A physico-chemical study of heparin: Evidence for a calcium-induced co-operative conformational transition

The conformations of heparin in aqueous solution in the presence of sodium, potassium, magnesium and calcium cations were studied using circular dichroism, optical rotation, nuclear magnetic resonance and equilibrium dialysis. Potassium and magnesium cations, when added to sodium heparinate solutions, cause small chiroptical changes. Binding of calcium ions gives rise to large changes in both optical rotation and circular dichroism. This is indicative of a major change in chain conformation, which is also manifest in ¹³C and ¹H n.m.r.⁴Equilibrium dialysis suggests one mole of calcium bound per mole of tetrasaccharide, which n.m.r. indicates to be appropriately sulphated iduronateglucosamine-iduronate-glucosamine. The calcium is chelated by two iduronate carboxyl groups. Proton-proton coupling constants, determined by convolution difference spectroscopy and Carr-Purcell sequences, indicate that, over the temperature range 285 to 353 K, the iduronate ring is best described as ¹C₄(l) and the glucosamine residue as ⁴C₁(d) for both sodium and calcium forms.The conformational change induced by calcium is ascribed to rotation around the glycosidic linkages. The binding process is co-operative and the binding constant of 10³ to 10⁴m^?1 is biologically significant. The findings are consistent with intramolecular binding. Hence, this study represents the first report of a polysaccharide undergoing a cation-induced intramolecular disorder-order process. The authors postulate that a function of the post-polymerization epimerization of d-glucuronate to l-iduronate is the attainment of the precise geometry required for co-operative calcium binding with consequent modulation of the flexibility of the tetrasaccharide units.