Influence of ionic strength and pH on the interaction between high-affinity heparin and antithrombin.
Binding constants for the binding of high-affinity heparin to antithrombin at different ionic strengths were determined by fluorescence titrations and were also estimated from dissociation curves of the heparin-antithrombin complex. These curves were monitored by near-ultraviolet circular dichroism or fluorescence. The dependence of the binding constant on the activity of NaCl suggested that maximally 5-6 charged groups are directly involved in the interaction between the two macromolecules. Major pH-dependent changes of the interaction, as evident by changes of the spectroscopic properties of the complex between the molecules, were found to occur below pH 5.5 and above pH 8.5. The acid change, which was irreversible, was most likely caused by an irreversible conformational change of antithrombin. At alkaline pH, however, the gross conformation of antithrombin was stable up to pH 12, while the at pH 8.5. The dissociation curve, which was reversible, had a midpoint around pH 9.5. This is compatible with the loss of affinity being caused by either a local conformational change, by ionization of tyrosine or by titration of one or more amino groups.
D000818:Animals; D000991:Antithrombins; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D006493:Heparin; D006863:Hydrogen-Ion Concentration; D009994:Osmolar Concentration; D012756:Sheep; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D000991:Antithrombins; D006493:Heparin