Quantitative Characterization of the Binding of Histamine by Heparin

Abstract

Tissue histamine is stored in mast cell granules, presumably as a histamine−heparin complex. Heparin is a polyelectrolyte, with a fraction of its anionic charge neutralized by condensed counterions. The interaction of heparin with histamine in aqueous solution was quantitatively characterized by ¹H nuclear magnetic resonance (NMR) spectroscopy. Binding constants were determined from chemical shift−pH titration data for the C2H proton of the imidazolium ring for a wide range of histamine, heparin, and Na⁺ concentrations. The results indicate a binding stoichiometry of 1 histamine per heparin disaccharide repeat unit. The binding is electrostatic, as indicated by the strong dependence of the binding constant on Na⁺ concentration. From an analysis of the binding constants using the counterion condensation theory of polyelectrolytes, it was determined that the binding of H₂A²⁺ results in displacement of 1.72 Na⁺ ions from the counterion condensation volume of heparin and that H₂A²⁺ makes 2 ionic interactions with heparin. The displacement of Na⁺ from the counterion condensation volume of heparin by H₂A²⁺ was also studied by ²³Na NMR. From ²³Na spin−lattice relaxation time data, it was determined directly that 1.78 Na⁺ ions are displaced per H₂A²⁺ bound by heparin. The results are discussed in terms of the ion exchange process which takes place when histamine is released by mast cells.