Factors Governing Autooxidation of Human Hemoglobin

Abstract

Determining mechanisms for the autooxidation of hemoglobin is required for understanding and treating unstable hemoglobinopathies and for developing more stable hemoglobin based O2 carriers. Previous studies suggested significant differences in autooxidation rates of α and β subunits. We used an azide reaction assay to measure the concentrations of ferric α and β chains at different time points during autooxidation. Our results showed no differences between the subunits. To obtain more accurate time courses for autooxidation, we deconvoluted observed spectra into the decay of HbO2, metHb appearance, hemichrome generation, and increases in turbidity due to hemin loss and apoprotein precipitation. The time courses for HbO2 decay at high concentrations (≥ 100µM heme) accelerate implying cooperative autooxidation, where as at low concentrations (≤ 10uM) the time courses are biphasic. These results suggest that the biphasic time courses at low hemoglobin are due to differences between tetramers and dimers. We have also measured autooxidation rates for a recombinant hemoglobin, rHb0.1, that contains a genetically crosslinked di-α subunit. This hemoglobin shows a monophasic time course for autooxidation at both high and low protein concentrations, and the azide binding assay showed equal amounts of ferric α and β subunits. We have also examined recombinant mutant hemoglobins to examine the structural factors that govern autooxidation. Increased rates of autooxidation were found for rHb Providence, rHb Bethesda, rHb Presbyterian, and rHb Kirklareli. We have also confirmed that the rate of autooxidation shows a bell-shaped dependence on oxygen concentration and increases markedly as the pH is decreased.Supported by NIH Grant P01 HL110900 and by Grant C-0612 from the Robert A. Welch Foundation.