Formation of Cross-Linked Asymmetrical Hybrid Hemoglobins by Double-Headed Aspirin

Double-headed aspirin [bis(3,5-dibromosalicyl)fumarate] selectively cross-links hemoglobin molecules between Lys 82 beta 1 and Lys 82 beta 2 and increases solubility of deoxy-Hb S (Walder et al., J. Mol. Biol., 141:195, 1980 and Kikugawa et al., J. Biol. Chem., 257:7525, 1982). We reacted this reagent with the mixture of Hb A and Hb S and the mixture of Hb S and Hb York (beta 146His replaced by Pro). Cross-linked asymmetrical hybrid hemoglobins (alpha 2 beta - beta S and alpha 2 beta Y - beta S) were produced in high yields in addition to the cross-linked parent hemoglobin molecules. Results on electrophoresis, gel electrofocusing, ion exchange column chromatography, mechanical stability and oxygen binding properties showed that the cross-linked asymmetrical hybrid hemoglobins had properties intermediate between those of the cross-linked parent hemoglobins. Oxygen affinities of the cross-linked asymmetrical hybrids were not affected by the addition of 2,3-diphosphoglycerate (DPG) or inositol hexaphosphate, probably due to the presence of a fumaryl group at the DPG binding site.