Monomerization and aggregation of β-lactoglobulin under adverse condition: A fluorescence correlation spectroscopic investigation

beta-Lactoglobulin is one of the major components of bovine milk and it remains in a dimeric form under physiological conditions. The present contribution elucidates the structural change of beta-lactoglobulin at pH 7.4 under the action of guanidine hydrochloride (GnHC1) and heat at the single molecular level. The only free cysteine (Cys-121) of beta-lactoglobulin has been tagged with 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) for this purpose. The dimeric structure of beta-lactoglobulin found to undergoes a monomerization prior to the unfolding process upon being subjected to GnHCI. The hydrodynamic diameter of the native dimer, native monomer and the unfolded monomer has been estimated as similar to 55 angstrom, similar to 29 angstrom and similar to 37 angstrom, respectively. The free energy change for the monomerization and denaturation are respectively 1.57 kcal mol(-1) and 8.93 kcal mol(-1). With change in temperature, development of two types of aggregates (small aggregates and large aggregates) was observed, which is triggered by the formation of the monomeric structure of j3-lactoglobulin. The hydrodynamic diameters of the smaller and larger aggregates have been estimated to be 77 A and similar to 117 angstrom, respectively. The formation of small aggregates turns out to be reversible whereas that of larger aggregates is irreversible. The free energy associated with these two steps are 0.69 kcal mol(-1) and 9.09 kcal mol(-1). Based on the size parameters, the smaller and larger aggregates have been proposed to contain similar to twenty and similar to sixty monomeric units. It has also been concluded that the monomeric subunits retain their native like secondary structure in these aggregates.