Evaluation of methemoglobin and polymerized methemoglobin for detoxification of cyanide, azide, and sulfide in in vitro binding models

Cyanide, azide, and sulfide are fast-acting cellular poisons that inhibit mitochondrial respiration by targeting metal centers within key enzymes, leading to rapid energy failure and potentially lethal metabolic collapse. Despite their clinical relevance, specific antidotes capable of broadly neutralizing these toxic anions remain limited. Methemoglobin (methHb), the ferric form of hemoglobin with high affinity for these ligands, has been recognized as a mechanistic antidote capable of sequestering toxic anions before they reach mitochondrial targets. Polymerized hemoglobin (PolyhHb) and its oxidized counterpart, polymerized methemoglobin (metPolyhHb), offer a promising strategy to potentially enhance the stability, circulation time, and safety profile of methHb-based detoxification systems. In this study, we characterize methHb and metPolyhHb as multi-ligand scavengers using in vitro binding models. High-performance liquid chromatography (HPLC) and dynamic light scattering (DLS) demonstrated that oxidation of PolyhHb does not alter its molecular size or distribution. Circular dichroism confirmed preservation of secondary structure following oxidation. Using UV-visible spectroscopy and stopped-flow kinetics, we show that both methHb and metPolyhHb rapidly and efficiently bind cyanide, azide, and sulfide. These findings demonstrate that PolyhHb retains structural integrity upon oxidation and maintains strong detoxification capability across multiple toxic anions, supporting its potential as a versatile platform for broad-spectrum antidotal development.