Autoxidation of the Site-Specifically PEGylated Hemoglobins: Role of the PEG Chains and the Sites of PEGylation in the Autoxidation

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Abstract
The PEGylated hemoglobin (Hb) has been evaluated as a potential blood substitute. In an attempt to understand the autoxidation of the PEGylated Hb, we have studied the autoxidation of the PEGylated Hb site-specifically modified at Cys-93(β) or at Val-1(β). PEGylation of Hb at Cys-93(β) perturbed the heme environment and increased the autoxidation rate of Hb, which is at a higher level than that caused by PEGylation at Val-1(β). The perturbation of the heme environment of Hb is attributed to the maleimide modification at Cys-93(β) and not due to conjugation of the PEG chains. However, the PEG chains enhance the autoxidation and the H2O2 mediated oxidation of Hb. Accordingly, the PEG chains are assumed to increase the water molecules in the hydration layer of Hb and enhance the autoxidation by promoting the nucleophilic attack of heme. The autoxidation rate of the PEGylated Hb does not show an inverse correlation with the oxygen affinity. The H2O2 mediated structural loss and the heme loss of Hb are increased by maleimide modification at Cys-93(β) and further decreased by conjugation of the PEG chains. The autoxidation of the PEGylated Hbs is attenuated significantly in the plasma, possibly due to the presence of the antioxidant species in the plasma. This result is consistent with the recent suggestion that there is no direct correlation between the in vitro and in vivo autoxidation of the PEGylated Hb. Therefore, the pattern of PEGylation can be manipulated for the design of the PEGylated Hb with minimal autoxidation.

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