Functional Properties of Human Hemoglobin Bound to the Erythrocyte Membrane

Studies are presented which deal primarily with the ligand binding kinetic properties of human oxyhemoglobin bound to the erythrocyte membrane. Static 90° relative light scattering measurements are also presented as a necessary preliminary to the functional studies. The light scattering measurements suggest that the dimer of oxyhemoglobin binds to the cytoplasmic surface of the membrane. Binding was apparently noncooperative with a constant of about 4 μM in dimer at pH 6, in 5 mM phosphate buffer at 23 °C. Further evidence for enhanced formation of the oxygenated dimer was obtained from kinetic measurements where oxygen was rapidly removed in the stopped-flow and the kinetics of CO binding studied. A substantial increase in the proportion of rapid CO binding component was observed with increasing ghost concentration. Complete reversibility of the increased fraction of rapid CO binding component was demonstrable upon addition of the enzyme glyceraldehyde-3-phosphate dehydrogenase (G3PD) after addition of oxyhemoglobin. This result may indicate that the oxygenated dimer binds reversibly to band 3 since G3PD has been shown to bind to this membrane protein (Yu, J., & Steck, T. L. (1975) J. Biol. Chem. 250, 9176). Oxygen release measurements in the absence and presence of CO indicate diminished cooperativity, as expected if the oxygenated dimer is stabilized. However, the initial rate constant for oxygen release from the β chain of the bound dimer was found to be about 20 times slower than in solution.