Ellipsometry and TIRF Studies of Enzymatic Degradation of Interfacial Proteinaceous Layers

Abstract

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.

Effect of the Polycation Nature on the Structure of Layer-by-Layer Electrostatically Self-Assembled Multilayers of Polyphenol Oxidase

“…We employed the values of d f and n f for PAH/PPO and PDDA/PPO films and assumed ∂ n /∂ c ≅ 0.177 cm 3 g -1 , obtained for GOx solutions of different concentrations, because extinction coefficients of PPO solution precluded the direct measurement for this enzyme. Similar values (0.19 cm 3 g -1 ) have been found for proteolytic enzymes and other proteins …”

“…The value of the ellipsometric areal mass density, m e , is usually obtained from d f and n f through the equation proposed by de Feijter et al where n o is the electrolyte solution refractive index and ∂ n /∂ c is the variation of the refractive index of the adsorbed substance with its concentration. This equation has been widely employed for protein films on planar surfaces and on colloids . As areal mass values were assessed from ellipsometric thickness, using a single layered model, the superficial mass concentrations involve averages values of surface roughness.…”

Unraveling the Spatial Distribution of Immunoglobulins, Enzymes, and Polyelectrolytes within Layer-by-Layer Self-Assembled Multilayers. Ellipsometric Studies

“…These coefficients have been widely employed for protein films on planar and on colloid surfaces . Similar values of ∂ n /∂ c ≅ 0.19 cm 3 g -1 have also been found for proteolytic enzymes and other proteins . Table summarizes the results for IgG, IgG-GOD, and the four layers of AP−PPO.…”

Redox Driven Swelling of Layer-by-layer Enzyme−Polyelectrolyte Multilayers

“…A lower limit, on the other hand, was assessed from the ellipsometric areal mass density, Δ m e , obtained from the optical film thickness, d f , and film refractive index, n f , through the de Feijter equation, which has been widely employed for protein films: where n o is the electrolyte refractive index and ∂ n /∂ c is the refractive index increment of the exchanged solution with concentration. For KNO 3 solutions in the concentration range of 0−0.27 g mL -1 , we have obtained a linear relationship with ∂ n /∂ c = 0.089 cm 3 g -1 (see Supporting Information).…”