SITE-DIRECTED MUTAGENESIS IN HEMOGLOBIN - EFFECT OF SOME MUTATIONS ATPROTEIN INTERFACES

The role of selected amino acid residues in the monomer monomer contac ts of Hb A has been studied by site-directed mutagenesis of the alpha chain bearing substitutions in the subunit surfaces. Mutation alpha38T hr-->Trp induced a stabilization of tetrameric Hb-CO with a decrease o f the K(d) for the equilibriUM alpha2beta2 double-line arrow pointing left and right 2alphabeta, but had no effect on ligand binding. Mutati on alpha40Thr-->Arg resulted in a complete loss of cooperativity in li gand binding. Mutation alpha103His-->Val had no noticeable effect. We also studied the behaviour of isolated, mutated alpha chains with resp ect to self association: compared to wt alpha chains, mutant alpha38Th r-->Trp showed stabilization of the dimeric state and (at high protein concentration) a detectable amount of tetramers. Mutant alpha103His-- >Val showed only a minor stabilization of the alpha2 dimer.