionic strength 0.1). These results are discussed in the light of presently available hydrodynamic theories relating the intrinsic, viscosity of DNA to its molecular extension.     

Polyelectroyte behavior of a bacterial polysaccharide from Xanthomonas campestris: Comparison with carboxymethylcellulose

The main physicochemical properties of the polysaccharide called Xanthan produced by Xanthomonas compestris are discussed: the activity coefficient of the counter-ion, the pK(α), and the ionic selectivity are investigated and compared to those of a carboxymetholcellulose. The weight-average molecular weight (M _w = 2 × 10⁶), the intrinsic viscosity and the constant of sedimentation are determined as a function of the ionic strength. It is proved that in dilute solution, there is no intermolecular association, whatever the ionic strength. The conformation is proposed to be a rigid rodlike molecule whose length is 6000 Å, independent of ionic strength > 10^?2N.     

The LiCl effect on the conformation of lentinan in DMSO

Lentinan (β‐(1→3)‐D ‐glucan) was found to be successfully fractionated by the mixture of dimethyl sulfoxide (DMSO) and lithium chloride (LiCl) as a solvent and acetone as a precipitant. Light scattering and viscosity measurements were made on solutions of fractionated samples in pure DMSO and 0.2M LiCl/DMSO in the range of the molecular weight M_w from 21.7 × 10⁴ to 84.7 × 10⁴. The values of M_w in both solvents were almost the same, but the remarkable difference between the values of intrinsic viscosity [η] demonstrated that the LiCl/DMSO solvent greatly enhances the stiffness of the lentinan backbone. The observed intrinsic viscosity [η] was analyzed by the Yoshizaki‐Nitta‐Yamakawa theory of a worm‐like chain, and the persistence length q and molecular weight per unit contour length M_L were determined roughly as 6.0 nm and 890 g nm^?1 in 0.2M LiCl/DMSO, and 5.1 nm and 890 g nm^?1 in pure DMSO, respectively. This slightly larger persistent length in 0.2M LiCl/DMSO also confirmed the higher stiffness of lentinan enhanced by the LiCl/DMSO solvent. The enhancement of the chain stiffness was ascribed to the electrostatic repulsion because of the hydrogen bonding of the hydroxyl protons of lentinan with the chloride ion, which is in turn associated with the Li⁺(DMSO)_n macrocation complex. © 2012 Wiley Periodicals, Inc. Biopolymers 97: 840–845, 2012.     

Viscosity and sedimentation study of sonicated DNA–proflavine complexes

The intrinsic viscosity of sonicated calf thymus DNA (molecular weight 4–5 × 10⁵) increases and the sedimentation constant decreases, with increasing binding of proflavine at 0. 2 ionic strength and at 25°C. The measurements correspond to a linear increase in length of the almost rodlike DNA molecules with the amount of proflavine bound; independent calculations from viscosity and sedimentation measurements yield almost identical results. Over the range of r (moles of proflavine bound per moles of nucleotides) equal to zero to r = 0.13, the length increases by about 20%. This extension is compatible with the intercalation hypothesis proposed by Lerman. Density increments at various values of r, at constant chemical potential of diffusible solutes, were determined. It was also found that, in addition to the known isosbestic point of DNA-proflavine complexes at 455.5 mμ, an additional isosbestic point exists at 225.5 mμ; this proved extremely useful for the evaluation of binding studies.     

Observations on the gel chromatography of collagen-like peptides and use of a simple calibration method for molecular weight determination.

Elution characteristics of collagen-derived polypeptides and of globular proteins were compared under identical experimental conditions with agarose gels. This comparison permitted calculation of the hydrodynamic radii of collagen polypeptide chains of different molecular weight, and these radii were shown to be in reasonable agreement with estimates made from intrinsic viscosity data. Two distinet linear relationships were observed for collagen polypeptide chains, relating logarithm of molecular weight to elution parameters. Peptide chains of MW 3300 and lower fell on a line of a steeper slope than did larger polypeptide chains.A simple procedure for molecular weight estimation of an unknown polypeptide chain of the collagen class is described, using only three commercially available standards for calibration: reduced, carboxymethylated Ascaris cuticle collagen, and the synthetic peptides (l-Pro-l-Pro-Gly)₁₀ and (l-Pro-L-Pro-Gly)₅.     

Calculated molecular weight of proteins in high ionic strengths: contribution of the apparent isopotential specific volume.

Recent sedimentation equilibrium measurements of the molecular weight of tail muscle lactate dehydrogenase from the North American East Coast lobster Homarus americanus show that this enzyme does not dissociate in buffer with high ionic strength (1.2 m ammonium sulfate). However, the apparent isopotential volume φ₂′ increases significantly with increasing ionic strength of the solution. Consequently, molecular weight estimates for proteins using an assumed apparent specific volume equal to that in low salt concentration solutions may lead to erroneously low values under experimental conditions of high ionic strength.     

Physical properties of the bovine encephalitogenic protein; molecular weight and conformation

Abstract— The highly basic encephalitogenic protein isolated from bovine spinal cord was studied by various physicochemical methods:

• 1 The molecular weight was determined by sedimentation equilibrium, by calculation from the data on sedimentation coefficients and intrinsic viscosities, by measurement of intrinsic viscosity in the presence of concentrated guanidine hydrochloride (according to the method of Tanford , Kawahara and Lapanie , 1967), and by exclusion chromatography on Sephadex G-100 columns. All values obtained were in good agreement and indicated a molecular weight of approximately 18,000–20,000.

• 2 Studies of sedimentation velocities in the presence and absence of 6 m -guanidine-HCl indicated that there was a significant difference in the values of sedimentation coefficients.

• 3 The same conditions were applied to the measurements of viscosity; the difference was small but significant. These findings and the magnitude of the intrinsic viscosity suggested that this protein was in a disordered configuration. From these data, it is concluded that the protein was apparently monodispersed, in the presence or absence of the denaturing agent. This protein behaved like a polyelectrolyte in neutral aqueous solution.

• 4 The measurements of optical rotatory dispersion also confirmed that this protein existed in a disordered configuration.

     

Selective antibody precipitation using polyelectrolytes: A novel approach to the purification of monoclonal antibodies

We evaluated the potential for polyelectrolyte induced precipitation of antibodies to replace traditional chromatography purification. We investigated the impact of solution pH, solution ionic strength and polyelectrolyte molecular weight on the degree of precipitation using the anionic polyelectrolytes polyvinylsulfonic acid (PVS), polyacrylic acid (PAA), and polystyrenesulfonic acid (PSS). As we approached the pI of the antibody, charge neutralization of the antibody reduced the antibody–polyelectrolyte interaction, reducing antibody precipitation. At a given pH, increasing solution ionic strength prevented the ionic interaction between the polyelectrolyte and the antibody, reducing antibody precipitation. With increasing pH of precipitation, there was an increase in impurity clearance. Increasing polyelectrolyte molecular weight allowed the precipitation to be performed under conditions of higher ionic strength. PVS was selected as the preferred polyelectrolyte based on step yield following resolubilization, purification performance, as well as the nature of the precipitate. We evaluated PVS precipitation as a replacement for the initial capture step, as well as an intermediate polishing step in the purification of a humanized monoclonal antibody. PVS precipitation separated the antibody from host cell impurities such as host cell proteins (HCP) and DNA, process impurities such as leached protein A, insulin and gentamicin, as well as antibody fragments and aggregates. PVS was subsequently removed from antibody pools to <1 µg/mg using anion exchange chromatography. PVS precipitation did not impact the biological activity of the resolubilized antibody. Biotechnol. Bioeng. 2009;102: 1141–1151. © 2008 Wiley Periodicals, Inc.     

Application of Polyelectrolyte Theory to the Study of the B-Z Transition in DNA (1)

Abstract

We have used the polyelectrolyte theory to study the ionic strength dependence of the B-Z equilibrium in DNA. A DNA molecule is molded as an infinitely long continuously charged cylinder of radius a with reduced linear charge density q. The parameters a and q for the B and Z forms were taken from X-ray data: a _B = 1nm, q _B = 4.2, a _z = 0.9 nm and q _z = 3.9. A simple theory shows that at low ionic strengths (when Debye screening length r _D>>a) the electrostatic free energy difference F ^el _Bz = F ^el _Z - F ^el _B increases with increasing ionic strength since q _B>q_z. At high ionic strengths (when r _D<<a) the F ^el _BZ would go on growing with increasing ionic strength if the inequality q _B/^a _B<q_z/a _z were valid. In the converse case when q _z/q _B<a_z/a _B the F ^el _BZ value decreases with increasing salt concentration at high ionic strength. Since X-ray data correspond to the latter case, theory predicts that the F ^el _BZ value reaches a maximum at an intermediate ionic strength of about 0.1 M (where r _D～a). We also performed rigorous calculations based on the Poisson-Boltzmann equation. These calculations have confirmed the above criterion of nonmonotonous behaviour of the F ^el _BZ value as a function of ionic strength. Different theoretical predictions for the B-Z transition in linear and superhelical molecules are discussed. Theory predicts specifically that at a very low ionic strength the Z form may prove to be more stable than the B form. Thus, one can observe the Z-B-Z transition with increasing ionic strength. In the light of our theoretical findings we discuss numerous experimental data on the B-Z transition in linear and superhelical DNA.     

Thermal stability and chain conformational studies of xanthan at different ionic strengths

The aim of the present study was to determine the influence of the ionic strength on the thermal stability of xanthan, i.e. xanthan resistance to chain breaking at high temperatures. Xanthan solutions of various ionic strengths were kept at 80, 90 and 95°C for periods up to 95 h. The thermal stability was determined by measuring the intrinsic viscosity after the heating periods. The experiments showed a critical ionic strength for the thermal stability of xanthan between 10 and 100 mm NaCl or KCl in this temperature range. Below the critical ionic strength the intrinsic viscosity was rapidly reduced, whereas above the critical ionic strength the intrinsic viscosity was virtually unaffected by heating.We then looked for a possible correlation between thermal stability and secondary structure of xanthan. The transition ionic strength (I_m) of xanthan solutions, i.e. where xanthan is midway between an ordered and a disordered structure, was determined by NMR at constant temperatures. I_m was found to be in the range of 24 mm at 80°C to 60 mm NaCl at 95°C, thus lying in the range of the critical ionic strength of the thermal stability. This suggests a close relationship between thermal stability and secondary structure of xanthan, indicated by the enhanced thermal stability in the ordered state. We believe this enhanced thermostability arises from a double-stranded conformation in the ordered state, as in DNA. The presence of double-stranded xanthan is also indicated by electron micrographs taken at both high and low ionic strengths.The transition temperature (T_m) of xanthan was determined by NMR and optical rotation measurements. At the ionic strength of 7·5 mm the two methods resulted in T_m values of 67 and 52°C respectively. This difference in T_m can possibly be due to the fact that the observed NMR and optical rotation (OR) effects are caused by different molecular phenomena.     

Conformation of the high molecular weight form ofDunaliella salina phosphofructokinase in solution by means of small-angle X-ray diffraction and viscosity measurements

Summary The intrinsic viscosity of phosphofructokinase fromDunaliella salina in different states of aggregation was determined. The instrinsic viscosity [], of the biologically active tetramer, with a molecular weight of 320,000, was found to be 6.5 ml·g^–1 at 4°C. Moreover, for the inactive dimer, with a molecular weight of 160,000, a value of []=8.0 ml·g^–1 was determined. The high molecular weight aggregate of phosphofructokinase fromDunaliella salina, that shows little activity, has an intrinsic viscosity of 23.2 ml·g^–1, which is significantly higher than that found for the active tetramer and the inactive dimer.Small angle X-ray scattering experiments in solution of this high molecular from of phosphofructokinase fromDunaliella salina reveal a radius of gyration of the cross section ofR _c=49.0 Å at an ionic strength of 0.15 M and_pH 7.2. Furthermore, a comparison of the values obtained for the tetramer and the radius of gyration (R _g=52.9 Å) with those of typical spherical proteins (3–4 ml·g^–1) shows that the values of [] andR _g are significantly larger for the high molecular weight form of phosphofructokinase than for the spherical proteins. The high intrinsic viscosity of the polymeric form of phosphofructokinase suggests an end-to-end aggregation consisting of monomeric units with heights,h=80–90 Å, and a cylindrical diameter of approximately 140.0 Å, resulting in a long rod of a total length of 1,800 Å and a molecular weight of two million. On the basis of the experimentally observedR _c and [] values, using a prolate ellipsoid of revolution as a model, the hydrodynamic volume and the hydration, the axial ratio could be determined to be 12. The native tetrameric form contains 0.4 g H₂O/g protein, whereas the higher aggregate structure corresponds to a hydration of 0.60 g H₂O/g protein.     

A comparative study on the hydrodynamic shape, conformation, and stability of E. coli ribosomal subunits in reconstitution buffer.

We have compared the hydrodynamic shape, conformation, and stabilities of active, unwashed ribosomal subunits, as well as their susceptibilities to changes in temperature and ionic strength. Both intrinsic viscosity and sedimentation velocity measurements indicate that the 30 S subunit has a more asymmetric hydrodynamic shape. The intrinsic viscosity of this subunit in reconstitution buffer has been found to be significantly larger than the value reported previously. While the RNA conformation in both subunits may be very similar as suggested by the near uv CD spectra, the average conformation of the protein in the two subunits is drastically different. The 30 S subunit has a lower T_m. The 50 S subunit is rather stable toward changes of ionic strength, whereas the 30 S subunit is quite susceptible to changes in ionic strength.     

Physicochemical investigation of k-carrageenan in the random state

Light-scattering, viscosity, and sedimentation experiments on aqueous solutions of k-carrageenan show that this sulfated polygalactose is an expanded flexible random coil. This expansion is due to long-range interactions that are predominantly electrostatic. Extrapolation of viscosity data to infinite ionic strength provided values for the intrinsic viscosity which were subjected to the Stockmayer-Fixman analysis, giving a value for the Mark-Houwink coefficient under theta-conditions, K_θ, of 0.27. The characteristic ratio, C_∞, under these conditions is 7.8, and the conformation factor σ is 2. In a solution of 0.118 ionic strength, where a Mark-Houwink exponent a_η of 0.86 is found, the radii of gyration calculated from viscosity data are lower than those found from the angular dependence of scattered light. On the other hand, the radius of gyration found from the sedimentation rate agrees well with the light-scattering radius. The relations between molecular parameters are corrected for the poly-dispersity of the sample.     

Polyelectrolyte and rheological studies on the polysaccharide welan

This paper investigates the polyelectrolyte behaviour of welan in aqueous solution. From conductivity and activity measurements it is demonstrated that a double-helical conformation is adopted irrespective of pH and ionic strength. The intrinsic pK of its carboxylic groups is found to be abnormally low (pK₀ = 2.2) and no conformational transition was observed during neutralization. The viscometric behaviour was studied as a function of degree of neutralization, polymer concentration, shear rate and ionic strength. The linear relation [η]=f(C_s^−1/2) allows the determination of B, the stiffness parameter from Smidsrod, whose value characterizes a very stiff molecule.     

Concentration and shear‐rate dependence of the viscosity of an exocellular polysaccharide

The viscosity of an exocellular polysaccharide (EPS) produced by the bacterium Lactococcus lactis subsp. cremoris B40 was studied in aqueous solution at an ionic strength of 0.10M. First, the zero‐shear viscosity was determined as a function of the concentration. From the data in the low concentration range, the intrinsic viscosity was determined. In addition, the shear‐thinning behavior was measured at several concentrations. By combining existing theories, a new equation is proposed that describes and predicts the intrinsic viscosity and the concentration dependence of the (zero‐shear) viscosity of B40 EPS solutions from the molar mass and the hydrodynamic radius of the polysaccharide. Based on the Rouse theory, the shear‐rate dependence of the viscosity also could be described and predicted from the molecular characteristics, i.e., molar mass and radius of gyration. It is shown that these equations can be applied to all random coil polysaccharides. © 1999 John Wiley & Sons, Inc. Biopoly 50: 641–646, 1999     

Intrinsic viscosity of native and single-stranded T7 DNA and its relationship to sedimentation coefficient

The intrinsic viscosity and sedimentation coefficient, of native and single-stranded T7 DNA have been determined at 25°C as a function of ionic strength in neutral and alkaline NaCl. The relationship between [η] and S_,w is well represented by the Mandelkern-Flory equation over the entire range of conditions between 0.0013 and 1M Na⁺. An apparent discrepancy between the two methods at moderate to high ionic strengths is probably due to a change in V with ionic strength. It appears that [η] is a more sensitive and reliable measure of molecular expansion for native DNA, S_,w but is a better index of conformational change in single strands, since [η] becomes too small to measure conveniently at high ionic strengths. At moderate to high ionic strengths, denaturation leads to a decrease in [η], although unfolded single strands retain considerable viscosity. At sufficiently low ionic strength, the intrinsic viscosity of the single strands becomes higher than that of native DNA, and the effective volume of a single strand approaches that of the native molecule.