Combining the influence of two low O₂ affinity-inducing chemical modifications of the central cavity of hemoglobin

HexaPEGylated hemoglobin (Hb), a non-hypertensive Hb, exhibits high O ₂ affinity, which makes it difficult for it to deliver the desired levels of oxygen to tissues. The PEGylation of very low O₂ affinity Hbs is now contemplated as the strategy to generate PEGylated Hbs with intermediate levels of O₂ affinity. Toward this goal, a doubly modified Hb with very low O₂ affinity has been generated. The amino terminal of the β-chain of HbA is modified by 2-hydroxy, 3-phospho propylation first to generate a low oxygen affinity Hb, HPPr-HbA. The oxygen affinity of this Hb is insensitive to DPG and IHP. Molecular modeling studies indicated potential interactions between the covalently linked phosphate group and Lys-82 of the trans β-chain. To further modulate the oxygen affinity of Hb, the αα-fumaryl cross-bridge has been introduced into HPPr-HbA in the mid central cavity. The doubly modified HbA (αα-fumaryl-HPPr- HbA) exhibits an O₂ affinity lower than that of either of the singly modified Hbs, with a partial additivity of the two modifications. The geminate recombination and the visible resonance Raman spectra of the photoproduct of αα-fumaryl-HPPr-HbA also reflect a degree of additive influence of each of these modifications. The two modifications induced a synergistic influence on the chemical reactivity of Cys-93(β). It is suggested that the doubly modified Hb has accessed the low affinity T-state that is non-responsive to effectors. The doubly modified Hb is considered as a potential candidate for generating PEGylated Hbs with an O₂ affinity comparable to that of erythrocytes for developing blood substitutes.