Mercury-binding Proteins Research Articles

Characterization of the mercury-binding proteins in tuna and salmon sashimi: Implications for health risk of mercury in food

Fish consumption is one of the major ways through which humans receive exposure to mercury (Hg). The existing forms of Hg in food, particularly Hg bound to proteins, may affect the absorption of Hg by humans and subsequently its potentially toxic effects. However, the knowledge regarding Hg-binding proteins in edible fish muscle is scarce. In the present study, salmon and tuna fish muscles, collected from seven different regions and countries, were analyzed using metallomics- and proteomics-based techniques. The concentration of Hg in sashimi samples ranged from 4.4 to 317.4 ng/g. Size exclusion chromatography (SEC) coupled with inductively coupled plasma mass spectrometer (ICP-MS) showed that beta-actin was a novel Hg-binding protein from the fish muscles, and this protein could also bind bismuth (Bi), silver (Ag), and copper (Cu). Hg bound to beta-actin accounted for approximately 30.2–37.6% of the total Hg in the tuna muscles and was significantly correlated to total Hg in the fish muscles (r = 0.98, p < 0.01) and in the fraction of soluble proteins (r = 0.94, p < 0.01). These findings suggest that proteins act as the main Hg accumulation sites in edible fish; thus, increasing human exposure to Hg following gastrointestinal digestion.

Metalloproteomic Strategies for Identifying Proteins as Biomarkers of Mercury Exposure in Serrasalmus rhombeus from the Amazon Region.

This manuscript describes the results of a metalloproteomic study of mercury in samples of muscle and liver tissue of the species Serrasalmus rhombeus, popularly known as black piranha and characterised as the most voracious and aggressive predator in the Brazilian Amazon. The metalloproteomic study involved using two-dimensional electrophoresis (2D PAGE) to fractionate the proteome of the muscle and liver tissue samples, along with atomic absorption spectrometry in a graphite furnace (GFAAS) to identify mercury associated with protein SPOTs and mass spectrometry with electrospray ionisation (ESI-MS/MS) to characterise the mercury-binding proteins. The protein SPOTs characterised showed concentrations in the order of 156 mg kg-1, which ranks as the highest concentrations of mercury determined so far in metalloproteomic studies involving fish species in the Amazon region. Based on FASTA sequences of proteins characterised by ESI-MS/MS, bioinformatics studies were performed that allowed identifying nine proteins with characteristics of biomarkers of mercury exposure. Of those proteins, glutathione peroxidase stands out as an enzyme of great importance in the antioxidant defence of organisms subjected to oxidative stress caused by xenobiotics.

Metalloproteomic approach of mercury-binding proteins in liver and kidney tissues of Plagioscion squamosissimus (corvina) and Colossoma macropomum (tambaqui) from Amazon region: Possible identification of mercury contamination biomarkers

Fish is an important source of protein, vitamins, and minerals. However, this food is also a major source of human exposure to toxic contaminants such as mercury. Thus, this paper aimed to evaluate mercury-binding proteins for possible application as biomarkers of mercury contamination in hepatic and renal tissues of Plagioscion squamosissimus (carnivorous fish) and Colossoma macropomum (omnivorous fish) from the Amazon region using metalloproteomic approach. The proteome of hepatic and renal tissues of fish species was separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and the mercury concentrations in protein spots were determined by graphite furnace atomic absorption spectrometry (GFAAS). Finally, the protein spots associated to mercury were characterized by electrospray ionization mass spectrometry (ESI-MS/MS). The activity of antioxidant enzymes (SOD, CAT, GPx, and GST) and lipid peroxidation (LPO) were also determined. The results showed that the highest concentrations of mercury were found in the carnivorous species (P. squamosissimus) and that the accumulation pattern of this metal was higher in hepatic tissues than in renal tissues for both species. A tendency was observed for greater enzymatic activity in the hepatic and renal tissues of P. squamosissimus, the species with the highest concentration of mercury. Only GPx activity in the kidney and GST in the liver were lower for the P. squamosissimus species, and this finding can be explained by the interaction of mercury with these enzymes. The data obtained by ESI-MS/MS allowed for the characterization of the protein spots associated to mercury, revealing proteins involved in energy metabolism, biomolecules transport, protein synthesis and degradation, cell differentiation, gene regulation, and the antioxidant system. The results obtained in the present study can contribute to understanding the physiological processes underlying mercury toxicity and have provided new perspectives on possible candidates for mercury contamination biomarkers in fish.

Characterization of mercury-binding proteins in rat blood plasma.

Mercury-binding protein profiles in rat plasma at different levels of mercury exposure in vitro and in vivo were systematically investigated using column gel electrophoresis coupled with inductively coupled plasma-mass spectrometry. The current finding provided various protein candidates that may play critical roles in mercury binding and transporting in plasma.

Characterization of mercury-binding proteins in human neuroblastoma SK-N-SH cells with immobilized metal affinity chromatography

Metal-binding proteins play important roles in biological functions of metals. However, only very limited mercury-binding proteins with high abundance were characterized in cells or organisms. Characterization of mercury-binding proteins in proteome-wide is important for elucidating mechanisms of mercury toxicity comprehensively. In this study, a method based on immobilized mercury ion affinity chromatography was developed for identification of putative mercury-binding proteins. The method was then successfully applied to profile mercury-binding proteins in human neuroblastoma SK-N-SH cells. In total, 38 proteins were identified as mercury-binding proteins, in which most of them were uncharacterized to associate with mercury in cells. The identified mercury-binding proteins did not show obvious relevance to protein abundance and were mainly involved in protein processing in endoplasmic reticulum, protein folding, and cytoskeleton organization. The newly built metalloproteomic approach provided valuable information on the possible molecular mechanisms and protein candidates for mercury transport and toxicity.

N-terminal domains of human copper-transporting adenosine triphosphatases (the Wilson's and Menkes disease proteins) bind copper selectively in vivo and in vitro with stoichiometry of one copper per metal-binding repeat.

N-terminal domains of the Wilson's and Menkes disease proteins (N-WND and N-MNK) were overexpressed in a soluble form in Escherichia coli as fusions with maltose-binding protein, purified, and their metal-binding properties were characterized. Both N-MNK and N-WND bind copper specifically as indicated by the results of metal-chelate chromatography, direct copper-binding measurements, and chemical modification of Cys residues in the presence of different heavy metals. When E. coli cells are grown in the presence of copper, N-MNK and N-WND bind copper in vivo with stoichiometry of 5-6 nmol of copper/nmol of protein. Copper released from the copper-N-MNK and copper-N-WND complexes reacts with the Cu(I)-selective chelator bicinchoninic acid in the absence of reducing agents. This suggests that in proteins, it is bound in reduced Cu(I) form, in agreement with the spectroscopic properties of the copper-bound domains. Copper bound to the domains in vivo or in vitro specifically protects the N-MNK and N-WND against labeling with the cysteine-directed probe; this indicates that Cys residues in the repetitive motifs GMTCXXCXXXIE are involved in coordination of copper. Direct involvement of the N-terminal domains in the binding of copper suggests their important role in copper-dependent functions of human copper-transporting adenosine triphosphatases (Wilson's and Menkes disease proteins).

Mercury binding proteins in liver and muscle of flat fish from the northern Tyrrhenian sea

1. The subcellular distribution of mercury and possible presence of Hg binding proteins of low molecular weight were investigated by ultracentrifugation and gel filtration in liver and muscle of the flat fish Citharus linguatula and Lepidorhombus boscii from the northern Tyrrhenian sea, heavily contaminated by the metal. 2. For both tissues, Hg contents were higher in the pellet than in the supernatant. 3. In the eluate of supernatant from Sephadex G-75 of both tissues, Hg was mainly bound to high molecular weight ligands. 4. Differently from the muscle eluate, that from liver also contained a consistent amount of Hg bound to low molecular weight ligands.

Mercury-Binding Proteins in the Slipper Limpet, Crepidula fornicata, Exposed to Increased Soluble Mercury

Crepidula fornicata were held in a flow-through bioassay system and exposed to sand-filtered seawater to which no soluble mercury (control) was added or to which either 5, 25, or 50 μg 1−1 soluble Hg was added. At specific intervals during the 16-week experiment, a group of limpets was removed from each tank; one subgroup was exposed for 48 h to high concentrations of Hg, and another was analyzed for Hg-binding proteins by gelpermeation chromatography and spectrometry. Mortality from exposure to Hg in the 48-h acute toxicity tests was related to concentrations of Hg experienced both during the long-term exposure period and the 48-h exposure period. Chronic exposure to low levels of Hg resulted in increased amounts of total Hg in the whole body and in the low-molecular-weight Hg-binding proteins. No evidence was found for increased tolerance to Hg with preexposure.

Mercury-binding proteins from the marine mussel, Mytilus edulis.

The marine mussel, Mytilus edulis, possesses low molecular weight, metal-binding proteins which can be induced by and, in turn, bind mercury when individuals are exposed to low, but elevated concentrations of mercury as HgCl2. Induction of the proteins by exposure of mussels to copper, cadmium, or mercury is associated with enhanced tolerance to mercury toxicity. Mercury-binding proteins isolated from gills of mussels occur as two molecular weight variants of about 20-25 and 10-12 kdaltons, respectively, on Sephadex G-75. These have been designated as HgBP20 and HgBP10 following the nomenclature used for cadmium-binding proteins. HgBP20 represents the primary mercury-binding species. These exist as dimers which can be dissociated into subunits by treatment with 1% 2-mercaptoethanol. Further purification of HgBP20 by DEAE-cellulose ion-exchange chromatography resulted in the resolution of three major mercury-binding protein peaks; analysis of two of these showed that both had similar amino acid compositions with 26% half-cystine, 16% glycine, and very low levels of the aromatic amino acids phenylalanine and tyrosine (0.3-0.5%), histidine (0.4%), methionine (about 0.5%), and leucine (about 1%). These are similar to the compositions of proteins reported as mussel thioneins by others. Separation of HgBP20 by anion-exchange high-performance liquid chromatography resulted in the resolution of six peaks, indicating a more complex situation than was evident from DEAE-cellulose separations. Although not completely purified, these also contain cysteine- and glycine-rich proteins.

Development of immunological procedures for the study of mercury-binding proteins of Mytilus edulis

Development of immunological procedures for the study of mercury-binding proteins of Mytilus edulis

Enhanced mercury tolerance in marine mussels and relationship to low molecular weight, mercury-binding proteins

Pre-exposure of marine mussels to low levels of mercury (0.5 μg l −1 and less) enhanced their tolerance to more toxic levels. Seawater mercury concentrations similar to our experimental pre-exposure levels have been reported in mercury-contaminated areas where mussels presently exist. A higher pre-exposure concentration (5 μg l −1) was not effective in inducing enhanced mercury tolerance. The relative effectiveness of pre-exposure concentration in inducing enhanced tolerance was related to induction of mercury-binding proteins, the degree of saturation of mercury on the proteins, and the extent of binding of mercury to other subcellular fractions.

Uptake and incorporation of mercury into mercury-binding proteins of gills of Mytilus edulis as a function of time

Individuals of Mytilus edulis (collected from Sequim Bay, Washington, in April and August, 1980) were exposed to 5 μg l-1 mercury as HgCl2 for 28 d. Gill mercury accumulation, mercury incorporation into the soluble fraction and low molecular weight, mercury-binding proteins of gills, and induction of these mercury-binding proteins were determined as a function of time. Short-term mercury uptake rates of excised gills were also determined for mussels sampled at intervals during exposure. Gill mercury accumulation occurred in three phases, represented by net uptake phases initially (up to Day 4) and toward the end of the exposure (Days 15 to 28), and an intermediate stable phase (Days 4 to 15). The stable phase was associated with induction of the predominant mercury-binding proteins and mercury incorporation into the proteins. After Day 15, the mercury-binding proteins were saturated and spillover of mercury into high molecular weight proteins had occurred. This was associated with saturation of the soluble fraction, increases of mercury on particulate fractions, and a loss of the ability of gills to maintain stable mercury concentrations. Mercury uptake rates of gills were not affected by the 28 d exposure of the whole organism.

Influence of mercaptoethanol on the isolation of mercury-binding proteins from the gills of Mytilus edulis

1. 1. Use of the reducing agent 2-mercaptoethanol during extraction and isolation of low mol. wt, mercury-binding proteins of mussel gills resulted in an appearance of significant quantities of mercury associated with substances of <3000 daltons. 2. 2. The source of this mercury was mercury originally bound to the pellet, high mol. wt proteins, and low mol. wt, mercury-binding proteins. 3. 3. This shift in mercury distribution is regarded as a procedural artifact caused by reduction of mercury-binding sites and possible association of released mercury with mercaptoethanol. 4. 4. Use of mercaptoethanol did not release significant amounts of the metal-binding proteins from the high mol. wt protein pool.

Characterization of mercury-binding proteins from the gills of marine mussels exposed to mercury

1. A low molecular weight, mercury-binding protein was isolated from the gills of Mytilus edulis and partially characterized. 2. This protein had a minimum mol. wt of 14,400 daltons and 115 amino acid residues per protein molecule. 3. The relatively low amounts of half-cystine (7.8 mol%) in this protein and the presence of aromatic amino acids and histidine were not characteristic of metallothionein. 4. The amino acid composition was similar to a cadmium-binding protein of mussels reported by other researchers.

Environmental Hygienic Studies on Mercury Compounds. IV. Mercury-binding Proteins in Human Placenta

In order to study the placental transfer of mercury compounds (methylmercury and inorganic mercury), mercury-binding proteins in human placenta were examined by comparing with proteins binding with Zn, Cu, Cd, or Se, and the following results were obtained. 1) The human placenta supernatants were fractionated by Sephadex G-75 column. Zn was mainly bound to high molecular weight (mol. wt.) proteins, but Cu was observed in several mol. wt. ranges. Se was bound both to high and low mol. wt. proteins. Cd was primarily found in low mol. wt. fractions (metallothionein) with only a minute amount in high mol. wt. fractions. Zn and Cu concentration in umbilical cord blood was lower than that in maternal blood. 2) Inorganic mercury was bound both to high (void volume to 40000) and low (about 1100, metallothionein-like protein) mol. wt. proteins. Methylmercury bound to high mol. wt. proteins (void volume to 40000), but components of low mol. wt. (only a few thousands) were also observed. It was noted that methylmercury-binding proteins were differrent from inorganicmercury-binding proteins and, therefore, it was suggested that methylmercury compounds were more transferable across the placenta than inorganic mercury compounds.