STRUCTURE AND OXYGEN-AFFINITY OF CRYSTALLINE DESARG141-ALPHA HUMAN HEMOGLOBIN-A IN THE T-STATE

The correlation of a protein structure determined crystallographically to its functional properties determined in solution can be an extreme ly complex problem due to potential differences of protein conformatio nal flexibility in the two physical states. A more direct approach to the correlation of structure with function is to examine both the stru cture and the function of a protein in the same crystalline environmen t. In this paper, the structural and functional properties of T state desArg hemoglobin (human hemoglobin modified by removal of the a-chain COOH-terminal residue, Arg141 alpha) have been studied in the same cr ystal form by high resolution X-ray diffraction methods and by polariz ed absorption microspectrophotometry Specifically, the crystal structu re of deoxygenated desArg human hemoglobin has been refined at a 2.1 A ngstrom resolution using crystals grown at low salt concentration from solutions of polyethylene glycol. The loss of Arg141 alpha and all of the salt bridges in which it participates is associated with subtle s tructural perturbations of the a-chains which include an increase in t he conformational flexibility of both the NH2 and COOH-terminal peptid es. Although the heme pockets appear unchanged and even the side-chain of Tyr140 is oriented nearly as in HbA, the functional characterizati on by microspectrophotometric measurements indicates that crystals of desArg hemoglobin bind oxygen with an affinity which is roughly 15-fol d greater than that of crystals of human hemoglobin A. There is no alk aline Bohr effect or effect of chloride ions, but an acid Bohr effect is observed. The oxygen affinities measured along two principal axes o f the crystals differ by 25%, indicating heterogeneity in the affiniti es of the oxygen binding sites. This finding and the measured Hill coe fficient of unity suggest significant cooperativity in the binding of oxygen in these crystals. The origins of the observed heterogeneity an d the implied cooperativity are unknown.