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.