Evidence that the induction time in the surface pressure evolution of lysozyme solutions is caused by a surface phase transition

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.