| The Molecular Basis of the Solution Properties of Hyaluronan Investigated by Confocal Fluorescence Recovery After Photobleaching Biophysical Journal, Volume 77, Issue 4, 1 October 1999, Pages 2210-2216 Philip Gribbon, Boon C. Heng and Timothy E. Hardingham Abstract Hyaluronan (HA) is a highly hydrated polyanion, which is a network-forming and space-filling component in the extracellular matrix of animal tissues. Confocal fluorescence recovery after photobleaching (confocal-FRAP) was used to investigate intramolecular hydrogen bonding and electrostatic interactions in hyaluronan solutions. Self and tracer lateral diffusion coefficients within hyaluronan solutions were measured over a wide range of concentrations (), with varying electrolyte and at neutral and alkaline pH. The free diffusion coefficient of fluoresceinamine-labeled HA of 500kDa in PBS was 7.9×10cms and of 830kDa HA was 5.6×10cms. Reductions in self- and tracer-diffusion with followed a stretched exponential model. Electrolyte-induced polyanion coil contraction and destiffening resulted in a 2.8-fold increase in self-diffusion between 0 and 100mM NaCl. Disruption of hydrogen bonds by strong alkali (0.5M NaOH) resulted in further larger increases in self- and tracer-diffusion coefficients, consistent with a more dynamic and permeable network. Concentrated hyaluronan solution properties were attributed to hydrodynamic and entanglement interactions between domains. There was no evidence of chain-chain associations. At physiological electrolyte concentration and pH, the greatest contribution to the intrinsic stiffness of hyaluronan appeared to be due to hydrogen bonds between adjacent saccharides. Abstract | Full Text | PDF (128 kb) |
| Interpretation of osmotic pressure in solutions of one and two nondiffusible components Biophysical Journal, Volume 16, Issue 1, 1 January 1976, Pages 43-57 M. Shaw Abstract Osmotic pressure data from aqueous solutions of nondiffusible serum albumin (BSA), chondroitin sulfate (CHS), and dextran T110 (D110), taken singly and in binary combinations, were interpreted in terms of excluded volume. The principal solvent was phosphate-buffered saline, pH 7.2, at 23 degrees C. Osmotic pressures were measured with a membrane osmometer fitted with Amicon PM-10 membranes. Data from each solution were fit by stepwise regression with a three- or four-term polynomial in integral powers of total nondiffusible solute concentration in accordance with the general solution theory of McMillan and Mayer (1945, J. Chem. Phys. 13:276) as extended by Yamakawa (1971, Modern Theory of Polymer Solutions, Harper & Row, New York). The date display a high internal consistency, and the results correlate well with published molecular weights and exclusion data where available. Number average molecular weights calculated from the "first virial coefficients" are: BSA, 67,000 +/- 11%; D110, 76,000 +/- 11%, CHS, 39,000 +/- 6%. Excluded volumes (in cubic centimeters per molecule) calculated from the "second virial coefficients" are: BSA, 0.97 X 10(-18); D110, 3.04 X 10(-18); CHS, 14.3 X 10(-18); BSA-D110, 6.8 X 10(-18); BSA-CHS, 7.8 X 10(-18). Uncertainty is about 30%. An empirical model for interpretation of calculated excluded volumes is proposed. It appears that CHS has the "largest" exclusion effect of the three molecules. Abstract | PDF (878 kb) |
| Measurements of Protein-Protein Interactions by Size Exclusion Chromatography Biophysical Journal, Volume 85, Issue 4, 1 October 2003, Pages 2619-2623 J. Bloustine, V. Berejnov and S. Fraden Abstract A method is presented for determining second virial coefficients () of protein solutions from retention time measurements in size exclusion chromatography. We determine by analyzing the concentration dependence of the chromatographic partition coefficient. We show the ability of this method to track the evolution of from positive to negative values in lysozyme and bovine serum albumin solutions. Our size exclusion chromatography results agree quantitatively with data obtained by light scattering. Abstract | Full Text | PDF (114 kb) |
Copyright © 1977 The Biophysical Society. All rights reserved.
Biophysical Journal, Volume 17, Issue 1, 47-55, 1 January 1977
doi:10.1016/S0006-3495(77)85626-9
Research Article
M. Shaw and A. Schy
Osmotic pressures of solutions of hyaluronate (HA) (mol wt 117,000) and mixtures of HA and bovine serum albumin (BSA) in phosphate-buffered saline, pH 7.2 were measured with a membrane osmometer. The data were fit with a virial expansion in integral powers of total nondiffusible solute concentration. Values of number average molecular weight were calculated for HA and the mixtures from the first virial coefficients. The excluded volume of HA in the single nondiffusible solute solution was calculated from the second virial coefficient extracted from the data on the HA solution. The excluded volume of HA with respect to BSA was estimated from the "osmotic parameters" of HA and BSA by an approach developed in 1976 by Shaw. The resulting excluded volume of HA with respect to BSA was compared with those obtained from a lightly cross-linked HA gel and from solutions of HA (mol wt 1.5 x 10(6)) studied in 1964 by Laurent. The development of this cross-linked HA gel and its subsequent calibration are described.