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Physicochemical Properties of Proteins

Q: What happens to the charge of a protein when an alkali is added?

In an alkaline environment, there is an excess of hydroxyl ions. They bind protons, stripping them from amino groups. As a result, the positive charge of the protein decreases, and it becomes more negative.

For medical students2 min readUpdated 2026-10-10

Proteins are complex macromolecules whose behavior in solution is strictly governed by the laws of physics and chemistry. The primary factors determining their stability and solubility are molecular weight, molecular shape, and, above all, their net electrical charge.

Molecular ChargeDepends on the balance of cationic and anionic amino acid residues within the protein.

DenaturationDuring denaturation, hydrophobic side chains are exposed to the surface, reducing solubility.

Isoelectric PointThe pH value at which the net charge of the protein is zero and it readily precipitates.

Gel FiltrationA method in which large molecules elute from the column first and small molecules last.

Solubility Factors and Electrical Charge

The ability of proteins to dissolve in water depends on both their intrinsic characteristics (the ratio of polar to non-polar residues, resistance to denaturation) and environmental parameters: temperature, salt composition, presence of organic substances, and pH.

The net charge of the molecule plays a key role. It is formed by the ionized groups of amino acids—negative carboxyl groups and positive amino groups. Changing the pH of the environment 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 stripped from amino groups, rendering them neutral. As a result, the protein loses positive charges 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 is balanced. This state is called the isoelectric point (pI).

At the isoelectric point, the net charge of the protein is zero. As a result, electrostatic repulsive forces between molecules disappear, causing them to aggregate and precipitate. Protein solubility is at its minimum precisely at the pI.

A classic example of this process is milk curdling. In fresh milk, casein protein molecules are negatively charged, forming a stable colloidal solution. As bacteria multiply, they produce lactic acid, which lowers the pH. When the acidity reaches the isoelectric point of casein (approximately 4.6), its charge is neutralized, molecules cease to repel each other, and the 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 workflow includes several stages:

Cell lysis to obtain a homogenate.
Fractionation (centrifugation) — separating 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 causes thermolabile impurities to denature and precipitate, leaving the target protein in solution.
Salting-out — adding salts (most commonly ammonium sulfate). Proteins with the lowest solubility precipitate at low salt concentrations. Gradually increasing the concentration allows the mixture to be separated into distinct fractions.

Separation of Proteins by Size and Mass

To separate macromolecules based on size and weight, ultracentrifugation, polyacrylamide gel electrophoresis, and gel filtration (size-exclusion chromatography) are used.

The principle of gel filtration is based on passing the mixture through a column packed with porous beads. Large proteins cannot physically penetrate the pores; they travel through the void space between beads and elute from the column first. Small molecules enter the pores, significantly lengthening their path, so they leave the column last.

Another essential method is zone electrophoresis, which separates proteins in an electric field. In healthy human serum, albumins migrate fastest toward the anode (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.

Mnemonic

To remember gel filtration elution order, imagine gel beads as houses with narrow doors. "Chunky" (large) molecules cannot enter the houses, so they run straight down the street and cross the finish line first. "Skinny" (small) molecules enter every door, wander around the rooms for a long time, and arrive last.

Frequently asked questions

What salts besides ammonium sulfate are used for protein salting-out?

For protein salting-out, sodium sulfate and sodium chloride are used in addition to ammonium sulfate.

Salting-out is the reversible precipitation of proteins from solution using high concentrations of neutral salts. Salt ions become hydrated, stripping water molecules from the protein shell and neutralizing their charge, which leads to protein aggregation and precipitation.

What chemical reagents cause irreversible protein precipitation?

Irreversible precipitation (denaturation) of proteins is caused by the following chemical reagents:

Acids and alkalis — alter the ionization of ionogenic groups, breaking ionic and hydrogen bonds.
Organic solvents (alcohol, phenol, chloroform) — disrupt hydrophobic interactions and hydrogen bonds.
Heavy metal salts — form insoluble protein salts.
Urea — disrupts intramolecular hydrogen bonds.
Detergents.

What accounts for the buffering capacity of protein solutions?

The buffering capacity of protein solutions is due to the presence of amino and carboxyl groups in their molecules, which can act as acids and bases.

A protein buffer system consists of the protein molecule (NH₂-Prot-COOH) and its salt (NH₂-Prot-COONa). When acid is added, the protein binds protons; when base is added, it releases protons, thereby maintaining the pH of the medium. The acid buffer capacity of proteins is greater than their alkali buffer capacity.

What types of chemical bonds are disrupted during thermal protein denaturation?

Thermal protein denaturation disrupts weak bonds that stabilize its tertiary and secondary structure.

These bonds include:

Hydrogen bonds.
Hydrophobic interactions.
Ionic bonds.

These bonds are disrupted due to increased chaotic thermal motion of molecules at high temperatures (above 60°C), while peptide bonds (primary structure) remain intact.

Why do denatured proteins precipitate more easily?

Denaturation disrupts the three-dimensional structure of the molecule, exposing hydrophobic amino acid side chains that are normally tucked inside to the surface. This sharply reduces the solubility of the protein in water.

What pH should be created for a peptide with a net 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 environment must be acidified, meaning the pI will be in an acidic medium (pH < 7).

What happens to the charge of a protein when an alkali is added?