The protein bCblC (bCblCpro) is a bovine homolog of a human B12 trafficking chaperone that is responsible for the processing of vitamin B12 and its escorted delivery in intracellular B12 metabolism. In this study, we found that bCblCpro is highly thermolabile with a Tm = 42.0 ± 0.2 °C as shown for the human homolog, suggesting thermal regulation of these proteins. Binding of the reduced form of glutathione (GSH) that is a predominant cellular thiol increased the Tm of bCblCpro from 42 °C to ~45 °C (ΔTm max = 3.1 ± 0.2 °C and AC50 = 2.1 ± 0.5 mM). Binding of vitamin B12 and its derivatives also stabilized bCblCpro increasing the Tm to a different extent and vitamin B12 (cyanocobalamin, CNCbl) was the least efficient (ΔTm max = 4.3 ± 0.3 °C and AC50 = 291 ± 36 μM). However, the stabilizing effect of CNCbl was significantly greater for GSH-bound bCblCpro (ΔTm max = 12.8 ± 0.6 °C and AC50 = 9.3 ± 1.6 μM) than for GSH-free bCblCpro. In addition, the stabilizing effect of GSH was also greater for CNCbl-bound bCblCpro (ΔTm max = 9.3 ± 0.3 °C and AC50 = 57.0 ± 6.8 μM). Limited proteolysis revealed that thermal stabilization of bCblCpro is derived from conformational changes of the protein induced by binding of the ligands. The results in this study indicate that GSH cooperates with vitamin B12 in thermal stabilization of bCblCpro and is a positive regulator of the protein.
