Protein Orientation

Lysozyme Orientation at the Silica-Water Interface

Lysozyme adsorption at the silica-water interface and subsequent layer structures were investigated. The build-up (and removal) of molecular layers adsorbing or reacting on a lightly doped silicon dioxide (silica) surface were measured in terms of thickness and refractive index changes over time. Lysozyme adsorption was monitored at a range of concentrations (from 0.03 g dm-3 to 4.0 g dm-3) and at both pH 4 and pH 7 in a flow-through cell arrangement. Adsorbed layers ranging from 14 to 43 ± lA in thickness and 0.21 to 2.36 ± 0.05 mg m-2 in terms of mass coverage were observed at pH 4 with increasing lysozyme concentration, indicating a strong deformation of the monolayer over the low concentration range and formation of an al-

Fig. 2. Schematic representation showing the physisorbed lysozyme at pH 4 and pH 7 at 1 g dm-3 and 20 °C at the silica-water interface

most complete sideways-on bilayer in which the molecules were adsorbed with their short axis normal to the silica surface at the highest concentrations. At pH 7, the thickness of adsorbed layers varied from 16 to 54 ± lA, with significantly higher surface coverage (0.74-3.29 ± 0.05 mg m-2) over the same lysozyme concentration range, again indicating structural deformation during the initial monolayer formation followed by a closely packed monolayer where the long axis of the lysozyme molecule was oblique to the silica surface at intermediate concentrations and a subsequent bilayer where the molecules of the second layer were oriented with the short axis normal to the silica surface at the highest concentrations. These experiments were repeated with a fixed lysozyme concentration of 1.0 g dm-3 whilst cycling the pH from 7 to 4 and back again or vice versa. Broadly reversible adsorption was observed, regardless of whether the pH was cycled or not. These observations agree with earlier studies undertaken using neutron reflection (Su et al. 1998a). A schematic representation of changes in orientation is shown in Fig. 2 (Freeman et al. 2004a).

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