Spectrofluorimetric study of porphyrin incorporation into membrane models--evidence for pH effects

The effects of hydrophobicity and charges of dicarboxylic porphyrins upon their interactions with membrane model systems are investigated. Four protonation steps are evidenced from fluorescence emission studies of hematoporphyrin IX and its more hydrophobic parent compound lacking of alcoholic chain, deuteroporphyrin IX. They are attributed to the successive protonations of the inner nitrogens of the porphyrin cycle (pK = 4.7 and 2.9 for hematoporphyrin and 4.4 and 2.7 for deuteroporphyrin) and successive deprotonations of propionic groups (pK approximately equal to 5.0 and 5.5 for hematoporphyrin and 5.4 and 6.0 for deuteroporphyrin). These porphyrins, as well as their dimethyl ester forms, are shown to incorporate as monomers into the hydrophobic bilayer of egg phosphatidylcholine small unilamellar vesicles, although the esterified forms are highly aggregated in aqueous solutions. In the case of the non-esterified forms, the incorporation of the porphyrins into the lipidic bilayer is reversible and strongly pH-dependent. A theoretical model is presented which takes into account the various protonation steps and the partition equilibria of the porphyrin between the vesicle lipidic phase and the water medium. The neutral form of the porphyrin (i.e., carboxylic groups protonated) presents the higher affinity, with constants of K approximately equal to 2 X 10(5) and K approximately equal to 6 X 10(6) M-1 (relative to lipid concentration) for hematoporphyrin and deuteroporphyrin, respectively. Protonation of one inner nitrogen leading to the monocationic form is sufficient to prevent incorporation into the hydrophobic bilayer. On the other hand, deprotonation of the peripheral propionic chains leading to anionic forms is less effective. These interactions between vesicles and porphyrins lead to shifts of the apparent pK of nitrogens and carboxylic groups, the latter one being now in the range of physiological pH. These results are discussed with regards to the hypothesis of a possible role of pH in the preferential uptake of porphyrins by tumors.