Ionic effects in semi-dilute biopolymer solutions: A small angle scattering study.

Systematic investigations using neutron and X-ray small angle scattering in near-physiological salt solutions were made to reveal the effect of polymer concentration, pH, and calcium ion concentration on the structure of semi-dilute solutions of four model biopolymers [polyaspartic acid, DNA, chondroitin sulfate, and hyaluronic acid (HA)] representing typical backbone structures. In the low q range (<0.01 Å-1), the scattering response I(q) is dominated by scattering from large clusters. In the intermediate q range, I(q) varies approximately as q -1, exposing the linear nature of the scatterers. In these polyelectrolyte solutions, the correlation length L displays a power law dependence on the polymer concentration c that resembles that of neutral polymer solutions. L increases with increasing calcium chloride concentration and with decreasing pH. The effect of the different divalent cations, Ba, Mg, Ca, Sr, and Mn, on the structure of DNA solutions is practically identical. However, in mixed salt conditions at the same ionic strength, the combined effect of mono- and divalent counter-ions on the structure of the polymer solutions deviates significantly from additivity. Anomalous small angle X-ray scattering observations on both DNA and HA solutions reveal that the divalent strontium counter-ions form a tight sheath around the polymer chain. The shape of the divalent ion cloud is similar in these two systems.

Ions in hyaluronic acid solutions.

Hyaluronic acid (HA) is an anionic biopolymer that is almost ubiquitous in biological tissues. An attempt is made to determine the dominant features that account for both its abundance and its multifunctional role, and which set it apart from other types of biopolymers. A combination of osmotic and scattering techniques is employed to quantify its dynamic and static properties in near-physiological solution conditions, where it is exposed both to mono- and divalent counterions. An equation of state is derived for the osmotic pressure Pi in the semidilute concentration region, in terms of two variables, the polymer concentration c and the ionic strength J of the added salt, according to which Pi=1.4x10(3)c(9/4)/J(3/4) kPa, where c and J are expressed in mole. Over the physiological ion concentration range, the effect of the sodium chloride and calcium chloride on the osmotic properties of HA solutions is fully accounted for by their contributions to the ionic strength. The absence of precipitation, even at high CaCl(2) concentrations, distinguishes this molecule from other biopolymers such as DNA. Dynamic light scattering measurements reveal that the collective diffusion coefficient in HA solutions exceeds that in aqueous solutions of typical neutral polymers by a factor of approximately 5. This property ensures rapid adjustment to, and recovery from, stress applied to HA-containing tissue. Small angle x-ray scattering measurements confirm the absence of appreciable structural reorganization over the observed length scale range 10-1000 A, as a result of calcium-sodium ion exchange. The scattered intensity in the transfer momentum range q>0.03 A(-1) varies as 1/q, indicating that the HA chain segments in semidilute solutions are linear over an extended concentration range. The osmotic compression modulus c partial differential Pi/partial differential c, a high value of which is a prerequisite in structural biopolymers, is several times greater than in typical neutral polymer solutions.

Using a Bayesian network to understand the importance of coastal storms and undeveloped landscapes for the creation and maintenance of early successional habitat.

Coastal storms have consequences for human lives and infrastructure but also create important early successional habitats for myriad species. For example, storm-induced overwash creates nesting habitat for shorebirds like piping plovers (Charadrius melodus). We examined how piping plover habitat extent and location changed on barrier islands in New York, New Jersey, and Virginia after Hurricane Sandy made landfall following the 2012 breeding season. We modeled nesting habitat using a nest presence/absence dataset that included characterizations of coastal morphology and vegetation. Using a Bayesian network, we predicted nesting habitat for each study site for the years 2010/2011, 2012, and 2014/2015 based on remotely sensed spatial datasets (e.g., lidar, orthophotos). We found that Hurricane Sandy increased piping plover habitat by 9 to 300% at 4 of 5 study sites but that one site saw a decrease in habitat by 27%. The amount, location, and longevity of new habitat appeared to be influenced by the level of human development at each site. At three of the five sites, the amount of habitat created and the time new habitat persisted were inversely related to the amount of development. Furthermore, the proportion of new habitat created in high-quality overwash was inversely related to the level of development on study areas, from 17% of all new habitat in overwash at one of the most densely developed sites to 80% of all new habitat at an undeveloped site. We also show that piping plovers exploited new habitat after the storm, with 14-57% of all nests located in newly created habitat in the 2013 breeding season. Our results quantify the importance of storms in creating and maintaining coastal habitats for beach-nesting species like piping plovers, and these results suggest a negative correlation between human development and beneficial ecological impacts of these natural disturbances.

Structure and Properties of Cartilage Proteoglycans.

The most abundant cartilage proteoglycan is aggrecan, a bottlebrush shaped molecule that possesses over 100 glycosaminoglycan (chondroitin sulfate and keratan sulfate) chains. The side-chains are linear sulfated polysaccharides that are negatively charged under physiological conditions. Aggrecan interacts with hyaluronic acid (HA) to form large aggregates. Osmotic pressure measurements and rheological measurements are used to study the static and dynamic behavior of aggrecan assemblies at the macroscopic length scales. The microscopic properties of aggrecan solutions are determined by small angle neutron scattering (SANS), and static and dynamic light scattering (SLS and DLS). In dilute solutions aggrecan forms microgels with a diffuse boundary, composed of loosely connected clusters. The osmotic pressure of the aggrecan-HA system decreases with increasing HA content. DLS yields a relaxation rate that varies as q3, arising from internal modes in the microgel. The relaxation rate in the solutions of the aggrecan-HA complex is slightly greater than in the pure aggrecan solution.

Effect of calcium/sodium ion exchange on the osmotic properties and structure of polyelectrolyte gels.

We discuss the main findings of a long-term research program exploring the consequences of sodium/calcium ion exchange on the macroscopic osmotic and elastic properties, and the microscopic structure of representative synthetic polyelectrolyte (sodium polyacrylate, (polyacrylic acid)) and biopolymer gels (DNA). A common feature of these gels is that above a threshold calcium ion concentration, they exhibit a reversible volume phase transition. At the macroscopic level, the concentration dependence of the osmotic pressure shows that calcium ions influence primarily the third-order interaction term in the Flory-Huggins model of polymer solutions. Mechanical tests reveal that the elastic modulus is practically unaffected by the presence of calcium ions, indicating that ion bridging does not create permanent cross-links. At the microscopic level, small-angle neutron scattering shows that polyacrylic acid and DNA gels exhibit qualitatively similar structural features in spite of important differences (e.g. chain flexibility and chemical composition) between the two polymers. The main effect of calcium ions is that the neutron scattering intensity increases due to the decrease in the osmotic modulus. At the level of the counterion cloud around dissolved macroions, anomalous small-angle X-ray scattering measurements made on DNA indicate that divalent ions form a cylindrical sheath enveloping the chain, but they are not localized. Small-angle neutron scattering and small-angle X-ray scattering provide complementary information on the structure and interactions in polymer solutions and gels.

Chondroitin Sulfate in Solution: Effects of Mono- and Divalent Salts.

Chondroitin sulphate (CS) is a linear sulfated polysaccharide found in cartilage and other tissues in the body. Small angle neutron scattering (SANS) and dynamic light scattering (DLS) measurements are made on semi-dilute CS solutions to determine ion induced changes in the local order of the CS chains and in their dynamic properties. In salt-free CS solutions SANS detects the correlation peak due to local ordering between adjacent chains in which the characteristic interchain distance is d ≈ 57 Å. In both monovalent and divalent salts (NaCl and CaCl2) aligned linear regions are distinguishable corresponding to distance scales ranging from the length of the monomer unit (8 Å) to about 1000 Å. With increasing calcium ion concentration, the scattering intensity increases. Even in the presence of 200 mM CaCl2, however, neither phase separation nor cross-linking occurs. DLS in the CS solutions reveals two characteristic relaxation modes, the fast mode corresponding to the thermal concentration fluctuations. The collective diffusion coefficient D decreases with increasing calcium ion concentration and exhibits a power law function of the single variable c/J, where c is the CS concentration and J is the ionic strength of the salt in the solution. This result implies that the effect of the sodium and calcium ions on the dynamic properties of CS solutions is fully accounted for by the ionic strength.

Hierarchical organization of cartilage proteoglycans.

The hierarchical organization of cartilage proteoglycans is investigated on different length and time scales using osmotic pressure measurements, small angle neutron scattering (SANS), small angle X-ray scattering (SAXS), static and dynamic light scattering and neutron spin echo techniques. Osmotic pressure measurements reveal association of aggrecan bottlebrushes into microgel-like assemblies. SAXS, SANS and light scattering results indicate weak interpenetration between neighboring aggrecan molecules. As opposed to DNA and many synthetic polyelectrolytes, which display great sensitivity to ion valence, aggrecan exhibits exceptional insensitivity to calcium ions in the physiological ion concentration range and beyond. This property allows aggrecan to play a role of ion reservoir that can mediate calcium metabolism in cartilage and bone.

Length scale dependence of the dynamic properties of hyaluronic acid solutions in the presence of salt.

In solutions of the charged semirigid biopolymer hyaluronic acid in salt-free conditions, the diffusion coefficient D(NSE) measured at high transfer momentum q by neutron spin echo is more than an order of magnitude smaller than that determined by dynamic light scattering, D(DLS). This behavior contrasts with neutral polymer solutions. With increasing salt content, D(DLS) approaches D(NSE), which is independent of ionic strength. Contrary to theoretical expectation, the ion-polymer coupling, which dominates the low q dynamics of polyelectrolyte solutions, already breaks down at distance scales greater than the Debye-Hückel length.

Counterion and pH-Mediated Structural Changes in Charged Biopolymer Gels.

DNA solutions and gels exhibit a wide range of phenomena, many of which have not yet been fully understood. In the presence of multivalent counterions, attraction between charged DNA strands occurs. Increasing the concentration of multivalent ions leads to a decrease of the osmotic pressure, and a sufficiently high ion concentration results in the precipitation of the polymer. Replacing the monovalent counterions by hydrogen ions (decreasing the pH) also produces a marked decrease of the osmotic pressure, and at low pH a phase transition takes place. In this paper we analyze osmotic swelling pressure measurements and small-angle neutron scattering (SANS) measurements made on chemically cross-linked DNA gels swollen in near physiological salt solutions. The effect of calcium ions is compared with that of decreasing the pH of the equilibrium salt solution. We demonstrate that both the concentration dependence of the osmotic pressure and the SANS response of DNA gels display significant differences in the two cases.

Insensitivity to salt of assembly of a rigid biopolymer aggrecan.

Many polyelectrolytes, ranging from sulfonated polystyrene to DNA, exhibit a strong sensitivity of their phase behavior to salt concentration, especially to higher valence salts, which often lead to phase separation. We show that the stiff polyelectrolyte aggrecan exhibits a qualitatively different behavior. Specifically, the scattering properties of aggrecan solutions are exceptionally insensitive to the addition of calcium salt, conferring on aggrecan the role of an ion reservoir mediating calcium metabolism in cartilage and bone, and also providing osmotic resilience to compressive load.

Gel-like behavior in aggrecan assemblies.

Aggrecan, a large biological polyelectrolyte molecule with a bottlebrush shape, forms complexes with hyaluronic acid (HA) that provide compressive resistance in cartilage. In solutions of aggrecan alone, the concentration dependence of the osmotic pressure Pi is marked by self-assembly of the molecules into aggregates. When HA is added to the solution at low aggrecan concentration c, the osmotic pressure is reduced, but in the physiological concentration range this trend is reversed. The osmotic modulus c partial differentialPi partial differentialc, which determines load bearing resistance, is enhanced in the HA-containing solutions. Dynamic light scattering (DLS) measurements show that the aggregates behave like microgels and that they become denser as the aggrecan concentration increases. The degree of densification is greatest at large distance scales in the microgels, but decreases at short distance scales. Measurements at higher resolution, involving small angle neutron scattering and small angle x-ray scattering (SAXS), confirm that at length scales shorter than 1000 angstroms, the density is independent of the concentration and that the individual bottlebrushes in the microgels retain their identity. The absence of collective diffusion modes in the relaxation spectrum, measured by DLS and neutron spin echo, corroborates the lack of interpenetration among the aggrecan subunits in the microgel. Complexation with HA modifies the long-range spatial organization of the microgels. Comparison of the scattering pattern of the individual aggrecan molecules obtained from SAXS measurements with that of the complexes measured by DLS shows that the aggrecan-HA structure is denser and is more uniform than the random microgels. This enhanced space-filling property allows higher packing densities to be attained, thus, optimizing resistance to osmotic compression.

Scaling equations for a biopolymer in salt solution.

The effect of the simultaneous presence of monovalent and divalent cations on the thermodynamics of polyelectrolyte solutions is an incompletely solved problem. In physiological conditions, combinations of these ions affect structure formation in biopolymer systems. Dynamic light scattering measurements of the collective diffusion coefficient D and the osmotic compressibility of semidilute hyaluronan solutions containing different ratios of sodium and calcium ions are compared with simple polyelectrolyte models. Scaling relationships are proposed in terms of polymer concentration and ionic strength J of the added salt. Differences in the effects of sodium and calcium ions are found to be expressed only through J.

Anomalous small angle x-ray scattering determination of ion distribution around a polyelectrolyte biopolymer in salt solution.

The distribution of counterions in solutions of high molecular mass hyaluronic acid, in near-physiological conditions where mono- and divalent ions are simultaneously present, is studied by small angle neutron scattering and anomalous small angle x-ray scattering. The solutions contain either sodium or rubidium chloride together with varying concentrations of calcium or strontium chloride. The effects of monovalent-divalent ion exchange dominate the amplitude and the form of the counterion cloud. In the absence of divalent ions, the shape of the anomalous scattering signal from the monovalent ions is consistent with the distribution calculated from the Poisson-Boltzmann equation, as found by other workers. In mixtures of monovalent and divalent ions, however, as the divalent ion concentration increases, both the diameter and the amplitude of the monovalent ion cloud decrease. The divalent counterions always occupy the immediate neighborhood of the charged polyanion. Above a given concentration their anomalous scattering signal saturates. Even in a large excess of divalent ions, ion exchange is incomplete.

Adsorption of divalent cations on DNA.

The distribution of divalent ions in semidilute solutions of high-molecular-mass DNA containing both sodium chloride and strontium chloride in near-physiological conditions is studied by small-angle x-ray scattering and by small-angle neutron scattering. Both small-angle neutron scattering and small-angle x-ray scattering reveal a continuous increase in the scattering intensity at low q with increasing divalent ion concentration, while at high q the scattering curves converge. The best fit to the data is found for a configuration in which DNA strands of cross-sectional radius 10 angstroms are surrounded by a counterion sheath of outer radius approximately 13.8 angstroms, independent of the strontium chloride concentration. When the strontium chloride is replaced by calcium chloride, similar results are obtained, but the thickness of the sheath increases when the divalent salt concentration decreases. These results correspond in both cases to partial localization of the counterions within a layer that is thinner than the effective Debye screening length.