Kinetic and structural characterization of adsorption-induced unfolding of bovine α-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 a-lactalbumin. Stopped-flow fluorescence spectroscopy reveals two kinetic phases during adsorption with rate constants k(1) similar to 50 s(-1) and k(2) similar to 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 a-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 a-lactalbumin.