THE DIMER-TETRAMER EQUILIBRIUM OF RECOMBINANT HEMOGLOBINS - STABILIZATION OF THE ALPHA(1)BETA(2) INTERFACE BY THE MUTATION BETA(CYS112-]GLY) AT THE ALPHA(1)BETA(1) INTERFACE

The dimer-tetramer association constants of several recombinant human hemoglobins (in the CO form) have been measured by differential gel fi ltration. Recombinant human hemoglobin prepared from recombinant beta- chains, and mutant hemoglobins where the substitution was on the surfa ce, beta(Thr4 --> Asp), in the heme pocket, beta(Val67 --> Thr), at th e 2,3-DPG binding site, beta(Val1 --> Met + His2del), had a twofold sm aller association with respect to natural hemoglobin. In a mutant at t he alpha(1) beta(2) interface, beta(Cys93 --> Ala), the association co nstant was decreased three-fold. Conversely, in a mutant at the alpha( 1) beta(1) interface, beta(Cys112 --> Gly), the association constant w as two- and four-fold increased with respect to natural and recombinan t human hemoglobin. These differences are energetically very small, co nsistent with the correct folding of the recombinant hemoglobins. The stabilization of the tetrameric structure by a mutation at the alpha(1 ) beta(1) interface indicates that structural changes at this interfac e can be propagated through the protein to the alpha(1) beta(1) interf ace and, thereby, exert an effect on the allosteric equilibrium.