Unexpected Displacement of the Equilibrium Between the Apo and the Holo Form during Ultrafiltration of the Metalloprotein α-lactalbumin
2012
Van Audenhaege , Marieke (INRA , Rennes (France). UMR 1253 Science et Technologie du Lait et de l'Oeuf) | Lambrouin , Fabienne (INRA , Rennes (France). UMR 1253 Science et Technologie du Lait et de l'Oeuf) | Piot , Michel (INRA , Rennes (France). UMR 1253 Science et Technologie du Lait et de l'Oeuf) | Gesan Guiziou , Genevieve(auteur de correspondance) (INRA , Rennes (France). UMR 1253 Science et Technologie du Lait et de l'Oeuf)
Ultrafiltration can be envisaged as an alternative to chromatography for the fractionation of metalloproteins provided the in-depth understanding of mechanisms involved during the process is completed. Considering the metalloprotein α-lactalbumin (α-LA), the role of its cofactor Ca2+ is predominant for a better control of final quality of protein fractions since it guarantees structure, bioactivity and stability of the protein. In this work, we have evidenced for the first time that protein conformation impacts UF performances of a metalloprotein: a higher transmission of apo-α-LA (calcium-depleted form) compared to holo-α-LA (native form) was observed during UF with pure solutions using a 30 kg mol−1 polyethersulfone membrane. Such a phenomenon was not controlled by electrostatic repulsions but was supposed to be linked to a distinct flexibility and/or hydrophobicity of the two conformations. Besides, we have shown that the behavior of the mixture of both conformations of α-LA did not reflect the behavior of individual conformations. During UF of the mixture, the higher transmission of the apo-α-LA was responsible for a displacement of the equilibrium between Ca2+ and the protein resulting in preferential permeation of free calcium. Then the Ca2+/α-LA molar ratio increased in permeate and reached values close to the molar ratio of the holo-α-LA form. This phenomenon was favored for membranes with a low cut-off compared to the molecular weight of α-LA and was confirmed by structural analyses of proteins before and after permeation.
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