Elucidating the binding thermodynamics of 8-anilino-1-naphthalene sulfonic acid with the A-state of alpha-lactalbumin: an isothermal titration calorimetric investigation.
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.
Singh
Sreelekha K
SK
Department of Chemistry, Indian Institute of Technology, Bombay, Powai, Mumbai-400 076, India.
Kishore
Nand
N
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry*; D000818:Animals; D001665:Binding Sites; D002151:Calorimetry; D002417:Cattle; D057927:Hydrophobic and Hydrophilic Interactions; D007768:Lactalbumin / Q000737:chemistry*; D011485:Protein Binding; D017510:Protein Folding; D013696:Temperature; D013816:Thermodynamics; D017186:Titrimetry
D000817:Anilino Naphthalenesulfonates; C027132:1-anilino-8-naphthalenesulfonate; D007768:Lactalbumin