Counting sulfhydryls and disulfide bonds in peptides and proteins using mercurial ions as an MS-tag.

Organic mercurial compounds are the most specific and sensitive reagents for reaction with the sulfhydryl groups (SHs) in peptides and proteins because of the strong mercury-sulfur affinity. Using the monofunctional organic mercury ion RHg(+) as a mass spectrometry (MS)-tag has the advantages of reacting with one sulfhydryl group, offering definite mass shift, and especially stable and characteristic nonradioactive isotopic distribution. Mass spectrometric analysis of derivatized sulfhydryls in peptides/proteins is thus an alternative for precisely counting the number of sulfhydryl groups and disulfide bonds (SS). Here the tags used include monomethylmercury chloride, monoethylmercury chloride, and 4-(hydroxymercuri) benzoic acid. The feasibility of this strategy is demonstrated using HPLC/ESI-MS to count SHs and SS in model peptides/proteins, i.e., glutathione, phytochelatins, lysozyme and beta-lactoglobulin, which contain increasing SHs and various SS linkages.

Comparative study on methyl- and ethylmercury-induced toxicity in C6 glioma cells and the potential role of LAT-1 in mediating mercurial-thiol complexes uptake.

Various forms of mercury possess different rates of absorption, metabolism and excretion, and consequently, toxicity. Methylmercury (MeHg) is a highly neurotoxic organic mercurial. Human exposure is mostly due to ingestion of contaminated fish. Ethylmercury (EtHg), another organic mercury compound, has received significant toxicological attention due to its presence in thimerosal-containing vaccines. This study was designed to compare the toxicities induced by MeHg and EtHg, as well as by their complexes with cysteine (MeHg-S-Cys and EtHg-S-Cys) in the C6 rat glioma cell line. MeHg and EtHg caused significant (p<0.0001) decreases in cellular viability when cells were treated during 30min with each mercurial following by a washing period of 24h (EC50 values of 4.83 and 5.05µM, respectively). Significant cytotoxicity (p<0.0001) was also observed when cells were treated under the same conditions with MeHg-S-Cys and EtHg-S-Cys, but the respective EC50 values were significantly increased (11.2 and 9.37µM). l-Methionine, a substrate for the l-type neutral amino acid carrier transport (LAT) system, significantly protected against the toxicities induced by both complexes (MeHg-S-Cys and EtHg-S-Cys). However, no protective effects of l-methionine were observed against MeHg and EtHg toxicities. Corroborating these findings, l-methionine significantly decreased mercurial uptake when cells were exposed to MeHg-S-Cys (p=0.028) and EtHg-S-Cys (p=0.023), but not to MeHg and EtHg. These results indicate that the uptake of MeHg-S-Cys and EtHg-S-Cys into C6 cells is mediated, at least in part, through the LAT system, but MeHg and EtHg enter C6 cells by mechanisms other than LAT system.

Thimerosal positivities: the role of organomercury alkyl compounds.

Contact allergy to thimerosal (TH) has not been considered a marker for mercury allergy, since there is a low degree of cross-sensitivity to inorganic as well as to organic mercury salts. 40 subjects, who previously gave a positive patch test reaction only to thimerosal 0.1% pet. (Hermal), when simultaneously repatch-tested to solutions containing TH, mersalyl acid, p-amino-phenylmercuric acid, mercuric acetate and thiosalicylic acid, respectively, gave positive reactions only to TH. 36 out of 40 subjects were divided into 2 groups of 18 subjects and simultaneously repatch-tested to solutions containing TH, methylmercury chloride (MeHgCl), thiosalicylic acid, and, ethylmercury chloride (EtHgCl), respectively. EtHgCl was tested in the 1st group at 0.031% and in the 2nd group at 0.015%. The results showed that all subjects gave concomitant positive reactions to TH, EtHgCl and MeHgCl. EtHgCl 0.031% gave a higher number of reactions than EtHgCl 0.015%, underlining the rôle of the solvent in these reactions. Patch test results in 300 consecutive patients to a standard series, to which MeHgCl was added, showed that MeHgCl and TH were never able to give isolated positive reactions, and that the concomitant positive reactions occurred in only 3.6% of subjects. In conclusion, our data seem to suggest that the positive reactions to TH found in our patients were due to EtHgCl, and that the structural similarities with MeHgCl were so close that the skin reacted against each as if they were identical.