MICROSPECTROFLUOROMETRY OF THE PROTONATION STATE OF ELLIPTICINE, AN ANTITUMOR ALKALOID, IN SINGLE CELLS

The protonation state and intracellular distribution of ellipticine we re investigated in single human mammary T47D cells by confocal laser m icrospectrofluorimetry. In the cell nucleus, only the protonated form of ellipticine was detected as a direct consequence of its apparent pK increase upon DNA binding. Both protonated and neutral forms were pre sent in the aqueous cytoplasm, where the pH is close to the drug pK. W hen cells were incubated in high concentrations of K+, a condition tha t depolarizes the plasma membrane potential, ellipticine cellular accu mulation was reduced. In the cytoplasm, ellipticine was mainly bound t o mitochondria, and its protonation equilibrium was shifted toward the neutral form. The fluorescence spectrum of ellipticine bound to mitoc hondria was insensitive to valinomycin, whereas it was markedly shifte d toward the protonated form after carbonyl cyanide p-trifluoromethoxy -phenylhydrazone or nigericin addition. Similar studies with elliptici ne bound to isolated mitochondria suggest that it behaves as a fluores cent probe of mitochondrial pH in both isolated mitochondria and singl e living cells.