Description of the thermodynamic incompatibility of the guar–dextran aqueous two-phase system by light scattering

The phase diagram of the guar–dextran aqueous two-phase system has been described on the basis of static light scattering measurements in the dilute regime. By determining the molar weight and second virial coefficient from the two single polymers and the second virial cross coefficient from mixtures at constant guar/dextran ratio (either 17/83 or 28/72), the thermodynamic models based on the virial expansion or the Flory–Huggins theory were successfully applied. The second virial coefficient of guar was difficult to estimate with enough accuracy by light scattering and therefore was obtained by adjustment using a simple criterion stating that the calculated spinodal passes through the experimental critical point. The obtained value was within the confidence interval given by light scattering. Virial expansion and Flory–Huggins approaches yielded quite similar theoretical phase diagrams that satisfactorily fitted the experimental one. The slight discrepancies observed on the position of binodals and critical points has been attributed to the polydispersity of guar or the difficulty in extrapolating from the dilute regime to the semidilute one. The slope of the tie-lines was predicted with a good accuracy, especially with the virial expansion model. The fact that both approaches gave such similar results is probably related to the fact that the expressions of chemical potentials are equivalent if the polymer concentrations are low enough. In this particular case, both models are based on excluded volume interactions and equally describe the phase behavior of the guar–dextran aqueous system.     

Graphical analysis of nonideal monomer N-mer, isodesmic, and type II indefinite self-associating systems by equilibrium ultracentrifugation.

A graphical procedure is described by which one can obtain in principle the monomer molecular weight, stoichiometry, equilibrium constant, and second virial coefficient of nonideal monomer N-mer, isodesmic, and type II indefinite self-associating systems. In addition, a method is presented for obtaining both the equilibrium constant and the second virial coefficient from the maximum in a plot of apparent molecular weight vs. concentration if the monomer molecular weight and stoichiometry are known. The usefulness and limitations of the methods are discussed, as well as the quality and range of data required for determination of the relevant parameters. The techniques described are applicable to analysis of self-associating systems by osmotic pressure and light scattering, as well as equilibrium ultracentrifugation measurements.     

Fractionation of whole histone by partition chromatography on Sephadex G-25.

Whole histone from calf thymus was fractionated by partition chromatography on the basis of distribution between an aqueous phase immobilized on Sephadex G-25 beads and mobile organic phases containing various concentrations of trichloroacetic acid. The chromatography was carried out by stepwise elution with five upper phases from water and butanol-2 solvent systems containing 4 M urea and 0.1-1.5% trichloroacetic acid, and finally water. Of the six peaks obtained, two (peaks 1 and 2) contained arginine-rich histones. Although these peaks were still heterogeneous electrophoretically, the band corresponding to F2al was observed only in the electrophoretic pattern of peak 1 and the main fraction in peak 2 was F3. A histone fraction having nearly equimolar amounts of arginine and lysine was obtained from peak 3. Its amino acid composition was similar to that of F2a2. Slightly lysine-rich histone obtained from peak 4 showed an amino acid composition typical of F2b. Peak 5 contained a histone fraction with a ratio of lysine/arginine of 6.14, showing a single band on gel-electrophoresis. Very lysine-rich histone (F1) was obtained from peak 6, and the electrophoretic pattern of this fraction showed a single band.     

Analysis of the thermodynamic non-ideality of proteins by sedimentation equilibrium experiments

This paper presents a modified method to determine experimentally the second virial coefficient of protein solutions by sedimentation equilibrium experiments. The improvement is based on the possibility of fitting simultaneously up to seven radial concentration distribution curves of solutions with different loading concentrations. The possibility of precise determination of the second virial coefficient allows estimation of the net charge and the excluded volume of a monomeric protein. Application of the method is demonstrated for lysozyme and ovalbumin. In 0.1 M sodium acetate buffer, pH 4.5, the second virial coefficient of hen egg white lysozyme amounts to 24 +/- 1 ml/g. Analysis based on spherical particle theory yield an excluded volume of 3.5 ml/g and a charge dependent value of 20.5 ml/g which is induced by a net charge number of 14.1 +/- 1. Under low salt conditions self-association processes on lysozyme are unfavorable due to electrostatic repulsion. To overcome these repulsive contributions, either a shift to neutral pH or addition of at least 2% NaCl is necessary. In this way the charge dependent contribution decreases below the value responsible for the excluded volume and allows crystallization of the protein. Similar effects can be observed with ovalbumin. The high virial coefficient observed at pH 8.5 is induced by the high net charge number of 27 +/- 1.     

The self-association of human spectrin at high concentration.

The self-association of purified human spectrin has been studied at sedimentation equilibrium over a wide range of concentration (0-20 g/L) at 30 degrees C and pH 7.5. Coincidence of apparent weight average molecular weight and omega (r) plots as a function of total spectrin concentration indicated that equilibrium was attained and that no significant concentration of solute was incapable of participating in the self-association reaction. Under these conditions, no significant dissociation of the heterodimer to component polypeptide chains could be detected. The behavior of spectrin between 0 and 20 g/L can be described reasonably well by a cooperative isodesmic model, in which the protomer for association is the alpha beta heterodimer. With this model, the equilibrium constant for the heterodimer-tetramer step, K24, is 2 x 10(6) M-1, and K(iso), the equilibrium constant describing all other steps, is approximately 0.2 x 10(6) M-1. The returned value of the second virial coefficient for this model, 1.0 x 10(-7) L mol g-2, is consistent with the lower limit of values calculated for the heterodimer from the charge and Stokes radius of spectrin. On the other hand, the attenuated indefinite association model fails to describe the self-association of spectrin adequately over the range 0-20 g/L. Systematic decreases in the estimates of the second virial coefficient and the equilibrium constants for association beyond the tetramer suggest that the assumption of a single value of the second virial coefficient may not be appropriate for spectrin, and that non-ideality would best be taken into account by consideration of the detailed solution composition.     

Lysine-rich histone H1 kinase from soybean hypocotyl.

A protein kinase (ATP: histone phosphotransferase) with high specificity for the phosphorylation of the very lysine-rich histone H1 has been partially purified and characterized from soybean hypocotyl. The enzyme has a molecular weight of about 48,500. Its activity and sedimentation behavior are refractory to cyclic nucleoside monophosphates. No significant amount of cyclic AMP or cyclic GMP binding activity could be detected in the crude or partially purified enzyme preparations. Km for ATP and histone H1 are 0.4 μM and 0.7 μM, respectively. The enzyme requires Mg²⁺ or Mn²⁺ for activity, while addition of 0.5 mM Ca²⁺, Zn²⁺ or Hg²⁺ results in 50% inhibition. Arginine-rich histones H3 and H4 are inhibitory to histone H1 phosphorylation; these histones affect the Vmax of the enzyme, but not the Km for histone H1.     

Structural characteristics of lysine-rich histone from the sperm of a mollusk Anodonta piscinalis

Sperm of freshwater bivalve mollusk Anodonta piscinalis was found to contain two fractions of lysine-rich histone: somatic histone H1 and sperm-specific protamine-like histone, named Hp. A detailed analysis of H1 and Hp structure was carried out by means of N-bromosuccinimide, chymotrypsin and pepsin cleavage followed by determination of the lysine residue number, positive charge and molecular length of obtained fragments by the method of incomplete succinylation. It has been shown, that Anodonta histone H1, like the avian histone H5, contains 3 tyrosine residues in the central hydrophobic domain of the molecule. Histone Hp contains 5 tyrosine residues, 3 of which are localized in the hydrophobic domain, while the rest two--in the COOH-terminal part of the molecule, characterized by a strong positive charge. Such unusual disposition of tyrosine residues in the lysine-rich histone has been found for the first time. All the regions of histone Hp molecule contain a great number of arginine residues. The only phenylalanine residue is localised approximately in the middle of the polypeptide chain for both H1 and Hp molecules. On the basis of structure homology between histones H1 and Hp the origin of Hp from H1 in the course of evolution is proposed.     

Tissue-specific histones in the erythrocytes of chicken and turtle

Mature erythrocytes from Leghorn chickens contain lysine-rich histone F1 and a tissue-specific histone F2c. The composition of the F1 fraction was found to be similar to the F1 histones in higher vertebrates. In the erythrocytes of a sea turtle (Chelonia mydas), only lysine-rich histones F1 could be detected. One of these fractions (F1b) differed in amino acid composition from the typical F1 histones described in the literature. The F1b histone fraction was not found in turtle liver. Chromatographic analysis of tryptic peptides of the chicken erythrocyte F1 and F2c histones and of the turtle erythrocyte F1a and F1b histones revealed considerable similarities between these four fractions, thus indicating their possible phylogenetic relationships.     

Laser light scattering measurements on vitreous and rooster comb hyaluronic acids

Molecular weights and translational diffusion coefficients have been measured for rooster comb and vitreous hyaluronic acid (HA) at pH 7.2 and 11. The results indicate that the molecular weight, second virial coefficient and translational diffusion coefficient for vitreous HA can be reversibly decreased by increasing the solution pH from 7.2 to 11, whereas the physical properties of rooster comb HA are independent of pH studied. In addition, it is reported that the second virial coefficient for vitreous HA is negative, suggesting intermolecular interactions exist in solution at both neutral and alkaline pH as opposed to rooster comb HA which exhibits a positive second vitrial coefficient associated with decreasing molecular weights may be related to the accessibility and number of hydrogen bond forming groups. Differences in the dependence of molecular weight on pH between vitreous and rooster comb HA may be due to differences in the number of intramolecular interactions per molecule. These studies indicate that molecules of low molecular weight HA are able to form higher molecular weight complexes and differences in the organization of the polysaccharide chains may contribute to the differences in molecular weight of HAs isolated from various tissues.     

Histone-acetylating enzyme of brain

1. Acetylation of histones by an enzyme system derived from rat brain and liver (histone acetylase) was studied by using [1-(14)C]acetyl-CoA as the acetyl group donor. 2. The activity of this enzyme was largely confined to the nucleus. 3. Histone-acetylating activity of cerebral nuclei purified by centrifugation through 1.9m-sucrose was not altered by the presence of the cytoplasmic fraction. 4. Cerebral nuclei from adult rats exhibited greater histone-acetylating activity than did the corresponding preparation from newborn animals. 5. Nuclear acetylating activity was higher in brain than in liver of adult rats but not in newborn animals. 6. The partially purified enzyme from cerebral nuclei, prepared by ammonium sulphate fractionation of an acetone-dried powder, specifically catalysed histone acetylation. 7. Polylysine, protamine, serum albumin and gamma-globulin were not enzymically acetylated by this preparation. 8. Soluble acetylating preparations from both brain and liver nuclei were more active towards arginine-rich F3 and slightly lysine-rich F2a and F2b histone fractions than towards the lysine-rich F1 fraction. 9. Enzymic acetylation of chromatin-bound proteins was much less extensive than that of free histones. 10. The high histone acetylase activity in mature brain may reflect the importance of this process in the genetic control of cerebral function.     

Second virial coefficient of alpha-crystallin

Light scattering studies were performed on bovine alpha-crystallin measuring the scattering intensities as a function of scattering angle, concentration, and temperature. The data yielded the molecular weight, radius of gyration, and second virial coefficient of alpha-crystallin at different temperatures. The second virial coefficient increased with increasing temperature. Both the enthalpy and entropy of solution of alpha-crystallin are positive. The Flory theta temperature was found to be 271 K.     

Studies on the myosin molecule from smooth muscle

The myosin molecule was extracted from the smooth muscle parts of horse esophagus and purified by ammonium sulfate fractionation. The schlieren pattern of the sedimentation velocity run showed a very sharp single peak of.5.9. S (s_20,w). Molecular weight of the protein was measured by means of the Archibald and sedimentation equilibrium methods, both in 0.5M KCI buffered by 1/150 M phosphate at pH 7.5 and at 5°C. The values obtained were 6.25 × 10⁵ and 5.81 × 10⁵respectively, for the two methods. The second virial coefficients were 1.1 × 10⁴ and 1.2 × 10^?4 ml/g. Denatured smooth muscle myosin was prepared in a solution of 5M guanidine HC1 containing 0.4 M KC1 and 0.2 M β-mercaptoet hanol buffered at p^H 8.0. The weight-average molecular weight of the denatured smooth muscle myosin was 2.24 × 10⁵ and the second virial coefficient was 7.6 × 10^?4 ml/g. The values described above are in good agreement with those reported for rabbit skeletal myosin with ammonium sulfate fractionation. The molecular dimension of the molecule is estimated as the value for an axial ratio of 100, assuming a rigid rod molecular model for this molecule, both the thermodynamical and hydrodynamical treatment being in a good agreement with this estimation.     

Effect of secondary structure on the potential of mean force for poly-L-lysine in the alpha-helix and beta-sheet conformations

Because poly-L-lysine (PLL) can exist in the alpha-helix or beta-sheet conformation depending on solution preparation and solution conditions, PLL is a suitable candidate to probe the dependence of protein interactions on secondary structure. The osmotic second virial coefficient and weight-average molecular weight are reported from low-angle laser-light scattering measurements for PLL as a function of NaCl concentration, pH, and alpha-helix or beta-sheet content. Interactions between PLL molecules become more attractive as salt concentration increases due to screening of PLL charge by salt ions and at low salt concentration become more attractive as pH increases due to decreased net charge on PLL. The experimental results show that interactions are stronger for the beta-sheet conformation than for the alpha-helix conformation. A spherically-symmetric model for the potential of mean force is used to account for specific interactions not described by DLVO theory and to show how differences in secondary structure affect PLL interactions.     

A cyclic adenosine 3',5'-monophosphate-dependent histone kinase from pig brain. Purification and some properties of the enzyme.

A cyclic adenosine 3',5'-monophosphate-dependent histone kinase (ATP: protein phosphotransferase, EC 2.7.1.37) was isolated from pig brain. The enzyme has been purified 1140-fold; it is homogeneous on polyacrylamide gel electrophoresis and gel filtration. The estimated molecular weight of the enzyme is 120 000. Histone kinase dissociates into a catalytic subunit and a regulatory one (molecular weights 40 000 and 90 000, respectively). The catalytic subunit has been obtained in homogeneous state as evidenced by sodium dodecylsulphate-polyacrylamide gel electrophoresis. At all purification steps, enzymatic activity is stimulated 5-fold by cyclic AMP. An apparent Km value for cyclic AMP is about 3.3 - 10- minus 7 M. In the presence of cyclic AMP(5 - 10- minus 6 M), the Km value for ATP and F1 histone were 1.2 - 10- minus five and 3 - 10- minus 5 M, respectively. Optimum pH value for histone kinase is 6.5, its isoelectric point is situated at pH 4.6. The purified enzyme displays high specificity for the lysine-rich and moderately lysine-rich histones F1, F2a2 and F2b. Arginine-rich histones and other known protein substrates for cyclic AMP-dependent protein kinases (casein, Escherichia coli RNA polymerase, etc.) are extremely poor substrates for this enzyme.     

Determination of the second virial coefficient of bovine serum albumin under varying pH and ionic strength by composition-gradient multi-angle static light scattering

Composition-gradient multi-angle static light scattering (CG-MALS) is an emerging technique for the determination of intermolecular interactions via the second virial coefficient B₂₂. With CG-MALS, detailed studies of the second virial coefficient can be carried out more accurately and effectively than with traditional methods. In addition, automated mixing, delivery and measurement enable high speed, continuous, fluctuation-free sample delivery and accurate results. Using CG-MALS we measure the second virial coefficient of bovine serum albumin (BSA) in aqueous solutions at various values of pH and ionic strength of a univalent salt (NaCl). The systematic variation of the second virial coefficient as a function of pH and NaCl strength reveals the net charge change and the isoelectric point of BSA under different solution conditions. The magnitude of the second virial coefficient decreases to 1.13 x 10⁻⁵ ml*mol/g² near the isoelectric point of pH 4.6 and 25 mM NaCl. These results illuminate the role of fundamental long-range electrostatic and van der Waals forces in protein-protein interactions, specifically their dependence on pH and ionic strength. Electronic supplementary material The online version of this article (doi:10.1007/s10867-014-9367-7) contains supplementary material, which is available to authorized users.     

Random coil scission rates determined by time-dependent total intensity light scattering: hyaluronate depolymerization by hyaluronidase

A recently developed theory of the light scattering by random coils undergoing random scission is applied to the digestion of hyaluronate by hyaluronidase. The time dependence of the scattered light from solutions undergoing digestion was monitored. Working at a high angle with high molecular weight hyaluronate allowed the use of a powerful approximation for determining initial velocities and the Henri-Michaelis-menten coefficients, without explicit knowledge of the hyaluronate molecular weight, radius of gyration, second virial coefficient, or polydispersity. Effects due to a molecular weight dependent second virial coefficient and to non-Gaussian behavior are briefly considered. Assays were performed over nearly two orders of magnitude in substrate concentration. The initial velocities are compared with those obtained by a standard reducing sugar assay, which was performed on identical samples. The main advantages of the light scattering assay procedure over the more traditional assays are that many relatively high-precision data points can be quickly and automatically collected with simple apparatus, and that the technique is most sensitive for the initial period of digestion, where the other assays are least sensitive. The shapes of the scattering curves also provide evidence that hyaluronate in these solutions is not a stable double strand and that the hyaluronidase cleaves bond randomly. The curves also indicate that enzyme deactivation occurs, which accounts for the lower velocities yielded by the slower reductimetric assay, which is measured over longer initial periods.     

Histones from Trypanosoma lewisi nuclei]

A preparation of total histones has been isolated for the first time from the purified fractions of T. lewisi cell nuclei and characterized in terms of its chemical composition and RNA-polymerase activity. A special attention during the isolation procedure was given to the repression of proteolytic degradation of the histones. The amount of protein in the chromatin is equivalent to that of DNA. The amino acid composition and heterogeneity of the protein during polyacrylamide gel electrophoresis in an acid system and in the presence of sodium dodecyl sulfate are typical for histones. Using two-dimensional electrophoresis, differential staining of electrophoregrams and ion-exchange chromatography on CM-cellulose the total preparation has been found to be made up of five fractions: two -- arginine-rich (one of them identical to histone H4, the other being similar to histone H3 from calf thymus); two -- moderately lysine-rich fractions, slightly differing in their properties from histones H2A and H2B from calf thymus, and one specific fraction with mol. weight of 16 000 and an extremely high positive charge. The above methods in combination with specific extraction have been used to demonstrate the absence of a typical lysine histone in the preparation, which is correlated with the absence of typical methaphase chromosomes during mitosis in T. lewisi.     

H5 Histone and DNA-relaxing enzyme of chicken erythrocytes. Interaction with superhelical DNA.

The interaction of closed circular duplex DNA with the lysine-rich H5 histone fraction of avian erythrocytes has been studied. H5, like H1 histone, interacts preferentially with superhelical DNA. The extent of interaction increases with increasing negative or positive superhelicity. Salt-extracted lysine-rich histones show the same specificity for interaction with superhelices as do acid-extracted preparations. Chicken erythrocyte nuclei contain DNA-relaxing enzyme. This enzyme is extracted from the nuclei at lower salt concentrations than those required to extract H1 and H5 histones and is, therefore, probably a function of a protein distinct from H1 and H5 histones.     

Inhibition of mammary gland cyclic AMP-dependent protein kinase by arginine-rich histones.

The cyclic AMP-dependent protein kinase activity from lactating bovine mammary gland efficiently phosphorylates lysine-rich histones but not arginine-rich histones. It is shown that arginine-rich histones in fact inhibit phosphorylation of lysine-rich histones. Polyarginine and a range of low molecular weight cationic molecules are also inhibitors. Inhibition of histone H2b phosphorylation by histones H4 and H3 is competitive with respect to H2b. This inhibition behaviour may be tissue-specific since the protein kinase activity in crude extracts from lactating bovine mammary gland, although heterogeneous, may be completely inhibited (>95%) by arginine-rich histones and polyarginine.     

Higher order chromatin structure determines double-nucleosome periodicity of DNA fragmentation

Double-nucleosome periodicity of DNA fragmentation with DNAse I in the nuclei of cells differing in size of the linker DNA length and lysine-rich histone composition was analyzed by means of nondenaturing agarose gel electrophoresis. DNAse I revealed this type of periodicity in rat thymus and CHO cell nuclei as well as in erythrocyte nuclei. It has been deduced that the so-called nucleodisome structure is also typical of cells possessing a usual DNA repeat length (200 bp or less) and lysine-rich histone H1. Two probably related events are important for establishing a clear double-nucleosome periodicity of DNA fragmentation: the replacement of H1 histone by a specific arginine-rich histone fraction (H5 histone in the case of erythrocyte) and the increase of the linker DNA length. The results are interpreted in terms of supranucleosomal organization of chromatin which may determine the dinucleosome periodicity of DNA fragmentation due to a specific packing of nucleosomes.