Immobilized protein films for assessing surface proteolysis kinetics

Enzymatic cleavage of protein substrates at solid surfaces is important in the food and detergent industries, and in biomedical applications. Creation of a reproducible protein substrate to study surface proteolysis is difficult as protein monolayers may not necessarily provide complete coverage of the surface, and protein multilayer systems are often unstable and nonuniform. We present a method to form a reproducible, immobilized, multilayer protein substrate. A 100-nm ovalbumin protein film is spin-cast onto an amine-functionalized silicon wafer and chemically cross-linked using glutaraldehyde to create a multilayer film. This protein film is stable in the presence of non-protease components such as detergents, and can be tailored to include different proteins and their mixtures, and varying degrees of susceptibility to proteolysis. Ellipsometry was used to measure the protein-film thickness as the substrate is cleaved by the protease subtilisin Carlsberg. The decrease in film thickness over time was found to be linear, indicating the depth-homogeneity of the model substrates. Lateral-homogeneity of the substrates was corroborated by atomic force microscopy (AFM) and by the reproducibility of the ellipsometric film thickness measured across different spots on the sample substrates. AFM of the multilayer protein surface before and after exposure to enzyme suggests uniform areal surface cleavage by the protease.