Determination of the sidedness of carbocyanine dye labeling of membranes

We have found that 2,4,6-trinitrobenzenesulfonate (TNBS) is an effective quencher of the fluorescence of the 1,1'-dialkyl-3,3,3',3'-tetramethylindocarbocyanines (CNdiI's). This quenching appears to occur by complex formation rather than a collisional mechanism. By use of this quenching, we have studied the transbilayer asymmetry of CNdiI labeling for large unilamellar membranes of egg phosphatidylcholine and the plasma membranes of human red blood cells and of ram epididymal spermatozoa. When CNdiI is added to membranes by ethanolic injection, only the outer leaflet labels. We have further shown that in large unilamellar vesicles of egg phosphatidylcholine, the CNdiI's do not appreciably "flip-flop" to the inner leaflet for at least 4 h at temperatures between 4 and 37 degrees C.