Physico-chemical properties of 2,6-TNS binding sites in squid giant axons: Involvement of water molecules in the excitation process

Abstract

Summary. The lifetime of the excited singlet state of 2,6-TNS (2-p-toluidinyl-6-naphthalene sulfonate) in the squid giant axon was measured by the use of the single photon counting technique. The accessibility of water molecules to the bound 2,6-TNS molecules by the use of heavy water indicated that these binding sites are only partially exposed to surrounding water molecules. Studies of the effect of temperature changes on the fluorescence of 2,6-TNS in axons suggested that relaxation of polar solvent molecules and/or neighboring polar side groups (during the lifetime of the excited dye molecule) plays a crucial role in the fluorescence emission from the bound 2,6-TNS. Time-resolved emission spectra suggested that the viscosity of the microenvironment of the axoplasmic bound dye molecules is high. The effect of replacing HzO with DzO in the medium on the fluorescence signals (intensity changes) from internally labeled squid giant axons during nerve excitation were investigated. The fluorescence signals (31) in the D20 were found to be smaller in size and longer in duration than those in HzO. The spectrum of A1 in DzO was similar to that in HzO; that is to say, blue-shifted and narrow as compared with the fluorescence spectrum from the axon at rest.The method of determining extrinsic fluorescence has been applied in the past as a means of studying structural changes of membrane macromolecules during the process of nerve excitation [17,19,20]. Among all the fluorescent probe-molecules so far employed, aminonaphthalene derivatives have yielded the most interesting results. Recently, particular attention has been given to the physico-chemical properties of 2-p-toluidinyl-6-naphthalene sulfonate (2,6-TNS) and emphasis placed on the information it can furnish about the macromolecular organization of the axonal membrane [ 15,16]. However, up