Ellipsometry and TIRF studies of enzymatic degradation of interfacial proteinaceous layers

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 w ell as the effects of addition of the proteolytic enzymes krillase and tryp sin, in relation to temperature, enzyme concentration, and enzymatic activi ty. The results for the hydrophilic substrates show that homogeneous and he terogeneous exchange occurs readily, as does autolysis of trypsin at the in terface. At the hydrophobic substrates, the effect of exchange is limited a nd a residual gelatin fraction is present at the interface throughout. The interfacial behavior of gelatin above and below the helix formation tempera ture (T-helix) shows that more extended surface layers are formed at both s ubstrates below Thelix At the hydrophilic substrates, the higher adsorbed l ayer thickness below Thelix is mainly due to the adsorption of more gelatin than at the higher temperature, whereas, at the hydrophobic substrates, th e increase in layer thickness below Thelix is due to a decrease in packing density. Enzyme addition to preadsorbed gelatin at methylated silica result s in the transition to a thinner and denser layer that contains both residu al gelatin and proteolytic enzymes (i.e., krillase or trypsin). At hydropho bic 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 Thelix The effect of trypsin addition to preadsorbe d gelatin is enhanced at T < T-helix, which is somewhat counterintuitive co nsidering the structure of gelatin in relation to temperature. Quantitative ly, the degree of gelatin degradation after addition of trypsin at T < T-he lix was found to be higher than after addition of a 500 times higher concen tration at T > T-helix Finally, the exposure of preadsorbed gelatin to inac tivated krillase showed a nearly complete elimination in the effects observ ed upon addition of intact krillase. This indicated that the enzymatic acti vity of krillase in its native form plays a major role for the interaction between krillase and preadsorbed gelatin.