Temperature dependence of dielectric constant at 10 GHz of Na-DNA gels

Permittivity ε′ and dielectric loss ε″ of aqueous Na-DNA gels have been measured at 10 GHz in the temperature interval ?15 to + 45°C. The experimental results are analyzed in terms of a three-component equation (Na-DNA, interfacial water, bulk water) and yield a value of 35 water molecules/nucleotide interacting with DNA. According to theoretical and experimental data the presence of strongly bonded and weakly bonded water is considered. The modified water exhibits a mean dielectric relaxation time two-or threefold greater than that of bulk water.     

Temperature dependence of the dielectric properties of blood

The aim of the work was to investigate the temperature effect in physiological range on the dielectric properties of a fish (Cyprinus carpio) blood. By applying an empirical non-damaging method and statistical analysis of the dielectric data, it is observed that a heating of carp blood induces an almost linear value variation of the electrical conductivity and permittivity of the intracellular matter of carp red blood cells. The dielectric data, however, reveal anomalous behaviour for conductivity and permittivity of the interior of carp erythrocytes at 23 degrees C and 23 degrees, 37 degrees C, respectively and for both volume fraction and dielectric loss factor tg delta at 30 degrees, 35 degrees, 37 degrees C. For the presented dielectric parameters the temperature coefficient and the Arrhenius activation energy were estimated. Moreover, this work led to conclusion that the changes of the volume fraction of carp erythrocytes induce corresponding variations of the maximum of the dielectric loss factor tg delta determined for a whole blood.     

Electric permittivity and dielectric dispersion of low-molecular weight DNA at low ionic strength.

The dielectric properties of sonicated calf-thymus DNA (MW approximately 3 X 10(5) g mol-1) have been investigated in a frequency range between a few kHz and 100 MHz. Two samples, sonicated in a different way were used after proper characterization including light-scattering, viscometry and contour length distribution by electron microscopy. Dielectric measurements were performed at several concentrations between 10(-4) and 3 X 10(-3) monomol 1-1 and 22 degrees C. Under all circumstances two separated dispersion regions were observed, the corresponding specific increments of which decreased with increasing concentration. The same was observed with the mean relaxation time of the high frequency dispersion. Both the frequency and concentration dependence was largely analogous to what is observed with other polyelectrolytes. Values of the dielectric parameters extrapolated to infinite dilution could also be interpreted in the same manner as for more simple, charged macromolecules and no specific effects had to be taken into account.     

Temperature dependence of DNA persistence length

We have determined the temperature dependence of DNA persistence length, a, using two different methods. The first approach was based on measuring the j-factors of short DNA fragments at various temperatures. Fitting the measured j-factors by the theoretical equation allowed us to obtain the values of a for temperatures between 5°C and 42°C. The second approach was based on measuring the equilibrium distribution of the linking number between the strands of circular DNA at different temperatures. The major contribution into the distribution variance comes from the fluctuations of DNA writhe in the nicked circular molecules which are specified by the value of a. The computation-based analysis of the measured variances was used to obtain the values of a for temperatures up to 60°C. We found a good agreement between the results obtained by these two methods. Our data show that DNA persistence length strongly depends on temperature and accounting for this dependence is important in quantitative comparison between experimental results obtained at different temperatures.     

Allowance for spatial dispersion of dielectric permittivity in polyelectrolyte model of DNA.

In order to allow for real dielectric properties of a solvent in calculating of electrostatic characteristics of strongly charged polyions such as DNA in salt solution we consider a simple model of linear dielectric response of a medium. The interactions between charged particles are treated in the framework of self-consistent-field approximation. The basic characteristic of the problem, electrostatic potential, can be found from the solution of non-linear integro-differential equation. Specifically we consider so-called quasimacroscopic model where dielectric response of a medium depends only on the distance from the polyion. Application of the approach for calculating of the B-to-Z free energy qualitatively retains the main conclusion obtained previously within the model with fixed dielectric constant: non-monotonous behavior of the free energy differences as a function of ionic strength. At the same time, essential sensitivity of the results to specific values of dielectric parameters is observed.     

Molecular-weight dependence of the low-frequency dielectric properties of aqueous solutions of gel-fractionated DNA

Combined three- and four-terminal AC bridge measurements have been made at frequencies from 10 Hz to 100 KHz on samples of DNA with different molecular weight in aqueous solution under varying conditions of DNA concentrations and added salt. A method is described for the separation of large quantities of DNA fractionated according to size. A complicated pattern of dependence of the specific dielectric increment on concentration is found, and the difficulties of comparing the results from sample to sample are discussed. The dielectric properties of the fractionated samples of DNA in aqueous solution are reported for solutions sufficiently dilute that specific dielectric increment is independent of concentration. The specific dielectric increment of the solutions (with concentration measured in moles of DNA molecules/liter) is found to increase as the square of the molecular weight. The results are compared with results of polyelectrolyte theories which deal explicitly with counterion fluctuations and interactions. The frequency dependence of the dispersion is much broader than for simple Debye relaxation. It is satisfactorily fitted by the empirical Cole–Cole circular are function and the breadth of the dispersion is found to be, if anything, less for the fractionated samples than for native DNA in solution.     

Temperature dependence of CD spectra of DNA from various sources

The CD spectra of DNA from various sources (T2; T4; C_d; Escherichia coli; calf thymus; Streptomyces chrysomalis) were investigated. A new band Δ_ε210 in the CD spectra of glucosylated DNA of the T even phages was found. The temperature dependence of the CD spectra of DNA was obtained over a wide range of temperatures, including those of the helix–coil transition. The band Δ_ε275 for all DNAs does not appreciably change in the range of the helix–coil transition. The monotonic increase of this band before melting, and its decrease after melting is observed with an increase in temperature. The amplitude of the CD band Δ_ε245 for all the DNAs studied and Δ_ε210 (glucosylated DNA) parallels the change of E₂₆₀ absorbance.     

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.     

Temperature dependence of the transition enthalpy of core particle DNA and of long DNA

The denaturation of short (145 base pairs) and long (about 8000 base pairs) DNA moelucules has been studied by adiabitic differential microcalorimetry in solutions with different NaCl content. It is found that the enthalpy of denaturation of short DNA is more sensitive to changes in T_m than that of long DNA. A comparison with other data is also given.     

Temperature dependence of ion permeation at the endplate channel

The dependence of acetylcholine receptor mean single-channel conductance on temperature was studied at garter snake twitch-muscle endplates using fluctuation analysis. In normal saline under conditions where most of the endplate current was carried by Na+, the channel conductance increased continuously from near 0 degrees C to approximately 23 degrees C with a Q10 of 1.97 +/- 0.14 (mean +/- SD). When 50% of the bath Na+ was replaced by either Li+, Rb+, or Cs+, the Q10 did not change significantly; however, at any temperature the channel conductance was greatest in Cs-saline and decreased with the ion sequence Cs greater than Rb greater than Na greater than Li. The results were fit by an Eyring-type model consisting of one free-energy well on the extracellular side of a single energy barrier. Ion selectivity appeared to result from ion-specific differences in the well and not in the barrier of this model. With a constant barrier enthalpy for different ions, well free-energy depth was greatest for Cs+ and graded identical to the permeability sequence. The correlation between increased well depth (i.e., ion binding) and increased channel conductance can be accounted for by the Boltzmann distribution of thermal energy.     

Temperature dependence of thermodynamic properties for DNA/DNA and RNA/DNA duplex formation.

A clear difference in the enthalpy changes derived from spectroscopic and calorimetric measurements has recently been shown. The exact interpretation of this deviation varied from study to study, but it was generally attributed to the non-two-state transition and heat capacity change. Although the temperature-dependent thermodynamics of the duplex formation was often implied, systemic and extensive studies have been lacking in universally assigning the appropriate thermodynamic parameter sets. In the present study, the 24 DNA/DNA and 41 RNA/DNA oligonucleotide duplexes, designed to avoid the formation of hairpin or slipped duplex structures and to limit the base pair length less than 12 bp, were selected to evaluate the heat capacity changes and temperature-dependent thermodynamic properties of duplex formation. Direct comparison reveals that the temperature-independent thermodynamic parameters could provide a reasonable approximation only when the temperature of interest has a small deviation from the mean melting temperature over the experimental range. The heat capacity changes depend on the base composition and sequences and are generally limited in the range of -160 to approximately -40 cal.mol-1.K-1 per base pair. In contrast to the enthalpy and entropy changes, the free energy change and melting temperature are relatively insensitive to the heat capacity change. Finally, the 16 NN-model free energy parameters and one helix initiation at physiological temperature were extracted from the temperature-dependent thermodynamic data of the 41 RNA/DNA hybrids.     

Temperature dependence of the gel electrophoretic mobility of superhelical DNA.

We have determined the gel electrophoretic behavior of closed circular plasmid pSM1 DNA (5420 bp) as a function of both temperature and of linking number (Lk). At temperatures below 37 degrees, the electrophoretic mobility first increases, then becomes constant as Lk is decreased below that of the relaxed closed DNA. As the temperature is increased above 37 degrees the electrophoretic mobility first increases as Lk decreases and then varies in a cyclic manner with further decreases in Lk. As the temperature is increased over the range 37 degrees - 65 degrees the cyclic behavior is manifested at progressively smaller decreases in Lk and the amplitude of the cycles increases. We interpret the results in terms of the early melting of superhelical DNA, in which the free energy associated with superhelix formation is progressively transferred to local denaturation. Using a two state approximation, we estimate the free energy change in the first cyclic transition to be 35 Kcal/mole DNA at 37 degrees and to decrease linearly with temperature. The free energy becomes equal to zero at a temperature of 71.6 degrees, which lies within 3 degrees of the melting temperature for the corresponding nicked circular DNA. From the slope of this relationship we estimate the apparent entropy and enthalpy of the first mobility transition to be 6.0 Kcal/mole base pair and 17.3 cal/mole base pair/degree, values consistent with duplex melting.     

Temperature dependence of accuracy of DNA polymerase I from Geobacillus anatolicus

Klenow-like DNA polymerase I fragment from Geobacillus anatolicus (GF) was cloned and purified. The accuracy of GF was measured in vitro at three different temperatures under single turnover conditions as well as using a forward mutation assay. In pre-steady-state kinetic measurements, when temperature was raised from 22 °C to 50 °C, the rate (k_pol) for cognate dTTP and non-cognate dATP nucleotide incorporations increased six- and four-fold, respectively, whereas the K_d for both nucleotide incorporations changed only slightly. As a result, the error frequency was remained constant (∼4 × 10⁻⁴) over this temperature range. The accuracy of GF was also measured using a forward mutation assay during a single cycle of DNA synthesis of the lacZα complementation gene in M13mp2 DNA. In this assay, which scores various types of replication errors, mutant frequency of GF was 5 × 10⁻³ at 72 °C which is four-fold higher than that of 37 °C.     

Temperature dependence and thermodynamics of Klenow polymerase binding to primed-template DNA

DNA binding of Klenow polymerase has been characterized with respect to temperature to delineate the thermodynamic driving forces involved in the interaction of this polymerase with primed-template DNA. The temperature dependence of the binding affinity exhibits distinct curvature, with tightest binding at 25-30 degrees C. Nonlinear temperature dependence indicates Klenow binds different primed-template constructs with large heat capacity (DeltaCp) values (-870 to -1220 cal/mole K) and thus exhibits large temperature dependent changes in enthalpy and entropy. Binding is entropy driven at lower temperatures and enthalpy driven at physiological temperatures. Large negative DeltaCp values have been proposed to be a 'signature' of site-specific DNA binding, but type I DNA polymerases do not exhibit significant DNA sequence specificity. We suggest that the binding of Klenow to a specific DNA structure, the primed-template junction, results in a correlated thermodynamic profile that mirrors what is commonly seen for DNA sequence-specific binding proteins. Klenow joins a small number of other DNA-sequence independent DNA binding proteins which exhibit unexpectedly large negative DeltaCp values. Spectroscopic measurements show small conformational rearrangements of both the DNA and Klenow upon binding, and small angle x-ray scattering shows a global induced fit conformational compaction of the protein upon binding. Calculations from both crystal structure and solution structural data indicate that Klenow DNA binding is an exception to the often observed correlation between DeltaCp and changes in accessible surface area. In the case of Klenow, surface area burial can account for only about half of the DeltaCp of binding.     

Temperature dependence of dielectric relaxations in alpha-elastin coacervate: evidence for a peptide librational mode

The dielectric permittivity of alpha-elastin coacervate is reported over the frequency range of 1 MHz to 1000 MHz and the temperature dependence from 6.8 degrees C to 70 degrees C is also reported. A temperature-dependent simple Debye-type relaxation is observed with a correlation time of 8 nsec (40 degrees C) which is similar to that of the polypentapeptide of elastin (i.e. 7 nsec at 40 degrees C) where the band has been assigned to a peptide librational mode. By analogy this allows for the first assignment of a peptide librational mode in a naturally occurring polypeptide or protein. The strong spectrally localized band indicates a regularity of structure. The low temperature dependence of the correlation time, giving a 1.7 kcal/mole enthalpy of activation, is consistent with torsional motions associated with a peptide librational mode.