Probing the characteristics of metal-binding proteins using high-performance liquid chromatography–atomic absorption spectroscopy and inductively coupled plasma mass spectrometry

Abstract

The primary structure of metallothionein (MT) and MT-like proteins (MLP) contains a high proportion of cysteine residues which are capable of complexing a variety of metals. Due to the unique metal-binding properties of MT and MLP, metal saturation assays have been used as an indirect method of quantifying the concentration of these proteins in various samples. In adapting Cd-saturation methodologies of MT from mammalian sources for MLP from freshwater mussels, the redox state of the MLP cysteine residues was indirectly monitored by adding Cd in the presence, or absence, of a reducing agent followed by high-performance liquid chromatography (HPLC)–microatomization (MA)–atomic absorption spectroscopy (AAS) and/or HPLC–inductively-coupled plasma–mass spectrometry (ICP–MS). The presence of 2-mercaptoethanol (2-MCE) in freshwater mussel extract results in a higher concentration of Cd present in the MLP fraction, indicating that these proteins may be partially oxidized in vivo and/or during isolation under anoxic conditions. Standard preparations of rabbit MT obtained by induced synthesis due to controlled exposure to Cd are fully reduced and saturated with metal (Cd and Zn) and, therefore, did not respond similarly to the reduction treatment used in these studies. Thus, as conventional metal saturation assays exclude a reduction step, they may be unsuitable for the quantitative analysis of MLP from molluscan tissues without further modification.