Refinement and evaluation of a model of Mg2+ buffering in human red cells

The total Mg2+ content of human red cells ([Mg](T,i)) is partitioned betwee n free and bound forms. The main cytoplasmic Mg2+ buffers are ATP and 2,3 b isphosphoglycerate. Haemoglobin binds free ATP and bisphosphoglycerate, pre ferentially in the deoxygenated state. Thus, the free ionized Mg2+ concentr ation ([Mg2+](i)) oscillates with the oxy-deoxy condition of the cells. The binding reactions are also modulated by the pH changes that accompany the oxygenation-deoxygenation transitions. The complex interactions between Mg2 +, its ligands and Hb can be encoded in a set of equilibrium equations repr esenting all the known binding reactions of the system. To develop a compre hensive understanding of the Mg2+ homeostasis of intact red cells it is nec essary to correct and refine the equations and parameters of the model by s ystematic comparisons between model predictions and measured cytoplasmic Mg 2+ buffering curves under a variety of experimental conditions. Earlier mod els largely underestimated total Mg2+ binding in intact cells. We carried o ut experiments in which [Mg](T,i) and [Mg2+](i) were controlled over a wide range ([Mg](T,i) between 0.1 and 23 mM) by the use of the ionophore A23187 , under diverse metabolic conditions, and the results were used to interpre t the adjustments required for good model fits. By the inclusion of low-aff inity Mg2+ binding to ATP and bisphosphoglycerate, and also binding of Mg2 to haemoglobin (four ions per tetramer) with an apparent dissociation cons tant of 45 mM we were able to realistically model, for the first time, all the experimentally observed changes in [Mg2+](i) in human red cells under d iverse metabolic conditions.