High and low oxygen affinity conformations of T state hemoglobin

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.