Influence of high hydrostatic pressure on protein clustering: Implications for processing and macroscopic crystallization.

Abstract

High pressure (HP) is used in various industrial applications to sterilize a product stream or to confer desired properties. HP is also present during freezing processes, yet relatively little is known about its effects on protein phases. Protein clustering has been well-documented as a precursor to liquid-liquid phase separation (LLPS) and macroscopic crystallization. Several globular proteins have been previously shown to salt-out in the presence of ammonium sulfate, with clustering observed when approaching a first aggregation boundary, phase separation beyond a second aggregation boundary, and gel bead nucleation between them. Nanocrystalline structures have been observed within the salted-out macroscopic gels, suggesting clusters below the first aggregation boundary are precursors to macroscopic crystals. The present work examines the influence of pressures up to 400 MPa on salt-induced clustering in ovalbumin solutions on either side of the first aggregation boundary. Small-angle x-ray scattering (SAXS) on aged solutions approaching the boundary reveals the presence of protein clusters, and a computational deconvolution into fractional oligomer population contributions to scattering was used to assess salt-induced clustering with similar characteristics on either side of the first aggregation boundary. Surprisingly, HP-SAXS on fresh samples reveals a sharp shift in pressure effects on clustering across the boundary. The discrepancy in pressure response suggests a fundamental difference in the clustering mechanism across the first aggregation boundary, and that clusters in this region are early nucleated gels rather than crystal precursors. The current results are of interest to protein processing and LLPS, providing insights into competing mechanisms of protein aggregation and suggesting pressure-dictated clustering as a potential tool for controlling macroscopic phase behavior.