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

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.
(Copyright 2001 John Wiley & Sons, Inc.)