A simple spectrophotometric method for metallothionein evaluation in marine organisms: an application to Mediterranean and Antarctic molluscs

A spectrophotometric method to evaluate the concentration of metallothioneins in the tissues of marine organisms has been developed. Metallothionein concentration was evaluated utilizing a partially purified metalloprotein fraction obtained by acidic ethanol/chloroform fractionation of the tissue homogenate. The procedure takes into account precautions to obtain a complete metallothionein precipitation and to avoid the oxidation of sulphydryl groups (SH), the contamination by soluble low molecular weight thiols and enzymatic protein degradation which can occur during sample preparation. In the extracts the concentration of metallothioneins, denatured by low pH and high ionic strength, was quantified spectrophotometrically utilizing the Ellman's SH reagent. The SDS-PAGE separation and the fluorimetric analysis of the proteins, labelled with the fluorescent SH reagent Thiolyte, indicated the presence of minimal amounts of SH-proteins different from metallothioneins in the preparation. The spectrophotometric method was applied to quantify the metallothionein concentration in both Mediterranean ( M. galloprovincialisis and Antarctic ( A. colbecki) molluscs either wild or experimentally exposed to heavy metals (Cu and Cd). Moreover, Zn and metallothionein content in the digestive gland of wild mussels during an annual cycle was evaluated indicating that metallothionein concentration varies with the Zn concentration in the tissues. In conclusion, this spectrophotometric method allows the simple, repeatable and low cost detection of minimal concentrations (nmoles) of metallothionein in biological samples and therefore it is suggested as a tool for metallothionein quantification in eco-toxicological investigations and biomonitoring programmes.