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Bovine ?-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 ?-lactoglobulin (?Lg) 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 ?Lg 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 ?Lg 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 ?M, and at ?45 ?M 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 ?Lg play a key role in the thermodynamics and kinetics of dimer association/dissociation.

SUBMITTER: Mercadante D 

PROVIDER: S-EPMC3400788 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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

Mercadante Davide D   Melton Laurence D LD   Norris Gillian E GE   Loo Trevor S TS   Williams Martin A K MA   Dobson Renwick C J RC   Jameson Geoffrey B GB  

Biophysical journal 20120717 2


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 hi  ...[more]

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2006-03-08 | GSE4370 | GEO