~ Pergamon 0305-0491(94)00235-5 Comp. Biochem. PhysioL Vol. IIIB, No. 1, pp. 17-25, 1995 Copyright © 1995 ElsevierScience Ltd Printed in Great Britain. All rights reserved 0305-0491/95 $9.50 + 0.00 Characterization of mammalian thioredoxin reductase, thioredoxin and glutaredoxin by immunochemical methods Emilia Martinez-Galisteo,* C. Alicia Padilla,* Arne Holmgren' and J. Antonio Bfircena* *Department of Biochemistry and Molecular Biology, Faculty of Veterinary, University of C6rdoba, 14071-C6rdoba, Spain; and ~'Medical Nobel Institute for Biochemistry, Karolinska Institute, Stockholm S- 17177, Sweden Specific polyclonai antibodies towards the oxidized form of bovine thioredoxin reduetase (TR) have been obtained in rabbits, and purified. The antigenicity was lost upon reduction of TR by NADPH indicating a large conformational change upon reduction of the redox-active disulfide in the enzyme. The antibodies did not cross-react with other bovine NADPH-dependent dehydrogenases. No reactivity was observed with TR from bacteria, yeast or rat and only a slight reaction was obtained with TR from horse. Immunoaifinity purified anti-thioredoxin and anti-qglutaredoxin antibodies were used to develop competitive indirect ELISA assays that were validated giving very good linearity, reproducibility, sensitivity and parallelism. The glutaredoxin (Grx) immunoassay is the first quantitative method described to measure the protein. When applied to a battery of calf tissues the contents of Grx varied from 7 to 120/~g per gram of fresh tissue. Skeletal and heart muscles gave the lowest values and spleen and salivary glands the highest. However, skeletal muscle showed the highest gluthathione-hydroxyethyl disulfide oxidoreductase specific activity. Key words: ELISA; Immunoassay; Thiol transferase; Redox-active disulfides; Flavoenzymes; Quantitative immtlnochemistry; Calf tissues; Glutathione-disulfide oxidoreductase. Comp. Biochem. Physiol. 111B, 17-25, 1995. Introduction Thioredoxin (Trx) and glutaredoxin (Grx) are two small proteins (MW 11,000-13,000) present in most living cells and capable of catalyzing thiol-disulfide oxido-reductions (Holmgren, 1985a, 1989). Their most apparent activity is to mediate the transfer of electrons from NADPH to disulfide substrates of very diverse nature. To achieve this function, each protein works in association with a flavoenzyme: thioredoxin re- Correspondence to: E. Martinez-Galisteo, Dept. of Bio- chemistry and Molecular Biology, Faculty of Veterinary, University of C6rdoba, 14071-C6rdoba, Spain. Received 18 November 1993; revised 18 October 1994; accepted 26 October 1994. Abbreviations: DTNB, 5,5'-dithiobis(2-nitrobenzoate); Grx, glutaredoxin; HED, hydroxyethyl disulfide; PMSF, phenyl-methyl sulphonyl fluoride; Trx, thioredoxin; TR, thioredoxin reductase. ductase (TR) for thioredoxin and glutathione reductase (GR) for glutaredoxin. In some pro- cesses, the glutaredoxin system is an alternative when the thioredoxin system is absent (Holm- gren, 1976), but their simultaneous presence in many cells suggests different functions for both proteins (Rozell et al., 1993). Although the structures of both Trx and Grx have been conserved during evol{ltion, no unique biological role has been found for any of them (Holmgren, 1989). On the contrary, a surprising variety of functions has been de- scribed and proposed for Trx and also for Grx, although the number of studies on Grx is still much lower. It has been pointed out that this may be a reflection of the general importance of thiol groups and disulfides in biochemical mechanisms and cellular regulation plus the 17