Oxygenation-linked subunit interactions in human hemoglobin: experimental studies on the concentration dependence of oxygenation curves

Abstract

An experimental study on the concentration dependence of oxygenation curves for human Hb was carried out between 4 .times. 10-8 M heme and 5 .times. 10-4 M heme in 0.1 M tris(hydroxymethyl)aminomethane hydrochloride, 0.1 M NaCl, 1 mM disodium EDTA, pH 7.4, 21.5.degree. C. With decreasing Hb concentration the curves show pronounced shifts in position and shape, consistent with dissociation of tetrameric Hb into dimeric species of high affinity and low cooperativity. Combination of these data with independently determined values of dissociation constants for unliganded and fully liganded Hb permits a resolution of the 7 parameters necessary to define the linked binding and subunit association processes. The total oxygenation-linked subunit dissociation energy (6.34 kcal) was resolved into intersubunit contact energy changes between .alpha..beta. dimers in tetrameric Hb which accompany binding of the 1st, middle 2 and last O molecules. The resolution is accurate to within approximately .+-.0.3 kcal. To within this limit, the isolated dimers bind O2 noncooperatively and with the same affinity as isolated .alpha. and .beta. chains. Equally good fits to the data are obtained when dimers are slightly anticooperative. At least 3 major energetic states are apparently assumed by Hb tetramers, with respect to the .alpha.1.beta.2 contact region, corresponding to unliganded, singly liganded and triply and quadruply liganded species. The results do not establish whether these states may be assumed by a single molecule, or whether they arise as averages over a distribution of conformational states. They do provide unequivocal evidence against a concerted transition at any particular binding step in a system with only 2 energetic states of tetramer (i.e., an all or none switchover between T and R states at a particular binding step).