Mechanical properties of DNA films

The Young's dynamical modulus (E) and the DNA film logarithmic decrement (theta) at frequencies from 50 Hz to 20 kHz are measured. These values are investigated as functions of the degree of hydration and temperature. Isotherms of DNA film hydration at 25 degrees C are measured. The process of film hydration changing with temperature is studied. It is shown that the Young's modulus for wet DNA films (E = 0.02-0.025 GN m-2) strongly increases with decreasing hydration and makes E = 0.5-0.7 GN m-2. Dependence of E on hydration is of a complex character. Young's modulus of denatured DNA films is larger than that of native ones. All peculiarities of changing of E and theta of native DNA films (observed at variation of hydration) vanish in the case of denatured ones. The native and denatured DNA films isotherms are different and depend on the technique of denaturation. The Young's modulus of DNA films containing greater than 1 g H2O/g dry DNA is found to decrease with increasing temperature, undergoing a number of step-like changes accompanied by changes in the film hydration. At low water content (less than 0.3 g H2O/g dry DNA), changing of E with increasing temperature takes place smoothly. The denaturation temperature is a function of the water content.     

Thermodynamic functions of biopolymer hydration. I. Their determination by vapor pressure studies,discussed in an analysis of the primary hydration process

The primary hydration process of native biopolymers is analyzed in a brief review of the literature, pertaining to various aspects of biopolymer–water systems. Based on this analysis, a hydration model is proposed that implies that the solution conformation of native biopolymers is stable at and above a critical degree of hydration (h_p′ = 0.06–0.1 g H₂O/g polymer). This water content corresponds to the fraction of strongly bound water, and amounts to ～20% of the primary hydration sphere. In order to test this model, detailed sorption–desorption scanning experiments were performed on a globular protein (α-chymotrypsin). The results obtained are consistent with the proposed hydration model. They show that under certain experimental conditions, sorption isotherms can be obtained that do not exhibit hysteresis. These data represent equilibrium conditions and are thus accessible to thermodynamic treatment. Valid thermodynamic functions, pertinent to the interaction of water with biopolymers in their solution state, can be obtained from these sorption experiments.     

Water adsorption isotherms of lipids

Hydration of bilayer lipids is a fundamental property of biological membranes. The available database of lipid hydration isotherms is fitted over the entire range of water activities by using a statistical mechanical approach that is an extension of the common Brunauer-Emmett-Teller model, to include differential energies of association for water molecules beyond the first strongly bound layer. Three-parameter fits are obtained that can be used to represent the experimental isotherms to a good degree of accuracy over the complete range of water-binding activities. Fits are also made in terms of the hydration pressure and correlation length of water ordering, by using the polarization theory of lipid hydration. The relationship of the latter approach to measurements of hydration forces between lipid bilayers is discussed.     

Study of the hydratation of the double-helical poly A-poly U complex by IR-spectroscopy and piezogravimetry methods

IR-spectra of a double-helical poly A-poly U complex and coiled poly U were studied at various r.h. in a 900-3800 cm-1 region. By the method of piezomicrobalance hydration isotherms for these polynucleotides were obtained. It is concluded that, as in the DNA case, simultaneous hydration of nucleic bases the backbone of polynucleotides occurs at lower r.h., and that the poly A-poly U hydration level is higher than poly A and poly U ones separately. Drastic changes in spectral parameters of poly A poly U uracil and adenine in-ring and out-of-ring absorption bands observed in 44-76% r.h. region were interpreted as a transition to helical conformation of the complex. Calculation of resonance frequencies for these normal vibrations in the dipole-dipole approximation agrees with the experimental data.     

Energetics of hydration of nucleic acids with various nucleotide composition

The energetics of hydration of natural DNA of different AT/GC content and model double-helical polyribonucleotides was studied. The results obtained by a new approach, which is based on calorimetric measurements of hydration-dehydration energy of nucleic acid-water systems at different relative humidities are presented. A correlation between the dehydration energy and the nucleotide composition of native DNA was found. The energetic characteristics of systems containing deoxynucleoside monophosphates and water clusters of different dimensions were obtained by the Monte Carlo method. The results of computer simulation correlate with the experimental calorimetric data.     

On the hydration of DNA. I. Preferential hydration and stability of DNA in concentrated trifluoroacetate solution

The hydration of DNA is an important factor in the stability of its secondary structure. Methods for measuring the hydration of DNA in solution and the results of various techniques are compared and discussed critically. The buoyant density of native and denatured T-7 bacteriophage DNA in potassium trifluoroacetate (KTFA) solution has been measured as a function of temperature between 5 and 50°C. The buoyant density of native DNA increased linearly with temperature, with a dependence of (2.3 ± 0.5) × 10^?4 g/cc-°C. DNA which has been heat denatured and quenched at 0°C in the salt solution shows a similar dependence of buoyant density on temperature at temperatures far below the T_m, and above the T_m. However, there is an inflection region in the buoyant density versus T curve over a wide range of temperatures below the T_m. Optical density versus temperature studies showed that this is due to the. inhibition by KTFA of recovery of secondary structure on quenching. If the partial specific volume is assumed to be the same for native and denatured DNA, the loss of water of hydration on denaturation is calculated to be about 20% in KTFA at a water activity of 0.7 at 25°C. By treating the denaturation of DNA as a phase transition, an equation has immmi derived relating the destabilizing effect of trifluoroacetate to the loss of hydration on denaturation. The hydration of native DNA is abnormally high in the presence of this anion, and the loss of hydration on denaturation is greater than in CsCl. In addition, trifluoroacetate appears to decrease the ΔHof denaturation.     

On the cooperative and noncooperative binding of ethidium to DNA.

The equilibrium binding of ethidium bromide to native DNAs and to poly(dG-dC) X poly(dG-dC) has been studied by both phase partition and direct spectrophotometric techniques. The binding isotherms obtained from both experimental techniques show that ethidium binds in a cooperative manner to E. coli DNA. On the other hand, no evidence of cooperative binding was observed in the binding isotherms obtained with calf thymus, C. perfringens, M. lysodeikticus, or poly(dG-dC) X (dG-dC) under the experimental conditions used (0.1 M NaCl).     

Isotherms of globular protein hydration under dynamic conditions]

The hydration isotherms of alpha-chymotrypsin, lysozyme, pork insulin, pork pepsin and serum albumin were obtained by means of dynamic method. The values of BET-monolayers for processes of water sorption leads to (h) and desorption comes from (h) do not depend on the static or dynamic way of achieving of hydration equilibrium in spite of difference in the shape of isotherms. The values of comes from h for proteins with known tertiary structure (alpha-chymotrypsin, lysozyme and insulin) coinside with the number of exposed polar amino acid side chains. The lowering of leads to h values in comparison with comes from h is correlated with inability of omega-amido groups of Asn and Gln residues and of ion pair-forming residues to take part in the formation of sorptive BET-monolayer. These rules for the interpretation of hydration isotherms were used to evaluate the numbers of exposed and buried polar side chains in proteins with unknown tertiary structure--pepsin and serum albumin.     

Volume changes accompanying the thermal denaturation of deoxyribonucleic acid. I. Denaturation at neutral pH

Dilatometric measurements were made to determine the change in apparent specific volume φ of DNA resulting from thermal denaturation in neutral solution, φ increased continuously with temperature in the range 10–85°C. No deviations from a monotonically rising curve were observed in the φ versus temperature profile in the region of the melting temperature. The results are interpreted in terms of a partial loss of the preferentially bound DNA hydration shell. The nature of the well known buoyant density difference between native and denatured DNA was investigated by evaluating the densities in a series of cesium salt gradients at constant temperature. Extrapolation of the results to zero water activity indicates that the partial specific volumes of anhydrous native and denatured DNA are equal. The density difference at nonzero water activities is attributed to decreased hydration in the denatured state. The absence of a related change in φ accompanying the denaturation in the dilatometric experiments suggests that the probable volume change associated with loss of bound water during denaturation is accompanied by other compensatory volume effects. The possible nature of these volume effects is discussed.     

Hydration force parameters of phosphatidylcholine lipid bilayers as determined from 2H-NMR studies of deuterated water.

The continuous decrease of the quadrupolar splitting of deuterated water interacting with phosphocholine lipid bilayers with growing water concentration is analyzed as a function of the water activity. From the apparent linear dependence on water activity a measure for hydration forces is obtained. The forces calculated are in the range of published data using sorption isotherms and osmotic stress technique in combination with SAXS. A simple interaction potential which includes orientational order of water adsorbed on surfaces gives a physical base for these findings. Therefore, deuterium NMR may become a powerful tool for hydration force analysis complementing well-known methods.     

The effect of the choline head group on phospholipid hydration.

The hydration behavior of three naturally occurring sphingomyelins (SM) has been studied. Adsorption isotherms for these SM, singly and in combination with other lipids, were obtained and the isotherms analyzed by the application of BET theory. The results are compared with those found previously by us for the phosphatidylcholines (PC). We find that, depending on the SM studied, both "weak" and "strong" water adsorption can be observed and thus, the presence of choline in the phospholipid head group, does not guarantee "strong" water adsorption. When the head group is removed from SM, the resulting ceramides are found to be very weak, water adsorbers. Cholesterol, when present in a mixture with SM, has a rather dramatic effect on the hydration of the SM. A mixed system of PC and SM exhibits water adsorption characteristics very similar to those exhibited by PC itself. We speculate that these hydration results may well play a role in the cell signaling action of SM and, moreover, may be closely associated with the hypothesized formation of lipid "rafts."     

Conformational state of DNA in chromatin subunits. Circular dichroism, melting, and ethidium bromide binding analysis.

This study compares some physical properties of DNA in native chromatin and mono-, di-, trinucleosomes obtained after mild micrococcal nuclease digestion. Melting curves and derivatives are shown to be very similar from one sample to another although a shift from 79 to 82 degrees C is observed between the mainly monophasic peak of multimers and chromatin. Careful analysis of the positive band of the circular dichroism spectra shows the appearance of a shoulder at 275nm, the intensity of which increases from the mono- to the di- and trinucleosome. This shoulder is maximum for native chromatin. At the same time binding isotherms of ethidium - bromide are characterized by two highly fluorescent binding sites for all the samples but the product KN of the apparent binding constant of the higher affinity binding sites by the apparent number of those sites increases from the mono- to the di- and trinucleosome. There again the valus is maximum for native chromatin. Such results strongly suggest that the native state of chromatin requires something more than the indefinite repeat of an elementary subunit.     

Thermal transition of DNA measured by NMR spin-echo technique

The thermal helix–coil transition of DNA can be studied by means of the spin-echo technique. The longitudinal spin–lattice relaxation time T1 and the transvense spin–spin relaxation time T2 of the DNA sample show a similar phase transition as observed spec-trophotometrically with increasing and decreasing temperatures. Four slopes on the T1 and T2 temperature relationship curves were found and interpreted as functions of nonrelational hydration of the DNA molecule. The T1 and T2 values differ depending on the native or denatured state of the DNA molecule. The importance of the dynamic equilibrium between water molecules in the hydration lattice and steps in the denaturation of the DNA molecule are discussed. This phenomenon may be directly related to the nonrotational hydration.     

Thermodynamic functions of biopolymer hydration. II. Enthalpy–entropy compensation in hydrophilic hydration processes

The thermodynamic functions of biopolymer hydration were investigated by multitemperature vapor pressure studies. Desorption measurements were performed that allowed determination of reversible isotherms in the hydration range of 0.1 to 0.3–0.5 g H₂O/g dry polymer. These isotherms are accessible to thermodynamic interpretation and are relevant to the interaction of water with biopolymers in their solution conformation. The results obtained on a series of different biopolymers (lysozyme, α-chymotrypsin, apo-lactoferrin, and desoxyribonucleic acid), show the following common features of interest: (1) The differential excess enthalpies (ΔH^e ) and entropies (ΔS^e ) are negative, and exhibit pronounced anomalies in a well-defined low-humidity range (approx. 0.1 g H₂O/g dry polymer). These initial extrema are interpretable by structural changes, induced in the native biopolymer structures by water removal below a critical degree of hydration. (2) The ΔH^e and ΔS^e terms exhibit statistically significant linear enthalpy–entropy compensation effects in all biopolymer–water systems investigated. The compensation temperatures \documentclass{article}\pagestyle{empty}\begin{document}$ \hat \beta = \overline {\Delta H} ^e /\overline {\Delta S} ^e $\end{document} are approximately identical for all biopolymers, ranging from 360 to 500 K. The compensation effects are attributable to phase transitions of water molecules between the bulk liquid and the inner-sphere hydration shell of native biopolymers. (3) The negative excess free energies (ΔG^e ) decrease monotonically with increasing water content and are close to zero at 0.3 to 0.5 g H₂O/g polymer. This result indicates that only transitions between the bulk liquid and the inner-sphere hydration shell are associated with significant net free energy effects. The outer-sphere hydration water is thermodynamically comparable to bulk water. The importance of the proportionality factor \documentclass{article}\pagestyle{empty}\begin{document}$ \hat \beta $\end{document} in the control of the free energy term is discussed.     

Hydration of polymeric components of cartilage--an infrared spectroscopic study on hyaluronic acid and chondroitin sulfate

Hydrated polysaccharides are major constituents of cartilage and play an important role in its water-binding properties. Infrared (IR) spectroscopy and sorption isotherms have been used to investigate the hydration behavior of the glycosaminoglycans hyaluronic acid and chondroitin sulfate. IR-dichroism of the vibrational modes of the pyranose ring is found at relative humidities (RH) smaller than 84%. The IR-dichroism data for the vibrational modes of the pyranose ring have been analyzed with respect to the helical structure of these polysaccharides. The orientation vanishes at higher relative humidities (>84%), because a strong increase in the water uptake occurs in the observed sorption isotherms. Differences in the IR-absorbance of the O-H stretching mode of sorbed water between hyaluronic acid and chondroitin sulfate are shown to be caused by the additional hydration of the sulfate groups. The corresponding H-bonds are weaker than those of the hydration shell of the pyranose rings.     

Isopycnic sedimentation of DNA in metrizamide: the effect of low concentrations of ions on buoyant density and hydration

Metrizamide, an inert, non-ionic organic compound, dissolves in water to give a dense solution in which DNA bands isopycnically at a density corresponding to that of fully hydrated DNA. Density-gradient centrifugation in solutions of metrizamide has been used to determine the effects of very dilute solutions of salts on the buoyant density of native and denatured DNA. It has been shown that the buoyant density of DNA is dependent on both the counter-cation and the anion present. Interpretation of the data in terms of the degree of hydration of the macromolecule indicates that (i), NaDNA is more highly hydrated than CsDNA; and (ii), the hydration of NaDNA varies with anion in the order sulphate< fluoride< chloride< bromide< iodide.     

Interaction of alkaline-earth metal ions with calf thymus DNA. Volume and compressibility effects in diluted aqueous solutions

The binding of Mg2+, Ca2+, Sr2+ and Ba2+ ions to calf thymus DNA in solutions has been investigated by ultrasonic and densimetric techniques. The obtained parameters, the apparent molar volume, phiV, and the apparent molar adiabatic compressibility, phiK(S), are very sensitive to hydration of investigated molecules. The interaction between the cations and DNA is accompanied by overlapping their hydration shells and consequently releasing the water molecules from hydration shells to bulk state. The change in the hydration is reflected in the measured parameters, phiV and phiK(S). The magnitude of these hydration changes is determined by the position of the cation relative to DNA atomic groups involved in the binding, and thus can characterize the structure of cation-DNA complexes. The values of the dehydration effects of the binding, deltaphiV and deltaphiK(S), correspond to two direct or higher number of indirect contacts between calf thymus DNA and the cations.     

Oxygen-17 and deuterium nuclear magnetic resonance studies of lysozyme hydration

Oxygen-17 and deuterium NMR studies of lysozyme hydration are reported for a wide range of lysozyme concentrations, and the relationship between water "activity" and water mobility in the lysozyme-water system as determined by high-field NMR is examined. In a first approximation, the effect of lysozyme activity on hydration is considered to be small because of the relatively low charge on lysozyme at pH 7 and the absence of salt in the lysozyme solutions. Correlation times are determined for tightly bound water, weakly bound water, and "multilayer" or trapped water in lysozyme at 20 degrees C. Hydration numbers are also determined for these three different water populations interacting with lysozyme. Good agreement is found between the hydration numbers determined by 17O NMR and the calculations based on the D'Arcy and Watt analysis of water sorption isotherms for proteins that considered three major water populations in hydrated lysozyme. A molecular interpretation for the three components in the D'Arcy and Watt theory of sorption isotherms is also proposed on the basis of our NMR results. Previous proton NMR spin-echo results are shown to be consistent with our findings by 17O NMR and support the view that there are at least four regions of distinct hydration behavior of lysozyme which span the whole range from solutions to solid powders.     

Hydration of denatured and molten globule proteins

The hydration of nonnative states is central to protein folding and stability but has been probed mainly by indirect methods. Here we use water 17O relaxation dispersion to monitor directly the internal and external hydration of alpha-lactalbumin, lysozyme, ribonuclease A, apomyoglobin and carbonic anhydrase in native and nonnative states. The results show that nonnative proteins are more structured and less solvent exposed than commonly believed. Molten globule proteins preserve most of the native internal hydration sites and have native-like surface hydration. Proteins denatured by guanidinium chloride are not fully solvent exposed but contain strongly perturbed occluded water. These findings shed new light on hydrophobic stabilization of proteins.     

Characteristic thermodynamic properties of hydrated water for 20 amino acid residues in globular proteins

Thermodynamic properties associated with hydrated water of proteins of known three-dimensional structure were computed and average values of hydration free energy, enthalpy, and heat capacity of unfolding for every amino acid residue were obtained. Each amino acid residue had characteristic values; in particular, the quantities for a side chain reflected the character of the amino acid, while those for the main chain were more or less the same except for glycine, alanine, and proline. The major contribution to the quantities was from the end group(s) of a side chain. The following interesting features were found. 1) The hydration quantity of unfolding derived from the native and extended conformations for a protein was approximately equal to the sum of the corresponding average quantities of component amino acid residues in the protein. 2) The profile of a quantity such as hydration free energy of unfolding along the sequence computed from the accessible surface areas of the native and extended conformations showed a strong correlation with the profile obtained by allocating the average value for the amino acid residue at every position on the sequence. The correlation coefficients between two profiles for unfolding quantities of hydration, i.e., free energy, enthalpy, heat capacity, and free energy of side chain are 0.72, 0.62, 0.80, and 0.75, respectively. Thus, every amino acid residue in the native conformation of a globular protein seems to be located in such a position that a thermodynamic quantity for each residue is approximately equal to its average value.