1,1,1,3,3,3-hexafluoroisopropanol induced thermal unfolding and molten globule state of bovine alpha-lactalbumin: calorimetric and spectroscopic studies.
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.
Department of Chemistry, Indian Institute of Technology, Bombay, Powai, Mumbai 400076, India.
Agnita
A
Kundu
Nand
N
Kishore
D000818:Animals; D002152:Calorimetry, Differential Scanning; D002417:Cattle; D002942:Circular Dichroism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007768:Lactalbumin / Q000737:chemistry*; D020005:Propanols / Q000494:pharmacology*; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects*; D012997:Solvents; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014867:Water / Q000737:chemistry; D006845:Hydrocarbons, Fluorinated
D007768:Lactalbumin; D020005:Propanols; D012997:Solvents; D014867:Water; C001337:hexafluoroisopropanol; D006845:Hydrocarbons, Fluorinated