The Characterization of Modified Human Hemoglobin

A dinitrophenyl derivative of human hemoglobin has been prepared and purified, in which the only site of reaction is the NH2 terminus of the α chain. Evidence for this comes from two sources: first, the identification of all of the tryptic peptides having absorption at 355 mµ; second, from experiments with 14C-labeled 1-fluoro-2,4-dinitrobenzene which show that the stoichiometry of the reaction is 1 dinitrophenylgroup per αβ subunit. Competition experiments with iodoacetamide show that 1-fluoro-2,4-dinitrobenzene does not react with the reactive sulfhydryl group on the β chain. This single substitution leads to quite drastic physical and functional changes. The modified hemoglobin has an increased oxygen affinity, an n value of 1 in the Hill equation, and no Bohr effect. In all dissociating solvents tested, the dinitrophenyl-hemoglobin is more highly associated than native hemoglobin.