A SIMPLE SPECTROPHOTOMETRIC METHOD FOR METALLOTHIONEIN EVALUATION IN MARINE ORGANISMS - AN APPLICATION TO MEDITERRANEAN AND ANTARCTIC MOLLUSKS

A spectrophotometric method to evaluate the concentration of metalloth ioneins in the tissues of marine organisms has been developed. Metallo thionein concentration was evaluated utilizing a partially purified me talloprotein fraction obtained by acidic ethanol/chloroform fractionat ion of the tissue homogenate. The procedure takes into account precaut ions to obtain a complete metallothionein precipitation and to avoid t he oxidation of sulphydryl groups (SH), the contamination by soluble l ow 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 qu antified spectrophotometrically utilizing the Ellman's SH reagent. The SDS-PAGE separation and the fluorimetric analysis of the proteins, la belled with the fluorescent SH reagent Thiolyte, indicated the presenc e of minimal amounts of SH-proteins different from metallothioneins in the preparation. The spectrophotometric method was applied to quantif y the metallothionein concentration in both Mediterranean (M. gallopro vincialis) and Antarctic (A. colbecki) molluscs either wild or experim entally exposed to heavy metals (Cu and Cd). Moreover, Zn and metallot hionein content in the digestive gland of wild mussels during an annua l cycle was evaluated indicating that metallothionein concentration va ries with the Zn concentration in the tissues. In conclusion, this spe ctrophotometric method allows the simple, repeatable and low cost dete ction of minimal concentrations (nmoles) of metallothionein in biologi cal samples and therefore it is suggested as a tool for metallothionei n quantification in eco-toxicological investigations and biomonitoring programmes. (C) 1997 Elsevier Science Ltd.