title: Asymmetric (deoxy dimer/azido-met dimer) hemoglobin hybrids dissociate within seconds.
abstract: Double mixing stopped-flow experiments have been performed to study the stability of asymmetric hemoglobin (Hb) hybrids, consisting of a deoxy and a liganded dimer. The doubly liganded [deoxy/cyano-met] hybrid (species 21) was reported to have an enhanced stability, with tetramer to dimer dissociation requiring over 100 seconds, based on a method that required an incubation of over two days. However, kinetic experiments revealed rapid ligand binding to species 21, as for triply liganded tetramers, which dissociate within a few seconds. For the present study, [deoxy dimer/azido-met dimer] hybrids are formed within 200 ms by stopped-flow mixing of dithionite with a solution containing oxyHb and azido-metHb. The dithionite scavenges oxygen, thus transforming oxyHb to deoxyHb, and the [oxy dimer/azido-met dimer] hybrid to the asymmetric [deoxy/azido-met] hybrid (species 21). After a variable aging time of the asymmetric hybrids, their allosteric state is probed by CO binding in a second mixing. As previously observed the freshly produced asymmetric hybrids bind CO rapidly as for R-state Hb. As the hybrids are aged from 0.1 to 10 seconds, the fraction of slow CO binding increases, consistent with a dissociation of the asymmetric hybrid to form the more stable deoxy Hb tetramer which reacts slowly with CO. Control experiments showed a predominantly slow phase for deoxy Hb, and fast rebinding for the symmetric hybrids. The kinetic data can be simulated with a tetramer to dimer dissociation rate for species 21 of 1.5/second at 100 mM NaCl (pH 7.2) and 1.9/second at 180 mM NaCl (pH 7.4). These values are similar to those reported for liganded Hb, as opposed to deoxy (T-state) tetramers which dissociate over four orders of magnitude more slowly. As expected from simulations of dimer exchange, the observed transition rate depends on the initial fractions of oxy- and metHb; this effect is not consistent with a slow R to T transition. These results, showing a lifetime of about one second for species 21, do not support the symmetry rule which is based on an enhanced stability of the asymmetric hybrid.
authors.affiliation: INSERM U473, 84 rue du Général Leclerc, Le Kremlin-Bicêtre Cedex, 94276, France.
authors.forename: L
authors.initials: L
authors.lastname: Kiger
authors.forename: M C
authors.initials: MC
authors.lastname: Marden
mesh_terms: D000494:Allosteric Regulation; D001386:Azides / Q000737:chemistry; D003198:Computer Simulation; D006454:Hemoglobins / Q000737:chemistry* / Q000235:genetics; D008024:Ligands; D009154:Mutation; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D055503:Protein Multimerization
chemical_list: D001386:Azides; D006454:Hemoglobins; D008024:Ligands