The application of covalent affinity chromatography with thiol-disulphide interchange for quantitative determination of metallothionein-specific protein from human fluids, as indicator of environmental exposure to heavy metals

The aim of this study was to present a new analytical method for the quantitative determination of metallothioneins (MT) protein in human body fluids and tissues, in order to determine the level of environmental and industrial exposition to heavy metals. For MTs isolation covalent affinity chromatography with thiol-di- sulphide interchange (CAC-TDI) was applied, which is a modern technique of separation of a high affinity, good repeatability and reproducibility, allowing specific isolation of the thiolproteins and metallothiolproteins. Fundamentals of indirect determination of the contents of metallothioneins protein were worked out throught estimation of the quantities of metals bound with metallothionein protein and adsorbed on covalent affinity chromatography gel as on solid-phase extraction support during a separating process. The (CAC-TDI) gel, specially prepared, was used as a solid phase extraction support (SPE) for preconcent- ration of Hg-Thionein (Hg-Th), Cd-thionein (Cd-Th), Zn-thionein (Zn-Th) and Cu-thionein (Cu-Th) proteins and Hg, Cd, Zn and Cu bonded with MTs from spiked water, urine, human plasma, breast milk and tissues' homogenates.