Dependence of laser light scattering of DNA on NaCl concentration

We show that the persistence length a of DNA, derived from total intensity laser light scattering of linear Col E₁ DNA and corrected for excluded-volume effects, varies from about 68 nm in 0.005M NaCl to about 40 nm in 0.2M NaCl, leveling off to a constant value (about 27 nm) at high NaCl (1–4M) concentration. These observations do not agree with current views on the effect of electrostatic charge and ionic conditions on DNA dimensions. The apparent diffusion constant D_app, determined from laser light scattering autocorrelation as a function of scattering vector q, at NaCl concentrations 0.005–4M, correctly yields the translational diffusion coefficient D_t at low values of q and scales with molecular dimensions rather than segment length at high values of q; thus, D_app/D_t yields a universal curve when plotted against q²R, where R_g is the radius of the gyration. The sedimentation coefficients s at 0.1 and 0.2M NaCl concentration closely agree with the well-tested empirical relations, and a combination of s, D_t, and the appropriate density increments yield correct molar masses over the whole salt concentration range. Approximate constancy of D_tR_g indicates limited draining in translational flow. We present some observations and thoughts on the regimes in which a dependence of the correlation decay times on q³ rather than q² applies. We conclude that quasielastic laser light scattering discloses little information about dynamics of internal motion of DNA chains.     

Translational and rotational diffusion of tobacco mosaic virus from polarized and depolarized light scattering

The translational and rotational dynamics of tobacco mosaic virus in sodium phosphate buffer (pH =7.5) solutions has been investigated by polarized and depolarized light scattering Rayleigh linewidth studies. For concentrations ranging from 1.75 × 10^?4 g ml^?1 to 0.25 × 10^?4 g ml^?1 the translational diffusion coefficient (D_T) has been found to be slightly concentration dependent and extrapolation to zero concentration gives D₀^20°C = 0.34 ± 0.01 × 10^?7 cm²S^?1. A full analysis of the polarized spectra obtained at high and low scattering angles and the depolarized spectra at near zero scattering angles has enabled these techniques to be compared and the rotational diffusion constant D_R to be determined. At a solution concentration of 1.75 × 10^?4 g ml^?1 a mean value is found to be D_R^20°C = 350 ± 30s^?1. These values of D_T and D_R are in approximate agreement with calculations based on models of the tobacco mosaic virus molecule as a cylindrical rod.     

Dynamic light-scattering studies of internal motions in DNA. I. Applicability of the rouse-zimm model

The intermediate scattering function G(K,t) for any polymer model obeying a linear separable Langevin equation can be expressed in terms of the eigenvalues and eigenvectors of its normal coordinate transformation. An algorithm for the extract numerical evaluation of G(K,t) for linear Rouse-Zimm chains in the presence of hydrodynamic interaction has been developed. The computed G(K,t)² were fit to C(t) = A exp(?t/τA) + B, and apparent diffusion coefficients calculated according to D_app ≡ 1/(2τ_AK²). G(K,t)² was surprisingly well-fit by single-exponential decays, especially at both small and large values of Kb, where K is the scattering vector and b the root-mean-squared subunit extension. Plots of D_app vs K² in-variably showed a sigmoidal rise from D₀ at K² = O up to a constant plateau value at large K²b². Analytical expression for G(K,t), exact in the limit of short times, were obtained for circular Rouse-Zimm chains with and without hydrodynamic interaction, and also for free-draining linear chains, and in addition for the independent-segment-mean-force (ISMF) model. The predicted behaviors for G(K,t) at large Kb (or KR_G) was found in all cases to be single-exponential with 1/τ ∝ K² at large Kb, in agreement with the computational results. A simple procedure for estamating all parameter of the Rouse-Zimm model from a plot of D_app vs K² is proposed. Experimental data for both native and pH-denatured calf-thymus DNA in 1.0M Nacl with and without EDTA clearly plateau behavior of D_app at large values of K, in harmony with the present Rouse-Zimm and ISMF theories, and in sharp contrast to previous predictions based on the Rouse-Zimm model.     

Effect of temperature on DNA secondary structure in the absence and presence of 0.5 M tetramethylammonium chloride

Changes in the average secondary structures of three different linear DNAs over the premelting region from 5 to 60°C were investigated by measuring their CD spectra and also their torsion elastic constants (〈α〉) by time-resolved fluorescence polarization anisotropy. For one of these DNAs, the HaeII fragment of pBR322, the apparent diffusion coefficients [D_app (k)] at small and large scattering vectors (k) were also measured by dynamic light scattering. With increasing temperature, all three DNAs exhibited typical premelting changes in their CD spectra, and these were accompanied by 1.4- to 1.7-fold decreases in 〈α〉. Also for the 1876 base pair fragment, D_app(k) at large scattering vectors, which is sensitive to the dynamic bending rigidity, decreased by 17%, even though there was no change at small scattering vectors, where D_app(k) = D₀ is the translational diffusion coefficient of the center-of-mass. These observations demonstrate conclusively that the premelting CD changes of these DNAs are associated with a significant change in average secondary structure and mechanical properties, though not in persistence length. In the presence of 0.5 M tetramethylammonium chloride (TMA-Cl) the premelting change in CD is largely suppressed, and the corresponding changes in 〈α〉 and D_app(k) at large scattering vectors are substantially diminished. These observations suggest that TMA-Cl, which binds preferentially to A · T-rich regions and stabilizes those regions (relative to G · C-rich regions) against melting, effectively stabilizes the prevailing low-temperature secondary structure sufficiently that the DNA is effectively trapped in that state over the temperature range observed. © 1998 John Wiley & Sons, Inc. Biopoly 45: 503–515, 1998     

Hydrodynamic properties of proteoglycan subunit from bovine nasal cartilage. Self-association behavior and interaction with hyaluronate studied by laser light scattering

Measurements of translational diffusion coefficients by quasielastic laser light scattering, sedimentation coefficients, and intrinsic viscosities at zero shear of proteoglycan subunit fraction A1-D1-D1 isolated from bovine nasal septa are reported. Molecular weights and hydrodynamic dimensions are compared with those expected on the basis of structural models previously proposed. Comparison of the concentration dependence of the diffusion coefficient in the presence of NaCl and GdnHCl leads to the conclusion that significant self-association behaviour of subunit occurs in the absence of GdnHCl. In the absence of added salt, anomalous nonlinear concentration dependence of D_t estimated from wide-angle light-scattering experiments is observed. In addition, D_t apparently becomes angle dependent. These results are interpreted in terms of the perturbation of normal translational diffusion of the monomer by strong repulsive intermolecular interactions due to the combined effects of long-range electrostatic forces and macromolecular congestion at higher concentrations. By carrying out experiments at small scattering angles, it is possible to determine D for proteoglycan subunit in the absence of supporting electrolyte. Titration of a dilute solution of subunit with hyaluronic acid results in a sigmoidal behaviour of the Stokes radius, indicating the formation of complexes of higher molecular weight results from the noncovalent association of proteoglycan subunits with hyaluronate. Observation of D_t appears to provide a useful method for studying the proteoglycan subunit–hyaluronate interactions.     

Conformational change of core particles studied by quasielastic light scattering

The diffusion translational coefficient D_T of core particles in monodisperse solutions has been measured by the quasielastic light scattering method in a large scale of salinities over the range 6.10⁻⁴ to 2M Na⁺ or K⁺. The observed values of D_T are independent of particle concentration in the range 0.1–2 mg/ml and do not vary with the scattering vector q corresponding to scattering angles between 40°–120°. When the salinity is progressively raised an increase of D_T from 1.9.10⁻⁷ cm²s⁻¹ to 3.2.10⁻⁷ cm²s⁻¹ was observed at about 2.10⁻³ M NaCl followed by a decrease of D_T beyond 0.6 M NaCl.The various possible causes of the changes of D_T such as interactions between particles or between particles and salt ions are discussed. We show that the single low ionic strength change is due to a conformational transition of the core particles, while the second variation of D_T accompanies the disorganization of the core particles.     

Li+ counterion self-diffusion in ordered DNA

The anisotropic self-diffusion coefficient of ⁷Li⁺ (I = 3/2) counterions has been studied in hydrated, macroscopically oriented Li-(B)DNA fibers at relatively high water contents, corresponding to approximate DNA-DNA helix axis distances of 22–35 Å, using the pulsed field gradient hmr spin-echo method. Self-diffusion coefficients parallel (D_∥) and perpendicular (D_?) to the DNA helix axis increase with increasing salt content and with increasing DNA-DNA helix axis distance. The observed anisotropy D_∥/D_? decreases from 1.6 to 1.2 with the DNA-DNA separation increasing from 22 to 35 Å in the salt-free sample. This result can be understood by the obstruction effect caused by the DNA molecules themselves. The values of the Li⁺ self-diffusion coefficients in the most water-rich system with no added salt (corresponding to an approximate distance of 35 Å between the DNA helix axes) were D_∥ ～ 1.15 × 10^?10 m² s^?1 and D_? ～ 0.98 × 10^?10 m² s^?1, compared to 9.14 × 10^?10 m² s^?1 for the diffusion of Li⁺ in an aqueous solution of LiCl (～ 2.1M). The possible occurrence of restriction effects in the DNA fibers have also been studied by determining the self-diffusion coefficient at different effective diffusion times. The self-diffusion coefficient of Li⁺ in the sample with the largest DNA-DNA helix axis distance seems to be independent of the effective diffusion time, which indicates that the lithium ions are not trapped within impermeable barriers. The possibility of diffusion through permeable barriers has also been investigated, and is discussed. © 1994 John Wiley & Sons, Inc.     

Diffusion NMR-based comparison of electrostatic influences of DNA on various monovalent cations

Counterions are important constituents for the structure and function of nucleic acids. Using ⁷Li and ¹³³Cs nuclear magnetic resonance (NMR) spectroscopy, we investigated how ionic radii affect the behavior of counterions around DNA through diffusion measurements of Li⁺ and Cs⁺ ions around a 15-bp DNA duplex. Together with our previous data on ²³Na⁺ and ¹⁵NH₄⁺ ions around the same DNA under the same conditions, we were able to compare the dynamics of four different monovalent ions around DNA. From the apparent diffusion coefficients at varied concentrations of DNA, we determined the diffusion coefficients of these cations inside and outside the ion atmosphere around DNA (D_b and D_f, respectively). We also analyzed ionic competition with K⁺ ions for the ion atmosphere and assessed the relative affinities of these cations for DNA. Interestingly, all cations (i.e., Li⁺, Na⁺, NH₄⁺, and Cs⁺) analyzed by diffusion NMR spectroscopy exhibited nearly identical D_b/D_f ratios despite the differences in their ionic radii, relative affinities, and diffusion coefficients. These results, along with the theoretical relationship between diffusion and entropy, suggest that the entropy change due to the release of counterions from the ion atmosphere around DNA is also similar regardless of the monovalent ion types. These findings and the experimental diffusion data on the monovalent ions are useful for examination of computational models for electrostatic interactions or ion solvation.     

Monomolecular condensation of lambda-DNA induced by cobalt hexamine

Measurements of static and dynamic light scattering have been used to distinguish between monomolecular DNA condensation and aggregation of condensed molecules. In low salt, using Co³⁺(NH₃)₆ as the condensing agent, and at λ-DNA concentrations below 0.2 μg/mL, the transition curves for monomolecular condensation and aggregation are well separated for times of 16 h. In these conditions, the intensity of scattered light (90°) and also the diffusion coefficient of the condensed DNA show reasonable values for monomolecular condensation that are independent of DNA concentration and also of Na⁺ Co³⁺(NH₃)₆ concentrations for which monomolecular condensation is complete. At higher Co³⁺(NH₃)₆ concentrations, which produce aggregation (as judged by the intensity of scattered light), the diffusion coefficient decreases sharply. The transition curve for monomolecular condensation is independent of DNA concentration but shows a hysteresis loop. The kinetics of condensation are slow in the forward direction and fast in the reverse direction, indicating that the actual transition curve is measured closely by reversal experiments. Aggregation is blocked kinetically in both the forward and reverse directions when Co³⁺(NH₃)₆ is the condensing agent at low Na⁺ concentrations. When spermine or spermidine is the condensing agent and observations are made at 16 h, it is not possible to separate the transition curves for monomolecular condensation and for aggregation in conditions that are successful with Co³⁺(NH₃)₆. Some interesting properties of monomolecular condensation are noted. (1) The transition is not a two-state reaction, as judged by measurements of the diffusion coefficient through the transition zone. (2) The transition for monomolecular condensation is diffuse. (3) The dimensions of the monomolecular condensates have been calculated from the translational diffusion coefficient for an assumed toroidal shape by the formula derived by Allison and coworkers [(1981) Biopolymers 20 , 469–488]. These dimensions are in reasonable agreement with ones deduced from electron microscopy by Chattoraj and coworkers [(1978) J. Mol. Biol. 121 , 327–337]. (4) The phase diagram relating the Na⁺ to the Co³⁺(NH₃)₆ concentrations needed for condensation has a slope of 0.6 in a log–log plot. According to numerical solutions of Manning's theory for the atmospheric binding of competing cations to DNA, this means that condensation occurs at a late stage in the replacement of Na⁺ by Co³⁺(NH₃)₆ around the DNA. The fraction of DNA phosphate charge neutralized at condensation is computed to be in the neighborhood of 0.9, as found by Wilson and Bloomfield [(1979) Biochemistry 18 , 2192–2196], but to vary with the Na⁺ concentration.     

The concentration jump method

Lipophilic cationic fluorescent dyes (D) specifically stain the mitochondria of living cells. A perfusion chamber for cell cultures is described, which can be used to determine the kinetics of vital staining of the mitochondria of single selected cells in situ. In these experiments styrylpyridinium dyes and cultures of HeLa cells were used. The dyes differ strongly in their lipophilic properties; R _m values and the partition coefficients P _o/w between n-octanol (o) and water (w) were determined in order to characterize their lipophilicity. In the thermostat-regulated chamber the concentration of the dye C _D can be increased from C _D=0 to C _D>0 within a few seconds (concentration jump). Thus, the time t=0 for the beginning of the vital staining and the dye concentration in the cell medium during the staining experiment, C _D=const., are unambiguously defined. The concentration of the dye, C _b, which is bound to the mitochondria (b), is proportional to the intensity of the fluorescence I _b. On the other hand, the free dye molecules (f) in the aqueous medium exhibit practically no fluorescence, I _fI _b. The intensity of the fluorescence I=I _b was measured as a function of time t; the measured values were corrected for photobleaching. The fluorescence intensity I(t) at first increases linearly with t and reaches a saturation value for t . In the linear range of I(t) the flow J _o=(dI/dt)_o of the dye into the cell depends strongly on the dye concentration and increases linearly with C _D. The concentration range C _D=10^–9–10^–5 M at 37° C was investigated. From the linear correlation between J _o and C _D it follows that the kinetics of the vital staining of mitochondria is controlled by diffusion. At t=0 the flow of the xenobiotic agent through the cell membrane determines the rate of staining. The slope dJ _o/dC _D of the plot J _o vs C _D describes the efficiency of dye accumulation at the mitochondria and strongly increases with increasing lipophilicity of the dye molecules. Thus lipophilic dyes pass through the cell membrane more easily than less lipophilic molecules.     

Dynamic light scattering from collagen solutions. I. Translational diffusion coefficient and aggregation effects

Solutions of native collagen extracted from rat tail tendons in neutral salt solution have been studied by dynamic light scattering. The spectra obtained are consistent with the presence in solution of both single rod-shaped collagen molecules and aggregates of molecules. No contribution to the spectrum has been detected at any scattering angle from rotational diffusion of single molecules, although a measurable broadening effect is expected at high angles. The translational diffusion coefficient D of single molecules, calculated from the broader spectral component, shows an anomalous dependence on collagen concentration with a maximum value of D_20,w = 8.6 ± 0.2 × 10^?12 m²/sec near the concentration 0.04% by weight. Above 0.05% D falls linearly with increasing concentration and takes the value D _20,w = 8.1 ± 0.2 × 10^?12 m²/sec at 0.064% collagen.     

Brownian Dynamics Simulations of Colloidal Liquids: Hydrodynamics and Stress Relaxation

Abstract

We describe the statistical mechanics background and additional algorithmic features of a recently proposed simple mean-field Brownian Dynamics algorithm formulated to include many-body hydrodynamics, using a local density approximation for the friction coefficient. We show that the equations of motion satisfy the incompressibility of phase space. We make further developments to the model, computing the hydrodynamic effects on the shear stress relaxation function. We show that stress relaxation takes place over two well-defined regimes, in both cases with and without mean field hydrodynamics, MFH. At short times ta ²/D ₀ < 10^?3, where a is the radius of the colloidal particle and D ₀ is the self-diffusion coefficient at infinite dilution, decay of the stress autocorrelation function, C_s(t) is essentially independent of volume fraction and does not fit to a simple analytic form. At longer times than ta ²/D ₀ < 10^?2 the decay has the fractional exponential form ～exp(-t ^β) with β ? 1. The transition between these two regimes coincides with a rapid fall in the time-dependent diffusion coefficient from the so-called short-time to long-time values. We do not find any evidence for power law decay in the C_s(t) as predicted by recent mode-coupling based analytical expansions.     

Theory of boundary spreading in velocity ultracentrifugation of polydisperse solutes

Under the assumptions that the rectangular approximation to the sector-shaped cell is valid and that both the sedimentation coefficient s and the diffusion coefficient D are independent of concentration, asymptotic solutions to the boundary spreading equation for velocity ultracentrifugation of polydisperse solutes have been derived for three cases: case A, D = constant for all s; case B, sD = constant; case C, \documentclass{article}\pagestyle{empty}\begin{document}$ \sqrt {sD} = {\rm constant} $\end{document}. Case A is the situation treated by all of the previous authors but supposed to be unrealistic for ordinary macromolecular solutes. Cases B and C may be associated with synthetic polymers under theta conditions and globular proteins in aqueous media, respectively. The solutions obtained have been used to explore the theoretical background of the empirical Gralén method for evaluating the distribution of s from sedimentation boundary curves, with special interest in the behavior of a plot for Sversus 1/t. Here S is the value of s for a fixed value of the apparent integral distribution of s and t is the time of centrifugation. It was found that when the distribution of s is Gaussian-like and fairly narrow, this plot becomes linear over a more extended range of t in the order case B > case C > case A.     

Molecular Dynamics Studies of Diffusion in Model Cylindrical Pores at Very Low Densities

Abstract

Molecular dynamics simulation has been used to study diffusion of methane at ambient temperature in cylindrical pores at very low densities. The cylinders were modelled as a continuum solid which interacts with the methane in the radial direction only. At the lowest densities, the VACF method does not yield reliable values of the self diffusion coefficient, D_s , but a suitable choice of time step and run length enables values of D_s to be found from MSD plots that are below the classical Knudsen diffusion coefficients. When density is increased, D_s passes through a maximum although the adsorption isotherm remains inside the Henry law region. Maxima are found for two cylinder radii and for two adsorbent field strengths. The existence of a maximum is attributed to transient intermolecular interactions. Analysis of a molecular trajectory demonstrates that long diffusion paths can be triggered by the rare event of an intermolecular encounter which forces a molecule into the repulsive part of the wall potential. At sufficiently high density, subsequent collisions quench the tendency towards long paths, and D_s decreases again. The issue of simulation artefact as a source of these observations is discussed.     

Dynamic light scattering from thin rigid rods: Anisotropy of translational diffusion of tobacco mosaic virus

An Exact theoretical expression for the apparent diffusion coefficient D_app(K) of a thin rigid rod with arbitrary anisotropy of its translational diffusion diffusion coefficient is derived from the first cumulant of its dynamic structure factor. D_app(K) is predicted to reach a limiting plateau value at extermely large values of KL, where K is the scattering vector and L the rod length. Howerver, that limiting plateau value is approached only very slowly along a quasi-plateau with a very gradual slope. Dynamic light-scattering studies have been performed on tobacco mosaic virus from K² = (0.4–20) × 10¹⁰ cm^?2 using 632-8-nm laser radiation. The present data yield D₀ = (4.19 ± 0.10) × 10^?8 cm²/s (corrected to 20,w conditions) and, with literature data to establish L = 2980 Å and the rotational diffusion coefficient D_R = 318s^?1, yield also Δ ≡ D_∥ ? D_⊥ = (1.79 ± 0.38) × 10^?8 cm²/s. The experimental data closely follow the curve of D_app(K) vs K² calcuated for these parameters. The present value of D₀ substantially exceeds all previous dynamic light-scattering values, but is in good aggreement with previous sedimentation data, which were confirmed for the presemt sample. The anisotropy ratio Δ/D₀ = 0.43 ± 0.09 is in accord with theoretical predictions based on the modified Kirkwood algorithm, despite the fact the D₀ lies significantly below its corresponding theoretical value. The present data largely predlude the possibility that both D₀ and Δ/D₀ could simultaneously match their theoretical predictions. We present a detailed comparison of the experimental data with the calculations of Tirado and Garcia de la Torre based on the modified Kirkwood algorithm and with the Broersma formulas.     

Transient state isoelectric focusing. Experimental determination of apparent diffusion coefficients in polyacrylamide gels

A photoelectric scanning assembly utilizing uv absorption optics and an on-line digital data acquisition and processing system has been used to follow kinetically zone spreading during the defocusing stage (absence of electric field) of transient state isoelectric focusing (TRANSIF) in polyacrylamide gels. Measurement of the variance (σ²) of a diffusing zone as a function of time yields a linear relationship, the slope of which corresponds to the apparent diffusion coefficient (D) of the protein. A linear relationship is also obtained when the logarithm of the apparent diffusion coefficients (logD) are plotted vs acrylamide concentration (T). This relationship can be used to extrapolate D to zero gel concentration. The apparent diffusion coefficient measured in this way is significantly larger than the true diffusion coefficient. The slope of the plot logD vs T, designated C_R, is expected to be a measure of molecular size related to the retardation coefficient in polyacrylamide gel electrophoresis.     

In situ effective diffusion coefficient profiles in live biofilms using pulsed‐field gradient nuclear magnetic resonance

Diffusive mass transfer in biofilms is characterized by the effective diffusion coefficient. It is well documented that the effective diffusion coefficient can vary by location in a biofilm. The current literature is dominated by effective diffusion coefficient measurements for distinct cell clusters and stratified biofilms showing this spatial variation. Regardless of whether distinct cell clusters or surface‐averaging methods are used, position‐dependent measurements of the effective diffusion coefficient are currently: (1) invasive to the biofilm, (2) performed under unnatural conditions, (3) lethal to cells, and/or (4) spatially restricted to only certain regions of the biofilm. Invasive measurements can lead to inaccurate results and prohibit further (time‐dependent) measurements which are important for the mathematical modeling of biofilms. In this study our goals were to: (1) measure the effective diffusion coefficient for water in live biofilms, (2) monitor how the effective diffusion coefficient changes over time under growth conditions, and (3) correlate the effective diffusion coefficient with depth in the biofilm. We measured in situ two‐dimensional effective diffusion coefficient maps within Shewanella oneidensis MR‐1 biofilms using pulsed‐field gradient nuclear magnetic resonance methods, and used them to calculate surface‐averaged relative effective diffusion coefficient (D_rs) profiles. We found that (1) D_rs decreased from the top of the biofilm to the bottom, (2) D_rs profiles differed for biofilms of different ages, (3) D_rs profiles changed over time and generally decreased with time, (4) all the biofilms showed very similar D_rs profiles near the top of the biofilm, and (5) the D_rs profile near the bottom of the biofilm was different for each biofilm. Practically, our results demonstrate that advanced biofilm models should use a variable effective diffusivity which changes with time and location in the biofilm. Biotechnol. Bioeng. 2010;106: 928–937. © 2010 Wiley Periodicals, Inc.     

Temperature dependence of the dynamic light scattering of linear phi 29 DNA: implications for spontaneous opening of the double-helix

The apparent diffusion coefficient D_app(K) of a single sample of linear ?29 DNA (M_r = 11.5 × 10⁶) has been measured as a function of K² from 0.21 × 10¹⁰ to 20 × 10¹⁰ cm^?2 at a variety of temperatures from ?0.5 to +70°C. D_app(K) scales closely as T/η at every value of K². All of these data are simulated by a particular Rouse-Zimm model comprised of a constant number of subchains with constant rms subchain extension b = 1057 Å and an apparent subchain diffusion coefficient D_plat that scales at T/η from ?0.5 to +70°C. It is inferred from these results that any temperature dependence of the flexural and torsional rigidities of DNA must be rather weak. A less firm inference is that these rigidities actually increase slightly with temperature, possibly in proportion to T, which is weak T dependence in this context. These findings eliminate the possibility that spontaneous transient opening of the DNA structure has any significant effect on the flexural and torsional rigidities of the DNA filament. A review of the most pertinent available data from other experiments concerning spontaneous transient opening of the DNA is presented. The formaldehyde kinetics data do not unequivocally implicate an open base-pair intermediate and provide only an upper limit to the fraction of open base pairs. An alternative nonopening model with a protonated doorway state is proposed to accommodate the hydrogen-exchange data. It is concluded that there is presently no incontrovertible evidence for a fraction of unstacked open base pairs greater than about 10^?4.     

Diffusion of DNA at very low concentrations

The diffusion behavior of DNA samples of molecular weights between 1 × 10⁶ and 25 × 10⁶ Daltons was investigated under standard conditions at mean concentrations c? between 0.0009 and 0.017 g/dl. Special techniques described previously were used and supplemented. The sensitivity required was accomplished by multiple passage through the sample cells (effective path length of 10–45 cm) and application of the Gouy interference method. The maximum DNA refraction index difference has been determined more precisely from Gouy interference fringes by applying a systematic variation procedure and a linear-plot criterion. Convection was prevented by a temperature constancy better than 0.002°C/day, vibrationless operation, and by application of a slight density gradient of heavy water, which also improved the boundary-forming procedure. The corresponding optical HDO gradient was compensated. The concentration dependence of the DNA diffusion coefficient average D_A was found to be positive and very small at extremely low concentrations, that is, below c? = 0.008 g/dl, for the sample of highest molecular weight investigated. With beginning penetration of different DNA molecules, D_A increases markedly. The diffusion constant averages of our polydisperse samples will be corrected for monodisperse subfractions in a following paper. The resulting molecular weights M from diffusion and sedimentation constants (D⁰, s⁰) together with data from literature are the basis of new s⁰–M, D⁰ ? M, and [η]–M relations for monodisperse DNA samples.     

Quasi-elastic light scattering study of salt induced conformational transitions of chromatin subunit

The translational diffusion coefficient D_T of monodisperse solutions of 146 base pairs (bp) core particles was studied by the quasi-elastic light scattering technique. When the salinity was raised a change of D_T from 1.9 × 10^?7 cm² s^?1 to 3.2 × 10^?7 cm² s^?1 was detected at about 2 mM NaCl, followed by a smooth decrease of D_T beyond 0.6 M NaCl. The measurements of particle concentration and scattering vector effects on the D_T showed that the influence of interactions between particles can be disregarded. The interaction between particles and counterions is also discussed and does not appear to be the origin of the actual changes in D_T. These transitions of D_T are hence related to changes of shape and size of the particles. It is shown that the single transition at low salinity corresponds to a conformational change while the variation of D_T at high salinity can be interpreted by a destabilization of the edifice. In different regions of salinities, the observed values of D_T can lead to reasonable hydrodynamic models.