Site-directed mutagenesis in hemoglobin. Effect of some mutations at protein interfaces.

The role of selected amino acid residues in the monomer-monomer contacts of Hb A has been studied by site-directed mutagenesis of the alpha chain bearing substitutions in the subunit surfaces. Mutation alpha 38Thr-->Trp induced a stabilization of tetrameric Hb-CO with a decrease of the Kd for the equilibrium alpha 2 beta 2<==>2 alpha beta, but had not effect on ligand binding. Mutation alpha 40Thr-->Arg resulted in a complete loss of cooperativity in ligand binding. Mutation alpha 103His-->Val had no noticeable effect. We also studied the behaviour of isolated, mutated alpha chains with respect to self association: compared to wt alpha chains, mutant alpha 38Thr-->Trp showed stabilization of the dimeric state and (at high protein concentration) a detectable amount of tetramers. Mutant alpha 103His-->Val showed only a minor stabilization of the alpha 2 dimer.