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A kinetic, chromatographic method for studying protein hydrodynamic behavior.

L A Larew¹, R R Walters

¹Department of Chemistry, Iowa State University, Ames 50011.

Analytical Biochemistry

|August 1, 1987

Summary

A new chromatographic method determines protein mass transfer coefficients using split-peak analysis. This technique accurately measures protein diffusion, unaffected by stationary phase interactions, and can track protein denaturation.

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Area of Science:

Biochemistry
Chromatography
Physical Chemistry

Background:

Chromatographic methods are essential for analyzing protein behavior.
Understanding mass transfer coefficients is crucial for predicting protein behavior in chromatographic systems.
Existing methods may be confounded by protein-stationary phase interactions.

Purpose of the Study:

To develop a novel chromatographic method for determining the coefficient of mass transfer of proteins.
To validate the method's theoretical underpinnings relating mass transfer to diffusion and particle size.
To demonstrate the method's utility in measuring protein diffusion coefficients and monitoring conformational changes, such as denaturation.

Main Methods:

Utilized a "split-peak" chromatographic behavior on small reversed-phase columns.

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Employed chromatographic theory to establish relationships between mass transfer, diffusion, and particle diameter.

Applied the method to measure changes in bovine serum albumin's mass transfer coefficient during denaturation induced by decreasing pH.

Main Results:

The coefficient of mass transfer was found to be linearly dependent on the protein translational diffusion coefficient.
The coefficient of mass transfer showed an inverse square relationship with the support particle diameter.
The method successfully monitored the denaturation of bovine serum albumin by tracking alterations in its mass transfer coefficient.

Conclusions:

The developed "split-peak" chromatographic method provides an accurate determination of protein mass transfer coefficients.
The method is robust as results are independent of protein-stationary phase interactions.
This technique offers a valuable tool for measuring protein diffusion coefficients and studying protein conformational dynamics.