Protein diffusion coefficients determined by macroscopic-gradient Rayleigh interferometry and dynamic light...

Area of Science:

• Biophysics
• Physical Chemistry

Background:

• Dynamic Light Scattering (DLS) is a common method for measuring macromolecule diffusion coefficients.
• DLS probes microscopic concentration fluctuations, differing from classical macroscopic gradient techniques.
• The accuracy and concordance of DLS with macroscopic methods require further investigation.

Purpose of the Study:

• To compare diffusion coefficients measured by DLS and Rayleigh interferometry for lysozyme in aqueous salt.
• To establish a new standard for DLS measurements using highly accurate interferometric data.
• To investigate the fundamental nature of DLS measurements in relation to diffusion coefficient matrices.

Main Methods:

• Measurement of lysozyme diffusion coefficients using both Dynamic Light Scattering (DLS) and Rayleigh interferometry.
• Rayleigh interferometry employed as a highly accurate macroscopic-gradient technique.
• Determination of the complete diffusion coefficient matrix for lysozyme-salt coupled diffusion.

Main Results:

• Unprecedented precision achieved in diffusion coefficient measurements.
• DLS-derived diffusion coefficients were consistently 2% higher than those obtained via interferometry.
• Experimental evidence shows DLS measures an eigenvalue, not the full protein diffusion coefficient.

Conclusions:

• Interferometry provides highly accurate protein diffusion data, serving as a benchmark for DLS quality control.
• DLS measurements do not directly yield the protein diffusion coefficient but an eigenvalue of the diffusion matrix.
• This study clarifies the interpretation of DLS data in complex systems like coupled diffusion.