Proteins undergo specific spatial conformation changes under certain physical and chemical factors. However, their primary structure or amino acids arrangement remains unchanged. They must function in an aqueous medium, so the forces that construct 4 levels structure not only exist in protein, but also include interactions with water.
The proteins structure is extremely delicate. Sometimes, even slight conformational changes can affect their function, for example, the loss of a secondary bond or a side chain group faces in another orientation. Denaturation involves the disappeared ordered structure in most cases. When completely denatured, it becomes a random coil of peptide chains. Some proteins unfold naturally, such as casein, which is difficult to denature due to the missing higher-order structures.
Conformational changes affect biological functions in a bidirectional manner. Their activity is completely lost or reduced in most cases. However, sometimes they not only maintain biological activity but even enhance it. This is because the conformation of non-essential regions only changes slightly; hidden active sites are exposed when unfolded, and the increased surface area makes them more accessible to other molecules; conformational changes can also form aggregates or complexes with new functions; in immunology, unfolded proteins may be more easily recognized by the immune system because more epitopes are brought to surface.
Whether the denaturation is reversible, it is closely related to protein complexity and degree of structural changes. Simple structures and mild changes make them likely to return to their original bio activities. Some complex proteins can’t spontaneously form their own structure and need the help of other molecules to fold. Their denaturation is often irreversible.
So, what factors affect protein denaturation?
High Temperature
It causes atoms that make up proteins to vibrate more violently. Water molecules also impact them more strongly and frequently. Van der Waals forces, hydrophobic interactions, and hydrogen bonds are sequentially broken. The molecules unfold in aqueous solution to leave some hydrophobic residues exposed, subsequently aggregation will happen between protein molecules. It is noteworthy that the high-energy disulfide bonds and peptide bonds are difficult to break, even if water is boiled. Frying or grilling can break these covalent bonds.
The reason why protein biological activity is very sensitive to temperature is that the secondary bond maintaining structure are weak. The Q10 coefficient in many chemical reactions is 3 to 4. That is, for every 10°C increase, the reaction will be accelerated by 3 to 4 times. This number in proteins ranges from 100 to 600. When temperature exceeds a threshold, the rate of denaturation raises by about 100 to 600 times for every 10°C.
This property is very important in food processing. For example, ultra-high temperature instantaneous sterilization (UHT) destroys biologically active proteins or enzymes in a short time. At the same time, other nutrients in food changes less.
pH
Hydrogen ions or hydroxide ions in water affects dissociation of side chain groups. They give protein's surface an extra positive or negative charge. If pH changes mildly, protein denaturation doesn’t occur, because the interaction of extra charges is less than other forces stabilizing their structure. However, situation is different in extreme pH conditions. For example, when pH drops to 2, R groups are generally positive, and negative charges are neutralized. Disappearance of attraction and enhancement of electrostatic repulsion cause protein molecules to unfold into chains. In addition, protein molecules will aggregate near the isoelectric point. Because at this time they carry zero net charge on surface, hydrophobic interactions and van der Waals forces gather them together.
The destructive effect of strong acids is particularly evident in scavenger stomach. Average PH value in vulture's stomach is as low as 1. They not only quickly dissolve muscles and bones but also eliminate pathogens.
High Concentration of Salt Solution (Non-heavy Metals)
If not much neutral salt is added, it will promote dissolution. This phenomenon is called "salting in." However, at high concentration, the solubility drops sharply and proteins can precipitate out. It is referred as "salting out." Their destructive effect is mainly manifested in following two aspects. The charge on protein surface attracts water molecules to form a hydration shell. High concentrations of salt water prevent the side chains from dissociating. Some partially charged atoms, such as oxygen and hydrogen, can’t compete with the fully charged salt ions for water molecules. Therefore, hydration shell will be thinner in salty water. Salt ions also adsorb on surface to neutralize charge. Proteins with weakened repulsive forces are more likely to aggregate and precipitate together. Their denaturation is temporary and bio activity is restored when diluted with distilled water. Therefore, it is commonly used for separation and preparation of bio-active proteins such as enzymes and hormones.
The tofu production is a classic example of salting out. Common salt coagulants include magnesium chloride and calcium sulfate. Metal ions make soy protein coagulate into a network structure to form solid tofu.
Organic Solvents and Detergents
Hydrophobic groups prefer to contact organic solvents to result in the destroyed hydrophobic interactions. Some subunits combined by this force separate. Groups buried inside are also exposed. A small dielectric constant means that organic solvents aren’t polarized easily and adsorbed on surface to shield electric field. As a result, it promotes intra-molecular attraction and repulsion, such as more alpha helices.
Detergents or surfactants are a combination of organic solvents and salts. Sodium dodecyl sulfate (SDS) has a hydrocarbon tail and a hydrophilic negatively charged head. The tail enters the interior to combine with hydrophobic part. The net charge in entire protein increases, so various parts repel each other to make it stretched. A complete and drastic structure change occurs with only a little SDS. Obviously, this denaturation in protein is irreversible.