Complex Equilibria, Speciation, and Heteroprotein Coacervation of Lactoferrin and β-Lactoglobulin

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.

Linear viscoelasticity of complex coacervates

“…The qualitative nature of turbidity-style measurements allows a phenomenological characterization of coacervate phase behavior, rather than a more direct quantification of the binodal phase space [8,15,71,[73][74][75][76][77]83,88,[91][92][93]104,105,108,112,114,. Typical characterization experiments include evaluation of the stoichiometric ratio of polycation to polyanion, the effect of increasing salt concentration, and the effect of variable pH.…”

Heteroprotein complex coacervation: A generic process

“…As illustrated in Fig. 2, LF exhibits two unequal positive domains on its surface capable of binding two β-LG 2 with different affinities [64].…”

“…These molecular species are attributed to different LF/β-LG complexes: LF(β-LG 2 ), LF(β-LG 2 ) 2 , LF 2 (β-LG 2 ) ranked in ascending Rh order. Only one of these complexes, LF(β-LG 2 ) 2 , was suggested to be involved in the coacervate structure [64]. By changing the protein mixing ratio away from the optimal value for coacervation, it was suggested that the stoichiometry of LF/β-LG complexes is modified and the coacervation yield decreases due to a lower abundance of LF(β-LG 2 ) 2 [64].…”

“…Only one of these complexes, LF(β-LG 2 ) 2 , was suggested to be involved in the coacervate structure [64]. By changing the protein mixing ratio away from the optimal value for coacervation, it was suggested that the stoichiometry of LF/β-LG complexes is modified and the coacervation yield decreases due to a lower abundance of LF(β-LG 2 ) 2 [64]. For certain LF/β-LG molar ratios, the charge and size compensation theory is not respected anymore, leading to the inhibition of the coacervation process.…”

Coacervates of whey proteins to protect and improve the oral delivery of a bioactive molecule

“…Recently the coacervation of two whey proteins, betalactoglobulin (BLG) and lactoferrin (LF), have been investigated in details (Anema & de Kruif, 2014;Flanagan et al, 2015;Yan et al, 2013). Beyond their ability to form coacervates, whey proteins display other features that are interesting from industrial and physicochemical standpoints.…”