Protein denaturation hydrochloride and urea have to say the difference
Protein is responsible for complex biochemical reactions, in the exercise of biological function, must have a specific three-dimensional spatial structure. After biosynthesis, the protein itself undergoes a complex physiological process. In biochemical trials, it is often necessary to study the variability of proteins, and guanidine hydrochloride and urea are commonly used reagents in protein variability, so what is the difference between the two? How should we choose in the experiment?
Due to the action of external factors, the conformation of natural protein molecules changes abnormally, which leads to the loss of biological activity and abnormal changes of physical and chemical properties, which is called protein degeneration. Denaturation can involve the fracture of secondary bond and disulfide bond, but it does not involve the fracture of peptide bond on the first stage structure.
Guanidine HydrochlorideAnd urea denatured protein consists of two mechanisms: a mechanism is the priority combination of denatured protein with guanidine hydrochloride and urea to form a complex, when the complex is removed, the reaction balance moves, with the increase of the concentration of denatured agent, the natural state of the protein is constantly transformed into a complex, resulting in complete protein degeneration; The second mechanism is the solubility of guanidine hydrochloride and urea to hydrophobic amino acid residues, because guanidine hydrochloride and urea have the ability to form hydrogen bonds, guanidine hydrochloride and urea can break the hydrogen bond in high concentration (4~8mol/l) aqueous solution, the results of guanidine hydrochloride and urea become a better solvent of non-polar residues, To increase the elongation and solubility of the hydrophobic residues inside the protein molecule, so that the protein can be denatured to varying degrees.
The difference between the two in protein degeneration:
Concentration: At room temperature, 3~4mol/l hydrochloride can change the spherical protein from the natural state to the midpoint of the degenerative state, usually increasing the concentration of denatured agent can improve the degree of degeneration, about 6mol/l hydrochloride can make the protein completely transformed into a denatured state. Guanidine hydrochloride is more denatured than urea because of its ionic properties. Some spherical proteins, even in 8mol/l urea solution, cannot be completely denatured, but in the 8mol/l hydrochloride solution, they generally exist in an irregular curl (completely denatured) conformation state.
Solubility: Urea dissolution capacity is slower and weaker than guanidine hydrochloride, solubility is 70%~90%, urea in the action time is longer or the temperature is higher when the formation of cyanate, the recombinant protein amino value modification, but it has the advantages of non-ionization, neutral, low cost and so on; Guanidine hydrochloride dissolution capacity of more than 95%, And the dissolution effect is fast without causing the covalent modification of recombinant protein, but the cost is higher than urea, easy to produce precipitation under acidic conditions, may interfere with subsequent experiments and other shortcomings.
In general, as a commonly used reagent in protein denaturation process, the advantages and disadvantages of guanidine hydrochloride and urea are: Guanidine hydrochloride dissolution ability and denaturation ability is relatively strong, will not cause covalent modification of recombinant protein, but has high cost, easy to produce precipitation under acidic conditions, interference with protein ion exchange chromatography and other shortcomings; Urea dissolution ability is relatively weak, but with non-ionization, neutral, low cost, protein complex will not cause a large number of protein precipitation and other advantages. In practical experiments, researchers need to choose according to conditions and purposes in order to obtain good experimental results.