An NMR and circular dichroism study of the interaction of thiocyanate with human and cross-linked hemoglobin: identification of Lys-α-99 as a possible dissociation linked binding site

Abstract

The interaction of thiocyanate with human native and cross-linked oxyhemoglobin (oxyHb), and methemoglobin (metHb) has been investigated by optical spectroscopy, circular dichroism (CD) and nuclear spin lattice relaxation rate measurements. The interaction of thiocyanate anion with human hemoglobin has been investigated by NMR measurements of the nuclear spin lattice relaxation rate of N¹⁵ labeled thiocyanate in the presence of cyanomethemoglobin and cross-linked cyanomethemoglobin. Results show that thiocyanate is located approximately 8.9 and 6.2 Å away from the heme group in cyanomethemoglobin and cross-linked cyanomethemoblobin, respectively. These results are consistent with the binding of SCN^- at the lys-α-99 in the unmodified hemoglobin. Since this site is blocked in the cross-linked hemoglobin, the binding site is different. Results show that one mole of SCN^- is binding to one mole of oxyhemoglobin suggesting that binding at the lys-α-99 is linked to dissociation of the hemoglobin tetramer into dimers due to its location at the α₁β₂ interface. Circular dichroism studies show that the interaction of thiocyanate with oxyHb decreases the optical rotation at 240 nm indicating a conformational change of the protein, which influences the electronic transitions of a number of peptide bonds or (and) a few aromatic side chains.