Dilution induced coacervation in polyelectrolyte-micelle and polyelectrolyte-protein systems.

"Self-suppression", the instability of complex coacervates at high concentration, is well-known for polycation-polyanion systems, but the transient nature of those complexes impedes development of a convincing model. The stable polyelectrolyte-micelle complexes of the polycation poly(diallyldimethylammonium chloride) (PDADMAC) with mixed micelles of sodium dodecyl sulfate (SDS)/Triton X-100 (TX100); and the stable complexes of PDADMAC with bovine serum albumin (BSA) can be characterized and identified as coacervate precursors. We observe liquid-liquid phase separation upon isoionic dilution, a common facet of self-suppression. While complex coacervation usually involves association of near-neutral inter-polymer complexes, dilution-induced coacervation (DIC) proceeds differently: for both systems studied, complex size decreases near the biphasic region: inter-macromolecular complexes with hydrodynamic radius Rh∼ 100 nm dissociate to intra-polyelectrolyte complexes with Rh≤ 30 nm. Such small complexes with ≤5 bound micelles are unlikely to be net neutral. In the polyelectrolyte-protein system, complexes are even less likely to be net neutral and the effect of dilution on size is less significant, with complex size diminishing from 50 nm to 35 nm.

Complex equilibria, speciation, and heteroprotein coacervation of lactoferrin and ß-lactoglobulin.

There has been a resurgence of interest in complex coacervation, a form of liquid-liquid phase separation (LLPS) in systems of oppositely charged macroions, but very few reports describe the somewhat anomalous coacervation between acidic and basic proteins, which occurs under very narrow ranges of conditions. We sought to identify the roles of equilibrium interprotein complexes during the coacervation of ß-lactoglobulin dimer (BLG2) with lactoferrin (LF) and found that this LLPS arises specifically from LF(BLG2)2. We followed the progress of complexation and coacervation as a function of r, the LF/BLG molar ratio, using turbidity to monitor the degree of coacervation and proton release and dynamic light scattering (DLS) to assess the stoichiometry and abundance of complexes. Isothermal titration calorimetry (ITC) showed that initial complex formation is endothermic, but a large exotherm related to coacervate formation obscured other regions. On the basis of turbidimetry, proton release, and DLS, we propose a speciation diagram that presents the abundance of various complexes as a function of r. Although multiple species could be simultaneously present, distinct regions could be identified corresponding to equilibria among particular protein pairs.

Structure of bovine ß-lactoglobulin-lactoferrin coacervates.

Lactoferrin (LF) and ß-lactoglobulin (BLG) are among the protein pairs that exhibit heteroprotein coacervation, a unique and relatively unexamined type of liquid-liquid phase separation (LLPS). In prior work we found that LF and BLG undergo coacervation at highly constrained conditions of pH, ionic strength and protein stoichiometry. The molar stoichiometry in coacervate and supernatant is LF : BLG2 1 : 2 (where BLG2 represents the 38 kDa BLG dimer), suggesting that this is the primary unit of the coacervate. The precise balance of repulsive and attractive forces among these units, thought to stabilize the coacervate, is achieved only at limited conditions of pH and I. Our purpose here is to define the process by which such structural units form, and to elucidate the forces among them that lead to the long-range order found in equilibrium coacervates. We use confocal laser scanning microscopy (CLSM), small angle neutron scattering (SANS), and rheology to (1) define the uniformity of interprotein spacing within the coacervate phase, (2) verify structural unit dimensions and spacing, and (3) rationalize bulk fluid properties in terms of inter-unit forces. Electrostatic modeling is used in concert with SANS to develop a molecular model for the primary unit of the coacervate that accounts for bulk viscoelastic properties. Modeling suggests that the charge anisotropies of the two proteins stabilize the dipole-like LF(BLG2)2 primary unit, while assembly of these dipoles into higher order equilibrium structures governs the macroscopic properties of the coacervate.

Heteroprotein complex coacervation: bovine ß-lactoglobulin and lactoferrin.

Lactoferrin (LF) and ß-lactoglobulin (BLG), strongly basic and weakly acidic bovine milk proteins, form optically clear coacervates under highly limited conditions of pH, ionic strength I, total protein concentration C(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 < 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 C(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.

pH-Dependent aggregation and disaggregation of native ß-lactoglobulin in low salt.

The aggregation of ß-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.

Complexation and coacervation of polyelectrolytes with oppositely charged colloids.

Polyelectrolyte-colloid coacervation could be viewed as a sub-category of complex coacervation, but is unique in (1) retaining the structure and properties of the colloid, and (2) reducing the heterogeneity and configurational complexity of polyelectrolyte-polyelectrolyte (PE-PE) systems. Interest in protein-polyelectrolyte coacervates arises from preservation of biofunctionality; in addition, the geometric and charge isotropy of micelles allows for better comparison with theory, taking into account the central role of colloid charge density. In the context of these two systems, we describe critical conditions for complex formation and for coacervation with regard to colloid and polyelectrolyte charge densities, ionic strength, PE molecular weight (MW), and stoichiometry; and effects of temperature and shear, which are unique to the PE-micelle systems. The coacervation process is discussed in terms of theoretical treatments and models, as supported by experimental findings. We point out how soluble aggregates, subject to various equilibria and disproportionation effects, can self-assemble leading to heterogeneity in macroscopically homogeneous coacervates, on multiple length scales.

Cluster formation in polyelectrolyte-micelle complex coacervation.

The temperature-induced liquid-liquid phase transition (complex coacervation) of a polycation-anionic/nonionic mixed micelle system was examined over a range of macroion concentrations and polycation molecular weights (MW) using turbidimetry and dynamic light scattering (DLS). DLS revealed a progressive increase in complex/aggregate size with temperature up to the phase transition at T(φ), followed by splitting of these clusters into respectively smaller and larger particles. We present two explanations: (1) large (200-400 nm) clusters (soluble aggregates) are necessary and sufficient coacervation precursors, and (2) the process of coacervation itself is accompanied by the expulsion of smaller aggregates to form submicrometer droplets. Although a reduction in T(φ) for higher MW appears to be correlated with larger clusters, in support of model 1, the opposite correlation between cluster size and T(φ) is seen upon isoionic dilution. We conclude that enhanced coacervation and increased cluster size at high polymer MW arise independently from increased intercomplex attractive forces. Dilution, on the other hand, leads to diminished cluster size, whereas the decrease in T(φ) on dilution is a reflection of coacervate self-suppression, previously observed for this system. The splitting of clusters into large and small species near T(φ) is explained by macroion disproportionation, as proposed by Shkolvskii et al for DNA condensation. We demonstrate and explain a similar phenomenon: broadening of the phase transition by an increase in cluster polydispersity, resulting from an increase in surfactant polydispersity.