Effect of ions on the adsorption of lysozyme protein below its isoelectric point on hydrophilic (OH–Si) and hydrophobic (H–Si) surfaces

Abstract

Lysozyme is a rigid globular protein with four disulfide bonds that is present in human tears, saliva, sweat and milk. In the present study, thin films of this protein are deposited on hydrophilic (OH–Si) and hydrophobic (H–Si) silicon surfaces using the dip coating method. In order to study the effect of ions, these films were also fabricated by varying the concentration of mono-(Na⁺), di-(Ca²⁺) and tri-(Y³⁺) valent ions. The structural and topographical characteristics of these films are investigated using X-ray reflectivity (XRR) and atomic force microscopy (AFM) techniques and are correlated with the surface hydrophobicity/hydrophilicity. On a OH–Si surface, a thick layer of lysozyme (≈47 Å) is adsorbed in the absence of ions. However, in the presence of 1–10 mM of ions the thickness varied from ≈40 to 61 Å. Conversely, the thickness of the lysozyme film in the absence of any ions on the hydrophobic H–Si surface is ≈57 Å, which varied in the range of ≈44 to 69 Å in the presence of ions. Mostly, a bi-molecular layer is formed on the substrate surface where the substrate-attached lower layer molecules are in the side-on orientation (≈24 Å) and on top of that one more layer is formed where the molecules are either in side-on orientation or tilted with respect to the lower layer. On the OH–Si surface, pure or ion-interacted lysozyme films are adsorbed in their native globular form with almost negligible structural modification. However, on the H–Si surface, the globular shape of the adsorbed lysozyme molecules is slightly elongated mostly due to the presence of the relatively higher hydrophobic interaction between the protein and H–Si surface. The hydrophobic nature of the protein films as obtained from the contact angle studies showed that on the OH–Si surface the contact angle of the lysozyme film interacting with Na⁺ ions is the highest, whereas it is intermediate and the lowest for Y³⁺ and Ca²⁺ ions, respectively. Moreover, the contact angle of the lysozyme film is the highest for Ca²⁺ and the lowest for Na⁺ ions on the H–Si surface. Therefore, the thicker protein films exhibit more hydrophobic nature and the contact angle varies in accordance with the electron density of the adsorbed films.