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

Publisher Website
Google Scholar
Abstract
The transfer enthalpy Q6 of α-lactalbumin from water to aqueous guanidine hydrochloride (GuHCl) solution at pH 6.0 and the transfer enthalpy Q62 from aqueous GuHCI 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 KN=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 ΔnD binding constant KD=1.2) were assumed to be exposed anew through the N⇄D transition. Also, from values of the transfer heat Q62° from pH 6.0 to pH 2.3 in 0.1 M NaC1 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, ΔnD and ΔnΔ (the total numbers of binding sites reexposed through the N⇉A and N⇉D 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].