LIGAND LINKED ASSEMBLY OF SCAPHARCA DIMERIC HEMOGLOBIN

The assembly of Scapharca dimeric hemoglobin as a function of ligation has been explored by analytical gel chromatography, sedimentation equ ilibrium, and oxygen binding experiments to test the proposal that its cooperativity is based on quaternary enhancement. This hypothesis pre dicts that the liganded form would be assembled more tightly into a di mer than the unliganded form and that dissociation would lead to lower oxygen affinity. Our experiments demonstrate that although the dimeri c interface is quite tight in this hemoglobin, dissociation can be cle arly detected in the liganded states with monomer to dimer association constants in the range of 10(8) M(-1) for the CO-liganded state and l ower association constants measured in the oxygenated state. In contra st, the deoxy dimer shows no detectable dissociation by analytical ult racentrifugation. Thus, the more highly hydrated deoxy interface of th is dimer is also the more tightly assembled. Equilibrium oxygen bindin g experiments reveal an increase in oxygen affinity and decrease in co operativity as the concentration is lowered (in the mu M range), These experiments unambiguously refute the hypothesis of quaternary enhance ment and indicate that, as in the case of human hemoglobin and other a llosteric proteins, quaternary constraint underlies cooperativity in S capharca dimeric hemoglobin.