Quantification of metallothioneins by a silver-saturation method.
The binding of Ag+ to metallothionein (MT) was investigated, and a Ag-saturation assay was developed for the measurement of MT in tissues. When samples of purified hepatic Zn-MT or Cd-MT were titrated with Ag+ followed by hemolysate-heat treatment, it was found that saturation of MT occurred at 17 to 18 g-atoms Ag+/mol protein. The rank order of potencies of metals to displace Ag+ from 110mAg-labeled Ag-MT was Ag+ greater than Cu2+ greater than Cd2+ greater than Hg2+ greater than Zn2+ at pH 8.5 in 0.5 M glycine buffer. When a Ag-saturation (Ag-hem) assay was compared with a Cd-saturation (Cd-hem) technique, excellent correspondence was obtained in the measurement of MT from various sources including purified Zn-MT (II), MT in human kidney and liver tissue samples, hepatic MT in Cd-injected adult rats, and renal MT in Hg-injected adult rats. However, in cases where significant amounts of Cu-MT were present, such as in the livers of Cu-injected rats or in the kidneys of Cd-injected rats, the Cd-hem assay consistently underestimated the MT concentrations compared to the Ag-hem method. This is attributed to the inability of Cd to displace Cu effectively from MT at pH 8.5. Thus the Cd-hem assay is not recommended for the measurement of metallothioneins containing a high Cu content. Under such conditions, the Ag-hem method seems superior.
D000818:Animals; D002104:Cadmium / Q000032:analysis / Q000378:metabolism; D055598:Chemical Phenomena; D002621:Chemistry; D002850:Chromatography, Gel; D006801:Humans; D007274:Injections, Intraperitoneal; D007668:Kidney / Q000032:analysis / Q000378:metabolism; D008099:Liver / Q000032:analysis / Q000378:metabolism; D008297:Male; D008628:Mercury / Q000032:analysis / Q000378:metabolism; D008668:Metallothionein / Q000378:metabolism*; D011868:Radioisotopes; D051381:Rats; D011919:Rats, Inbred Strains; D012834:Silver / Q000378:metabolism; D014018:Tissue Distribution
D011868:Radioisotopes; C007121:copper thionein; C021267:zinc thionein; D002104:Cadmium; D012834:Silver; D008668:Metallothionein; D008628:Mercury