An Improved Method for the Purification and Characterization of a 54 kDa Protein in Rat Liver Which Has Recently Been Identified as a Selenium-Binding Protein

Abstract

A 54kDa protein, which is induced by treatment with 3, 3', 4, 4', 5-pentachlorobiphenyl, 3-methylcholanthrene and butylated hydroxytoluene, was purified from rat liver by conventional methods involving DEAE-Sephacel column chromatography and processing using a Rotofor Cell^TM. This method does not require a denaturation or solubilization step and yields a single protein on SDS-PAGE. The molecular mass of the purified protein was 54.7kDa. We also characterized the 54kDa protein by immunoblot analysis and measured the binding ratio of selenium. The purified 54kDa protein was stained with antiserum which had been previously obtained, and the molecular ratio of selenium to the 54kDa protein was 0.014. Glutathione peroxidase (GPx) is a typical selenoprotein which has a selenocysteine amino acid sequence. We measured the binding ratio of selenium with commercial GPx as a positive control and obtained a value of 1.214. The binding ratio of selenium to 54kDa protein was much lower than that of GPx, and this led us to believe that there is no selenocysteine in the 54kDa protein. In this report, we have developed a convenient purification procedure and measured the purification factors and binding ratio of selenium associated with a 54kDa protein in rat liver which is homologous with a selenium-binding protein in the mouse.