Influence of acid-induced conformational variability on protein separation in reversed phase high performance liquid chromatography

• Unusual, ion-pairing related RP-chromatographic behavior of proteins was observed.
• Ion-pairing induced conformation change influences protein elution.
• This finding could be utilized in separation of proteins.

Influence of acid concentration in the mobile phase on protein separation was studied in a wide concentration range using trifluoroacetic acid (TFA) and formic acid (FA). At low, 0.001–0.01 (v/v%) TFA concentration and appropriate solvent strength proteins elute before the column's dead time. This is explained by the proteins having a structured, but relatively extended conformation in the eluent; and are excluded from the pores of the stationary phase. Above ca. 0.01–0.05 (v/v%) TFA concentration proteins undergo further conformational change, leading to a compact, molten globule-like structure, likely stabilized by ion pairing. Proteins in this conformation enter the pores and are retained on the column. The results suggest a pore exclusion induced separation related to protein conformation. This effect is influenced by the pH and type of acid used, and is likely to involve ion-pair formation. The TFA concentration needed to result in protein folding (and therefore to observe retention on the column) depends on the protein; and therefore can be utilized to improve chromatographic performance. Conformation change was monitored by circular dichroism spectroscopy and mass spectrometry; and it was shown that not only TFA but FA can also induce molten globule formation.