COOPERATIVE LIGAND-BINDING OF CROSS-LINKED HEMOGLOBINS AT VERY HIGH-TEMPERATURES

Human hemoglobin was reacted with the bifunctional reagent bis(3,5-dibromosalicyl) fumarate to yield a derivative (Hb αα̂) crosslinked between the two α-chains; when the reaction was carried out with HbA already crosslinked between the two β-chains by 2-nor-2-formylpyridoxal 5′-phosphate, a doubly crosslinked derivative (Hb αα̂ββ̂) was obtained. We have observed that both modified hemoglobins are extremely stable up to temperatures of at least 85°C. The carbon monoxide binding kinetics of both crosslinked hemoglobins, studied at temperatures between 15 and 85°C, by means of stopped flow and flash photolysis techniques, show that the ligand-linked allosteric transition is maintained even at the highest temperatures. These results are also relevant to the mechanism of thermal unfolding of human hemoglobin, since they show that dissociation into αβ dimers (and exposure of the relatively hydrophobic dimer-dimer interfaces) is an obligatory step in the irreversible denaturation of deoxy and carbon monoxy hemoglobin. © 1990 Academic Press Limited.