title: Application of a modified 203Hg binding assay for metallothionein.
abstract: A sensitive and rapid method to estimate concentrations of functional metallothionein in small biological samples, based upon the acid stability of 203Hg binding and solubility of this protein in trichloroacetic acid is described. Sephadex G-10 minicolumns supported in centrifuge tubes afforded separation and quantitation of isotope bound metallothionein from unbound metal. Elution of metallothionein bound 203Hg was achieved by short term-low speed centrifugation that segregated chelator-ligand complex into the eluate while unbound ligand remained in the gel. A well characterized standard of pure metallothionein protein was utilized to verify the specificity and sensitivity of the modified assay. Metallothionein levels were estimated by 203Hg binding in extracts of wild type and cadmium resistant Chinese hamster ovary cells treated with maximum tolerable concentrations of CdCl2. Similar separation methods demonstrated [35S]-cysteine incorporation into induced metallothionein. Additionally, induction of metallothionein was observed after treatment with particulate CdS but not crystalline NiS particles. These results demonstrate that the modified assay system is easily applied to serial measurement of metallothionein levels in multiple small biological samples.
mesh_terms: D000818:Animals; D002104:Cadmium / Q000494:pharmacology; D019256:Cadmium Chloride; D002460:Cell Line; D002850:Chromatography, Gel; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D008628:Mercury / Q000378:metabolism*; D008631:Mercury Radioisotopes / Q000378:metabolism; D008667:Metalloproteins / Q000032:analysis*; D008668:Metallothionein / Q000032:analysis*; D008722:Methods; D010053:Ovary / Q000032:analysis / Q000187:drug effects
chemical_list: D008631:Mercury Radioisotopes; D008667:Metalloproteins; D002104:Cadmium; D008668:Metallothionein; D008628:Mercury; D019256:Cadmium Chloride