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Protein thermal stability and phospholipid–protein interaction investigated by capillary isoelectric focusing with whole column imaging detection
Author(s) -
Bo Tao,
Pawliszyn Janusz
Publication year - 2006
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.200500456
Subject(s) - chemistry , phospholipid , chromatography , isoelectric focusing , phosphatidylcholine , denaturation (fissile materials) , vesicle , isoelectric point , thermal stability , protein–protein interaction , biophysics , biochemistry , organic chemistry , membrane , biology , nuclear chemistry , enzyme
CIEF with whole column imaging detection (WCID) is an attractive technique for studying protein reaction and protein–ligand interaction due to its fast separation, simple operation, and high efficiency. In this study, two interesting applications by the CIEF‐WCID were developed, involving the study of protein thermal stability and phospholipid–protein interaction. Four proteins (β‐lactoglobulin B, trypsin inhibitor, phosphorylase b, and trypsinogen) with different p I , and two types of phospholipids, including phosphatidylcholine (PC) and phosphatidylserine (PS), were used for this purpose. First, the altered CIEF profiles of four proteins were exhibited due to conformational changes resulting from protein denaturation induced by a high incubation temperature at 60°C. It was demonstrated that the addition of a zwitterionic phospholipid (PC) played a crucial role in the thermal stability of targeted proteins, especially for trypsin inhibitor whose thermal stability was promoted with the addition of the PC vesicles at 60°C. Second, the zwitterionic (PC) and acidic (PS) phospholipids displayed completely different interactions with the proteins. The addition of PS vesicles modified the zwitterionic phospholipids to carry negative charges, which correspondingly changed the interaction between the phospholipid and the protein. Our study demonstrates that the CIEF‐WCID is a powerful approach to study protein reaction and protein–ligand interaction with high efficiency, high selectivity, and fast separation.