Purification, cloning and expression of dehydroascorbic acid-reducing activity from human neutrophils: identification as glutaredoxin

Abstract

Dehydroascorbic acid-reducing activity in normal human neutrophil lysates was characterized and identified by activity-based purification and measurement of newly synthesized ascorbate by HPLC. The initial reducing activity was non-dialysable and could not be accounted for by the activity of glutathione as a reducing agent. The reducing activity was purified to homogeneity as an 11 kDa protein. The protein had a specific activity of 3 μmol/min per mg of protein and was glutathione dependent. Kinetic experiments showed that the protein had a K_m for glutathione of 2.0 mM and a K_m for dehydroascorbic acid of 250 μM. Dehydroascorbic acid reduction by the purified protein was pH dependent and was maximal at pH 7.5. Peptide fragments from the purified protein were analysed for amino acid sequence and the protein was identified as glutaredoxin. By using degenerate oligonucleotides based on the amino acid sequence, glutaredoxin was cloned from a human neutrophil library. Expressed purified glutaredoxin displayed reducing activity and kinetics that were indistinguishable from those of native purified enzyme. Several approaches indicated that glutaredoxin was responsible for the most of the protein-mediated dehydroascorbic acid reduction in lysates. From protein purification data, glutaredoxin was responsible for at least 47% of the initial reducing activity. Dehydroascorbic acid reduction was at least 5-fold greater in neutrophil lysates than in myeloid tumour cell lysates, and glutaredoxin was detected in normal neutrophil lysates but not in myeloid tumour cell lysates by Western blotting. Glutaredoxin inhibitors inhibited dehydroascorbic acid reduction in neutrophil lysates as much as 80%. These findings indicate that glutaredoxin plays a major role in dehydroascorbic acid reduction in normal human neutrophil lysates, and represent the first identification of dehydroascorbic acid reductase in human tissue by activity-based purification.