In general, guanidine hydrochloride and urea can be used as protein denaturant. Guanidine hydrochloride and urea are generally used to obtain an estimate of protein conformational stability (the difference between proteins in their native and denatured states). Is the result obtained using these two denaturants consistent? Let's take a look at the results of Monera et al. [1].

Although urea and guanidine hydrochloride are commonly used as denaturants for proteins, they differ in their basic molecular structure. Urea is a neutral molecule, and guanidine hydrochloride is ionic nature. Based on the above facts, in the early stages of conducting specific experiments, they speculated that the use of guanidine hydrochloride and urea as denaturant to estimate protein stability may be affected by the stabilizing effect of electrostatic interactions on proteins. To this end, they specifically designed four coiled-coil analogs, which gradually changed the intrachain and interchain electrostatic attractions to repulsions. Then, the data were detected using guanidine hydrochloride and urea as denaturants. The experimental results showed that for the four analogues, the value of [hydroguanidine hydrochloride] 1/2 was very close (3.5 M), and the difference of stability energy for the four analogues was close to 0 (0.2 kcal/mol); The value of [urea]1/2 gradually decreases proportionately with the stepwise change from electrostatic attractions to repulsions, and the difference in energy value that characterizes the stability of the protein analogue is also greater than zero. These results show that the ionic nature of guanidine hydrochloride can masks electrostatic interactions in these model proteins while the neutral urea has no shielding effect.

From the above experiments designed by Monera et al., while estimating the conformational stability of a protein, we first need to confirm that the effect of electrostatic interaction on the conformational stability of a protein under investigation. If the electrostatic interaction contributes more to the conformational stability of the protein, the experimental results obtained using guanidine hydrochloride as a denaturant will have a large error. Therefore, in the protein denaturation experiment, we need to comprehensively take into account the experimental subjects and the denaturant itself.

Reference:

[1] O.D. Monera, C. M. Kay, R. S. Hodges, Protein Science, 1994, 3, 1984-1991.

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