Quaternary enhancement in binding of oxygen by human hemoglobin

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Abstract
The linkage between dimer-tetramer association and O2 binding in human Hb revealed that triliganded tetramers .alpha.2.beta.2(O2)3 have an affinity for O2 that is significantly higher than that of .alpha.1.beta.1 dimers (superscripts denote intersubunit contacts). This conclusion was based on a newly determined series of accurate O2 binding isotherms, which were analyzed in conjunction with independently determined values of the dimer-tetramer equilibrium constants in the unliganded and fully oxygenated states. In the molecule .alpha.2.beta.2(O2)3 the interactions at the .alpha.1.beta.2 intersubunit contacts are apparently propagated to the unliganded heme in a manner that increases its affinity for O2. This effect contrasts sharply with the well-known reduction in O2 affinity arising from these same contacts when unliganded dimers are assembled to form unliganded .alpha.2.beta.2 tetramers. The magnitude of the enhancement in affinity at the unliganded site in triliganded tetramers (0.81 kcal, 3.39 kJ) is approximately 1/4 as great as the reduction in affinity on each heme site that arises from subunit assembly of the unliganded tetramer. The terms quaternary constraint and quaternary enhancement are employed to describe those oppositely directed effects of intersubunit interaction on heme-site affinity. Dimers apparently bind O2 with a higher affinity than monomeric .alpha. and .beta. chains do under the same temperature and buffer conditions (21.5.degree. C, pH 7.40, 0.1 M Tris .cntdot. HCl/0.1 M NaCl/1 mM Na2EDTA). Thus quaternary enhancement may be manifested at the .alpha.1.beta.1 contacts. Implications of these results for models of the cooperative mechanism were discussed.