Biophysical and Biochemical Characterization of PEGylated High-Molecular-Weight Relaxed and Tense Quaternary State Polymerized Human Hemoglobin

Abstract

This study investigates the biophysical properties of PEGylated hemoglobin-based oxygen carriers (HBOCs), including PEGylated human hemoglobin (hHb) and tense (T) and relaxed (R) quaternary state polymerized human hemoglobins (PolyhHbs), to potentially improve their safety and efficacy as red blood cell substitutes. PEGylation slightly increased autoxidation in all variants compared to their precursors (hHb and T- and R-state PolyhHb). PEG-hHb showed elevated heme release, while PEGylated PolyhHbs retained a similar heme release compared to their precursors. The thermal stability of PEGylated variants was maintained compared to their precursors, indicating a preserved structural integrity. PEG-T-state PolyhHb maintained a low oxygen affinity (P₅₀: 46.6 mmHg) and PEG-R-state PolyhHb showed a high affinity (P₅₀: 1.9 mmHg) similar to their precursors, reflecting quaternary structural effects. Oxygen offloading kinetics were quaternary structure-dependent and unaffected by PEGylation. The Bohr effect was significantly reduced in PEG-hHb, T-state PolyhHb, and PEG-T-state PolyhHb compared to hHb. Catalase activity remained consistent post-PEGylation, and both PEG-T- and R-state PolyhHbs exhibited reduced haptoglobin binding compared to that of their precursors, suggesting prolonged circulation potential. Resistance to H₂O₂-induced oxidation was also preserved. Overall, PEGylation modulated key HBOC properties, positioning PEG-T-state polyhHb as a promising candidate for further HBOC development.