TABLE 2.
O2 and CO binding parameters for subunits in the R-state form of tetrameric rHb Kirklareli, WT rHbA, and native HbA in 0.1 m sodium phosphate at pH 7.0, 20 °C, taken from Birukou et al. (2)
The symbols k′, k, and K represent association rate, dissociation rate, and association equilibrium constants, respectively, for either CO or O2 binding to the subunits of hemoglobin. P50 is defined as 1/K and represents an equilibrium dissociation constant. M is the ratio of CO to O2 affinities.
| Subunit | kO2′ | kO2 | 1/KO2 (P50 R state) | kCO′ | kCO | 1/KCO (P50 R state) | M (KCO/KO2) |
|---|---|---|---|---|---|---|---|
| μm−1s−1 | s−1 | μm | μm−1s−1 | s−1 | μm | ||
| β native | 82 ± 15 | 28 ± 6 | 0.32 ± 0.10 | 7.1 ± 2.4 | 0.007 ± 0.003 | 0.0010 ± 0.0006 | 320 ± 140 |
| β WT | 60 ± 12 | 31 ± 13 | 0.26 ± 0.04 | 7.1 ± 2.0 | 0.008 ± 0.001 | 0.0011 ± 0.0006 | 230 ± 84 |
| α native | 32 ± 4 | 13 ± 2.9 | 0.39 ± 0.06 | 2.9 ± 0.5 | 0.005 ± 0.002 | 0.0017 ± 0.0007 | 230 ± 75 |
| α WT | 29 ± 11 | 14 ± 8 | 0.53 ± 0.14 | 4.0 ± 1.1 | 0.011 ± 0.004 | 0.0028 ± 0.0015 | 190 ± 82 |
| α H58L | 100 ± 60 | 680 ± 180 | 7.1 ± 5.1 | 22 (20)a,b | 0.0019a | 0.00009a | 80,000a |
a The CO binding experiments for tetrameric rHb α(H58L)β(WT) were only done two times independently in Birukou et al. (2). However, experiments were also done with isolated α(H58L) monomers, and similar rate parameters were observed so the estimated errors for the CO binding parameters for the mutant α subunits are assumed to be ≤30%.
b A sample of purified HbCO Kirklareli from the patient was examined in microsecond flash photolysis experiments, and a fast phase representing bimolecular rebinding to the mutant α subunit was observed with a rate constant equal to ∼20 μm−1 s−1 for CO binding to the R state, as was observed for α(H58L) subunits in both recombinant Hb tetramers and monomers (2).