Asymmetric [deoxy dimer/azido-met dimer] hemoglobin hybrids dissociate within seconds

Double mixing stopped-flow experiments have been performed to study the sta bility of asymmetric hemoglobin (Hb) hybrids, consisting of a deoxy and a l iganded dimer. The doubly liganded [deoxy/cyano-met] hybrid (species 21) wa s reported to have an enhanced stability, with tetramer to dimer dissociati on requiring over 100 seconds, based on a method that required an incubatio n of over two days. However, kinetic experiments revealed rapid ligand bind ing to species 21, as for triply liganded tetramers, which dissociate withi n a few seconds. For the present study, [deoxy dimer/azido-met dimer] hybrids are formed wit hin 200 ms by stopped-flow mixing of dithionite with a solution containing oxyHb and azido-metHb. The dithionite scavenges oxygen, thus transforming o xyHb to deoxyHb, and the [oxy dimer/azidomet dimer] hybrid to the asymmetri c [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 se cond 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 disso ciation of the asymmetric hybrid to form the more stable deoxy Hb tetramer which reacts slowly with CO. Control experiments showed a predominantly slo w phase for deoxy Hb, and fast rebinding for the symmetric hybrids. The kinetic data can be simulated with a tetramer to dimer dissociation rat e for species 21 of 1.5/second at 100 mM NaCl (pH 7.2) and 1.9/second at 18 0 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 ord ers of magnitude more slowly. As expected from simulations of dimer exchang e, the observed transition rate depends on the initial fractions of oxy- an d 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 sup port the symmetry rule which is based on an enhanced stability of the asymm etric hybrid. (C) 1999 Academic Press.