The structure of denatured α-lactalbumin elucidated by the technique of disulfide scrambling -: Fractionation of conformational isomers of α-lactalbumin

The structure of denatured alpha -lactalbumin (alpha -LA) has been characterized using the method of disulfide scrambling. Under denaturing conditions (urea, guanidine hydrochloride, guanidine thiocyanate, organic solvent or elevated temperature) and in the presence of thiol initiator, alpha -LA denatures by shuffling its four native disulfide bonds and converts to a mixture of fully oxidized scrambled structures. Analysis by reversed-phase HPLC reveals that the denatured alpha -LA comprises a minimum of 45 fractions of scrambled isomers. Among them, six well populated isomers have been isolated and structurally characterized. Their relative concentrations, which represent the fingerprinting of the denatured alpha -LA, vary substantially under different denaturing conditions. These results permit independent plotting of the denaturation and unfolding curves of alpha -LA Most importantly, unique isomers of partially unfolded alpha -LA were shown to populate at mild and selected denaturing conditions. Organic solvent disrupts preferentially the hydrophobic alpha -helical domain, generating a predominant isomer containing two native disulfide bonds at the beta -sheet domain and two scrambled disulfide bonds at the alpha -helical region. Thermal denaturation selectively unfolds the beta -sheet domain of alpha -LA, producing a prevalent isomer that exhibits structural characteristics of the molten globule state of alpha -LA.