Calorimetric Study on the Conformational Transition of α-Lactalbumin Induced by Guanidine Hydrochloride

Abstract

The transfer enthalpy Q₆ of α-lactalbumin from water to aqueous guanidine hydrochloride (GuHCl) solution at pH 6.0 and the transfer enthalpy Q₆₈ from aqueous GuHCl at pH 6.0 to pH 2.3 were measured. The dependence of Q₆ on the denaturant activity indicated that (1) guanidinium ions bind to the protein in the native (N) state with a binding constant of K_N=0.31, (2) the transition enthalpy from the N to the denatured (D) state in the absence of denaturant is 25.4 kcal/mol, where the binding sites (total number Δn_D, binding constant K_D=1.2) were assumed to be exposed anew through the N_??_D transition. Also, from values of the transfer heat Q⁰₆₂ from pH 6.0 to pH 2.3 in 0.1M NaCl solution and of the calculated protonation enthalpy of the protein in the pH range of 6.0-2.3, the transition enthalpy from the N to the acid (A) state was determined to be 24±3 kcal/mol in the absence of denaturant.
The van't Hoff enthalpies of both the N_??_A and N_??_D transitions of the protein determined from CD and difference spectra measurements were compared with the calorimetric values. Each transition in the three-state transition processes of the protein was concluded to satisfy the two-state hypothesis of Lumry et al. in the absence of the denaturant.
On the other hand, Δn_D and Δn_D (the total numbers of binding sites reexposed through the N_??_D and N_??_A transitions, respectively) were estimated to be about 23 and 10, respectively, in fair agreement with the values previously determined from spectroscopic measurements.
The calorimetric transition enthalpies and the binding parameters for α-lactalbumin obtained in the present study are compared with those for lysozyme [EC 3. 2. 1. 17].