STRUCTURE AND FUNCTION IN HEMOGLOBINS

The linkage between cofactor and oxygen binding by hemoglobin will be analyzed under conditions approximating physiological ones, i.e. high hemoglobin concentration and ratios of allosteric effector to hemoglobin equal to and less than 1. Oxygen equilibrium curves will be determined as a function of cofactor concentration, cofactor binding constants will be measured by the proton association method and the results analyzed by non-linear least square fitting to the theoretical relation recently derived by Gary Ackers. The concentration dependence of DPG binding to hemoglobin will be investigated using two methods, one of which measures DPG activity and the other DPG concentration. This will permit determination of the thermodynamic as well as the apparent binding constants and yield the activity coefficient of DPG as a function of solution conditions. The activity coefficients will then be used to refine the analysis of the multiple linkage between oxygen, proton and cofactor binding by hemoglobin. Hemoglobin tetramers with one or more of the subunits in the methemoglobin cyanide form will be prepared by intramolecular crosslinking. The oxygenation properties of these molecules, as well as the polymerization of corresponding ones with beta S chains, will be investigated in order to probe the effect of individual subunits "frozen" in the r state on the allosteric transitions of the tetramer.