Author(s): Miller YI, Smith A, Morgan WT, Shaklai N
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 O2 binding (n1/2=0.88, P1/2=0.33 mm Hg in inorganic phosphate buffer at pH 7 and 25 °C). Rapid-reaction kinetics also exhibited active-site heterogeneity, with a slower process of O2 dissociation and a faster process of CO binding relative to uncomplexed Hb. Deoxygenated Hb–Hp had significantly reduced absorption at the λmax of 430 nm relative to uncomplexed Hb, as occurs for isolated Hb subunits that lack T-state stabilization. Under comparable experimental conditions, the redox potential (E1/2) of Hb–Hp was found to be +54 mV, showing that it is much more easily oxidized than uncomplexed Hb (E1/2=+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 Hb-induced oxidative damage afforded by formation of the Hb–Hp complex.
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