1,1,1,3,3,3‐hexafluoroisopropanol induced thermal unfolding and molten globule state of bovine α‐lactalbumin: Calorimetric and spectroscopic studies

Abstract

The thermal denaturation of alpha-lactalbumin was studied at pH 7.0 and 9.0 in aqueous 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) by high-sensitivity differential scanning calorimetry. The conformation of the protein was analyzed by a combination of fluorescence and circular dichroism measurements. The most obvious effect of HFIP was lowering of the transition temperature with an increase in the concentration of the alcohol up to 0.30M, beyond which no calorimetric transition was observed. Up to 0.30M HFIP the calorimetric and van't Hoff enthalpy remained the same, indicating the validity of the two-state approximation for the thermal unfolding of alpha-lactalbumin. The quantitative thermodynamic parameters accompanying the thermal transitions have been evaluated. Spectroscopic observations confirm that alpha-lactalbumin is in the molten globule state in the presence of 0.50M HFIP at pH 7.0 and 0.75M HFIP at pH 9.0. The results also demonstrate that alpha-lactalbumin in the molten globule state undergoes a noncooperative thermal transition to the denatured state. It is observed that two of four tryptophans are exposed to the solvent in the HFIP induced molten globule state of alpha-lactalbumin compared to four in the 8.5M urea induced denatured state of the protein. It is also observed that the HFIP induced molten globule states at the two pH values are different from the acid induced molten globule state (A state) of alpha-lactalbumin.

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

“…However, it is also known that a negative contribution from the exposure of the polar groups is expected to offset partially the positive contribution (DC pol < 0; DC apolar > 0) [29,30]. In the absence of any additive, the value of DC p for a-lactalbumin is known to be (4.6 ± 0.3) kJ Á K À1 Á mol À1 [16]. Lowering the value of DC p for a-lactalbumin under these experimental conditions indicates enhanced hydrophobic interactions between the non-polar regions of the unfolded protein and the alkyl groups of isopropanol and -CF 3 groups of HFIP, respectively, leading to reduction of the extent of reordering of water molecules surrounding the hydrophobic regions of the protein.…”

“…This is also supported by our fluorescence experiments where a very low intensity of binding is observed. The sulphonate group of ANS could possibly be forming an ion pair with guanidine group of GndSCN, leading to a lesser TABLE 7 Association constant (K), enthalpy (DH), and entropy (DS) of binding of ANS to a-lactalbumin in the presence of the 0.25 mol Á dm À3 (HFIP + GndSCN) equimolar mixture and 0.85 mol Á dm À3 (isopropanol + GndSCN) equimolar mixture at pH 7.0 and T = 298.15 K according to two sequential binding sites model described by equations (15) and (16) 0.25 mol Á dm À3 (HFIP + GndSCN) 0.85 mol Á dm À3 (isopropanol + GndSCN)…”

“…Fluoro-substituted alcohols have been shown to exhibit structure stabilizing effects on proteins and peptides [13][14][15]. In recent years, use of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) has been reported in the generation of intermediate conformations and several other applications in biochemical studies [16][17][18][19][20][21]. The biological implication of HFIP has been reported in the investigation of prion diseases and Alzhiemer's amyloid peptides [22,23].…”

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

“…Xie et al 38 further suggested that the thermodynamic parameters obtained for the intermediate state of a-LA 36 are consistent with the presence of a sizeable hydrophobic core and a significant amount of secondary structure and reported that some MG-states do indeed undergo cooperative thermal transition and they are enthalpically different from the unfolded and N-states. Based upon the differential scanning calorimetric and ultraviolet (UV)-visible thermal unfolding studies of a-LA 39 and concanavalin A, 40 we have observed that the MG-state of these proteins do not show any endotherm in the entire range of the thermal scans. These observations match the findings that the MG-states do not undergo cooperative thermal transitions to the D-state.…”

Biophysical analysis of partially folded state of α-lactalbumin in the presence of cationic and anionic surfactants

“…The DC p of native a-LA is 4.7 ± 0.3 kJ K À1 mol À1 [35]. Assuming that both polar and apolar accessible surface areas do not change significantly, the overall increase in the DC p in the presence of HTAB and SDS may be attributed to the enhanced hydrophobic interactions between the non-polar regions of the unfolded proteins and the alkyl groups of HTAB and SDS preceded by drastic exposure of the apolar accessible surface area.…”