Structure-function relation in hemoglobin Köln (β98 val → met)

Hemoglobin Köln, an unstable hemoglobin resulting from the substitution of normal valine by methionine at FG 5 (β98) is the most commonly encountered unstable hemoglobin. In Hb Köln from a hitherto undetected family, we confirmed earlier observations of heme depletion and high oxygen affinity, with the absence of co-operative interactions. In an effort to elucidate the basis of the altered oxygen equilibria, sedimentation velocity and the kinetics of the reactions of the abnormal hemoglobin with ligands were studied. The results of ultracentrifuge studies indicated that at pH 7 hemoglobin Köln, in the liganded as well as in the deoxy form, existed largely as dimers. The ratio of optical densities at 540 nm and 280 nm indicate that the abnormal β chains were heme depleted. Hb Köln reacted with CO (Hb Köln + CO → l′ Hb Köln CO) approximately 20 times faster than did hemoglobin A (Hb A). However, the corresponding rate constants for O₂ dissociation (Hb Köln O₂ → k Hb Köln + O₂) are similar for Hb Köln and Hb A. For Hb Köln the two rate constants, l′ and k show little pH or concentration dependence. Thus, the high oxygen affinity of Hb Köln (P _{1 2} = 0.2 mm Hg at 10 °C, pH 6.8) arises in part from a larger combination rate constant for the reaction with oxygen. Addition of a 20-fold excess of 2,3-diphosphoglyceric acid did not affect the kinetics of CO-combination. However, in the presence of a sixfold excess of heme, the fast monophasic CO-combination reaction was replaced by a biphasic one. The rate constant of the slow phase was approximately the same as the corresponding rate constant for Hb A. The fast and slow phases were presumably due to the reaction of CO with Hb Köln dimers (α^h/gb^o† † Abbreviation used: ^o as a superscript, heme-depleted chains; Hb A, adult hemoglobin. and hemesaturated tetramers, respectively. The results of the present study are explained in terms of weakened α₁-β₂ and heme-globin contacts in the mutant.