Abstract
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.
Highlights
The structure of denatured ␣-lactalbumin (␣-LA) has been characterized using the method of disulfide scrambling
In the presence of denaturant and thiol initiator, the native ␣-LA denatures by shuffling its native disulfide bonds and converts to a mixture of fully oxidized scrambled isomers that are trapped by non-native disulfide bonds. ␣-LA contains four disulfide bonds and may adopt 104 possible scrambled isomers
The Structure of Denatured ␣-Lactalbumin and heterogeneity of denatured ␣-LA, 2) to elucidate the unfolding pathway of ␣-LA denatured under increasing concentrations of selected denaturants, and 3) to characterize the molecular structure of the molten globule state of ␣-LA
Summary
Materials—Calcium-depleted bovine ␣-LA (L-6010) was used throughout this study and was obtained from Sigma. The major criteria to ensure that denaturation has reached equilibrium is time-course kinetics analysis, which indicates that conversion of the native ␣-LA to the scrambled species has reached a plateau, and the ratio of scrambled to the native species remains constant during prolonged sample incubation. Another method to verify the state of equilibrium of denaturation is to renature (refold) the extensively unfolded ␣-LA under the same conditions that denature the native ␣-LA, for instance, by allowing the sample denatured with 8 M GdmCl to refold after dilution of the denaturant. X-␣-LA-a represents species “a” of scrambled ␣-LA
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