Elucidating the binding thermodynamics of 8‐anilino‐1‐naphthalene sulfonic acid with the A‐state of α‐lactalbumin: An isothermal titration calorimetric investigation

Abstract

Isothermal titration calorimetry has been used to demonstrate that the heat profile associated with the binding of 8-anilino-1-naphthalene sulfonic acid (ANS) with the acid induced molten globule state (A-state) of alpha-lactalbumin (alpha-LA) is different from that with the native and denatured states of the protein. The results corroborate the spectroscopic observations that ANS binds more strongly to the partially folded states of the protein compared to that with the native and denatured states. ANS binds to the A-state of alpha-LA at two independent binding sites that remain nearly the same in the temperature range of 10-35 degrees C. The number of moles of ANS binding at site 1 at 10 degrees C is 14.0+/-0.2 and remains nearly the same with rise in temperature. However, the number of moles of ANS molecules binding at site 2 show an increase from 1.6+/-0.2 at 10 degrees C to 4.1+/-0.1 at 35 degrees C. The deviation of the slope of enthalpy-entropy compensation plot from unity and nonadherence to van't Hoff dictates implies that the binding sites on the A-state of alpha-LA for ANS are not well defined and specific; rather, these binding sites are formed due to greater exposure of hydrophobic clusters in the A-state of the protein. The results for the first time demonstrate the use of isothermal titration calorimetry in characterizing the A-state of alpha-LA both qualitatively and quantitatively.

Isothermal titration calorimetric and spectroscopic studies on (alcohol+salt) induced partially folded state of α-lactalbumin and its binding with 8-anilino-1-naphthalenesulfonic acid

“…This further corroborates that in the presence of the 0.25 mol Á dm À3 (HFIP + GndSCN) and 0.85 mol Á dm À3 (isopropanol + GndSCN) mixtures a-LA exists in partially folded conformations which are different from the conventional MG state where a very strong binding with ANS has been observed [40].…”

1-Anilino-8-Naphthalene Sulfonate (ANS) Is Not a Desirable Probe for Determining the Molten Globule State of Chymopapain

“…The total heat content of the solution is given by:where Mt is the total concentration of the macromolecule, V0 is the active cell volume, n1 and n2 represent the number of ligand molecules bound to first and second set of sites respectively, ΔH1 and ΔH2 correspond to the enthalpy change associated with respective binding sites. The heat released ΔQ(i) from the ith injection for an injection volume dVi is given by:which is then used in the Marquardt minimization algorithm to obtain best fitting values until constant χ2 values were achieved [39], [40].…”

A thermodynamic characterization of the interaction of 8‐anilino‐1‐naphthalenesulfonic acid with native globular proteins: the effect of the ligand dimerization in the analysis of the binding isotherms

“…This technique provides us a full thermodynamic description of the process and also allows us to complete the information obtained by means of other techniques. Thus, in addition to the fluorescence studies, ITC experiments have been used to evaluate the binding of ANS to proteins (Matulis and Lovrien, 1998;Celej et al, 2005;Banerjee and Kishore, 2006;Singh and Kishore, 2006;Kinsley et al, 2008). In these studies, the binding isotherms obtained at acidic pH values show a complex behavior and their analysis is not always possible (Sharma and Kishore, 2009).…”