Bovine β-lactoglobulin is dimeric under imitative physiological conditions: dissociation equilibrium and rate constants over the pH range of 2.5-7.5

Biophys J. 2012 Jul 18;103(2):303-12. doi: 10.1016/j.bpj.2012.05.041. Epub 2012 Jul 17.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Cattle
  • Hydrogen-Ion Concentration
  • Kinetics
  • Lactoglobulins / chemistry*
  • Lactoglobulins / metabolism*
  • Models, Molecular
  • Molecular Weight
  • Protein Binding
  • Protein Multimerization*
  • Protein Stability
  • Static Electricity
  • Thermodynamics
  • Ultracentrifugation


  • Lactoglobulins