The reaction of metallothionein with mercuribenzoate. A dialysis and 113Cd-n.m.r. study (original) (raw)
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European journal of biochemistry / FEBS, 2001
The reaction of Cd5Zn2-metallothionein (MT) with 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) has been studied at different reagent stoichiometries, pH and temperature conditions and in the presence of several ligands. At stoichiometries of Nbs2 to MT from 0.5 to 5, the reaction followed first order kinetics. The first order rate constants obtained were independent from the concentration of Nbs2 but were linearly dependent on the concentration of MT. At higher Nbs2/MT stoichiometries, the reaction deviates from first order kinetics and the observed rate constant increases. The reactivity of MT towards Nbs2 has been probed at 4 microM concentration of both reagents where the reaction is monophasic and is characterized by a linear Arrhenius plot (Ea = 45.8 +/- 2.7 kJ.mol-1). It has been demonstrated that metal release at low pH or subtraction from MT by EDTA substantially increases the reactivity of MT towards Nbs2. At the same time, a number of nonmetallic ligands moderately accele...
Comparative biochemistry and physiology. C, Comparative pharmacology and toxicology, 1988
1. Metallothionein behavior in SDS-PAGE has been characterized. 2. It has been found that metallothionein behavior in this electrophoretic system depends upon the reducing environment. Migration as a well-defined protein band is only achieved in the presence of 2-100 mM 2-mercaptoethanol. 3. Within those 2-mercaptoethanol levels, both rat and dogfish metallothionein migrate as a protein with a molecular weight several times higher than that expected by amino acid analyses. This is not due to molecule oxidations, since this effect is promoted by the presence of 2-mercaptoethanol. 4. No effect of 2-mercaptoethanol on metallothionein behavior is found in conventional PAGE. 5. The present results suggest that to study the effect of 2-mercaptoethanol in SDS-PAGE is a simple and accurate way to identify a protein as metallothionein. 6. It has also been found that metallothionein aggregates naturally in the absence of ionic strength.
Biochemical mechanism of metallothionein-carbon tetrachloride interaction in vitro
Biochemical Pharmacology, 1990
To elucidate the mechanism underlying the protective effect of metallothionein (MT) against carbon tetrachloride (CCL) toxicity, in uitro experiments were carried out to study the interaction of metallothionein and CCL,. Results from this study showed that incubation of Cd,Zn-MT with CCI, in the presence of hepatic microsomes and NADPH resulted in a time-dependent depletion of MT thiols with a concurrent reduction in the metal-binding sites of the protein. Moreover, this reaction also released Zn and Cd from MT. Results from experiments conducted to determine whether or not the CC&-induced decrease in MT-thiol content was due to the scavenging of Ccl, metabohte(s) showed that the trichloromethyl radical, chloroform and phosgene as well as the products of CCL-induced microsomal lipid peroxidation were not directly involved. Although covalent binding of r4CC1, to MT was detected following incubation in the presence of a microsomal bioactivation system. it did not account for the CC&-induced loss of MT thiol groups for the following reasons: (i) prior oxidation of sulfhydryl groups of MT by hydrogen peroxide did not alter the binding; and (ii) anaerobiosis did not alter the extent of covalent binding but obliterated the inhibitory effect of CC& on MT thiol content. Measurement of the thiol content of C&-treated MT after treatment with 1,4-dithiothreitol revealed that all the thiol groups that were lost subsequent to CC& treatment could be regenerated. These data suggest that CCL,-linked oxidation of MT, rather than the covalent binding of 14CC14 metabolite(s), may be responsible for the CC&-induced loss of metal binding sites of MT with the concurrent release of Zn and Cd. However. the orecise role of the metal released during the oxidation of MT in CC& toxicity remains to be defined. L
Ferritin and metallothionein: dangerous liaisons
Chemical Communications, 2011
Reaction of ferritin with MTs: Horse spleen ferritin (56 mg/ml) was obtained from Sigma-Aldrich and passed down a Sephadex G-25 column to remove any free iron. Aqueous solutions of proteins were prepared using water purified through the Milli-Q system. Ferritin (0.1 mg/ml, 21 μM in Fe) was incubated at room temperature with each Zn-MTx preparation (1 μM) in the presence of an excess of ferrozine. The experiments were carried out in anaerobic conditions in a glove box and the solutions transferred to screw-cap quartz cuvettes. The development of the iron(II)-ferrozine complex was followed by UV-visible spectroscopy using a Thermospectronic UV300 spectrophotometer against reference containing solutions containing Milli-Q water. Spectra were recorded during 24 h. The concentrations of [Fe(ferrozine) 3 ] 4after 24 h were calculated from the UV-Vis absorbance values at 562 nm using ε 562 = 27.900 mM-1 cm-1. To determine the Zn(II) concentration, the resulting Ft-MTx solutions were filtered and then exhaustively dialyzed at 4 ºC for two days against several changes of Milli-Q water using a dialysis bag with a molecular weight cutoff of 5.000 Da. After 48 h, the water of the dialysis reservoirs (100 ml) were collected and used to determine the Zn(II) concentration by atomic absorption spectrometry. Likewise the Zn(II) concentration of the dialyzed solution was also determined by atomic absorption spectrometry. Two blank experiments were performed at the same conditions either in the absence of ferritin or the Zn-MT complex. In the absence of Zn-MT, the ferritin-ferrozine solution did not show any absorption at 562 nm in the UV-Visible spectrum. Likewise, anaerobic solutions of the Zn-MTx species did not change over time as corroborated by ESI-MS.
Chemical foundation of the attenuation of methylmercury(II) cytotoxicity by metallothioneins
2004
To elucidate the chemical interactions underlying the role of metallothioneins (MTs) in reducing the cytotoxicity caused by MeHg(II), we monitored in parallel by electronic absorption and CD spectroscopies the stepwise addition of MeHgCl stock solution to mammalian Zn 7 -MT1 and the isolated Zn 4 -aMT1 and Zn 3 -bMT1 fragments. The incorporation of MeHg + into Zn 7 -MT and Zn 3 -bMT entails total displacement of Zn(II) and unfolding of the protein.