Hollmann, O. ; Steitz, R. ; Czeslik, C.: Structure and dynamics of alpha-lactalbumin adsorbed at a charged brush interface. Physical Chemistry Chemical Physics 10 (2008), p. 1448-1456
10.1039/b716264b
Abstract:
We have studied the adsorption of a-lactalbumin at a planar poly(acrylic acid) (PAA) brush using neutron reflectometry (NR) and total internal reflection fluorescence (TIRF) spectroscopy. The PAA brush has been prepared by spin-coating silicon or quartz plates with a hydrophobic poly(styrene) film and by transferring the copolymer poly(styrene)–poly(acrylic acid) onto the modified surface. In the case of NR, the poly(styrene) film and the poly(styrene) chain ends of the copolymer were perdeuterated in order to generate a high contrast to the non-deuterated PAA brush. a-Lactalbumin was chosen as the model protein because it is a relatively small globular protein with a negative net charge at neutral pH-values, as chosen in the experiments. Thus, it is interacting with the PAA brush on the ‘wrong’ side of its isoelectric point. In addition, the effects of temperature on the volume fraction profile and the reorientational mobility of the protein within the PAA brush were determined. From the analysis of the NR data, it has been found that despite of its negative net charge, a-lactalbumin is penetrating into the PAA brush. Its volume fraction profile parallels that of the PAA brush, indicating an exclusive interaction between the protein and the PAA. No protein accumulation is found at the inner poly(styrene) or the outer solution interface of the PAA brush. When increasing the temperature from 20 to 40 1C, less protein is adsorbed, suggesting the presence of enthalpic interaction contributions. From the analysis of the TIRF data, a high degree of reorientational mobility of a-lactalbumin within a PAA brush can be inferred. The reorientational correlation time of a-lactalbumin labeled with the Alexa Fluor 488 dye was found to increase from 5.5 to 32 ns upon adsorption, which can well be explained by the higher viscosity inside the PAA brush. Overall, the results of this study quantify for the first time the molecular details of the unique interaction of a protein on the ‘wrong’ side of its isoelectric point with a planar charged brush interface. It is concluded that the high mobility of a-lactalbumin within a PAA brush can partially be understood by the presence of repulsive electrostatic interactions. There is no ‘freezing’ of the protein dynamics, which is a precondition for biological activity.