Degree of exposure of membrane proteins determined by fluorescence quenching.
Quaternary salts of 4-picoline are shown to act as efficient quenchers of tryptophan fluorescence in membrane proteins. Fluorescence quenching determinations of sarcoplasmic reticulum membranes from rabbit muscle and of human erythrocyte membranes of different cholesterol to phospholipid mole ratios (C/PL) were carried out with quaternary picolinium salts in phosphate-buffered saline (PBS) and in 2,2,2-trifluorethanol (TFE)-water 2:1 (v/v), where the membrane is presumably completely disintegrated. In both solvent systems, the tryptophan quenching characteristics were typical of heterogenous systems and were analyzed as such. The ratio of the fraction of fluorescence intensity available for quenching with N-methylpicolinium perchlorate in PBS and in 2:1 TFE-water, (formula: see text), was taken as an index for the bulk degree of exposure of the membrane proteins to the aqueous surrounding. This value was found to increase with C/PL which is in line with the notion that increase in lipid microviscosity results in increase of exposure of membrane proteins. Analogous experiments were performed with N-hexyl- and N-benzylpicolinium, which can quench tryptophyl residues in both the aqueous phase and the hydrocarbon-water interface, and with N-hexadecylpicolinium which is dissolved in the membrane lipid layer and acts mostly as a static quencher of tryptophan at the hydrocarbon-water interface. With these quenchers the complementary indices (formula: see text) and (formula: see text), which represent the fraction of the protein mass located in the hydrocarbon-water interface and in the hydrocarbon layer, respectively, could be semiquantitatively resolved.
D000818:Animals; D004910:Erythrocyte Membrane / Q000648:ultrastructure*; D004912:Erythrocytes / Q000648:ultrastructure*; D006801:Humans; D008433:Mathematics; D008565:Membrane Proteins / Q000097:blood; D009132:Muscles; D010848:Picolinic Acids; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000648:ultrastructure*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000032:analysis
D008565:Membrane Proteins; D010848:Picolinic Acids; D014364:Tryptophan