Comprehensive Biophysical Characterization of Multi-Arm PEGylated Apohemoglobin as a Potential Synthetic Heme Scavenger

Abstract Cell-free heme released during hemolysis is highly toxic and pro-oxidant, necessitating the development of effective heme scavengers. In this study, four hemoglobin (Hb)-derived constructs, apohemoglobin (apoHb), linear poly(ethylene glycol) (PEG) surface conjugated apoHb (PEG-apoHb), multi-arm PEG surface conjugated apoHb (MA-PEG-apoHb), and heme-removed multi-arm PEG-Hb (HR-MA-PEG-Hb) were systematically evaluated to identify an optimal heme-scavenging candidate based on production feasibility, physicochemical characteristics, structural homogeneity, and heme-binding properties. ApoHb exhibited moderate yield (53 ± 11%), high activity (71.9 ± 7.1%), the strongest apparent ferric heme affinity (Kd,app = 10.7 ± 0.7 μM), rapid heme acquisition from heme-albumin (heme-HSA) (0.017 ± 0.0003 s−1), and fast haptoglobin (Hp) binding. However, its small hydrodynamic diameter (∼3.9 nm) suggests rapid renal clearance, limiting its potential therapeutic utility. PEG-apoHb exhibited the highest yield (82 ± 6%) and increased hydrodynamic size (10.3 ± 0.4 nm), but reduced heme-binding activity, slower heme acquisition, weaker ferric heme affinity (Kd,app = 12.6 ± 0.7 μM), accelerated heme transfer to hemopexin, and markedly diminished Hp binding. In contrast, both MA-PEG-apoHb and HR-MA-PEG-Hb exhibited larger hydrodynamic diameters (18.8 ± 1.1 and 12.1 ± 0.7 nm, respectively), reduced Hp-binding kinetics, efficient heme acquisition from heme-HSA (0.015 ± 0.002 and 0.013 ± 0.001 s−1, respectively), and markedly faster heme-binding kinetics (kfast ≈ 5−7 × 107 M−1 s−1) than apoHb and PEG-apoHb, while exhibiting only modest reductions in ferric heme affinity. SEC-HPLC, DLS, and SDS-PAGE analysis further showed that HR-MA-PEG-Hb formed a more homogeneous conjugate with a narrower molecular-weight (MW) distribution and lower polydispersity than MA-PEG-apoHb, which exhibited a broader MW distributions and greater heterogeneity. Overall, HR-MA-PEG-Hb, and to a lesser extent MA-PEG-apoHb, demonstrated the most favorable combination of structural homogeneity, physicochemical properties, heme-binding affinity, and kinetic performance, thus identifying HR-MA-PEG-Hb as the most promising candidate for the treatment of heme toxicity.