Formation of the intrachain disulfide bond in the constant fragment of the immunoglobulin light chain

Abstract

Regeneration by glutathione of the constant fragment of the immunoglobulin light chain was studied in the absence and presence of 8 m-urea. The species that appeared during the reaction of the reduced constant fragment with oxidized glutathione were trapped by alkylation with iodoacetamide and identified by electrophoresis in 15% polyacrylamide gel at pH 9.5. The kinetics of the reactions starting from various species formed during the reaction of the reduced constant fragment were also studied, and the overall reaction kinetics of the formation of the intrachain disulfide bond in the constant fragment were established in the absence and presence of urea. The reaction of the reduced constant fragment with oxidized glutathione was much slower but the yield of the constant fragment with the disulfide bond was much higher in the absence than in the presence of 8 m-urea. The slowness of the reaction in the absence of urea is due to the two cysteinyl residues of the reduced constant fragment being buried in the interior of the molecule and because oxidized glutathione is capable of reacting with the thiols only in the opened form of the protein molecule. The high yield is due to the cysteinyl thiol and the mixed disulfide in the intermediate forming an intrachain disulfide bond through thiol-disulfide interchange, the reaction sites being exposed to solvent and located at the appropriate proximity. These findings indicate first, that the appropriate proximity of a pair of cysteinyl residues is essential to form a disulfide bond and second, that they are not easily oxidized to disulfide if they are buried in the interior of the protein molecule.