Protein Chemistry. Structure, Properties, Research Methods - Shendryk A.N. 2022

Methods for Experimental Investigation of Protein Structure
Methods for Determining Protein Molecular Weight
Determination of Molecular Weight by Sedimentation - Diffusion and Diffusion Coefficient

When two gases or liquids are mixed, their molecules tend to distribute evenly throughout the entire volume of the vessel. If the vessel is divided into two parts by an impermeable partition, with one half filled with one substance and the other half with another, removing the partition will cause the molecules of both substances to spread throughout the entire vessel. This process is known as diffusion. The direction of diffusion is always the same: from the region of higher component concentration toward the region of lower concentration, i.e., along the concentration gradient. In the simplest case, The rate of linear diffusion is described by Fick's first law:

Class="center">ds/dt = -Da(dc/dx)

This law states that The amount of dissolved substance (ds) diffusing over a time dt through an area A, oriented perpendicularly to the direction of diffusion, is proportional to the concentration gradient (dc/dx) in that direction. The proportionality constant, D, is called the diffusion coefficient. It is defined as the amount of substance diffusing through a surface of [1 unit of length] area at a unit concentration gradient. Because diffusion proceeds in the direction of decreasing concentration, a minus sign appears in Fick's law equation.

Diffusion coefficients of Proteins are determined in diffusion Cells by measuring the rate of Displacement of the protein-solvent interface. Such a boundary is formed within the diffusion Cell by carefully layering the solvent over the protein solution. The movement of the boundary is monitored optically. For very large protein molecules, There is a slight dependence of the diffusion rate on the molecular weight (MM). However, this dependence is relatively weak. For instance, the MM of serum albumin is twice that of ß-lactoglobulin, yet the difference in their diffusion coefficients is only 16%. Consequently, direct determination of D does not, strictly speaking, allow for a sufficiently accurate estimation of MM. Nevertheless, knowing this coefficient is essential for determining MM via sedimentation velocity (see above).