Analysis of hemoglobin oxygen equilibrium curves. Are unique solutions possible?

Abstract

As a hemoglobin tetramer is oxygenated, it converts from a form with low ligand affinity to a high-affinity structure. This allosteric transition occurs in partially liganded molecules, typically after two or three ligands are bound. As a result of the co-operative nature of the process, the populations of the partially liganded forms are low. The relative proportions and precise properties of these intermediate substrates are therefore difficult to measure and are subject to controversy. The problem is compounded by compensating effects; for example, over-estimation of the oxygen affinity of triply liganded forms will result in under-estimation of the proportion of these species. Specifically, published values for the oxygen affinity of the triply liganded species vary for identical conditions by a factor of more than 6. In analyses based on the highest affinity values, the triply liganded species virtually disappears. However, this affinity is usually calculated from the last few per cent of the oxygenation curve, and this part of the curve is extremely sensitive to the normalization of the data. We conclude that unique solutions for the ligand affinity and substrate populations may be impossible to achieve, and that unusual mechanisms based on particular combinations of parameters, therefore, should be viewed with caution.