Measuring O2-Binding Properties.

O₂ equilibrium curves were measured using a blood oxygen-binding system (BOBS; Loligo Systems) at 37 °C. The pH of whole-blood samples was set by measuring curves in the presence of 45 torr CO₂, whereas the pH of Hb solutions was set with Hepes buffer (see above). Each whole-blood sample and Hb solution was sequentially equilibrated with an array of oxygen tensions (PO₂), while the sample absorbance was continually monitored at 430 nm (deoxy peak) and 421 nm (oxy/deoxy isobestic point). Each equilibration step was considered complete when the absorbance at 430 nm had stabilized (2 to 4 min). Only PO₂ values yielding 30 to 70% Hb O₂ saturation were used in subsequent analyses. Hill plots (log[fractional saturation/[1 − fractional saturation]] vs. logPO₂) were constructed from these measurements. A linear regression was fitted to these plots and used to determine the PO₂ at half-saturation (P₅₀) and the cooperativity coefficient (n₅₀), where the x-intercept and slope of the regression line represent the P₅₀ and n₅₀, respectively. Values for whole-blood samples (n = 3) are presented as mean ± SE. For Hb solutions, a linear regression was fit to plots of logP₅₀ vs. pH, and the resulting equation was used to estimate P₅₀ values at pH 7.40 (± SE of the regression estimate). We did not make direct comparisons between native Hb and recombinantly expressed Hb, because recombinant Hb often exhibits slightly lower P₅₀ values due to an increased rate of autoxidation. Thus, all inferences are based on comparisons among native Hb samples from extant species or on comparisons between recombinant Hb samples representing reconstructed ancestors