Protein-Selective Coacervation with Hyaluronic Acid

Selective coacervation with hyaluronic acid (HA), a biocompatible and injectable anionic polysaccharide, was used to isolate a target protein, bovine serum albumin (BSA), with 90% purity from a 1:1 mixture with a second protein of similar pI, β-lactoglobulin (BLG). This separation was attributed to the higher HA-affinity of BSA, arising from its more concentrated positive domain. The values of pH corresponding respectively to the onset of complex formation, coacervation, precipitation, and redissolution (pH_c, pH_ϕ, pH_p, and pH_d) were determined as a function of ionic strength <i>I</i>. These pH values were related to critical values of protein charge, <i>Z</i>, and their dependence on <i>I</i> provided some insights into the mechanisms of these transitions. The higher polyanion binding affinity of BSA, deduced from its higher values of pH_c, was confirmed by isothermal titration calorimetry (ITC). Confocal laser microscopy clearly showed time-dependent coalescence of vesicular droplets into a continuous film. Comparisons with prior results for the polycation poly­(diallyldimethylammonium chloride) (PDADMAC) show reversal of protein selectivity due to reversal of the polyelectrolyte charge. Stronger binding of both proteins to PDADMAC established by ITC may be related to the higher chain flexibility and effective linear charge density of this polycation

Multimerization and Aggregation of Native-State Insulin: Effect of Zinc

The aggregation of insulin is complicated by the coexistence of various multimers, especially in the presence of Zn²⁺. Most investigations of insulin multimerization tend to overlook aggregation kinetics, while studies of insulin aggregation generally pay little attention to multimerization. A clear understanding of the starting multimer state of insulin is necessary for the elucidation of its aggregation mechanism. In this work, the native-state aggregation of insulin as either the Zn–insulin hexamer or the Zn-free dimer was studied by turbidimetry and dynamic light scattering, at low ionic strength and pH near pI. The two states were achieved by varying the Zn²⁺ content of insulin at low concentrations, in accordance with size-exclusion chromatography results and literature findings (Tantipolphan, R.; Romeijn, S.; Engelsman, J. d.; Torosantucci, R.; Rasmussen, T.; Jiskoot, W. J. Pharm. Biomed. 2010, 52, 195). The much greater aggregation rate and limiting turbidity (τ_∞) for the Zn–insulin hexamer relative to the Zn-free dimer was explained by their different aggregation mechanisms. Sequential first-order kinetic regimes and the concentration dependence of τ_∞ for the Zn–insulin hexamer indicate a nucleation and growth mechanism, as proposed by Wang and Kurganov (Wang, K.; Kurganov, B. I. Biophys. Chem. 2003, 106, 97). The pure second-order process for the Zn-free dimer suggests isodesmic aggregation, consistent with the literature. The aggregation behavior at an intermediate Zn²⁺ concentration appears to be the sum of the two processes

pH-Dependent Aggregation and Disaggregation of Native β‑Lactoglobulin in Low Salt

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 pH_max (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 pH_max 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

Effect of Heparin on Protein Aggregation: Inhibition versus Promotion

The effect of heparin on both native and denatured protein aggregation was investigated by turbidimetry and dynamic light scattering (DLS). Turbidimetric data show that heparin is capable of inhibiting and reversing the native aggregation of bovine serum albumin (BSA), β-lactoglobulin (BLG), and Zn–insulin at a pH near pI and at low ionic strength <i>I</i>; however, the results vary with regard to the range of pH, <i>I</i>, and protein–heparin stoichiometry required to achieve these effects. The kinetics of this process were studied to determine the mechanism by which interaction with heparin could result in inhibition or reversal of native protein aggregates. For each protein, the binding of heparin to distinctive intermediate aggregates formed at different times in the aggregation process dictates the outcome of complexation. This differential binding was explained by changes in the affinity of a given protein for heparin, partly due to the effects of protein charge anisotropy as visualized by electrostatic modeling. The heparin effect can be further extended to include inhibition of denaturing protein aggregation, as seen from the kinetics of BLG aggregation under conditions of thermally induced unfolding with and without heparin

Precipitate–Coacervate Transformation in Polyelectrolyte–Mixed Micelle Systems

The polycation/anionic-nonionic mixed micelle, poly­(diallyldimethylammonium chloride)-sodium dodecyl sulfate/Triton X-100 (PDADMAC-SDS/TX100), is a model polyelectrolyte-colloid system in that the micellar mole fraction of SDS (<i>Y</i>) controls the micelle surface charge density, thus modulating the polyelectrolyte-colloid interaction. The exquisite temperature dependence of this system provides an important additional variable, controlling both liquid–liquid (L–L) and liquid–solid (L–S) phase separation, both of which are driven by the entropy of small ion release. In order to elucidate these transitions, we applied high-precision turbidimetry (±0.1 %), isothermal titration calorimetry, and epifluorescence microscopy which demonstrates preservation of micelle structure under all conditions. The L–S region at large <i>Y</i> including precipitation displays a remarkable linear, inverse <i>Y-</i>dependence of the L–S transition temperature <i>T</i>_s. In sharp contrast, the critical temperature for L–L coacervation <i>T</i>_φ, shows nearly symmetrical effects of positive and negative deviations in <i>Y</i> from the point of soluble complex neutrality, which is controlled in solution by the micelle charge and the number of micelles bound per polymer chain <i>n</i> (<i>Z</i>_complex = <i>Z</i>_polymer + <i>nZ</i>_micelle). In solid-like states, <i>n</i> no longer signifies the number of micelles bound per polymer chain, since the proximity of micelles inverts the host–guest relationship with each micelle binding multiple PE chains. This intimate binding goes hand-in-hand with the entropy of release of micelle-localized charge-compensating ions whose concentration depends on <i>Y.</i> These ions need not be released in L–L coacervation, but during L–S transition their displacement by PE accounts for the inverse dependence of <i>T</i>_<i>s</i> on micelle charge, <i>Y.</i

Modulation of Polyelectrolyte–Micelle Interactions via Zeta Potentials

The onset of soluble complex formation between polycations and nonionic/anionic mixed micelles was found to occur at well-defined micelle surface charge density, σ_c, which could be modulated via <i>Y</i>, the mole fraction of anionic surfactant in the mixed micelle. Critical values of <i>Y</i> were detected by precision turbidimetry for two polycations, each combined with any of the four mixed micelles formed from two anionic and two nonionic surfactants. The values of <i>Y</i>_c observed for each of the resultant eight ternary polycation/anionic–nonionic combinations were used as surrogates for polycation binding affinity: for a given polycation and a given value of <i>Y</i>, micelles with <i>Y</i>_c < <i>Y</i> will bind, while those with <i>Y</i>_c > <i>Y</i> will not. The polycation affinity of micelles correlated with their “zeta potentials” (ζ), measured by electrophoretic light scattering, and their average surface potentials (ψ₀), measured by potentiometric titration of a comicellized probe. For a given polycation at a fixed ionic strength, we found that the critical zeta potential (ζ_c) measured at <i>Y</i>_c was independent of the surfactant pair chosen. This potential at the micelle “shear plane” is thus interpreted as the potential experienced by a bound polycation. The binding affinity was furthermore found to be stronger for polycations with higher linear charge density as well as for micelles with higher axial ratio, attributed respectively to an increase in the number of micelle-bound charged polycation repeat units and to the higher surface potential for micelles with lower surface curvature

Heteroprotein Complex Coacervation: Bovine β‑Lactoglobulin and Lactoferrin

Lactoferrin (LF) and β-lactoglobulin (BLG), strongly basic and weakly acidic bovine milk proteins, form optically clear coacervates under highly limited conditions of pH, ionic strength <i>I</i>, total protein concentration <i>C</i>_P, and BLG:LF stoichiometry. At 1:1 weight ratio, the coacervate composition has the same stoichiometry as its supernatant, which along with DLS measurements is consistent with an average structure LF­(BLG₂)₂. In contrast to coacervation involving polyelectrolytes here, coacervates only form at <i>I</i> < 20 mM. The range of pH at which coacervation occurs is similarly narrow, ca. 5.7–6.2. On the other hand, suppression of coacervation is observed at high <i>C</i>_P, similar to the behavior of some polyelectrolyte–colloid systems. It is proposed that the structural homogeneity of complexes versus coacervates with polyelectrolytes greatly reduces the entropy of coacervation (both chain configuration and counterion loss) so that a very precise balance of repulsive and attractive forces is required for phase separation of the coacervate equilibrium state. The liquid–liquid phase transition can however be obscured by the kinetics of BLG aggregation which can compete with coacervation by depletion of BLG

Mass Spectrometry Reveals a Multifaceted Role of Glycosaminoglycan Chains in Factor Xa Inactivation by Antithrombin

Factor Xa (fXa) inhibition by antithrombin (AT) enabled by heparin or heparan sulfate is critical for controlling blood coagulation. AT activation by heparin has been investigated extensively, while interaction of heparin with trapped AT/fXa intermediates has received relatively little attention. We use native electrospray ionization mass spectrometry to study the role of heparin chains of varying length [hexa-, octa-, deca-, and eicosasaccharides (dp6, dp8, dp10, and dp20, respectively)] in AT/fXa complex assembly. Despite being critical promoters of AT/Xa binding, shorter heparin chains are excluded from the final products (trapped intermediates). However, replacement of short heparin segments with dp20 gives rise to a prominent ionic signal of ternary complexes. These species are also observed when the trapped intermediate is initially prepared in the presence of a short oligoheparin (dp6), followed by addition of a longer heparin chain (dp20), indicating that binding of heparin to AT/fXa complexes takes place after the inhibition event. The importance of the heparin chain length for its ability to associate with the trapped intermediate suggests that the binding likely occurs in a bidentate fashion (where two distinct segments of oligoheparin make contacts with the protein components, while the part of the chain separating these two segments is extended into solution to minimize electrostatic repulsion). This model is corroborated by both molecular dynamics simulations with an explicit solvent and ion mobility measurements in the gas phase. The observed post-inhibition binding of heparin to the trapped AT/fXa intermediates hints at the likely role played by heparan sulfate in their catabolism