Formation and Degradation of Intermediate Hemoglobin Polymers are Responsible for the Cooperativity of Oxygen Binding Isotherms

Abstract

According to the MWC model, cooperativity is due to the equilibrium between the lower affinity T form and the higher affinity R form at the beginning and end of oxygen binding isotherm. The model disregards the energy barrier necessary for preventing the T and R forms relaxing into a single conformation system. Also, the difference between the binding Gibbs free energy of the two affinities at pH 9.0 is ΔG=2000 cal/mole. It means that at equilibrium the energy level of the T form increases by 2.0 Kcal/mole. This increase is inconsistent with the zero sum of heat exchanges needed for equilibriums. To solve these problems we tested the response of the isotherm to osmotic pressure, heat, and protein concentration. Osmotic stress reveals a higher volume/surface ratio of the T form, suggesting a more spherical shape. Increasing protein concentration decreases the oxygen affinity of the system indicating formation of transient polymeric forms, with low oxygen affinity. Enthalpies of the intermediates show a strong absorption of heat at the third oxygenation step. This is the heat necessary for polymerizing tetrameric Hb into quinary structures, as in the fibers of HbS, The net absorption and release of heat, of the enthalpies of the conformational changes, is a negative ΔG=-2000 cal/mole, reflecting the degradation of the quinary polymers into the quaternary T and R forms. This heat released to the system compensates to zero the energy absorbed by the T form upon deoxygenation. It should be stressed that the high energy level of polymeric intermediates is the energy barrier preventing the direct relaxation of the T and R conformations into one another. This molecular mechanics explains the cooperativity of oxygen binding isotherm.