Characterization of phospholipid-protein interactions by capillary isoelectric focusing with whole-column imaging detection.
The integration of functional proteins in the phospholipid bilayer is one of the most crucial features of biological membrane architecture. Phospholipid-protein interactions play an important role in the functions of bounded proteins in the phospholipid membrane. When the phospholipid-protein interactions occur, the protein structure tends to alter, which can result in a change in the isoelectric points (pI) of protein. Capillary isoelectric focusing (cIEF) with whole-column imaging detection (WCID) is an attractive technique that has the features of simple operation, high resolution, and fast separation without focused band mobility for detection of amphoteric biomolecules. In this study, a cIEF-WCID method was developed to characterize the phospholipids-protein interactions by monitoring the protein cIEF profiles. Seven proteins with different pI and molecular mass , and a zwitterionic phosphatidylcholine (PC) with zwitterionic properties, were used to evaluate the feasibility of the cIEF-WCID approach in the study of phospholipid-protein interactions. The cIEF profiles changed in response to the changes in protein conformation, clearly exhibiting interactions between the PC vesicles and the targeted proteins. The formation of PC-protein complex was observed in the cIEF electropherograms. It was demonstrated that seven proteins displayed distinct interactions with the PC vesicles due to their different chemical and physical properties. The influences of the PC concentration, incubation time, and incubation temperature on the phospholipids-protein interactions were investigated. This study validated a novel analytical approach for the characterization of phospholipid-protein interactions.
Bo
Tao
T
Department of Chemistry, University of Waterloo, Ont., Canada.
Pawliszyn
Janusz
J
D000818:Animals; D002021:Buffers; D004285:Dogs; D019075:Electrophoresis, Capillary / Q000379:methods*; D006454:Hemoglobins / Q000737:chemistry; D007525:Isoelectric Focusing / Q000379:methods*; D007526:Isoelectric Point; D007782:Lactoglobulins / Q000737:chemistry; D008566:Membranes / Q000737:chemistry*; D009211:Myoglobin / Q000737:chemistry; D010713:Phosphatidylcholines / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry*; D010763:Phosphorylase b / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000737:chemistry; D014361:Trypsin Inhibitors / Q000737:chemistry; D014362:Trypsinogen / Q000737:chemistry
D002021:Buffers; D006454:Hemoglobins; D007782:Lactoglobulins; D009211:Myoglobin; D010713:Phosphatidylcholines; D010743:Phospholipids; D011506:Proteins; D014361:Trypsin Inhibitors; D012710:Serum Albumin, Bovine; D014362:Trypsinogen; D010763:Phosphorylase b