Structure of bovine b-lactoglobulin–lactoferrin coacervates† Ebru Kizilay, a Daniel Seeman, * a Yunfeng Yan, a Xiaosong Du, b Paul L. Dubin, a Laurence Donato-Capel, c Lionel Bovetto c and Christophe Schmitt c Lactoferrin (LF) and b-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 : BLG 2 1 : 2 (where BLG 2 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(BLG 2 ) 2 primary unit, while assembly of these dipoles into higher order equilibrium structures governs the macroscopic properties of the coacervate. Introduction Complex coacervation, a spontaneous liquid–liquid phase sepa- ration, can be exhibited under a wide range of conditions by a variety of systems including: oppositely charged polyelectrolytes (PE), 1 or PE's in combination with oppositely charged macroions including proteins 2–4 micelles 5,6 or dendrimers. 7 These phenomena all differ from coacervation of oppositely charged proteins 8–10 which is known to arise from the tendency of those macroions to assemble as dense, homogeneous uids under a very limited range of stoichiometry, ionic strength and pH. 9,11 Heteroprotein coacervation is still a largely unexplored phenomenon, with the majority of publications originating from a single group, in many cases not explicitly identied as complex coacervation. 12–14 The lactoferrin–b-lactoglobulin system serves as one example of a heteroprotein coacervation with character- istic dependence on the aforementioned variables. 11 The relatively few papers reporting on heteroprotein coac- ervation reveal substantial differences from classic examples of complex coacervation. Soluble complexes have been established as precursors in nearly all typical forms of macroionic coacer- vation 15 and in some cases, particularly those involving poly- electrolyte–colloid systems, these complexes and aggregates thereof have been well characterized by techniques such as light scattering 16 and electrophoresis 17,18 and neutron scattering. 19 Evidence for analogous primary complexes in heteroprotein systems is currently very limited. Charge stoichiometry ([+]/[]) appears to play a different role for heteroprotein vs. PE–PE or PE–colloid coacervation, oen appearing in the latter case as the dominant factor determining coacervation yield, but apparently a necessary but not sufficient condition for hetero- protein coacervation. 20 The range of charge stoichiometry over which this form of coacervation can occur seems more narrow than for the more typical macroionic systems. 21 The difference between highly exible polyions and conformationally rigid globular proteins would of course be expected to have dramatic consequences because of the limitations of ion-pairing on intermolecular mixing in the protein–protein case, leading to a greatly reduced role for counterion release and congurational entropy in the heteroprotein system. Phenomenological studies of the conditions under which heteroprotein systems coacervate have illuminated some of these issues. 22,23 a Department of Chemistry, University of Massachusetts Amherst, Amherst, Massachusetts 01003, USA. E-mail: dseeman@chem.umass.edu b Department of Polymer Science and Engineering, University of Massachusetts Amherst, Amherst, Massachusetts 01003, USA c Department of Food Science and Technology, Nestl´ e Research Center, Vers-chez-les- Blanc, CH-1000 Lausanne 26, Switzerland † Electronic supplementary information (ESI) available. See DOI: 10.1039/c4sm01333f Cite this: Soft Matter, 2014, 10, 7262 Received 20th June 2014 Accepted 10th July 2014 DOI: 10.1039/c4sm01333f www.rsc.org/softmatter 7262 | Soft Matter, 2014, 10, 7262–7268 This journal is © The Royal Society of Chemistry 2014 Soft Matter PAPER Published on 10 July 2014. Downloaded by University of Massachusetts - Amherst on 28/01/2015 21:56:38. View Article Online View Journal | View Issue