PROTONATION EQUILIBRIA OF HEMATOPORPHYRIN AS STUDIED BY FLUORESCENCE LIFETIME ANALYSIS

Fluorescence decay of haematoporphyrin in aqueous solutions has been examined at pH values ranging from 0.8 to 10 and at concentrations between 5 x 10(-7) and 2 x 10(-5) mol dm-3. Above pH 6.5, the fluorescence decay was mainly monoexponential with a lifetime of 15.1 +/- 0.2 ns, characteristic of the neutral form of the porphyrin. Between pH 6.5 and 3.5, fluorescence decays are mainly biexponential. The relative weight of the 15.1 ns component decayed for the benefit of a well defined, short-lived component (7.6 +/- 0.4 ns), which was assigned to the porphyrin cation protonated at one nitrogen. Theoretical analysis of the data makes it possible to assign a pK of 5.00 +/- 0.04 to the porphyrin in the ground state. Between pH 3 and 0.8, decays mainly consist of the short-lived component, the lifetime of which slightly decreases to 6 ns at acidic pH. A small contribution from a 0.7 ns component was also observed. These changes are tentatively attributed to protonation equilibria at the excited state. Changing the porphyrin concentration has little effect on the general features described above, reflecting the major contribution of monomeric species to the fluorescence observed.