The content of metals in metallothioneins of the bivalve mollusk Unio tumidus depen­ding on different in situ and in vitro exposures

Abstract

Metallothioneins are low-molecular-weight proteins involved in homeostasis and detoxification of the transition metals. The bivalve mollusks are known to be the most effective filter feeding aquatic organisms that provide the accumulation of toxic metals from the environment. The analytical methods for detection and quantitative determination of metallothioneins in the biological samples must be improved to adjust the correct conditions for detecting their metal composition. The sodium azide is frequently utilized to conserve the Sephadex and prevent the microbial growth. The aim of this study was to select the conditions for isolation of the metallothioneins of the bivalve mollusk that would ensure a stability of their metal-binding forms.We examined the metallothioneins of mollusks from four rural sites in the basin of Dniester River, upstream and downstream the dam of two hydropower plants (small HPP in the Kasperivtsy, the Seret River and micro HPP in the Krasnostavtsy, the Zhvanchyk River. The mollusks from the most polluted site (the reservoir of small HPP) were subjected to depuration in the laboratory during 21 days, and the metallothioneins from their digestive gland were compared in different conditions of elution. Four compositions of the eluent based on 10 mM Tris-HCl buffer, pH 8.0 were applied: without additions; containing 10 mM 2-mercaptoethanol and 5 mM sodium azide; containing 5 mM sodium azide; and containing 10 mM 2-mercaptoethanol. The obtained results indicated the distortion of the chromatographic and spectral features of metallothioneins in the presence of sodium azide in the eluent. The detecting of the metal composition of the metallothioneins demonstrated that they predominantly bind Zn. In field conditions of two HPPS, the highest level of Cu in the digestive gland tissue and in the metallothioneins was detected in the mollusk from the reser­voir of small HPP. The depuration of the specimens caused the elimination of Cu from the metallothioneins. The ratio of Zn and Cd was rather stable in all studied conditions of the elution. The function of detoxification of the metallothioneins regarding Cd and Cu was less revealed in mollusks from the reservoir of small HPP. The results accented a necessity of adjusting of correct conditions of elution in the investigation of the metallothioneins in mollusks.

Keywords

• metallothioneins
• bivalve mollusks
• copper
• zinc
• cadmium
• gel-permeation chromatography