Purification and properties of a glutathione peroxidase from Southern bluefin tuna (Thunnus maccoyii) liver.
A glutathione peroxidase (GPX) protein was purified approximately 1000-fold from Southern bluefin tuna (Thunnus maccoyii) liver to a final specific activity of 256 micromol NADPH oxidised min(-1) mg(-1) protein. Gel filtration chromatography and denaturing protein gel electrophoresis of the purified preparation indicated that the protein has a native molecular mass of 85 kDa and is most likely a homotetramer with subunits of approximately 24 kDa. The Km values of the purified enzyme for hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide and glutathione were 12, 90, 90 and 5900 microM, respectively. The Km values for cumene hydroperoxide and t-butyl hydroperoxide were approximately 8-fold greater than the Km value for hydrogen peroxide. Thus, the SBT liver GPX has a considerably greater affinity for hydrogen peroxide than for the other two substrates. The pH optimum of the purified enzyme was pH 8.0. Immunoblotting experiments with polyclonal antibodies, raised against a recombinant human GPX, provided further evidence that the purified SBT enzyme is a genuine GPX.
School of Biological Sciences, Flinders University and Aquafin CRC, GPO Box 2100, Adelaide, South Australia 5001, Australia.
Janene L
JL
Thompson
Philip M
PM
Thomas
Kathryn A
KA
Schuller
D000818:Animals; D001555:Benzene Derivatives / Q000378:metabolism; D002850:Chromatography, Gel; D005979:Glutathione Peroxidase / Q000302:isolation & purification* / Q000502:physiology*; D006801:Humans; D006861:Hydrogen Peroxide; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D008099:Liver / Q000201:enzymology*; D013379:Substrate Specificity; D014413:Tuna; D020122:tert-Butylhydroperoxide / Q000378:metabolism
D001555:Benzene Derivatives; D020122:tert-Butylhydroperoxide; D006861:Hydrogen Peroxide; D005979:Glutathione Peroxidase; C007164:cumene hydroperoxide