Lipid Binding to the Amphipathic Membrane Protein Cytochrome b5

The lipid binding properties of the membrane protein cytochrome b₅ (detergent-extracted from calf liver microsomal preparations) were characterized by studying the interaction of spin-labeled lipids (5-, 12-, and 16-doxylstearic acid and 5- and 16-doxylphosphatidyl-choline, where doxyl refers to the nitroxide moiety) with cytochrome b₅, using electron spin resonance spectroscopy. The intact cytochrome b₅ molecule immobilizes all of the lipid spin labels, while the segment of cytochrome b₅ released by trypsin does not affect lipid mobility. The immobilization of lipid spin labels on the hydrophobic surface of intact cytochrome b₅ is not appreciably altered by associating the protein with liposomes. Differences in polarity of the lipid binding sites between cytochrome b₅ and phospholipid vesicles were also observed. The lipid binding sites on cytochrome b₅ are hydrophobic by conventional criteria, but are more polar than the interior of fluid phospholipid bilayers.