Separation of proteins from low molecular weight impurities
Membrane sieve method (dialysis)
A dialysis membrane is used, which is a polymer and has pores of a certain size. Small molecules (low molecular weight impurities) pass through the pores in the membrane, and large molecules (proteins) are retained. In this way, the proteins are washed away from impurities.
Separation of proteins by molecular weight
Gel chromatography
The chromatographic column is filled with gel granules (Sephadex), which has pores of a certain size. A mixture of proteins is added to the column. Proteins whose size is smaller than the size of the Sephadex pores are retained in the column, as they are “stuck” in the pores, while the rest freely exit the column. The size of a protein depends on its molecular weight.
Ultracentrifugation
This method is based on different rates of sedimentation (precipitation) of protein molecules in solutions with different density gradients (sucrose buffer or cesium chloride).
Electrophoresis
This method is based on different rates of migration of proteins and peptides in an electric field depending on the charge.
Gels, cellulose acetate, and agar can serve as carriers for electrophoresis. The molecules being separated move in the gel depending on their size: those that are large will be delayed as they pass through the pores of the gel. Smaller molecules will encounter less resistance and therefore move faster. As a result, after electrophoresis, large molecules will be closer to the start than smaller ones.
Electrophoresis can be used to separate proteins by molecular weight. To do this, PAGE electrophoresis is used in the presence of sodium dodecyl sulfate (SDS-Na).
DDS-Na is a diphilic substance and contains a charged group and a hydrophobic one. Proteins bind to SDS-Na with their hydrophobic radicals and in the process become denatured. In this way, the proteins are aligned in shape and charge. After this, the mobility of the protein during electrophoresis depends only on its molecular weight.
salting out: precipitation with salts of alkali, alkaline earth metals (sodium chloride, magnesium sulfate), ammonium sulfate; the primary structure of the protein is not disrupted;
deposition: use of water-removing substances: alcohol or acetone at low temperatures (about –20 С).
When using these methods, proteins lose their hydration shell and precipitate in solution.
Denaturation- violation of the spatial structure of proteins (the primary structure of the molecule is preserved). It can be reversible (the protein structure is restored after removing the denaturing agent) or irreversible (the spatial structure of the molecule is not restored, for example, when proteins are precipitated with concentrated mineral acids, salts of heavy metals).
Methods for protein separation Separation of proteins from low molecular weight impurities
Dialysis
A special polymer membrane is used, which has pores of a certain size. Small molecules (low molecular weight impurities) pass through the pores in the membrane, and large molecules (proteins) are retained. Thus, proteins are washed away from impurities.
Separation of proteins by molecular weight
Gel chromatography
The chromatographic column is filled with gel granules (Sephadex), which has pores of a certain size. A mixture of proteins is added to the column. Proteins whose size is smaller than the size of the Sephadex pores are retained in the column, as they are “stuck” in the pores, while the rest freely exit the column (Fig. 2.1). The size of a protein depends on its molecular weight.
Rice. 2.1. Protein separation by gel filtration
Ultracentrifugation
This method is based on different rates of sedimentation (precipitation) of protein molecules in solutions with different density gradients (sucrose buffer or cesium chloride) (Fig. 2.2).
Rice. 2.2. Separation of proteins by ultracentrifugation
Electrophoresis
This method is based on different rates of migration of proteins and peptides in an electric field depending on the charge.
Gels, cellulose acetate, and agar can serve as carriers for electrophoresis. Separated molecules move in the gel depending on their size: those that are larger will be delayed as they pass through the pores of the gel. Smaller molecules will encounter less resistance and therefore move faster. As a result, after electrophoresis, larger molecules will be closer to the start than smaller ones (Fig. 2.3).
Rice. 2.3. Protein separation by gel electrophoresis
Electrophoresis can also be used to separate proteins by molecular weight. For this use PAGE electrophoresis in the presence of sodium dodecyl sulfate (SDS-Na).
Isolation of individual proteins
Affinity chromatography
The method is based on the ability of proteins to bind strongly to various molecules through non-covalent bonds. Used for the isolation and purification of enzymes, immunoglobulins, and receptor proteins.
Molecules of substances (ligands), to which certain proteins specifically bind, are covalently combined with particles of an inert substance. The protein mixture is added to the column, and the desired protein is firmly attached to the ligand. The remaining proteins leave the column freely. The retained protein can then be washed out of the column using a buffer solution containing the free ligand. This highly sensitive method allows very small amounts of protein to be isolated in pure form from a cell extract containing hundreds of other proteins.
Isoelectric focusing
The method is based on different IET values of proteins. Proteins are separated by electrophoresis on a plate with ampholine (this is a substance in which a pH gradient in the range from 3 to 10 is pre-formed). During electrophoresis, proteins are separated according to their IET value (in IET, the charge of the protein will be zero, and it will not move in the electric field).
2D electrophoresis
It is a combination of isoelectric focusing and electrophoresis with SDS-Na. Electrophoresis is first carried out in the horizontal direction on a plate with ampholine. Proteins are separated according to charge (IET). Then the plate is treated with a SDS-Na solution and electrophoresis is carried out in the vertical direction. Proteins are separated based on molecular weight.
Immunoelectrophoresis (Western blot)
An analytical method used to identify specific proteins in a sample (Figure 2.4).
Isolation of proteins from biological material.
Separation of proteins by molecular weight by electrophoresis in PAGE with SDS-Na.
Transfer of proteins from the gel to a polymer plate to facilitate further work.
Treatment of the plate with a solution of nonspecific protein to fill the remaining pores.
Thus, after this stage, a plate is obtained, the pores of which contain separated proteins, and the space between them is filled with a nonspecific protein. Now we need to determine whether among the proteins there is the one we are looking for that is responsible for some disease. Antibody treatment is used for detection. Primary antibodies are antibodies to the protein of interest. Secondary antibodies mean antibodies to primary antibodies. An additional special label (so-called molecular probe) is added to the secondary antibodies so that the results can then be visualized. A radioactive phosphate or enzyme tightly bound to a secondary antibody is used as a label. Binding first to primary and then to secondary antibodies has two purposes: standardization of the method and improvement of results.
Treatment with a solution of primary antibodies binding occurs in the place of the plate where the antigen (the desired protein) is located.
Removal of unbound antibodies (washing).
Treatment with a solution of labeled secondary antibodies for subsequent development.
Removal of unbound secondary antibodies (washing).
Rice. 2.4. Immunoelectrophoresis (Western blot)
If the desired protein is present in the biological material, a band appears on the plate, indicating the binding of this protein to the corresponding antibodies.
The study of physicochemical properties, chemical composition and structure is possible only by studying a purified protein preparation. To isolate and fractionate individual proteins, the following are used: salting out, precipitation with organic solvents, gel filtration, electrophoresis, ion exchange chromatography, affinity chromatography.
Salting out proteins based on the dependence of protein solubility on the properties of the medium. Proteins are less soluble in distilled water than in weak salt solutions, since low ion concentrations maintain their hydration shells. But at high salt concentrations, protein molecules lose their hydration shells, aggregate, and a precipitate forms. After the salt is removed, the proteins return to solution, maintaining their native properties and conformation.
Changes in solubility at different salt concentrations and pH are used to isolate individual proteins. Most often, solutions of ammonium sulfate of different concentrations are used to salt out proteins.
Precipitation of proteins from solution without their denaturation is carried out using dehydrogenating agents - organic solvents (ethanol, acetone).
Gel filtration based on the separation of proteins according to the size and shape of the molecule. The separation is carried out in chromatographic columns filled with porous gel granules (Sephadex, agarose) in a buffer solution with a certain pH value. Gel granules are permeable to proteins thanks to internal channels (pores) with a certain average diameter, the size of which depends on the type of gel (Sephadex G-25, G-200, etc.). The protein mixture is added to the column and then washed out (eluted) with a buffer solution with a certain pH value. Large protein molecules do not penetrate the pores of the gel and move at high speed along with the solvent. Small molecules of a low molecular weight impurity (salt) or other protein are retained by the gel granules and are washed out of the column more slowly (Fig. 1.29). At the column outlet, the solution (eluate) is collected in the form of separate fractions.
Rice. 1.29. Protein separation by gel filtration
Electrophoresis is based on the property of charged protein molecules to move in an electric field at a speed proportional to their total charge. Proteins that have a total negative charge at a given pH value move toward the anode, and a positive charge moves toward the cathode. Electrophoresis is carried out on different media: paper, starch gel, polyacrylamide gel, etc. The speed of movement depends on the charge, mass and shape of protein molecules. After completion of electrophoresis, the protein zones on the carrier are stained with special dyes (Fig. 1.30, A).
The resolution of electrophoresis in a gel is higher than on paper, so when electrophoresing blood serum proteins on paper, 5 fractions are isolated (albumin, α 1 -, α 2 -, β-, γ-globulins), and in a polyacrylamide gel - up to 18 fractions ( Fig. 1.30, B).
Rice. 1.30. Electropherogram of blood serum proteins of a healthy person
A- electropherogram of blood serum proteins on paper;
B- the amount of plasma proteins of different fractions.
I - γ-globulins; II - β-globulins; III - a 2 -globulins;
IV - a 1 -globulins; V - albumins
Ion exchange chromatography based on the separation of proteins that differ in total charge. A protein solution with a certain pH value is passed through a chromatographic column filled with a solid porous sorbent, while some of the proteins are retained as a result of electrostatic interaction. Ion exchange substances are used as sorbents: anion exchangers (containing cationic groups) to isolate acidic proteins; cation exchangers (containing anionic groups) to isolate essential proteins.
When passing a protein through a column, the strength of its binding to the ion exchanger depends on the magnitude of the charge opposite to the charge of the sorbent. Proteins adsorbed on an ion exchange sorbent are eluted with buffer solutions with different salt concentrations and pH, obtaining different protein fractions.
Affinity chromatography is based on the binding specificity of the protein to the ligand attached to the solid support. Enzyme substrates, prosthetic groups of holoproteins, antigens, etc. are used as ligands. When passing a mixture of proteins through the column, only the complementary protein attaches to the ligand (Fig. 1.31, A), all the others come out along with the solution. The adsorbed protein is eluted with a solution with a different pH value (Fig. 1.31, B). This method is highly specific and allows one to obtain highly purified protein preparations.
Protein isolation and purification usually takes place in several steps using different methods. The sequence of steps is selected empirically and may vary for different proteins. A high degree of protein purification is very important both when using them as medicines (the hormone insulin, etc.) and when diagnosing various diseases by changing the protein composition of tissues, blood, saliva, etc.
The set of proteins in the cells of various organs of an adult is individual and is maintained relatively constant throughout life. Specialized tissues may contain specific proteins, such as hemoglobin in red blood cells, actin and myosin in muscles, rhodopsin in the retina, and different types of collagen in bone and connective tissues. Some proteins are found in many tissues, but in different quantities. Selected composition changes
Rice. 1.31. Separation of proteins by affinity chromatography
A- binding of the isolated protein to a specific ligand attached to a neutral carrier; B- obtaining a solution of individual protein
proteins of tissues and blood are possible and are associated primarily with diet, food composition, and physical activity of a person.
In diseases, the protein composition of blood and tissue cells can change significantly; deficiency of any protein or a decrease in its activity often develops - proteinopathy. Therefore, the determination of pronounced changes in the protein composition of blood and tissues is used to diagnose various diseases in clinical studies.
After extracting a mixture of proteins from biological material, it is separated into individual protein fractions. Several protein fractionation methods have been developed, based on different physicochemical properties of proteins.
– precipitation of proteins at the isoelectric point – the method is based on the property of proteins to precipitate at the isoelectric point due to neutralization of the charge of the protein molecule. For each protein, the value of the isoelectric point is strictly individual, so this method makes it possible to isolate individual proteins (for more information about the method, see laboratory work No. 3 in the “Biochemistry” course).
– protein fractionation by salting out method – based on the different solubility of proteins in concentrated solutions of neutral salts, depending on the molecular weight (for more information about the method, see laboratory work No. 3 in the “Biochemistry” course).
– electrophoretic separation method proteins into fractions - described in the section physicochemical properties of proteins.
In addition to the methods presented above, they are widely used to separate proteins into fractions. chromatographic methods . Column chromatography is most often used.
The peculiarity of this method is that a mixture of molecules of various proteins and peptides is passed through a column containing a solid porous material (matrix). As a result of interaction with the matrix, different proteins pass through the column at different rates. After the proteins reach the bottom of the column in a certain sequence, they are collected in separate fractions into test tubes.
Allocate three main types column chromatography:
– ion exchange – for protein chromatography, ion exchangers based on cellulose or other hydrophilic polymers are used, for example, diethylaminoethylcellulose (DEAE-cellulose), containing cationic groups (negative charge) or containing carboxymethylcellulose (CM-cellulose), containing amine groups (positive charge):
The stronger the binding of proteins to DEAE-cellulose, the higher the number of carboxyl groups in the protein molecule. Proteins adsorbed to DEAE cellulose can be washed (eluted) from the column with solutions of increasing concentrations of sodium chloride. Loosely bound proteins are eluted first, and as the salt concentration increases, other proteins are eluted, in order of increasing affinity for DEAE-cellulose.
CM cellulose is used similarly, but the affinity of proteins for it is directly proportional to the number of amino groups in the protein molecule.
To remove bound protein, the pH of the eluent is also changed.
– gel filtration chromatography – has the second name “molecular sieve method”. Sephadex (polysaccharide dextran treated with epichloride hydrin) is used as sieves. Sephadex grains swell in water and form a gel. The swollen granules have pores of a certain diameter.
The separation is based on the fact that Sephadex grains (the pores of the granules) are impermeable or limitedly permeable to substances with a large molecular weight, and small molecules freely diffuse (penetrate) into the pores of the grains.
A gel-like mass of swollen Sephadex is placed in a glass column (tube), a layer of protein solution is applied to the surface of the gel (Fig. 12, a) and then a buffer solution (eluting liquid) is passed through the column. Proteins pass along the column between granules the more slowly, the lower their molecular weight, since protein molecules with an even lower molecular weight diffuse more easily into the granules (into the pores) (Fig. 12).
Rice. 12. Protein fractionation by gel filtration
Proteins are washed out (eluted) from the column in descending order of molecular weight. Consequently, large protein molecules are eluted first (Fig. 12, b), which do not diffuse into the grains, then small molecules and, lastly, low-molecular impurities.
This method is used not only for fractionating proteins by molecular weight, but also for purifying them from low molecular weight impurities.
– affinity chromatography – or affinity chromatography. The principle of the method is that selective interaction of proteins occurs with specific substances - ligands attached to carriers (Fig. 13).
Cyanogen bromide-activated Sepharose is used as a carrier. Ligands of various origins are attached to Sepharose - a substrate, or an antigen, or a receptor, which will affinity bind only one protein from the mixture:
– substrate → enzyme;
– antigen → antibody;
– hormone → receptor for a given hormone.
Other proteins that are not bound to the ligand are removed by washing the column.
Rice. 13. Mechanism of affinity chromatography
Removal of affinity-bound protein from the column is carried out using a buffer solution (eluent). A detergent is introduced into the buffer, which weakens the bonds between the protein and the ligand, or a solution with a high concentration of free ligand is passed through the column. In this case, the protein binds more easily to the free ligand and is washed out (eluted) from the column.
Laboratory work No. 8
ELECTROPHORETIC SEPARATION OF PROTEINS
The method is based on the fact that protein molecules have an electrical charge, the magnitude and sign of which are determined by the amino acid composition of the protein, pH and ionic strength of the environment. Under the influence of an external electric field, charged molecules move in the solution towards the oppositely charged pole. The speed of movement of protein particles is proportional to the magnitude of their charge and inversely proportional to the size of the particles and the degree of their hydration.
The so-called “zonal electrophoresis” is currently widespread - electrophoresis on a solid carrier (on paper strips, agar, starch, acrylamide) impregnated with a buffer solution with the desired pH value. The position of proteins on paper or gel is determined by fixing and then staining them with one or another dye (usually bromophenol blue, amide black or Coomassie blue). The amount of protein in each fraction can be approximately determined by the color intensity of the associated dye. This definition does not give a strictly quantitative ratio of protein fractions, since the amount of dye bound by different proteins is not the same.
ELECTROPHORESIS HA PAPER
The separation of the analyzed mixture occurs on certain types of chromatographic paper impregnated with a buffer solution in electrophoresis devices. Proteins are separated at voltages up to 500 V.
The electrophoresis chamber consists of a plexiglass bath and a lid fitted to it (1). The bath has 2 electrode compartments (2), each of which is divided by a longitudinal partition (3) into two compartments communicating with each other. Bo the inner compartments of the compartments lower the electrodes, and the ends of the paper strips into the outer compartments (4), the main part of which is placed on a horizontal plate with spikes (5), located in the central part of the chamber. There are sticks between the horizontal plate and the outer compartment of the electrode compartments (6),
Fig.2. Device diagram for low-voltage electrophoresis
through which paper strips are thrown and which serve to support them. Under the top cover of the chamber there is a plate made of plexiglass with large round holes (7), on which sheets of filter paper soaked in distilled water and folded 4-5 times are placed on top. These sheets help to increase the tightness of the chamber and, as a result, reduce the evaporation of liquid from electropherograms during electrophoresis.
Using paper electrophoresis, students are asked to separate blood serum proteins. Using this method, blood serum can be divided into 5 - 9 fractions and the relative protein content in each of them can be determined. The separation is carried out in a buffer solution (pH 8.6 - 8.9) with a potential gradient of 3 - 5 V/cm (120 - 350 V for strips 40 - 45 cm long) at room temperature. The current should not exceed 0.1 - 0.3 mA for each centimeter of the cross-section of the paper strip. Increasing the current strength by more than 2 times is unacceptable, since this will result in excessive heating, a significant increase in evaporation and V ultimately - burning of paper
Reagents:
1. Buffer solution. Can be used:
a) veronal-medinal buffer (pH 8.6): dissolve 10.32 gmedinal (sodium salt of veronal) in 300 ml of distilled water, add 1.84 gveronal, heat with stirring in a water bath until dissolved and add water to 1 liter;
b) veronal-acetate buffer (pH 8.6): 4.3 veronal, 0.95 sodium hydroxide and 3.24 sodium acetate are dissolved in 300 ml of distilled water. Add 30 ml of 0.1 M HCl solution to the solution and add water to 1 liter;
c) Tris buffer (pH 8.9): 60.5 g of Tris, 6 ethylenediaminetetraacetic and 4.6 gboric acid are dissolved in 1 distilled water.
2. Solutions for coloring electropherograms:
a) acidic blue-black dye (similar to amide black 10 B) -0.2 g of mixture: acetic acid (glacial) - 100 ml + methyl alcohol - 900 ml;
b) bromophenol blue -0.5 g, mercuric chloride -10 g, acetic acid (glacial) - 20 ml, distilled water - 980 ml;
c) bromophenol blue - 0.1 g, ZnSO 4 7H 2 O -50 g, acetic acid (glacial) 50 ml, distilled water - 900 ml.
3. Solutions for washing electropherograms from dye not bound to the protein and fixing the dye on the protein:
a) acetic acid - 2% solution;
b) sodium acetate - 2% solution prepared with a 10% solution of acetic acid.
4. Solutions for the elution of colored products from electropherograms:
a) to extract bromophenol blue -0.01 M NaOH solution;
b) to extract the acidic blue-black dye -0.1 M NaOH solution.
Equipment: test tubes; cuvettes, spectrophotometer, electrophoresis device, chromatographic paper: FN4, FN5, Whatman 3, Whatman 3MM, etc.
Obtaining blood serum. 2 - 3 ml of blood is drawn into a dry centrifuge tube and left for 1/2 - 1 hour. Using a thin glass rod, carefully circle the walls of the tube to separate the clot from them, centrifuge and pour the serum into a clean tube.
Preparing the camera. The electrode compartments are filled with buffer solution to the same level (to avoid buffer overflow), approximately 800 ml in each compartment. Bo the internal parts of the electrode compartments immerse the electrodes. On a sheet of chromatographic paper (18x45 cm) (when using thin types of paper, it is better to apply samples on separate strips 4-5 cm wide) at a distance of 15 cm from one of its narrow sides, use a simple soft pencil (graphite prevents the spreading of liquid) to outline the places for applying samples. They are rectangles (2 x 0.3 cm), the large sides of which are perpendicular to the length of the paper strip. The distance between the starting zones and the edges of the electropherogram is 2 cm. The electropherogram is impregnated with the buffer in which electrophoresis will take place. To do this, it is pulled through a cuvette with a buffer solution. The ends of the paper strips (6-8 cm) are not wetted. Excess buffer is removed by blotting the strips between two or three sheets of filter paper. The wet electropherogram is placed in the chamber on the central horizontal plate (5), and the ends are lowered into the outer compartments of the electrode compartments. The device is tightly closed with a lid, under which there are sheets of filter paper moistened with water.
Carrying out electrophoresis. After the paper strips are completely saturated with the buffer solution, samples of 0.01 - 0.02 ml (1 - 2 mg of protein) of serum are applied to the marked areas using a 0.1 ml pipette. The chamber is closed with a lid and the current is turned on. The duration of electrophoresis is 22 - 24 hours at a voltage of 200 - 300 V
Fixation and staining of electropherograms. At the end of electrophoresis, turn off the current and immediately remove the electropherograms from the device. They are placed on a special stand and dried in air under draft, then in an oven at 105 ºC for 20 minutes to fix the proteins on paper, after which they are placed in an enamel cuvette, filled with dye and left for 2 - 3 hours or more. The dye is drained and the electropherograms are washed from its excess by pouring 3-4 times with a 2% solution of acetic acid, each time for 5-10 minutes. Areas of paper that do not contain protein must be completely free of dye. To fix the colored products, electropherograms are filled with a 2% solution of sodium acetate for 2 minutes and dried in air under suction.
Determination of the ratio of individual protein fractions. At pH 8.6, serum proteins are negatively charged and move in the electric field towards the anode. The fraction that moves fastest to the anode is albumin, followed by α 1 -, α 2 -, β- and γ-globulins (see Fig. 3) . Sections of paper tapes on which protein stains have appeared are divided by transverse lines with a simple pencil into strips 3-5 mm wide and cut along these lines. Each strip is crushed and placed in a separate numbered test tube, filled with 3 ml of 0.01 M NaOH solution, left for an hour to remove the paint from the paper, and then the optical density value is found for each solution on a photocolorimeter (spectrophotometer) at 612 nm.
Rice. 3. Electropherogram of human blood serum and distribution curve of protein fractions
A control sample is processed in parallel. For it, a strip is cut out from the unstained areas of the electropherogram.
Based on the data obtained, a distribution curve of colored products is plotted on the electropherogram. The numbers of the tubes are marked on the abscissa axis, and the corresponding optical density value is marked on the ordinate axis (see Fig. 3) . The percentage of protein fractions in blood serum is calculated. To do this, the drawn curve is divided according to the minimums into a number of sections corresponding to individual fractions. The size of the area of each section is proportional to the amount of paint combined with the protein of a given fraction. The ratio between these areas is calculated by weight (the weight of sections of paper is proportional to their area), the entire area is taken as 100%. If you have a densitometer, the ratio of protein fractions in blood serum can be determined from a densitogram.
Having previously determined the protein content in the whey, its amount is calculated for each fraction.
