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In the less extensive technique of equilibrium unfolding, the fractions of folded and unfolded molecules (denoted as and , respectively) are measured as the solution conditions are gradually changed from those favoring the native state to those favoring the unfolded state, e.g., by adding a denaturant such as guanidinium hydrochloride or urea.
For alkaline buffers, a strong base such as sodium hydroxide may be added. Alternatively, a buffer mixture can be made from a mixture of an acid and its conjugate base. For example, an acetate buffer can be made from a mixture of acetic acid and sodium acetate. Similarly, an alkaline buffer can be made from a mixture of the base and its ...
The Charlot equation, named after Gaston Charlot, is used in analytical chemistry to relate the hydrogen ion concentration, and therefore the pH, with the formal analytical concentration of an acid and its conjugate base. It can be used for computing the pH of buffer solutions when the approximations of the Henderson–Hasselbalch equation ...
The sum of the two rates is the observed relaxation rate. An agreement between equilibrium m-value and the absolute sum of the kinetic m-values is typically seen as a signature for two-state behavior. Most of the reported denaturation experiments have been carried out at 298 K with either urea or guanidinium chloride (GuHCl) as denaturants.
Denaturation midpoint of a protein is defined as the temperature (T m) or concentration of denaturant (C m) at which both the folded and unfolded states are equally populated at equilibrium (assuming two-state protein folding). T m is often determined using a thermal shift assay.
The Van 't Hoff equation relates the change in the equilibrium constant, K eq, of a chemical reaction to the change in temperature, T, given the standard enthalpy change, Δ r H ⊖, for the process. The subscript r {\displaystyle r} means "reaction" and the superscript ⊖ {\displaystyle \ominus } means "standard".
Once the mutants have been established, two methods can be employed to calculate the free energy associated with a salt bridge. One method involves the observation of the melting temperature of the wild-type protein versus that of the three mutants. The denaturation can be monitored through a change in circular dichroism. A reduction in melting ...
The Henderson–Hasselbalch equation can be used to estimate the pH of a buffer solution by approximating the actual concentration ratio as the ratio of the analytical concentrations of the acid and of a salt, MA. The equation can also be applied to bases by specifying the protonated form of the base as the acid.