Article pubs.acs.org/Langmuir
Complex Equilibria, Speciation, and Heteroprotein Coacervation of Lactoferrin and β‑Lactoglobulin Sean E. Flanagan,† Alexander J. Malanowski,† Ebru Kizilay,† Daniel Seeman,*,† Paul L. Dubin,† Laurence Donato-Capel,‡ Lionel Bovetto,‡ and Christophe Schmitt‡ †
Department of Chemistry, University of MassachusettsAmherst, Amherst, Massachusetts 01003, United States Department of Food Science and Technology, Nestlé Research Center, Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland
‡
ABSTRACT: 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 β-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.
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points.2,4,11 Bouhallab et al. determined that this condition ensures that the complex can attain net neutrality, a prerequisite of coacervation,2,4 and empirically observed that the requirement of charge neutrality in the complex equates to an excess of the smaller protein such that each protein occupies approximately half of the volume of the complex.2,4 The aforementioned observations have led to a model in which size compensation and electroneutrality within the complex are requirements for the formation of a dense liquid phase.2,10,12 Although PE−protein coacervation has been studied for decades13−17 and models have emerged that relate phase behavior to molecular interactions and complex formation, HP coacervation is less well understood. For example, the formation of inter- and intrachain complexes between proteins and polymers prior to coacervation is readily seen in PE− protein systems,12 but such precursors have been less evident for HP coacervation. Different equilibria in the latter case might explain the appearance of coacervates4,5,10 rather than the precipitates more commonly encountered in the use of polyelectrolytes for protein purification.18−23 In this study we seek (1) to characterize the molecular processes that lead to LLPS in HP systems, analogous to those surmised for PE− protein coacervation, and (2) to expand the current model for
INTRODUCTION Many proteins can exhibit liquid−liquid phase separation (LLPS), but heteroprotein (HP) coacervation constitutes a special case in which the dense liquid phase is composed of interprotein complexes.1−4 Many of these proteins exhibit significant electrostatic and geometric anisotropy,5 features in common with monoclonal antibodies that undergo LLPS.6−9 HP complexes are in equilibrium with both the coacervate and free proteins,3,10 but the molecular interactions that determine these equilibria are not well understood. As a consequence, the effects of key experimental variables on phase behavior are difficult to explain. Milk proteins β-lactoglobulin (BLG2) and lactoferrin (LF) comprise a model system for examining how the experimental variables (pH, ionic strength, protein concentration, and bulk stoichiometry) influence the charges on the proteins, the stoichiometry of the complex, and thus consequent phase states.1,5 Coacervates in HP systems are composed of stoichiometric protein complexes held together by electrostatic interactions.1,3,5,10 Evidence for the presence of these interprotein complexes comes from fluorescence microscopy of the apo-αlactalbumin/lysozyme system that indicated through FRET (Förster resonance energy transfer) that the protein molecules were less than 10 nm apart.1 Thus, HP coacervates are not a simple mixture of proteins but instead can comprise one or more fixed stoichiometries1,5,11 reflecting interactions of opposite charges in the pH region between the two isoelectric © 2015 American Chemical Society
Received: October 10, 2014 Revised: January 5, 2015 Published: January 7, 2015 1776
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Figure 1. Addition of 100 g/L LF to 1 g/L BLG (both in pure water at initial pH 6.0). (a) pH shift and (b) proton release as a function of r, the LF/ BLG mole ratio. Closed symbols in (b) correspond to biphasic systems, as determined from turbidity (Figure 2). Results at r ≥ 1.0 are not included in (b) because the gradual increase in pH at larger r is attributed to the buffering capacity of added pH 6.0 LF solution. Proton Release. pH was recorded upon the addition of LF to BLG, with a delay time of 1 min between increments. The pH of the LF titrant was continuously monitored as pH 6.0 throughout the experiment. Dynamic Light Scattering (DLS). DLS was performed with a Malvern Zetasizer ZS equipped with a 633 nm He−Ne laser and aligned for backscattering at 173° on samples prepared by mixing BLG and LF solutions, both at pH 6.0, in 0.010 M NaCl. Biphasic samples at r ≥ 0.05 were filtered (0.22 μm) to remove coacervate prior to measurements. Multiple (two to six) samples were prepared identically at each value of r, and the results reported below represent mean or standard deviations. Assuming diffusive relaxations, translational diffusion coefficients (DT) were obtained from the fitting of DLS autocorrelation functions with non-negative constrained least squares (NNLS). DT was converted into the hydrodynamic radius (RH = kT/ (6πηDT)) where k is the Boltzmann constant, T is the absolute temperature, and η is the solvent viscosity, here taken as that of water. For purposes of comparison, a single DLS measurement was performed with a Brookhaven Instruments BI-200SM goniometer equipped with a PCI BI-9000AT digital correlator, a temperature controller, and a solid-state laser (model 25-LHP-928-249, 633 nm). Isothermal Titration Calorimetry (ITC). ITC was performed using a VP-ITC (Microcal Inc., Northampton, MA). LF was added to BLG in 60 s intervals, with each injection lasting 1.2 s. Modeling. DelPhi V. 4r1.1 was used to model the electrostatic potential around the protein at pH 6, I = 5 mM. PDB 1BLF (diferric bovine LF) and 1BEB (BLG dimer) were taken from the RCSB Protein Data Bank (http://www.rcsb.org). Amino acid charges were generated using the spherical-smeared-charged model proposed by Tanford on the basis of titration curves of BLG25 and LF.5
HP coacervates by relating the conditions required for coacervation to equilibria among complexes and free proteins. We find that a pH shift upon the addition of one protein to the other can be used to identify complex formation because the release of protons indicates interactions and complex formation between BLG2 and LF. This pH shift is analogous to that seen upon protein binding to a polycation,24 but has a more clear stoichiometric meaning in the present case in that there are only three states of complexed BLG, as opposed to the polydispersity in the number of proteins bound per polycation chain. Complementary experiments by isothermal titration calorimetry (ITC) gave additional insight into successive binding events. Lastly, the stoichiometries of the resultant complexes were inferred from sizes obtained by dynamic light scattering. These results were combined to construct a speciation diagram for BLG2/LF as a function of r, the LF/BLG molar ratio.
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MATERIALS AND METHODS
Materials. Bovine β-lactoglobulin powder (BLG from Davisco Foods International, Inc., batch number JE 001-8-415) and lactoferrin powder (LF, from DMV, batch number 10444427) were supplied by Nestlé (Lausanne, Switzerland). (We used “BLG” synonymously with β-lactoglobulin but “BLG2” where appropriate to indicate the dimer as the predominant form in our study, at 5 < pH < 7). The powder composition was the following (g/100 g of wet powder): BLG2: Na 0.554, K 0.004, Mg 0.002, Ca 0.018, P 0.054, Cl 0.047, protein 89.3, of which 97% was β-lactoglobulin. LF: Na 0.087, K 0.001, Mg 0.0003, Ca 0.002, Fe 0.014, P 0.021, Cl 0.920, protein 93.1 (Kjeldhal, Nx6.38), of which 97% was lactoferrin. Milli-Q water was used in all sample preparation. Sodium chloride and standard HCl and NaOH solutions were purchased from Fischer Scientific. Methods. All studies involved the progressive addition of LF 100 g/L to BLG 1 g/L, both in pure water at pH 6.0 unless otherwise noted. Turbidimetry. Transmittance was measured using a Brinkmann PC 800 colorimeter equipped with a 470 nm filter and a 1.0 cm path length fiber optics probe calibrated to 100% transmittance with MilliQ water. The turbidity was reported as 100 − %T, which is linear with the true turbidity of τ = −ln T for 100 − %T < 15. The pH was measured with a Corning 240 pH meter calibrated with pH 4.0 and 7.0 buffers.
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RESULTS pH Shift, Proton Release, and Turbidity. The addition of LF to BLG can result in a rapid drop in pH from 6.0 to 5.2. This is shown incrementally in Figure 1(A), where the pH is plotted as a function of r = LF/BLG mol/mol, here linear with the concentration of added LF. Lactoferrin is positively charged at pH 6.0 (3 pH units below its pI of ∼8.7) so that the formation of complexes of LF with BLG can stabilize the conjugate base of the BLG (pI ∼5.2) acidic residues, leading to a reduction in their pKa. Similar behavior for mixtures of lysozyme or LF and different caseins was introduced by de Kruif and coworkers as evidence of interprotein interactions.26,27 Figure 1(B), which presents the results as n, the 1777
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Langmuir number of moles of protons [H+] released per mole of BLG, shows clearly well-delineated regions in which n varies linearly with the number of moles of added LF. The slope n/r increases strongly at r = 0.25 and then decreases at r = 0.6, attaining a near-plateau at LF/BLG = 0.7. This corresponds to the region of almost constant pH in Figure 1(A), although further diminution of pH at high r in Figure 1(A) could be masked by the increase in pH due to additional LF at pH 6.0. Anema et al.4 observed a very similar pH drop upon mixing BLG and LF, deducing from the turbidity maximum a single stoichiometry of 3:1 BLG/LF, a species that we do not identify in the present work, in part because the dimer is the dominant form of BLG. Ratio 3:1 for BLG:/LF might indeed correspond to some type of average of the three relevant species that we identify by DLS coupled with proton release vide inf ra. The turbidity maximum in Figure 2 at r = 0.22 corresponds to the junction of regions A and B in Figure 1(B), whereas the
relatively high. It is remarkable that the abrupt phase change at r = 0.1 produces no corresponding feature in Figure 1(A); similarly, as shown in Figure 1(B), filled and empty symbols (biphasic and monophasic) are colinear. These data need to be considered in conjunction with the pH shift shown in Figure 1(A). The turbidity onset and maxima of Figure 2 do not correspond to any prominent feature in Figure 1(B) because the pKa shift that accompanies complex formation is not directly related to the aggregation and phase behavior of the complexes. Although phase separation and proton release are both consequences of complexation, they are separate phenomena. Dynamic Light Scattering. In an attempt to correlate complex stoichiometry with bulk (mixing) stoichiometry (r), we measured the complex size by dynamic light scattering as a function of added LF, using filtered samples representing the continuous phase. Figure 4 shows the results for the dominant
Figure 2. Turbidity of the BLG solution (initially 1 g/L) as a function of added LF (100 g/L). Both protein solutions have no added salt and are initially adjusted to pH 6.0. Error bars indicate run-to-run reproducibility.
Figure 4. Apparent size of fast mode species of the supernatant of the BLG/LF mixture (10 mM NaCl, pH 6) as a function of r. Error bars indicate the standard deviation among independently prepared samples, largely related to the nonrobust nature of the autocorrelogram fitting.
fast mode (weakly scattering slow modes corresponding to minor components with apparent radii of 100−200 nm, not reported). The error bars, indicating means or standard deviations of identically prepared samples, are seen to increase significantly as r changes from zero (pure BLG2) to higher species. The variation in RH was found to be particularly large among multiple DLS runs on a given sample, particularly for the region of 0.4 < r < 0.7. This run-to-run variability, greater than sample-to-sample variability, is related to the presence of multiple species vide infra. Comparisons between the values of Figure 4 and expected sizes of LF(BLG2)2, LF(BLG2), and LF2(BLG2) have to be tempered by uncertainties in this averaging of signals and the inexact dimensions of the complexes, absent crystal structures. The ultimate value of RH ≈ 11.5 nm corresponds to the formation of the largest species, LF2BLG2, at large r where the system again becomes monophasic (Figure 1(B)). The apparent gradual changes between these states arise from unresolved signals of coexisting species, e.g., BLG2 and LF(BLG2)2 (the limit of complete resolution in RH is roughly a factor of 2). Extrapolation to r = 0 gives as expected RH = 3.8 nm for the BLG dimer, and the limiting value of 11 nm at r = 1 corresponds to LF2(BLG2) at
midpoint of coacervate redissolution in Figure 2 corresponds to the junction of regions B and C. The rapid increase in turbidity at r > 0.1 is due to the onset of coacervation. This is evident from low-angle laser light scattering seen in Figure 3, even though the measured transmittance under these conditions is
Figure 3. Low-angle laser light scattering from coacervate suspension at r ≈ 0.1. The grainy appearance in the beam is characteristic of stable suspensions of coacervate droplets. 1778
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increased repulsion between like-charged (positive/positive) groups on BLG2 and LF. The depletion of higher-affinity binding sites on LF leads to the formation of species with multiple BLG2 molecules bound to LF as seen at low to intermediate r (Figure 4). However, at large values of r, an excess of LF and thus of high-affinity binding sites relative to the total amount of BLG2 present, potentially allow for multiple LFs to bind to a single BLG2 (Scheme 1, eq 3). In contrast to LF(BLG2)2, which as the coacervating species would be expected to be near charge neutrality, complexes that do not lead to coacervation have no reason to be charge neutral. The pH dependence of BLG2 and LF charges (Figure 6A) is not consistent with the formation of neutral species, and a drop in pH from 6 to 5.2 (pH ≈ BLG pI) would be expected to bring complexes further from charge neutrality.
excess LF. Even though LF(BLG2)2 is being converted to LF(BLG2) in region B, only a modest decrease in RH is observed at r > 0.45 because overlapping equilibria also allow for the depletion of LF(BLG2) in this region. Disparities between the apparently well-defined regions derived from proton release in Figure 2 and the much more gradual transitions appearing from DLS are discussed further in the section of speciation (below). Isothermal Titration Calorimetry. Figure 5 juxtaposes the ITC result with those from turbidimetry and proton release.
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DISCUSSION Previous work identified LF(BLG2)2 as the precursor of coacervation on the basis of compositional analysis (size exclusion chromatography) and dynamic light scattering of both the one-phase system at incipient coacervation and the supernatant of the subsequent coacervate.11 LF and BLG2 conform to the size complementarity of coacervating protein complexes presented by Bouhallab and coworkers, and at pH 6.0, the LF(BLG2)2 complex is close to electrical neutrality.1,2 Its formation from LF and BLG2 must be governed by an equilibrium constant, and its partitioning between the coacervate and supernatant can be considered to be analogous to a solubility product. Here, proton release, turbidity, DLS, and ITC as a function of r are used to describe speciation and complex equilibrium between LF and BLG as a function of r. Proton Release Correlates with Binding Events Regardless of Phase Changes. Figure 1(B) indicates that the number of protons released per LF added (slope) is constant within each region; this corresponds to a fixed complex stoichiometry over each range of r. This analysis is based on the assumption that the number of moles of H+ released is the product of (10−pH−10−6) and the total volume, even though the pH measured at r > 0.1 is that of the continuous phase. This would be valid if both the measured pH and its relationship to [H+] were the same in both phases. However, one might question whether the activity coefficients in the two phases are identical, and it might be proposed that the pKa shift that occurs when the two proteins are mixed might lead to the expulsion of H+ into the dilute phase. However, Figure 1(A,B) shows no discontinuities in pH or n at r = 1.0 or 0.1at which point the coacervate either dissolves or (abruptly) appearswhich would be expected as a consequence of unequal partitioning of H+ between the two phases. This appears to justify the assumption that the measured pH describes both phases. The pH shifts in Figure 1(A) corresponding to regions A−C arise from a reduction in the pKa of BLG2 carboxyl groups complexed close to LF positive domains, analogous to the increased acidity of BSA upon binding to a strong polycation.24 This analogy is appropriate, as the BLG2 carboxyl groups are under the influence of a positive electrostatic potential because of the nearby positive domain of LF at pH 6 that is large compared to the smaller negative domain of BLG. The four states of BLG [BLG2, LF(BLG2)2, LF(BLG2), and LF2(BLG2)] are related by their coupled equilibria, and the values of n in Figure 1(A) are in fact continuous because the equilibrium constants are not extremely large. Finally, the
Figure 5. (A) Isothermal titration calorimetry as heat released or absorbed per mole of added lactoferrin as a function of LF/BLG stoichiometry r in comparison to (B) proton release and turbidity from Figure 1.
Four regions exist in the thermogram of Figure 5(A): (1) an initial endotherm at r < 0.2, (2) an exotherm at 0.25 < r < 0.45, (3) an exotherm with positive slope at 0.45 < r < 0.65, and (4) an asymptotic approach to zero at r > 0.65. The first and last regions can be ascribed to the initiation or termination of complexes, respectively, but coacervation appears to affect the intermediate signal, from its onset at r ≈ 0.025 to its reversal at r > 0.5 vide inf ra. Note that r = 0.25 corresponds to the transition from endotherm to exotherm, near the transition from coacervate formation to dissolution and close to the junction of regions A and B, where the production of LF(BLG2)2 is replaced by its consumption to form LF(BLG2). This correlation of results from ITC, proton release, and turbidimetry will be discussed below. Electrostatic Modeling. Isopotential contours displayed with a cutoff of 0.5 kT/e show that LF (Figure 6(A)) is highly anisotropic with respect to its distribution of positive charge. It can be inferred that LF contains two unequal positive domains capable of binding to the negative domain of BLG2 with different affinities. The first BLG2 would be expected to bind preferentially near the more positive region of LF. The lesspositive end of LF should represent a lower affinity site for the binding of BLG2 because of two factors: (1) lessened attraction between the respective BLG2 and LF domains and (2) 1779
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Figure 6. (A) BLG2 and LF shown at pH 6.0, I = 0.005 M and isopotential contours generated at +0.5kT/e (blue) and −0.5kT/e (red). Illustration of the unequal affinity of LF (center) for the negative domains of BLG2, where the size of the dashed arrows indicates the relative strength of electrostatic attraction. (B) The confinement of BLG2 near LF’s strongly positive potential should shift the pK’s of acidic groups in the negative domain of BLG2, increasing its acidity and resulting in the release of protons.
coexist at those junctions. Remarkably, the features in either figure are unaffected by a substantial and abrupt phase change occurring at 0.1 < r < 0.2 (Figure 2) where the grainy appearance is characteristic of submicrometer coacervate droplets (Figure 3) suggesting that proton release may yield identical values of [H+] in both phases. Nevertheless, the small error bars in Figure 2 indicate a high level of run-to-run reproducibility, showing that these systems are indeed metastable and not time-dependent liquid−liquid suspensions. This reproducibility at r ≥ 0.4 indicates that droplet dissolution or dispersion is remarkably consistent, although its mechanism can have little resemblance to the far more abrupt droplet formation. In Figure 5(B) the gradual slope of turbidity versus r at r = 0.5 does not reflect the junction between regions B and C in Figure 1(B). The reduction in slope at this junction can reflect the formation of LF2(BLG2) from LF(BLG2) at r > 0.6 as suggested by results from DLS presented below. If LF(BLG2)2 is the species essential to coacervate formation, vide inf ra, then its depletion in the dilute phase with increasing r could be coupled with coacervate dissolution via {LF(BLG2)2}c = {LF(BLG2)2}d, where subscripts represent the coacervate and dilute phases. This leads to diminished proton release (region C) and terminal values of turbidity (r ≥ 0.8) when LF(BLG2)2 is present in neither coacervate nor supernatant. Although the formation and subsequent consumption of LF(BLG 2 ) 2 accounts for the proton release features in regions A and B in Figure 1(B), the ultimate conversion to LF2(BLG2) might be large enough to be responsible for the terminal values of T ≈ 93% . DLS Reveals the Formation of Distinct but Coexistent Complexes. With the exception of the value for r ≈ 0 (the BLG dimer), the radii reported in Figure 4 represent averages arising from coexistent species with sizes too close for DLS
Scheme 1. Equilibria among Species Present in the Continuous Phase or Supernatant
terminal plateau corresponds to the appearance of excess LF; the number of protons released per LF added is zero. As noted above, the discontinuity in Figure 1(B) at r = 0.22 corresponds to a near doubling of the total number of protons released per LF added. In region A with excess [BLG2], the predominant process is LF + 2(BLG2) = LF(BLG2)2, followed in region B by LF + LF(BLG2)2 = 2LF(BLG2) as a consequence of diminishing [BLG2]. When two BLG2’s share a single LF in region A, it is not possible for both dimers to reside in the LF region of maximum positive charge (Figure 6(A)). Consequently, their susceptibility to an electrostatically induced pKa shift (Figure 6(B)) is less than that of the single BLG dimer in LF(BLG2). This enhanced acidity of the dimer in LF(BLG2) appears as a 2-fold increase in proton release per added LF. In contrast to Figure 5(B), Figure 1(B) is drawn to emphasize the discontinuities at r = 0.22 and 0.5, but Figure 5(B) reflects the fact that two or even three species typically 1780
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During isenthalpic coacervate dissolution, the turbidity change may correspond to kinetically controlled changes in droplet size with no associated release of protons. The conversion from endotherm to exotherm centered around r = 0.25 is then the change from formation to consumption of LF(BLG2)2, which is causally related to both the exotherm and coacervate dissolution. It is not surprising that coacervate dissolution coincides with the depletion of LF(BLG2)2. For the transition near the junction of regions A and B in Figure 1(A) at r ≈ 0.22, we can write LF + LF(BLG2)2 → 2 LF(BLG2), i.e., LF binding to two BLG2’s is replaced by LF binding to only a single BLG2. As noted above, the charge anisotropy of LF (Figure 6(A)) indicates two BLG2 binding sites with different affinities. Designating BLG2 bound to the high-affinity site as BLG2″ and BLG2 bound to the weak binding site as BLG2′, we have the conversion of (BLG″ + BLG′) to 2(BLG″) for every LF added. This exchange could account for the exotherm that begins at r = 0.2 in region B (Figure 4). Beyond r = 0.4, no weak binding sites are eliminated, but with the formation of LF2(BLG2), a single BLG dimer now acts as a bridge between the more positive domains of two LFs. Repulsion between those two domains can reduce the magnitude of the favorable enthalpy compared to that of the previous process (0.2 < r < 0.4), resulting in the return of ΔH to zero. The gradual change from positive to negative ΔH suggests considerable overlap of regions A and B, i.e., coexistence of LF(BLG2)2 and LF(BLG2) over a wide region of r. Thus, whereas Figure 1(B) is drawn to emphasize regions in which one reaction is dominant, Figure 5(B) more realistically reflects gradual transitions. The interactions described above differ dramatically from those involved in PE−protein complexation and coacervation. In that case, a single protein-binding site on the polyelectrolyte interacts with an identifiable PE-binding site on the protein, giving rise in at least one case to a negative ΔH whose magnitude correlates with the strength of the interaction.36 Polyelectrolyte−protein systems also exhibit complexities in microcalorimetry: successive changes in the sign of ΔH during stoichiometric titrations have been observed for a number of protein−polyelectrolyte systems. McClements et al.36 found both an endotherm and exotherm in the region of coacervate formation and redissolution, with the sign of ΔH being strongly dependent on pH, but the influence of a phase change on the sign or magnitude of ΔH was not discussed. Stoichiometry Controls Speciation. The relationship among the species present with increasing r is described by the reactions and corresponding equilibrium constants in Scheme 1. The absence of any significant contribution of BLG dimer (RH = 3.8 nm) to any of the DLS signals presented in Figure 4 indicates K1 ≫ 1. We can also conclude that K1 > K2 from the presence of LF(BLG2)2 in amounts large enough for extensive coacervation. We further propose that K2 itself is large compared to unity because LF(BLG2) with RH ≈ 8 nm, along with LF2(BLG2), appears to be a significant species in the region centered around r ≈ 0.65. LF(BLG2)2 detected by DLS in fact represents only the continuous phase, with the majority of this coacervate species residing in the dense phase. Above r = 0.9, the value of RH ≈ 11.5 is consistent with LF2(BLG2) as the terminal product, with all LF(BLG2) having been consumed, so that additional LF appears in the uncomplexed state. Although values of RH beyond this point might provide further evidence, measurements were terminated at the point at which further titration with pH 6.0 LF trivially elevated the pH.
resolution. Indeed, the regions defined in Figure 1(B) correspond to the dominance but not exclusivity of a particular reaction (with corresponding proton release per molecular event). Thus, multiple species exist to some extent at most values of r. DLS radii in Figure 4 reflect this heterogeneity but nevertheless are consistent with the consumption of BLG2 to form LF(BLG2)2 corresponding to the continued increase in size between r = 0 and r = 0.2. Although LF(BLG2)2 is expected from modeling to have a radius of 11 to 12 nm, measured radii at 0.2 < r < 0.45 do not attain this value because of contributions from BLG2 and LF(BLG)2, both smaller species. Thus, the shallow maximum at r = 0.45 indicates a condition at which the sum of the contributions of these three species yields RH ≈ 10.5; the subsequent decline in RH (0.5 < r < 0.7) is due to the consumption of LF(BLG2)2 to form LF(BLG2). The coexistence of simultaneously present species, i.e., LF(BLG2)2, LF(BLG2), and LF2(BLG)2, results in large “error bars” in this region related to the sensitivity of the deconvolution program to minor variations in the autocorrelation function for multiple unresolved modes. That this lack of robustness is related to a distribution of species was supported by a single Brookhaven DLS measurement indicating weakly multiple modes at r ≈ 0.4 (run-to-run variability greater than sample-to-sample variability). The value of 11.5 nm reported a r = 1 indicates that terminal product LF2BLG2 (the complex with the largest plausible expected size RH ≥ 12 nm) must coexist with the smaller LF(BLG2)2. It was expected that the successive formation of LF(BLG2)2, LF(BLG2), and LF2(BLG2) with increasing r should be reflected in corresponding exotherms or endotherms. ITC is Consistent with Proton Release and DLS but Complicated by Phase Separation. The analysis of ITC results in Figure 5(A) requires the consideration of both changing complex stoichiometry and also phase states as shown by turbidity in Figure 5(B). ITC is typically capable of providing quantitative binding data when fit to an appropriate model. While very common for simple 1:1 binding of small molecules, fitting can be quite complex when applied to either phase-separating systems28 or overlapping multispecies equilibria.29 This is the case for protein−protein,30 protein− polyelectrolyte,31,32 and peptide−peptide interactions33 as well as for interactions in mixed surfactant systems.34,35 Here, we use it only to confirm the regions of LF(BLG2)2 formation and the termination of complex formation. Only for the first, at r ≤ 0.1 is it possible to disregard phase separation and attribute the initial endotherm to the formation of LF(BLG2)2. Interestingly, the subsequent change from positive to negative enthalpy at r ≈ 0.25 corresponds to the onset of coacervate dissolution (Figure 5(B)) and the transition from region A (formation of LF(BLG2)2) to region B (its conversion to LF(BLG2)). This isenthalpic region diminishes and disappears at 0.45 < r < 0.65, i.e., within region B where LF(BLG2) is converted to LF2(BLG2) and turbidity continues to diminish, perhaps because the net positive charge of this complex opposes self-association. However, the continued dissolution of coacervate at r > 0.6, where no calorimetric signal appears, makes it difficult to attribute this ITC signal to phase behavior. The fact that coacervate dissolution occurs here without an ITC signal suggests that the exotherm beyond r = 0.25 and completed at r = 0.6 arises solely from the conversion of LF(BLG2)2 (RH = 12 nm) to LF(BLG2) (RH= 8 nm) so that LF binds a single BLG dimer that then experiences a larger pKa shift. 1781
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Results published elsewhere40 have shown that the macroion concentration of the PE−protein dense phase is not dependent on the initial concentration or the concentration of the supernatant after phase separation. The present system resembles that of PE−protein and PE−micelle systems in that a shift in bulk stoichiometry, i.e., to “excess” PE or colloid, can bring the system from the two-phase to the one-phase regime; here such a shift is accomplished with excess LF.
The coexistence of equilibrium species is typically represented by speciation plots such as Figure 7 and is
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CONCLUSIONS Heteroprotein coacervation of lactoferrin (LF) and βlactoglobulin (BLG) involves equilibria among free proteins and complexes; those that meet certain requirements of size complementarity and charge neutrality can form coacervates. This occurs when conditions of pH, ionic strength, and stoichiometry result in the abundance of such coacervate precursors. Various species can be characterized by dynamic light scattering, and their formation as a function of BLG/LF stoichiometry can be established from proton release at fixed ionic strength. A speciation diagram qualitatively exhibits the coexistence of these complexes but does not fully take into account the biphasic nature of the system.
Figure 7. Tentative speciation diagram for BLG2 as a function of r. BLG2 first interacts with LF to form LF(BLG2)2, which is depleted as more LF is added to form 2 equiv of LF(BLG2), which is subsequently depleted to form LF2(BLG2). Values of r consistent with DLS and ITC results and the maximum for LF(BLG2)2 at r = 0.4 correspond to the midpoint of region B. This plot is consistent with the proton release and DLS data presented above and the foregoing conclusions about the relative magnitudes of K1 and K2.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected].
commonly associated with metal−ligand complexes37,38 or metal−protein complexes.39 In the present case, lacking the ability to resolve and quantitate coexistent species and thus measure the corresponding equilibrium constants, it is possible to specify only the state of BLG at r = 0 and 1 (pure BLG2 and LF2(BLG2), respectively). Identification of the latter is based on taking the minimum pH in Figure 1(A) to correspond to the termination of proton release and is consistent with the size measured in Figure 4 and the full dissolution of coacervate r = 1 in Figure 2, while disregarding the less easily interpretable ITC results. The continuous increase of measured sizes at lower values of r in Figure 4indicate that the equilibria in Scheme 1 overlap. The relative amounts of other species at intermediate r indicated in Figure 7 are approximations consistent with, although not derived from, data in Figures 1 and 4. The appearance of free LF at r > 0.65 (only when all BLG has been consumed) is consistent with K2 ≫ 1. The limiting value of RH ≈ 11 nm appearing at r > 0.6 in Figure 4 corresponds to the end of the titration (consumption of all BLG2 to yield LF2(BLG2)) identified with K3. The terminal reaction drives the disappearance of coacervate through the consumption of LF(BLG2)2 in the supernatant, thus depleting the system of this primary unit of the coacervate. It is necessary to consider the relationship between speciation and the biphasic nature of the system. Although Figure 7 represents the way in which BLG2 is distributed among its various stoichiometric forms, the dense phase actually contains mainly LF(BLG2)2. For metal−ligand speciation, insoluble (solid) species are treated as having unit activity, with the amount controlled by Ksp.37 In the present case, a coacervate−supernatant partition coefficient might describe the relationship between species in the coacervate, LF(BLG2)2(c), and species in the supernatant, LF(BLG2)2(s). This is not analogous to a solubility product because the coacervate is thought to arise from a single soluble molecular species. At the present time, it is not clear whether such a partition coefficient should reflect the supernatant concentration of total protein or only LF(BLG2)2.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We acknowledge the support of Nestlé and the National Science Foundation under grants CBET-0966923 and CBET1133289. A.J.M. acknowledges a Jack Ragle Summer Research Fellowship in Chemistry from the University of MassachusettsAmherst.
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