Reactions of fluorescent probes with normal and chemically modified myelin

The fluorescent probes 8-anilino-1-naphthalenesulfonate (ANS) and 2-p-toluidinylnaphthalene-6-sulfonate (TNS) bind to highly purified myelin membranes obtained from bovine brain white matter. Binding of the dyes was markedly increased by environmental conditions which reduce the negative surface potential of the membrane, i.e., cations (La-3+ is greater than Ca-2+ is greater than Na-+,K-+), H-+, local anesthetics, and the antibiotic polymyxin B. Chemical alteration of accessible membrane charged groups affected dye binding in a manner consistent with the hypothesis that such binding is primarily dependent upon the membrane surface potential. Thus, binding was increased by blocking of carboxyl groups via carbodiimide activation and subsequent coupling with neutral amino acid esters, and even more so with a basic amino acid ester (e.g., arginine methyl ester). Dye binding was reduced by succinylation of amino groups, and by hydrolysis of choline and ethanolamine head groups of phospho- and sphingolipids by phospholipase C. Phospholipase C treatment of myelin, or sphingomyelin vesicles, reduced or abolished the augmentation of ANS and TNS binding due to cations, local anesthetics, or polymyxin B. Energy transfer from myelin tryptophan residues to bound ANS occurs, but with low efficiency. Oxidation of membrane tryptophan residues with N-bromosuccinimide, or alkylation with 2-hydroxy (or methoxy)-5-nitrobenzyl bromide, markedly reduced intrinsic membrane fluorescence and energy transfer to bound ANS, but did not significantly affect dye binding or the quantum yield of ANS fluorescence when excitation was at 380nm. Proteolytic digestion removed 6-30% of myelin protein, depending upon the enzyme used, but had no effect on fluorescent dye binding. It is concluded that the binding of the anionic fluorescent probes ANS and TNS to myelin is primarily a function of the membrane surface charge density and net surface potential, as is the case with other biological membranes. Conclusions about the degree of dye binding to membrane lipids or membrane proteins cannot be drawn unless additional studies are carried out on isolated water soluble membrane proteins.