Kinetic and structural characterization of adsorption-induced unfolding of bovine alpha -lactalbumin.
Conformational changes of bovine alpha-lactalbumin induced by adsorption on a hydrophobic interface are studied by fluorescence and circular dichroism spectroscopy. Adsorption of bovine alpha-lactalbumin on hydrophobic polystyrene nanospheres induces a non-native state of the protein, which is characterized by preserved secondary structure, lost tertiary structure, and release of calcium. This partially denatured state therefore resembles a molten globule state, which is an intermediate in the folding of bovine alpha-lactalbumin. Stopped-flow fluorescence spectroscopy reveals two kinetic phases during adsorption with rate constants k(1) approximately 50 s(-1) and k(2) approximately 8 s(-1). The rate of partial unfolding is remarkably fast and even faster than unfolding induced by the addition of 5.4 m guanidinium hydrochloride to native alpha-lactalbumin. The large unfolding rates exclude the possibility that unfolding of bovine alpha-lactalbumin to the intermediate state occurs before adsorption takes place. Stopped-flow fluorescence anisotropy experiments show that adsorption of bovine alpha-lactalbumin on polystyrene nanospheres occurs within the dead time (15 ms) of the experiment. This shows that the kinetic processes as determined by stopped-flow fluorescence spectroscopy are not affected by diffusion or association processes but are solely caused by unfolding of bovine alpha-lactalbumin induced by adsorption on the polystyrene surface. A scheme is presented that incorporates the results obtained and describes the adsorption of bovine alpha-lactalbumin.
Engel
Maarten F M
MF
Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, Wageningen, The Netherlands. maarten.engel@nmr.bc.wau.nl
van Mierlo
Carlo P M
CP
Visser
Antonie J W G
AJ
D000327:Adsorption; D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D024202:Electrophoretic Mobility Shift Assay; D005454:Fluorescence Polarization; D007700:Kinetics; D007768:Lactalbumin / Q000737:chemistry*; D008863:Microspheres; D011487:Protein Conformation; D011489:Protein Denaturation
D007768:Lactalbumin