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

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 H2O with D2O in the medium on the fluorescence signals (intensity changes) from internally labeled squid giant axons during nerve excitation were investigated. The fluorescence signals (DeltaI) in the D2O were found to be smaller in size and longer in duration than those in H2O. The spectrum of DeltaI in D2O was similar to that in H2O; that is to say, blue-shifted and narrow as compared with the fluorescence spectrum from the axon at rest.