Hemoglobin and dendrimer conjugates

In this dissertation, we used the hemoglobin and PAMAM dendrimer conjugates to study the oxygen binding behavior of hemoglobin.; Hemoglobins are crowded in red blood cells. The proximity of hemoglobins to one another could elicit transient and frequent protein-protein interactions affecting hemoglobin's oxygen binding and release. To emulate the proximity effect of red cells, a hemoglobin cluster was prepared by conjugating several site-specifically cross-linked hemoglobins to a common core, the PAMAM dendrimer. The resulting protein cluster allows us to study the proximal protein-protein interactions in a structurally-defined manner.; The effect of the conjugated dendrimer on hemoglobin's structure/function was studied by attaching one equivalent of hemoglobin to the dendrimer. CD, NMR and resonance Raman measurements were applied to search for the structural perturbations caused by dendrimer conjugation. Conjugated dendrimer reduces the steric interference effect of the distal ligand binding site, leading to increased oxygen affinity of hemoglobin. These results further our understanding of the mechanism by which hemoglobin controls its oxygen uptake and release.; Without direct conjugation, the PAMAM dendrimer could influence hemoglobin's oxygen affinity by perturbing the activity of heterotropic effectors. At low dendrimer concentration, the oxygen affinity of hemoglobin increases because the positively charged dendrimer competes with hemoglobin for anionic effectors. At high dendrimer concentration, the oxygen affinity of hemoglobin decreases. The decreased oxygen affinity of hemoglobin is an interesting phenomenon, because the polycationic dendrimer has the same effect on hemoglobin as the anionic effectors. Our dynamic laser scattering results showed that the PAMAM dendrimer was an osmolyte lowering water's activity. Water is an effector that preferentially binds to the R state of hemoglobin. The lowered water activity destabilizes the R state, leading to decreased oxygen affinity of hemoglobin. These findings expand our knowledge of hemoglobin's heterotropic effects.