Solubility factors and electrical charge
The ability of proteins to dissolve in water depends both on their intrinsic characteristics (ratio of polar to nonpolar residues, resistance to denaturation) and environmental parameters: temperature, salt composition, presence of organic substances, and acidity (pH).
A key role is played by the net charge of the molecule. It is formed by ionized amino acid groups — negative carboxyl groups and positive amino groups. A change in environmental pH shifts this balance:
- In an acidic environment (with an excess of hydrogen protons), the dissociation of carboxyl groups is suppressed. The protein loses negative charges and becomes more positive.
- In an alkaline environment (with an excess of hydroxyl ions), protons are removed from amino groups, turning them into neutral ones. As a result, the protein loses its positive charge and becomes more negative.
Isoelectric point (pI)
There is a specific pH value at which the number of positively and negatively charged groups in a protein molecule equalizes. This state is called the isoelectric point.
In the isoelectric state, the net charge of the protein is zero. As a result, electrostatic repulsion forces between molecules disappear, they begin to stick together (aggregate) and precipitate. It is at the pI that protein solubility is minimal.
A striking example of this process is milk curdling. In fresh milk, casein protein molecules are negatively charged and form a stable colloidal solution. As bacteria multiply, lactic acid is released, which lowers the pH. When acidity reaches the isoelectric point of casein (about 4.6), its charge is neutralized, molecules stop repelling each other, and milk curdles.
Principles of protein isolation and purification
To study the properties of a specific protein, it must be extracted from cells and purified. The standard algorithm includes several stages:
- Cell lysis to obtain a homogenate.
- Fractionation (centrifugation) — separation of the mixture into nuclear, mitochondrial, cytosolic, and other fractions to isolate the one containing the target protein.
- Selective thermal denaturation — brief heating. If the target protein is thermostable, heating will cause thermolabile impurities to denature and precipitate, leaving the target protein in solution.
- Salting out — addition of salts (most often ammonium sulfate). Proteins with the lowest solubility precipitate at low salt concentrations. By gradually increasing the concentration, the mixture can be separated into individual fractions.
Separation of proteins by mass
To separate macromolecules based on their size and weight, ultracentrifugation, polyacrylamide gel electrophoresis, and gel filtration (molecular sieve chromatography) are used.
The principle of gel filtration is based on passing a mixture through a column packed with porous beads. Large proteins physically cannot penetrate the gel pores; they travel through the void space between the beads and elute from the column first. Small molecules penetrate into the pores, significantly lengthening their path, so they leave the column last.
Another important method is paper electrophoresis, which separates proteins in an electric field. In the blood serum of a healthy person, albumins move toward the anode the fastest (the largest fraction, 54–58%). They are followed by globulins (alpha-1, alpha-2, and beta). Gamma-globulins have the lowest mobility, forming the cathodic fraction.
Briefly: answers to topical questions
What salts besides ammonium sulfate are used for protein salting out?
For protein salting out, besides ammonium sulfate, sodium sulfate and sodium chloride are used.
Salting out is the reversible precipitation of a protein from solution using high concentrations of neutral salts. Salt ions become hydrated, stripping water molecules away from protein molecules and neutralizing their charge, which leads to protein aggregation and precipitation.
What chemical reagents are used for irreversible protein precipitation?
Irreversible precipitation (denaturation) of proteins is carried out using the following chemical reagents:
- Acids and alkalis — alter the ionization of ionogenic groups, breaking ionic and hydrogen bonds.
- Organic solvents (alcohol, phenol, chloramine) — disrupt hydrophobic interactions and hydrogen bonds.
- Heavy metal salts — form insoluble protein salts.
- Urea — disrupts intramolecular hydrogen bonds.
- Detergents.
What determines the buffer properties of protein solutions?
The buffer properties of protein solutions are due to the presence of amino and carboxyl groups in their molecules, which can act as acids and bases.
The composition of the protein buffer system includes a protein molecule (NH₂-Prot-COOH) and its salt (NH₂-Prot-COONa). When an acid is added, the protein binds protons, and when bases are added, it donates protons, thereby maintaining the pH of the medium. The buffer capacity of proteins is greater for acids than for alkalis.
What types of chemical bonds are disrupted during thermal denaturation of a protein?
During thermal denaturation of a protein, weak bonds stabilizing its tertiary and secondary structure are disrupted.
These bonds include:
- Hydrogen bonds.
- Hydrophobic interactions.
- Ionic bonds.
The disruption of these bonds occurs due to increased chaotic molecular motion at high temperatures (above 60°C), while peptide bonds (primary structure) remain intact.
To avoid confusion with gel filtration results, imagine that the gel beads are houses with narrow doors. "Fat" (large) molecules cannot enter the house, so they quickly run along the street and reach the finish line first. "Thin" (small) molecules enter every door, wander through the rooms for a long time, and arrive last.
Class and exam questions
Why do denatured proteins precipitate more easily?
During denaturation, the spatial structure of the molecule is disrupted, and the hydrophobic amino acid radicals, which are normally hidden inside, end up on the surface. This sharply reduces the solubility of the protein in water.
What pH value must be established for a peptide with a charge of -1 to reach its isoelectric point?
Since the peptide charge is negative, it must be neutralized by adding protons (positive charges). To do this, the medium must be acidified, meaning the pI will lie in an acidic environment (pH < 7).
What happens to the protein charge when alkali is added?
In an alkaline environment, an excess of hydroxide ions is present. They bind protons by stripping them from amino groups. As a result, the positive charge of the protein decreases, and it becomes more negative.
What else to study on this topic
- Rules for calculating the net peptide charge based on amino acid residues
- Proteinogram of a healthy individual: percentage ratio of fractions
- Principles of polyacrylamide gel electrophoresis (in the presence of SDS)
- Molecular mechanisms of reversible protein salting-out
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