A Mercury saturation assay for measuring metallothionein in fish

An accurate, rapid, sensitive, and simple method using mercury saturation for quantifying metallothionein (MT) is described. A complex solution (“pseudocytosol”) of enzymatic and non-enzymatic thiols, including rabbit liver MT-II, and supernatants from homogenized samples of rainbow trout liver were incubated in the presence of 203Hg in 10% trichloroacetic acid Excess Hg was bound to and removed by chicken egg albumin, which denatured on contact with the acidic assay medium. After centrifugation, MT labeled with 203Hg remained in the TCA supernatant and was estimated using known stoichiometry for Hg-MT binding. A dilution series was used to establish that nonspecific metal binding, a common problem with other metal saturation assays, is negligible. Analysis of hepatic MT with high Cu content from rainbow trout demonstrated virtually complete displacement of Cu, Cd, and Zn by Hg. When compared to other metal-saturation assays developed for vertebrates, this method requires the least number of technical steps, and one-third or less of total preparatory and analytical time.