Structural modifications of globular proteins in an ultrafiltration loop as evidenced by intrinsic fluorescence and reverse-phase liquid chromatography. - Université de Rennes Accéder directement au contenu
Article Dans Une Revue Separation and Purification Technology Année : 2012

Structural modifications of globular proteins in an ultrafiltration loop as evidenced by intrinsic fluorescence and reverse-phase liquid chromatography.

Résumé

Occurrence of denaturation of proteins during ultrafiltration (UF) was already discussed in literature but it must be underlined that no general trends were drawn explaining the versatile obsd. behaviors. This paper aims at proposing a complementary approach focused on slight denaturation of proteins due to mech. stress in a UF loop. This methodol. has required powerful anal. tools as it aims at revealing slight denaturations leading to proteins remaining in sol. states and not to pptn. that is quite easily shown. Intrinsic fluorescence and Reverse Phase HPLC (RP-HPLC) techniques were selected to highlight subtle changes in protein structure. Mainly, only highly enriched fractions of proteins were available (with a more than 95% purity), but it is a limitation to the use of intrinsic fluorescence that is very sensitive to occurrence of minor protein contaminants. Then RP-HPLC was used not only for quantification of remaining sol. proteins but also to show slight changes in the protein peak shape, only evidenced with elution gradients specially established for this study to reach the goal. The main novelties of this paper deal with: (i) the use of a wide variety of globular proteins. A set of five proteins with different structural characteristics (mol. wt., overall hydrophobicity, occurrence of quaternary structures or not) was used for the demonstration, and (ii) the selection of specific RP-HPLC elution conditions able to highlight protein structural evolution with respect to UF requirement, esp. concerning the use of low concns. (1 g L-1) to avoid denaturation due to highly conc. media. An in-depth discussion based on protein peak shape was proposed and (iii) the establishment of model expts. that voluntary led to denatured proteins only obtained by mech. stress at 45 °C, conceptually different than denaturation obtained by external physico-chem. variations such as addn. of chem. compds. modifying protein structure as commonly used to denature proteins for anal. purpose. These model expts. were based on establishment of various shear stresses obtained by more or less quick stirring from zero to several thousand rotations per min allowing establishing a ref. scale of protein denaturation states with a Reynolds no. varying in a wide range. Cross-flow UF of each single protein soln. was performed in batch mode during 5-6 h at 45 °C, retentates were analyzed and compared to the ref. scale of mech. denaturation obtained from the model expts. As a general conclusion, it seemed that proteins can be classified according to a "soft/hard" criterion depending on the protein own ability to be denatured by contact with a surface. The "soft" proteins were partly denatured by the combination of shear stress and temp. in model expts. and in UF, whereas the "hard" proteins of quite stable structure were not modified. The behavior during UF was sometimes much more complex, esp. when the protein were capable of crossing the membrane. [on SciFinder(R)]

Domaines

Autre

Dates et versions

hal-01070077 , version 1 (30-09-2014)

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Citer

Jihane Belmejdoub, Murielle Rabiller-Baudry, David Delaunay, Geneviève Gésan-Guiziou. Structural modifications of globular proteins in an ultrafiltration loop as evidenced by intrinsic fluorescence and reverse-phase liquid chromatography.. Separation and Purification Technology, 2012, 96, pp.274--288. ⟨10.1016/j.seppur.2012.06.010⟩. ⟨hal-01070077⟩
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