CHARACTERIZATION OF MAMMALIAN THIOREDOXIN REDUCTASE, THIOREDOXIN AND GLUTAREDOXIN BY IMMUNOCHEMICAL METHODS

Specific polyclonal antibodies towards the oxidized form of bovine thi oredoxin reductase (TR) have been obtained in rabbits, and purified. T he antigenicity was lost upon reduction of TR by NADPH indicating a la rge conformational change upon reduction of the redox-active disulfide in the enzyme, The antibodies did not cross-react with other bovine N ADPH-dependent dehydrogenases. No reactivity was observed with TR from bacteria, yeast or rat and only a slight reaction was obtained with T R from horse, Immunoaffinity purified anti-thioredoxin and anti-glutar edoxin antibodies were used to develop competitive indirect ELISA assa ys that were validated giving very good linearity, reproducibility, se nsitivity and parallelism, The glutaredoxin (Grx) immunoassay is the f irst quantitative method described to measure the protein. When applie d to a battery of calf tissues the contents of Grx varied from 7 to 12 0 mu g per gram of fresh tissue. Skeletal and heart muscles gave the l owest values and spleen and salivary glands the highest. However, skel etal muscle showed the highest gluthathione-hydroxyethyl disulfide oxi doreductase specific activity.