Thermal Aggregation of Hen Egg White Proteins in the Presence of Salts

Hen egg white contains more than 40 kinds of proteins with concentrations reaching 100 mg/mL. Highly concentrated protein mixtures are common in the food industry, but the effects of a crowded environment containing salts on protein stability and aggregation have only been investigated using pure protein solutions. Here, we investigated the thermal aggregation of hen egg white protein (EWP) at various concentrations in the presence of inorganic salts by solubility measurements and SDS-PAGE. EWP at 1 mg/mL formed aggregates with increasing temperature above 55 °C; the aggregation temperatures increased in the presence of inorganic salt with the Hofmeister series. Namely, the chaotrope 0.5 M NaSCN completely suppressed the thermal aggregation of 1 mg/mL EWP. As the protein concentration increased, NaSCN unexpectedly enhanced the protein aggregation; the aggregation temperature of 10 and 100 mg/mL EWP solutions were dramatically decreased at 62 and 47 °C, respectively. This decrease in aggregation temperatures due to the chaotrope was described by the excluded volume effect, based on a comparative experiment using Ficoll 70 as a neutral crowder. By contrast, the kosmotrope Na2SO4 did not affect the aggregation temperature at concentrations from 1 to 100 mg/mL EWPs. The unexpected fact that a chaotrope rather enhanced the protein aggregation at high concentration provides new insight into the aggregation phenomena with the Hofmeister effect as well as the crude state of highly concentrated proteins.