Langmuir monolayer of lysozyme at variable subphase pH conditions: a comprehensive study on structure, morphology and hysteresis behaviour

Formation of a pure Langmuir monolayer of lysozyme at the air–water interface and its investigation by means of a surface pressure (π)–mean molecular area (A) isotherm has been accomplished under different subphase pH conditions. A normalized area–time curve confirms the stable nature of the lysozyme monolayer whose compressibility variation with an increased surface pressure at specific subphase pH has also been studied from π–A isotherms. The monolayers exhibit irreversible hysteresis behaviour irrespective of subphase pH conditions, as evidenced from successive compression–expansion π–A isotherm cycles. Comparison of surface thermodynamics under hysteresis with subphase pH variation confirms that the monolayer at subphase pH ≈ 4.0 involves a greater amount of energy to attain and retain the ordered and compact monolayer than the other two pH conditions (pH ≈ 7.0 and 9.5). In situ visualization of lysozyme monolayers by Brewster angle microscopy suggests the homogeneous and stripe-like pattern formation at lower and higher surface pressure respectively. Further investigations of lysozyme films at solid surfaces have been carried out with atomic force microscopy and X-ray reflectivity (XRR) analysis. Structural reversibility of lysozyme molecules under compression–expansion–compression of the monolayer is revealed from the comparison of height profiles of AFM images and electron density profiles as extracted from XRR analysis of the films deposited during both first and second compressions of the monolayer. The mechanism of the structural rearrangement of lysozyme molecules with surface pressure variation at different subphase pH is explored, correlating macroscopic and microscopic information.