To help understand the nature of physical/chemical interactions in biomolecules and biomolecular assemblies, we have developed an experimental approach to study their structure (morphology) and thermodynamic properties simultaneously as a function of the length scale (spatial resolution). The method combines macroscopic osmotic swelling pressure measurements and small angle neutron scattering (SANS). Macroscopic swelling pressure measurements probe the system in the large length scale range, thus providing information on the overall thermodynamic response. The SANS measurement simultaneously provides information about the size of different structural elements and their respective contribution to the osmotic properties. Combining these measurements allows us both to separate the scattering intensity arising from thermodynamic concentration fluctuations from the intensity scattered by large static superstructures (e.g., aggregates), and to determine the length scales relevant to the macroscopic thermodynamic properties. This thermodynamic and structural information cannot be obtained by other techniques.? ? Specifically, we have applied this approach to studying the interactions of multivalent cations, particularly Calcium, on the structure and morphology of various biomolecules. Divalent cations are ubiquitous in the biological milieu, yet existing theories do not adequately explain their effect on and interactions with charged polymers or biomolecules. Moreover, experiments to study these interactions are difficult to perform, particularly in solution, because above a low concentration threshold multivalent cations generally cause phase separation or precipitation of charged molecules. Since macroscopic phase separation does not occur in cross-linked gels, we have overcome this limitation by cross-linking our biopolymers, extending the range of ion concentrations over which the system remains stable and can be studied. In pilot studies, this new method has been applied to investigate cross-linked gels of a model synthetic polymer, polyacrylic acid, and of DNA. Concentrated DNA solutions and novel DNA gels have never before been investigated using SANS in conjunction with osmotic measurements. The proposed method also provides a unique framework for analyzing the osmotic and scattering behavior of other biomolecular systems.? ? In pilot studies, this new non-destructive procedure has been applied to investigate cross-linked gels of a model synthetic polymer, polyacrylic acid, and different biopolymers such as DNA and hyaluronic acid to determine the size of the structural elements that contribute to the osmotic concentration fluctuations. We have combined small-angle X-ray scattering (SAXS) and SANS to estimate the osmotic modulus of hyaluronic acid solutions in the presence of monovalent and divalent counterions. We also investigate the diffusion processes in biopolymer solutions using DLS to determine the osmotic modulus independently from the dynamic response. Concentrated biopolymer solutions and gels have never before been investigated using SANS and SAXS in conjunction with osmotic measurements.

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U.S. National Inst/Child Hlth/Human Dev
United States
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Horkay, Ferenc; Basser, Peter J (2008) Ionic and pH effects on the osmotic properties and structure of polyelectrolyte gels. J Polym Sci B Polym Phys 46:2803-2810
Van Thienen, T G; Horkay, F; Braeckmans, K et al. (2007) Influence of free chains on the swelling pressure of PEG-HEMA and dex-HEMA hydrogels. Int J Pharm 337:31-9
Horkay, Ferenc; Basser, Peter J (2004) Osmotic observations on chemically cross-linked DNA gels in physiological salt solutions. Biomacromolecules 5:232-7
Morfin, Isabelle; Horkay, Ferenc; Basser, Peter J et al. (2004) Adsorption of divalent cations on DNA. Biophys J 87:2897-904
Stubbe, Barbara G; Horkay, Ferenc; Amsden, Brian et al. (2003) Tailoring the swelling pressure of degrading dextran hydroxyethyl methacrylate hydrogels. Biomacromolecules 4:691-5
Han, In Suk; Han, Man-Hee; Kim, Jinwon et al. (2002) Constant-volume hydrogel osmometer: a new device concept for miniature biosensors. Biomacromolecules 3:1271-5
Dimitriadis, Emilios K; Horkay, Ferenc; Maresca, Julia et al. (2002) Determination of elastic moduli of thin layers of soft material using the atomic force microscope. Biophys J 82:2798-810