Precision determination and Adair scheme analysis of oxygen equilibrium curves of concentrated hemoglobin solution. A strict examination of Adair constant evaluation methods

To examine the validity of the recent finding by Gill et al. (S.J. Gill, E. Di Cera, M.L. Doyle, G.A. Bishop and C.H. Robert, Biochemistry 26 (1987) 3995) that the third overall Adair constant (A3) for human hemoglobin tetramers (Hb A) is too small to be determined and therefore that the contribution of the triply ligated species in the oxygenation process is negligibly small, highly accurate oxygen equilibrium curves for concentrated pure Hb A solutions were determined with an automatic oxygenation apparatus and analyzed by a least-squares curve-fitting method with various options. The present results indicate that an appropriate choice of weighting for data points is the key to the correct evaluation of the Adair constants and the present experimental data cannot accommodate the Adair scheme with A3 = 0, giving distinctly positive values for A3. Several criteria for correct determination of the Adair constants are presented.