Oxygen Offloading and Haptoglobin Binding Kinetics

PolyhHb and hHb O₂ offloading and haptoglobin (Hp) binding kinetics were measured using an SX-20 micro-volume stopped-flow apparatus (Applied Photophysics, Leatherhead, U.K.). The O₂ dissociation kinetics were monitored by absorbance changes at 437.5 nm in 0.1 M PBS, pH 7.4. A 15 µM (heme basis) oxygenated hHb/PolyhHb solution was rapidly mixed with equal volumes of 1.5 mg/mL sodium dithionite^{43 (link)}. The average of eight kinetic traces were fit to exponential equations using Applied Photophysics software to regress the first order oxygen dissociation rate constant (k_O2,off)^{31 (link)}. The Hp binding kinetics were measured and analyzed as described previously by Belcher et al.^{12 (link)}