title: Bovine β-lactoglobulin is dimeric under imitative physiological conditions: dissociation equilibrium and rate constants over the pH range of 2.5-7.5.
abstract: The oligomerization of β-lactoglobulin (βLg) has been studied extensively, but with somewhat contradictory results. Using analytical ultracentrifugation in both sedimentation equilibrium and sedimentation velocity modes, we studied the oligomerization of βLg variants A and B over a pH range of 2.5-7.5 in 100 mM NaCl at 25°C. For the first time, to our knowledge, we were able to estimate rate constants (k(off)) for βLg dimer dissociation. At pH 2.5 k(off) is low (0.008 and 0.009 s(-1)), but at higher pH (6.5 and 7.5) k(off) is considerably greater (>0.1 s(-1)). We analyzed the sedimentation velocity data using the van Holde-Weischet method, and the results were consistent with a monomer-dimer reversible self-association at pH 2.5, 3.5, 6.5, and 7.5. Dimer dissociation constants K(D)(2-1) fell close to or within the protein concentration range of ∼5 to ∼45 μM, and at ∼45 μM the dimer predominated. No species larger than the dimer could be detected. The K(D)(2-1) increased as |pH-pI| increased, indicating that the hydrophobic effect is the major factor stabilizing the dimer, and suggesting that, especially at low pH, electrostatic repulsion destabilizes the dimer. Therefore, through Poisson-Boltzmann calculations, we determined the electrostatic dimerization energy and the ionic charge distribution as a function of ionic strength at pH above (pH 7.5) and below (pH 2.5) the isoelectric point (pI∼5.3). We propose a mechanism for dimer stabilization whereby the added ionic species screen and neutralize charges in the vicinity of the dimer interface. The electrostatic forces of the ion cloud surrounding βLg play a key role in the thermodynamics and kinetics of dimer association/dissociation.
authors.affiliation: Riddet Institute, Massey University, Palmerston North, New Zealand.
authors.forename: Davide
authors.initials: D
authors.lastname: Mercadante
authors.forename: Laurence D
authors.initials: LD
authors.lastname: Melton
authors.forename: Gillian E
authors.initials: GE
authors.lastname: Norris
authors.forename: Trevor S
authors.initials: TS
authors.lastname: Loo
authors.forename: Martin A K
authors.initials: MA
authors.lastname: Williams
authors.forename: Renwick C J
authors.initials: RC
authors.lastname: Dobson
authors.forename: Geoffrey B
authors.initials: GB
authors.lastname: Jameson
mesh_terms: D000818:Animals; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007782:Lactoglobulins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008970:Molecular Weight; D011485:Protein Binding; D055503:Protein Multimerization; D055550:Protein Stability; D055672:Static Electricity; D013816:Thermodynamics; D014461:Ultracentrifugation
chemical_list: D007782:Lactoglobulins