Evaluation of the Methemoglobin-Haptoglobin Protein Complex for Detoxification of Cyanide, Azide, and Sulfide in In Vitro Binding Models

Cyanide, azide, and sulfide are potent anion inhibitors of mitochondrial enzymes, causing rapid disruption of cellular energy metabolism and potentially fatal outcomes. Current treatments for these toxic anions are limited, highlighting the need for broadly effective antidotes. Methemoglobin (methHb), the ferric form of hemoglobin (Hb), can bind these ligands with high affinity, acting as a mechanistic scavenger that prevents their interaction with mitochondrial targets. Binding methHb to haptoglobin (Hp), an endogenous Hb-binding plasma protein, offers a strategy to enhance methHb stability and potentially increase circulation time while reducing potential oxidative side effects. Hence, in this work, we examine the detoxification potential of both cell-free methHb and methHb-Hp protein complexes against these anions using in vitro binding assays. Analyses by size exclusion high-performance liquid chromatography (SEC-HPLC) and dynamic light scattering (DLS) indicate that methHb-Hp complex formation exhibits a similar increase in molecular size relative to the Hp solution, while circular dichroism spectroscopy confirms maintenance of protein secondary structure. Kinetic studies using UV-visible spectroscopy and stopped-flow techniques reveal rapid and efficient sequestration of cyanide, azide, and sulfide by both methHb and methHb-Hp. These results demonstrate that Hp association stabilizes methHb without compromising its multiligand binding capacity, supporting its promise as a flexible platform for developing broad-spectrum toxic anion antidotes.