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Thiol-induced oligomerization of α-lactalbumin at high pressure

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Abstract

Denaturation and aggregation ofα-lactalbumin at high pressure (up to 10 kbar, 1000 MPa) were studied by means of circular dichroism, gel-permeation chromatography, sodium dodecyl sulfate and gel electrophoresis. It was found that the unfolding ofα-lactalbumin at high pressure is reversible even in basic pH and at a protein concentration as large as 10%. In these conditions only a negligible fraction of the protein is denatured irreversibly and aggregates. The rate of aggregation ofα-lactalbumin at high pressure increases significantly in the presence of low-molecular reducing agents such as cysteine, 2-mercaptoethanol, and dithiothreitol. Maximal yield ofα-lactalbumin oligomerization (over 90%) was achieved in the presence of cysteine at the molar cysteine/protein ratioq=2 and atpH 8.5. Apparent molecular weight of the obtained oligomers was over 500 kDa. It was shown that the size distribution of oligomers can be modulated by varyingpH and reducing agent. The size distribution shifts in the direction of very large, poorly soluble particles whenpH decreases. Maximal content of the insoluble fraction (about 30%) can be reached at pH 5.5 when cysteine (q=2) is used as reducing agent. The oligomers ofα-lactalbumin are stabilized mainly by nonnative interchain disulfide bridges. Circular dichroism measurements point to an additional mechanism of cohesion of polypeptide chains in the oligomers, which is formation of intermolecularβ-sheets.

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Authors and Affiliations

  1. Institut National de la Recherche Agronomique, B.P. 1627, 44316, Nantes, Cedex 03, France

    Marianne Jegouic & Thomas Haertlé

  2. Institute of Biochemical Physics, Russian Academy of Sciences, 117813, Moscow GSP-1, Russia

    Valerij Ya. Grinberg

  3. Faculté des Sciences, Laboratoire de Synthesé Organique, 44072, Nantes, Cedex 03, France

    André Guingant

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  1. Marianne Jegouic
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  2. Valerij Ya. Grinberg
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  3. André Guingant
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  4. Thomas Haertlé
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Jegouic, M., Grinberg, V.Y., Guingant, A. et al. Thiol-induced oligomerization of α-lactalbumin at high pressure. J Protein Chem 15, 501–509 (1996). https://doi.org/10.1007/BF01908531

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  • DOI: https://doi.org/10.1007/BF01908531

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