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

Methods for Experimental Investigation of Protein Structure
Methods for Determining the Molecular Weight of Proteins

The Physicochemical Methods used to determine the molecular weight (MW) of Proteins are divided into two groups. One group comprises Methods for determining the number-average molecular weight (M_n):

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where n_i is the number of moles of protein with a Molecular Weight of M_i.

The number-average MW is essentially the arithmetic mean MW. These methods are based on measurements of colligative properties of solutions, such as osmotic pressure, which is determined by the concentration of the dissolved substance.

The other group brings together methods for determining the weight-average molecular weight (M_W):

where c_i = n_iM_i /V is the weight concentration in g/cm³. Or

These methods are based on the direct measurement of mass.

The fundamental difference between these two groups of methods is as follows. If a protein solution is monodisperse (all protein molecules are of the same size), both groups of methods will yield the same MW value. However, if the solution is polydisperse (protein molecules vary in size), the weight-average MW turns out to be greater than the number-average MW. There are various reasons why molecules of the same protein in solution may differ in size. Most commonly, this is due to dissociation into subunits or the association of several molecules into protein aggregates. Therefore, comparing the weight-average and number-average MW serves as one of the ways to detect supramolecular Structure/178.html">Protein Complexes.

According to the measurement techniques used, methods for determining protein MW are subdivided into static and dynamic. The static group consists of Methods Based on measuring:

> osmotic pressure;

> saturated vapor pressure;

> light scattering.

Dynamic methods include those based on:

> studying diffusion;

> measuring solution viscosity;

> sedimentation analysis;

> Gel Chromatography;

> Electrophoresis.

In the early Stages of Protein chemistry, an analytical method was used to determine molecular weight. It was based on available data regarding The chemical composition of the protein molecule. For example, it is known that a Myoglobin molecule contains a single prosthetic group with an iron atom. The mass of this element accounts for 0.335% of the total mass of the protein sample analyzed. Hence, the minimum MW of a single myoglobin molecule is determined as:

Currently, this method is practically no longer used.