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

Methods for Experimental Study of Protein Structure
Protein Separation Methods
Experimental Methods of Electrophoresis

There are three MAIN TYPES OF Electrophoresis (EP):

> frontal;

> zonal;

> stationary.

In frontal EP, an electric field is applied to the system once a distinct, well-defined boundary has formed between the solution of charged particles and the medium (solvent or buffer system). During the course of electrophoresis, this boundary moves.

Zonal EP relies on The Use of a stabilized porous medium (such as paper, gel, or blocks of granulated material). During electrophoresis, molecules of the same type cluster into distinct zones.

Stationary (or displacement) EP is characterized by the establishment of a state of equilibrium some time after Separation begins.

Let us examine the aforementioned types of electrophoretic separation in greater detail using protein mixtures as an example.

Extensive research into the electrochemical Properties of Proteins was conducted during the 1920s and 1930s. This work established that protein molecules are amphoteric polyelectrolytes, meaning they carry both positive and negative charges simultaneously. When applied to proteins, the term "amphoteric" indicates that they can be titrated with both acids and alkalis. In other words, over a fairly wide pH range, a protein molecule bears both positive and negative charges concurrently. In strongly acidic environments (pH = 1), virtually all proteins acquire a maximum positive charge, whereas in strongly alkaline media (pH = 13), they reach a maximum negative charge. There are, however, exceptions; Histones, for instance, retain a positive charge even at pH = 11.

Differences in the charges of protein molecules were first utilized to separate protein mixtures in 1937 by the Swedish researcher Arne Tiselius, who designed the first apparatus for performing electrophoresis. In recognition of his achievements in this field, Tiselius was awarded the Nobel Prize in 1948.

For a long time, electrophoresis remained virtually the sole quantitative method for protein analysis. It continues to be widely used today, although it has been superseded in certain Applications by modern physicochemical techniques, notably Chromatography, Gel filtration, ultracentrifugation, and density-gradient centrifugation.

Protein molecules contain both basic and acidic ionizable groups, including carboxyl, hydroxyl, amino, guanidino, phenolic, imidazole, and thiol groups. These are ionizable (ionogenic) groups. For every protein, There is a distinct, characteristic pH value at which the net charge of the protein molecule is zero—this is the isoelectric point (pI). At this point, the protein molecule contains an equal number of positive and negative charges, a distinction that is crucial to understand. A zero net charge does not mean the absence of charges; rather, it signifies that the number of positively and negatively charged groups within the same molecule is equal. At pH values above the pI, a protein carries a net negative charge, while below the pI, its net charge is positive. Most Serum proteins have their isoelectric points within the pH range of 4–6. In an electric field, positively charged particles migrate toward the cathode, while negatively charged particles move toward the anode.