Uptake of metallic mercury and mercuric ion by human erythrocytes

Uptake of metallic mercury (Hg degrees) and mercuric ion (Hg2+) by erythrocytes was studied by incubating erythrocytes with various concentrations of radioactive metallic mercury and mercuric ion in phosphate-buffered saline (pH 6.8) or plasma at 25 degrees C for 30 min. Radioactivity taken up in the cytosol (endsome) and stroma were determined with a gamma scintillation counter. The radioactivity ratio of the mercury recovered in the cytosol fraction to metallic mercury incubated in the saline was significantly higher than the ratio of that to mercuric ion. Similar findings were observed in erythrocytes incubated with metallic mercury and mercuric ion in plasma, although the recovered radioactivity of mercury in the cytosol of erythrocytes incubated with metallic mercury or mercuric ion in plasma was less than that incubated in phosphate-buffered saline. Thus, erythrocytes incubated with metallic mercury took up a larger amount of mercury than those incubated with mercuric ion. Discussion is made on these findings.     

Specific labeling and partial inactivation of cytochrome oxidase by fluorescein mercuric acetate

Addition of 1 eq of fluorescein mercuric acetate (FMA) to beef heart cytochrome oxidase was found to inhibit the steady-state electron transfer activity by 50%, but further additions up to 10 eq had no additional effect on activity. The partial inhibition caused by FMA is thus similar to that observed with other mercury compounds (Mann, A. J., and Auer, H. E. (1980) J. Biol. Chem. 255, 454-458). The fluorescence of FMA was quenched by a factor of 10 upon binding to cytochrome oxidase, consistent with the involvement of a sulfhydryl group. However, addition of mercuric chloride to FMA-cytochrome oxidase resulted in an increase in fluorescence, suggesting that FMA was displaced from the high affinity binding site. Cytochrome c binding to FMA-cytochrome oxidase resulted in a 10% decrease in the fluorescence, possibly caused by Forster energy transfer from FMA to the cytochrome c heme. The binding site for FMA in cytochrome oxidase was investigated by carrying out sodium dodecyl sulfate gel electrophoresis under progressively milder dissociation conditions. When FMA-cytochrome oxidase was dissociated with 3% sodium dodecyl sulfate and 6 M urea, FMA was predominantly bound to subunit II following electrophoresis. However, when the dissociation was carried out at 4 degrees C in the absence of urea with progressively smaller amounts of lithium dodecyl sulfate, the labeling of subunit II decreased and that of subunit I increased. These experiments demonstrate that mercury compounds bind to a high affinity site on cytochrome oxidase, possibly located in subunit I, but then migrate to subunit II under the normal sodium dodecyl sulfate gel electrophoresis conditions. A definitive assignment of the high affinity binding site in the native enzyme cannot be made, however, because it is possible that mercury compounds can migrate from one sulfhydryl to another under even the mildest electrophoresis conditions.     

Levels of metallic mercury and mercuric ion in the venous and arterial bloods of normal and acatalasemic mice following exposure to mercury vapor

Levels of metallic mercury and mercuric ion in the arterial and venous bloods of normal and acatalasemic mice exposed to metallic mercury vapor in vitro and in vivo were investigated. Mercury uptake in venous blood from air saturated with mercury vapor with or without hydrogen peroxide in vitro was determined. Level of mercuric ion in venous blood of normal mice was significantly higher than that of acatalasemic mice. By contrast, metallic mercury in venous blood of acatalasemic mice was elevated relative to level in normal mice. Metallic mercury level in red blood cells and plasma was also significantly higher in acatalasemic mice. The ratio of metallic mercury to total mercury (Hg degrees + Hg2+) in the arterial and venous bloods of acatalasemic mice exposed to metallic mercury vapor was increased relative to normal mice. This ratio in red blood cells and plasma in the venous bloods of acatalasemic mice in vivo was also significantly higher than those of normal mice. The significance of metallic mercury in plasma for distribution of mercury in organs is discussed.     

Bacterial removal of mercury from sewage

Mercury-resistant bacteria, which are able to reduce mercuric ion (Hg(2+)) to metallic mercury (Hg(0)), were examined for their ability to remove mercury from waste-water aerobically. Growth studies in artificial medium indicated that mercury increases the lag phase, but does not effect the growth rate of these bacteria. Further studies demonstrated that growth was minimal during a phase of rapid mercury removal, after which growth resumed. Small but significant amounts of carbohydrates are required for the mercuric ion reduction. Prolonged periods of bacterial growth under nonsterile conditions was accomplished without the loss of the mercuric reducing ability of the culture. A continuous culture of the resistant organism was maintained on raw sewage for two weeks, during which time relatively high concentrations of mercury (70 mg/L) were removed from the sewage at a rate of 2.5 mg/L h and at efficiencies exceeding 98%.     

Fluorescence labeling of the human erythrocyte anion transport system. Subunit structure studied with energy transfer

The anion transport system of human red cells was isolated in vesicles containing the original membrane lipids and the 95 000 dalton polypeptides (band 3) by the method of Wolosin et al. (J. Biol. Chem. (1977) 252, 2419–2427). The vesicles have a functional anion transport system since they display sulfate transport that is inhibited by the fluorescent probe 8-anilinonaphthalene 1-sulfonate (ANS) with similar potency as in red cells. The vesicles were labeled with the SH-specific probe fluorescein mercuric acetate (FMA). Labeling lowers FMA fluorescence, and is prevented or reversed by dithiothreitol, suggesting that the reaction is with a thiol group on the protein. Fluorescence titrations show a maximum labeling stoichiometry of 1.3 ± 0.4 mol FMA/mol 95 000 dalton polypeptide. The polarization of bound FMA fluorescence is high indicating that the probe is highly immobilized. Pretreatment with Cu²⁺ + o-phenanthroline under conditions that crosslink band 3 in ghosts decreases FMA labeling 50%. Differences in kinetics of FMA labeling in sealed and leaky vesicles suggest that the reactive SH group is located in the intravesicular portion of the protein (corresponding to the cytoplasmic surface of the red cell) and that FMA can cross the membrane. Inhibitors of anion transport have no effect on FMA labeling kinetics suggesting it is not transported via the anion     

A fluorescent histochemical method for the detection of tissue disulphide groups

Summary Bennett and Yphantis (1948) introduced into histochemistry the use of organic mercurials for the detection of sulphydryl groups. The new fluorescent method which is now reported is similarly based upon the reaction of mercaptans with an organic mercurial, fluorescein mercuric acetate (FMA). This method is sensitive; moreover it is not as elaborate as the dihydroxy-dinaphthyl-disulphide (DDD) technique of Barrnett and Seligman (1952).     

Foetal distribution of inhaled mercury vapor in normal and acatalasaemic mice

Levels of mercury distribution in placenta, amniotic sac and foetus and those in brain and liver of maternal acatasaemic mice were higher than those of normal, respectively. The levels of mercury distribution in the blood and lungs of maternal acatalasaemic mice exposed to metallic mercury vapor were also lower than those of normal. Mercury concentrations in placenta and foetus of acatalasaemic mice following exposure to metallic mercury vapor were higher than those of normal. Maternal acatalasaemic mice had decreased levels of mercury in the blood than those of normal mice. Thus, the placenta/blood or foetus/blood ratio of mercury concentration in acatalasaemic mice was significantly higher than that in normal mice. Similarly the brain/blood or liver/blood ratio of maternal acatalasaemic mice was higher than that of normal mice. These results suggest that metallic mercury in the blood readily passed through the blood-brain, blood-foetus barriers. In contrast to the results on exposures of mice to metallic mercury, the foetus/maternal blood ratio of mercuric concentration in the acatalasaemic mice following injection of mercuric chloride was similar to that in the normal mice. Moreover, the foetus/maternal blood ratio of mercury concentration in acatalasaemic or normal mice injected with mercuric chloride was lower than those in acatalasaemic or normal mice exposed to metallic mercury.     

Mercury and Organomercurial Resistances Determined by Plasmids in Pseudomonas

Mercury and organomercurial resistance determined by genes on ten Pseudomonas aeruginosa plasmids and one Pseudomonas putida plasmid have been studied with regard to the range of substrates and the range of inducers. The plasmidless strains were sensitive to growth inhibition by Hg(2+) and did not volatilize Hg(0) from Hg(2+). A strain with plasmid RP1 (which does not confer resistance to Hg(2+)) similarly did not volatilize mercury. All 10 plasmids determine mercury resistance by way of an inducible enzyme system. Hg(2+) was reduced to Hg(0), which is insoluble in water and rapidly volatilizes from the growth medium. Plasmids pMG1, pMG2, R26, R933, R93-1, and pVS1 in P. aeruginosa and MER in P. putida conferred resistance to and the ability to volatilize mercury from Hg(2+), but strains with these plasmids were sensitive to and could not volatilize mercury from the organomercurials methylmercury, ethylmercury, phenylmercury, and thimerosal. These plasmids, in addition, conferred resistance to the organomercurials merbromin, p-hydroxymercuribenzoate, and fluorescein mercuric acetate. The other plasmids, FP2, R38, R3108, and pVS2, determined resistance to and decomposition of a range of organomercurials, including methylmercury, ethylmercury, phenylmercury, and thimerosal. These plasmids also conferred resistance to the organomercurials merbromin, p-hydroxymercuribenzoate, and fluorescein mercuric acetate by a mechanism not involving degradation. In all cases, organomercurial decomposition and mercury volatilization were induced by exposure to Hg(2+) or organomercurials. The plasmids differed in the relative efficacy of inducers. Hg(2+) resistance with strains that are organomercurial sensitive appeared to be induced preferentially by Hg(2+) and only poorly by organomercurials to which the cells are sensitive. However, the organomercurials p-hydroxymercuribenzoate, merbromin, and fluorescein mercuric acetate were strong gratuitous inducers but not substrates for the Hg(2+) volatilization system. With strains resistant to phenylmercury and thimerosal, these organomercurials were both inducers and substrates.     

Reduction of mercuric ion in vitro by superoxide anion

The reduction rate of mercuric ion to metallic mercury by a superoxide anion produced by a xanthine-xanthine oxidase system increased with an increased concentration of xanthine oxidase in the presence of enough xanthine. The reduction rate of mercuric ion by a superoxide anion in the presence of nitroblue tetrazolium (NBT) was proportional to the concentration of NBT. The result suggests that NBT was reduced to diformazan by a superoxide anion produced by a xanthine-xanthine oxidase system and that mercuric ion will be reduced to metallic mercury by diformazan. The reduction rate of mercuric ion was also indicative that a superoxide anion produced by an NADH-phenazine methosulfate (PMS) system increased with an increased concentration of PMS.     

Fluorescein mercuric acetate specifically displaces zinc from cytochrome oxidase

Treatment of beef heart cytochrome oxidase with fluorescein mercuric acetate (FMA) was found to specifically displace zinc from the enzyme and inhibit the steady-state activity in a parallel fashion. The native cytochrome oxidase preparation contained 2.3 Cu: 2.0 Fe: 1.1 Zn: 0.9 Mg. Addition of 2 equivalents of FMA inhibited the activity by 50% and displaced 60% of the zinc from the enzyme, but did not affect the copper, iron or magnesium content. The pre-steady-state reduction of cytochrome oxidase by ferrocytochrome c was not affected by the FMA treatment, in contrast to the inhibition of steady state activity. These results suggest a possible structural or functional role for zinc in cytochrome oxidase.     

The ecology of mercury-resistant bacteria in Chesapeake Bay

Total ambient mercury concentrations and numbers of mercury resistant, aerobic heterotrophic bacteria at six locations in Chesapeake Bay were monitored over a 17 month period. Mercury resistance expressed as the proportion of the total, viable, aerobic, heterotrophic bacterial population reached a reproducible maximum in spring and was positively correlated with dissolved oxygen concentration and sediment mercury concentration and negatively correlated with water turbidity. A relationship between mercury resistance and metabolic capability for reduction of mercuric ion to the metallic state was established by surveying a number of HgCl₂-resistant cultures. The reaction was also observed in microrganisms isolated by differential centrifugation of water and sediment samples. Mercuric ion exhibited an average half-life of 12.5 days in the presence of approximately 10⁵ organisms/ml. Cultures resistant to 6 ppm of mercuric chloride and 3 ppm of phenylmercuric acetate (PMA) were classified into eight generic categories.Pseudomonas spp. were the most numerous of those bacteria capable of metabolizing both compounds; however, PMA was more toxic and was more selective forPseudomonas. The mercury-resistant generic distribution was distinct from that of the total bacterial generic distribution and differed significantly between water and sediment, positionally and seasonally. The proportion of nonglucose-utilizing mercury-resistantPsuedomonas spp. was found to be positively correlated with total bacterial mercury resistance. It is concluded from this study that numbers of mercury-resistant bacteria as established by plate count can serve as a valid index ofin situ Hg²⁺ metabolism.     

Absorption, fluorescence, and linear dichroism spectra of fluorescein mercuric acetate (FMA) bound to F-actin. Two kinds of effects of divalent cations.

Two kinds of F-actin were prepared; one binds magnesium at the unique divalent cation binding site of actin protomer and the other binds calcium at this site. They were designated as F(Mg)-actin and F(Ca)-actin, respectively. The binding of fluorescein mercuric acetate (FMA) to F(Mg)-actin and F(Ca)-actin was studied spectroscopically. The absorption and fluorescence spectra of bound FMA differed slightly but distinctly between F(Mg)-actin and F(Ca)-actin. Moreover, FMA bound to F(Mg)-actin showed linear dichroism in the presence of 2 mM MgCl2 (or 2mM CaCl2) in the solvent, while the dichroism was abolished by the removal of divalent cations from the solvent. In contrast, FMA bound to F(Ca)-actin did not show any appreciable linear dichroism irrespective of the presence (or absence) of divalent cations in the solvent. These results suggest that the structure of F-actin is characteristically regulated by divalent cations in a dual mode.     

Metallic mercury in the arterial blood of normal and acatalasemic mice exposed to metallic mercury vapor

Concentration of metallic mercury in the arterial blood was higher in acatalasemic mice after exposure to 3.45 mg/m3 for 10 minutes in comparison with normal mice, whereas concentration of mercuric ion was lower in acatalasemic mice than in normal mice. Thus, the ratio of metallic mercury to total mercury in the arterial blood of acatalasemic mice was 5.86 +/- 3.61%, which was statistically significantly higher than the value (1.36 +/- 0.65%) of corresponding normal mice. The mercury concentration and distribution in the brain and liver of acatalasemic mice were higher than those in normal mice. Data indicate that the concentration of metallic mercury in the arterial blood of acatalasemic mice was higher than that of normal mice and that metallic mercury soluble in lipids is likely transferred to the brain and liver from the blood. Conclusively, metallic mercury in the arterial blood is the biologically active form for transferring mercury from blood to organs.     

The ecology of mercury-resistant bacteria in Chesapeake Bay

Total ambient mercury concentrations and numbers of mercury resistant, aerobic heterotrophic bacteria at six locations in Chesapeake Bay were monitored over a 17 month period. Mercury resistance expressed as the proportion of the total, viable, aerobic, heterotrophic bacterial population reached a reproducible maximum in spring and was positively correlated with dissolved oxygen concentration and sediment mercury concentration and negatively correlated with water turbidity. A relationship between mercury resistance and metabolic capability for reduction of mercuric ion to the metallic state was established by surveying a number of HgCl₂-resistant cultures. The reaction was also observed in microrganisms isolated by differential centrifugation of water and sediment samples. Mercuric ion exhibited an average half-life of 12.5 days in the presence of approximately 10⁵ organisms/ml. Cultures resistant to 6 ppm of mercuric chloride and 3 ppm of phenylmercuric acetate (PMA) were classified into eight generic categories.Pseudomonas spp. were the most numerous of those bacteria capable of metabolizing both compounds; however, PMA was more toxic and was more selective forPseudomonas. The mercury-resistant generic distribution was distinct from that of the total bacterial generic distribution and differed significantly between water and sediment, positionally and seasonally. The proportion of nonglucose-utilizing mercury-resistantPsuedomonas spp. was found to be positively correlated with total bacterial mercury resistance. It is concluded from this study that numbers of mercury-resistant bacteria as established by plate count can serve as a valid index ofin situ Hg²⁺ metabolism.     

Demonstration of protein-bound sulfhydryl and disulfide groups with fluorescent mercurials

Summary Four mercurials, mercury orange, 4-(p-dimethylamino benzene azo) phenyl mercuric acetate, fluorescein mercuric acetate, and mercurochrome were tested and found to be successful reagents for demonstrating protein-bound SH and SS groups. Of this group, 4-(p-dimethylamino benzene azo) phenyl mercuric acetate gave the weakest fluorescence and mercurochrome gave the most useful contrast by direct microscopy. While no quantitative data are yet available, qualitatively reliable patterns were obtained after one hour in the staining solutions.Binding of the dyes was influenced by both fixation and conditions of staining. These factors are discussed in the context of the experimental design.     

Transformation of peanut using a modified bacterial mercuric ion reductase gene driven by an actin promoter from Arabidopsis thaliana

In order to test an alternative selectable marker system for the production of transgenic peanut plants (Arachis hypogaea), the bacterial mercuric ion reductase gene, merA, was introduced into embryogenic cultures via microprojectile bombardment. MerA reduces toxic Hg(II) to the volatile and less toxic metallic mercury molecule, Hg(0), and renders its source Gram-negative bacterium mercury resistant. A codon-modified version of the merA gene, MerApe9, was cloned into a plant expression cassette containing the ACT2 promoter from Arabidopsis thaliana and the NOS terminator. The expression cassette also was inserted into a second vector containing the hygromycin resistance gene driven by the UBI3 promoter from potato. Stable transgenic plants were recovered through hygromycin-based selection from somatic embryo tissues bombarded with the plasmid containing both genes. However, no transgenic somatic embryos were recovered from selection on 50-100 micromol/L HgCl2. Expression of merA as mRNA was detected by Northern blot analysis in leaf tissues of transgenic peanut, but not in somatic embryos. Western blot analysis showed the production of the mercuric ion reductase protein in leaf tissues. Differential responses to HgCl2 of embryo-derived explants from segregating R1 seeds of one transgenic line also were observed.     

Mercury resistance in a plasmid-bearing strain of Escherichia coli

A strain of Escherichia coli carrying genes determining mercury resistance on a naturally occurring resistance transfer factor (RTF) converts 95% of 10(-5)m Hg(2+) (chloride) to metallic mercury at a rate of 4 to 5 nmoles of Hg(2+) per min per 10(8) cells. The metallic mercury is rapidly eliminated from the culture medium as mercury vapor. The volatilizing activity has a temperature dependence and heat sensitivity characteristic of enzymatic catalysis and is inducible by mercuric chloride. Ag(+) and Au(3+) are markedly inhibitory of mercury volatilization.     

INHIBITION OF OLFACTION IN THE MOTH HELIOTHIS VIRESCENS BY THE SULFHYDRYL REAGENT FLUORESCEIN MERCURIC ACETATE

A combined electrophysiological, behavioral, and biochemicalstudy was initiated to determine the effects of the sulfhydryl-specificreagent fluorescein mercuric acetate (FMA) on olfaction in thetobacco budworm moth Heliothis virescens. The electroantennogram(EAG) response to the standard odorant n-pentyl acetate showedboth a time and concentration dependent inhibition by FMA. Treatmentof insect antennae with 2.52 x 10^–5 M FMA for 2 min reducedthe EAG by 50%, while treatment for 17 min reduced the EAG by80%. Incubation of antennae for 7 min with 2.52 x 10^–6M FMA resulted in 30% inhibition, while incubation with 2.52x 10^–6 M FMA for 7 min resulted in 65% inhibition. Antennalgrooming behavior was inhibited by FMA in a similar time andconcentration dependent manner as the EAG. Regeneration of previouslyinhibited behavioral and EAG responses has been observed withina 24-hr period. The interaction of protein, obtained by sonicatingintact antennae in phosphate buffer, with FMA was monitoredfluorometrically. Successive additions of antennal sonicateto FMA resulted in stepwise decreases in fluorescence. Partialrecovery of fluorescence was obtained by addition of cysteineto the FMA-antennal sonicate solution. The polarization of theFMA-antennal sonicate fluorescence decreased upon addition ofcysteine. These data indicate that FMA is reacting with a relativelylarge antennal protein (s) by mercaptide linkage and blockingthe olfactory transduction process.     

Development of a biological mercury removal-recovery system

A mercury removal-recovery system was developed for collection of elemental mercury volatilized by biological mercuric ion reduction. Using the mercury removal-recovery system, removal of mercuric chloride from mercury-containing buffer without nutrients by resting cells of mercury-resistant bacterium, Pseudomonas putida PpY101/pSR134 was tested. Optimum temperature, pH, thiol compounds and cell concentration on removal of mercuric chloride were determined, and 92 to 98% of 40 mg Hg l^–1 was recovered in 24 h. The efficiency of mercuric chloride removal from river water and seawater was as high as that observed when using a buffered solution.     

Characterization of a mercury-reducing Bacillus cereus strain isolated from the Pulicat Lake sediments, south east coast of India

Pulicat Lake sediments are often severely polluted with the toxic heavy metal mercury. Several mercury-resistant strains of Bacillus species were isolated from the sediments and all the isolates exhibited broad spectrum resistance (resistance to both organic and inorganic mercuric compounds). Plasmid curing assay showed that all the isolated Bacillus strains carry chromosomally borne mercury resistance. Polymerase chain reaction and southern hybridization analyses using merA and merB3 gene primers/probes showed that five of the isolated Bacillus strains carry sequences similar to known merA and merB3 genes. Results of multiple sequence alignment revealed 99% similarity with merA and merB3 of TnMERI1 (class II transposons). Other mercury resistant Bacillus species lacking homology to these genes were not able to volatilize mercuric chloride, indicating the presence of other modes of resistance to mercuric compounds.