The replacement of beta 5(A2)Pro by Arg in Hb Warwickshire appears to
be without an effect on the functional properties of human Hb A, despi
te adding two external positive charges close to the central cavity of
the hemoglobin tetramer, along the dyad axis. To clarify the role of
this portion of the molecule involved in oxygen-linked anion binding,
we have engineered the recombinant hemoglobin alpha(2) beta(2)5(A2)Pro
-->Ala[rH beta 5(A2)Pro-->Ala]. The rHb beta 5(A2)Pro-->Ala exhibits a
n increased oxygen affinity compared to Hb A, with normal heterotropic
effects in standard conditions. The increased oxygen affinity may be
attributed to the absence of proline, which would render the A helix m
ore flexible, thus destabilizing the T structure. The normal functiona
l properties of Hb Warwickshire may be due to the regulation of oxygen
affinity by electrostatic effects involving diffusible anions not bou
nd to any specific site.