Modification of hysteresis behaviors of protein monolayer and the corresponding structures with the variation of protein surface charges.

Successive compression-decompression cycles of the surface pressure (π) - specific molecular area (A) isotherms of protein (BSA) monolayers show that reversible hysteresis persists if the protein molecules contain effective positive or negative surface charges. However, for neutral condition, i.e., close to the isoelectric point of the protein, irreversibility in the hysteresis behaviour dominates. Out-of-plane structures obtained from the X-ray reflectivity analysis suggest that at lower surface pressure monomolecular layer of BSA is formed on the water surface. With increasing surface pressure, molecules start to lift-up from the water surface in such a way that semi-major axis makes an angle with the water surface. Depending on the surface pressure and surface charge of BSA, monomolecular or bimolecular layer of tilted BSA molecules is formed on the water surface, however, formation of bimolecular layer is observed when the pH is relatively closer to the BSA isoelectric point. After complete decompression, tilted monomolecular or bimolecular structures again transform into monomolecular layer as evidenced from the structural analysis of the films deposited at lower surface pressures in the second compression, however, structural hysteresis varies depending upon the subphase pH or protein surface charge. Structures obtained from the films deposited at first and second compressions at lower pressure implies that although structural dissimilarity is present but structural hysteresis is only present near the isoelectric point of BSA and becomes negligible below and above that pH. Competitive electrostatic and van der Waals interactions are responsible for such hysteresis behaviours and structural modifications.