Methods for finding the molecular weight of a protein are chemical, physico-chemical, and physical. The most common physicochemical methods are gel chromatography (both column and thin layer) and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. They do not require sophisticated equipment and large quantities of test material.
Protein Characterization
Proteins are high molecular weight polymers of biological origin. They consist of amino acids connected in series by peptide bonds. The sizes of proteins depend on the amount of these amino acids. The average elemental composition of proteins in%:
- carbon in the range of 50.6-54.5;
- oxygen - in the range of 21.5-23.5;
- nitrogen - about 15.0-17.6;
- hydrogen in the range of 6.5-7.3;
- sulfur - in the range of 0.3-2.5;
- mineral substances - not more than 0.5.
Proteins are divided into simple, consisting only of amino acid residues, and complex, including prosthetic groups. Non-protein components can be carbohydrates, lipids, nucleic acids, derivatives of vitamins, metal ions, heme, etc. There are four protein structures.
The amino acid composition of proteins is found by acid hydrolysis, combined with the subsequent separation of the released amino acids using ion exchange chromatography.
The quantitative indicators of each amino acid are determined by the nin-hydrin method. Identification of the placement of amino acids in a protein molecule is carried out by sequential cleavage of terminal amino acids using enzymes and their identification, which allows to determine the structure of the protein molecule. Identification is based on their various physicochemical properties. Often, color reactions to certain amino acids and chromatography are used for this.
Column gel chromatography
This method is based on a linear dependence of the logarithm of the molecular weight of many globular proteins and the volume of elution with a filled gel (a certain pore size). Thus, to determine the molecular weight of a protein, one only needs to find the volume of its elution from a previously calibrated column. Calibration is performed by passing through a column of proteins with previously known masses of molecules and measuring the volumes of elution of each of them. If Sephadex G-75 is used as a filler, the molecular weight is calculated according to the equation found experimentally:
log M = 5.624 - 0.752 · (V e / V o ),
where M is the desired molecular weight; V e - leaving the column volume of the solution with the test substance; V o is the volume of the free column.
For analysis, you will need a solution of blue dextran (1%), NaCl solution (0.1 mol / l), Sephadex G-75 (4 g), hemoglobin solution (1%).
Analysis
The prepared column is filled with Sephadex G-75 gel and washed with a NaCl solution. After the NaCl solution, rising above the gel level, is poured, 0.5 ml of a blue dextran solution is carefully placed on its surface. Then, the liquid leaving the column is collected in a measuring cylinder, where they are stored until the end of the experiment. A solution beginning to flow out with a blue color is collected 20 drops into prepared test tubes. The eluate from them from the first to the most intensely colored is poured into the same graduated cylinder, in which unpainted fractions are already collected. The volume of the tubes of the weakening color of the solution is not taken into account.
The volume of fluid collected in the measuring cylinder is made up of the free volume of the column (V o ). Next, repeat the filling of the column, but instead of blue dextrin, a hemoglobin solution is used. The volume of the eluate in the graduated cylinder until the solution with the maximum pink color is V e . The found values of V o and V e substitute in the corresponding equation and calculate the mass of the protein. The amino acid composition of proteins is found by similar methods.
Thin layer gel chromatography
The principle of this method is that during the advancement of the protein solution on the plate with the thinnest layer of Sephadex, their mixture is distributed unevenly. The distance traveled by each of the proteins from the original starting line, find the logarithms of their molecular masses. To determine the molecular mass of a protein by thin-layer gel chromatography, a calibration graph is constructed that reflects the dependence of the distances traveled by marker proteins on the logarithms of their molecular weights. Already on it find the path length of the studied protein and the mass of its molecule.
To perform the experiment, you will need a table with a varying angle of inclination, as well as a chromatographic chamber. Of the reagents you will need Sephadex G-200 or G-150, sodium phosphate buffer, pH 7.4 (0.1 mol / l), a solution of bromphenol blue (0.1%), a solution of CH 3 COOH (5%), a solution CH 3 COONa (2%), marker protein kit, chromatographic paper.
Experiment
First, it is necessary to prepare Sephadex gel, for which its dry weight of 4 g is suspended in an excess of sodium phosphate buffer, and then left to swell for 3 days at n.o. A glass plate with sides of 20x40 cm is thoroughly washed. Before applying Sephadex to it, the buffer is decanted over it, and then the gel is mixed well. It is applied to a horizontally located plate with a porcelain spoon, and then distributed by rolling a glass rod 1x22 cm in size. The rolling is repeated until the gel layer is evenly distributed without lumps and bubbles 1 mm thick. The prepared plate is dried in air for 15 minutes, and then placed in a chromatographic chamber.
Phosphate buffer is poured into the extreme tanks, the gel is combined with buffer with chromatographic paper. Next, the camera is closed and placed on a special stand. The angle of its inclination is set at 7-10 °. For saturation, the camera is left overnight.
Protein solutions, the molecular weight of which is known, as well as the test samples, are applied with a micropipette, the volume of each portion should be 0.02 ml. The plate is returned to a horizontal position and put portions of protein at specific points. The distance between them and from the upper edge should be 3 cm. Then the camera is closed and placed at an angle of 7-10 °. Gel chromatographic analysis is carried out for 4 hours
After the allotted time, the camera is placed horizontally, chromatographic paper is removed. The glass plate is placed on a stand in a horizontal position and a "replica" is removed on chromatographic paper. To do this, it is rolled up into a tube and applied to a thin gel layer, gradually unfolding. At the same time, the paper should stick to it, but it must be done carefully to keep it intact. The paper is left on the surface of the plate for 1 minute, then dried at a temperature of 90 ° C for 20 minutes. and placed in a special ditch for staining.
Deciphering the results
To identify protein zones, a "replica" is placed in a solution of bromphenol blue for 3 minutes. Next, the dye must be washed twice with a solution of acetic acid and fixed with sodium acetate. After that, the chromatographic paper is washed thoroughly in cold running water and dried. Then proceed to measure the distance traveled by each protein from the starting point to the center of the spot.
According to the data obtained, a calibration graph is built up, by plotting along the abscissa axis l a / l st (where index a refers to the analyzed one and st to the standard protein), along the ordinate axis - logM. By measuring the distance of the protein region of the test sample from the start, the molecular weight and the estimated structure of the protein molecule are determined from the built-in graph.