Non-hypertensive tetraPEGylated canine haemoglobin: Correlation between PEGylation, O₂ affinity and tissue oxygenation

TetraPEGylated canine Hb, [SP (succinimidophenyl)-PEG5K] ₄-canine-Hb, with PEGylation at its four reactive cysteine residues (α₁₁₁ and β₉₃) has been prepared and characterized. The hydrodynamic volume and the molecular radius of (SP-PEG5K)₄-canine-Hb are intermediate to those of di- and hexaPEGylated human Hb as expected. However, the COP (colloidal osmotic pressure) of tetraPEGylated canine Hb is closer to that of hexaPEGylated human Hb than to that of diPEGylated human Hb. The O₂ affinity of tetraPEGylated canine Hb is higher than that of canine Hb and comparable with that of hexaPEGylated Hb. The O₂ affinity of tetraPEGylated canine Hb is not responsive to the presence of DPG (diphosphoglycerate) or chloride, but it retains almost full response to L-35, an allosteric effector that interacts at the αα-end of the central cavity. The tetraPEGylated canine Hb is vasoinactive in hamster in 10% top load infusion studies. It is also essentially non-hypertensive in an extreme exchange haemodilution protocol in hamster just as di- and hexaPEGylated human Hb. The O₂ delivery by tetraPEGylated canine Hb is comparable with that of hexaPEGylated Hb but not as efficient as diPEGylated Hb. These results demonstrate that PEGylation-induced solution properties of PEG [poly(ethylene glycol)]-Hb conjugates are dictated by the level and chemistry of PEGylation and the interplay of these plays a critical role in tissue oxygenation. The studies imply the need to establish the right level (and/or pattern) of PEGylation and O₂ affinity of Hb-PEG adducts in designing O₂-carrying plasma volume expanders, and this remains the primary challenge in the design of PEGylated Hb as blood substitutes.