The denaturation of beta-lactoglobulin-A at pH 2.

The denaturation of beta-lactoglobulin-A by heat and guanidine hydrochloride at pH 2 has been investigated. The effect of ethylene glycol on the thermal denaturation at this pH has also been studied. The conditions of the experiments have been chosen so as to eliminate complications arising out of disulfide interchange, changes in the degree of association of the protein during denaturation, and intermolecular aggregation. The physical parameters characterizing the denatured states of the protein which are produced by heat and guanidine hydrochloride have been determined. The thermodynamic parameters for these transitions have been estimated using a two-state hypothesis in each case. Both the physical and thermodynamic parameters indicate that the heat-denatured state of beta-lactoglobulin-A retains about 15-20% of residual structure which is destroyed on adding guanidine hydrochloride.

The quantitative comparison of the effects of denaturants on the stability of proteins.

A novel quantitative comparison of denaturants involving the complete reversible unfolding of proteins is presented. Ribonuclease A was denatured with guanidinium chloride in the presence of low fixed concentrations of various partial denaturants, with the unfolding process being monitored by circular dichroism and difference spectroscopy. The major advantage of this method is that it allows a direct quantitative comparison of the effects of denaturants on the stability of proteins. The effect on the stability of ribonuclease A was shown to be linearly dependent upon the concentration of denaturant. An investigation of the constitutive ions of salts revealed that their effects were additive only in the case of salts that have no specific binding capability. This method can also be useful in detecting the specific binding of salts.

Inorganic salt denaturants stabilize ribonuclease against denaturation by urea.

The isothermal denaturation of ribonuclease A by mixed denaturant systems was investigated at 25 degrees C. It was observed that low concentrations of lithium chloride stabilize the protein against denaturation by urea, even though the salt itself is a denaturant. This study also provides, for the first time, the most convincing evidence that the lithium chloride denatured ribonuclease A contains some of the native secondary and tertiary structure.

Estimation of the free energy of stabilization of ribonuclease A, lysozyme, alpha-lactalbumin, and myoglobin.

The denaturation of ribonuclease A, lysozyme alpha-lactalbumin, and myoglobin by urea, guanidine hydrochloride, and guanidine thiocyanate has been followed with the use of difference spectral measurements. The free energy of stabilization (delta GH2OD) has been determined by the linear extrapolation of the free energy of denaturation to zero denaturant concentration. The values of delta GH2OD are 7.3 +/- 0.2, 8.9 +/- 0.1, 4.3 +/- 0.1;, and 7.9 +/- 0.2 kcal/mol at 25 degrees C for ribonuclease A (pH 7.0), lysozyme (pH 7.0), alpha-lactalbumin (pH 7.0), and myoglobin (pH 6.6), respectively. The dependence of the free energy of denaturation on concentration ranges from 0.88 to 2.08 kcal/mol.M for urea and from 1.27 to 4.22 kcal/mol.M for guanidine hydrochloride for four proteins. The ratio of this dependence in guanidine hydrochloride to that in urea may depend on the polarity of the amino acid residues in the unfolding unit.

Free energy changes in alpha-lactalbumin denaturation.

Previous work has shown that native alpha-lactalbumin (N) is completely denatured by the addition of guanidine hydrochloride (conformation D) but that partially denatured conformations appear in other denaturants. In particular, conformation I appears when the pH is lowered from 5.5 to 2.2 (I2.2) or when LiClO4 is added at pH 5.5 (I5.5). We have now determined the free energy changes for the processes N leads to I5.5, N leads to D5.5, and I2.2 leads to D2.2. We have also estimated the maximum value of the free energy change for the process N leads to I.22, and this allows us to estimate the changes for all conformational changes between any two of these five conformations.

Free energy changes in lysozyme denaturation.

Previous work has shown that native lysozyme (N) is completely denatured by the addition of guanidinium chloride (conformation D) but that partially denatured conformations appear in other denaturants. Conformation I appears when LiClO4 is added to the protein, and conformation II is caused by heating. We have now determined the apparent free energy changes for the reversible processes between N and the three unfolded conformations and for the process between II and D. This allows us to estimate the apparent free energy changes for a process between any two of these four conformations.

The thermal denaturation of bovine cardiac G-actin.

The thermal denaturation of bovine cardiac G-actin has been studied by ultraviolet difference spectroscopy and circular dichroism between pH 7.5 and 10.5. As with proteins previously studied, thermal unfolding is incomplete compared with unfolding by urea or GuHCl. However, the same conformational change is observed over the pH range studied, and the available evidence indicates it is a two-state transition. Thermodynamic analysis of the data shows that deltaHo and deltaSo are strongly dependent on the temperature, that deltaCp is 1300 cal deg-1 mol-1, and that G-actin has a temperature of maximum stability near -5 degrees C.