Irreversible self-assembly of ovalbumin into fibrils and the resulting network rheology

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Abstract

The self-assembly of ovalbumin into fibrils and resulting network properties were investigated at pH 2, as a function of ionic strength. Using transmission electron microscopy (TEM), the effect of ovalbumin concentration on the contour length was determined. The contour length was increasing with increasing ovalbumin concentration. TEM micrographs were made to investigate the effect of ionic strength on the contour length. In the measured ionic strength regime (0.01–0.035 M) fibrils of approximately equal length (±200 nm) were observed. TEM micrographs showed that the contour length of the fibrils, versus time after dilution, remained constant, which indicates that the self-assembly of ovalbumin is irreversible. Using the results of rheological measurements, we observed a decreasing critical percolation concentration with increasing ionic strength. We explain this result in terms of an adjusted random contact model for charged semiflexible fibrils. Hereby, this model has now been proven to be valid for fibril networks of β-lg, BSA and, currently, for ovalbumin.

Introduction

Ovalbumin belongs to a family of more than 20 homologous proteins, the serpins, found in animals, plants and viruses [1]. Most of these proteins are serine protease inhibitors, but for ovalbumin no inhibitory activity is known [1]. Ovalbumin is a major globular protein component in egg white, and has multi-functional properties, like its ability to foam and to form gels upon heating [2], [3], [4], [5]. An ovalbumin monomer consists of 385 amino acid residues, and has a molecular weight of 45,000 Da [2], [3], [6]. One monomer has an ellipsoidal shape with dimensions of 7nm×4.5nm×5 nm [7]. One internal S–S bridge is present in ovalbumin, and four free sulfhydryl groups [3]. Its secondary structure is composed of 30.6% α-helices and 31.4% β-sheets [1], [8]. This secondary structure content is almost the same for pH 2 and pH 7. The native tertiary structure is disrupted at acidic pH, which indicates the presence of a molten globule state at acidic pH [9].

The formation of heat-induced ovalbumin gels takes place in three steps: denaturation, aggregation and the formation of a gel network [10]. In the denaturation process, a change from the native protein conformation to a more unfolded conformation takes place, which makes the protein molecules more reactive [11], [12]. Some of the hydrophobic areas that have been buried in the interior of the globular molecule are exposed on the surface, and may act as binding sites for aggregation of thermally denatured ovalbumin molecules [2], [12]. The denaturation temperature of ovalbumin at pH 7 is 80 °C, whereas a denaturation temperature of 63.9 °C was found at pH 3 [13], [14].

After denaturation, intermolecular bonds between heat-denatured ovalbumin monomers are formed, when the protein concentration is high enough. This aggregation process is governed by a balance between attractive hydrophobic and repulsive electrostatic interactions [3], [12], [13], [15].

At pH values far from the isoelectric point and at low ionic strength, electrostatic repulsive forces hinder the formation of random aggregates, and linear fibrils are formed [16], [17], [18]. At high ionic strength, and a pH near the pI, random aggregates are formed because of a weaker electrostatic repulsion [16], [17], [18].

The final step in the gelation process is the gel formation. When the protein concentration is high enough, and when there is a balance between attractive and repulsive forces between the fibrils, a well ordered tertiary network can be formed [12], [14], [15], [16], [19].

The presence of fibrillar structures was observed using transmission electron microscopy (TEM) [13], [20], [21], [22]. Koseki et al., and Doi et al. performed experiments on ovalbumin at pH 7 and low ionic strength [13], [21]. They both observed a diameter of the fibrils of 5 nm. The contour length of fibrils was increasing with increasing heating time, and increasing protein concentration. For 0.5% ovalbumin, a contour length of approximately 180 nm was found when the solution was heated at 85 °C for 20 min. A contour length of about 200±50 nm was found for 1% ovalbumin heated at 75 °C for 2 h, or 0.5% ovalbumin heated at 75 °C for 24 h [13], [21]. Weijers et al. [20] performed TEM experiments on 2% ovalbumin at acidic pH. For pH 2 a contour length of 25 nm was found for 0.01 M ionic strength, and a length of about 140 nm was observed for 0.025 M ionic strength [20].

In this study, we investigated the self-assembly of ovalbumin into fibrils and the resulting network rheology at conditions far below the isoelectric point. It is important to get insight in the self-assembly process and network rheology far below the isoelectric point, because these differ significantly from those at pH values above the isoelectric point. The effect of ionic strength on the critical percolation concentration of ovalbumin gels at pH 2 was investigated. TEM experiments were performed near the percolation concentration to estimate the effect of ionic strength on the contour length of the fibrils. The assembly mechanism of ovalbumin is not evident. To get more insight in the self-assembly process of ovalbumin, the effect of protein concentration, and the effect of time after dilution on the contour length was investigated, using TEM experiments. Results from rheological measurements can be used to determine the critical percolation concentration, cp, which is the minimum concentration for gelation, as a function of ionic strength. Percolation theory assumes the following relation: G′∼(ccp)t, where G′ is the storage modulus, c the monomer concentration, and t a scaling exponent dependent on the Hamiltonian of the system [23]. The dependence of cp on ionic strength was explained using an adjusted random contact model for charged semiflexible fibrils [24].

Section snippets

Sample preparation

Ovalbumin was obtained from Sigma (A5503, lot no.120k7001) with a purity of at least 98%. The protein was dissolved in bidistilled water, of set ionic strength (between 0.01 and 0.035 M) and was stirred for 30 min. The pH was adjusted to pH 2, and after that stirred for 1.5 h. The ovalbumin solutions were centrifuged at 20,000×g for 30 min, to remove any traces of undissolved material. The supernatant was filtered through a protein filter (FP 030/2, 0.45 μm, Schleicher and Schuell). The

Effect of ovalbumin concentration on the contour length

The effect of the ovalbumin concentration on the contour length was investigated for 2–7% ovalbumin at pH 2 and an ionic strength of 0.01 M (Fig. 1a–f). All samples were heated at 80 °C for 1 h. An increasing contour length with increasing ovalbumin concentration was observed. Fig. 2 shows the average value (based on the average of 10 single fibrils) of the contour length at various ovalbumin concentrations.

Effect of ionic strength on the contour length

Fig. 3a–f shows the effect of ionic strength (0.01–0.035 M) on the contour length of

Conclusion

The effect of ovalbumin concentration on the contour length was investigated using TEM. An increasing contour length with increasing ovalbumin concentration was observed. Investigation of the effect of ionic strength on the contour length showed that for all measured conditions (0.01–0.035 M) fibrils of approximately equal length were formed. Dilution of heated ovalbumin samples showed that the fibrils did not fall apart up to 24 h after dilution. This result indicates that the aggregation

Acknowledgements

The authors thank Harry Baptist for his assistance with transmission electron microscopy, and Jan van Lent from the Laboratory of Virology, Wageningen University, for his technical advice on transmission electron microscopy.

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