Vol. 107, No. 4, 1982 August 31, 1982 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1475-1481 PHOTO-LABELLING OF GLUTAREDOXIN FROM ESCHERICHIA COLI Jan-Olav H~g 1, Kenneth T. Douglas 2' , Claudius D'Silva 2 1 and Arne Holmgren iDepartment of Chemistry, Karolinska Institutet, S-IO40l Stockholm, Sweden 2Department of Chemistry, University of Essex, Colchester, Essex CO4 3SQ, U.K. Received June 21, 1982 Summary: N,N'-bis (4-azidobenzoyl) glutathione disulphide (!) was a strong inhibitor of glutaredoxin in the dark. The presence of 1 caused the velocity versus glutathione concentration curves of the glutaredoxin activity as assayed by E. coli ribonucleotide reductase to change from hyperbolic (in its absence) to sigmoidal. Under conditions in which glutaredoxin itself was photostable co-irradiation of glutaredoxin and 1 led to 32.7~2.5% covalent inhibition of glutaredoxin, which was not protected against by the presence of glutathione disulphide. Thus, !, is a photo-label of E. coli glutaredoxin. For the reduction of ribonucleotides to the corresponding deoxyribonucleotides, ribonucleotide reductase requires a hydrogen donor (1,2). This function can be fulfilled not only by thioredoxin, the first-discovered dithiol protein hyodrogen-donor, but also by glutaredoxin, which couples the oxidation of reduced glutathione (GSH) to the reduction of the ribonucleotide (3). Glutaredoxin, a small protein, enablesmonothiolic GSH to act as an hydrogen donor for ribonucleotide reductase by a combination of reactions (2). glutaredoxin/ ribonucleotide 2GSH + rNDP redUctase > GSSG +dNDP +H20 GSSG + NADPH+ H + qlutathione -reductase ) 2GSH + NADP + Person to whom reprint requests should be addressed at the University of Essex. 1475 0006-291X/82/161475-07501.00/0 Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.