The aggregation of β-lactoglobulin (BLG) near its isoelectric point was studied as a function of ionic strength and pH. We compared the behavior of native BLG with those of its two isoforms, BLG-A and BLG-B, and with that of a protein with a very similar pI, bovine serum albumin (BSA). Rates of aggregation were obtained through a highly precise and convenient pH/turbidimetric titration that measures transmittance to ±0.05 %T. A comparison of BLG and BSA suggests that the difference between pHmax (the pH of the maximum aggregation rate) and pI is systematically related to the nature of protein charge asymmetry, as further supported by the effect of localized charge density on the dramatically different aggregation rates of the two BLG isoforms. Kinetic measurements including very short time periods show well-differentiated first and second steps. BLG was analyzed by light scattering under conditions corresponding to maxima in the first and second steps. Dynamic light scattering (DLS) was used to monitor the kinetics, and static light scattering (SLS) was used to evaluate the aggregate structure fractal dimensions at different quench points. The rate of the first step is relatively symmetrical around pHmax and is attributed to the local charges within the negative domain of the free protein. In contrast, the remarkably linear pH dependence of the second step is related to the uniform reduction in global protein charge with increasing pH below pI, accompanied by an attractive force due to surface charge fluctuations.
Protein native state aggregation, a major problem in pharmaceutical and biological processes, has been addressed pharmacologically by the addition of protein-binding excipients. Heparin (Hp), a highly sulfated polysaccharide, interacts with numerous proteins with moderate to high affinity, but reports about its effect on protein aggregation are contradictory. We studied the pH dependence of the aggregation of antithrombin (AT) and bovine serum albumin (BSA) in the presence and absence of heparin. High-precision turbidimetry showed strong aggregation for both AT and BSA in I = 10 mM NaCl, conditions at which electrostatically driven Hp binding and aggregation both occur, with more obvious aggregation of heparin-free AT appearing as larger aggregate size. Aggregation of AT was dramatically inhibited at Hp: protein 6:1 (mole ratio); however, the effect at 0.5:1 Hp:protein was greater for BSA. Frontal analysis capillary electrophoresis showed a much larger equilibrium association constant Kobs between Hp and AT, in accord with the onset of Hp binding at a higher pH; both effects are explained by the higher charge density of the positive domain for AT as revealed by modeling with DelPhi. The corresponding modeling images showed that these domains persist at high salt only for AT, consistent with the 160-fold drop in Kobs at 100 mM salt for BSA-Hp binding. The smaller inhibition effect for AT arises from the tendency of its uncomplexed monomer to form larger aggregates more rapidly, but the stronger binding of Hp to AT does not facilitate Hp-induced aggregate dissolution which occurs more readily for BSA. This can be attributed to the higher density of AT aggregates evidenced by higher fractal dimensions. Differences between inhibition and reversal by Hp arise because the former may depend on the stage at which Hp enters the aggregation process and the latter on aggregate size and morphology.
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