title: Pressure-induced subunit dissociation and unfolding of dimeric beta-lactoglobulin.
abstract: 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.
authors.affiliation: Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-590, Brazil.
authors.forename: V L
authors.initials: VL
authors.lastname: Valente-Mesquita
authors.forename: M M
authors.initials: MM
authors.lastname: Botelho
authors.forename: S T
authors.initials: ST
authors.lastname: Ferreira
mesh_terms: D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002417:Cattle; D019281:Dimerization; D004355:Drug Stability; D019791:Guanidine; D006874:Hydrostatic Pressure; D066298:In Vitro Techniques; D007782:Lactoglobulins / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013696:Temperature
chemical_list: D007782:Lactoglobulins; D019791:Guanidine