To understand the interplay between tertiary and quaternary transitions ass
ociated with hemoglobin function and regulation, oxygen binding curves were
obtained for hemoglobin A fixed in the T quaternary state by encapsulation
in wet porous silica gets. At pH 7.0 and 15 degreesC, the oxygen pressure
at half saturation (p50) was measured to be 12.4 +/- 0.2 and 139 +/- 4 torr
for hemoglobin gels prepared in the absence and presence of the strong all
osteric effectors inositol hexaphosphate and bezafibrate, respectively. Bot
h values are in excellent agreement with those found for the binding of the
first oxygen to hemoglobin in solution under similar experimental conditio
ns. The corresponding Hill coefficients of hemoglobin gels were 0.94 +/- 0.
02 and 0.93 +/- 0.03, indicating, in the frame of the Monod, Wyman, and Cha
ngeux model, that high and low oxygen-affinity tertiary T-state conformatio
ns have been isolated in a pure form. The values, slightly lower than unity
, reflect the different oxygen affinity of alpha- and beta -hemes. Signific
antly, hemoglobin encapsulated in the presence of the weak effector phospha
te led to gels that show intermediate oxygen affinity and Hill coefficients
of 0.7 to 0.8. The heterogeneous oxygen binding results from the presence
of a mixture of the high and low oxygen-affinity T states. The Bohr effect
was measured for hemoglobin gels containing the pure conformations and foun
d to be more pronounced for the high-affinity T state and almost absent for
the low-affinity T state. These findings indicate that the functional prop
erties of the T quaternary state result from the contribution of two distin
ct, interconverting conformations, characterized by a 10-fold difference in
oxygen affinity and a different extent of tertiary Bohr effect. The very s
mall degree of T-state cooperativity observed in solution and in the crysta
lline state might arise from a ligand-induced perturbation of the distribut
ion between the high- and low-affinity T-state conformations.