An electrochemical approach to quantitation and characterization of metallothioneins

Olafson, Robert W.; Sim, Rob

doi:10.1016/0003-2697(79)90239-2

Cited by 182 publications

(53 citation statements)

References 8 publications

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“…Then, cytosol was submitted to heat-denaturation (95°C, 15 min) and centrifuged (10000 G, 15 min) in order to separate thermostable from thermolabile proteins (e.g., Temara et al, 1997). From newly obtained supernatants, the concentrations of metallothionein (MT) were determined by differential pulse polarography through quantitification of the cysteinic residues (Olafson and Sim, 1979, modified by Thompson and Cosson, 1984), using a PARC Model 174A analyzer and a PARC EG&G Model 303 static mercury drop electrode (SMDE). The quantification of MTs was based on the standard addition method using rabbit liver MT (M-7641, Sigma) and on the variation of height of the "B" peak which is the more electronegative peak following the reduction of cobalt wave.…”

Section: Metallothionein Analysismentioning

confidence: 99%

Bioaccumulation and detoxification processes of Hg in the king scallop Pecten maximus: Field and laboratory investigations

Métian

Warnau

Cosson³

et al. 2008

Aquatic Toxicology

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wt), they did for the digestive gland and the gills. According to the experimental results, a higher exposure to dissolved Hg might occur in the site close to the estuary whereas Hg would be mainly incorporated via the dietary pathway in the site away from the estuary.Within the cells of wild scallops, Hg was mainly associated to the cytosolic fraction in the digestive gland and gills (60-100%). However, the lack of relationship between Hg and MT levels suggests that Hg detoxification in P. maximus involves other, non-MT, soluble compounds. In kidneys, insoluble compounds played an important role in Hg sequestration.No effect of scallop age was observed neither on Hg and MT concentrations nor on the subcellular distribution of the metal. Finally, according to FAO/WHO recommendations (maximum weekly Hg intake), our results clearly indicate that the low Hg contents in the edible part of the king scallops from the Bay of Seine prevent any risk for Human consumers.

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Section: Metallothionein Analysismentioning

confidence: 99%

Bioaccumulation and detoxification processes of Hg in the king scallop Pecten maximus: Field and laboratory investigations

Métian

Warnau

Cosson³

et al. 2008

Aquatic Toxicology

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“…The aliquot for metallothionein determination was heated at 95 "C for 4 min, re-centrifuged at 10 000 X g and the supernatant stored at -20 "C. Metallothionein was determined in the heat-stable extract by differential pulse polarography using a modification of the Brdicka (1933) method as described by Olafson & Sim (1979) and Thompson & Cosson (1984). A static mercury drop electrode (EG and G PARC, model 303A) was linked to a polarographic analyser (Princeton Applied Research, model 174A) and analysis was carried out in 10 m1 of supporting electrolyte (1.0 M NH,OH, 1.0 M NH,CI and 1.2 mM [Co(NH,),]Cl,), with addition of 100 p1 of Triton X-100 (0.0125 % v/v) to the sample cup.…”

Section: Methodsmentioning

confidence: 99%

Changes in branchial Na+,K+-ATPase, metallo-thionein and P450 1A1 in dab Limanda limanda in the German Bight: indicators of sediment contamination?

Stagg¹,

Goksøyr²,

Rodger³

1992

Mar. Ecol. Prog. Ser.

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Na+,K+-ATPase activity, ouabain binding, metallothionein and P450 1Al were measured in gill tissue from the dab Lirnanda limanda caught from a contaminant gradient in the German Bight during the Bremerhaven Workshop. Metallothionein levels were highest at the most contaminated sites closest to the mouth of the Elbe and lowest at the outermost station on the Dogger Bank. Tissue levels of zinc and cadmium were also elevated at these sites but copper levels did not change along the gradient. P450 1Al levels, measured with an antibody probe, showed a complex pattern of induction which suggests an inhibitory effect at the most contaminated stations. Na+,Kc-ATPase was similar in fish from all of the stations but measurement of ouabain binding show that levels of the enzyme are elevated at the innermost and outer stations. Calculation of activity per binding site indicates that the turnover rates of the enzyme were lower at the most contaminated stations. The results show that the role of adaptive responses in the face of inhibition by contaminants is an important consideration in identifying biological effects. The data were analysed with respect to the contaminant burdens in the total and fine particulate fraction (< 63 pm) of the sediment and in the liver of dab caught at each station. The utility of biological effects measurement in the gill as an indicator of environmental contamination is discussed.

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“…A variety of techniques are available ranging from colorimetric methods such as the Lowry method (14), thiol titration (15), and polarographic methods (16), calculation of molar extinction coefficients (17) for apo-MT and immunochemical procedures (18). Each of these approaches to protein quantitation has its advantages and potential problems.…”

Section: Protein Quantitationmentioning

confidence: 99%

Techniques and problems in metal-binding protein chemistry and implications for proteins in nonmammalian organisms.

Winge¹,

Brouwer²

1986

Environ Health Perspect

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Proper isolation and purification techniques for metallothionein (MT) and related proteins in nonmammalian species are essential to the study of these molecules at all levels of biological organization. There are a number of basic approaches to preparing these proteins from biological tissues that have both positive and negative aspects depending upon the subsequent scientific questions under investigation. A general flow diagram for the isolation of MT and related proteins is given in Figure 1. The following discussion will attempt to examine each of the steps in this general isolation procedure and point out both the advantages and known disadvantages in each procedure. Homogenization ProceduresIf tissues are to be stored frozen, this should be done at -70°C. Inhibition of endogenous tissue proteases during homogenization procedures is essential to preventing degradation of MT and related proteins. A number of protease inhibitors, such as phenylmethylsulfonyl fluoride (PMSF) or a2-macroglobulin have been commonly used to inhibit protein degradation with varying degrees of success. PMSF dissolved in ethanol and added to a final concentration of 0.5 to 1.0 nM is usually effective in preventing proteolysis by serine proteases.Since PMSF hydrolyzes quickly, it should be added directly to sample. In addition, utilization of reducing agents such as ,B-mercaptoethanol (P-ME) or dithiothreitol (DTT) to prevent copper oxidation of protein SH groups may be helpful. In the case of p-ME 5 to 20 mM final concentration is sufficient, whereas only 0.5 to 1.0 mM DTT is necessary (1) because of its higher reducing potential.

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An electrochemical approach to quantitation and characterization of metallothioneins

Cited by 182 publications

References 8 publications

Bioaccumulation and detoxification processes of Hg in the king scallop Pecten maximus: Field and laboratory investigations

Bioaccumulation and detoxification processes of Hg in the king scallop Pecten maximus: Field and laboratory investigations

Changes in branchial Na+,K+-ATPase, metallo-thionein and P450 1A1 in dab Limanda limanda in the German Bight: indicators of sediment contamination?

Techniques and problems in metal-binding protein chemistry and implications for proteins in nonmammalian organisms.

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