Although stability is critical for in vivo application of immunotoxins, a thermodynamic description of their folding/stability is still lacking. We applied differential scanning calorimetry (DSC) to RNase-based immunofusion comprising barnase, cytotoxic RNase from Bacillus amyloliquefaciens, fused to the light chain variable domain (VL) of anti-human ferritin antibody F11. By analyzing DSC curves recorded with or without preheating and addition of the barnase-stabilizing ligand guanosine 3′-monophosphate, we (i) assigned two well-resolved thermal transitions to the VL and barnase modules of VL-barnase, (ii) demonstrated independent folding of these two modules, and (iii) showed altered stability of the barnase module, which resulted from the dimeric state of VL-barnase.
