Evaluation of PEGylated Methemoglobin for Sequestration of Cyanide, Azide, and Sulfide in In Vitro Binding Models

Cyanide, azide, and sulfide are potent inhibitors of mitochondrial enzymes that disrupt cellular energy metabolism and can cause rapid, potentially fatal outcomes. Effective treatments for these toxic anions remain limited, underscoring the need for broadly acting antidotal strategies. Methemoglobin (methHb), the ferric form of hemoglobin (hHb), binds these ligands with high affinity and acts as a mechanistic scavenger by sequestering toxic anions before they can interact with mitochondrial targets. PEGylation of methHb (met-PEG-hHb) offers a strategy to improve methHb molecular stability and may reduce potential oxidative side effects while altering protein interactions that could influence future in vivo pharmacokinetic behavior. In this study, we characterize various met-PEG-hHbs as multiligand scavengers using in vitro binding models. Size-exclusion high-performance liquid chromatography (SEC-HPLC) and dynamic light scattering (DLS) confirm that PEGylation preserves molecular size and homogeneity after oxidation, while circular dichroism spectroscopy demonstrates maintenance of protein secondary structure. UV-vis spectroscopy and stopped-flow kinetics reveal that met-PEG-hHb rapidly and efficiently binds cyanide, azide, and sulfide. These findings show that PEGylation stabilizes methHb while preserving its ability to bind cyanide, azide, and sulfide in vitro, supporting met-PEG-hHb as a promising platform for further preclinical evaluation as a broad-spectrum toxic anion scavenger.