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

TitleInfluence of acid-induced conformational variability on protein separation in reversed phase high performance liquid chromatography.
Publication TypeJournal Article
Year of Publication2014
AuthorsBobály, B, Tóth, E, Drahos, L, Zsila, F, Visy, J, Fekete, J, Vékey, K
JournalJ Chromatogr A
Volume1325
Pagination155-62
Date Published2014 Jan 17
ISSN1873-3778
KeywordsChromatography, High Pressure Liquid, Chromatography, Reverse-Phase, Formates, Ions, Mass Spectrometry, Protein Conformation, Proteins, Solvents, Trifluoroacetic Acid
Abstract

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.

DOI10.1016/j.chroma.2013.12.022
Alternate JournalJ Chromatogr A
PubMed ID24373532