Oxidative aggregation of hemoglobin–a mechanism for low-temperature plasma-mediated wound healing

Plasma medicine is a field that utilizes reactive species generated from atmospheric low-temperature plasmas for applications such as sterilization, blood coagulation, and cancer therapy. Commercial plasma devices are available for wound healing, but research on the chemical modifications induced by these plasmas is scarce. This study explores the chemical modifications in hemoglobin when exposed to a helium plasma dielectric barrier discharge, with the aim of explaining the potential mechanisms through which it contributes to blood coagulation and enhances wound healing. Optical microscopy of cold atmospheric plasma (CAP) treated whole capillary blood showed an increase in red blood cell (RBC) size and the formation of rouleaux structures. The treatment of whole blood leads to hemolysis of RBCs and the release of intracellular protein content. We then treated purified hemoglobin protein at physiological concentrations, which led to the formation of aggregates that could be observed using ion mobility mass spectrometry (IM–MS), size exclusion chromatography, and optical microscopy. The aggregates formed fibril-like structures as observed using atomic force microscopy. The formation of hemoglobin aggregates is hypothesized to be the result of new intermolecular interactions formed following the CAP-mediated protein oxidation. We studied the changes to hemoglobin structure after treatment with a CAP using high-resolution MS and found that the hemoglobin subunits are oxidized with the addition of at least 4 oxygen atoms each. The intact tetrameric hemoglobin structure remains unchanged; however, the monomeric and dimeric proteins adopt a more compact structure, as observed by IM–MS. We propose that CAP treatment of fresh blood leads to hemolysis, and that the extracellular protein, primarily hemoglobin, is oxidized leading to the formation of aggregates.