Perturbation of the intermolecular contact regions (molecular surface) of hemoglobin S by intramolecular, low-O-2-affinity-inducing central cavity cross-bridges

The general assumption among researchers on hemoglobin is that the intramol ecular central cavity cross-bridging of Hb does not result in any generaliz ed perturbations at the protein surface. A corollary of this is that centra l cavity cross-bridges are unlikely to influence the polymerization of deox y HbS, since polymerization is a protein surface phenomenon involving the p articipation of multiple protein surface amino acid residues. In an attempt to evaluate this experimentally, we have introduced two low-O-2-affinity-i nducing central cavity cross-bridges into HbS, beta beta-sebacyl [between t he two Lys-82(beta) residues] and alpha alpha-fumaryl [between the two Lys- 99(alpha) residues], and investigated their influence on the polymerization of the deoxy protein. The O-2 affinities of the cross-bridged HbS exhibite d sensitivity toward the buffer ions and pH in a cross-link-specific fashio n. The modulation of the O-2 affinity of these cross-bridged HbS in the pre sence of allosteric effecters, DPG and L-35, is also very distinct, reflect ing the differences in the conformational features these two cross-bridges induce within the central cavity at the respective effector-binding domains . In addition, the alpha alpha-fumaryl cross bridge inhibited the polymeriz ation, reflecting the perturbation of the microenvironment of one or more i ntermolecular contact residues, protein surface residues, as a consequence of the central cavity crossbridge. On the other hand, the beta beta-sebacyl cross-bridge exerted a slight potentiating effect on the polymerization of HbS. This reflects the fact that the perturbations at the protein surface are limited and favor polymerization. The results presented demonstrate tha t the structural changes induced by the central cavity cross-bridges are ve ry specific and not simply restricted to the sites of modification, but are propagated to distant sites/domains, both within and outside the central c avity. It is conceivable that other surface regions that are not involved i n the polymerization could also experience similar structural/conformationa l consequences. These results should be taken into consideration in designi ng intramolecularly cross-bridged asymmetric hybrid HbS for mapping the con tribution of the intermolecular contact residues in the cis and trans dimer s of deoxy HbS during polymerization.