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Phase equilibria in the lysozyme-ammonium sulfate-water system
J J Moretti1, S I Sandler, A M Lenhoff
1Center for Molecular and Engineering Thermodynamics, Department of Chemical Engineering, University of Delaware, Newark, Delaware 19716, USA.
Biotechnology and Bioengineering
|October 24, 2000
Summary
This study maps lysozyme phase diagrams in ammonium sulfate solutions, revealing protein crystals as the true equilibrium state. Unequal salt partitioning highlights complexities in protein phase behavior.
Area of Science:
- Biophysics
- Crystallography
- Solution Chemistry
Background:
- Protein crystallization is crucial for structural biology.
- Understanding protein phase diagrams aids in optimizing crystallization conditions.
- Lysozyme and ammonium sulfate are common model systems for studying protein phase behavior.
Purpose of the Study:
- To construct ternary phase diagrams for lysozyme in ammonium sulfate solutions at pH 4 and 8.
- To investigate the formation of different protein phases (crystals, gels, flocs).
- To determine the true equilibrium state of the protein system and analyze salt partitioning.
Main Methods:
- Measurement of ternary phase diagrams using UV spectroscopy, barium chloride titration, and lyophilization.
- Assaying independent mass fractions of lysozyme, ammonium sulfate, and water.
- Observation of crystal growth from gel and supernatant phases.
Main Results:
- Protein crystals, flocs, and gels were identified in distinct regions of the phase diagrams.
- Crystal phase was confirmed as the true equilibrium state, evidenced by solubility discontinuities and growth observations.
- Ammonium sulfate partitioning between supernatant and dense phases was unequal, contradicting common assumptions.
Conclusions:
- Protein phase diagrams are complex and exhibit rich behavior.
- The assumption of equal ammonium sulfate partitioning is often invalid in protein phase equilibrium studies.
- Slow equilibration can introduce uncertainties in protein phase diagram studies.