Biotoxicity of mercury as influenced by mercury(II) speciation

Integration of physicochemical procedures for studying mercury(II) speciation with microbiological procedures for studying the effects of mercury on bacterial growth allows evaluation of ionic factors (e.g., pH and ligand species and concentration) which affect biotoxicity. A Pseudomonas fluorescens strain capable of methylating inorganic Hg(II) was isolated from sediment samples collected at Buffalo Pound Lake in Saskatchewan, Canada. The effect of pH and ligand species on the toxic response (i.e., 50% inhibitory concentration [IC50]) of the P. fluorescens isolated to mercury were determined and related to the aqueous speciation of Hg(II). It was determined that the toxicities of different mercury salts were influenced by the nature of the co-ion. At a given pH level, mercuric acetate and mercuric nitrate yielded essentially the same IC50s; mercuric chloride, on the other hand, always produced lower IC50s. For each Hg salt, toxicity was greatest at pH 6.0 and decreased significantly (P = 0.05) at pH 7.0. Increasing the pH to 8.0 had no effect on the toxicity of mercuric acetate or mercuric nitrate but significantly (P = 0.05) reduced the toxicity of mercuric chloride. The aqueous speciation of Hg(II) in the synthetic growth medium M-IIY (a minimal salts medium amended to contain 0.1% yeast extract and 0.1% glycerol) was calculated by using the computer program GEOCHEM-PC with a modified data base. Results of the speciation calculations indicated that complexes of Hg(II) with histidine [Hg(H-HIS)HIS+ and Hg(H-HIS)2(2+)], chloride (HgCl+, HgCl2(0), HgClOH0, and HgCl3-), phosphate (HgHPO4(0), ammonia (HgNH3(2+), glycine [Hg(GLY)+], alanine [Hg(ALA)+], and hydroxyl ion (HgOH+) were the Hg species primarily responsible for toxicity in the M-IIY medium. The toxicity of mercuric nitrate at pH 8.0 was unaffected by the addition of citrate, enhanced by the addition of chloride, and reduced by the addition of cysteine. In the chloride-amended system, HgCl+, HgCl2(0), and HgClOH0 were the species primarily responsible for observed increases in toxicity. In the cysteine-amended system, formation of Hg(CYS)2(2-) was responsible for detoxification effects that were observed. The formation of Hg-citrate complexes was insignificant and had no effect on Hg toxicity.     

Biotoxicity of mercury as influenced by mercury(II) speciation.

Integration of physicochemical procedures for studying mercury(II) speciation with microbiological procedures for studying the effects of mercury on bacterial growth allows evaluation of ionic factors (e.g., pH and ligand species and concentration) which affect biotoxicity. A Pseudomonas fluorescens strain capable of methylating inorganic Hg(II) was isolated from sediment samples collected at Buffalo Pound Lake in Saskatchewan, Canada. The effect of pH and ligand species on the toxic response (i.e., 50% inhibitory concentration [IC50]) of the P. fluorescens isolated to mercury were determined and related to the aqueous speciation of Hg(II). It was determined that the toxicities of different mercury salts were influenced by the nature of the co-ion. At a given pH level, mercuric acetate and mercuric nitrate yielded essentially the same IC50s; mercuric chloride, on the other hand, always produced lower IC50s. For each Hg salt, toxicity was greatest at pH 6.0 and decreased significantly (P = 0.05) at pH 7.0. Increasing the pH to 8.0 had no effect on the toxicity of mercuric acetate or mercuric nitrate but significantly (P = 0.05) reduced the toxicity of mercuric chloride. The aqueous speciation of Hg(II) in the synthetic growth medium M-IIY (a minimal salts medium amended to contain 0.1% yeast extract and 0.1% glycerol) was calculated by using the computer program GEOCHEM-PC with a modified data base. Results of the speciation calculations indicated that complexes of Hg(II) with histidine [Hg(H-HIS)HIS+ and Hg(H-HIS)2(2+)], chloride (HgCl+, HgCl2(0), HgClOH0, and HgCl3-), phosphate (HgHPO4(0), ammonia (HgNH3(2+), glycine [Hg(GLY)+], alanine [Hg(ALA)+], and hydroxyl ion (HgOH+) were the Hg species primarily responsible for toxicity in the M-IIY medium. The toxicity of mercuric nitrate at pH 8.0 was unaffected by the addition of citrate, enhanced by the addition of chloride, and reduced by the addition of cysteine. In the chloride-amended system, HgCl+, HgCl2(0), and HgClOH0 were the species primarily responsible for observed increases in toxicity. In the cysteine-amended system, formation of Hg(CYS)2(2-) was responsible for detoxification effects that were observed. The formation of Hg-citrate complexes was insignificant and had no effect on Hg toxicity.     

Polynuclear complexes of a dissociative excited state formed in the [Ru(bpy)2(CN)2]-HgCl2 system.

The complex cis-dicyanobis(2,2'-bipyridine)ruthenium(II) forms various bimetallic complexes with mercury(II)chloride, such as [(NC)Ru(bpy)2(CN)-HgCl2], [Cl2Hg-(NC)Ru(bpy)2(CN)-HgCl2-(NC)Ru(bpy)2(CN)-HgCl2] and [Cl2Hg-(NC)Ru(bpy)2(CN)-(HgCl2)] in CH3CN. These polynuclear complexes of the equilibrium system have been identified and characterized by their formation constants and absorption spectra. Excitation of bimetallic complexes produces the MLCT state localized on [Ru(bpy)2(CN)2] ligand, resulting in the cleavage of the bond formed between the nitrogen atom of the coordinated cyanide ligand and the Hg(II) central atom in ground state. Unlike many photoinduced metal ligand dissociations, the dissociated fragment remains in a luminescent excited state.     

Transformation of mercuric chloride and methylmercury by the rumen microflora

The microflora in strained rumen fluid did not methylate or volatilize 203Hg2+ at detectable rates. However, there was an exponential decay in the concentration of added CH3Hg+, which was attributed to demethylation. The major product of demethylation was metallic mercury (Hg0), and it was released as a volatile product from the reaction mixture. Demethylation occurred under both anaerobic and aerobic conditions. The rate of demethylation was proportional to the concentration of added CH3Hg+-Hg from 0.02 to 100 microgram of Hg per ml. The presence of HgCl2 had almost no inhibitory effect on the rate of cleavage of the carbon-mercury bond of CH2HgCl, but it completely inhibited volatilization of the Hg formed, when the concentration of HgCl2-Hg reached 100 micrograms/ml. Three of 11 species of anaerobic rumen bacteria catalyzed demethylation. These were Desulfovibrio desulfuricans, Selenomonas ruminantium, and Megasphaera elsdenii. None of the 11 species caused detectable methylation, and only two caused limited volatilization of Hg2+. Three species of bacteria out of 90 fresh aerobic isolates from rumen contents were demethylators: two were identified as Pseudomonas sp., and the third was a Micrococcus sp. Demethylation by the rumen microflora appeared to be carried out by both aerobic and anaerobic bacteria and, on the basis of Hg2+ sensitivity, probably resulted from the activity of two enzymes, a CH3-Hg+ hydrolase and a Hg2+ reductase.     

Transformation of mercuric chloride and methylmercury by the rumen microflora.

The microflora in strained rumen fluid did not methylate or volatilize 203Hg2+ at detectable rates. However, there was an exponential decay in the concentration of added CH3Hg+, which was attributed to demethylation. The major product of demethylation was metallic mercury (Hg0), and it was released as a volatile product from the reaction mixture. Demethylation occurred under both anaerobic and aerobic conditions. The rate of demethylation was proportional to the concentration of added CH3Hg+-Hg from 0.02 to 100 microgram of Hg per ml. The presence of HgCl2 had almost no inhibitory effect on the rate of cleavage of the carbon-mercury bond of CH2HgCl, but it completely inhibited volatilization of the Hg formed, when the concentration of HgCl2-Hg reached 100 micrograms/ml. Three of 11 species of anaerobic rumen bacteria catalyzed demethylation. These were Desulfovibrio desulfuricans, Selenomonas ruminantium, and Megasphaera elsdenii. None of the 11 species caused detectable methylation, and only two caused limited volatilization of Hg2+. Three species of bacteria out of 90 fresh aerobic isolates from rumen contents were demethylators: two were identified as Pseudomonas sp., and the third was a Micrococcus sp. Demethylation by the rumen microflora appeared to be carried out by both aerobic and anaerobic bacteria and, on the basis of Hg2+ sensitivity, probably resulted from the activity of two enzymes, a CH3-Hg+ hydrolase and a Hg2+ reductase.     

Differential toxicities of mercury to bacteria and bacteriophages in sea and in lake water.

Mixtures of anionic HgCl3-/HgCl4(2)-complexes were less toxic to terrestrial bacteria (Erwinia herbicola, Agrobacterium tumefaciens), to marine bacteria (Acinetobacter sp., Aeromonas sp.), and to bacteriophages (phi 11 M 15 of Staphylococcus aureus and P1 of Escherichia coli) than were equivalent concentrations of Hg as cationic Hg2+. The toxicity of 1 ppm Hg to A. tumefaciens. Aeromonas sp., and phi 11 M 15 was less in seawater than in lake water. Inasmuch as the Hg-Cl species are formed in environments of high chloride concentration, it was postulated that the lower toxicity of Hg in seawater was a result of the formation of HgCl3-/HgCl4(2)-complexes.     

Methylation and demethylation of mercury under controlled redox, pH and salinity conditions.

In estuarine sediments, the microbially mediated processes of methylation, demethylation, and volatilization determine the state and overall toxicity of mercury pollutants. The effects of redox potential (Eh) and salinity on the above microbial processes were investigated in reactors constructed to allow for continuous monitoring and adjustment of the pH (6.8) and Eh of freshly collected estuarine sediments. For measurements of methylation and demethylation activity, sediment slurries adjusted to appropriate salinity were spiked with HgCl2 or CH3HgCl, respectively, and were incubated in the reactors. Methylmercury was measured by gas chromatography. Volatilized elemental mercury (Hg0) was trapped and determined by cold vapor atomic absorption spectrometry. Volatilization of Hg0 and CH3HgCH3 were found to be minimal. Methylation of Hg2+ was favored at Eh-220 mV as compared to +110 mV. At -220 mV, high salinity (2.5%) inhibited methylation, and low salinity (0.4%) favored it. At +110 mV, the salinity effect was less pronounced. Demethylation of CH3HgCl was favored at +110 mV regardless of the salinity level. Low redox potential under low salinity conditions inhibited demethylation, but high salinity reversed this inhibition. These findings are helpful for interpreting and predicting the behavior of mercury pollutants in estuarine sediments.     

Inhibition by methyl mercury chloride and mercuric chloride of the in vitro polymerization of microtubules

Mercury was tested at the same concentration but under two different forms, organic CH3HgCl and inorganic HgCl2, in order to compare its relative inhibitory effect on in vitro microtubules polymerization. Induced by GTP and glycerol 8 M, tubulin polymerization was completely inhibited by HgCl2 10(-3) M while a 75.8% inhibition was measured for CH3HgCl2 10(-3) M.     

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.     

Heavy metal-nucleotide interactions. III. The participation of amino groups in the binding of methylmercury (II) to cytidine and adenosine 5'-phosphate in aqueous solution: studies by Raman difference spectrophotometry.

Raman difference spectrophotometry has been used to study the interaction of CH3Hg(II) with cytidine and Ado-5'-P at high pH. In contrast to the binding reactions which occur at lower pH or in non-aqueous solvents such as dimethyl sulfoxide, a proton is transferred from the amino group; and the complexes are CH3HgCydH-1 and CH3HgAdoH-1-5'-P. The spectra are significantly different from those of the cationic complexes. The integrated intensities of ligand modes which shift upon metalation can be used to measure the concentration of unreacted ligand and consequently the extent of the reaction. Equilibrium constants for the reactions CH3HgOH + L yields CH3HgLH-1 + H2O were estimated to be log KCyd equals 0.63 plus or minus 0.05 and log KAdo-5'-P equals 0.85 plus or minus 0.05, in fair agreement with values determined under very different conditions by ultraviolet spectrophotometry. The vibrational spectrum of the ligand in CH3HgCydH-1 is virtually the same as that of UrdH-1- which is isoelectronic. The spectrum of the ligand in CH3HgAdoH-1-5'-P is more similar to the isoelectronic base InoH-1-than to Ado-5'-P, although the resemblance is not so close as in the CydH-1---UrdH-1-case. The structures of these complexes are discussed on the basis of their vibrational spectra and similarities in the spectra of related compounds. It is concluded that the CH3Hg(II) binds to the amino nitrogen at high pH with both cytidine and Ado-5'-P. In neutral solution with excess CH3Hg(II), metalation occurs on the amino groups, on the ring, and also on the ribose.     

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.     

1H-NMR study of the removal of methylmercury from intact erythrocytes by sulfhydryl compounds

The effectiveness of penicillamine, N-acetylpenicillamine, meso-2,3-dimercaptosuccinic acid, 2,3-dimercaptopropanesulfonic acid, and dithioerythritol for removing methylmercury (CH3Hg(II) from intact human erythrocytes has been studied by 1H-nuclear magnetic resonance spectroscopy. The removal of CH3Hg(II) was monitored by measuring the chemical shift of the resonance for the proton on the alpha-carbon of the cysteinyl residue of intracellular glutathione in 1H-NMR spectra of intact, CH3Hg(II)-containing erythrocytes in suspensions to which the sulfhydryl ligands were added. Because exchange of intracellular glutathione between its free and CH3Hg(II) complexed forms is fast, the chemical shift of the cysteinyl resonance provides a direct, noninvasive measure of the fraction of intracellular glutathione that is complexed. The sulfhydryl ligands were found to remove CH3Hg(II) from intact erythrocytes in the order 2,3-dimercaptosuccinic acid greater than 2,3-dimercaptopropane sulfonic acid greater than dithioerythritol greater than penicillamine approximately N-acetylpenicillamine, which also is the order of the conditional formation constants of the CH3Hg(II) complexes at pH 7.4. All five ligands removed CH3Hg(II) from intact erythrocytes much more rapidly than can be accounted for by a mechanism in which the ligand crosses the membrane, combines with the CH3Hg(II), and then transports it out of the cell. An alternative mechanism is proposed in which the ligand reacts with CH3Hg(II) which is complexed by sulfhydryl groups of the membrane, which in turn react with the intracellular CH3Hg(II) to bring more CH3Hg(II) into the membrane, where it can react with the added sulfhydryl ligand.     

A methylene blue-mediated enzyme electrode for the determination of trace mercury(II), mercury(I), methylmercury, and mercury-glutathione complex

A methylene blue-mediated enzyme biosensor has been developed for the detection of inhibitors including mercury(II), mercury(I), methylmercury, and mercury-glutathione complex. The inhibition to horseradish peroxidase was apparently reversible and noncompetitive in the presence of HgCl2 in less than 8 s and irreversibly inactivated when incubated with different concentrations of HgCl2 for 1-8 min. The binding site of horseradish peroxidase with HgCl2 probably was a cysteine residue SH. Mercury compounds can be assayed amperometrically with the detection limits 0.1 ng ml(-1) Hg for HgCl2 and methylmercury, 0.2 ng ml(-1) Hg for Hg2(NO3)2 and 1.7 ng ml(-1) Hg for mercury glutathione complex. Inactivation of the immobilized horseradish peroxidase was displayed in the AFM images of the enzyme membranes.     

Co-ordination of transition metal ions by galactaric acid: a potentiometric and spectroscopic study

A solution study on the ability of galactaric acid [GalaH(2), HOOC(CH)(4)COOH] in the complexation of biological metal ions such as Co(II) and Ni(II) and toxic metal ions such as Cd(II), Pb(II) and Hg(II), is reported. The stability constants of the complex species are determined by means of potentiometric measurements. Galactaric acid behaves as chelate ligand through carboxylic oxygen and alpha-hydroxy group towards Co(II) and Ni(II), while in the Pb(II) and Cd(II) containing system it co-ordinates the metal ion with carboxylic oxygen and two alcoholic hydroxy groups. The prevailing species at acidic or neutral pH is [MGala] which is also isolated in the solid state and characterized by means of IR spectroscopy. On increasing pH, the [MGalaH(-1)](-) species is also formed where the co-ordinated OH group undergoes deprotonation in all metal ion complexes except those with Hg(II), where the co-ordination of hydroxide ion is suggested as the precipitation of the metal hydroxide occurs at pH 7.     

不同汞化合物对水稻、小麦的影响及作物对汞的吸收积累

本试验研究了5种汞化合物(HgS,HgO,CH_3HgCl,HgCl_2,C_8H_8O_2Hg)对水稻、小麦生长发育的影响及作物对汞的吸收、积累。结果表明,C_8H_8O_2Hg对作物的危害比HgCl_2和CH_3HgCl大,HgS的危害最轻。不同汞化合物对水稻蒸腾作用的抑制程度看出,C_8H_8O_2Hg的毒性大,HgS的毒性最小;抑制小麦光合作用的程度看出,HgCl_2的毒性大、HgS的毒性小。不同汞化合物处理的土壤中,水稻、小麦的含汞量是随着汞化合物的浓度增加而增加,以C_8H_8O_2Hg处理的土壤,作物吸收的汞最多,转移到地上部的汞最多,HgS处理的土壤,汞转移到地上部最少;小麦吸收的汞大部积累在根中,地上部(茎叶)的含汞量显著比水稻少;各处理的土壤总汞含量与水稻的含汞量相关性显著。土壤中的HgS、HgCl_2可以转化为CH_3HgCl,并转运到植物体各器官。 本试验是用盆栽试验的方法,土壤用不同浓度不同汞化台物处理。 用的“称重法”测定了水稻的蒸腾作用。用FQW-CO_2红外气体分析仪测定了小麦的光合强度。用F-732测汞仪测定了水稻、小麦不同器官和土壤中的总汞含量。用巯基棉气相色谱法测定了甲基汞的含量。     

Differential effects of methylmercuric chloride and mercuric chloride on the histochemistry of rat thyroid peroxidase and the thyroid peroxidase activity of isolated pig thyroid cells

This study was designed to characterize the interaction of CH3HgCl or HgCl2 with thyroid peroxidase (TPO). Two types of experiments were performed. First, the thyroids from rats that were given 5.6 mg/kg/day of either CH3HgCl or HgCl2 for 2 weeks by intubation were subjected to histochemical treatment and then to electron microscopy. TPO activities in all cell compartments were inhibited by HgCl2 but not by CH3HgCl. Morphological observation showed that taller epithelia were induced by HgCl2, whereas flattened epithelia forming large follicles were induced by CH3HgCl. The serum thyrotropin level was substantially lowered by CH3HgCl but was unchanged by HgCl2. Second, the guaiacol oxidation by TPO in isolated and ruptured pig thyroid cells was spectrophotometrically monitored in the presence of either CH3HgCl or HgCl2. The TPO was not inhibited by CH3HgCl but was inhibited by HgCl2. These results indicated that CH3HgCl induced a hypothyroid state without affecting TPO, whereas HgCl2 inhibited TPO and induced a hypertropic state owing to compensation for loss of enzyme activity, and that the lack of inhibitory activity of CH3HgCl was not due to the inability to penetrate the cells. Therefore, there appeared to be a differential interaction of organic and inorganic forms of mercurials with the thyroid.     

Iron oxidation stimulates organic matter decomposition in humid tropical forest soils

Humid tropical forests have the fastest rates of organic matter decomposition globally, which often coincide with fluctuating oxygen (O₂) availability in surface soils. Microbial iron (Fe) reduction generates reduced iron [Fe(II)] under anaerobic conditions, which oxidizes to Fe(III) under subsequent aerobic conditions. We demonstrate that Fe (II) oxidation stimulates organic matter decomposition via two mechanisms: (i) organic matter oxidation, likely driven by reactive oxygen species; and (ii) increased dissolved organic carbon (DOC) availability, likely driven by acidification. Phenol oxidative activity increased linearly with Fe(II) concentrations (P < 0.0001, pseudo R² = 0.79) in soils sampled within and among five tropical forest sites. A similar pattern occurred in the absence of soil, suggesting an abiotic driver of this reaction. No phenol oxidative activity occurred in soils under anaerobic conditions, implying the importance of oxidants such as O₂ or hydrogen peroxide (H₂O₂) in addition to Fe(II). Reactions between Fe(II) and H₂O₂ generate hydroxyl radical, a strong nonselective oxidant of organic compounds. We found increasing consumption of H₂O₂ as soil Fe(II) concentrations increased, suggesting that reactive oxygen species produced by Fe(II) oxidation explained variation in phenol oxidative activity among samples. Amending soils with Fe(II) at field concentrations stimulated short‐term C mineralization by up to 270%, likely via a second mechanism. Oxidation of Fe(II) drove a decrease in pH and a monotonic increase in DOC; a decline of two pH units doubled DOC, likely stimulating microbial respiration. We obtained similar results by manipulating soil acidity independently of Fe(II), implying that Fe(II) oxidation affected C substrate availability via pH fluctuations, in addition to producing reactive oxygen species. Iron oxidation coupled to organic matter decomposition contributes to rapid rates of C cycling across humid tropical forests in spite of periodic O₂ limitation, and may help explain the rapid turnover of complex C molecules in these soils.     

Microbiology and chemistry of acid lakes in Florida: I. Effects of drought and post-drought conditions

Short term changes in acid loading and dissolved organic carbon (DOC) content were studied in relation to water column bacteria of ten acid lakes on the Katharine Ordway Preserve, Florida. Five clear oligotrophic lakes and five dark dystrophic lakes were sampled during and after a drought period in July and September, 1985. Water column bacterial densities, light extinction, chlorophyll a, DOC, pH, dissolved oxygen, nutrients, and other chemical variables were measured. Significant positive correlations existed among DOC, chlorophyll a, pH, and water column bacterial densities during the drought period.There were no significant changes in water column bacterial densities or pH of clear lakes in the post-drought period, despite a 4.6 fold increase in acid loading from rainfall. A 3 fold increase of DOC, a decline in pH, and decreased bacterial densities in dark lakes suggested inhibition of bacteria by DOC and pH. A decrease in the relationship of DOC to bacterial numbers in all lakes was also noted. The correlations among DOC, chlorophyll a, and pH were no longer significant.Using data from both time periods significant polynomial regressions were observed between DOC and bacterial density and DOC and chlorophyll a. Maximum bacterial numbers occurred at 20 mg C 1^–1 of DOC. Above this bacterial numbers decreased also suggesting an inhibitory effect of DOC. Because pH was lower after DOC had increased in the dark lakes, the increase in acid conditions may have enhanced this inhibitory effect. The short term effects of DOC on the dark-lake bacteria greatly exceeded the influence of acid loading on clear-lake bacteria.     

Thermodynamic model for the stabilization of trigonal thiolato mercury(II) in designed three-stranded coiled coils.

A thermodynamic model is presented that describes the binding of Hg(II) to de novo designed peptides, Tri L9C and Baby L9C, which were derived from the Tri family. The Tri peptides are based on the parent sequence Ac-NH-G(LKALEEK)(x)()G-CONH(2) and are known to form two-stranded coiled coils at low pH (pH <4) and three-stranded coiled coils at high pH (pH >7). Tri L9C (x = 4) contains a four heptad repeat sequence with cysteine in position 9 and leucines in the other a and d positions; Baby L9C (x = 3), which also has a cysteine in position 9 but is one heptad shorter than Tri L9C, was designed to form less stable helical coiled coils in solution. The free energies of coiled coil formation for Tri, Tri L9C, Baby Tri, and Baby L9C at pH 2.5 and 8.5 were determined by guanidinium denaturation titrations; Tri L9C was observed to be highly helical in the absence of denaturant at pH 8.5 while Baby L9C contained <20% helical content at pH 8.5, indicating a weakly associated or unassociated coiled coil. Size-exclusion chromatography (SEC) verified that Baby L9C was a monomer at pH 8.5. The helicity of Baby L9C was induced by addition of HgCl(2). The subsequent formation of a trigonal thiolato Hg(II) in the interior of a three-stranded coiled coil was verified by the presence of a characteristic HgS(3) UV band at 248 nm. Titrations of Tri L9C and Baby L9C into solutions of HgCl(2) at pH values between 7 and 9 were performed to extract binding constants. Global fits to the data employed a mechanism that involved initial binding of mercury to the peptides forming a two-stranded coiled coil with linear thiolato Hg(II) at [peptide]/[Hg] <2, followed by addition of a more weakly associated third helix to generate a three-stranded coiled coil. This mechanism would require the deprotonation of the third cysteine thiol to generate the trigonal thiolato Hg(II) at pH >7.5 [the pK(a) of the cysteine thiol in the presence of Hg(II)]. Support for this mechanism was given by the observation of a three-stranded coiled coil by SEC in a solution of Tri L9C at pH 7.0.     

Heavy metal-nucleotide interactions. IX. Raman difference spectroscopic studies on the binding of CH3Hg(II) to 1-methylthymine, thymidine-5'-monophosphate, DNA models and native DNA.

Raman spectra have been obtained for dTMP and its complex with CH3Hg (II) in aqueous solution as a function of pH. Difference spectroscopy is employed to increase the sensitivity of the Raman technique. The binding reaction is essentially quantitative from pH 3 to 9, and the value of the equilibrium constant for CH3HgOH2+ + dThd in equilibrium CH3Hg(dThdH--1) + H30+ is estimated from intensity measurements to be 0.6 in reasonable agreement with an earlier value based upon uv spectrophotometric data. Binding is to N(3) with substitution of CH3Hg+ for the proton. A similar reaction occurs with 1-MeThy. Raman spectra for aqueous and crystalline 1-MeThy and for the complex CH3Hg(1-MeThyH--1) are reported. The spectrum of crystalline Hg(1-MeThyH--1)2, for which the crystal structure is known, also was obtained for comparison. Raman difference spectroscopy was used to confirm that CH3Hg (II) binds to N(3) of dTMP and N(1) of GMP at r = 0.2 (MeHg+: phosphate) ratios with mixtures of GMP + CMP + AMP + dTMP. In contrast, native calf thymus DNA does not appear to bind CH3Hg(II) at these sites at r = 0.15, although no significant amount of free CH3HgOH is present. With r = 0.3, extensive binding occurs both to the Thy and Gua bases. Raman difference spectroscopy is a valuable technique for studying the binding of ions and molecules to polynucleotides in moderately dilute aqueous solution.