Electrostatically Driven Protein Aggregation:  β-Lactoglobulin at Low Ionic Strength

Abstract

The aggregation of beta-lactoglobulin (BLG) at ambient temperature was studied using turbidimetry and dynamic light scattering in the range 3.8<pH<5.2 in 0.0045 M NaCl, and in the ionic strength range 0.0045-0.5 M at fixed pH=5.0. The initial rate of aggregation, taken as the initial slope of turbidity vs time, (dtau/dt)0, indicated maximum aggregation near pH 4.6 (below the isoelectric point of 5.2), but the dependence of the initial rate of aggregation on pH was highly asymmetric. At pH 5.0, (dtau/dt)0 strongly increased with a decrease in ionic strength I from 0.1 to 0.0045 M and was found to be nearly linear with 1/I. DLS measurements revealed an increase in particle size with time, with the appearance of bimodal distributions in which the fast and slow modes corresponded, respectively, to a BLG dimer and to larger aggregates in the 100-800 nm range. At conditions of slower aggregation, DLS revealed the consumption of dimers to form higher order aggregates with no intermediate species. Computer modeling (Delphi) was used to visualize the electrostatic potential around the dimer to elucidate the pH and ionic strength dependence of the initial aggregation rates. The aggregation process appears to comprise an initial fast consumption of the dimer, whose dependence on pH and I arises from the interaction of the positive and negative domains of interacting dimers, followed by the slow formation of much larger aggregates with relatively little sensitivity to pH and I. The open-ended nature of BLG aggregation is thought to arise from the asymmetry of the dimer charge distribution.

The effect of charge mutations on the stability and aggregation of a human single chain Fv fragment

“…This behaviour is consistent with the increase in the k D value for 5E when adding sodium chloride. Previous studies have found a strong correlation between increased aggregation rates and electrostatic selfassociation [13,55,80,99], which might also explain the increased aggregation of 5E.…”

“…For 5E, the salt-induced increase in k D reflects the presence of an electrostatic attraction between proteins [48,55,71]. This behaviour occurs when proteins have near net neutral charge and large anisotropic charge distributions, which likely arises in 5E, from engineering the negative charge patch on a positively charged scaffold.…”

“…However, care must be taken when engineering in charged mutations. If the protein net charge is close to zero, an anisotropic charge distribution can cause protein self association [13,14,71,99], which has been correlated with increased aggregation propensity [55,80]. To avoid increasing protein charge anisotropy, charged mutations should carry the same sign as the net charge on the corresponding protein scaffold [31,100].…”

Suppression of Insulin Aggregation by Heparin

“…The mechanisms of aggregation are very different, with Zn-free insulin at nonextreme pH conditions forming predominantly dimers, tetramers, hexamers, and higher order multimers, 59 while BLG forms dimers in equilibrium with much larger aggregates. 49 The possibility that these differences arise from the higher degree of charge asymmetry for BLG at pH near pI (a distinct positive domain coupled with a large and more diffuse negative domain, which leads to a tendency for highly nonlinear aggregation 49 ) is certainly a hypothesis worth further examination.…”

Accurate determination of key surface properties that determine the efficient separation of bovine milk BSA and LF proteins

“…Although increases in the protein concentration commonly result in an increase in protein aggregation [7,27], some authors consider the opposite behavior due to the repulsion-attraction forces. The increase in concentration reduces the attraction forces, resulting in decreased protein-protein interactions and therefore the formation of aggregates [30].…”