X-ray fibre diffraction study of conformational changes in hyaluronate induced in the presence of sodium,potassium and calcium cations

Hyaluronate purified from all cations by ion exchange chromatography was introduced to the cations sodium, potassium and calcium in a controlled way. The conformations formed in the presence of these ions were studied as a function of ionic strength, hydrogen ion activity, humidity and temperature using X-ray fibre diffraction. In sodium hyaluronate above pH 4.0 a contracted helix is found which approximates to a four-fold helix with an axial rise per disaccharide of 0.84 nm. There is no requirement for water molecules in the unit cell as the Na⁺ can be coordinate by the hyaluronate chains alone. On crystallizing hyaluronate below pH 4.0 an extended 2-fold helix with an axial rise per disaccharide of 0.98 nm is formed. In the presence of potassium above pH 4.0 a conformation similar, but not identical, to that of sodium was found where the helix backbone is again four-fold with an axial rise per disaccharide h=0.90 nm. To maintain the coordination of the potassium ion, four water molecule/disaccharide are required and on removal of these the conformation is destabilized going to a new helix where n = 4 and h = 0.97 nm. Below pH 4.0 the conformation is a contracted 4-fold helix with h = 0.82 nm. In this structure two antiparallel chains intertwine to form a double helix. The packing of the double helical units is stabilized by water molecules, the unit cell requiring 8 water molecules/disaccharide. Formation of the calcium hyaluronate complex above pH 3.5 yields a three-fold helix with h = 0.95 nm. The requirement for water in the unit cell to maintain full crystallinity is high, at 9 water molecules/disaccharide; however, on removal of this water, though the crystallinity is disrupted, the conformation remains constant. The acid form of calcium-hyaluronate yields an equivalent conformation to that of sodium under the same condition, i.e. a helix with n = 2, h = 0.98 nm. The presence of small quantities of calcium in what are otherwise potassium or sodium solutions of hyaluronate yield the 3-fold conformation for hyaluronate. Thus calcium has an important role to play in deciding the dominating conformation present in hyaluronate. The variety of conformations yielded by the different cations indicates a subtle interaction between hyaluronate and its environment, in which the balance between the cations will control to some degree the interactions between hyaluronate chains and thus affect the mechanical properties of the matrix which they form. The conformations of individual chains are all stabilized in varying degrees by intra-chain hydrogen bonds.