Poly(acrylic acid) (PAA) brushes are well-known to interact with proteins in an ionic strength-dependent way. Moreover， they provide a native-like environment that largely maintains the secondary structure and biological activity of adsorbed proteins. Recently， it has been shown that the application of high pressure can lead to a reduced protein adsorption at a PAA brush in the case of a positively charged protein. Here， we analyze the effect of pressure on the interactions between a protein and a PAA brush in more detail. We use calmodulin as model protein that has a negative net charge at neutral pH-value and determine the degree of adsorption at a planar PAA brush applying total internal reflection flu... More
Poly(acrylic acid) (PAA) brushes are well-known to interact with proteins in an ionic strength-dependent way. Moreover， they provide a native-like environment that largely maintains the secondary structure and biological activity of adsorbed proteins. Recently， it has been shown that the application of high pressure can lead to a reduced protein adsorption at a PAA brush in the case of a positively charged protein. Here， we analyze the effect of pressure on the interactions between a protein and a PAA brush in more detail. We use calmodulin as model protein that has a negative net charge at neutral pH-value and determine the degree of adsorption at a planar PAA brush applying total internal reflection fluorescence (TIRF) spectroscopy. Remarkably， the degree of calmodulin adsorption at a PAA brush is increasing with increasing pressure， when the protein is negatively charged. However， at low pH-value， where calmodulin is positively charged， high pressure leads to a partial desorption of the protein. Moreover， in the presence of trifluoperazine， which binds to calmodulin as a ligand， the pressure effect is diminished. The results of this study indicate that protein adsorption at a PAA brush at the "wrong" side of the isoelectric point， i.e. under net electrostatic repulsion， can involve a volume reduction that is favored under high pressure. It is suggested that this volume reduction is related to a hydration of counterions that are released from the PAA chains and the protein surface. In contrast， at pH-values close to the isoelectric point， the obtained data are consistent with a charge regulation mechanism that involves a volume increase. Thus， the application of high pressure in combination with pH-variation， as carried out in this study， provides the volume changes of adsorption that need to be consistent with any proposed mechanism of protein interaction with a PAA brush.
