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Electrochemical quantification and characterisation of metallothioneins

This presentation deals with the electrochemical approach to the quantification and characterization of metallothioneins, isolated from the marine invertebrates i.e. the common mussel Mytilus galloprovincialis. Applying the electrochemical technique the protein content is determined by a modified Brdička reaction, which is based on the catalytic hydrogen evolution in the presence of proteins containing SH-groups. The signal height of the catalytic wave is evaluated and the MT concentration determined from the calibration line. The amplitude of the catalytic signal CAT2 is related to the dissolved amount of MTs. The advantage of the Brdička reaction is that it gives information on the overall MT content. The polarographic response is independent of the type and the concentration of the metal bound to SH-functional groups of the MTs. Proteins of higher molar mass than MTs, which contain SH-groups, contribute to the overall polarographic signal. Therefore, the reliability of the modified Brdička method depends on the sample pretreatment previous to the analysis. In that respect it is essential to define the validated isolation and purification procedure for MTs of marine invertebrate origin. The sensitivity of the modified Brdička method can be enhanced by quantifying the Cat1 signal, which should first be resolved by deconvolution (Raspor et al., 1994). Chemical reactivity of MTs, isolated from the digestive gland of cadmium-exposed M. galloprovincialis has been studied applying the voltammetric method in order to characterise the chemical forms of this specific invertebrate metal-binding protein, which has been extensively studied. The characterisation is based on the quantification of the specific signal of the Cd-thionein complex. A defined concentration of purified MT component, isolated from M. galloprovincialis has been titrated with the defined concentrations of CdCl2. The molar ratio of Cd(II)-ions with respect to MT concentration has been defined as 5, irrespective of the MT concentration. Nased on our data it has been concluded that the stoichiometric ratio of Cd(II) atoms bound per molecule of in vivo induced mussel MT is 5 (Raspor and Pavičić, 1996). References B. Raspor, I. Pižeta, M. Branica, Anal.Chim.Acta, 285 (1994) 103-111. B. Raspor, J. Pavičić, Fresenius J.Anal.Chem. 354 (1996) 529-534.

metallothioneins; quantification of; characterisation of

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