Protein molecules are amphoteric bases because they contain free amino and carboxy groups. At a pH of a solution of less than 7, they have a negative charge, and at a pH of more than 7, they have a positive charge. With the equality of these charges, which is achievable when a certain acidity or alkalinity is established in the solution, the so-called isoelectric point of the protein is established.
The concept of isoelectric point
Proteins are made up of amino acids. Some of these compounds (arginine, aspartic acid, histidine, glutamic acid, lysine) are presented in the form of radicals containing ionic groups, that is, such groups that are capable of ionization. In addition to them, alpha-carboxyl and amino groups located on the carbon and nitrogen ends of polypeptide chains are capable of ionization. If the pH of the solution is 7 or close to this mark, then all ionogenic groups are in the ionized state. As the pH moves away from a given pH value in one direction or another, and mainly in the acidic one, the protein begins to transition to the isoelectric state, in which the molecule of this substance becomes electrically neutral, the number of ionized groups tends to zero. The pH at which the proteins go into an isoelectric state is called the isoelectric point of proteins (IET).
Physico-chemical nature of proteins
Due to the fact that carboxy and amine groups are part of proteins, they can dissociate as bases and as acids. During dissociation, the free carboxyl group gives off a positively charged hydrogen ion and COO– anion. As a result, the hydrogen ion joins the amino group, which characterizes the basic properties of the protein, resulting in the formation of protein particles with negative and positive charges. When a protein is placed in an acidic solution, its acid dissociation will be suppressed due to the significant presence of hydrogen cations. Conversely, when placed in a basic solution, its main dissociation will be suppressed due to the presence of COO– anions.
Passing an electric current through a protein solution will cause the anions to move toward the cathode, and the cations to the anode. In any protein there is a certain pH value at which the movement of ions during the passage of current will not occur. In this case, they speak of the equality of multidirectional ions and the equality of different degrees (basic and acidic) in the protein molecule, which is characterized by an isoelectric state.
As you know, water is a dipole, so it places its particles around a protein molecule, depending on how it is charged. At the isoelectric point, the protein molecule does not have a hydrate-oriented shell. If protein precipitation is carried out, it is necessary, first of all, to destroy the hydration shell by removing the electric charge.
Use of IET in industry
In the isoelectric state, some properties of the protein solution, such as swelling, viscosity, osmotic pressure, light transmission, have minimum values, while the refractive index and optical density reach, on the contrary, the maximum value. The isoelectric point of the protein can be determined empirically by determining the dependence of the above properties of the protein solution: on the pH value, while the IET is determined by the position of the extrema in the graphs. In the isoelectric state, casein is able to precipitate, which is used in the production of cheeses and dairy products, to obtain casein from skim milk, as a raw material in various industries (casein glues, artificial foods, etc.). The measurement of IET allows us to assess the quality of the protein, in particular, the dairy product for the presence of impurities. This is relevant today, since the introduction of herbal supplements into the milk base allows replacing part of the animal protein with vegetable protein, which is cheaper.
In addition, the isoelectric point of the protein can be used in wastewater treatment from poultry farms. Thus, the main share of wastewater pollution from the slaughterhouse of the poultry farm is accounted for by blood proteins. Considering that the IET of most proteins is in the zone of a weakly acid reaction of the medium, the most complete extraction of proteins will occur during the weakly acid reaction of the medium at a pH value that tends to IET.
Factors Affecting IET
IET is influenced by several factors. Consider what the isoelectric point of proteins depends on. First of all, it is determined by the predominance of amine or carboxy groups in the protein molecule. Most of the proteins are stronger acids compared to bases, therefore there is less than 7 for them. There is a group of proteins that are stronger than acids, for them more than 7. There is a strong correlation between the protein isoelectric point and ion content salts in solution. The concentration of protein has no effect on this indicator. The factors considered allow us to understand why the isoelectric point is different for different proteins.
Examples of IET proteins:
- pepsin has an IET value of about 1;
- casein and gelatin - 4.7;
- egg albumin - 4.8;
- mucin - 2.7;
- pepsinogen - 3.7;
- albumin - 4.6;
- insulin 5.3;
- oxyhemoglobin - 6.8;
- carboxyhemoglobin - about 6.9;
- myoglobin - 7.0;
- chymotrypsin - 8.6;
- cytochrome C - 10.5;
- salmin - 12.
IET and its definition
All methods for determining the isoelectric point of proteins are based on the preparation of buffer solutions having a different reaction medium. In all these solutions, equal weighed portions of the studied protein are placed, which can be either in dry form or in the form of a solution. Various methods for determining IET are used. How to determine the isoelectric point of a protein?
The main methods for determining IET are electrophoresis, which minimizes viscosity and is associated with the use of water-taking substances. Some other methods may be used, such as determining the degree of swelling of the dry protein, the speed of gelation, but they are less accurate and require a large amount of protein.
Electrophoresis
When using this method, strips of chromatographic or filter paper moistened with a specific buffer solution are placed in the device for its implementation. A pencil mark is made in the middle of each strip, into which one drop of the studied protein solution is applied with a pipette. Then the device is turned on and electric current is passed through these strips. Macromolecules change their charge depending on the pH of the buffer solution. If the pH exceeds IET, then a negative charge of macromolecules is observed, and vice versa.
If the pH is IEI, then the macromolecules become neutrally charged. After a certain time, the current supply ceases, the strips of paper are removed from the device and dried, after which the protein spots are sprayed with ninhydrin for their manifestation. IET is installed on a buffer solution of a strip of paper, where the protein stain remains in the same place where the drop was applied. If necessary, this method can also be used for fine fractionation of proteins.
The use of other methods to determine IET
When in the isoelectric state, the protein molecules are less hydrated, therefore, the isoelectric point of the protein can be determined using the minimum viscosity method. For its use, a viscometer is required. Using this device, the relative viscosity of the buffer solutions is determined. The molecules of the protein in the isoelectric state are folded, so the solution will have the smallest viscosity, in which its pH will coincide with the IET.
The method associated with the action of dewatering agents is based on the same property. Such agents may be acetone, ether or alcohol. The selection of proteins from the corresponding solutions potentially occurs the faster and more complete, the more fully corresponds to the reaction of the IET medium. At the isoelectric point, protein solutions are unstable.
Thus, there are various methods for determining the isoelectric point of a protein. And its determination should be carried out depending on the available equipment, materials, the amount of protein.
IET protein stability
At the isoelectric point of the protein, the repulsive forces between the protein particles in the macromolecule are weakened, due to which the aggregation of these molecules occurs and the protein precipitates. This indicates that the protein in the IET is unstable due to the loss of charge, which is a factor in the stabilization of aqueous protein solutions. If acid or base is added to the protein, the molecules are recharged, the protein makes a transition to the solution.
Finally
Thus, the isoelectric point of the protein is the value of the reaction of the medium (pH) at which the equality of the multidirectional (negative and positive) charges and the equality of the different degrees (basic and acid) of dissociation are observed in the protein molecule. At this point, the protein loses its charges and becomes unstable, as a result of which it precipitates. The protein molecule coagulates, while it carries certain charges, it is straightened in the form of a thread.