Ellipsometry and total internal reflectance fluorescence spectroscopy (TIRF) have been employed to
investigate the layer structure of gelatin adsorbed from aqueous solutions onto silica/glass and methylated
silica/glass, as well as the effects of addition of the proteolytic enzymes krillase and trypsin, in relation
to temperature, enzyme concentration, and enzymatic activity. The results for the hydrophilic substrates
show that homogeneous and heterogeneous exchange occurs readily, as does autolysis of trypsin at the
interface. At the hydrophobic substrates, the effect of exchange is limited and a residual gelatin fraction
is present at the interface throughout. The interfacial behavior of gelatin above and below the helix formation
temperature (T
helix) shows that more extended surface layers are formed at both substrates below T
helix.
At the hydrophilic substrates, the higher adsorbed layer thickness below T
helix is mainly due to the adsorption
of more gelatin than at the higher temperature, whereas, at the hydrophobic substrates, the increase in
layer thickness below T
helix is due to a decrease in packing density. Enzyme addition to preadsorbed gelatin
at methylated silica results in the transition to a thinner and denser layer that contains both residual
gelatin and proteolytic enzymes (i.e., krillase or trypsin). At hydrophobic surfaces, a faster and more
extensive degradation of the gelatin layer is observed with increasing krillase concentration, the effect of
which is similar above and below T
helix. The effect of trypsin addition to preadsorbed gelatin is enhanced
at T < T
helix, which is somewhat counterintuitive considering the structure of gelatin in relation to
temperature. Quantitatively, the degree of gelatin degradation after addition of trypsin at T < T
helix was
found to be higher than after addition of a 500 times higher concentration at T > T
helix. Finally, the
exposure of preadsorbed gelatin to inactivated krillase showed a nearly complete elimination in the effects
observed upon addition of intact krillase. This indicated that the enzymatic activity of krillase in its native
form plays a major role for the interaction between krillase and preadsorbed gelatin.