Fast Microdialysis Buffer Exchange to Study Poly(glutamate)/Lysozyme Coacervates in Concentrated Conditions

Area of Science:

• Biomaterials Science
• Polymer Chemistry
• Biophysics

Background:

• Complex coacervation of proteins and polyelectrolytes is crucial for biological systems and biomedical applications.
• Studying protein coacervates is challenging due to limited sample sizes and sensitivity to pH and ionic strength.
• Existing methods lack precise control over microvolumes and environmental conditions.

Purpose of the Study:

• To introduce a microdialysis chip for in situ control of microvolumes in protein coacervate studies.
• To investigate the effects of ionic strength and chirality on lysozyme/polyglutamate coacervates.
• To characterize the phase behavior and material properties of protein coacervates.

Main Methods:

• Utilized a microdialysis chip for precise control of ionic strength and microvolumes.
• Studied model coacervates formed from lysozyme and polyglutamate.
• Employed rapid equilibration with phosphate buffers of varying concentrations.
• Used Fluorescence Recovery After Photobleaching (FRAP) to assess diffusivity and material transitions.

Main Results:

• Fine control of ionic strength is critical, influencing the phase diagram above 80 g/L lysozyme.
• Phase separation was observed to be reversible.
• Chirality of polyglutamate was found to impact coacervation.
• Annealing coacervates into films enabled FRAP measurements, showing a transition from fluid-like to solid-like states below 60 mM phosphate.

Conclusions:

• The microdialysis chip offers a powerful tool for overcoming experimental constraints in protein coacervate research.
• Ionic strength, reversibility, and polyglutamate chirality are key factors governing protein coacervate formation and properties.
• Protein coacervates exhibit tunable material properties, transitioning from fluid to solid-like states with implications for biomimetic materials.