Preparation of a fluorescent derivative of cytochrome b5 and its interaction with phospholipids.
A fluorescence derivative of bovine cytochrome b5 was prepared by using 5-(dimethylamino)naphthalene-1-sulfonyl chloride (dansyl chloride) in deoxycholate. Reaction conditions were established to specifically label the hydrophobic membrane-binding domain of the protein at a ratio of 0.9 +/- 0.1 dansyl group per cytochrome b5. Fluorescence measurements on the dansyl-labeled protein reflected the state of aggregation of the protein and its binding to lipids. The cytochrome b5 derivative was a sensitive probe for the detection of phospholipid phase transitions in reconstituted phospholipid vesicles. The rotational relaxation time of the labeled protein was strongly influenced by the phospholipid composition and the cholesterol content of the lipid bilayer, but it was largely insensitive to the integrity of the hydrophilic domain of the protein. When the membrane-binding domain of cytochrome b5 was bound to phospholipid vesicles, a preferential association with either the gel or the liquid-crystalline phase was not observed. The results suggest that the two domains of cytochrome b5 undergo predominantly independent motion and that the motion of the dansyl-labeled membrane-binding domain directly reflects the properties of the bulk lipids in the bilayer.
D000818:Animals; D002417:Cattle; D003580:Cytochromes / Q000378:metabolism*; D015786:Cytochromes b5; D003619:Dansyl Compounds; D003840:Deoxycholic Acid; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D008081:Liposomes / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013696:Temperature
D003580:Cytochromes; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D008081:Liposomes; D010446:Peptide Fragments; D010743:Phospholipids; D003840:Deoxycholic Acid; D015786:Cytochromes b5; C029038:dansyl chloride