Monomer concentrations and dimerization constants in crystallizing lysozyme solutions by dialysis kinetics

Area of Science:

• Protein Crystallization
• Biophysical Chemistry
• Solution Behavior

Background:

• Lysozyme crystallization is crucial for structural biology.
• Understanding protein aggregation mechanisms is vital for controlling crystal growth.
• Ionic strength is a known factor influencing protein solubility and aggregation.

Purpose of the Study:

• To investigate the effect of ionic strength on lysozyme dimerization.
• To determine the relationship between ionic strength and lysozyme aggregation.
• To model the early stages of lysozyme crystallization.

Main Methods:

• Dialysis kinetics measurements using 25,000 molecular weight cutoff (MWCO) membranes.
• Flux measurements of lysozyme in varying NaCl concentrations (1-7%) at pH 4.0.
• Modeling of monomer-dimer equilibrium and higher-order aggregation models (Li model).

Main Results:

• Dimerization constants increased with ionic strength, from 265 M⁻¹ at 3% NaCl to 7879 M⁻¹ at 7% NaCl.
• Monomer and dimer lysozyme species showed distinct escape velocities from dialysis membranes.
• A monomer <-> dimer <-> tetramer <-> octamer <-> 16-mer model (Li model) accurately described flux data at high supersaturation.

Conclusions:

• Lysozyme dimerization is enhanced by increasing ionic strength, indicating electrostatic interactions moderate aggregation.
• Simple monomer-dimer models are insufficient at high protein concentrations; higher-order aggregates are present.
• The study provides insights into the aggregation pathway of lysozyme during crystallization.