New mercury transporting protein in human blood identified

Organic mercury species such as Methylmercury and Ethylmercury are widely known to be highly toxic. Humans are exposed to Methylmercury mainly via consumption  of marine food, especially of carnivorous fish species that have bioaccumulated methylmercury and biomagnified its concentration along the food-chain. Moreover, thimerosal (THI) is another mercury containing substance, which is used as a preservative in multidose ampullae of vaccines and other drugs. The antimicrobial and toxic effects of THI are based on its decomposition in aqueous media releasing a (mono)-ethylmercury cation (EtHg+). The molecular mechanisms of MeHg+ and EtHg+ toxicity and toxic kinetics are not fully understood and still are a topic of ongoing research. By entering the body, the organic mercury species are distributed via the blood stream to the different organs. Due to the high affinity of mercury towards sulfur, reactions with free thiol groups of peptides and proteins with different mercury species may occur. It is assumed that the interaction of mercury species with thiols plays an important role for mercury transport, overcoming certain barriers such as the blood/brain barrier and for their toxic mechanisms of action. Prior studies have shown that most of the mercury in blood is bound to the erythrocytes and one of the binding partners, namely hemoglobin, has been identified.

Researchers from the University of Münster now investigated whether further biomolecules interact with the organic mercury species. They concentrated their attention towards another highly abundant protein in blood, namely the carbonic anhydrase (CA). This enzyme catalyzes the reversible hydration of carbon dioxide and is found in all animals and photosynthesizing organisms. Using in vitro experiments under physiological conditions, the researchers indeed were able to identify the adduct formation between CA and the organic mercury species by means of LC/ESI-ToF-MS and LC/ICP-MS.

Figure 1: The adduct formation between CA1 and Methylmercury shows up in the ESI-MS spectrum by the addition of 214.7 DA. After enzymatic digestion of the CA, the methylmercury binding site can be identified by the Mercury isotopic pattern within the peptide-adduct.

Adduct formation between CA and mercury was found for both methylmercury as well as ethylmercury. The adduct could be identified by its exact mass as well as the isotope fingerprint of mercury. To specify the expected binding site, an enzymatic digest was performed. Trypsin  and  chymotrypsin  were used  in  parallel  for  the digestion to obtain different peptide patterns. The tryptic digest of CA1 results in 24 specific peptides, in which the peptide T19 (#174–213)  is  the  peptide  featuring  the  only cysteine residue and consists of 40 amino acids. The digestion with chymotrypsin produced 45 fragments from CA1. The peptide C40, which contains the cysteine, consists of 14 amino acids (#210–223). In both digestion experiments, mercury binding was found at the only cysteine containing peptide. In this way it was verified that carbonic anhydrase, and not only hemoglobin, provides such binding site for organic mercury species and forms the related adducts. Since both proteins have a high abundance in blood, they both are expected to play a role in the
transport of mercury in blood.

 

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