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.

Kinetics of Protein Complex Dissociation Studied by Hydrogen/Deuterium Exchange and Mass Spectrometry

“…This value is close to the value of 0.007 s −1 that was obtained previously by analytical ultracentrifugation (AUC). 28 Our value is slightly higher than the previously reported value, but this reflects the lower ionic strength used in our experiments. Under low pH conditions, the βLG dimer is well known to be destabilized and to dissociate more rapidly at low ionic strengths.…”

“…Here, we describe our initial work using the β-lactoglobulin (βLG) dimer as a well-studied model system. 28–30 We perform the deuterium labeling reaction as the dimer dissociates into monomer. The resulting exchange patterns for specific protein regions are then measured and fit to a kinetic model to obtain rate information.…”

Characterising the secondary structure changes occurring in high density systems of BLG dissolved in aqueous pH 3 buffer

“…Studies have found that BLG at 100 mM is dimeric at 25 C over a pH range of 2.5e7.5. Estimations of the dimer dissociation rate constants (k off ) at pH 2.5 were low (0.008 and 0.009 s À1 ) but considerably higher at pH (6.5 and 7.5) >0.1 s À1 (Mercadante et al, 2012). Furthermore, dimer dissociation constants (K D ) fell close to or within the protein concentration range of~5e~45 mM, whereas at 45 mM the dimer predominated (Mercadante et al, 2012).…”

The complex of hypericin with β-lactoglobulin has antimicrobial activity with potential applications in dairy industry

“…β-Lactoglobulin forms dimers (2β-LG) under conditions close to those encountered physiologically, with a dimerization constant of K D = 8.6 µM at pH 7.5 (Mercadante et al, 2012). β-Lactoglobulin is shaped to accommodate small hydrophobic compounds, such as retinol and the aliphatic chains of FA, in the lipocalintype β-barrel.…”

“…β-Lactoglobulin is a small, homodimeric protein of 162 amino acids (~18,400 Da) belonging to the lipocalin family (Åkerström et al, 2006;Mercadante et al, 2012). Owing to their ability to bind small hydrophobic molecules, while simultaneously being recognized by cell surface receptors, lipocalins were suggested as potential systems for drug delivery (Åkerström et al, 2006).…”