Thioredoxin-catalyzed refolding of disulfide-containing proteins.
Thioredoxin, a known catalyst for reducing protein disulfides, was shown to catalyze efficiently the refolding of pancreatic RNase either from the reduced, denatured form or from the scrambled form containing oxidized but incorrectly paired disulfides. Thioredoxin was 1000-fold more efficient on a molar basis than the model dithiol, dithiothreitol, in reactivating reduced, denatured RNase, suggesting that thioredoxin acts as an efficient catalyst for disulfide interchange. Starting with reduced, denatured RNase, enzyme activity was recovered quantitatively with a t1/2 of 30 hr with 100 microM thioredoxin compared to only a 10-20% recovery of activity in the control using air oxidation. Oxygen further stimulated the effectiveness of thioredoxin severalfold. Thioredoxin was most effective in reactivating inactive scrambled RNase, which contained mispaired disulfides, showing a t1/2 of 2 hr. Reduced thioredoxin was optimal for catalyzing disulfide interchange in scrambled RNase, whereas oxidized thioredoxin was required for reactivation of the reduced, denatured species. Optimal reactivation of scrambled RNase required a mixture of reduced and oxidized thioredoxin. Addition of reduced thioredoxin after initiating refolding of reduced denatured RNase with oxidized glutathione effected a rapid reactivation of RNase, suggesting a two-step model for protein refolding in which the monothiol catalyzes the rapid initial formation of protein disulfides and thioredoxin catalyzes the second step of disulfide interchange. Arguments are presented suggesting that thioredoxin may serve an in vivo role analogous to the protein disulfide-isomerase (EC 5.3.4.1).
Pigiet
V P
VP
Schuster
B J
BJ
D001426:Bacterial Proteins / Q000494:pharmacology*; D004220:Disulfides / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007535:Isomerases / Q000494:pharmacology; D010084:Oxidation-Reduction; D010100:Oxygen / Q000494:pharmacology; D011489:Protein Denaturation; D019704:Protein Disulfide-Isomerases; D011506:Proteins / Q000378:metabolism*; D012260:Ribonucleases; D013879:Thioredoxins / Q000494:pharmacology*
D001426:Bacterial Proteins; D004220:Disulfides; D011506:Proteins; D013879:Thioredoxins; D012260:Ribonucleases; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D010100:Oxygen; D004229:Dithiothreitol