An improved method for isolation and identification of Zn‐metallothionein from cadmium‐induced rat liver

Abstract

Zn-metallothioneins (MT-1 and MT-2) were isolated and purified from Wistar rat liver induced by subcutaneous injection with cadmium chloride over a short time. Instead of Sephadex G-50 and DEAE Sephadex A-50, new chromatographic media produced by Pharmacia, Sephacryl S-200, S-100 and DEAE Sepharose Fast Flow were used in the purification of metallothioneins. The time required for purification with the new method was only 1/3 that required with the usual method and had the same purification effect and rate of recovery. The number of mercapto groups measured with modified Ellman's reagent and cysteine as standard is 20 in MT molecules. Zn and Cd concentrations in each fraction were measured by single sweep polarography rather than atomic absorption spectrophotometry. MT-1 and MT-2 contained 6 gram atoms of zinc, but no cadmium. Purified MT-1 and MT-2 were shown by high performance liquid chromatographic analysis to be highly homogeneous and had an amino acid composition similar to that of Cd-MT.

Purification and characterization of hedgehog liver metallothioneins

“…The difference in microheterogeneity between hedgehog and rabbit MTs might depend upon the species difference. The amino acid composition of hedgehog liver MT-1 and MT-2 (Table 1) resembles that of liver MTs from rabbit and other species (Pan et al, 1992). The molecular weight of hedgehog liver MT-1 and MT-2 measured by HPLC was about 10,000 ( fig.…”

Bio-inorganic speciation analysis by hyphenated techniques

“…In metalloprotein biochemistry the technique has been widely used for the fractionation of metallothionein 122,123 and serum proteins. In the high-performance version (sometimes called ion chromatography, IC) the polymer beads are coated with an ion exchanger.…”

“…As a result, two fractions, a soluble one (cell supernatant, cytosol) and a particulate one (cell membranes and organelles), are obtained. The selectivity in terms of MT-1 and MT-2 classes could be achieved by anion-exchange HPLC with AAS 122,123,339,344 or radioactivity detection 117 or RP-HPLC-FAAS. This procedure allows the protein load on the HPLC column to be decreased not only improving the separation of MT isoforms but also prolonging the column lifetime.…”

“…Both cation and anion exchangers are widely used for the separation of metal species, especially of organoarsenic and organoselenium compounds. In metalloprotein biochemistry the technique has been widely used for the fractionation of metallothionein 122,123 and serum proteins. 80,83,124 Separations on ion exchange resins require drastic elution conditions with acids, bases or high salt concentrations in order to elute multicharged anions and cations in a reasonable time.…”

“…112 As can be seen from Table 4, SE HPLC-ICP-MS has been the most widely used technique for the quantification of MTs. The selectivity in terms of MT-1 and MT-2 classes could be achieved by anion-exchange HPLC with AAS 122,123,339,344 or radioactivity detection 117 or RP-HPLC-FAAS. 164 The highest resolution was reported to be given by microbore RP-HPLC with DIN-ICP-MS and ESMS detection 141 and CZE-ESMS 219 which were shown to be able to separate the particular subisoforms within each of the classes of MT isoforms.…”