The advantages and disadvantages, differences, denaturation mechanism and precautions of common protein denaturants urea, guanidine hydrochloride and guanidine isothiocyanate

The advantages and disadvantages, differences, denaturation mechanism and precautions of common protein denaturants urea, guanidine hydrochloride and guanidine isothiocyanate

Protein denaturants are a class of reagents that can denaturate proteins. Protein denaturation refers to the process in which the original structure and properties of proteins are changed when they are affected by physical or chemical factors. It is generally believed that the secondary structure and tertiary structure of proteins have been changed or destroyed, which is the result of denaturation.

Common protein denaturants include strong acid, strong base, heavy metal salt, acetone, urea, guanidine hydrochloride and guanidine isothiocyanate. In biochemical experiments, protein denaturation research is often needed. Generally, we commonly use urea, guanidine hydrochloride and guanidine isothiocyanate as three protein denaturants. Today we will talk about the advantages and disadvantages of these three protein denaturants, differences, denaturation mechanisms and precautions.

Advantages and disadvantages of urea, guanidine hydrochloride and guanidine isothiocyanate:

The solubility and denaturation capacity of urea is relatively weak, but it has the advantages of non-ionization, neutral, low cost, and will not cause a large amount of protein precipitation after protein renaturation.

The solubility and denaturation ability of guanidine hydrochloride are relatively strong, but the cost is higher than that of urea, it is easy to precipitate under acidic conditions, and may interfere with protein ion exchange chromatography.

The solubility and denaturation capacity of guanidine isothiocyanate is the strongest of these three protein denaturants, but also the most expensive.

The difference between urea, guanidine hydrochloride and guanidine isothiocyanate:

Concentration: At room temperature, 4 to 6mol/L urea and 3 to 4mol/L guanidine hydrochloride can make the spherical protein from the natural state to the denaturation state of the midpoint, usually increasing the denaturation agent concentration can improve the degree of denaturation, in general, 8mol/L urea and 6mol/L guanidine hydrochloride can make the protein completely transformed into a denaturation state. However, there are some spherical proteins, even in 8mol/L urea and 6mol/L guanidine hydrochloride solution can not be completely denatured, however, continue to increase the concentration of the solution, in 8mol/L guanidine hydrochloride solution, the protein generally exists in a random coiled (fully denatured) conformation.

Solubility: The solubility of urea is slower and weaker than guanidine hydrochloride, and the solubility of guanidine hydrochloride is slower and weaker than guanidine isothiocyanate. The solubility of urea is 70%~90%, and it will split to form cyanate when the action time is long or the temperature is high, which can covalently modify the amino group of recombinant protein. The solubility of guanidine hydrochloride is more than 95%, and the dissolution is fast without covalent modification of recombinant protein.

Mechanism of degeneration caused by urea and guanidine hydrochloride:

The denatured protein can preferentially bind to urea and guanidine hydrochloride to form the denatured protein-denatant complex, which causes the N→D reaction equilibrium to shift to the right when the complex is removed. With the increase of denaturant concentration, the protein in its natural state is continuously transformed into a complex, which eventually leads to complete denaturation of the protein. However, the binding of the denaturant to the denaturated protein is very weak. Therefore, only high concentration of denaturants can cause complete denaturation of proteins;

Solubilization of hydrophobic amino acid residues by urea and guanidine hydrochloride. Because both urea and guanidine hydrochloride have the ability to form hydrogen bonds, urea and guanidine hydrochloride can destroy the hydrogen bond structure of water in a high concentration (4-8 mol/L) aqueous solution. As a result, urea and guanidine hydrochloride become a better solvent for non-polar residues, which increases the stretch and solubility of hydrophobic residues inside the protein molecules, thus denaturating proteins to varying degrees.

Protein denaturation caused by urea and guanidine hydrochloride is usually reversible. However, in some cases, because a portion of urea can be converted to cyanate and ammonia, the amino group of the protein can react with the cyanate, causing a change in the protein charge distribution. Therefore, protein denaturation caused by urea is sometimes difficult to fully renaturate. The use of some reducing agents (cysteine, ascorbic acid, β-mercaptoethanol and DTT) can reduce disulfide bonds and facilitate the renaturation of denatured proteins.

Precautions for the use of urea, guanidine hydrochloride and guanidine isothiocyanate:

According to the experimental conditions and objectives, the appropriate protein denaturant was selected and the appropriate concentration of the solution was allocated.

Urea, guanidine hydrochloride, and guanidine isothiocyanate are all chemical agents. Inhalation or ingestion may cause injury. Wear gloves and safety goggles when operating.

In the actual experiment, researchers can choose which protein denaturant to use according to the conditions and purposes to obtain the best experimental results.