- Español
- Português
- русский
- Français
- 日本語
- Deutsch
- tiếng Việt
- Italiano
- Nederlands
- ภาษาไทย
- Polski
- 한국어
- Svenska
- magyar
- Malay
- বাংলা ভাষার
- Dansk
- Suomi
- हिन्दी
- Pilipino
- Türkçe
- Gaeilge
- العربية
- Indonesia
- Norsk
- تمل
- český
- ελληνικά
- український
- Javanese
- فارسی
- தமிழ்
- తెలుగు
- नेपाली
- Burmese
- български
- ລາວ
- Latine
- Қазақша
- Euskal
- Azərbaycan
- Slovenský jazyk
- Македонски
- Lietuvos
- Eesti Keel
- Română
- Slovenski
- मराठी
- Srpski језик
Advantages, disadvantages, differences, denaturation mechanisms and precautions of common protein denaturants urea, guanidine hydrochloride and guanidine isothiocyanate
2022-03-09
Protein denaturants are a class of reagents that can denature proteins. Protein denaturation refers to the process in which the original structure and properties of a protein are changed when the protein is affected by physical or chemical factors. It is generally believed that the change or destruction of the secondary and tertiary structures of proteins is the result of denaturation.
Common protein denaturants include strong acids, strong bases, heavy metal salts, acetone, urea, guanidine hydrochloride and guanidine isothiocyanate. In biochemical experiments, protein denaturation is often required. Generally, we commonly use three protein denaturants, urea, guanidine hydrochloride and guanidine isothiocyanate. Today we will talk about the advantages and disadvantages, differences, denaturation mechanisms and precautions of these three protein denaturants.
Advantages and disadvantages of urea, guanidine hydrochloride and guanidine isothiocyanate:
Urea has relatively weak dissolving ability and denaturing ability, but has the advantages of non-ionization, neutrality, low cost, and no protein precipitation after protein renaturation.
Guanidine hydrochloride has relatively strong dissolving ability and denaturing ability, but has disadvantages such as higher cost than urea, easy precipitation under acidic conditions, and possible interference with protein ion exchange chromatography.
The solubility and denaturing ability of guanidine isothiocyanate is the strongest among the three protein denaturants, and it is also the most expensive.
The difference between urea, guanidine hydrochloride and guanidine isothiocyanate:
Concentration: At room temperature, 4~6mol/L urea and 3~4mol/L guanidine hydrochloride can make the globular protein change from the natural state to the midpoint of the denatured state, usually increasing the concentration of the denaturant can improve the degree of denaturation, generally 8mol /L urea and 6mol/L guanidine hydrochloride can completely transform the protein into a denatured state. However, there are also some globular proteins that cannot be completely denatured even in 8 mol/L urea and 6 mol/L guanidine hydrochloride solutions, but continue to increase the solution concentration. The conformational state exists.
Solubility: The solubility of urea is slower and weaker than that of guanidine hydrochloride, and the solubility of guanidine hydrochloride is slower and weaker than that of guanidine isothiocyanate. The solubility of urea is 70%~90%, it will crack to form cyanate when the action time is long or the temperature is high, and the amino group of the recombinant protein is covalently modified; the solubility of guanidine hydrochloride is more than 95%, and the dissolution effect Fast without causing covalent modification of recombinant proteins.
Mechanisms of denaturation induced by urea and guanidine hydrochloride:
The denatured protein can preferentially combine with urea and guanidine hydrochloride to form a denatured protein-denaturant complex, which, when the complex is removed, causes the N→D reaction equilibrium to shift to the right. As the concentration of the denaturant increases, the protein in its native state is continuously transformed into a complex, eventually resulting in complete denaturation of the protein. However, 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;
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 when they are in a high concentration (4~8mol/L) aqueous solution. As a result, urea and guanidine hydrochloride become non-polar residues It is a better solvent to make the hydrophobic residues inside the protein molecule stretch and increase the solubility, so that the protein can be denatured to different degrees.
Protein denaturation by urea and guanidine hydrochloride is usually reversible. However, in some cases, since a part of urea can be converted into cyanate and ammonia, and the amino group of protein can react with cyanate, resulting in the change of protein charge distribution. Therefore, urea-induced protein denaturation is sometimes difficult to fully renature. The use of some reducing agents (cysteine, ascorbic acid, β-mercaptoethanol and DTT) can reduce disulfide bonds and can help renaturation of denatured proteins.
Precautions for the use of urea, guanidine hydrochloride and guanidine isothiocyanate:
According to the conditions and purposes of the experiment, select the appropriate protein denaturant and configure the solution with the appropriate concentration.
Urea, guanidine hydrochloride and guanidine isothiocyanate are all chemical reagents. Human inhalation, ingestion, and skin contact may cause injury. Wear gloves and goggles during operation.
Common protein denaturants include strong acids, strong bases, heavy metal salts, acetone, urea, guanidine hydrochloride and guanidine isothiocyanate. In biochemical experiments, protein denaturation is often required. Generally, we commonly use three protein denaturants, urea, guanidine hydrochloride and guanidine isothiocyanate. Today we will talk about the advantages and disadvantages, differences, denaturation mechanisms and precautions of these three protein denaturants.
Advantages and disadvantages of urea, guanidine hydrochloride and guanidine isothiocyanate:
Urea has relatively weak dissolving ability and denaturing ability, but has the advantages of non-ionization, neutrality, low cost, and no protein precipitation after protein renaturation.
Guanidine hydrochloride has relatively strong dissolving ability and denaturing ability, but has disadvantages such as higher cost than urea, easy precipitation under acidic conditions, and possible interference with protein ion exchange chromatography.
The solubility and denaturing ability of guanidine isothiocyanate is the strongest among the three protein denaturants, and it is also the most expensive.
The difference between urea, guanidine hydrochloride and guanidine isothiocyanate:
Concentration: At room temperature, 4~6mol/L urea and 3~4mol/L guanidine hydrochloride can make the globular protein change from the natural state to the midpoint of the denatured state, usually increasing the concentration of the denaturant can improve the degree of denaturation, generally 8mol /L urea and 6mol/L guanidine hydrochloride can completely transform the protein into a denatured state. However, there are also some globular proteins that cannot be completely denatured even in 8 mol/L urea and 6 mol/L guanidine hydrochloride solutions, but continue to increase the solution concentration. The conformational state exists.
Solubility: The solubility of urea is slower and weaker than that of guanidine hydrochloride, and the solubility of guanidine hydrochloride is slower and weaker than that of guanidine isothiocyanate. The solubility of urea is 70%~90%, it will crack to form cyanate when the action time is long or the temperature is high, and the amino group of the recombinant protein is covalently modified; the solubility of guanidine hydrochloride is more than 95%, and the dissolution effect Fast without causing covalent modification of recombinant proteins.
Mechanisms of denaturation induced by urea and guanidine hydrochloride:
The denatured protein can preferentially combine with urea and guanidine hydrochloride to form a denatured protein-denaturant complex, which, when the complex is removed, causes the N→D reaction equilibrium to shift to the right. As the concentration of the denaturant increases, the protein in its native state is continuously transformed into a complex, eventually resulting in complete denaturation of the protein. However, 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;
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 when they are in a high concentration (4~8mol/L) aqueous solution. As a result, urea and guanidine hydrochloride become non-polar residues It is a better solvent to make the hydrophobic residues inside the protein molecule stretch and increase the solubility, so that the protein can be denatured to different degrees.
Protein denaturation by urea and guanidine hydrochloride is usually reversible. However, in some cases, since a part of urea can be converted into cyanate and ammonia, and the amino group of protein can react with cyanate, resulting in the change of protein charge distribution. Therefore, urea-induced protein denaturation is sometimes difficult to fully renature. The use of some reducing agents (cysteine, ascorbic acid, β-mercaptoethanol and DTT) can reduce disulfide bonds and can help renaturation of denatured proteins.
Precautions for the use of urea, guanidine hydrochloride and guanidine isothiocyanate:
According to the conditions and purposes of the experiment, select the appropriate protein denaturant and configure the solution with the appropriate concentration.
Urea, guanidine hydrochloride and guanidine isothiocyanate are all chemical reagents. Human inhalation, ingestion, and skin contact may cause injury. Wear gloves and goggles during operation.
In actual experiments, researchers can choose which protein denaturant to use according to conditions and purposes to obtain optimal experimental results.
