Cytochrome c-dimyristoylphosphatidylglycerol interactions studied by asymmetrical flow field-flow fractionation.
Lipid membranes are well recognized ligands that bind peripheral and integral proteins in a specific manner and regulate their function. Cytochrome c (cyt c) is one of the partner peripheral protein that binds to the lipid membranes via electrostatic and hydrophobic interactions. In this study, asymmetrical flow field-flow fractionation (AsFlFFF) was used to compare the interactions of cyt c with the acidic phospholipid 1,2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol (DMPG), oleic acid (OA), and sodium dodecyl sulfate (SDS). The influence of pH and the cyt c-lipid molar mass ratios were evaluated by monitoring the diffusion coefficients and particle diameter distributions obtained for the free and lipid-bound protein. The hydrodynamic particle diameter of cyt c (pI 10) was 4.1 nm at pH 11.4 and around 4.2 nm at pH 7.0 and 8.0. Standard molar mass marker proteins were used for calibration to obtain the molar masses of free cyt c and its complexes with lipids. AsFlFFF revealed the binding of cyt c to DMPG and to OA to be mainly electrostatic. In the absence of electrostatic interactions, minor complex formation occurred, possibly due to the extended lipid anchorage involving the hydrophobic cavity of cyt c and the hydrocarbon chains of DMPG or SDS. The possibility of the formation of the molten globule state of cyt c, induced by the interaction between cyt c and lipids, is discussed.
Yohannes
Gebrenegus
G
Laboratory of Analytical Chemistry, Department of Chemistry, University of Helsinki, P.O. Box 55, 00014 Helsinki, Finland.
Wiedmer
Susanne K
SK
Tuominen
Esa K J
EK
Kinnunen
Paavo K J
PK
Riekkola
Marja-Liisa
ML
D045304:Cytochromes c / Q000032:analysis / Q000737:chemistry*; D031521:Fractionation, Field Flow / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D008055:Lipids / Q000737:chemistry; D019301:Oleic Acid / Q000032:analysis / Q000737:chemistry; D010316:Particle Size; D010715:Phosphatidylglycerols / Q000032:analysis / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry; D012967:Sodium Dodecyl Sulfate / Q000032:analysis / Q000737:chemistry; D055672:Static Electricity
D008055:Lipids; D010715:Phosphatidylglycerols; D011506:Proteins; D019301:Oleic Acid; D012967:Sodium Dodecyl Sulfate; D045304:Cytochromes c; C002773:dimyristoylphosphatidylglycerol