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The second virial coefficient as a predictor of protein aggregation propensity: A self-interaction chromatography study.

Quigley A, Williams DR - Eur J Pharm Biopharm (2015)

Bottom Line: The second osmotic virial coefficients (b2) of four proteins - lysozyme, recombinant human lactoferrin, concanavalin A and catalase were measured by self-interaction chromatography (SIC) in solutions of varying salt type, concentration and pH.Aggregate sizes of <∼10nm, indicative of non-aggregated protein systems, were consistently observed to have b2 values >0.It is concluded that the quantification of protein-protein interactions using SIC based b2 data is a potentially valuable screening tool for predicting protein aggregation propensity.

View Article: PubMed Central - PubMed

Affiliation: Surfaces and Particle Engineering Laboratory, Department of Chemical Engineering, Imperial College London, London SW7 2BY, UK.

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The reduced osmotic virial coefficient (b2) for catalase (A), lactoferrin (B), lysozyme (C) and con A (D) (measured by SIC) as a function of increasing ionic strength and type (20 mM sodium phosphate buffer pH 7.0 for rhLf, catalase and con A, and 50 mM sodium acetate buffer pH 4.5 for lysozyme, 0.5 mL/min, 10 μL injections).
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f0010: The reduced osmotic virial coefficient (b2) for catalase (A), lactoferrin (B), lysozyme (C) and con A (D) (measured by SIC) as a function of increasing ionic strength and type (20 mM sodium phosphate buffer pH 7.0 for rhLf, catalase and con A, and 50 mM sodium acetate buffer pH 4.5 for lysozyme, 0.5 mL/min, 10 μL injections).

Mentions: Fig. 2 illustrates that the effect of the ionic strength and salt type in a solution is very much protein dependent. For reasons of clarity, error bars are not shown for b2 values but are typically close in size to the data point symbols used. Lysozyme b2 values (Fig. 2C) decrease with increasing ionic strength, indicating that attractive interactions are becoming increasingly dominant. This trend has been widely reported and although b2 does not provide any information on the physical origins of the experimentally observed interaction patterns, this trend is commonly interpreted as resulting from increased screening of the protein’s surface charges with increasing ionic strength, a classic electrical double layer effect [44], [43]. It appears as though this trend is somewhat unique, with most other proteins exhibiting very different interaction behaviour, possibly reflecting more complex protein structures and chemistry for these other larger proteins [19]. Indeed, lysozyme has a history of displaying atypical protein interaction behaviour and was the first protein demonstrated to follow an inverse Hofmeister series of reactivity in salt solutions at a pH below the pI of the protein; a trend which can be seen in the measured b2 values in Fig. 2C [45], [65], [9]. This reversal has since been shown to apply not only to lysozyme but also to a range of other small proteins including α-crystallins, ATCase and BMV [20]. The SIC data for lactoferrin, catalase and con A indicate that the greatest changes in b2 values occurred with the addition of salts with more strongly hydrated anions (SO2− and Cl−) in the given range of concentrations examined. Indeed, the b2 trend for lactoferrin shows that this protein also follows an inverse Hofmeister series at this pH, which is not unexpected as lactoferrin is positively charged under these conditions. The SIC data for con A and catalase are more complex. Both proteins are negatively charged at pH 7 and the investigations demonstrated that below salt concentrations of around 0.5–0.8 M these proteins follow a direct Hofmeister series trend. At higher salt concentrations these proteins undergo a reversal and these proteins then follow the inverse Hofmeister series. The exact physical origin of the inverse and direct Hofmeister series still remains challenging area of study but the effect is thought to stem from an interplay of ionic sizes, hydration phenomena and dispersion forces as well as the specific chemical and physical properties of the peptides and proteins themselves [50], [9], [39].


The second virial coefficient as a predictor of protein aggregation propensity: A self-interaction chromatography study.

Quigley A, Williams DR - Eur J Pharm Biopharm (2015)

The reduced osmotic virial coefficient (b2) for catalase (A), lactoferrin (B), lysozyme (C) and con A (D) (measured by SIC) as a function of increasing ionic strength and type (20 mM sodium phosphate buffer pH 7.0 for rhLf, catalase and con A, and 50 mM sodium acetate buffer pH 4.5 for lysozyme, 0.5 mL/min, 10 μL injections).
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4644993&req=5

f0010: The reduced osmotic virial coefficient (b2) for catalase (A), lactoferrin (B), lysozyme (C) and con A (D) (measured by SIC) as a function of increasing ionic strength and type (20 mM sodium phosphate buffer pH 7.0 for rhLf, catalase and con A, and 50 mM sodium acetate buffer pH 4.5 for lysozyme, 0.5 mL/min, 10 μL injections).
Mentions: Fig. 2 illustrates that the effect of the ionic strength and salt type in a solution is very much protein dependent. For reasons of clarity, error bars are not shown for b2 values but are typically close in size to the data point symbols used. Lysozyme b2 values (Fig. 2C) decrease with increasing ionic strength, indicating that attractive interactions are becoming increasingly dominant. This trend has been widely reported and although b2 does not provide any information on the physical origins of the experimentally observed interaction patterns, this trend is commonly interpreted as resulting from increased screening of the protein’s surface charges with increasing ionic strength, a classic electrical double layer effect [44], [43]. It appears as though this trend is somewhat unique, with most other proteins exhibiting very different interaction behaviour, possibly reflecting more complex protein structures and chemistry for these other larger proteins [19]. Indeed, lysozyme has a history of displaying atypical protein interaction behaviour and was the first protein demonstrated to follow an inverse Hofmeister series of reactivity in salt solutions at a pH below the pI of the protein; a trend which can be seen in the measured b2 values in Fig. 2C [45], [65], [9]. This reversal has since been shown to apply not only to lysozyme but also to a range of other small proteins including α-crystallins, ATCase and BMV [20]. The SIC data for lactoferrin, catalase and con A indicate that the greatest changes in b2 values occurred with the addition of salts with more strongly hydrated anions (SO2− and Cl−) in the given range of concentrations examined. Indeed, the b2 trend for lactoferrin shows that this protein also follows an inverse Hofmeister series at this pH, which is not unexpected as lactoferrin is positively charged under these conditions. The SIC data for con A and catalase are more complex. Both proteins are negatively charged at pH 7 and the investigations demonstrated that below salt concentrations of around 0.5–0.8 M these proteins follow a direct Hofmeister series trend. At higher salt concentrations these proteins undergo a reversal and these proteins then follow the inverse Hofmeister series. The exact physical origin of the inverse and direct Hofmeister series still remains challenging area of study but the effect is thought to stem from an interplay of ionic sizes, hydration phenomena and dispersion forces as well as the specific chemical and physical properties of the peptides and proteins themselves [50], [9], [39].

Bottom Line: The second osmotic virial coefficients (b2) of four proteins - lysozyme, recombinant human lactoferrin, concanavalin A and catalase were measured by self-interaction chromatography (SIC) in solutions of varying salt type, concentration and pH.Aggregate sizes of <∼10nm, indicative of non-aggregated protein systems, were consistently observed to have b2 values >0.It is concluded that the quantification of protein-protein interactions using SIC based b2 data is a potentially valuable screening tool for predicting protein aggregation propensity.

View Article: PubMed Central - PubMed

Affiliation: Surfaces and Particle Engineering Laboratory, Department of Chemical Engineering, Imperial College London, London SW7 2BY, UK.

Show MeSH
Related in: MedlinePlus