Observation of liquid–liquid phase separation for eye lens S-crystallin

S-crystallin ( S) is an important human and bovine eye lens protein involved in maintaining the transparency of the eye. By adding small amounts of polyethylene glycol (PEG) to the binary aqueous bovine S solutions, we have observed liquid–liquid phase separation (LLPS) at 8°C and revealed that, in the binary S–water system, this phase transition would occur at 28°C. We have measured both the effect of PEG concentration on the LLPS temperature and protein PEG partitioning between the two liquid coexisting phases. We use our measurements of protein PEG partitioning to determine the nature and the magnitude of the S-PEG interactions and to quantitatively assess the effectiveness of PEG as a crystallizing agent for S. We use our measurements of LLPS temperature as a function of protein and PEG concentration to successfully determine the location of the critical point for the binary S-water system. This phase transition cannot be observed in the absence of PEG because it is inaccessible due to the freezing of the system. Our findings indicate that the effective interactions between S molecules in the binary S–water system are attractive. We compare the magnitude of the attraction found for S with the results obtained for the other -crystallins for which the critical temperature is located above the freezing point of the system. This work suggests that PEG can be used to reveal the existence of LLPS for a much wider range of binary protein– water systems than known previously.