Self-association of haemoglobin Olympia (alpha 2 beta 2 20 (B2) Val----Met). A human haemoglobin bearing a substitution at the surface of the molecule.

Oxygenation measurements at equilibrium were carried out for solutions of pure haemoglobin (Hb) Olympia (alpha 2 beta 2 20 (B2) Val----Met) at 200 microM (haem) and revealed a high oxygen affinity (P50 = 4.2 torr at pH 7.20, 25 degrees C) compared to HbA (P50 = 5.6 torr), with the Hill coefficient (eta H) reduced from the normal value of 2.9 to 2.5 for Hb Olympia at neutral pH. 2,3-Diphosphoglycerate and chloride effects were normal, but measurements of the alkaline Bohr effect indicated an excess Bohr effect of about 20% for Hb Olympia. Precise determinations of the oxygen binding curves gave the unexpected finding of a dependence of co-operativity on pH with eta H rising from 2.4 at pH 6.8 to 3.0 at pH 8. Moreover, the Hill coefficient was dependent upon the concentration at alkaline pH and fell to 1.8 in low concentration solutions (approximately 30 microM-haem) of the haemoglobin variant; at this concentration the Bohr effect was normal. This effect of concentration on co-operativity could be accounted for fully by the allosteric model, with introduction of Hb Olympia self-association. In this case the allosteric constant L' for the supramolecular species has the value of the allosteric constant L for the tetramer species, raised to a power equal to the number of molecules in the aggregates and modulated by the ratio of the dissociation constants of the aggregates, DNR/DNT. Model curves with N tetramers per aggregate (where N approximately 2 at pH 7.5 and N approximately 4 at pH 8.0) fully represented the concentration dependence for Hb Olympia of the eta H values and the detailed shape of the experimental curves for eta H as a function of log[y/(1-y)], the first derivative of the Hill plot. These curves are much steeper when supramolecular species are present. Direct measurements of the protein aggregation by centrifugation confirmed the presence of aggregates in the solutions of Hb Olympia. Hb Olympia is therefore one of the few examples of mutant human haemoglobins that self-associate with functional consequences in terms of oxygen binding properties.(ABSTRACT TRUNCATED AT 400 WORDS)