Effects of ionic strength and mobile phase pH on the binding orientation of lysozyme on different ion-exchange adsorbents

Dismer, Florian; Petzold, Martin; Hubbuch, Jürgen

doi:10.1016/j.chroma.2007.12.085

Cited by 110 publications

(71 citation statements)

References 33 publications

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“…This set is consistent with results obtained by Aizawa et al who propose that the potential adsorption site is composed from Lys1, Lys13 and Arg14 [18]. The composition of the main adsorption site found by Dismer et al [19] also agrees with our selection of residues. Therefore, while the 20 ns MD trajectories are too short to test for protein unfolding at the Table 1.…”

Section: Molecular Dynamics Simulationssupporting

confidence: 82%

Protein adsorption mechanisms on solid surfaces: lysozyme-on-mica

Mulheran

Kubiak

2009

Molecular Simulation

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A methodology for discovering the mechanisms and dynamics of protein clustering on solid surfaces is reviewed and complemented by atomistic molecular dynamics (MD) simulations. In situ atomic force microscopy images of the early stages of protein film formation are quantitatively compared with Monte Carlo simulations, using cluster statistics to differentiate various growth models. We have studied lysozyme adsorption on mica as a model system, finding that all surface-supported clusters are mobile with diffusion constant inversely related to cluster size. Furthermore, our results suggest that protein monomers diffusing to the surface from solution only adhere to the bare surface with a finite probability. Fully atomistic MD simulations reveal that the lysozyme does indeed have a preferred orientation for binding to the surface, so that proteins with incorrect orientations move away from the surface rather than towards it. Agreement with experimental studies in the literature for the residues involved in the surface adsorption is found.

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Section: Molecular Dynamics Simulationssupporting

confidence: 82%

Protein adsorption mechanisms on solid surfaces: lysozyme-on-mica

Mulheran

Kubiak

2009

Molecular Simulation

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“…Equivalent conclusions resulted from an experimental search for favourable binding orientations of lysozyme on negatively charged surfaces, which also indicated that pH may have a pronounced inuence. 51 Obviously, from the present results we cannot decide which of the basic amino acids forms the binding site on the lysozyme molecule.…”

Section: Bridging Aggregationmentioning

confidence: 61%

Bridging interactions of proteins with silica nanoparticles: The influence of pH, ionic strength and protein concentration

et al. 2014

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Charge-driven bridging of nanoparticles by macromolecules represents a promising route for engineering functional structures, but the strong electrostatic interactions involved when using conventional polyelectrolytes impart irreversible complexation and ill-defined structures. Recently it was found that the electrostatic interaction of silica nanoparticles with small globular proteins leads to aggregate structures that can be controlled by pH. Here we study the combined influence of pH and electrolyte concentration on the bridging aggregation of silica nanoparticles with lysozyme in dilute aqueous dispersions. We find that protein binding to the silica particles is determined by pH irrespective of the ionic strength. The hetero-aggregate structures formed by the silica particles with the protein were studied by small-angle X-ray scattering (SAXS) and the structure factor data were analyzed on the basis of a short-range square-well attractive pair potential (close to the sticky-hard-sphere limit). It is found that the electrolyte concentration has a strong influence on the stickiness near pH 5, where the weakly charged silica particles are bridged by the strongly charged protein. An even stronger influence of the electrolyte is found in the vicinity of the isoelectric point of the protein (pI ¼ 10.7) and is attributed to shielding of the repulsion between the highly charged silica particles and hydrophobic interactions between the bridging protein molecules.

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“…Nodake et al [11] reported the same conclusion, although in their study PEGylated lysozyme eluted as a single broad peak, which might have contained PEGylated lysozyme isomers. The research group of Hubbuch investigated the binding mechanism of lysozyme on cation exchange chromatography media using florescent dyes, and found that the dye was easily attached to Lys33 and Lys96 [25][26][27]. It is, therefore, quite likely that peak 1 and 2 in Fig.…”

Section: Number Of Binding Sites (B)mentioning

confidence: 99%

Interaction mechanism of mono‐PEGylated proteins in electrostatic interaction chromatography

Abe

Akbarzaderaleh

Hamachi

et al. 2010

Biotechnology Journal

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The retention and binding mechanisms in electrostatic interaction-based chromatography (ion-exchange chromatography) of PEGylated proteins (covalent attachment of polyethylene glycol chains to protein) were investigated using our previously developed model. Lysozyme and bovine serum albumin were chosen as model proteins. The retention volume of PEGylated proteins shifted to lower elution volumes with increasing PEG molecular weight compared with the non-modified (native) protein retention volume. However, PEGylation did not affect the number of binding sites appreciably. The enzyme activity of PEGylated lysozyme measured with a standard insoluble substrate in suspension decreased considerably, whereas the activity with a soluble small-molecule substrate did not drop significantly. These findings indicate that when a protein is mono-PEGylated, the binding site is not affected and the elution volume reduces due to the steric hindrance between PEGylated protein and ion-exchange ligand.

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Effects of ionic strength and mobile phase pH on the binding orientation of lysozyme on different ion-exchange adsorbents

Cited by 110 publications

References 33 publications

Protein adsorption mechanisms on solid surfaces: lysozyme-on-mica

Protein adsorption mechanisms on solid surfaces: lysozyme-on-mica

Bridging interactions of proteins with silica nanoparticles: The influence of pH, ionic strength and protein concentration

Interaction mechanism of mono‐PEGylated proteins in electrostatic interaction chromatography

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