Equilibrium and kinetics of the unfolding of alpha-lactalbumin by guanidine hydrochloride (IV): dependence of the N equilibrium A transconformation on the temperature.
The reversible unfolding from the native (N) state to the acid (A) state of alpha-lactalbumin by guanidine-HCl (0.8-2.0 M) was studied at 10-35 degrees C by means of difference spectra and pH-jump measurements. At each temperature, all points plotted as the logarithmic equilibrium constant log KNA of the N equilibrium A process against pH could fall on a curve independent of the denaturant concentration by shifting each point along the log KNA axis, where the shift factor f did not depend on temperature. Moreover, by shifting the points at each temperature along the log (KNA/f) axis, a master curve, independent of both temperature and the denaturant concentration, could be obtained for the pH-dependence of log KNA. From the dependence of the logarithmic rate constants on pH, master curves independent of both temperature and the denaturant concentration could also be made for the N leads to A and the A leads to A processes, where A mean the activated state. The results show the two-state character of the N equilibrium A process. The enthalpy changes and the differences in heat capacity for the N equilibrium A, N equilibrium A and A equilibrium A processes were determined from the accurate measurements of temperature dependence of the unfolding at pH 4.3 and 1.0 M guanidine-HCl. The results show that the disruption of hydrophobic interaction is caused mainly in the A leads to A process, while most of the changes in the pK values of the ionizing groups are caused in the N leads to A process.
D006146:Guanidines / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007768:Lactalbumin; D009113:Muramidase; D011489:Protein Denaturation / Q000187:drug effects; D013696:Temperature; D013816:Thermodynamics
D006146:Guanidines; D007768:Lactalbumin; D009113:Muramidase