The protein, bCblCpro, is a bovine B-12 trafficking chaperone involved in intracellular B-12 metabolism. bCblCpro is highly thermolabile (T-m = similar to 42 degrees C) and the reduced form of glutathione, GSH, has been found to stabilize bCblCpro as a positive regulator. In this study, we discovered that the oxidized form of glutathione, GSSG, destabilizes bCblCpro, which is derived from changes in the conformation of the protein upon GSSG binding. The binding affinity for GSSG was determined to be similar with the affinity for GSH. The AC(50) = 2.8 +/- 0.4 mM of GSSG for destabilization of bCblCpro was consistently similar with the AC(50) = 2.1 +/- 0.5 mM of GSH for stabilization of the protein. These results suggest that GSSG is a negative regulator of bCblCpro and that the molar ratio of[GSH]/[GSSG] in cells may determine the stability of the B-12 trafficking chaperone. (C) 2012 Elsevier Inc. All rights reserved.