Comprehensive Biophysical and Biochemical Characterization of Multi-Arm Maleimide-Poly(ethylene glycol) Conjugated Bovine and Human Hemoglobin as Potential Red Blood Cell Substitutes

Abstract This study examines bench-scale synthesis and the physicochemical and functional properties of hemoglobin (Hb) formulations, including bovine (bHb), human (hHb), and pyridoxylated (PLP, pyridoxal-5′-phosphate) human Hb (PLP-hHb), along with their poly(ethylene glycol) (PEG)-conjugated counterparts. These PEGylated Hb constructs were synthesized via an inside-out strategy targeting the β-Cys93 residue of Hb using a four-arm PEG-maleimide molecule (MW = 20 kDa per arm; 80 kDa total), producing multi-arm PEG conjugated Hbs (MA-PEG-Hbs). The precursor Hbs exist as α2β2 tetramers (MW ∼64–65 kDa; diameter ∼5 nm), whereas MA-PEG-Hbs showed increased size (320–345 kDa; ∼14 nm), consistent with 3–4 Hb tetramers per PEG scaffold. This expansion reduced haptoglobin binding kinetics by ∼6–9-fold, potentially prolonging circulation and reducing renal clearance. Circular dichroism confirmed preservation of α-helical structure and heme integrity, while thermal stability decreased but remained within 63–66 °C. Bench-scale synthesis demonstrated scalability, with MA-PEG-hHb achieving the highest yield (∼84 ± 8%). Oxygen-binding studies showed preserved cooperativity and increased affinity (lower P50): MA-PEG-bHb (16.2 ± 0.6 mm Hg), MA-PEG-hHb (6.88 ± 0.10 mm Hg), and MA-PEG-PLP-hHb (8.87 ± 0.27 mm Hg), with cooperativity coefficients of 2.1 ± 0.1, 2.2 ± 0.01, and 1.7 ± 0.03. These contrast with Hemospan (P50 = 6 ± 2 mm Hg; cooperativity 1.2 ± 0.5). Hemospan was synthesized by surface PEGylation of thiolated hHb with 5 kDa maleimide-PEG, suggesting that high oxygen affinity HBOCs such as the MA-PEG-Hbs variants may benefit in targeting ischemic tissues with extremely low oxygen tension. Oxidative analysis showed increased auto-oxidation after PEGylation (bHb: 0.010 → 0.026 h−1; hHb: 0.007 → 0.013 h−1; PLP-hHb: 0.011 → 0.015 h−1), comparable to Hemospan (0.007 → 0.021 h−1). Overall, multi-arm PEGylation enhances the molecular size, increases oxygen affinity, and circulation potential while maintaining oxidative behavior within ranges observed for commercial HBOCs, highlighting a balance between functional performance and biochemical stability.