Interaction of thimerosal with hemoglobin causes alteration of structure and loss of oxygen binding capacity

Thimerosal (TH, Fig. 1a) is an organomercurial used as an antibacterial in vaccines. Recent studies have shown that EtHg+ are absorbed by red blood cells and can bind to hemoglobin (Hb, Fig. 1b).1 This protein is fundamental for the oxygen transport and is the major component of erythrocytes.2 Due to human exposure to these species potentially toxic, we evaluate the interaction of TH with hemoglobin using spectroscopic techniques. Initially, a spectrofluorometric titration was performed exploring intrinsic Hb fluorescence. The increasing of TH concentration leaded to a gradual reduction of analytical signal, indicating that interaction process occurs. Table 1 shows that Kb and Ksv values decreased with temperature increasing, characteristic of static quenching mechanism. Kb values indicates that the Hb-TH presents a moderate interaction and this interaction is stabilized by electrostatic interactions (ΔH < 0 and ΔS > 0). Synchronous fluorescence assay shown that polarity in the microenvironment near of Trp was more changed. These data corroborate with the results obtained by 3D fluorescence. The intermolecular distance between Hb-TM was less than 8 nm (r0 = 2.63 nm) based on FRET studies. Finally, hemoglobin O2 binding capacity decreased 52% comparing free Hb (38.05 ± 2.26 nmol O2 / 10 nmol Hb) with Hb-TM (18.14 ± 0.34 nmol O2 / 10 nmol Hb) at ratio 1:1. Finally, the loss O2 binding capacity must be associated with the transfer of EtHg+ from the TM to the free Cys residue in the Hb β-chain.