The carbamate equilibrium of bovine hemoglobin at 37 °c

Abstract

Hemoglobin-bound CO 2 was estimated by a procedure first described by Rossi-Bernardi et al. (1969) in which the carbamate compound is stabilized by rapid mixing with alkali and then separated from other CO 2 constituents in solution by gel filtration and ion-exchange chromatography. Carbamate equilibrium of bovine hemoglobin was studied at constant P co 2 (44 mm Hg) and varying pH as well as at constant pH (7.4) and varying P co 2 (ionic strength 0.18, temperature 37.0 °C). The difference in Z (ΔZ) between hemoglobin and oxyhemoglobin appeared to be 0.11 ± 0.04 (pH = 7.40; P co 2 = 44 mm Hg) i.e. about half the value observed in human hemoglobin. ΔZ was shown to account completely for the difference in CO 2 content (C co 2 ) between hemoglobin and oxyhemoglobin when in total equilibrium with CO 2. Carbamate determinations on bovine hemoglobin specifically modified at all terminal amino groups (double-blocked carbamylated derivative) did not show any CO 2 binding at all, thus giving a final proof for the exclusive role of the terminal amino groups in CO 2 binding under physiological conditions. Attempts to calculate the ionization constant (K z) and the carbamate equilibrium constant (K c) of the terminal amino groups failed, suggesting that both terminal groups are not equivalent in their CO 2 binding properties. This was confirmed by the fact that earbamate data obtained at constant P co 2 and varying pH fitted binding curves derived from two sets of independent but non-equivalent binding sites. Association constants for both kinds of binding sites appeared to differ by a factor of at least 3 in hemoglobin and of about 10 in oxyhemoglobin. From determinations of hemoglobin-bound CO 2 and CO 2 content of hemoglobin and oxyhemoglobin solutions in total equilibrium with CO 2. the apparent first dissociation constant of carbonic acid was calculated as 5.71 + 0.061 pH and found to be independent of the oxygénation state of hemoglobin. In contrast with hemoglobin of other species bovine hemoglobin appeared to be not influenced by the presence of 2,3-diphosphoglycerate as far as its CO 2-binding properties are concerned.