Kinetic trapping of a key hemoglobin intermediate.

The complete binding cascade of human hemoglobin consists of a series of partially ligated intermediates. The individual intermediate binding constants cannot be distinguished in O(2) binding curves, however, each constant can be determined from the O(2)-induced change in assembly constant for the α(2)β(2) tetramer from its constituent αβ dimers. The characterization of these O(2) binding constants has shown the Hb cascade to be asymmetric in nature, with binding dependent upon the specific distribution of O(2) among the four hemesites. A stopped-flow approach to measuring the dissociation constant of a key doubly ligated intermediate, that in which one dimer is oxygenated and the other is not, is described. The intermediate is transiently formed in the absence of O(2) and then allowed to dissociate in the presence of O(2). The free dimers thus released are trapped by the plasma protein haptoglobin, the rate limiting step being that of tetramer dissociation. The kinetic constant observed for the dissociation of this intermediate confirms the value for its equilibrium O(2) binding constant, previously determined under equilibrium conditions by subzero isoelectric focusing.