Phytoremediation of Mercury- and Methylmercury-Polluted Soils Using Genetically Engineered Plants

Inorganic mercury in contaminated soils and sediments is relatively immobile, though biological and chemical processes can transform it to more toxic and bioavailable methylmercury. Methylmercury is neurotoxic to vertebrates and is biomagnified in animal tissues as it is passed from prey to predator. Traditional remediation strategies for mercury contaminated soils are expensive and site-destructive. As an alternative we propose the use of transgenic aquatic, salt marsh, and upland plants to remove available inorganic mercury and methylmercury from contaminated soils and sediments. Plants engineered with a modified bacterial mercuric reductase gene, merA, are capable of converting Hg(II) taken up by roots to the much less toxic Hg(0), which is volatilized from the plant. Plants engineered to express the bacterial organo-mercurial lyase gene, merB, are capable of converting methylmercury taken up by plant roots into sulfhydryl-bound Hg(II). Plants expressing both genes are capable of converting ionic mercury and methylmercury to volatile Hg(0) which is released into an enormous global atmospheric Hg(0) pool. To assess the phytoremediation capability of plants containing the merA gene, a variety of assays were carried out with the model plants Arabidopsis thaliana, and tobacco (Nicotiana tabacum).     

Ascorbate promotes emission of mercury vapour from plants

Mercury vapour (Hg°) emission from plants contributes to the atmospheric mercury cycle. Although a part of this Hg° emission originates from Hg(II) uptake by the roots, the question how terrestrial plants reduce Hg(II) has not been addressed so far. Young barley plants grown on a hydroponic cultivation containing Hg(II) increased the Hg° emission significantly. Homogenates of barley leaves added to dissolved Hg(II) induced a powerful volatilization at alkaline but not at acidic pH. The same pH dependence and emission kinetic together with the highest reduction capacity was observed for ascorbic acid as compared to other phytoreductants. The electrochemical potentials of the reactions involved suggest an electron transfer from NADPH via GSH and ascorbate to Hg(II). The results support the assumption of a novel mechanism how plants transfer reduction equivalents from the antioxidative defense system via ascorbate to reduce Hg(II) ions, thus counteracting mercury toxicity by volatilizing the metal. This effect appears to be assisted by other light-dependent processes such as transpiration and ascorbate synthesis.     

Acidithiobacillus ferrooxidans fixation on mercuric surfaces and its application in stripping voltammetry

The adsorption (fixation) of bacteria Acidithiobacillus ferrooxidans on Hg and Cu metallic surfaces was qualitatively studied owing to two independent methods: frequency measurement using a quartz crystal microbalance and light absorption measuring at the Hg/bacterial culture interface. A method using a dropping mercury electrode (DME) allowed quantifying this bacterial fixation. Determining the superficial tension at Hg/bacterial culture interface led to determine bacteria adsorption on Hg through the Gibbs isotherm. A modified stripping voltammetry was proposed taking benefit of both bacterial adsorption on Hg surface and metal fixation capacity on biomass. Metal preconcentration on the biologically modified Hg electrode appeared to improve the measurement sensitivity of differential pulse anodic stripping voltammetry (DPASV). The height of the detected peaks was thus increased of 17.6% for copper, 48.4% for lead, and 132% for cadmium determinations compared to those obtained with an unmodified mercury electrode. Such augmentation depended on bulk bacteria concentration and bacteria preconcentration.     

Stoichiometry and kinetics of mercury uptake by photosynthetic bacteria

Mercury adsorption on the cell surface and intracellular uptake by bacteria represent the key first step in the production and accumulation of highly toxic mercury in living organisms. In this work, the biophysical characteristics of mercury bioaccumulation are studied in intact cells of photosynthetic bacteria by use of analytical (dithizone) assay and physiological photosynthetic markers (pigment content, fluorescence induction, and membrane potential) to determine the amount of mercury ions bound to the cell surface and taken up by the cell. It is shown that the Hg(II) uptake mechanism (1) has two kinetically distinguishable components, (2) includes co-opted influx through heavy metal transporters since the slow component is inhibited by Ca²⁺ channel blockers, (3) shows complex pH dependence demonstrating the competition of ligand binding of Hg(II) ions with H⁺ ions (low pH) and high tendency of complex formation of Hg(II) with hydroxyl ions (high pH), and (4) is not a passive but an energy-dependent process as evidenced by light activation and inhibition by protonophore. Photosynthetic bacteria can accumulate Hg(II) in amounts much (about 10⁵) greater than their own masses by well-defined strong and weak binding sites with equilibrium binding constants in the range of 1 (μM)^?1 and 1 (mM)^?1, respectively. The strong binding sites are attributed to sulfhydryl groups as the uptake is blocked by use of sulfhydryl modifying agents and their number is much (two orders of magnitude) smaller than the number of weak binding sites. Biofilms developed by some bacteria (e.g., Rvx. gelatinosus) increase the mercury binding capacity further by a factor of about five. Photosynthetic bacteria in the light act as a sponge of Hg(II) and can be potentially used for biomonitoring and bioremediation of mercury-contaminated aqueous cultures.     

Biosorption of mercury by the inactivated cells of pseudomonas aeruginosa PU21 (Rip64)

Biomass of a mercury-resistant strain Pseudomonas aeruginosa PU21 (Rip64) and hydrogen-form cation exchange resin (AG 50W-X8) were investigated for their ability to adsorb mercury. The maximum adsorption capacity was approximately 180 mg Hg/g dry cell in deionized water and 400 mg Hg/g dry cell in sodium phosphate solution at pH 7.4, higher than the maximum mercury uptake capacity in the cation exchange resin (100 mg Hg/g dry resin in deionized water). The mercury selectivity of the biomass over sodium ions was evaluated when 50 mM and 150 mM of Na(+) were present. Biosorption of mercury was also examined in sodium phosphate solution andphosphate-buffered saline solution (pH 7.0), containing 50mM and 150 mM of Na(+), respectively. It was found that the presence of Na(+) did not severely affect the biosorption of Hg(2+), indicating a high mercury selectivity ofthe biomass over sodium ions. In contrast, the mercury uptake by the ion exchange resin was strongly inhibited by high sodium concentrations. The mercury biosorption was most favorable in sodium phosphate solution (pH 7.4), with a more than twofold increase in the maximum mercury uptake capacity. The pH was found to affect the adsorption of Hg(2+)bythe biomass and the optimal pH value was approximately 7.4. The adsorption of mercury on the biomass and the ion exchange resin appeared to follow theLangmuir or Freundlich adsorption isotherms. (c) 1994 John Wiley & Sons, Inc.     

Methylmercury accumulation and fluxes across the intestine of channel catfish,Ictalurus punctatus

The excised intestines of channel catfish, Ictalurus punctatus, were perfused at 20 or 4 degrees C for 1 h 45 min, with methylmercury (CH(3)HgCl) alone, or in the presence of excess L-cysteine (L-Cys), D-cysteine (D-Cys), L-methionine (L-Met); or with ouabain or probenecid to identify the potential CH(3)Hg(II) uptake pathways in fish intestines. A temperature effect was noted, with CH(3)Hg(II) concentrations in tissues perfused at 20 degrees C being higher than at 4 degrees C, substantiating the idea that mechanisms requiring metabolic energy are involved in CH(3)Hg(II) uptake in fish intestines. The results indicate that, when CH(3)Hg(II) is complexed as the CH(3)Hg-L-Cys complex, it is taken up via an L-neutral amino acid carrier and rapidly transported to the serosal side of the intestine. Methylmercury uptake could be inhibited by probenecid and ouabain, although probenecid had less impact on CH(3)Hg(II) uptake than ouabain. Our results for CH(3)Hg(II) uptake in the presence of D-Cys, L-Met in excess of L-Cys, or with a metal mixture further established that CH(3)Hg(II) uptake across fish intestines occurs via a variety of pathways, including an energy-dependent L-neutral amino acid carrier, and that the route and amount of accumulation were a function of CH(3)Hg(II) speciation in the digestive tract of the fish.     

Distribution and partitioning of mercury in a river catchment impacted by former mercury mining activity

Mercury distribution and partitioning was studied in the River Idrijca system, draining the area of the former Idrija mercury mine, Slovenia. Mercury dynamics were assessed by speciation analysis of mercury in water and river bed sediment samples during a 2-year study at locations on the River Idrijca and its major tributaries. Simultaneously, the influence of some major physico-chemical parameters that influence the fate of mercury in the aquatic environment was investigated. The distribution of mercury species in the River Idrijca catchment indicated contamination from mine tailings distributed in the town of Idrija and erosion of contaminated soils. The partitioning between dissolved and particulate mercury phases in river water was found to be mostly controlled by the variable content of suspended solids resulting from changing hydrological conditions and complexation with various ligands present in river water, among which dissolved organic carbon (DOC) seems to be the most important. Overall results indicate that mercury is transported downstream from the mining area mainly as finely suspended material including colloids rather than in the dissolved phase. This riverine transport occurs mostly during short, but extreme hydro-meteorological conditions when remobilization of mercury from the river bed sediments occurs. A significant part of the mercury particulate phase in water corresponds to cinnabar particles. During its transport, important Hg transformation mechanisms that increase the risk of mercury uptake by biota take place, evidenced by the increase in the relative contribution of reactive mercury (Hg_R), dissolved gaseous mercury (DGM) and monomethylmercury (MeHg) downstream from the Idrija mine. However, our data revealed relatively low methylation efficiency in this contaminated river system. We attribute this to the site specific physico-chemical conditions responsible for making inorganic mercury unavailable and limiting the capacity of methylating bacteria.     

Using Earthworms to Assess Hg Distribution and Bioavailability in Gold Mining Soils

Between 1980 and 2000, the municipality of Cachoeira do Piriá, located in Pará State, Brazil, experienced an intense gold rush with approximately 5,000 artisanal miners discharging more than four tonnes of mercury into soils, air and aquatic systems. Mercury is dispersed across an area of approximately 2,100 ha and concentrations in soils and sediments frequently exceed 1,000 μg.kg^?1. The metallic mercury discharged by miners into the environment has the potential to be transformed into a highly toxic form of mercury, methylmercury. A 28-day bioassay with the earthworm Eisenia fetida was used to assess mercury bioavailability in mine tailings, soils, and sediments. Experiments indicated that the highest Hg concentration in earthworms was associated with low-Hg-organic-rich soils collected from densely vegetated areas despite higher mercury concentrations in organic-poor tailings. This indicates that reaction with organic acids is an important pathway for mercury incorporation into food chains. The quick, inexpensive, and simple bioassay also provided a means to evaluate remedial measures (i.e. by capping “hotspots” with local soils). Earthworm experiments indicate that covering “environmental hotspots” (sites with high Hg bioavailability) with local clay-rich sediments is very effective in terms of preventing uptake of mercury from tailings, while organic-rich sediments are relatively ineffective.     

The Use of a Mercury Biosensor to Evaluate the Bioavailability of Mercury-Thiol Complexes and Mechanisms of Mercury Uptake in Bacteria

As mercury (Hg) biosensors are sensitive to only intracellular Hg, they are useful in the investigation of Hg uptake mechanisms and the effects of speciation on Hg bioavailability to microbes. In this study, bacterial biosensors were used to evaluate the roles that several transporters such as the glutathione, cystine/cysteine, and Mer transporters play in the uptake of Hg from Hg-thiol complexes by comparing uptake rates in strains with functioning transport systems to strains where these transporters had been knocked out by deletion of key genes. The Hg uptake into the biosensors was quantified based on the intracellular conversion of inorganic mercury (Hg(II)) to elemental mercury (Hg(0)) by the enzyme MerA. It was found that uptake of Hg from Hg-cysteine (Hg(CYS)₂) and Hg-glutathione (Hg(GSH)₂) complexes occurred at the same rate as that of inorganic complexes of Hg(II) into Escherichia coli strains with and without intact Mer transport systems. However, higher rates of Hg uptake were observed in the strain with a functioning Mer transport system. These results demonstrate that thiol-bound Hg is bioavailable to E. coli and that this bioavailability is higher in Hg-resistant bacteria with a complete Mer system than in non-resistant strains. No difference in the uptake rate of Hg from Hg(GSH)₂ was observed in E. coli strains with or without functioning glutathione transport systems. There was also no difference in uptake rates between a wildtype Bacillus subtilis strain with a functioning cystine/cysteine transport system, and a mutant strain where this transport system had been knocked out. These results cast doubt on the viability of the hypothesis that the entire Hg-thiol complex is taken up into the cell by a thiol transporter. It is more likely that the Hg in the Hg-thiol complex is transferred to a transport protein on the cell membrane and is subsequently internalized.     

Effects of acidification on mercury methylation, demethylation, and volatilization in sediments from an acid-susceptible lake.

The effect of experimental acidification on mercury methylation, demethylation, and volatilization was examined in surficial sediment samples from a weakly buffered northern Wisconsin lake. All mercury transformations were measured with radioisotopic tracers. Acidification of sediment pH with H2SO4, HCl, or HNO3 significantly decreased 203Hg(II) methylation. Acidification of pH 6.1 (ambient) sediments to pH 4.5 with either H2SO4 or HCl inhibited methylation by over 65%. The decreased methylation was due to the increased hydrogen ion concentration because methylation was not affected by concentrations of Na2SO4 or NaCl equimolar to the amount of acid added. Inhibition of methylation was observed even after prolonged acidification of sediments to pH 5.0 for up to 74 days. Acidification of sediments to pH 5.5, 4.5, and 3.5 with HNO3 resulted in a near complete inhibition of methylation at each pH. Similarly, the addition of equimolar amounts of NaNO3 resulted in a near complete inhibition of methylation, indicating that the inhibition was due to the nitrate ion rather than to the acidity. Demethylation of methyl mercury was not affected by pHs between 8.0 and 4.4, but sharply decreased below pH 4.4. Volatilization of 203Hg(II) from surface sediments was less than 2% of methylation activity and was not significantly different from that in killed sediments. This study indicated that acidification of sediments inhibits mercury methylation and that the observed increase in the mercury burdens in fish from low pH lakes is not due to increased production of methylmercury in sediments.     

Effects of acidification on mercury methylation, demethylation, and volatilization in sediments from an acid-susceptible lake

The effect of experimental acidification on mercury methylation, demethylation, and volatilization was examined in surficial sediment samples from a weakly buffered northern Wisconsin lake. All mercury transformations were measured with radioisotopic tracers. Acidification of sediment pH with H2SO4, HCl, or HNO3 significantly decreased 203Hg(II) methylation. Acidification of pH 6.1 (ambient) sediments to pH 4.5 with either H2SO4 or HCl inhibited methylation by over 65%. The decreased methylation was due to the increased hydrogen ion concentration because methylation was not affected by concentrations of Na2SO4 or NaCl equimolar to the amount of acid added. Inhibition of methylation was observed even after prolonged acidification of sediments to pH 5.0 for up to 74 days. Acidification of sediments to pH 5.5, 4.5, and 3.5 with HNO3 resulted in a near complete inhibition of methylation at each pH. Similarly, the addition of equimolar amounts of NaNO3 resulted in a near complete inhibition of methylation, indicating that the inhibition was due to the nitrate ion rather than to the acidity. Demethylation of methyl mercury was not affected by pHs between 8.0 and 4.4, but sharply decreased below pH 4.4. Volatilization of 203Hg(II) from surface sediments was less than 2% of methylation activity and was not significantly different from that in killed sediments. This study indicated that acidification of sediments inhibits mercury methylation and that the observed increase in the mercury burdens in fish from low pH lakes is not due to increased production of methylmercury in sediments.     

Voltammetry on mercury and carbon electrodes as a tool for studies of metallothionein interactions with metal ions.

Rabbit liver Cd-metallothionein (CdMT) and Cd-complex of synthetically prepared pentapeptide (gamma-Glu-Cys)2-Gly were studied as examples of animal and plant metallothioneins. Using hanging mercury electrode, cathodic stripping voltammetry after adsorptive accumulation of the Cd(II)-SR complex at different potentials, is suitable for estimating changes occurring in metal coordination due to the presence of metal ions such as Zn2+, Cu2+, Hg2+ or excessive Cd2+. Conditions under which similar behaviour can be observed for both CdMT and Cd-pentapeptide complex are specified. On carbon electrodes, detailed study of reduction processes of Cd(II)-SR complexes is prevented by occurrence of a large catalytic current; oxidation processes are more suitable for study at these electrodes. Carbon composite paste electrode (10% SiO2) allows deposition of Cd(II)-SR complex during its reduction, as was demonstrated with Cd-cysteine, CdMT or Cd-pentapeptide complex. After deposition, oxidation peak of the uncomplexed Cd2+ ions and one or two oxidation peaks corresponding to a formation of the RS-Cd(II) complex are observed. Also, similarly as on Hg electrode, it was observed that excessive Cd2+ or Zn2+ ions influence oxidation peaks of the RS-Cd(II) complex formation. Combination of measurements on mercury electrode and composite paste electrode is recommended for studies of metallothionein interactions with metal ions or other metal complexes.     

Synthesis and characterization of gum acacia inspired silica hybrid xerogels for mercury(II) adsorption

In a sol-gel process, gum acacia inspired silica xerogels have been synthesized from tetraethylorthosilicate. Besides showing photoluminescence under ultraviolet excitation, the hybrid xerogels were very efficient in capturing mercury(II) from synthetic solution. To synthesize the optimum sample (in terms of Hg(II) uptake), different ratios of H(2)O:TEOS:EtOH were taken at fixed GA and catalyst concentrations where 4:1:1 ratio was found to be most favorable. Calcination in air further enhanced the mercury binding capacity of this sample. Optimum sample (H4) was obtained on calcination of the gel at 600°C. The hybrids have been structurally characterized using Infrared spectroscopy, X-ray diffraction, scanning electron microscopy, thermo gravimetric analysis, photoluminescence spectroscopy and Brunauer-Emmett-Teller analysis. In a preliminary batch adsorption experiment, H4 was evaluated to be highly efficient in the removal Hg(II) from synthetic aqueous solution.     

Toxicity of organic and inorganic mercury species in differentiated human neurons and human astrocytes

Organic mercury (Hg) species exert their toxicity primarily in the central nervous system. The food relevant Hg species methylmercury (MeHg) has been frequently studied regarding its neurotoxic effects in vitro and in vivo. Neurotoxicity of thiomersal, which is used as a preservative in medical preparations, is to date less characterised. Due to dealkylation of organic Hg or oxidation of elemental Hg, inorganic Hg is present in the brain albeit these species are not able to readily cross the blood brain barrier. This study compared for the first time toxic effects of organic MeHg chloride (MeHgCl) and thiomersal as well as inorganic mercury chloride (HgCl₂) in differentiated human neurons (LUHMES) and human astrocytes (CCF-STTG1). The three Hg species differ in their degree and mechanism of toxicity in those two types of brain cells. Generally, neurons are more susceptible to Hg species induced cytotoxicity as compared to astrocytes. This might be due to the massive cellular mercury uptake in the differentiated neurons. The organic compounds exerted stronger cytotoxic effects as compared to inorganic HgCl₂. In contrast to HgCl₂ exposure, organic Hg compounds seem to induce the apoptotic cascade in neurons following low-level exposure. No indicators for apoptosis were identified for both inorganic and organic mercury species in astrocytes. Our studies clearly demonstrate species-specific toxic mechanisms. A mixed exposure towards all Hg species in the brain can be assumed. Thus, prospectively coexposure studies as well as cocultures of neurons and astrocytes could provide additional information in the investigation of Hg induced neurotoxicity.     

Expression of mercuric ion reductase in Eastern cottonwood (Populus deltoides) confers mercuric ion reduction and resistance

Mercury is one of the most hazardous heavy metals and is a particular problem in aquatic ecosystems, where organic mercury is biomagnified in the food chain. Previous studies demonstrated that transgenic model plants expressing a modified mercuric ion reductase gene from bacteria could detoxify mercury by converting the more toxic and reductive ionic form [Hg(II)] to less toxic elemental mercury [Hg(0)]. To further investigate if a genetic engineering approach for mercury phytoremediation can be effective in trees with a greater potential in riparian ecosystems, we generated transgenic Eastern cottonwood (Populus deltoides) trees expressing modified merA9 and merA18 genes. Leaf sections from transgenic plantlets produced adventitious shoots in the presence of 50 microm Hg(II) supplied as HgCl2, which inhibited shoot induction from leaf explants of wild-type plantlets. Transgenic shoots cultured in a medium containing 25 microm Hg(II) showed normal growth and rooted, while wild-type shoots were killed. When the transgenic cottonwood plantlets were exposed to Hg(II), they evolved 2-4-fold the amount of Hg(0) relative to wild-type plantlets. Transgenic merA9 and merA18 plants accumulated significantly higher biomass than control plants on a Georgia Piedmont soil contaminated with 40 p.p.m. Hg(II). Our results indicate that Eastern cottonwood plants expressing the bacterial mercuric ion reductase gene have potential as candidates for in situ remediation of mercury-contaminated soils or wastewater.     

Experiments with large enclosed ecosystems.

Three of the major advantages of enclosure experiments are that they ensure (1) that the same populations are sampled over a long period; (2) that populations of at least three trophic levels are initially enclosed in naturally occurring proportions and that they are self sustaining over a long experimental period; and (3) that replicate enclosed populations can be experimentally manipulated. There are two disadvantages which must be mentioned. These are (1) that vertical mixing, which may be reduced by as much as an order of magnitude compared to the open sea, will undoubtedly affect the sinking rates of phytoplankton and may influence the structure of the population; and (2) that as a general rule the larger and therefore more expensive the enclosures become, the more difficult it is to run sufficient replicates. An experiment is described in which 1 microgram Hg/l was added to two 95 m3 bags (3 mdiameter by 17 m deep) and the response of the pelagic population monitored over the following 20 days. A further 10 micrograms Hg/l was then added to each enclosure and the response measured for a further 20 days. The results indicated that: (i) inorganic mercury added to the water column is very rapidly transformed into 'bound' or 'non-reactive' mercury and that about 25% of the mercury added was recovered associated with the organic material settling to the bottom of the bags; (ii) the response of the biological population to 1 microgram Hg/l was very limited and in fact a transient reduction in photosynthetic carbon uptake per unit chlorophyll was the only noticeable effect and there were no changes in population size or structure that could be attributed to mercury; (iii) at 10 micrograms Hg/l the zooplankton population was reduced markedly and this did produce changes in the structure of both the zooplankton and phytoplankton populations. These results are similar to the results of a comparable experiment carried out in Vancouver Island (Cepex) and point to the conclusion that the levels of mercury found in surface waters around the coast of the U.K. (0.001--0.022 microgram Hg/l) are one or two orders of magnitude below the levels at which a response of the biological population can be demonstrated. The usefulness of large scale enclosed ecosystems for further pollution research is discussed and it is concluded that those facilities that provided a link between the water column and the sediments would be most useful since they would (1) enable estimates to be made of the flux rates of pollutants from the water column to the sediments; and (2) allow experiments to be carried out with the pollutant in contact with sediment in its natural form.     

Interaction of Mercuric Ions with Different Marine Algal Species

The use of different seaweeds such as Sargassum sp., Turbinaria conoides, and Ulva sp. in removing mercury(II) from aqueous solutions were investigated. The initial experimental runs, conducted at different equilibrium pH conditions, demonstrated that brown seaweeds outperformed green seaweed in Hg(II) biosorption at all pH conditions. In particular, at pH 5, maximum biosorption capacities of 170.3 and 145.8 mg/g were recorded for the brown seaweeds T. conoides and Sargassum sp., respectively, compared with 138.4 mg/g for the green seaweed Ulva sp. Isotherm data were modeled and interpreted using the Langmuir, Freundlich, Redlich-Peterson, and Toth models, with the latter described the Hg(II) isotherms with high correlation coefficients and low % error values. The kinetic data indicate the rapidity of the biosorption process, with the equilibrium achieved within 90 min. Several models, including the Elovich, pseudo-first-order, and pseudo-second-order models, were examined for their suitability with the present data; the correlation coefficient and % error values, along with better prediction of equilibrium uptake values, favored the pseudo-first-order model. The desorption experiments were highly successful for T. conoides biomass with 0.05 M HCl, whereas for the other two seaweeds, 0.05 M HCl resulted in high biomass weight loss. Reusing T. conoides biomass in three successive sorption-desorption cycles resulted in only 8.8% reduction in Hg(II) biosorption capacity compared with its original uptake.     

Octanol/water partition coefficients as a model system for assessing antidotes for methylmercury(II) poisoning, and for studying mercurials with medicinal applications

1-Octanol/water partition coefficients, [HgII]octanol/[HgII]water, provide a simple but limited model system for aspects of the biological behavior of methylmercury(II) and commonly used organomercury(II) medicinal compounds. In an octanol/water system some widely studied antidotes for mercury poisoning at least partly displace the biological thiols L-cysteine and glutathione from binding to MeHgII at pH 6.9. Addition of the antidote meso-dimercaptosuccinic acid to MeHgII in the presence of glutathione results in formation of metallic mercury. For RHgII derivatives of L-cysteine and glutathione, octanol/water partition coefficients follow the order Ph greater than Et greater than Me. An exceptionally high value for diphenylmercury, compared with PhHgII derivatives of L-cysteine and glutathione, is consistent with reported results of the distribution of mercury compounds in rats. Ethylmercury(II) is partly displaced from thimerosal by L-cysteine and glutathione in the octanol/water system, indicating that the active form of thimerosal in vivo may involve binding of EtHgII to biological ligands.     

Effect of solid-phase chemical modification on the features of the lipase from Thermomyces lanuginosus

The lipase from Thermomyces lanuginosus (TLL) was immobilized on octyl Sepharose and further modified with ethylenediamine (EDA) after activation of the carboxylic groups with carbodiimide. Different degrees of modification of the carboxyl groups were carried out by controlling the concentration of carbodiimide (10%, 50% or 100%). Subsequently, the effect of incubation of the modified preparations on hydroxylamine to recover the modified tyrosine was also studied. The modified enzymes exhibited a mobility in native electrophoresis quite different from that of the unmodified lipase (as expected by the changes in charge), and required higher concentrations of cationic detergent to become desorbed from the support. Interestingly, the chemical modification of the immobilized TLL produced an improvement in its activity, proportional to the amination degree. This increase in activity was much more significant at pH 10, where the fully modified preparation increased the activity by a factor of 10 as compared to the unmodified preparation. Moreover, the incubation of the chemically aminated preparations in a hydroxylamine solution improved the activity by an additional factor of 1.2. The fully aminated and incubated in hydroxylamine preparation exhibited a thermostability higher than that of the unmodified preparation, mainly at pH 5 (almost a 30 fold factor). In the presence of tetrahydrofurane, some stabilization was observed at pH 7, while at pH 9 the stability of the modified enzyme decreased (under all the assayed amination degrees) when compared to that of the unmodified enzyme. Thus, this simple protocol may be a rapid and efficient way of preparing a TLL biocatalyst with higher activity and stability, although this will depend on the inactivation conditions.