Opticohydrodynamic properties of high-molecular-weight DNA. III. The effects of NaCl concentration

Excluded volume and persistence length of high-molecular-weight DNA from T2 bacteriophage have been evaluated over a range of NaCl concentrations from 0.005 to 2.0M using low-shear flow-birefringence and intrinsic-viscosity data. Uncertainty in persistence length due to ambiguity in the assignment of intrinsic birefringence has been avoided by calibrating the data at 0.2M NaCl using a recently reported persistence-length value based upon photon correlation spectroscopy [Jolly, D. & Eisenberg, H. (1976) Biopolymers 15 , 61]. Results at high salt concertrations are in satisfactory agreement with other estimates of excluded volume and chain flexibility in the literature, but at very low salt concentrations they reflect greater chain expansion than has heretofore been reported. The extinction-angle data imply a transition from a nondraining chain with excluded volume at 0.1M NaCl to an almost freely draining chain at 0.005M NaCl. Over this same salt range, the experimental persistencelength data agree very well with Flory's thermodynamic chain-expansion theory [Flory, P. J. (1953) J. Chem. Phys. 21 , 162], but are in generally poor agreement with other theoretical treatments. A detailed comparison of the results with other data in the literature suggests that the combined flow-birefringence-intrinsic-viscosity technique employed here may be more sensitive to the distribution of chain stiffness and excluded volume in polyelectroytechain expansion of DNA than are othe rhydrodynamic methods such as sedimentation or intrinsic viscosity alone.     

Dilute solution of amylose in dimethylsuldoxide

Measurements of light scatting, sedimentation equilibrium, sedimentation velocity, and viscosity were carried out on fractions of native amylose in dimethylsulfoxide at 25°C. The data for statistical radius as a function of weight-average molecular weight M_w suggested a stiff nature of this biopolymer in the solvent studied when interpreted in terms of Kirste's recent calculations with a stiff chain model. The data for sedimentation coefficient were consistent with this suggestion, and when analyzed in terms of the theory Hearst and Stockmayer for wormlike chain, a value of 233 Ų was obtainedd for a/λ, where a is the length of a monomer unit projected on the chain axis and (2λ)^?1is the persistence length of the wormlike chain. The intrinsic viscosity data gave a high a value as 0.91 for the exoponent in the Houwink-Mark-Sakuarada equation, in Substantial agreement with Cowie's prenious work. We attempted to interpret these data by use of the Eizner-Ptitsyn equation for wormlike chains, with omission of the free-drainage term and introduction of the a/λ value obtained from sedimentation data. It was found that, except in the region of M_w above one million, the observed values were fitted well by the E-P theory with a = 1.4 Å and (2λ)^?1 = 87 Å. The disagreement in the high-molecular-weight region was tentatively attributed to excluded volume effect. The a value obtained suggests that the molecular conformation of amylose in dimethylsulfoxide is predominantly helical, in contrast to that of the same polymer in aqueous solutions of simple electrolyte. It was also found that a similar value of a was derived from our data for the second virial coefficient and partial specific volume if the molecule was assumed to be essentially rodlike.     

Hydrodynamic characteristics and equilibrium rigidity of pullulan molecules

The hydrodynamic characteristics of the polysaccharide pullulan (polymaltotriose) in water have been investigated and its molecular characteristics have been determined. Experimental values varied over the following ranges: velocity sedimentation coefficient (S): 0.9 < S < 11.2, translational diffusion coefficient (10⁷ cm² s⁻¹): 1.1 < D < 14.7 and intrinsic viscosity (cm³ g⁻¹): 6.7 < [η] < 164, which corresponds to a change in molecular weight (× 10³) in the range 3.9 < M_SD < 644. On the basis of analysis of the literature and our experimental data, excluded volume effects have been shown to have a prevailing influence on the chain length of these polysaccharides. The equilibrium rigidity and hydrodynamic chain diameter of pullulan were evaluated on the basis of the theory of hydrodynamic properties of a wormlike necklace, taking into account excluded volume effects. At low M (< 30 × 10³) the translation friction data (in contrast to viscometric data) cannot be described in the framework of the theory of linear molecules.     

The molecular size and shape of the Folch-Pi apoprotein in aqueous and organic solvents.

In 1% acetic acid, sedimentation velocity measurements and equilibrium ultracentrifuge experiments demonstrate that the Folch-Pi apoprotein is not monodisperse. The weight-average molecular weight calculated from ultracentrifuge experiments and combining sedimentation coefficient and viscosity measurements, ranged from 64000 to 80000. The intrinsic viscosity value suggests an asymetric shape for the apoprotein if a low value of hydration is considered. In dioxan/1% acetic acid (2:3, v/v) a smaller sedimentation coefficient was found, the intrinsic viscosity value remaining identical to that in 4% acetic acid. In pure 2-chloroethanol, light-scattering experiments led to a molecular weight of 165000 indicating that even in this solvent the protein is not monomeric. Intrinsic viscosity and light scattering measurements on the one hand, primary sequence on the other hand (six proline residues per monomer of Mr 23500) suggest that the molecule in 2-chloroethanol may consist of rod-like segments with flexible junctions.     

Donnan membrane equilibrium,sedimentation equilibrium,and coil expansion of DNA in salt solutions

This is a review of applications of the McMillan-Mayer-Hill virial theory and the ionic double-layer theory to dilute colloidal solutions, in particular, solutions of DNA. Interactions of highly charged colloidal rods are developed in terms of the second virial coefficients between two rods, and between one rod and one small co-ion. The relevant cluster integrals are evaluated with interaction potentials based on the Poisson-Boltzmann equation. The treatment is extended to the intrachain repulsion responsible for the statistical swelling of coiled DNA (excluded volume effect). The theory is compared with three sets of experimental data: The salt distribution in Donnan membrane equilibria of DNA-salt solutions, sedimentation equilibria of short DNA fragments at different ionic strengths, and the intrinsic viscosity of T7 DNA in NaCl solutions. In all cases the theory agrees well with the experiments. The agreement is not convincing for the sedimentation equilibrium at low ionic strength, because here the experimental DNA concentration is too high for the truncated dilute solution expansion of the DNA-salt repulsion.     

Molecular Weight and Chemical-Modifications of the Gum from the Seeds of Sesbania cannabina (RETZ.) PERS

The water-soluble polysaccharide is isolated and purified from the seeds of Sesbania cannabina (Retz.) Pers. Its physiochemical properties are investigated. The purified preparation seems to be homogeneous after ultra-centrifugation. The sedimentation coefficient of the gum obtained from alcoholic purification is 4.71 S, its intrinsic viscosity is 5.93 dl/g and its molecular weight is estimated to be 391,000. When it is obtained from copper alcoholic purification, however, its sedimentation coefficient is 2.82 S, its intrinsic viscosity 2.44 dl/g and its molecular weight 206,000. Several derivatives of the gum have been prepared, including carboxymethylated gum, hydroxyethylated gum and carboxymethylhydroxyethylated gum.     

Pressure cell assisted solution characterization of galactomannans. 3. Application of analytical ultracentrifugation techniques

The pressure heating cell approach previously applied to galactomannans in two earlier studies is now used to prepare samples for characterization using the analytical ultracentrifuge. Sedimentation velocity data were obtained for both guar gum and locust bean gum samples. These were compared to our earlier light scattering and intrinsic viscosity measurements on samples prepared using identical temperature and pressure profiles. A number of methods were then employed to obtain chain persistence lengths, including the Hearst-Stockmayer and Bohdanecky wormlike chain approaches. These results were compared to earlier results obtained using methods appropriate for excluded volume coil and rodlike chains, respectively.     

Light scattering from wormlike chains with excluded volume effects

This paper reports a calculation of the angular dependence of light scattering from wormlike chains with excluded volume effects. The Daniels distribution function, modified for excluded volume effects, is used to compute averages for scattering elements separated by contour lengths which are long with respect to the persistence length of the chain. An expansion in terms of exactly known moments of the distribution for the wormlike coil without excluded volume is used for short contour lengths. The results are applied to scattering from calf thymus (M = 18.1 × 10⁶) and T7 (M = 25.4 × 10⁶) DNA. It is demonstrated that the same values of excluded volume parameter (ε = 0.11) and statistical segment length (1/λ′ = 900 Å) which explain the sedimentation and viscosity behavior of DNA also account satisfactorily for the scattering behavior. Molecular weights and root-mean-square radii estimated by extrapolation from scattering data obtained in the angular region from 10° to 25° will be 5–10% too large for DNA of molecular weight 20 × 10⁶–30 × 10⁶.     

Sedimentation and intrinsic viscosity behavior of PM2 bacteriophage DNA in alkaline solution

The sedimentation coefficient and intrinsic viscosity of nicked and closed circular PM2 bacteriophage DNA have been measured as a function of pH in the alkaline region. A gradual increase in the sidimentation coefficient, and a corresponding decrease in the intrinsic viscosity, are observed for the superhelical (closed) circle in the pH region from 10.5 to about 10.9. This has been tentatively interpreted in terms of the known dependence of sedimentation coefficient upon the number of superhelical turns. At slightly higher pH values, the curve passes through the minimum (sedimentation coefficient) and maximum (intrinsic viscosity) expected when the superhelical turns present at neutral pH are unwound by partial alkaline denaturation. Sedimentation studies of the relaxed (nicked) circular species have revealed the existence of DNA forms in the pH region from 11.27 to 11.37 which sediment considerably faster than the closed circle in the same pH region. These have been identified as partially denatured nicked circles, in which varying fractions of the duplex structure have undergone alkaline denaturation, but strand separation has not yet occurred. Varying fractions of a slower species, either undenatured or completely denatured nicked circles, are also observed in some of these experiments. A corresponding result is observed in the intrinsic viscosity vs. pH curve. When nicked circular PM2 DNA is exposed to various alkaline pH's, rapidly neutralized, and sedimented at neutral pH, the expected sharp transition from native to denatured (strand-separated) molecules is seen. However, a very narrow pH range is noted in which native and denatured forms coexist in a single experiment. The above experiments carried out upon the closed form also reveal a narrow pH range in which the bulk of the transition from native closed circles to the collapsed cyclic coil takes place, in acccord with an earlier study on a different DNA. This transition is shown never to be completely effected, however, as there is a fraction (7–8%)of the closed circles which renature to the native form, regardless of the alkaline pH employed. This same phenomenon was not observed in the case of artificially closed λb₂b₅c DNA circles. Possible explanations for some of the above results are discussed.     

Rapid characterization of protein molecular weights and hydrodynamic structures by quasielastic laser-light scattering

Two methods for the characterization of protein molecular weights from their diffusion coefficients are discussed. These measurements can be made quickly and reliably at low concentrations using quasielastic light-scattering techniques. First, an empirical calibration of the diffusion coefficient at infinite dilution of denatured random coils against molecular weight is reported. The second method combines the measurement of D₀ with the intrinsic viscosity [η]. This D₀–[η] relationship proves to be very insensitive to polymers structure or solvent type. The data indicate that the ratio of the hydrodynamic radius measured by viscosity to the hydrodynamic radius measured by diffusion is about 15% smaller than that predicted by theoretical models. The nature of the molecular-weight average obtained for polydisperse systems is defined for a Schulz distribution. These hydrodynamic methods have also been used to demonstrate the presence of chain branching in the glycoprotein ovomucoid. In addition, a method is proposed by which the effective segment length and an excluded volume parameter for random coils may be evaluated for diffusion measurements.     

Viscoelastic behavior of fractionated ovine submaxillary mucins.

Solution properties of fractionated ovine submaxillary mucin (OSM) and asialo OSM (aOSM) in aqueous guanidine hydrochloride have been investigated using light scattering and rheological methods. For the first time we present viscometric evidence in both dilute and concentrated solution that the molecular structure of OSM is that of a wormlike chain. The intrinsic viscosity shows molecular weight dependence consistent with the linear extended chain conformation observed by light scattering measurements. The viscoelastic behavior of the OSM fractions in aqueous guanidine hydrochloride was further examined above the overlap concentration as a function of molecular weight and temperature. Under these solvent conditions in which the role of nonbonding intermolecular interactions is minimized, OSM shows predominantly fluid like behavior. However, high molecular weight OSM shows evidence of the existence of an entanglement network at high concentration. The frequency-dependent shear storage and loss moduli at all concentrations and molecular weights can be scaled to yield a master curve by incorporating typical viscoelastic shift parameters. The entanglement molecular weight and concentration are consistent with literature data for extended, semiflexible wormlike chains. The behavior of aOSM is similar to that of intact OSM at comparable degrees of coil overlap, indicating that the terminal sialic acid residue on the carbohydrate side chain has no effect on the rheology of concentrated OSM solutions beyond that due to an increase in the hydrodynamic volume.     

Persistence length of DNA

In the wormlike chain (Kratky-Porod) model of DNA the stiffness of the chain is determined by the persistence length, a. The persistence length may be evaluated from light-scattering measurements of the molecular weight and the mean-square radius if the samples are not polydisperse or if the polydispersity can be quantitatively determined. The persistence length can also be evaluated with the aid of hydro dynamic theory from measurements of intrinsic viscosity and sedimentation coefficient. Data taken from the literature and from other studies by the authors are examined by these methods. The light-scattering method yields a value of a of 900 ± 200 Å the hydrodynamic data yield 600 ± 100 Å. These values are considerably larger than those obtained by most previous authors.     

The conformation of histone f2al (histone IV) in solution at low ionic strength

Hydrodynamic studies of histone f2a1 are performed in dilute salt solution. Protein sedimentation is shown to be dependent upon the partial specific volume and molarity of the supporting electrolyte. To compensate for the secondary chage effect, a solvent of dilute tetramethylammonium chloride, for which (1 ? V?_saltρ) ? 0, is used. To correct for the primary charge effect in sedimentation, a special linear extrapolation to infinte dilution of the protein is employed. Measurements of intrinsic viscosity are interpreted in terms of an increase in molecular dimension with a decrease in ionic strength. The conformation of histone f2a1 in dilute salt solution is interpreted from sedimentation and viscosity data to be that of a highly charged random coil possessing 20–30% of nuclear globular structure.     

Single-stranded RNA molecular weight and shape determination by differential pressure capillary viscometry, sedimentation velocity, and gel electrophoresis.

Determination of the size of a population of nucleic acids can be achieved by several distinct methods. Most of these methods are cumbersome and require complicated equipment or techniques. We demonstrate here the use of a differential pressure capillary viscometer for the rapid and simple determination of RNA molecular weight. This highly sensitive viscometer allowed single viscosity determinations on dilute solutions of RNA, providing a direct measure of the intrinsic viscosity without the need to extrapolate from several concentrations. The molecular weights and conformations of the linear single-stranded RNA homopolymer poly(inosinic acid) (poly(I] and the single-stranded RNA (ssRNA) copolymer poly(cytidylic acid:uridylic acid, 12:1) (poly(C12,U], were determined. The ssRNAs were synthesized in a range of sizes (100 to 100,000 bases). They were widely polydisperse. The Mandelkern-Flory equation (1952, J. Chem. Phys. 20, 212-214), which requires both the intrinsic viscosity and sedimentation coefficient of a macromolecule, was used to calculate the molecular weights. The molecular weights determined by agarose gel electrophoresis were compared to those determined by intrinsic viscosity plus sedimentation coefficient. The correlation between the molecular weights determined by these two methods was good, at R2 greater than or equal to 0.92. The conformations of the RNAs were determined by application of the Mark-Houwink equation. The Mark-Houwink exponents for poly(I) and poly(C12,U) intrinsic viscosities were 0.90 and 0.84, respectively. When compared to other nucleic acid polymers, for which the conformations have been established by several methods, poly(I) and poly(C12,U) are rigid, extended random coils, in a low-salt buffer (15 mM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes of the oligomeric structure of legumin from pea (Pisum sativum L.) after succinylation

The influence of various levels of succinylation on the structure of the legumin from pea seed has been studied by the techniques of sedimentation velocity, viscometry, fluorescence and circular dichroism spectroscopy, as well as dynamic light scattering. The protein dissociates gradually into the 3S subunit forming a 7S intermediate. At a level of 75-80% succinylation, sudden unfolding of the protein occurs characterized by drastic changes in viscometric and spectroscopic properties. The fluorescence spectra point to the formation of a novel organized structure at a moderate degree of modification before the molecular unfolding takes place. The succinylated subunit was shown to have a sedimentation coefficient of 3.2S, a diffusion coefficient of 5.03 x 10(-7) cm2 . s-1 a Stokes' radius of 4.24 nm, a partial specific volume of 0.703 ml/g, an intrinsic viscosity of 0.13 dl/g, a molar mass of 52.2 kDa and a frictional ratio of 1.74.     

Determination of molecular weight and related hydrodynamic parameters of high molecular weight protein fraction from a few oilseeds

The hydrodynamic parameters of the major protein fraction, viz. arachin from groundnut, alpha-globulin from sesame seed, brassin (M) from mustard seed and helianthinin from sunflower seed, have been determined in a single solvent system (0.05 M Tris-HCl buffer, pH 7.5 containing 0.5 M sodium chloride): sedimentation coefficient (s0(20,w)) and diffusion coefficient (D0(20,w)) by analytical ultracentrifugation, intrinsic viscosity [eta] by Ostwald viscometry and partial specific volume (V) by densimetry. The molecular weights (M) of the four proteins, calculated using the sedimentation-viscosity and sedimentation-diffusion coefficient methods, were found close to each other. The values have been compared with those in the literature and the reasons for discrepancies have been discussed.     

Some physical and chemical properties of trypsin-digested nucleoprotein

A DNA-peptide complex that is soluble in 0.2m-sodium chloride can be prepared by trypsin digestion of calf thymus nucleoprotein. The trypsin-digested nucleoprotein molecule contains about 70% of DNA and 30% of peptides by weight, and consists of one DNA molecule associated with arginine-rich peptides. A series of trypsin-digested nucleoprotein preparations differing only in molecular weight were prepared by blending. The intrinsic viscosity and average sedimentation coefficient were determined for each of these preparations. Then the DNA was isolated from each preparation and the hydrodynamic measurements were repeated on the DNA. From a comparison of these results it was concluded that the presence of the complex-forming peptides causes a large decrease in intrinsic viscosity of the DNA and an increase in sedimentation coefficient. In addition, the hydrodynamic data indicate that the DNA-peptide complex behaves like a coil in solution but is more compact than the same length of DNA. The ;melting' profiles, streptomycin precipitation curves and maximum viscosities obtained with ethidium bromide binding for the trypsin-digested nucleoprotein are similar to those of purified DNA, and markedly different from those of undigested nucleoprotein. These findings suggest that the peptides are not strongly associated with the DNA, and that secondary valency forces are involved in the binding.     

Chain stiffness of heteropolysaccharide from Aeromonas gum in dilute solution by dynamic light scattering

Dynamic light scattering measurements have been made on 15 fractions of aeromonas (A) gum, an extracellular heteropolysaccharide produced by the strain Aeromonas nichidenii, with dimethylsulfoxide containing 0.2M lithium chloride as the solvent at 25 degrees C. Data for the translational diffusion coefficient D covering a molecular weight range from 4.5 x 10(5) to 2.1 x 10(6) and ratios of the z-average radius of gyration (z) (1/2) to the hydrodynamic radius R(H) (calculated with previous (z) data) suggest that the polymer behaves like a semiflexible chain in this solvent similar to the stiffness of cellulose derivatives. Thus the D data are analyzed on the basis of the Yamakawa-Fujii theory for the translational friction coefficient of a wormlike cylinder by coarse-graining the heteropolysaccharide molecule. Excluded-volume effects are taken into account in the quasi-two-parameter scheme, as was done previously for (z) and [eta] (the intrinsic viscosity) of A gum in the same solvent. The molecular weight dependence of R(H) is found to be explained by the perturbed wormlike chain with a persistence length of 10 nm, a linear mass density of 1350 nm(-1), an excluded-volume strength parameter of 1.3 nm, and a chain diameter of 2.8 nm. These parameters are in substantial agreement with those estimated previously from (z) and [eta] data, demonstrating that the solution properties (D, (z), and [eta]) of the heteropolysaccharide are almost quantitatively described by the current theories for wormlike chains in the molecular weight range studied.     

Viscoelastic properties of an exopolysaccharide: Aeromonas gum, produced by Aeromonas nichidenii 5797

The viscoelastic properties of Aeromonas (A) gum in water were investigated by using the Rheometric Scientific ARES controlled strain rheometer. An intrinsic viscosity of 8336 ml/g was obtained according to the Fuoss-Straus equation. The effect of salt concentration on intrinsic viscosity revealed that the A gum exists as semiflexible chain. Typical shear-thinning (pseudoplastic) behavior was observed at concentrations higher than 0.52%. The zero shear viscosity (eta(0)) increased with increasing polysaccharide concentration (c) showing a gradient of approximately 1.0, 2.9 and 4.8 in different concentration domains. The critical concentrations c* and c**, at which the transitions from a dilute solution of independently moving chains to semidilute and then concentrated domains occurred, were determined roughly to be 1.2% and 3.5%. The results from dynamic experiments revealed that the A gum solution shows characteristics of polymer solutions without any evidence of gel-like character. All the results from steady and dynamic tests suggest that the A gum is a non-gelling polysaccharide. The temperature dependence of apparent viscosity was described by Arrhenius equation and the flow activation energy was estimated to be 45.2 kJ/mol, which is independent on polymer concentration.     

Effect of molecular weight distribution on the solution properties of sodium hyaluronate in 0.2M NaCl solution

Various molecular parameters, which characterize sodium hyaluronate in 0.2M NaCl solution, were obtained at 25°C by means of the static and dynamic light scattering and low shear viscometry over the molecular weight range of 5.94–627 × 10⁴. Molecular weight distribution was obtained by using the Laplace inversion method of the autocorrelation function of the scattered light intensity and by Yamakawa theory for the wormlike chain with the stiff chain parameters for sodium hyaluronate in 0.2M NaCl (persistence length, chain diameter, molar mass per unit contour length, and the excluded‐volume strength). The molecular weight distribution thus obtained reproduced the solution properties of sodium hyaluronate well. Especially, the intrinsic viscosity showed a good agreement over four orders of molecular weight with Yamakawa theory combined with the Barrett function. Sodium hyaluronate in 0.2M NaCl solution is well expressed by the wormlike chain model affected by the excluded‐volume effect with the persistence length of 4.2 nm. © 1999 John Wiley & Sons, Inc. Biopoly 50: 87–98, 1999