Protein phase behavior in aqueous solutions: crystallization, liquid-liquid phase separation, gels, and aggregates

Biophys J. 2008 Jan 15;94(2):570-83. doi: 10.1529/biophysj.107.116152.


The aggregates and gels commonly observed during protein crystallization have generally been considered disordered phases without further characterization. Here their physical nature is addressed by investigating protein salting-out in ammonium sulfate and sodium chloride for six proteins (ovalbumin, ribonuclease A, soybean trypsin inhibitor, lysozyme, and beta-lactoglobulin A and B) at 4 degrees C, 23 degrees C, and 37 degrees C. When interpreted within the framework of a theoretical phase diagram obtained for colloidal particles displaying short-range attractive interactions, the results show that the formation of aggregates can be interpreted theoretically in terms of a gas-liquid phase separation for aggregates that are amorphous or gel-like. A notable additional feature is the existence of a second aggregation line observed for both ovalbumin and ribonuclease A in ammonium sulfate, interpreted theoretically as the spinodal. Further investigation of ovalbumin and lysozyme reveals that the formation of aggregates can be interpreted, in light of theoretical results from mode-coupling theory, as a kinetically trapped state or a gel phase that occurs through the intermediate of a gas-liquid phase separation. Despite the limitations of simple theoretical models of short-range attractive interactions, such as their inability to reproduce the effect of temperature, they provide a framework useful to describe the main features of protein phase behavior.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Cattle
  • Chickens
  • Crystallization
  • Gels
  • Muramidase / chemistry
  • Ovalbumin / chemistry
  • Protein Structure, Quaternary
  • Proteins / chemistry*
  • Ribonuclease, Pancreatic / chemistry
  • Solubility
  • Solutions
  • Temperature
  • Time Factors


  • Gels
  • Proteins
  • Solutions
  • Ovalbumin
  • Ribonuclease, Pancreatic
  • Muramidase