Denaturation equilibrium

The use of differential scanning microcalorimetry for measuring the thermal denaturation of proteins is described in Chapter 17, section Ale. Typically, 0.5-1 mg of protein in 1 mL of buffer, or 0.1-0.2 mg in 0.5 mL with the most sensitive apparatus, is required for an accurate determination of the enthalpy of denaturation. The thermodynamics of dissociation of a reversibly bound ligand may be calculated from its effects on the denaturation curve of a protein.14 The binding of ligands always raises the apparent Tm (temperature at 50% denaturation) of a protein because of the law of mass action the ligand does not bind to the denatured state of the protein, and so binding displaces the denaturation equilibrium toward the native state. 

Figure 3. Denaturation equilibrium curves obtained from fluorescence intensity ( A ), activity measurements ( and difference spectroscopy ( a ) at 23 C, in the presence of different sucrose concentrations a) no sucrose, b) 0.6 M sucrose (data for 0.3 M sucrose not shown). The solid lines are approximate fits for comparison of the experimental data.

The denaturation equilibrium experiments described in this paper show that p-lactamase becomes unfolded in the presence of more than 1.25 M guanidine-HCl. The noncoincidence of the difference spectra and fluorescence emission at guanidine-HCl concentrations between 0.3 and 0.75 M suggests the presence of an intermediate which is populated at equilibrium. 

The fact that the pressure effect on the alcohol inhibition tends to disappear at the higher temperatures indicates that the volume change in the equilibrium combination between the alcohol and the enzyme protein is very small, the pressure effects at lower temperatures being mediated through the ordinary temperature- and pressure-sensitive denaturation equilibrium. 

The effect of a co-solvent on protein stability is expressed by its effect on the standard free energy, AG° N D of the denaturation equilibrium, N D. It is given by ... 

John F. Brandts measured the equilibrium constants for the denaturation over a range of pH and temperatures. The data for pH 3 ... 

The equilibrium constants determined by Brandts at several temperatures for the denaturation of chymotrypsinogen (see previous Example) can be used to calculate the free energy changes for the denaturation process. For example, the equilibrium constant at 54.5°C is 0.27, so... 

For biochemical reactions in which hydrogen ions (H ) are consumed or produced, the usual definition of the standard state is awkward. Standard state for the ion is 1 M, which corresponds to pH 0. At this pH, nearly all enzymes would be denatured, and biological reactions could not occur. It makes more sense to use free energies and equilibrium constants determined at pH 7. Biochemists have thus adopted a modified standard state, designated with prime ( ) symbols, as in AG°, AH°, and so on. For values determined... 

The ROA spectra of partially unfolded denatured hen lysozyme and bovine ribonuclease A, prepared by reducing all the disulfide bonds and keeping the sample at low pH, together with the ROA spectra of the corresponding native proteins, are displayed in Figure 5. As pointed out in Section II,B, the short time scale of the Raman scattering event means that the ROA spectrum of a disordered system is a superposition of snapshot ROA spectra from all the distinct conformations present at equilibrium. Because of the reduced ROA intensities and large... 

The cold-denatured state clearly has residual structure in that it is collapsed and exhibits a fairly globular Kratky scattering profile. Truncated, equilibrium unfolded states appear similarly compact (and have been shown by other spectroscopic means to have structure). But what of the highly expanded, urea, GuHCl, or thermally denatured states ... 

McGhee JD, von Hippel PH. Formaldehyde as a probe of DNA structure. 3. Equilibrium denaturation of DNA and synthetic polynucleotides. Biochemistry 1977 16 3267-3275. 

Zerovnik, E., Jerala, R., Kroon-Zitko, L., Turk, V., and Lohner, K. (1997). Characterization of the equilibrium intermediates in acid denaturation of human stefin B. Eur.J. Biochem. 245, 364-372. 

Where Dx is a general term for denatured protein X of all kinds. The inhibitor (and all other non-enzymatic components in the detergent) was assumed to be stable in the detergent, i.e. no inactivation of the inhibitor occurs. Reaction III is expected to be much faster than the other reactions and equilibrium is assumed in the calculations. Reaction IV was excluded from the system to reduce the... 

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