Plasma membrane associated location of sulfonated meso-tetraphenylporphyrins of different hydrophilicity probed by total internal reflection fluorescence spectroscopy

Sulfonated meso-tetraphenylporphyrins of different hydrophilicity were micr ospectrofluorimetrically examined in endothelial cells using total infernal reflection (TIR) illumination or epi-illumination, Since the penetration d epth of the evanescent held during TIR illumination is limited to a few hun dred nanometers, photosensitizers were almost selectively examined in close vicinity to the plasma membrane. Pronounced fluorescence signals during TI R illumination were observed for the hydrophilic compounds meso-tetraphenyl porphyrin tetrasulfonate (TPPS4) and meso-tetraphenylporphyrin trisulfonate (TPPS3), whereas the more lipophilic compounds meso-tetraphenylporphyrin d isulfonate (TPPS2a) and meso-tetraphenylporphyrin monosulfonate (TPPS1) cou ld only be detected under epi-illumination. Irradiation of TPPS1 and TPPS2a in the Soret band led to an increase in fluorescence intensity and formati on of a photoproduct with an emission maximum around 610 mn, which was limi ted to intracellular compartments, In contrast, fluorescence spectra of TPP S3 and TPPS4 obtained by TIR and epi-illumination remained almost unchanged after irradiation in the Soret band. Extralysosomal location of TPPS3 and TPPS4 in close proximity to the plasma membrane was deduced from experiment s with the lysosomal markers acridine orange (AO) or lysotracker yellow (LY ), which were not detectable under TIR illumination. In conclusion, these r esults provide for the first time direct evidence for a plasma membrane-ass ociated fraction of the hydrophilic compounds TPPS3 and TPPS4 in living cel ls.