Preparation of metallothionein from rat liver and studies of its properties with respect to use as a standard in gel permeation chromatography, polyacrylamide gel systems, autoradiography and Western blotting.
1. A simple method for preparation of metallothionein (Mt) I and II has been developed for the purpose of making standards for use in various biochemical systems and in antibody production. 2. The theoretical content of SH groups in a Mt protein; assuming the mol. wt to be 10,000 and each molecule to contain 20 SH groups was found to be 7.1 and 7.7 times higher than for our purified Mt I and II, respectively. 3. In our native polyacrylamide gel system Mt I ran ahead of Mt II, while the two Mt forms were not separated in the Laemmli SDS system in which it behaved as a protein with mol. wt 10,000. In both gel systems, however, Mt I stained as a very faint band in comparison to Mt II, despite equal absorbance at 254 nm and Cd binding capacity. 4. Compared to staining of polyacrylamide gels with Coomassie Brilliant Blue less than 1/50 parts (1 ng) of the protein could be easily seen after silver staining. 5. It was found that Mt may undergo spontaneous modification, polymerization and loss of metal binding properties. 6. Spontaneous modification and polymerization reduced the antigenic properties of our purified Mt. Only Mt II appeared to be immunologically active.
Andersen
R A
RA
Department of Toxicology, National Institute of Public Health, Oslo, Norway.
Daae
H L
HL
D000818:Animals; D001345:Autoradiography / Q000379:methods; D002104:Cadmium / Q000494:pharmacology; D019187:Cadmium Compounds; D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008668:Metallothionein / Q000096:biosynthesis / Q000302:isolation & purification*; D008970:Molecular Weight; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D012015:Reference Standards; D013431:Sulfates
D019187:Cadmium Compounds; D013431:Sulfates; D002104:Cadmium; D008668:Metallothionein; C037123:cadmium sulfate