The Effect of Temperature on the Oxygen-linked Ionizations of Hemoglobin

Abstract On the assumption that only two of the acid groups per heme are affected by oxygenation, the difference in electrical charge, ΔX, between oxygenated and deoxygenated hemoglobin (O2Hb and Hb) at the same pH (as determined from the difference in their titration curves) is given by the equation. [see PDF for equation] where h = hydrogen ion concentration and Ko, K'o, Kr, and K'r are the ionization constants of these "Bohr groups" in O2Hb and Hb, respectively. The above equation has been found to fit (within the limits of experimental error) the data on the "difference" titration curves for bovine, human, and horse O2Hb and Hb over the pH range 5.0 to 9.3 at 25–37°; "best" (i.e. least mean square) values of the ionization constants have been estimated statistically by means of an automatic computer program, written in Fortran language. Thus, for human hemoglobin at 25°, pKr = 7.84 (±0.006), pKo = 6.84 (±0.009), pK'r = 5.13 (±0.04), and pK'o = 5.60 (±0.03). These figures differ by up to 0.7 pK unit from those obtained by other recent workers, who did not, however, analyze their data statistically. Values for the corresponding heats of ionization, Qo, Q'o, Qr and Q'r, have been calculated from the effect of temperature on the respective ionization constants. The most accurate results have been obtained in the case of human hemoglobin, wherein it is found that (Qr - Qo) is 5,000 (±800) cal. The actual value of Qr is of the order of 11,000 cal and thus lies outside the usual range for the heat of ionization of imidazole and its derivatives. These conclusions are based upon the experimental data not only of the present paper but also of the recent paper (11) of Antonini, Wyman, Brunori, Fronticelli, Bucci, and Rossi-Fanelli, the concordance between these two independent sets of results, when statistically analyzed, being very satisfactory. (Q'o - Q'r) has also been found to be of the order of 5000 cal, but this figure is subject to a wide margin of uncertainty, owing to the difficulty in obtaining accurate estimates of pK'o and pK'r from data in the acid pH range, wherein hemoglobin is unstable. Preliminary experiments have shown that at pH below 6.2 "rapid" titration figures (i.e. within 10 msec) differ significantly from the results obtained by the customary slow titration procedure, which takes several minutes. The results of this paper are discussed in light of current views on the chemical and x-ray structure of O2Hb and Hb