Sx 20 micro volume stopped flow apparatus

A SX‐20 micro‐volume stopped‐flow apparatus (Applied Photophysics) was used to measure the O₂ offloading kinetics and the haptoglobin (Hp) binding kinetics of hHb/PolyhHb. For the O₂ offloading kinetics, the absorbance at 437.5 nm was measured when a 12.5 µM (heme‐basis) solution of oxygenated hHb/PolyhHb in PBS was rapidly mixed with a 1.5 g/L solution of sodium dithionite in degassed PBS. The average of the kinetic traces was fit to an exponential function using the Applied Photophysics software to regress the first‐order O₂ dissociation rate constant (ko2,off) in s⁻¹.
Hp binding kinetics was monitored by measuring the fluorescence emission at 310 nm when Hp binds to hHb/PolyhHb. A 5, 2.5, 1.25, and 0.625 µM solution of hHb/PolyhHb sample in PBS was rapidly mixed with a 0.25 µM solution of Hp. The average of the kinetic traces was fit to an exponential function and a pseudo‐first‐order rate constant was regressed at each concentration. The pseudo‐first‐order rate constants were plotted against hHb/PolyhHb concentration and fit to a linear function to regress the second‐order Hp binding rate constant (k_Hp‐Hb) in µM⁻¹ s⁻¹.