Protein folding occurs simultaneously with disulfide bond formation. In general, the in vitro folding of proteins containing disulfide bond(s) is carried out in the presence of redox reagents, such as glutathione, to permit native disulfide pairing to occur. It is well known that the formation of a disulfide bond and the correct tertiary structure of a target protein are strongly affected by the redox reagent used. However, little is known concerning the role of each amino acid residue of the redox reagent, such as glutathione. Therefore, we prepared glutathione derivatives - glutamyl-cysteinyl-arginine (ECR) and arginyl-cysteinyl-glycine (RCG) - and examined their ability to facilitate protein folding using lysozyme and prouroguanylin as model proteins. When the reduced and oxidized forms of RCG were used, folding recovery was greater than that for a typical glutathione redox system. This was particularly true when high protein concentrations were employed, whereas folding recovery using ECR was similar to that of the glutathione redox system. Kinetic analyses of the oxidative folding of prouroguanylin revealed that the folding velocity (KRCG = 3.69 Ã - 10-3 s-1) using reduced RCG/oxidized RCG was approximately threefold higher than that using reduced glutathione/oxidized glutathione. In addition, folding experiments using only the oxidized form of RCG or glutathione indicated that prouroguanylin was converted to the native conformation more efficiently in the case of RCG, compared with glutathione. The findings indicate that a positively charged redox molecule is preferred to accelerate disulfide-exchange reactions and that the RCG system is effective in mediating the formation of native disulfide bonds in proteins.