Voltammetric methods in isolation and identification of plant metallothioneins from alga Chlorella

Abstract

In response to the environmental pollution by metals such as Cd, Cu, Pb, Ni, and As, special thiol-rich peptides are synthesized in plants and certain fungi. Their general structure is (γ-Glu-Cys)_n-R, where R is an amino acid residue (Gly, Ala, Ser, Glu). These peptides are known as plant metallothioneins, class III metallothioneins or γ-EC peptides [1]. Cd and Cu are the metals most frequently bound to plant metallothioneins from different sources and are well characterized by UV spectroscopy due to special absorption of the Cd-S and Cu-S bonds. Another possibility is offered by voltammetric methods which can distinguish between free metal ion and metal ion bound in complexes of different stability. For Cd complexes of γ-EC peptides structural models have been proposed [2] in which each cadmium ion is coordinated by four sulfur atoms. No voltammetric studies have been performed with γ-EC peptides so far. With animal metallothioneins, where tetracoordinated cadmium is present in molecules consisting of 60-62 amino acids, several polarographic and voltammetric studies have been performed. In our first study [3], we worked with rabbit liver metallothionein-2, prepared from rabbits exposed to cadmium salt, and with Cd-MT and apo-MT prepared from rabbit. We compared the voltammetric behavior on several types of carbon electrodes with that on the hanging mercury drop electrode (HMDE); active role of mercury electrode, confirmed also in other papers, can be best demonstrated with cyclic voltammetry (Fig. 1). An extreme reactivity of thiols with mercury is well known and detailed studies on reactions of cystine [4] and cysteine [5] have been performed. Also, it has been demonstrated, that under certain conditions, compact triphenyphosphine oxide films formed at the Hg/solution interface, can block thiol or disulfide adsorption on Hg electrodes [4].