Thermodynamics of thermal unfolding of bovine apo‐α‐lactalbumin

Abstract

Thermal unfolding of bovine α‐lactalbumin in 10 mM borate buffer at pH 8.0 in the presence of 0.01–1.0 M NaCl was studied in terms of CD ellipticity. The apoprotein changes the conformation from a native‐like (N) to an unfolded (U) form, which has an appreciable amount of the secondary structure but no tertiary structure, in the two‐state type. Various thermodynamic parameters of the transition were analyzed. The differences in enthalpy and heat capacity between the N and U states are similar to the corresponding differences of the holoprotein obtained with the calorimetric method by Pfeil. It is shown that one Na+ binds with a binding constant larger than 102– 103 M‐1 to a specific site (probably to the Ca2+‐binding site) in the molecule and the bound Na+ stabilizes the N form of the apoprotein.

Moisture‐Induced Aggregation of Alpha‐Lactalbumin: Effects of Temperature, Cations, and pH

“…It would compete for the cation binding sites on apo ‐ alpha ‐lactalbumin. Although its binding affinity was lower than that of calcium, the interactions of Mg 2+ with lactalbumin still helped to stabilize the conformation of protein molecule (Hiraoka and Sugai 1984; Permyakov and others 1985; Kronman 1989; Relkin and others 1993; Boye and others 1997). The stronger an ion binds to alpha ‐lactalbumin, the more stable the protein becomes (Permyakov and Berliner 2000).…”

“…As a Ca 2+ ‐binding protein (Hiraoka and others 1980; Kronman 1989), alpha ‐lactalbumin may exist in 2 different forms at neutral pH: a Ca 2+ ‐bound form ( holo form) or a Ca 2+ ‐free form ( apo form). There are 2 cation‐binding sites on alpha ‐lactalbumin, a strong binding site and a weak one (Hiraoka and others 1980, 1984; Permyakov and others 1985; Kronman 1989; Berliner and others 1991). The folding properties and thermal stability of lactalbumin are significantly affected by its specific interactions with calcium ion (Hiraoka and Sugai 1984; Permyakov and others 1985; Pfeil and Sadowski 1985; Kuwajima and others 1990).…”

“…There are 2 cation‐binding sites on alpha ‐lactalbumin, a strong binding site and a weak one (Hiraoka and others 1980, 1984; Permyakov and others 1985; Kronman 1989; Berliner and others 1991). The folding properties and thermal stability of lactalbumin are significantly affected by its specific interactions with calcium ion (Hiraoka and Sugai 1984; Permyakov and others 1985; Pfeil and Sadowski 1985; Kuwajima and others 1990). The removal of bound calcium from lactalbumin results in a partly unfolded state of protein molecule, due to the strong repulsive forces between negative charges at the calcium‐binding site, which greatly decreases the thermal stability of lactalbumin (Griko and others 1994; Hendrix and others 2000).…”

Influence of Ca2+ binding on the structure and stability of bovine α‐lactalbumin studied by circular dichroism and nuclear magnetic resonance spectra

“…Recently, Hiraoka 8c Sugai (17,18) studied the thermal transition of apo-a-LA. The apoprotein can assume a native-like structure at a low temperature as suggested by similarity in the CD spectrum t o that observed in the native holo-protein that has one bound Ca2+ per the molecule.…”

“…Since addition of Ca2+ ion t o a-LA solution is known t o stabilize the native structure extensively (8,16) and also since existence of more than one metal-binding site on a-LA has been proposed (1 1 -13), it is important t o investigate the effect of CaZ+ ion on thc stability and structure of a-LA and to compare the results with those known for the apo-protein. In the present study, the CD spectrum a t 2 7 0 n m that was previously used in the apo-protein studies (8,(16)(17)(18) was also used to follow the thernial unfolding a t different concentrations of Ca2+. Comparison o f the structures of the native-like apo-protein and tlie native holo-protein was made by nieans of 500-MHz proton n.ti1.r.…”