Reversible self-association of ovalbumin at air-water interfaces and the consequences for the exerted surface pressure

Abstract

In this study the relation between the ability of protein self-association and the surface properties at air-water interfaces is investigated using a combination of spectroscopic techniques. Three forms of chicken egg ovalbumin were obtained with different self-associating behavior: native ovalbumin, heat-treated ov-albumin-being a cluster of 12-16 predominantly noncovalently bound proteins, and succinylated ovalbumin, as a form with diminished aggregation properties due to increased electrostatic repulsion. While the bulk diffusion of aggregated protein is clearly slower compared to monomeric protein, the efficiency of transport to the interface is increased, just like the efficiency of sticking to rather than bouncing from the interface. On a timescale of hours, the aggregated protein dissociates and adopts a conformation comparable to that of native protein adsorbed to the interface. The exerted surface pressure is higher for aggregated material, most probably because the deformability of the particle is smaller. Aggregated protein has a lower ability to desorb from the interface upon compression of the surface layer, resulting in a steadily increasing surface pressure upon reducing the available area for the surface layer. This observation is opposite to what is observed for succinylated protein that may desorb more easily and thereby suppresses the buildup of a surface pressure. Generally, this work demonstrates that modulating the ability of proteins to self-associate offers a tool to control the rheological properties of interfaces.

Figure 1.

(A) FTIR-ATR spectra of N-OVA and T-OVA. The film was dried from 100 μL of 10 mg/mL solution of the protein in 10 mM phosphate buffer (pH 6.9). (B) Enlargement of panel A showing the amide I region of N-OVA and T-OVA. The dashed lines illustrate the frequency assignment of the secondary structure types present. (β) β-strand; (α) α-helix; (r.c.) random coil; (anti-β) antiparallel β-strand.

Figure 2.

The overlaying experimental and simulated IRRAS spectra of N-OVA and T-OVA adsorbed from a bulk protein concentration of 10 mg/mL in 10 mM phosphate buffer (pH 6.9). The equilibration time for adsorption was 60 min.

Figure 3.

The dependencies of (A) the surface layer thickness (d), (B) protein concentration in the surface layer (c₁), and (C) the ratio of the protein concentration in the surface layer and the subphase (c₂) as a function of adsorption time. The data are derived from global analysis of IRRAS spectra for N-OVA (open circles) and T-OVA (closed circles). The protein bulk concentration was 10 mg/mL in 10 mM phosphate buffer (pH 6.9).

Figure 4.

The dependencies of the surface pressure on time for N-OVA and T-OVA upon adsorption at the air/water interface measured by the automated drop tensiometer. The protein bulk concentration was 0.1 mg/mL in 10 mM phosphate buffer (pH 6.9).

Figure 5.

The amide I region of IRRAS spectra of N-OVA (A) and T-OVA (B) adsorbed at the air/water interface and estimation of the secondary structure from the band-shape analysis of the spectra. The protein bulk concentration was 10 mg/mL in 10 mM phosphate buffer (pH 6.9). The adsorption equilibration time was 60 min. (exp) Curves correspond to experimental IRRAS spectra; (sim) curves correspond to simulated IRRAS spectra.

Figure 6.

Disaggregation of T-OVA upon adsorption at the air/water interface. (A) Amide I region of IRRAS spectra of T-OVA for various times of adsorption. (B) The degree of aggregation of T-OVA as a function of the time as derived by spectral simulation of IRRAS spectra, where the ATR spectrum of T-OVA was taken as input spectrum. The protein bulk concentration was 10 mg/mL in 10 mM phosphate buffer (pH 6.9). The initial degree of aggregation was estimated from gel-permeation chromatography.

Figure 7.

(A) Example of a depth profile of the protein concentration obtained for N-OVA by confocal laser scanning the sample from the glass bottom (just below 5.9 mm) to the air/water interface and measuring the fluorescence intensity. The protein bulk concentration was 10⁻⁴ mg/mL in 10 mM phosphate buffer (pH 6.9). (B) Calibration curve of the observed fluorescence intensity in the bulk phase as a function of the protein bulk concentration. (C) The dependence of the protein surface concentration on the protein bulk concentration estimated from the depth profiles of N-OVA using the calibration curve.

Figure 8.

Autocorrelation curves obtained by fluorescence correlation spectroscopy in the bulk (curves 1; see arrow in Fig. 7A ▶) and at the interface (curves 2/3) for N-OVA (A) and T-OVA (B). In all panels the fitted (thick lines) and experimental (thin lines) curves are shown. For T-OVA two populations are found at the interface.

Figure 9.

Pressure–area isotherms for ovalbumin obtained by compression of the surface layer after adsorption during 60 min. The protein bulk concentration was 0.1 mg/mL in 10 mM phosphate buffer (pH 6.9). (A) N-OVA; (B) T- OVA; and (C) suc-OVA.

Figure 10.

T-OVA concentration in the (A) surface layer (c₁) and (B) surface layer thickness (d) evaluated from IRRAS as a function of the surface area upon compression of the surface layer. The protein bulk concentration was 0.1 mg/mL in 10 mM phosphate buffer (pH 6.9).