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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

100%

Kabzinski A. K. M.

Polish Journal of Environmental Studies

1997

tom 06

nr 5

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.

Sepharose-DTNB covalent affinity chromatography of hepatic metallothionein from Cu-treated rats

84%

Kabziński A. K. M. , Takagi T. , Tanaka K.

Chemia Analityczna

1999

tom Vol. 44, No. 5

805-823

Covalent affinity chromatography with thiol-disulphide interchange (CAC-- TDI) on Sepharose-DTNB gel was employed for separation of Cu-thionein (Cu-Th) from the łivers of Cu induced white rats (3.0 mg of Cu per kg body weight, administered subcutaneously in physiological saline). Cu-thionein was prepared by gel permeation chro-matography (GPC) on Sephadex G-200 geł. The metallothionein protein (MT) was fractionated by two-, three- and four-step elutions. The CU(2+) ion dissociated fi.om Cu-thionein and protein aggregated by thiol-thiol intermolecular oxidation and mercaptide bonds (oligoaggregates and polyaggregates) and intramolecular formation of disulphide and mercaptide bridges in the lightmass monomer resulted from the contact with cova-lent affinity chlomatography support, inferred from rechromatoglraphy on Sephadex G-200. The dissociated CU(2+) appeared as a light complex with DTNB in fractions about 2000-2800 Da mass.

Metodę chromatografii powinowactwa kowalencyjnego z wymianą mostków disiarczkowych (CAC- TDI) na złożu Sepharose-DTND stosowano do rozdziału Cu-tioneiny (Cu- Th) z wątroby białych szczurów eksponowanych przy użyciu Cu (3.0 mg Cu na kg wagi ciała, w postaci CUSO4 podawanego podskórnie w roztworze soli fizjologicznej). Cu-tioneina była otrzymywana metodą klasycznej chromatografii żelowej (GPC) na żelu Sephadex G-200. Białko metalotioneinowe frakcjonowano przy użyciu dwu-, trój- i czterostopniowej elucji. Podczas rozdziału jony CU2+ dysocjowały z Cu-tioneiny i powstały agregaty białkowe w wyniku utleniania grup tiolowych oraz tworzenia się wiązań merkaptydowych (oligoagaty i poliagregaty). W wyniku kontaktu ze złożem do chromatografii powinowactwa kowalencyjnego powstawały także wewnątrzcząsteczkowe mostki disiarczkowe oraz merkaptydowe w monomerach o niskiej masie cząsteczkowej, co potwierdzano rechromatografując powstałe frakcje na żelu Sephadex G-200. Dysocjujące jony Cu(2+) występowały w niskocząsteczkowej frakcji, jako kompleksy z DTNB o masie w granicach 2000 --28OO Da.

Investigation of metallothionein concentration with preconcentration by solid phase extraction on covalent affinity chromatography gel for quantitative determination of exposure to heavy metals

84%

Kabzinski A. K. M.

Polish Journal of Environmental Studies

1996

tom 05

nr 5

The aim of this study was to test a new method for the quantitative determination of metallo- thionein (MT), in order to determine the level of exposure to heavy metals. For MT preconcentration, the solid phase extraction (SPE) method with covalent affinity chromatography with thiol-disulphide interchange (CAC-TDI) gel was applied. In experiment Cd-thionein (Cd-Th) obtained from rats exposed to cadmium salt was used. Protein concentration was determined by indirect method based on metal analysis, bonded with MT protein by atomic absorption spectrometry (AAS). The results showed satisfactory correlation between Cd-Th concentration and the level of exposition for heavy metals.