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

Abstract

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.

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

“…Nevertheless, the most likely explanation for hydrogen exchange protection in our study would seem to be preservation of native structure, since even small fluctuations from native conformations increase hydrogen exchange rates dramatically. The similarity between many of the protection patterns observed here and in our related studies (McNay and Fernandez, 2001) suggest that residual native structure is an important factor in hydrogen exchange. This is further supported by the similarity between the protected regions in HEWL adsorbed on RPC surfaces (McNay and Fernandez, 1999) and in other denaturing environments (Buck et al, 1993;Chang and Fernandez, 1998).…”

Combined effects of potassium chloride and ethanol as mobile phase modulators on hydrophobic interaction and reversed-phase chromatography of three insulin variants

“…Nonetheless, the hydrophobic interactions between solute and ligand in both HIC and RPC can be manipulated by salts as well as organic solvents. However, few studies have been carried out on the effect of alcohols on HIC [10][11][12][13], and of inorganic salts on RPC [14,15], and those that have been performed were either focused on a certain separation problem [10,11,15], or on phenomena other than retention, such as protein unfolding during chromatography [14], or peak shape and carryover [12].…”

The effects of ligand chain length, salt concentration and temperature on the adsorption of bovine serum albumin onto polypropyleneglycol–Sepharose

“…It has been argued that hydrophobic interactions can be strongly influenced by conformational changes in both the protein (Oscarsson, 1995;McNay et al, 2001) and ligand (Yarovsky et al, 1995). As reported earlier (Dias-Cabral et al, 2003), experimental data indicate that changes in protein conformation influence the process.…”