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Phenylboronate chromatography selectively separates glycoproteins through the manipulation of electrostatic, charge transfer, and cis ‐diol interactions
Author(s) -
Carvalho Rimenys J.,
Woo James,
AiresBarros M. Raquel,
Cramer Steven M.,
Azevedo Ana M.
Publication year - 2014
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201400170
Subject(s) - chemistry , adsorption , biomolecule , glycan , electrostatics , protein adsorption , glycoprotein , static electricity , surface charge , ligand (biochemistry) , chromatography , biochemistry , organic chemistry , receptor , electrical engineering , engineering
Phenylboronate chromatography (PBC) has been applied for several years, however details regarding the mechanisms of interactions between the ligand and biomolecules are still scarce. The goal of this work is to investigate the various chemical interactions between proteins and their ligands, using a protein library containing both glycosylated and nonglycosylated proteins. Differences in the adsorption of these proteins over a pH range from 4 to 9 were related to two main properties: charge and presence of glycans. Acidic or neutral proteins were strongly adsorbed below pH 8 although the uncharged trigonal form of phenylboronate (PB) is less susceptible to forming electrostatic and cis ‐diol interactions with proteins. The glycosylated proteins were only adsorbed above pH 8 when the electrostatic repulsion between the boronate anion and the protein surface was mitigated (at 200 mM NaCl). All basic proteins were highly adsorbed above pH 8 with PB also acting as a cation‐exchanger with binding occurring through electrostatic interactions. Batch adsorption performed at acidic conditions in the presence of Lewis base showed that charge‐transfer interactions are critical for protein retention. This study demonstrates the multimodal interaction of PBC, which can be a selective tool for separation of different classes of proteins.