In situ detection of ALA-stimulated porphyrin metabolic products in Escherichia coli B by fluorescence line narrowing spectroscopy

Abstract

In a recent work [Photochem. Photobiol. B: Biol. 50 (1999) 8] the successful photodynamic inactivation of Escherichia coli bacteria by visible light was reported based on δ-aminolevulinic acid (ALA)-induced endogenous porphyrin accumulation. In this work, the identification of these porphyrin derivatives in intact bacteria was performed by low-temperature conventional fluorescence and fluorescence line narrowing (FLN) techniques. Conventional fluorescence emission spectroscopy at cryogenic temperatures revealed the presence of the free-base porphyrins, identified earlier by high-performance liquid chromatography analysis of disintegrated bacterial cells after ALA induction; however, emission maxima characteristic for metal porphyrins were also observed. We demonstrated that the primary reason for this signal is that metal porphyrins are formed from free-base porphyrins by Mg2+ ions present in the culturing medium. Incorporation of Zn ions originating from the glassware could also be supposed. In the FLN experiment, the energy selection effect could be clearly demonstrated for (0,0) emissions of both the free-base and the metal porphyrins. The comparison of the conventional emission spectra and the bands revealed by the FLN experiment show that the dominant monomeric structural population is that of metal porphyrins. The intensity and the shape of the FLN lines indicate an aggregated population of the free-base porphyrins, beside a small monomeric population.