Dielectric relaxation of DNA in aqueous solutions.

Using a four-electrode cell and a new electronic system for direct detection of the frequency differences specturm of solution impedance, the complex dielectric constant of calf thymus DNA (M_r = 4 × 10⁶) in aqueous NaCl at 10°C is measured at frequencies ranging from 0.2 Hz to 30 kHz. The DNA concentrations are C_p = 0.01% and 0.05%, and the NaCl concentrations are varied from C_s = 10^?4 M to 10^?3 M. A single relaxation regions is found in this frequency range, the relaxation frequency being 10 Hz at C_p = 0.01% and C_s = 10^?3 M. At C_p = 0.05% it is evidenced that the DNA chains have appreciable intermolecular interactions. The dielectric relaxaton time τ_d at C_p = 0.01% agrees well with the rotational relaxation time estimated from the reduced visocisty on the assumption that the DNA is not representable as a rigid rod but a coiled chain. It is concluded that the dielectric relaxiatioinis ascribed to the rotation of the molecule. Observed values of dielectric increment and other experimental findings are reasonably explained by assuming that the dipole moment of DNA results from the slow counterion fluctuation which has a longer relaxation time than τ_d.     

Proton relaxation in aqueous DNA solutions

By use of D₂O we found that the shortening of the longitudinal proton relaxation time which occurs in the investigated aqueous yeast DNA solutions (≦ 2.4% with 2% protein) was not based on a hydration effect, but was caused by magnetic impurities only. An estimate shows that the mobility of the hydrated water molecules is reduced by less than two orders of magnitude in comparison with the free water molecules.     

Sodium ion and solvent nuclear relaxation results in aqueous solutions of DNA

The field-dependent ²³Na nuclear relaxation in aqueous DNA solutions has been obtained for a range of temperatures, including the DNA melting region. At least two correlation times are needed to characterize the spectral density function for the ²³Na relaxation. For the slow process (with the largest correlation time), the temperature dependence of the coupling constant and the correlation time were determined, and important premelting effects were observed. Possible origins of the slow process are discussed. The last process is shown to be correlated with the properties of the hydration water of DNA as reflected by the ¹⁷O relaxation rates in these solutions. The influence of the polyelectrolyte and NaCl concentrations on the ²³Na relaxation rate is compared with previous results from solutions of linear flexible polyelectrolytes.     

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.     

Dielectric relaxation of DNA solutions. II

Real and imaganiry parts of complex dielectric constant of dilute solutions of DNA in 10^?3M NaCl with molecular weight ranging from 0.4 × 10⁶ to 4 × 10⁶ were measured at frequencies from 0.2 Hz to 30 kHz. Dielectric increments Δε were obtained from Cole-Cole plots and relaxation times τ_D from the loss maximum frequency. The τ_D of all samples agrees well with twice of the maximum viscoelastic relexation time in the Zimm theory, indicating that the low-frequency dielectric relaxiation should be ascribed to be the rotation of DNA. The rms dipole moment, which was obtained from Δε, agree well with that calculated from the counterion fluctuation theory. The dielectric increment was found to be greatly depressed in MgCl₂, which is resonably interpreted in terms of a strong binding of Mg⁺⁺ ions with DNA.     

Dielectric relaxation of collagen in aqueous solutions

The complex dielectric constant of collagen in aqueous solutions (polymer concentration, C_p = 0.02–0.2%) was measured at 10°C in the frequency range from 3 Hz to 30 kHz. The loss peak for C_p = 0.02% is located at 90 Hz and the dielectric relaxation time τ_D is estimated to be 1.8 ± 0.3 msec. The τ_D agrees well with the rotational relaxation time estimated from the reduced viscosity, and the relaxation is ascribed to the end-over-end rotation of the molecule. The C_p dependence of τ_D and the dielectric increment Δε are interpreted in terms of the aggregation of molecules. The dipole moment of a molecule, obtained from Δε at C_p = 0.02% and pH 6.5, is (5.2 ± 0.2) × 10⁴D, which is explained by the asymmetrical distribution of the ionized side chains of the molecule.     

High electric field dielectric studies of aqueous myoglobin solutions.

High field dielectric measurements of the Piekara factor deltaepsilon/E2 have been carried out for a range of concentrations of horse heart myoglobin in water at 293K. Using the literature value for the dipole moment of myoglobin and the established theory for the classical orientational dipolar non-linear effect predicts a value of deltaepsilon/E2 one order of magnitude greater than that for water. The measured effect, however, was found to be one order of magnitude less than for water. This difference is explained as being most probably due to the existence of antiparallel molecular dipole pairs in the myoglobin solution. The possibility of a positive deltaepsilon due to a field induced conformational change of the myoglobin cannot, however, be ruled out.     

Theory of dipolar relaxation in aqueous macromolecular solutions

Recent advances in dielectric theory are applied to two models representing an aqueous solution of dipolar macromolecules. In one model the water is treated as a dielectric continuum and the macromolecule as a finite-sized sphere; in the other both components are represented as point dipoles suspended in a background dielectric. The predicted frequency dependences of the complex permittivity in these two cases agree and the validity of the dielectric technique for estimating macromolecular size and shape is established. The model in which water is treated as a dielectric continuum predicts a larger dielectric dispersion in the radio frequency region, which is consistent with the experimental data available for myoglobin. The validity of the Debye formula for relaxation time and the effect of “dielectric friction” in macromolecular solutions are also discussed.     

Atomic force microscopy of DNA in aqueous solutions.

DNA on mica can be imaged in the atomic force microscope (AFM) in water or in some buffers if the sample has first been dehydrated thoroughly with propanol or by baking in vacuum and if the sample is imaged with a tip that has been deposited in the scanning electron microscope (SEM). Without adequate dehydration or with an unmodified tip, the DNA is scraped off the substrate by AFM-imaging in aqueous solutions. The measured heights and widths of DNA are larger in aqueous solutions than in propanol. The measured lengths of DNA molecules are the same in propanol and in aqueous solutions and correspond to the base spacing for B-DNA, the hydrated form of DNA; when the DNA is again imaged in propanol after buffer, however, it shortens to the length expected for dehydrated A-DNA. Other results include the imaging of E. coli RNA polymerase bound to DNA in a propanol-water mixture and the observation that washing samples in the AFM is an effective way of disaggregating salt-DNA complexes. The ability to image DNA in aqueous solutions has potential applications for observing processes involving DNA in the AFM.     

Effect of ions on the dielectric relaxation of DNA

The dielectric relaxation of DNA solutions has been investigated with and without extraneous ions covering a wide frequency range. The effect of monovalent ions such as Na, K, and Li as well as divalent ions such as Mg, Ca, and Hg have been included in the study. These ions are found to have a profound effect on the dielectric increment and the relaxation time without affecting the molecular dimension drastically. This dielectric effect is interpreted as indicating the importance of counterion fluctuation on the low frequency dielectric constant of DNA in solution. The effect of an organic ion, tetra-methylammoniun bromide, has also been studied. This ion has no noticeable effect. A simple theory is derived on the basis of a microscopic model to account for the effect of external ions on the dielectric behavior of solutions of DNA.     

Microwave dielectric absorption of DNA in aqueous solution

The dielectric properties of aqueous solutions of DNA were measured at frequencies ranging from 0.1 to 12 GHz. The results are analyzed using the Maxwell mixture theory and yield a value for the hydration of the DNA of about 0.4 g/g, which is in the range observed in other investigations. No evidence was found for an additional absorption effect at microwave frequencies, which has been predicted to occur in certain DNA analogs due to the vibrational excitation of the double helix by the applied microwave field.     

Monoalkyl phosphodiesters: Synthesis and dielectric relaxation of solutions

Chromatographically pure hexadecylphosphocholine, -(N,N-dimethyl)-ethanolamine, -(N-N-methyl)-ethanolamine and -ethanolamine have been synthesized. Aqueous solutions of these phospholipids have been prepared for the purpose of measuring their dielectric spectra. Micellar solutions appropriate for the dielectric studies were obtained with the choline and the (N,N-dimethyl)-ethanolamine head groups.The dielectric spectra of these phospholipid/water systems are evaluated in terms of phenomenologically introduced sum of Cole-Cole relaxation functions and also on the basis of a model relaxation function which has regard to internal depolarizing fields of the colloidal solutions. Parameters reflecting the motions of the dipolar head groups and of the hydration water molecules of the synthetic monoalkyl phosphodiesters are discussed and are compared with those for egg lysolecithin.The mobility of the dipolar phospholipid head groups and the number of influenced water molecules per zwitterion decreases when changing from lysolecithin to hexadecylphosphocholine and further to hexadecylphospho-(N,N-dimethyl)-ethanolamine, while the relaxation time of the hydration water increases. These results indicate the micellar surface to get less porous within the above series of lipids.     

Motion of the lengthened zwitterionic head groups of C16-lecithin analogues in aqueous solutions as studied by dielectric relaxation measurements

Phosphatidyl choline analogues with increased phosphate-trimethylammonium distance were synthesized and aqueous solutions of these bilayer forming phospholipids were prepared. Dielectric spectra of the solutions were measured at several temperatures around the crystalline/liquid-crystalline phase transition temperature of the samples. The observed data are treated in terms of a Debye relaxation function and also of a relaxation function based on a theoretical model of the aqueous solutions of multibilayer vesicles. As a noteworthy result, a pronounced cooperativity effect in the diffusive motions of the zwitterionic head groups emerges. The degree of cooperativity depends on the radius of curvature of the multibilayer vesicles and also on the length of the phospholipid zwitterions. The values for the mobility of the trimethylammonium group are of the same order of magnitude as those for the mobility of whole phospholipid molecules in its lateral diffusive motion. Indications for a phase transition at a temperature above the main transition temperature are found with solutions of C_16⁻ lecithin analogues with 9 and 10 methylene groups between the phosphate and the trimethylammonium group.     

The contribution of proton fluctuation to dielectric relaxation in protein solutions

Equations are derived which account for the effect of an applied electric field on the fluctuation of protons associated with a macromolecule. The contribution of this proton polarization to the complex permittivity of the macromolecules is evaluated in terms of its effect on both the dispersion due to proton fluctuation and the dispersion caused by dipolar rotation. An expression for the fluctuation correlation time τiδ is derived in terms of the mean lifetimes of the ionised and unionised state τ0 and τ+. If τiδ is very much less than the dipolar correlation time τi, the fluctuation dispersion will occur at much higher frequencies than the dispersion due to orientation polarization; hence the dispersions will be well separated. If τiδ » τi the two regions would overlap and would be indistinguishable as separate entities. At present insufficient data are available to test rigorously these conclusions but the potentialities of the theory in relation to small globular proteins is shown.     

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).     

Frequency dependence of the proton magnetic relaxation in aqueous solutions iof haemoproteins

The longitudinal proton magnetic relaxation times T₁ were measured for ferri (met)-and carbonmonoxy-bovine haemoglobin and equine myoglobin in 0.1 M KH₂PO₄ aqueous solutions near pH 6 at 5°C and 35°C from 1.5- to 60-MHz Larmor frequencies. It is concluded that the correlation time τ_C for the dipole–dipole interaction of electron and nuclear spins is in fact the electron (ferric) spin relaxation time τ_S being close to 1.5 × 10^?10 sec for both metHb and metMb at 5°C. At 35°C the paramagnetic relaxation rates are not determined solely by the relaxation of protons exchanging from the haem pocket with bulk solvent. Hence, τC at 35°C cannot be calculated from the dispersion data obtained at this temperature. The relevance of this for the determination of interspin distances r is discussed.     

Volatile transport in frozen aqueous solutions. I. Development of a mechanism

Frozen aqueous butanol solutions are equilibrated at constant subzero temperature over activated charcoal. A fraction of the butanol is lost within 24 hr, the remainder being retained for over 350 hr. The retained butanol is lost only with the simultaneous loss of water. Pure ice is demonstrated to be permeable to the transport of butanol. Based on experiments which remove, the free surface, the butanol loss that is independent of water loss is shown to originate from a surface layer postulated to form during freezing of the solution.Three types of butanol-ice interactions are postulated: (1) Butanol in the surface layer; (2) butanol entrapped in interdendritic spaces; (3) butanol present in pores and cracks after sorption from the vapor state.     

A proton magnetic relaxation study of ferrimyoglobin in aqueous ionic solutions

Proton magnetic longitudinal T₁ relaxation times have been measured for acid (horse) ferrimyoglobin solutions [0.1 M NaCl and KH₂PO₄, 2 M NaCl and 1 M MgCl₂] from 5°C to 35°C in dependence on myoglobin concentration up to 6 mM. The enhancement of the relaxation rate due to the paramagnetic haem iron. which is observed in this temperature range is compared with analogous data for the ferrihaemoglobin solution. The conclusion is that the protons exchanging from the haem pocket with bulk solvent are not those from the water molecule at the sixth ligand site of haem iron. The exchanging protons are more than 4 Å away from the haem iron being closer to it in ferrimyoglobin than in ferrihaemogiobin. This distance becomes larger in solutions with higher salt concentration, the largest difference between 0.1 M NaCl and 1 M MgCl₂ being over one Angstrom unit. This indicates a conformational change of the haem pocket, possibly its tightening.