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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

Publication Date
Jan 01, 2008
DSpace at IIT Bombay
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Interaction of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) and isopropanol in the presence of equimolar quantities of guanidine thiocyanate (GndSCN) with bovine α-lactalbumin (α-LA) has been investigated by using a combination of isothermal titration calorimetry, circular dichroism, fluorescence, and ultra-violet spectroscopies at in 20 · 10−3 mol · dm−3 phosphate buffer pH 7.0. All the thermal unfolding transitions, in the presence of both the (alcohol + salt) mixtures were found to be reversible as judged by the same values of absorbance observed at different temperatures during cooling after the completion of thermal unfolding. In the presence of the 0.25 mol · dm−3 (HFIP + GndSCN) equimolar mixture and 0.85 mol · dm−3 (isopropanol + GndSCN) equimolar mixture, α-lactalbumin was observed to be in the partially folded state with significant loss of native tertiary structure. Intrinsic fluorescence results, acrylamide and potassium iodide quenching, 8-anilino-1-naphthalenesulfonic acid (ANS) binding, and energy transfer results also corroborate the presence of partially folded states of α-lactalbumin. Apart from the generation of the partially folded states, it was also observed that destabilizing action of GndSCN is reduced in the presence of isopropanol compared to that in HFIP. Isothermal titration calorimetry has been used to characterize the energetics of ANS binding to the partially folded states of the protein. ITC results indicate that ANS binds to these partially folded states at pH 7.0 due to the presence of two sequentially binding sites on the protein under the solvent conditions employed. For example, ANS binds to the 0.25 mol · dm−3 (HFIP + GndSCN) induced partially folded state with affinity constants K1 = (858 ± 220), K2 = (1.12 ± 0.25) · 103; enthalpies of binding ΔH1 = (4.4 ± 1.0) kJ · mol−1, ΔH2 = (2.1 ± 0.2) kJ · mol−1; and entropies of binding ΔS1 = 70 J · K−1 · mol−1 and ΔS2 = 65 J · K−1 · mol−1, respectively at these two sequential binding sites. In light of the fluorescence results, possible binding sites where ANS can bind to the protein have also been suggested.

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