To better understand the fragmentation processes of the metal-biothiol conjugates and their possible significance in biological terms, an energy-resolved mass spectrometric study of the glutathione conjugates of heavy metals, of several thiols and disulfides of the glutathione metaboloma has been carried out. The main fragmentation process of gamma-glutamyl compounds, whether in the thiol, disulfide, thioether or metal-bis-thiolate form, is the loss of the gamma-glutamyl residue, a process which ERMS data showed to be hardly influenced by the sulfur substitution. However, loss of the gamma-glutamyl residue from the mono-S-glutathionyl-mercury (II) cation is a much more energetic process, possibly pointing at a strong coordination of the carboxylic group to the metal. Moreover, loss of neutral mercury from ions containing the gamma-glutamyl residue to yield a sulfenium cation was a much more energetic process than those not containing them, suggesting that the redox potential of the thiol/disulfide system plays a role in the formal reduction of the mercury dication in the gas phase. Occurrence of complementary sulfenium and protonated thiol fragments in the spectra of protonated disulfides of the glutathione metaboloma mirrors the thiol/disulfide redox process of biological importance. The intensity ratio of the fragments is proportional to the reduction potential in solution of the corresponding redox pairs. This finding has allowed the calculation of the previously unreported reduction potentials for the disulfide/thiol pair of cysteinylglycine, thereby confirming the decomposition scheme of bis- and mono-S-glutathionyl-mercury (II) ions. Finally, on the sole basis of the mass spectrometric fragmentation of the glutathione-mercury conjugates, and supported by independent literature evidence, an unprecedented mechanism for mercury ion-induced cellular oxidative stress could be proposed, based on the depletion of the glutathione pool by a catalytic mechanism acting on the metal (II)-thiol conjugates and involving as a necessary step the enzymatic removal of the glutamic acid residue to yield a mercury (II)-cysteinyl-glycine conjugate capable of regenerating neutral mercury through the oxidation of glutathione thiols to the corresponding disulfides.
a b s t r a c tSecondary metabolites (SMs) are organic compounds of low molecular mass that represent a vast chemical diversity. In plants, one of their preeminent roles is their repellent activity against predators. The Eurasian red squirrels (Sciurus vulgaris) is one of the most important predators of conifer tree seeds in boreal and alpine forests in Europe. Its population dynamics and space use are strongly affected by the size of Norway spruce (Picea abies) seed-crops, but not by silver fir (Abies alba) seed-crops. Moreover, squirrel heavily feed on spruce seeds but tend to avoid fir seeds, although the latter has a higher seed-energy content per cone. We tested the hypothesis that a higher concentration of some SMs in fir than in spruce seeds and/or cone scales, the protective tissue of seeds, was related with squirrel feeding preferences. We determined terpene concentrations in the cyclohexane extract of seeds and scales by gas-chromatography-electron ionisation mass-spectrometry (GC/EI-MS), and measured the protein precipitation activity of tannin contained in spruce and fir seeds and scales. Of the nearly 300 separated chemical entities, only limonene, ␣-and -pinene and myrcene occurred in all samples and their levels accounted for 80% or more of all cyclohexane-extractable materials of fir and for less than 50% of that of spruce. In fir, limonene was by far the most abundant compound, and fir scales had a 13 times higher concentration of ␣-pinene than spruce scales. Fir seeds had much higher limonene concentrations than fir scales and than seeds or scales of spruce. Tannin concentrations were higher in cone scales than in seeds, with no differences between the two tree species. This study suggests that high concentrations of limonene might reduce the feeding activity of red squirrels on fir seeds, and that the preferred spruce seeds had much lower monoterpene concentrations. Feeding trials using food items treated with different concentrations of limonene will be carried out to confirm this hypothesis.
A series of 19 compounds of general formula R1S-Cd-SR2, R1, and R2, being some biologically relevant thiol amino acids and peptides, were prepared by direct reaction of cadmium(II) ions and thiols in water at millimolar concentration. The obtained products were characterized by electrospray ionization and triple quadrupole tandem mass spectrometry. The source spectra of stoichiometric 1:2 Cd-thiol systems containing either an individual thiol or equimolar mixtures of two different thiols featured several Cd-containing signals, although at much lesser intensity than in the previously reported experiments with mercury(II) (J. Am. Soc. Mass Spectrom. 2004, 15, 288-300). Also, the relative intensity of the homo- and heterodimeric thiolates were significantly different from the theoretically expected 1:2:1 ratio, thus pointing at some degree of discrimination between the different thiols. In particular, homo-cysteine showed much less reactivity than cysteine, and penicillamine and cysteine methyl ester much less than the free amino acid. The fragment spectra show structure-specific ions for the different ligands bound to the metal ion and allow a stand-alone determination of the connectivity also of isomeric pairs. The fragmentation pathways are similar to those observed for the corresponding mercury(II) analogues, with the addition of further intense and specific fragments, one formally carrying a Cd-bound OH ligand and one connected as a five-membered oxazolone carrying a cadmium-bis-thiolate side chain, both formed with a high intensity. Energy-resolved fragmentation data show that metal-free ions can be generated from cysteine but not from glutathione conjugates and point to the possibility of unveiling differences in the biochemical behavior of the conjugates of different heavy metals through the detailed study of their mass spectrometric fragmentation.
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