Ergothioneine exhibits selective, capacity-limited localization within a hemoglobin-associated microenvironment.

Ergothioneine (EGT) is a diet-derived sulfur-containing small molecule that accumulates in red blood cells and has been reported to protect hemoglobin (Hb). However, the concentration of Hb in erythrocytes far exceeds that of EGT, raising the question of how low-abundance EGT can effectively protect Hb. Here, we show that EGT is selectively recovered with Hb under trichloroacetic acid (TCA) precipitation conditions, whereas this behavior was not observed in plasma or non-globin proteins. This selective recovery was reproduced with purified Hb and myoglobin. Recombinant cytoglobin and neuroglobin showed much weaker effects. Analyses of EGT analogs suggested the importance of a mercaptoimidazole-related structure. UV-visible spectroscopy showed no detectable alteration of heme absorption, and ITC did not support a simple high-affinity binding interaction. In contrast, Hofmeister salt effects and ultrafiltration experiments supported the involvement of weak hydrophobic association between EGT and Hb. Together, the TCA coprecipitation and ultrafiltration results suggested that the TCA-coprecipitated population may include both directly Hb-associated EGT and proximal EGT. This TCA-coprecipitated population showed a nonlinear, capacity-limited profile. These findings support a model in which EGT is dynamically localized near Hb through weak hydrophobic association rather than stable occupancy of discrete binding sites. Such localization may help explain how EGT influences Hb despite its much lower concentration in erythrocytes.