Hydrophobic and electrostatic forces control the retention of membrane peptides and proteins with an immobilised phosphatidic acid column.
The retention behaviour of four membrane-associated peptides and proteins with an immobilized phosphatidic acid (PA) stationary phase was evaluated. The solutes included the cytolytic peptides gramicidin A and melittin, the integral membrane protein bacteriorhodpsin and cytochrome c, a peripheral membrane protein. Gramicidin has no nett charge and exhibited normal reversed phase-like behaviour which was largely independent of mobile phase pH. In contrast, melittin, which has a positively charged C-terminal tail, exhibited reversed phase like retention at pH 5.4 and 7.4, and was not retained at pH 3 reflecting the influence of electrostatic interactions with the negatively charged phosphatidic acid ligand. Bacteriorhodpsin was eluted at high acetonitrile concentrations at pH 3 and 5.4 and cytochrome c was only eluted at pH 3. Moreover, cytochrome c eluted in the breakthrough peak between 0 and 100% acetonitrile, demonstrating the role of electrostatic interactions with the PA surface. Overall, the results demonstrate that pH can be used to optimize the fractionation and separation of membrane proteins with immobilized lipid stationary phases.
Davies-Tuck
Miranda
M
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Vic 3800, Australia.
Lee
Tzong-Hsien
TH
Apffel
Alex
A
Aguilar
Marie-Isabel
MI
D001436:Bacteriorhodopsins / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006096:Gramicidin / Q000302:isolation & purification; D057927:Hydrophobic and Hydrophilic Interactions; D008051:Lipid Bilayers; D008555:Melitten / Q000302:isolation & purification; D008565:Membrane Proteins / Q000302:isolation & purification*; D010455:Peptides / Q000302:isolation & purification*; D010712:Phosphatidic Acids / Q000737:chemistry*; D055672:Static Electricity
D008051:Lipid Bilayers; D008565:Membrane Proteins; D010455:Peptides; D010712:Phosphatidic Acids; D006096:Gramicidin; D008555:Melitten; D001436:Bacteriorhodopsins