Pressure-induced subunit dissociation and unfolding of dimeric beta-lactoglobulin

Effects of hydrostatic pressure on dimeric beta-lactoglobulin A (beta-Lg) were investigated. Application of pressures of up to 3.5 kbar induced a significant red shift ( approximately 11 nm) and a 60% increase in intrinsic fluorescence emission of beta-Lg. These changes were very similar to those induced by guanidine hydrochloride, which caused subunit dissociation and unfolding of beta-Lg. A large hysteresis in the recovery of fluorescence parameters was observed upon decompression of beta-Lg. Pressure-induced dissociation and unfolding were not fully reversible, because of the formation of a nonnative intersubunit disulfide bond that hampered correct refolding of the dimer. Comparison between pressure dissociation/unfolding at 3 degrees C and 23 degrees C revealed a marked destabilization of beta-Lg at low temperature. The stability of beta-Lg toward pressure was significantly enhanced by 1 M NaCl, but not by glycerol (up to 20% v/v). These observations suggest that salt stabilization was not related to a general cosolvent effect, but may reflect charge screening. Interestingly, pressure-induced dissociation/unfolding was completely independent of beta-Lg concentration, in apparent violation of the law of mass action. Possible causes for this anomalous behavior are discussed.