On the Origin of Hemoglobin Cooperativity under Non-equilibrium Conditions

Abnormal hemoglobins can have major consequences for tissue delivery of oxygen. Correct diagnosis of hemoglobinopathies with altered oxygen affinity requires a determination of hemoglobin oxygen dissociation curve, which relates the hemoglobin oxygen saturation to the partial pressure of oxygen in the blood. Determination of the oxygen dissociation curve of human hemoglobin is typically carried out under conditions in which hemoglobin is in equilibrium with O2 at each partial pressure. However, in the human body due to the fast transit of red blood cells through tissues hemoglobin oxygen exchanges occur under non-equilibrium conditions. We describe the determination of non-equilibrium oxygen dissociation curve and show that under these conditions the true nature of hemoglobin cooperativity is revealed as emerging solely from the consecutive binding of oxygen to each one of the four subunits of hemoglobin until the entire tetramer is saturated. We call this form of cooperativity the sequential cooperativity of hemoglobin and define the simplest model that includes it as the minimalist model of hemoglobin. A single instantiation of this model accounts for ~70% of hemoglobin cooperativity under non-equilibrium conditions. The total cooperativity of hemoglobin can be viewed more correctly as the summation of two instantiations of the minimalist model (each one corresponding to a tetramer of low and high affinity for O2, respectively) in equilibrium with each other, as in the Monod-Wyman-Changeux model of hemoglobin. In addition to offering new insights on the nature of hemoglobin reaction with oxygen, the methodology described here for the determination of hemoglobin non-equilibrium oxygen dissociation curve provides a simple, fast, low-cost alternative to complex spectrophotometric methods, which is expected to be particularly valuable in regions where hemoglobinopathies are a significant public health problem, but where highly specialized laboratories capable of determining a traditional oxygen dissociation curve are not easily accessible.