Heteroprotein Complex Coacervation: Bovine β-Lactoglobulin and Lactoferrin

Abstract

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, 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.

Heteroprotein complex coacervation: A generic process

“…Below 10 g L -1 , β-LG and LF mixture does not lead to coacervate in agreement with Tavares et al [61]. The disappearance of the coacervates at high protein concentration was linked to the self-aggregation of β-LG leading to a progressive depletion of free β-LG [57]. The protein concentration domain for LF/β-LG coacervation was larger for β-LG variant A than for β-LG variant B. LF/CNs coacervates were observed at much lower concentration than for LF/β-LG coacervates.…”

“…The LF/β-LG complex coacervation was found to be highly sensitive to ionic strength and occurs for values below 25 mM [57,61]. However, other results showed the occurrence of such coacervation at ionic strength as high as 100 mM [58].…”

“…However, for Yan et al [57], coacervates were formed at protein concentration between 10 g L -1 and 40 g L -1 at the optimal protein mixing ratio. Below 10 g L -1 , β-LG and LF mixture does not lead to coacervate in agreement with Tavares et al [61].…”

“…The decrease of the pH after mixing negatively charged protein solutions and LF solution in water suggests a displacement of the pK of carboxylic amino acids (aspartic and glutamic acids) consecutive to protein association. The differences in the pH range for LF/β-LG coacervation reported by Yan et al [57] and Anema and de Kruif [58] could be explained by i-different protein concentrations investigated and consequently different buffering capacity of the system modulating pH drift, ii-and/or certain specificity linked to the β-LG variant used. Working in buffer to prevent any protein association induced pH drift, Tavares et al [61] observed that LF/β-LG coacervates were formed in the pH range 5.5 -6.0 using a mixture of variant A and B of β-LG.…”

“…For Yan et al [57], the LF/β-LG coacervate are formed in the pH range 5.7 -6.2 with optimal pH condition at 6.0. At pH lower than 6.0 the LF/β-LG coacervation was in competition with β-LG self-aggregation approaching its pI.…”