Abstract
Abstract A procedure for the isolation of bovine extracellular glutathione peroxidase (eGSHPx) from plasma was developed. The purification method included ammonium sulphate fractionation and column chromatography using hydrophobic interaction, gel filtration and ion-exchange. The purified protein was visualised as one band on SDS-PAGE and identified as eGSHPx based on its amino acid composition and peroxidase activity. Mass spectrometry analysis showed a subunit weight between 21 and 23 kDa for different preparations. Polyclonal antibodies to the purified eGSHPx were raised in rabbits and an enzyme-linked immunosorbent assay (ELISA) was developed. The ELISA method was used to measure eGSHPx in bovine milk. The mean concentration in 15 milk samples was 77 ng mL −1 , and in the corresponding whey samples 51 ng mL −1 . The eGSHPx content was significantly correlated with that of selenium in both whey and milk. The new ELISA assay will be a valuable tool in future studies on eGSHPx in dairy products.
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