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How to choose protein denaturants?

2019-07-08 来源:亚科官网
Proteins are responsible for complex biochemical reactions that must have a specific three-dimensional structure when performing biological functions. After biosynthesis, the protein itself undergoes a complex physiological process. In biochemical tests, it is often necessary to study the renaturation of proteins. Hydroquinone hydrochloride and urea are the most commonly used reagents for protein renaturation.
Guanidine hydrochloride and urea
Urea and guanidine hydrochloride can break hydrogen bonds in high concentration (4~8mol/L) aqueous solution, which causes protein to be denatured to varying degrees. At the same time, it is also possible to reduce the hydrophobic interaction by increasing the solubility of the acidic residue of the hydrophobic group in the aqueous phase. At room temperature, 4~6 mol/L urea and 3~4 mol/L guanidine hydrochloride can change the globular protein from the natural state to the midpoint of the denaturing state. Usually, the denaturant concentration can increase the degree of denaturation. Some globular proteins were not completely denatured even in the 8 mol/L urea solution, whereas in the 8 mol/L guanidine hydrochloride solution, they were generally present in a random coiled (fully denatured) conformation state.
Degeneration mechanism caused by urea and guanidine hydrochloride:
1) The denatured protein can preferentially bind with urea and guanidine hydrochloride to form a denatured protein-denaturing agent complex, and when the complex is removed, the N→D reaction equilibrium shifts to the right. As the concentration of the denaturant increases, the protein in its natural state continues to transform into a complex, which ultimately leads to complete denaturation of the protein. However, since the binding of the denaturant to the denatured protein is very weak. Therefore, only high concentrations of denaturant can cause complete denaturation of the protein;
2) Solubilization of hydrophobic amino acid residues by urea and guanidine hydrochloride. Because both urea and guanidine hydrochloride have the ability to form hydrogen bonds, when they are at high concentrations, they can destroy the hydrogen bond structure of water. As a result, urea and guanidine hydrochloride become better solvents for non-polar residues, making the protein molecules internal. The hydrophobic residue stretches and the solubility increases. The denaturation caused by urea and guanidine hydrochloride is usually reversible. However, in some cases, a portion of the urea can be converted to cyanate and ammonia, and the amino group of the protein can react with the cyanate, causing a change in the charge distribution of the protein. Therefore, protein denaturation caused by urea is sometimes difficult to completely renature. The use of some reducing agents (cysteine, ascorbic acid, β-mercaptoethanol, and DTT) can reduce disulfide bonds and contribute to the renaturation of denatured proteins.
In general, as a commonly used reagent in the process of protein denaturation, the advantages and disadvantages of guanidine hydrochloride and urea are: guanidine hydrochloride has relatively strong dissolving ability and denaturing ability, and does not cause covalent modification of recombinant protein, but it has high cost. It is easy to produce precipitate under acidic conditions and interfere with protein ion exchange chromatography; while urea has relatively weak solubility, but it has the advantages of non-ionization, neutrality, low cost, and no protein precipitation after protein renaturation. In actual experiments, researchers can choose based on conditions and objectives to obtain optimal experimental results.
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