Evidence that the Induction Time in the Surface Pressure Evolution of Lysozyme Solutions Is Caused by a Surface Phase Transition

Abstract

Induction periods have been reported in the surface pressure evolution of a wide variety of proteins. In this work, this induction period is shown to be caused by a first-order phase change from a surface gaseous to a liquid-expanded state as the protein lysozyme adsorbs and decreases the mean area per molecule. The evolution of this transition is studied using concomitant fluorescence microscopy and surface pressure measurements. The fluorescent images are obtained by using spread films of the dye NBD-HDA at the air-liquid interface. This dye fluoresces when in contact with hydrophobic moieties and is quenched when in contact with water. The hydrophobic liquid-expanded domains therefore appear bright on a dark background. Lysozyme initially adsorbs to establish a surface gaseous phase, and the interface appears dark. The surface tension is fairly insensitive to changes in area per molecule in this state. Once the adsorbed concentration of lysozyme reaches the surface gaseous binodal, protein interactions drive a first-order phase change. Domains of liquid expanded phase grow at the interface at the expense of the surface gaseous phase. During this phase change, the surface tension remains constant. Only after the interface is covered in the bright, liquid-expanded phase does the surface tension decrease from the clean interface value. These results are discussed in terms of the surface coverage and orientation of lysozyme.

Dilational rheology of protein films adsorbed at fluid interfaces

“…In several cases, considerable adsorptions were measured at nearzero surface pressures [18,19]. Such behaviour was observed with both small-area surfactants [19] and proteins [20,21]. This indicates that long induction periods can be compatible with diffusioncontrolled adsorption [2 •• ,18].…”

Phases and phase transitions in Gibbs monolayers of an alkyl phosphate surfactant

“…Surface pressure was then followed with time, and simultaneously the change in surface morphology was observed by BAM. explanation is that the induction time during adsorption of a surfactant is caused by a first-order phase transition from a gaseous state to a higher density phase [10][11][12][13]47,48]. After the induction time, the surface pressure rises with time, indicating that continuous adsorption of the molecules takes place during the experiment.…”

“…Any conspicuous cusp point in these π -t curves indicates a first-order phase transition [5][6][7][8][9]. Most claims of first-order phase transition in Gibbs monolayers are based on the existence of such a cusp point in the π -t curves followed by a plateau or on the surface tension relaxation in the γ -t curves [10][11][12][13]. Although, previously, there were some controversies over the existence of phase transitions, a first-order phase transition from a lower density state to a higher density state in Gibbs monolayers is now well established [5][6][7][8][9][10][11][12][13][14][15].…”

“…The gaseous state reflects little light and appears dark, while the disordered LE and ordered LC phases change the refractive index so that these phases appear bright, with the LC phase brighter than the LE phase. Among the variety of mesophases in 2D systems, BAM can, at least, clearly visualize the two-phase coexistent state of G-LC [24,25], G-LE [12,13,26], and/or LE-LC [26][27][28][29] phase transitions.…”

The adsorption and unfolding kinetics determines the folding state of proteins at the air–water interface and thereby the equation of state

“…Some authors suggest that only an unfolded protein will adsorb at the interface, thereby relating structural stability to the initial adsorption [1,8,9]. Other authors claim that protein unfolding is required to increase the surface pressure [10][11][12]. In these models the conformational state of the proteins is often assumed to change with the surface pressure.…”