4.1.3. Conformation Changes upon Immobilization

While electrostatic interactions between the enzyme and the support may drive the adsorption process and prevent enzyme leaching, strong interactions between two charged entities can affect the structure of the enzymes. Flattening of the structure was for example observed when proteins were immobilized on gold metal [118], while no change in the secondary structure was detected when immobilization occurred on a gold surface modified by a self-assembled-monolayer (SAM) of thiol [116]. In the course of adsorption, protein density increases on the surface, and enzyme/enzyme interactions may surpass enzyme/support interactions. Low surface coverage can induce modification of the conformation because the enzyme molecules maximize their contact surface. At higher enzyme loading, it can be expected that protein-protein interactions decrease the interaction with the surface and preserve enzyme conformation. Protein–protein interactions may otherwise induce repulsion between the neighbors which increases with the decrease of the distances between the adsorbed proteins. A variety of methods exists to gain information on the conformation of the enzyme in the immobilized state including CD, surface plasmon resonance (SPR), ellipsometry, fluorescence, and Raman and FTIR spectroscopies. However, characterization methods that can separate effects due to complex coupled mechanisms upon enzyme immobilization are lacking, and data often result in spatial and temporal averages.
