Protein unfolding during reversed‐phase chromatography: I. Effect of surface properties and duration of adsorption

McNay, Jennifer L. M.; Fernandez, Erik J.

doi:10.1002/bit.10015

Cited by 56 publications

(35 citation statements)

References 32 publications

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“…A branched C4-alkyl modified silica RPC surface with 300 Å pores was selected as representative of RPC surfaces commonly used in protein separations. The interaction of BPTI with this surface in the absence of salt was previously documented (McNay and Fernandez, 2001), so the effect of salt could be clearly identified. Furthermore, it was shown that BPTIstructure was not influenced by pore size effects on these C4 surfaces (McNay and Fernandez, 2001).…”

Section: Introductionmentioning

confidence: 92%

“…The flow rate for all surface-labeling and control experiments was 2.0 mL/min. After loading, the protein was held on the surface for either 5 min or 2 h. Subsequent aspects of the protocol are as previously described (McNay and Fernandez, 2001) except that the indicated salt concentration was maintained in addition to the buffer system used to control pH in all RPC mobile phases (i.e., label buffer, quench buffer, and elution mixture). Any added salt and elution solvents were removed in the size-exclusion step via buffer exchange into 10 mM sodium acetate (NaAc), pH* 2.8.…”

Section: Hydrogen Exchange Labeled Samplesmentioning

confidence: 99%

“…BPTI was selected as a model protein since the two-dimensional NMR peak assignments are available (Wagner and Wüthrich, 1982a) and we have successfully used this protein in previous surface-adsorption experiments with isotope-labeling techniques (McNay and Fernandez, 2001). A branched C4-alkyl modified silica RPC surface with 300 Å pores was selected as representative of RPC surfaces commonly used in protein separations.…”

Section: Introductionmentioning

confidence: 99%

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Protein unfolding during reversed‐phase chromatography: II. Role of salt type and ionic strength

McNay

O’Connell

Fernandez

2001

Biotech & Bioengineering

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While reversed-phase chromatography (RPC) may be a powerful method for purification of proteins at the analytical scale, both preparative and analytical applications have been hindered by the complex chromatographic behavior of proteins compared to small molecules. Further, preparative applications have been limited because of poor yields caused by the denaturing conditions involved. One means for modulating both the stability and chromatographic behavior of proteins is through the use of added salt. In this investigation, we show how salt type and ionic strength affect protein conformation on RPC surfaces. Exposure of amide groups of adsorbed BPTI was monitored using nuclear magnetic resonance (NMR) spectroscopy and hydrogen-deuterium isotope exchange. Sodium chloride, sodium acetate, and ammonium sulfate were studied at ionic strengths up to I = 0.375, with adsorption hold times being 5 min and 2 h. We found that increasing ionic strength decreased exposure of the exchange reporter groups in essentially all cases. However, even at the same ionic strength the level and distribution of residue protection varied with salt type and hold time. NaCl does not protect certain reporter groups at all, while those that it does protect to some degree at short hold times can exchange slightly more at longer times. The pattern and level of protection for NaAc at short times is similar to that for NaCl, but at longer times more uniform protection is seen as the reporter groups completely exposed at short times become more protected. For (NH(4))(2)SO(4) the pattern of protection at short hold time is similar to those of the other salts, although it protects all groups much more. This would be expected from the Hofmeister series. However, at longer times the level of protection with (NH(4))(2)SO(4) decreases below that of the other salts, while it uniquely protects all groups to nearly the same level. Such subtle variations in the protein structure would not have been detected without the measurements and analysis used here. Chromatographic retention times and peak shapes were obtained for the above systems. Variations of behavior were seen that could not be correlated with any of the above protection patterns and levels or even with heuristics such as the Hofmeister series. This suggests further conformational changes upon elution may be critical to the retention process. However, an excellent correlation was found between peak width at half-height and the average degree of unfolding, as indicated by the average level of isotopic exchange. Thus, while further studies are needed to definitively determine the connection between protein unfolding and retention, use of this correlation may improve designing and screening for chromatographic conditions that minimize protein unfolding.

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Section: Introductionmentioning

confidence: 92%

Section: Hydrogen Exchange Labeled Samplesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Protein unfolding during reversed‐phase chromatography: II. Role of salt type and ionic strength

McNay

O’Connell

Fernandez

2001

Biotech & Bioengineering

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“…Such unfolding has resulted in an increased apparent footprint of the protein (Wertz and Santore, 1999), increased solvent accessibility (McNay and Fernandez, 2001), and decreased density of adsorbed protein molecules (Vörös, 2004).…”

Section: Effect Of Protein Loading Adsorption Time and Mobile Phasementioning

confidence: 99%

QCM‐D sensitivity to protein adsorption reversibility

Jordan

Fernandez

2008

Biotech & Bioengineering

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Using a quartz crystal microbalance with dissipative monitoring (QCM-D) we have determined the adsorption reversibility and viscoelastic properties of ribonuclease A adsorbed to hydrophobic self-assembled monolayers. Consistent with previous work with proteins unfolding on hydrophobic surfaces, high protein solution concentrations, reduced adsorption times, and low ammonium sulfate concentrations lead to increased adsorption reversibility. Measured rigidity of the protein layers normalized for adsorbed protein amounts, a quantity we term specific dissipation, correlated with adsorption reversibility of ribonuclease A. These results suggest that specific dissipation may be correlated with changes in structure of adsorbed proteins.

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“…There are also examples in which protein adsorption is a desired feature, as long as this can be done in a controlled manner, so that the structural and functional integrity of the protein is maintained. Examples of such areas are the production of combined, adsorbed vaccines (3) and the development of chromatography material (4,5).…”

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confidence: 99%

Reduction of Irreversible Protein Adsorption on Solid Surfaces by ProteinEngineering for IncreasedStability

Karlsson

Ekeroth

Elwing

et al. 2005

Journal of Biological Chemistry

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The influence of protein stability on the adsorption and desorption behavior to surfaces with fundamentally different properties (negatively charged, positively charged, hydrophilic, and hydrophobic) was examined by surface plasmon resonance measurements. Three engineered variants of human carbonic anhydrase II were used that have unchanged surface properties but large differences in stability. The orientation and conformational state of the adsorbed protein could be elucidated by taking all of the following properties of the protein variants into account: stability, unfolding, adsorption, and desorption behavior. Regardless of the nature of the surface, there were correlation between (i) the protein stability and kinetics of adsorption, with an increased amplitude of the first kinetic phase of adsorption with increasing stability; (ii) the protein stability and the extent of maximally adsorbed protein to the actual surface, with an increased amount of adsorbed protein with increasing stability; (iii) the protein stability and the amount of protein desorbed upon washing with buffer, with an increased elutability of the adsorbed protein with increased stability. All of the above correlations could be explained by the rate of denaturation and the conformational state of the adsorbed protein. In conclusion, protein engineering for increased stability can be used as a strategy to decrease irreversible adsorption on surfaces at a liquid-solid interface.

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Protein unfolding during reversed‐phase chromatography: I. Effect of surface properties and duration of adsorption

Cited by 56 publications

References 32 publications

Protein unfolding during reversed‐phase chromatography: II. Role of salt type and ionic strength

Protein unfolding during reversed‐phase chromatography: II. Role of salt type and ionic strength

QCM‐D sensitivity to protein adsorption reversibility

Reduction of Irreversible Protein Adsorption on Solid Surfaces by ProteinEngineering for IncreasedStability

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