title: Influence of acid-induced conformational variability on protein separation in reversed phase high performance liquid chromatography.
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.
authors.affiliation: Research Centre for Natural Sciences, Hungarian Academy of Sciences, Pusztaszeri út 59-67, Budapest 1025, Hungary; Budapest University of Technology and Economics, Department of Inorganic and Analytical Chemistry, Szt. Gellért tér 4, Budapest 1111, Hungary.
authors.forename: Balázs
authors.initials: B
authors.lastname: Bobály
authors.affiliation: Research Centre for Natural Sciences, Hungarian Academy of Sciences, Pusztaszeri út 59-67, Budapest 1025, Hungary.
authors.forename: Eszter
authors.initials: E
authors.lastname: Tóth
authors.affiliation: Research Centre for Natural Sciences, Hungarian Academy of Sciences, Pusztaszeri út 59-67, Budapest 1025, Hungary.
authors.forename: László
authors.initials: L
authors.lastname: Drahos
authors.affiliation: Research Centre for Natural Sciences, Hungarian Academy of Sciences, Pusztaszeri út 59-67, Budapest 1025, Hungary.
authors.forename: Ferenc
authors.initials: F
authors.lastname: Zsila
authors.affiliation: Research Centre for Natural Sciences, Hungarian Academy of Sciences, Pusztaszeri út 59-67, Budapest 1025, Hungary.
authors.forename: Júlia
authors.initials: J
authors.lastname: Visy
authors.affiliation: Budapest University of Technology and Economics, Department of Inorganic and Analytical Chemistry, Szt. Gellért tér 4, Budapest 1111, Hungary.
authors.forename: Jenő
authors.initials: J
authors.lastname: Fekete
authors.affiliation: Research Centre for Natural Sciences, Hungarian Academy of Sciences, Pusztaszeri út 59-67, Budapest 1025, Hungary. Electronic address: vekey.karoly@ttk.mta.hu.
authors.forename: Károly
authors.initials: K
authors.lastname: Vékey
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D005561:Formates / Q000737:chemistry; D007477:Ions / Q000737:chemistry; D013058:Mass Spectrometry; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D012997:Solvents / Q000737:chemistry; D014269:Trifluoroacetic Acid / Q000737:chemistry
chemical_list: D005561:Formates; D007477:Ions; D011506:Proteins; D012997:Solvents; C030544:formic acid; D014269:Trifluoroacetic Acid
keywords: Protein conformation; Protein separation mechanism; RP-HPLC; TFA–protein adduct