Exploring Bovine Pancreatic Trypsin Inhibitor Phase Transitions

Abstract

This paper presents an investigation of the phase diagram of BPTI (bovine pancreatic trypsin inhibitor)/350 mM KSCN at pH 4.9 by direct observation and numerical simulations. We report optical microscopy and light and X-ray scattering experiments coupled with theoretical data analysis using numerical tools. The phase diagram is thoroughly determined, as a function of temperature. Two polymorphs are observed by video microscopy and their solubility measured. In this phase diagram, the liquid-liquid phase separation (LLPS) is metastable with respect to the solid-liquid phase separation. Above the T(L-L) boundary curve, solutions are composed of a mixture of BPTI monomers and decamers. Attractive interactions are stronger between decamers than between monomers. Below the T(L-L) boundary curve, the dense phase is highly concentrated in protein and composed of BPTI decamers alone. Thus, the driving force for liquid-liquid or liquid-solid phase separation is the attraction between decamers at low pH. The structure factors of the dense phases are characteristic of repulsive dense phases because of a hard sphere repulsion core, meaning that in the dense phase proteins are actually in contact (interparticle distance of 53 A). In agreement with the Oswald rule of stages, LLPS occurs prior to and impedes the solid nucleation.