Little is known about the changes in protein conformation that occur after displacement of a protein from an interface. Here, results are presented that give insight into the conformation of bovine a-lactalbumin (BLA) molecules that are displaced from a hydrophobic polystyrene interface. After the BLA molecules are adsorbed on polystyrene nanospheres, they are displaced from these nanospheres using two surfactants: Tween 20 and CHAPS. The properties of displaced BLA depend on the concentration of the surfactant used to displace the protein and on the incubation time during displacement, as can be concluded from intrinsic fluorescence spectroscopy, circular dichroism spectroscopy, and nondenaturing gel electrophoresis. CHAPS is more effective in displacing adsorbed BLA than Tween 20. The largest amount of displaced BLA (90% recovery) is obtained at a CHAPS concentration of 2 mM or higher. At a surfactant concentration of 1 or 2 mM, displaced BLA contains calcium and has native spectroscopic properties, indicating that BLA, which has a molten globulelike conformation in the adsorbed state, refolds to its native state upon displacement from the surface. However, non-native properties of displaced BLA are observed at a low surfactant concentration (0.3 mM) after prolonged incubation times. Under these conditions, the ensemble of displaced BLA molecules contains calcium, has a nativelike secondary structure, has a non-native tertiary structure, and contains a population of molecules that has a higher electrophoretic mobility on nondenaturing gels compared to that of native BLA. Intramolecular disulfide shuffling can cause the observed conformational changes. The disulfide shuffling is initiated by a few reactive groups on the surface of the nanospheres. It occurs during the homomolecular exchange of proteins at a surfactant concentration of 0.3 mM and is time dependent. Both Tween 20 and CHAPS are good candidates for the removal of proteins from interfaces, as long as the incubation time is short and the surfactant concentration is above a certain threshold. The displacement procedure presented here is essential for the future study of the atomic details of the conformation of proteins adsorbed on interfaces using NMR spectroscopy in combination with H/D exchange measurements.