Characterization of hen phosvitin in aqueous salt solutions: Size, structure, and aggregation
Takeuchi, Machi; Mashima, Tsuyoshi; Sztucki, Michael; Petukhov, Andrei V.; Vis, Mark; Friedrich, Heiner; Tuinier, Remco
(2022) Food Hydrocolloids, volume 129
(Article)
Abstract
Phosvitins is a key egg yolk protein and can often be found in food emulsions. It is highly phosphorylated and hence phosvitins contain a large number of negatively charged amino acid groups, for pH > pI. Due to the presence of these phophoserines, phosvitins bind to positively charged multivalent ions.
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Its amphipolar structure makes phosvitin also an efficient emulsion stabilizer. The ion binding and emulsifying abilities of phosvitins are influenced by environmental conditions such as pH and ionic strength. Various physicochemical properties of phosvitins such as size and charge under various conditions, and how they self-assemble via multivalent ions are not well-understood. To gain more insight into these physical characteristics, we performed high brilliance synchrotron small angle X-ray scattering (SAXS) on phosvitin solutions. The structure factor S(q) obtained from the SAXS profiles showed that the double layer interactions between charged phosvitin assemblies are strongly affected by pH and ionic strength of the buffer. The effects of multivalent ions (Mg2+, Fe3+) on the size and structure of phosvitin were also investigated. Our results revealed that the aggregation of phosvitin mediated by metal ions is induced by electrostatic attraction and only occurs beyond a threshold cation concentration, where phosvitin loses long-range electrostatic double layer repulsions. These findings help understanding the effects of metal ions and pH on phosvitin in more complex environments such as food emulsions.
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Keywords: Electrostatic interaction, Phosvitin, Small angle X-ray scattering, Structure factor, Food Science, General Chemistry, General Chemical Engineering
ISSN: 0268-005X
Publisher: Elsevier
Note: Funding Information: This research was financially supported by the Netherlands Organisation for Scientific Research in the framework of the Innovation Fund for Chemistry and from the Ministry of Economic Affairs in the framework of the “TKI/PPS-Toeslagregeling”, grant number 731.017.301 . We acknowledge the European Synchrotron Radiation Facility for provision of synchrotron radiation facilities and we would like to thank Dr. Theyencheri Narayanan for enabling the execution of the experiments at beamline ID02. We thank Dr. Naomi Elstone for helping with the scattering data analysis and Joeri Opdam for useful discussions. We thank Prof. Dr. John van Duynhoven (Unilever) for useful discussions. Funding Information: Purification of phosvitin was performed with anion exchange chromatography (AEC) following a previously published (Zhang, Qiu, Geng, & Ma, 2011) purification protocol. Zhang et al. (Zhang et al., 2011) employed ethylenediaminetetraacetic acid disodium salt (Na2EDTA) during AEC to obtain metal-free phosvitin. We did not use Na2EDTA during purification of phosvitin to study the protein in its native state as used in food emulsions. Besides the above purification, size exclusion chromatography (SEC) (Bio-Rad NGC chromatography system with GE healthcare HiLoad Superdex 200 16/600, eluent: 100 mM potassium phosphate buffer, pH 7) was performed to further purify phosvitin. Analytical SEC was performed to estimate the molar mass of purified phosvitin (see the Supporting Information, section S1). Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) was conducted to evaluate the purity of the obtained phosvitin. Samples were heated at 95 °C for 5 min in sample buffer (Laemmli Sample Buffer (Bio-Rad) and 50 mM DTT) and run on pre-cast 4–20% Mini-PROTEAN® TGX gels (Bio-Rad) in running buffer (Tris/Glycine/SDS Buffer, Bio-Rad) for 35 min at 180 V. Precision Plus Protein™ All Blue Standards (Bio-Rad) was used as a reference. Gels were stained with Coomassie Brilliant Blue G-250 (Bio-Rad). An image of the gel was acquired using an ImageQuant 400 Digital Imager (GE Healthcare). The purified phosvitin solution was rapidly frozen by liquid nitrogen and stored at a temperature T = − 20 °C prior to further use.To obtain high-purity phosvitin for the scattering studies, size exclusion chromatography (SEC) was performed to remove impurities from phosvitin solutions after isolating phosvitin from egg with anion-exchange chromatography. The SEC trace showed a clear peak at the elution volume of 66–86 mL (Fig. 1A). Purity of the eluted fraction was analyzed by SDS-PAGE (Fig. 1B). The band corresponding to about 35 kDa is considered to correspond to individual phosvitin molecules (Huopalahti et al., 2007). In contrast, the eluted solution without SEC had multiple bands in the SDS-PAGE electrophoretogram, which were remaining impurities from egg yolk. We used the high-purity phosvitin obtained from elution fraction I in the SEC trace to prepare dispersions for the scattering measurements. To evaluate the molar mass (M) of the obtained phosvitin, analytical SEC was conducted (for details see the Supporting Information, section S1). The trace of analytical SEC revealed that the molar mass M of the purified phosvitin after SEC was about 140 kDa. Therefore, the purified phosvitin is expected to be α-phosvitin composed of 3–4 individual phosvitin subunits of 35 kDa (Huopalahti et al., 2007).This research was financially supported by the Netherlands Organisation for Scientific Research in the framework of the Innovation Fund for Chemistry and from the Ministry of Economic Affairs in the framework of the “TKI/PPS-Toeslagregeling”, grant number 731.017.301. We acknowledge the European Synchrotron Radiation Facility for provision of synchrotron radiation facilities and we would like to thank Dr. Theyencheri Narayanan for enabling the execution of the experiments at beamline ID02. We thank Dr. Naomi Elstone for helping with the scattering data analysis and Joeri Opdam for useful discussions. We thank Prof. Dr. John van Duynhoven (Unilever) for useful discussions. Publisher Copyright: © 2022 The Author(s)
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