Haptoglobin alters oxygenation and oxidation of hemoglobin and decreases propagation of peroxide-induced oxidative reactions

We compared oxygenation and anaerobic oxidation reactions of a purified complex of human hemoglobin (Hb) and haptoglobin (Hb-Hp) to those of uncomplexed Hb. Under equilibrium conditions, Hb-Hp exhibited active-site heterogeneity and noncooperative, high-affinity O-2 binding (n(1/2)=0.88, P-1/2=0.33 mm Hg in inorganic phosphate buffer at pH 7 and 25 degrees C). Rapid-reaction kinetics also exhibited active-site heterogeneity, with a slower process of O-2 dissociation and a faster process of CO binding relative to uncomplexed Hb. Deoxygenated Hb-Hp had significantly reduced absorption at the lambda(max) of 430 nm relative to uncomplexed Fib, as occurs for isolated Hb subunits that lack T-state stabilization. Under comparable experimental conditions, the redox potential (E-1/2) of Hb-Hp was found to be +54 mV, showing that it is much more easily oxidized than uncomplexed Fib (E-1/2 =+125 mV). The Nernst plots for Hb-Hp oxidation showed no cooperativity and slopes less than unity indicated active-site heterogeneity. The redox potential of Hb-Hp was unchanged by pH over the range of 6.4-8.3. Exposure of Hb-Hp to excess hydrogen peroxide (H2O2) produced ferryl heme, which was found to be more kinetically inert in the Hb-Hp complex than in uncomplexed Hb. The negative shift in the redox potential of Hb-Hp and its stabilized ferryl state may be central elements in the protection against Fib-induced oxidative damage afforded by formation of the Hb-Hp complex. Published by Elsevier Inc.