Caged DNA does not aggregate in high ionic strength solutions.

The assembly of DNA into compact particles that do not aggregate in physiologic salt solution occurs naturally in chromatin and viral particles but has been challenging to duplicate using artificial constructs. Cross-linking amino-containing polycations in the presence of DNA with bisimidoester cross-linker leads to the formation of caged DNA particles that are stable in salt solutions. This first demonstration of caged DNA provides insight into how natural condensation processes avoid aggregation and a promising avenue for developing nonviral gene therapy vectors.     

Gel electrophoresis measurement of counterion condensation on DNA

We used agarose gel electrophoresis to measure the effective charge neutralization of DNA by counterions of different structure and valence, including Na⁺, Mg²⁺, Co(NH₃), and sperinidine³⁺, which competed for binding with an excess of Tris acetate buffer. Linear DNA molecules ranged in size from 1 to 5 kilobases, and supercoiled plasmid pUC18 was also measured. In all cases, the results were in good agreement with theoretical predict ions from counterion condensation theory for two-counterion mixtures. © 1995 John Wiley & Sons, Inc.     

Barium modulates the gating of batrachotoxin-treated Na+ channels in high ionic strength solutions.

Batrachotoxin-activated rat brain Na+ channels were reconstituted in neutral planar phospholipid bilayers in high ionic strength solutions (3 M NaCl). Under these conditions, diffuse surface charges present on the channel protein are screened. Nevertheless, the addition of extracellular and/or intracellular Ba2+ caused the following alterations in the gating of Na+ channels: (a) external (or internal) Ba2+ caused a depolarizing (or hyperpolarizing) voltage shift in the gating curve (open probability versus membrane potential curve) of the channels; (b) In the concentration range of 10-120 mM, extracellular Ba2+ caused a larger voltage shift in the gating curve of Na+ channels than intracellular Ba2+; (c) voltage shifts of the gating curve of Na+ channels as a function of external or internal Ba2+ were fitted with a simple binding isotherm with the following parameters: for internal Ba2+, delta V0.5,max (maximum voltage shift) = -11.5 mV, KD = 64.7 mM; for external Ba2+, delta V0.5,max = 13.5 mV, KD = 25.8 mM; (d) the change in the open probability of the channel caused by extracellular or intracellular Ba2+ is a consequence of alterations in both the opening and closing rate constants. Extracellular and intracellular divalent cations can modify the gating kinetics of Na+ channels by a specific modulatory effect that is independent of diffuse surface potentials. External or internal divalent cations probably bind to specific charges on the Na+ channel glycoprotein that modulate channel gating.     

Approach to the limit of counterion condensation

According to counterion condensation theory, one of the contributions to the polyelectrolyte free energy is a pairwise sum of Debye-Hückel potentials between polymer charges that are reduced by condensed counterions. When the polyion model is taken as an infinitely long and uniformly spaced line of charges, a simple closed expression for the summation, combined with entropy-derived mixing contributions, leads to the central result of the theory, a condensed fraction of counterions dependent only on the linear charge density of the polyion and the valence of the counterion, stable against increases of salt up to concentrations in excess of 0.1 M. Here we evaluate the sum numerically for B-DNA models other than the infinite line of B-DNA charges. For a finite-length line there are end effects at low salt. The condensation limit is reached as a flat plateau by increasing the salt concentration. At a fixed salt concentration the condensation limit is reached by increasing the length of the line. At moderate salt even very short B-DNA line-model oligomers have condensed fractions not far from the infinite polymer limit. For a long double-helical array with charge coordinates at the phosphates of B-DNA, the limiting condensed fraction appears to be approached at low salt. In contrast to the results for the line of charges, however, the computed condensed fraction varies strongly with salt in the range of experimentally typical concentrations. Salt invariance is restored, in agreement with both the line model and experimental data, when dielectric saturation is considered by means of a distance-dependent dielectric function. For sufficiently long B-DNA line and helical models, as typical salt concentrations, the counterion binding fraction approaches the polymer limit as a linear function of 1/P, where P is the number of phosphate groups of B-DNA.     

A new model of counterion condensation in polyelectrolyte solutions. III. Theoretical predictions of competitive condensation between counterions of different valences

Our model has been extended for theoretical estimation of competitive condensation of counterions of different valences onto polyelectrolytes in solution. The estimations are compared with those obtained from Manning theory and with experimental data on counterion activity coefficients. The agreement with the data for sodium polystyrenesulfonate/MgCl2, CaCl2 is satisfactory.     

Limiting laws and counterion condensation in polyelectrolyte solutions: V. Further development of the chemical model

Counterion binding to polyelectrolyte chains is formulated as a chemical reaction M^z(free) → M^z(bound). Expressions for the chemical potentials of free and bound counterions are set equal to obtain the reaction equilibrium. The results are equivalent to those in the previous paper of this series. An additional result obtained here is that a polyion holds its bound counterion layer with a strength on the order of 100 $\text{kcal}\text{(mole cooperative unit)}$. The method is then applied to the calculation of the polarizability along the chain due to the bound (condensed) counterions.     

Thermodynamics of the interaction of sodium-n-dodecyl sulphate with Aspergillus niger catalase in high ionic strength aqueous solutions

The thermodynamic parameters for the interaction between sodium n-dodecyl sulphate (SDS) and Aspergillus niger catalase in aqueous solution at pH 3.2 and 6.4 have been measured by microcalorimetry and equilibrium dialysis over a range of ionic strength from 0.05 to 0.2 at 25 degrees C. Binding isotherms have been interpreted in terms of theoretical models (Hill equation and Wyman binding potential). The Gibbs energies of interaction become increasingly negative with increase in ionic strength and the entropies of interaction become increasingly positive. The ionic strength dependence of the Gibbs energies are much greater than predicted by the Debye-Hückel limiting law indicating a strongly ionic strength dependent hydrophobic contribution to the interactions.     

Viscosity of chromatin solutions increases with increasing ionic strength

Increasing the ionic strength of rat liver chromatin solutions above 0.4 M causes increasing viscosity. This indicates transformation of the compact chromatin molecules to more elongated forms. In the range of 0.4–0.5 M ionic strength histone H1 is dissociating continuously from the chromatin and the quaternary structure chromatin unravels. At ionic strength higher than 0.5 M the viscosities of chromatin solutions are furthermore increasing due to structural deformation. Near 0.7 M ionic strength the core histones H2A and H2B begin to dissociate from the chromatin, and the opening of the nucleosome cores leads to increasing elongation of the chromatin molecules.     

Trivalent counterion condensation on DNA measured by pulse gel electrophoresis

Pulse gel electrophoresis was used to measure the reduction of mobilities of λ-DNA-Hind III fragments ranging from 23.130 to 2.027 kilobase pairs in Tris borate buffer solutions mixed with either hexammine cobalt(III), or spermidine³⁺ trivalent counterions that competed with Tris⁺ and Na⁺ for binding onto polyion DNA. The normalized titration curves of mobility were well fit by the two-variable counterion condensation theory. The agreement between measured charge fraction neutralized and counterion condensation prediction was good over a relatively wide range of trivalent cation concentrations at several solution conditions (pH, ionic strength). The effect of ionic strength, trivalent cation concentration, counterion structure, and DNA length on the binding were discussed based on the experimental measurements and the counterion condensation theory. © 1996 John Wiley & Sons, Inc.     

Evaluation of the counterion condensation theory of polyelectrolytes.

We compare free energies of counterion distributions in polyelectrolyte solutions predicted from the cylindrical Poisson-Boltzmann (PB) model and from the counterion condensation theories of Manning: CC1 (Manning, 1969a, b), which assumes an infinitely thin region of condensed counterions, and CC2 (Manning, 1977), which assumes a region of finite thickness. We consider rods of finite radius with the linear charge density of B-DNA in 1-1 valent and 2-2 valent salt solutions. We find that under all conditions considered here the free energy of the CC1 and the CC2 models is higher than that of the PB model. We argue that counterion condensation theory imposes nonphysical constraints and is, therefore, a poorer approximation to the underlying physics based on continuum dielectrics, point-charge small ions, Poisson electrostatics, and Boltzmann distributions. The errors in counterion condensation theory diminish with increasing distance from, or radius of, the polyion.     

Immobilization of polyribonucleotides in agarose gels at high ionic strength

Purine polyribonucleotides poly(A), poly(G), and poly(I) associate reversibly with agarose gels at high NaCl molarities over the pH range 6–10, at 20°?40°C. Pyrimidine polyribonucleotides poly (C) and poly(U) could not be immobilized in agarose gels under the above conditions. However, poly(C) could be immobilized in agarose without precipitation between pH 3.2 and 4.0. Association of poly(G) and poly(I) with agarose appears to decrease progressively with deprotonation of their purine residues, and both polymers interact with the gel very weakly above pH 10 regardless of NaCl concentration. The binding to agarose of these polymers at pH 7.5 is also strongly influenced by temperature in the range 20°?40°C. The association of single-stranded poly(A) is only shifted toward higher NaCl molarities by increased pH; its binding is also little affected by temperature in the above range. At NaCl molarities effecting the saturating retention in agarose and at neutral pH, the immobilization of several polynucleotides could be prevented by urea in a concentration-dependent manner. The corresponding profiles of urea molarity appear to disclose a number of hydrophobic interactions between polynucleotides and agarose, some of which could be relatively strong, especially in the case of poly(A).     

Effect of the ionic strength of solutions on the size of ganglioside mycelia

Gangliosides in aqueous media of low ionic strength (2-5 mM NaCl) and in concentrations over the critical ones (10(-5) M) form micellas which do not differ from liposomes as regards the chromatographic behavior on Sepharose 4R, with a molecular weight of greater than or equal to 10(7) dalton. In aqueous media of a higher ionic strength (greater than or equal to 20 mM NaCl), gangliosides form micellas which are eluted during chromatography in far later fractions than liposomes 70-80 nm in diameter, with a molecular weight of (1-5) X 10(5) dalton. It is assumed that the conclusions about ganglioside incorporation into the liposomal membrane made on the basis of their peaks coincidence are correct, provided that ganglioside-containing liposomes are obtained and chromatographed under high ionic strength (greater than or equal to 20 mM NaCl).     

Mechanoformation of neutral giant phospholipid vesicles in high ionic strength solution

We present new and simple method for formation of giant unilamellar vesicles (GUVs) in high ionic strength solutions, such as phosphate buffered saline (PBS). Mechanoformation method is an alternative method to electroformation method. The advantage of the mechanoformation procedure is that there are no limitations with respect to the ionic strength of the aqueous solutions, because there is no applied electric potential thus no current flow through the formation cell and no electrolysis is induced.     

Effect of heparin polyanion on chromatin preparations obtained in solutions of low ionic strength]

DNP obtained in low ionic strength solutions (0.7 mM Na-phosphate buffer, pH 7.0) was found to be dissociated under the effect of heparin. The dissociation order of the three histone fractions was established: H2a, H1, H4. The following order of histones is assumed: H2a, H2b, H1, H3, H4. Activation of the DNA and RNA synthesis in the eucaryotic cells, their nuclei and chromatin under the effect of low heparin doses should be associated not with the H1 histone dissociation, but with the dissociation of histones moderately rich in lysine--H2a, and, probably, H2b.     

Influence of temperature and ionic strength on the low-frequency dielectric dispersion of DNA solutions

The dielectric properties of DNA solutions at low frequencies (5 Hz to 2 kHz) have been measured by means of a four-terminal bridge method utilized to minimize electrode polarization errors. At 24°C native salt-free DNA has a very large specific dielectric increment, Δε/c = 9.8 × 10⁶ l/mol and a very low frequency relaxation centered at 18 Hz. Both the dielectric increment and the relaxation time are greatly decreased by partial heat denaturation at temperatures above 60°C or by addition of salt, the effects being much larger for divalent anions. These results are shown to be in qualitative agreement with theoretical treatments of counterion fluctuation polarization by McTague and Gibbs for the equilibrium case and by Mandel for relaxation. The ratio of the relaxation time for the low-frequency process to that previously observed at much higher frequencies suggests that these relaxations result from counterion fluctuations along the longitudinal and transverse axes of the molecule, respectively.     

Polyamine addition to preparation media induces chromatin condensation, irreversibly at low ionic strength

Polyamines (spermine, spermidine) are commonly used as stabilizing cations in the chromatin preparation media. Their residual binding to chromatin is not easily reversed at low ionic strength, even after extensive dialysis, as evidenced by the use of labelled spermidine. Electric dichroism measurements show that their presence interferes with the physico-chemical characterization of chromatin by maintaining a condensed structure. These results give a definite answer to the controversy about the sign of optical anisotropy of chromatin determined by electric and flow dichroism techniques.