Transformations of inorganic mercury by Candida albicans and Saccharomyces cerevisiae

Saccharomyces cerevisiae and Candida albicans were incubated with 0.25, 0.5, or 0.75 micrograms of Hg (as HgCl2) per ml of Nelson's medium in the presence of trace amounts of oxygen at 28 degrees C for 12 days. Two control media were used, one without added Hg and one without yeast inoculum. Yeast cell growth was estimated after 1, 2, 3, and 8 days of incubation. The contents of organomercury in the system and of elemental mercury released from the media and collected in traps were determined at the end of the experiments. The results were as follows. (i) C. albicans was the more mercury-resistant species, but both yeast species failed to grow in the media containing 0.75 micrograms of Hg per ml. (ii) The amounts of organomercury produced by the two species were proportional to the amount of HgCl2 added to the medium. In all cases C. albicans produced considerably larger amounts of methylmercury than S. cerevisiae. (iii) The amounts of elemental Hg produced were inversely proportional to the HgCl2 level added in the case of S. cerevisiae but were all similar in the case of C. albicans. (iv) Neither organomercury nor elemental Hg was produced in any of the control media.     

Differential mercury volatilization by tobacco organs expressing a modified bacterial merA gene

INTRODUCTIONEnvironmental pollution is an increasing prob-lem both fOr developing and developed countries.Mercury, both in organic and ionic fOrm, is one of themost hazardous pollutants among the heavy met-als[l]and its accumuIation in human body results ininactivation of metabolic enzymes and structuralproteins[2, 3] giving rise to serious health problems(Minamatasyndrome).Usually mercury pollution is caused by indus-trial and agricultural activities, releasing mercuryinto air, water an…     

Transformations of inorganic mercury by Candida albicans and Saccharomyces cerevisiae.

Saccharomyces cerevisiae and Candida albicans were incubated with 0.25, 0.5, or 0.75 micrograms of Hg (as HgCl2) per ml of Nelson's medium in the presence of trace amounts of oxygen at 28 degrees C for 12 days. Two control media were used, one without added Hg and one without yeast inoculum. Yeast cell growth was estimated after 1, 2, 3, and 8 days of incubation. The contents of organomercury in the system and of elemental mercury released from the media and collected in traps were determined at the end of the experiments. The results were as follows. (i) C. albicans was the more mercury-resistant species, but both yeast species failed to grow in the media containing 0.75 micrograms of Hg per ml. (ii) The amounts of organomercury produced by the two species were proportional to the amount of HgCl2 added to the medium. In all cases C. albicans produced considerably larger amounts of methylmercury than S. cerevisiae. (iii) The amounts of elemental Hg produced were inversely proportional to the HgCl2 level added in the case of S. cerevisiae but were all similar in the case of C. albicans. (iv) Neither organomercury nor elemental Hg was produced in any of the control media.     

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.     

Effects of dissolved organic carbon and salinity on bioavailability of mercury.

Hypotheses that dissolved organic carbon (DOC) and electrochemical charge affect the rate of methylmercury [CH3Hg(I)] synthesis by modulating the availability of ionic mercury [Hg(II)] to bacteria were tested by using a mer-lux bioindicator (O. Selifonova, R. Burlage, and T. Barkay, Appl. Environ. Microbiol. 59:3083-3090, 1993). A decline in Hg(II)-dependent light production was observed in the presence of increasing concentrations of DOC, and this decline was more pronounced at pH 7 than at pH 5, suggesting that DOC is a factor controlling the bioavailability of Hg(II). A thermodynamic model (MINTEQA2) was used to select assay conditions that clearly distinguished among various Hg(II) species. By using this approach, it was shown that negatively charged forms of mercuric chloride (HgCl3-/HgCl(4)2-) induced less light production than the electrochemically neutral form (HgCl2), and no difference was observed between the two neutral forms, HgCl2 and Hg(OH)2. These results suggest that the negative charge of Hg(II) species reduces their availability to bacteria and may be one reason why accumulation of CH3Hg(I) is more often reported to occur in freshwater than in estuarine and marine biota.     

A new method for direct selection of plasmid-free segregants using mercury hypersensitivity as a phenotypic marker of bacterial plasmids

A new method was developed for direct selection of plasmid-free segregants using mercury hypersensitivity (Hg(ss)) as a phenotypic marker of bacterial plasmids. The Hg(ss) marker originated from the 4.8-kb EcoRI fragment H of the R-factor R100. Since the EcoRI fragment H spans the majority of the mercury resistance operon (mer), but lacks the intact merA gene coding for the mercury reductase enzyme, this fragment conferred the Hg(ss) phenotype. The Hg(ss) marker was introduced into high-copy-number plasmids pUC18, pBR322, and pHSG298. Segregational loss of the Hg(ss) plasmids caused a significant increase of resistance to Hg(2+), and this allowed direct selection of plasmid-free segregants on nutrient agars containing 1-2 mug HgCl(2) ml(-1). Plasmid-loss segregants were estimated to appear at frequencies ranging from 10(-3) to 10(-7) for the tested high-copy-number plasmids. THe Hg(ss) marker proved to be useful for direct selection of plasmid-free segregants from a mixed population of plasmid-harboring and plasmid-free cells.     

Metalloantibodies: mercury(II)-dependent acyl transferases.

A new approach for the elicitation of metal-dependent catalytic antibodies for ester hydrolysis is described. A coordinatively unsaturated mercury complex 1-(Hg), has been utilized as a hapten to elicit antibodies that incorporate mercury(II) as a Lewis acid cofactor. From a panel of monoclonal antibodies generated to 1-(Hg), antibody 38G2 was found to hydrolyze the ester 3 in the presence of HgCl(2) [K(m)app(3)=345 microM; K(m)app(Hg(2+))=87 microM; k(cat)app/k(uncat)=3 x 10(2)]. This is the first example of a biocatalyst that enlists mercuric ion as a cofactor and it is anticipated that this approach will open new avenues for exploitation of metals thought previously beyond the scope of protein catalysts.     

Mechanisms of cholinesterase inhibition by inorganic mercury

The poorly known mechanism of inhibition of cholinesterases by inorganic mercury (HgCl2) has been studied with a view to using these enzymes as biomarkers or as biological components of biosensors to survey polluted areas. The inhibition of a variety of cholinesterases by HgCl2 was investigated by kinetic studies, X-ray crystallography, and dynamic light scattering. Our results show that when a free sensitive sulfhydryl group is present in the enzyme, as in Torpedo californica acetylcholinesterase, inhibition is irreversible and follows pseudo-first-order kinetics that are completed within 1 h in the micromolar range. When the free sulfhydryl group is not sensitive to mercury (Drosophila melanogaster acetylcholinesterase and human butyrylcholinesterase) or is otherwise absent (Electrophorus electricus acetylcholinesterase), then inhibition occurs in the millimolar range. Inhibition follows a slow binding model, with successive binding of two mercury ions to the enzyme surface. Binding of mercury ions has several consequences: reversible inhibition, enzyme denaturation, and protein aggregation, protecting the enzyme from denaturation. Mercury-induced inactivation of cholinesterases is thus a rather complex process. Our results indicate that among the various cholinesterases that we have studied, only Torpedo californica acetylcholinesterase is suitable for mercury detection using biosensors, and that a careful study of cholinesterase inhibition in a species is a prerequisite before using it as a biomarker to survey mercury in the environment.     

A direct competitive enzyme-linked immunosorbent assay (ELISA) for determination of praziquantel concentration in serum

A simple and reproducible enzyme-linked immunosorbent assay (ELISA) for the determination of the concentration of praziquantel in the serum was developed. Since praziquantel has no functional group to conjugate with carrier protein, praziquantel was first converted to a compound with an amino group similar to praziquantel. This compound was then conjugated to bovine serum albumin for use as an immunogen, and to horseradish peroxidase, as enzyme-labeled praziquantel, respectively. The conjugate of praziquantel-bovine serum albumin conjugate was used to raise anti-praziquantel antiserum in mice. The direct competitive ELISA was conducted by simultaneously incubating praziquantel and horseradish peroxidase-labeled praziquantel conjugate with anti-praziquantel antiserum over a second antibody and the enzyme activity of the remaining horseradish peroxidase-labeled praziquantel conjugate was measured. The intra- and inter-assay coefficient of variation was < 10% in the range of 1.0 to 30 ng ml(-1), and the limit of the detection was 0.3 ng ml(-1). The cross reactivities of anti-praziquantel antibody with compounds related to praziquantel were negligible. Using this ELISA, serum levels of praziquantel were easily determined in male Wistar rats up to 8 h following a single intraperitoneal injection at 2 mg kg(-1) of body weight.     

Inhibition of the human thioredoxin system. A molecular mechanism of mercury toxicity

Mercury toxicity mediated by different forms of mercury is a major health problem; however, the molecular mechanisms underlying toxicity remain elusive. We analyzed the effects of mercuric chloride (HgCl(2)) and monomethylmercury (MeHg) on the proteins of the mammalian thioredoxin system, thioredoxin reductase (TrxR) and thioredoxin (Trx), and of the glutaredoxin system, glutathione reductase (GR) and glutaredoxin (Grx). HgCl(2) and MeHg inhibited recombinant rat TrxR with IC(50) values of 7.2 and 19.7 nm, respectively. Fully reduced human Trx1 bound mercury and lost all five free thiols and activity after incubation with HgCl(2) or MeHg, but only HgCl(2) generated dimers. Mass spectra analysis demonstrated binding of 2.5 mol of Hg(2+) and 5 mol of MeHg(+)/mol of Trx1 with the very strong Hg(2+) complexes involving active site and structural disulfides. Inhibition of both TrxR and Trx activity was observed in HeLa and HEK 293 cells treated with HgCl(2) or MeHg. GR was inhibited by HgCl(2) and MeHg in vitro, but no decrease in GR activity was detected in cell extracts treated with mercurials. Human Grx1 showed similar reactivity as Trx1 with both mercurial compounds, with the loss of all free thiols and Grx dimerization in the presence of HgCl(2), but no inhibition of Grx activity was observed in lysates of HeLa cells exposed to mercury. Overall, mercury inhibition was selective toward the thioredoxin system. In particular, the remarkable potency of the mercury compounds to bind to the selenol-thiol in the active site of TrxR should be a major molecular mechanism of mercury toxicity.     

Monoclonal antibody-based enzyme immunoassay for mercury(II) determination

A monoclonal antibody (K3C6) was developed against Hg(II) and applied in different enzyme immunoassay (EIA) formats to determine the test system with the highest sensitivity. A detection limit of 1. 0 microg/L Hg(II) could be achieved with a competitive format in contrast to a detection limit of 2.1 microg/L Hg(II) with a noncompetitive EIA. A competitive displacement EIA yielded the best detection limit of 0.4 microg/L Hg(II) and was well suited to measuring real samples. For this purpose different water samples were diluted at least 1:10 to avoid matrix effects and subsequently spiked with 1 microg/L HgCl(2). Recovery of the spiked samples was between 80 and 120%.     

Capillary electrophoretic immunoassay for alpha-fetoprotein with chemiluminescence detection

A capillary electrophoretic immunoassay with chemiluminescence detection (CEIA-CL) using a non-competitive format for analyzing tumor marker alpha-fetoprotein (AFP) has been developed. In this method, antigen (Ag) AFP reacts with an excess amount of horseradish peroxidase (HRP)-labeled antibody (Ab*). The free Ab* and the bound Ab*-Ag complex produced in the solution are separated by CE in a separation capillary. Then they catalyze the reaction of enzyme substrate luminol and H(2)O(2) in a reaction capillary following the separation capillary. Parameters affecting the CE separation and CL detection were investigated. Under the optimal conditions, the free Ab* and the Ab*-Ag complex were well separated within 4 min, the linear range and the detection limit (S/N=3) for AFP were 5-500 ng/ml and 0.85 ng/ml (1.2 x 10(-11)M), respectively. The proposed method has been applied satisfactorily in the analysis of human sera samples.     

Cobalamin-mediated mercury methylation by Desulfovibrio desulfuricans LS.

The prominence of sulfate reducers in mercury biomethylation prompted the examination of the methyl carrier and mercury methylation activity of Desulfovibrio desulfuricans LS. There was a low degree of mercury tolerance and a high degree of methylation during fermentative growth; the opposite was true during sulfate reduction. During 2 days of fermentative growth, up to 37% of HgCl2 was methylated at 0.1 micrograms/ml, but only 1.5% was methylated at 10.0 micrograms/ml. Less than 1% of the added HgCl2 was methylated under sulfate-reducing conditions. D. desulfuricans LS radioimmunoassay results were positive for cobalamin. The addition of CoCl2 and benzimidazole to fermentative cultures increased methylation activity. From D. desulfuricans LS grown in the presence of (57)CoCl2, a corrinoid was extracted and purified. High-performance liquid chromatography analysis of the purified extract yielded a single peak with the retention time of cobalamin, and 97% of the (57)Co radioactivity was associated with this peak. Fast atom bombardment and UV and visible spectra of the isolated corrinoid matched those of cobalamin. When methylated with (14)CH3I, the isolated corrinoid methylated Hg(2+) with a 93.9% preservation of (14)C specific activity. We conclude that D. desulfuricans LS methylates mercury via cobalamin (vitamin B12). Under physiological conditions, the enzymatic catalysis of this reaction is likely.     

Methylmercury Resistance in Desulfovibrio desulfuricans Strains in Relation to Methylmercury Degradation

Two strains of Desulfovibrio desulfuricans, one known to synthesize monomethylmercury from ionic mercury, were grown to determine methylmercury toxicity and for comparison with an anaerobic strain of Clostridium pasteurianum, a H₂ producer, and with the broad-spectrum mercury-resistant Pseudomonas putida strain FB-1, capable of degrading 1 μg of methylmercury to methane and elemental mercury in 2 h. The CH₃HgCl resistance of D. desulfuricans strains was 10 times that of P. putida FB-1 and 100 times that of C. pasteurianum. The methylmercury resistance of D. desulfuricans was related to the disappearance of methylmercury from cultures by transformation to dimethylmercury, metacinnabar, methane, and traces of ionic mercury. During a 15-day experiment the kinetics of the two volatile compounds dimethylmercury [(CH₃)₂Hg] and methane were monitored in the liquid by a specific new technique with purge-and-trap gas chromatography in line with Fourier transform infrared spectroscopy and in the headspace by gas chromatography with flame ionization detection. Insoluble metacinnabar (cubic HgS) of biological origin was detected by X-ray diffractometry in the gray precipitate from the insoluble residue of the pellet of a 1-liter culture spiked with 100 mg of CH₃HgCl. This was compared with a 1-liter culture of D. desulfuricans LS spiked with 100 mg of HgCl₂. In a further experiment, it was demonstrated that insoluble, decomposable, white dimethylmercury sulfide [(CH₃Hg)₂S] formed instantly in the reaction of methylmercury with hydrogen sulfide. This organomercurial was extracted with chloroform and identified by gas chromatography in line with mass spectrometry. The D. desulfuricans strains were resistant to high concentrations of methylmercury because they produced insoluble dimethylmercury sulfide, which slowly decomposed under anaerobic conditions to metacinnabar and volatilized to dimethylmercury and methane between pHs 6.2 and 6.5 for high (4.5-g · liter^-1) or low (0.09-g · liter^-1) sulfate contents. Methane was produced from CH₃HgCl at a lower rate than by the broad-spectrum Hg-resistant P. putida strain FB-1.     

Low nanomolar concentration of mercury chloride increases vascular reactivity to phenylephrine and local angiotensin production in rats

Exposure to mercury at nanomolar level affects cardiac function but its effects on vascular reactivity have yet to be investigated. Pressor responses to phenylephrine (PHE) were investigated in perfused rat tail arteries before and after treatment with 6 nM HgCl2 during 1 h, in the presence (E+) and absence (E-) of endothelium, after L-NAME (10(-4) M), indomethacin (10(-5 )M), enalaprilate (1 microM), tempol (1 microM) and deferoxamine (300 microM) treatments. HgCl2 increased sensitivity (pD2) without modifying the maximum response (Emax) to PHE, but the pD2 increase was abolished after endothelial damage. L-NAME treatment increased pD2 and Emax. However, in the presence of HgCl2, this increase was smaller, and it did not modify Emax. After indomethacin treatment, the increase of pD2 induced by HgCl2 was maintained. Enalaprilate, tempol and deferoxamine reversed the increase of pD2 evoked by HgCl2. HgCl2 increased the angiotensin converting enzyme (ACE) activity explaining the result obtained with enalaprilate. Results suggest that at nanomolar concentrations HgCl2 increase the vascular reactivity to PHE. This response is endothelium mediated and involves the reduction of NO bioavailability and the action of reactive oxygen species. The local ACE participates in mercury actions and depends on the angiotensin II generation.     

Mercury induces cell cytotoxicity and oxidative stress and increases beta-amyloid secretion and tau phosphorylation in SHSY5Y neuroblastoma cells

Concentrations of heavy metals, including mercury, have been shown to be altered in the brain and body fluids of Alzheimer's disease (AD) patients. To explore potential pathophysiological mechanisms we used an in vitro model system (SHSY5Y neuroblastoma cells) and investigated the effects of inorganic mercury (HgCl2) on oxidative stress, cell cytotoxicity, beta-amyloid production, and tau phosphorylation. We demonstrated that exposure of cells to 50 microg/L (180 nM) HgCl2 for 30 min induces a 30% reduction in cellular glutathione (GSH) levels (n = 13, p<0.001). Preincubation of cells for 30 min with 1 microM melatonin or premixing melatonin and HgCl2 appeared to protect cells from the mercury-induced GSH loss. Similarly, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assays revealed that 50 microg/L HgCl2 for 24 h produced a 50% inhibition of MTT reduction (n = 9, p<0.001). Again, melatonin preincubation protected cells from the deleterious effects of mercury, resulting in MTT reduction equaling control levels. The release of beta-amyloid peptide (Abeta) 1-40 and 1-42 into cell culture supernatants after exposure to HgCl2 was shown to be different: Abeta 1-40 showed maximal (15.3 ng/ml) release after 4 h, whereas Abeta 1-42 showed maximal (9.3 ng/ml) release after 6 h of exposure to mercury compared with untreated controls (n = 9, p<0.001). Preincubation of cells with melatonin resulted in an attenuation of Abeta 1-40 and Abeta 1-42 release. Tau phosphorylation was significantly increased in the presence of mercury (n = 9, p<0.001), whereas melatonin preincubation reduced the phosphorylation to control values. These results indicate that mercury may play a role in pathophysiological mechanisms of AD.     

Endogenous antioxidant defence system in rat liver following mercury chloride oral intoxication

Mercury is a highly toxic metal which induces oxidative stress. Superoxide dismutases, catalase, and glutathion peroxidase are proteins involved in the endogenous antioxidant defence system. In the present study rats were administered orally, by gavage, a single daily dose of HgCl2 for three consecutive days. In order to find a relation between the proteins involved in the antioxidant defence and mercury intoxication, parameters of liver injury, redox state of the cells, as well as intracellular protein levels and enzyme activities of Mn-dependent superoxide dismutase (MnSOD), Cu-Zn-dependent superoxide dismutase (CuZnSOD), catalase, and glutathione peroxidase (GPx) were assayed both in blood and in liver homogenates. HgCl2 at the doses of 0.1 mg/kg produced liver damage which that was detected by a slight increase in serum alanine aminotransferase and gamma glutamyl transferase. Hepatic GSH/GSSG ratio was assayed as a parameter of oxidative stress and a significant decrease was detected, as well as significant increases in enzyme activities and protein levels of hepatic antioxidant defence systems. Changes in both MnSOD and CuZnSOD were parallel to those of liver injury and oxidative stress, while the changes detected in catalase and GPx activities were progressively increased along with the mercury intoxication. Other enzyme activities related to the glutathione redox cycle, such as glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PDH), also increased progressively. We conclude that against low doses of mercury that produce a slight oxidative stress and liver injury, the response of the liver was to induce the synthesis and activity of the enzymes involved in the endogenous antioxidant system. The activities of all the enzymes assayed showed a rapidly induced coordinated response.     

The accumulation of mercury by the thornback ray, Raja clavata L.

The accumulation of inorganic and organic mercury by the thornback ray has been studied using ²⁰³HgCl₂ and CH₃²⁰³HgCl. Observations have been made on the rates of intake and loss of ²⁰³Hg, from both labelled food and sea water, and the internal distributions of the isotope compared with that of total mercury. Both forms were readily absorbed from sea water. Retention of the two forms from food was, however, dissimilar in that, in contrast to inorganic mercury, methylmercury was readily absorbed and only slowly eliminated. The results are compared with data on the accumulation of mercury by the plaice.     

Homogeneous immunoenzyme analysis of human immunoglobulin G using horseradish peroxidase

The author studied the steady-state kinetics of cooxidation of 4-aminoantipyrine (AAP) with phenol and its derivatives--alpha-naphthol, o- and m-acetylaminophenols--by horseradish peroxidase and its conjugate with human immunoglobulins IgG (HRP-IgG). When phenol and AAP were used as peroxidase substrates, anti-human IgG antibodies stimulated the HPR-IgG enzyme activity in the presence of excess H2O2. A homogeneous enzyme immunoassay of human IgG was developed on the basis of this stimulating effect. The kinetics of the interactions between immunological reagents was studied. In the presence of 3% polyethylene glycol 6000, a complete antibody-antigen interaction proceeds at 37 degrees for 15-20 min. The sensitivity of the enzyme immunoassay is 350 ng/ml IgG, and the dynamic detection range is 0.35-15.0 mg/ml.