A flow procedure to determine oxygen binding isotherms for low affinity and easily oxidized hemoglobins

1. 1. The determination of binding isotherms for low affinity hemoglobins is particularly difficult because of rapid oxidation. 2. 2. In carp Hb (pH 6 + 1HP, 25°C), one-quarter of the hemes are oxidized within 3 min, preventing the accurate determination of even P₅₀ or X. 3. 3. We circumvent this problem by rapidly flowing HbO₂, initially at pH 9 (X = 1.4 μM), against a low pH buffer to bring the system rapidly to equilibrium in the low affinity form. Diode-array spectrophotometry allows a complete spectrum to be obtained < 5 sec, after flow ceases, before significant oxidation has occurred. In tandem with the stopped flow apparatus is an oxygen electrode to measure O₂ activity. 4. 4. At 22°C, the half-saturation oxygen activity (X) is 227 μM, and the Hill number is 0.91, for carp Hb (T state) implying significant differences in the O₂ affinities of the alpha and beta chain hemes or, two different T states.