Thioredoxin-catalyzed refolding of disulfide-containing proteins.

Abstract

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 til2 of 30 hr with 100 ,AM 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 til2 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).Thioredoxin(s) have been implicated in a variety of physiological roles, including serving as the hydrogen donor for the reduction of ribonucleotides and sulfoxides (1,2) and for the activation of enzymes (3) and serving as a co-factor for T7 phage DNA polymerase (4) and assembly of filamentous phage (5

Arabidopsis Cotyledon-Specific Chloroplast Biogenesis Factor CYO1 Is a Protein Disulfide Isomerase

“…We measured the reduction of insulin (130 mM Renaturation of reduced, denatured RNase A, which contains eight sulfhydryl groups, involves the oxidation of its thiol groups followed by rearrangement of the disulfides to the native conformation (with four disulfide bridges) (Anfinsen and Scheraga, 1975). As reported previously (Pigiet and Schuster, 1986), the spontaneous refolding of reduced, denatured RNase A was negligible in air ( Figure 8B). Addition of D 30 CYO1 or DnaJ to the reaction stimulated RNase A renaturation.…”

“…Reduced RNase A (Sigma-Aldrich) was prepared as described previously (Pigiet and Schuster, 1986). PDI activity was determined by measurement of the reactivation of reduced RNase A (Hasegawa et al, 2003).…”

Controlling the speed of hirudin folding

“…On the other hand, the folding mechanism of hirudin is essentially accordant with that of ribonuclease (Hantgan et al, 1974;Pigiet and Schuster, 1986). Both engage an exceedingly large number of folding intermediates (Scheraga et al, 1984;Rothwarf and Scheraga, 1993;Chatrenet and Chang, 1993) and in both cases disulphide formation occurred much more rapidly than the recovery of their biological activity (Anfinsen et al, 1961;Chatrenet and Chang, 1992).…”

“…There are few proteins, except for bovine pancreatic trypsin inhibitor (BPTI) (Creighton, 1978;Weissman and Kim, 1991) and ribonuclease (Hantgan et al, 1974;Scheraga et al, 1984;Pigiet and Schuster, 1986), which have had their folding mechanisms investigated in great detail at the molecular level. Of those which were set to refold in the presence of GSH/GSSG or PDI (Saxena and Wetlaufer, 1970;Pigiet and Schuster, 1986;Lyles and Gilbert, 1991), none has claimed to achieve efficiency comparable with those achieved in the cell. With only limited examples, it is premature to make any generalization.…”

Isolation of a thermostable enzyme catalyzing disulfide bond formation from the archaebacterium Sulfolobus solfataricus

“…It is a multifunctional protein which catalyzes disulfide reduction and participates in other redox processes. Pigiet and Schuster [3] demonstrated that TH can reactivate both Abbreviurions: EDTA, ethylene diamine tetraacetic acid; DTNB, 5,5'-dithio-his-nitrohenzoic acid; DTT, dithiothreitoi; SDS-PAGE, sodium dodecyl sulfate-polyacrylamidc gel electrophoresis.…”