Nicola Angelini

SPECTROSCOPIC AND PHOTOACOUSTIC STUDIES OF HYPERICIN EMBEDDED IN LIPOSOMES AS A PHOTORECEPTOR MODEL

Abstract— In photoresponsive ciliates, like Blepharisma japonicum and Stentor coeruleus, the photoreceptor pigments responsible for photomotile reactions are hypericin‐type chromophores packed in highly osmiophilic subpellicular granules. Liposomes loaded with hypericin can constitute a simple model system, appropriate for understanding the primary light‐induced molecular events triggering the sensory chain in these microorganisms. Optical absorption, steady‐state and time‐resolved fluorescence and pulsed photoacoustic calorimetry have been used to measure spectral distributions, fluorescence lifetimes, radiative and radiationless transition quantum yields of hypericin when assembled into egg L‐a‐phosphatidylcholine liposomes. With respect to hypericin ethanol solutions, both absorption and fluorescence maxima are 5 nm red shifted when the pigment is inserted into the lipidic microenvironment, regardless of the hypericin local concentration. Increasing by 100 times the hypericin local concentration decreases the relative fluorescence quantum yield by a factor of around 150 and the fraction of thermally released energy, conversely, increases from 0.6 to 0.9. From the analysis of fluorescence lifetimes and their relative amplitudes it appears that a subnanosecond living component is predominant at the highest hypericin local concentrations.

Electron Transfer Fluorescence Quenching of Blepharisma japonicum Photoreceptor Pigments

Abstract— The hypericin analogs blepharismin (BP), oxyblepharismin (OxyBP) and stentorin (ST), the photosensing chromophores responsible for photomotile reactions in the ciliates Blepharisma japonicum (red and blue cells) and Stentor coeruleus, represent a new class of photoreceptor pigments whose chemical structures have recently been determined. In the case of ST it has been shown that the first excited singlet state can be deactivated by donation of an electron to an appropriate acceptor molecule (e.g. a quinone molecule). This charge transfer can be considered a possible mechanism for the primary photoprocess for the photomotile responses in S. coeruleus. To determine whether an electron transfer process also occurs in the deactivation of excited blepharismin, we studied the fluorescence quenching of OxyBP in dimethyl‐sulfoxide (DMSO) and in ethanol using electron acceptors with different reduction potentials. Under our experimental conditions ground state and excited state complexes (like fluorescent exciplexes) are not formed between the fluorophore and the quenchers. In DMSO the bimolecular quenching constant values (kq) calculated on the basis of the best fitting procedures clearly show that the quenching efficiency decreases with the quencher negative reduction potential, E0. The kq (M‐1 s‐1) and E0 (V) values are, respectively, 7.8 times 109 and ‐0.134 for 1,4‐benzoquinone, 8.9 times 109 and ‐0.309 for 1,4‐naphthoquinone, 2.4 times 109 and ‐0.8 for nitrobenzene, 0.009 times 109 and ‐1.022 for azobenzene and 0 and ‐1.448 for benzophenone. These findings point to the conclusion that upon formation of the encounter complex between OxyBP and the quencher, an electron is released from excited OxyBP to the quencher, similar to what happens in ST. It is suggested that in the pigment granules such a light‐induced charge transfer from excited blepharismin to a suitable electron acceptor triggers sensory transduction processes in B. japonicum.

Tetrakis(4-pyridyl)porphyrin Supramolecular Complexes with Cyclodextrins in Aqueous Solution

Abstract The formation of inclusion complexes of hydroxypropyl-β-cyclodextrin, heptakis(2,6-di-O-methyl)-β-cyclodextrin and heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin with 5,10,15,20-tetrakis(4-pyridyl)porphyrin (TpyP) has been studied in aqueous buffer solution (phosphate buffer pH = 7 and I = 0.01 M) to give a structural and spectroscopic characterization of a new class of potential sensitizers for photodynamic therapy. The interaction was investigated by a combination of UV/Vis absorption, fluorescence anisotropy, time-resolved fluorescence and circular dichroism. The experimental results point to the presence of the pigment in water in a monomeric complexed form. The fluorescence anisotropy measurements suggest that TpyP forms 1:1 complexes with heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin and hydroxypropyl-β-cyclodextrin, while 1:2 complexes are obtained with heptakis(2,6-di-O-methyl)-β-cyclodextrin.

Uncharged water-soluble porphyrin tweezers as a supramolecular sensor for α-amino acids

The binding between uncharged cobalt porphyrin tweezers and L-amino acids in aqueous solutions is studied by means of UV–vis and circular dichroism spectroscopy. By varying the length of the aliphatic bridge between the two porphyrin units, the number of cobalt ions in the porphyrin cores and the pH of the solution, the chirality induction phenomenon has been investigated. The binding of the amino acid to the porphyrin seems to occur via a coordination mechanism between the metal and the nitrogen of the amino group; the steric, hydrophobic and π–π interactions operate to stabilize the complexes. The chirogenesis displays an opposite behaviour in the presence of aromatic guests with respect to the non-aromatic ones. Moreover, the UV–vis and the induced circular dichroism spectral changes suggest that the amino acid arrangement in the tweezers is determined by many factors, so that, unlike in organic solvent, the porphyrin tweezers in aqueous solution allow for two different arrangements of the same aromatic amino acid. The experimental findings indicate that the porphyrins tweezers reported in the paper are promising in opening perspectives toward their application as a selective molecular sensor in aqueous solutions directly.

Artificial models of biological photoreceptors: effect of quenchers on the fluorescence properties of hypericin embedded in liposomes

Abstract Liposomes loaded with hypericin at different local concentrations were used as artificial models of biological light sensors. The steady state and time-resolved fluorescence of the chromophore were investigated in the presence of fluorescence quenchers which penetrate differently into and diffuse differently through lipid bilayers: iodide (I−), 9,10-anthraquinone-2,6-disulphonate (AQDS2−) and 9,10-anthraquinone-2-sulphonate (ACMS−). The results of the experiments with I− indicate that two of the three fluorescent species detected in our system, the long-lived and intermediate-lived species, are relatively close to the lipid-water interface. Anthraquinone compounds, which partially penetrate into the liposome, not only significantly quench the chromophore fluorescence emission, but also, apparently, affect the distribution of hypericin molecules in the vesicle, shifting the long-lived fluorescent molecules towards chemical-physical configurations characterized by shorter fluorescence lifetimes.

IN VIVO SPECTROSCOPIC STUDY OF PHOTORECEPTOR PIGMENTS OF BLEPHARISMA JAPONICUM RED AND BLUE CELLS

Abstract In the coloured ciliate Blepharisma japonicum , step-up photophobic responses are triggered by the endogenous pigment blepharismin. Blepharismin, red in dark-grown cells, is intracellularly photooxidized into a blue form (oxyblepharismin), still acting as photosensing pigment. With the aim of correlating the spectroscopic properties of blepharismin and oxyblepharismin in vivo with their photophysiological features, optical absorption, steady-state and time-resolved fluorescence spectra have been measured on cell suspensions. Both in blepharismin and oxyblepharismin in their physiological molecular environment, three fluorescent species have been observed, with virtually the same lifetimes ( ∼ 0.2 ns, ∼ 1.0 ns, ∼ 3.5 ns), but significantly different relative amplitudes. In red cells the long-living component has a very low relative amplitude (∼ 4%) and the short-living one is largely predominant (> 78%), whereas in blue cells the slowly decaying species has a slightly higher relative amplitude (∼ 40%) than the intermediately (∼ 31%) and the fast decaying species (∼ 29%). Together with the spectral width of time-gated spectra, these data are discussed in connection with current hypotheses on the structures of the chromophores. No meaningful difference in the above-mentioned spectroscopic parameters was observed after 30 min of UV-B irradiation, showing that no significant difference exists between red and blue blepharismin as far as UV-B lability is concerned.

Spectroscopic study of the chromophore--protein association and primary photoinduced events in the photoreceptor of Blepharisma japonicum.

Blepharisma japonicum is a ciliated protozoan exhibiting a strong step-up photophobic response upon illumination. The photoreceptor chromophores responsible for this response have been identified to be hypericin-like chromophores (blepharismin and oxyblepharismin), complexed to a 200 kDa non-water-soluble protein. The present work opens up new perspectives on the primary phototransduction steps of B. japonicums light perception through a joined approach by steady-state fluorescence spectroscopy, time-resolved fluorescence anisotropy and sub-picosecond transient absorption spectroscopy. The free chromophore of the light-adapted form of the cell (oxyblepharismin) was studied in various solvents and its spectroscopic properties, as well as its primary excited-state reactivity, compared with those of the corresponding pigment-protein complex, extracted by phosphate-concentration-step chromatography on a hydroxyapatite column. Fluorescence anisotropy together with SDS PAGE electrophoresis results confirm that oxyblepharismin is non-covalently bound to the apoprotein and show that, in the excited state, it is free to rotate in all directions within the binding site where it experiences a large local viscosity. Time-resolved anisotropy measurements on aromatic amino acids confirm that the molecular weight of the protein is of the order of 200 kDa. Although showing very similar steady-state spectra, free oxyblepharismin and its protein complex have noticeably different excited-state behaviours. In particular, the protein complex exhibits a pronounced short-lived absorption feature in the 640--750 nm range, decaying biexponentially in 4 ps and 56 ps. Those decays, also observed in other spectral regions, are not found in the corresponding kinetics of the isolated pigment in solution. This early behaviour of the protein complex might be the signature of the primary phototransduction process, possibly involving an electron transfer from the pigment to a neighbouring protein acceptor residue as it had been suggested in previous studies.

Circular Dichroism of the Photoreceptor Pigment Oxyblepharismin

Abstract Circular dichroism (CD) was used to study the structure of oxyblepharismin (OxyBP), the photoreceptor chromophore for the photophobic response of the blue form of Blepharisma japonicum. Both the chromophore associated to its native protein and the free chromophore in ethanol solution were investigated. CD spectra in the far-UV range indicate that OxyBP induces a slight increase in the α-helix content of the protein matrix. CD spectra in the near-UV and visible region of the spectrum show that OxyBP adopts a chiral conformation with a preferential geometry not only when associated to its protein matrix, but also when isolated and dissolved in ethanol. This experimental result is related to the existence of a high-energy interconversion barrier between two enantiomeric structures of the molecule and discussed on the basis of an asymmetric biosynthesis of its precursor, blepharismin.

Spectroscopic investigation and molecular modeling on porphyrin/PAMAM supramolecular adduct.

Noncovalent adducts (TPPC@PAMAM) between meso‐tetrakis(4‐carboxyphenyl)porphyrin (TPPC) and polyamidoamine PAMAM dendrimer (generation 2.0) have been obtained by simply mixing the two components at different stoichiometric amount. The resulting species are readily soluble and stable in aqueous solution up to millimolar concentration. Electrostatic interactions between the anionic carboxylate groups of TPPC and the protonated amino groups of the PAMAM dendrimer play an important role in the stabilization of these adducts. UV/Vis absorption, steady state and time‐resolved fluorescence emission and anisotropy measurements suggest the presence of equilibria involving different species as function of the [PAMAM]/[TPPC] ratio. At low ratios the observed spectroscopic behavior evidence the presence of H‐aggregates, while at higher ratios well‐defined species containing monomeric TPPC strongly interacting with the charged dendrimer are formed. Docking of the binary supramolecular adduct further supports the experimental results showing a favorable interaction with the porphyrin being completely included in the dendrimer. The interaction of the binary TPPC@PAMAM adduct (1/1 ratio) with calf‐thymus DNA has been investigated through spectroscopic and photophysical techniques. All the experimental results point to the formation of a ternary complex between the binary adduct and the DNA backbone.

Molecular Basis of Photoreception

Any physiological phenomenon which is driven, triggered or modulated by sunlight in living organisms (from vision to photosynthesis, from photoreactivation to vitamin D biosynthesis, from phototropism to seed germination) is the final result of a series of physical, chemical, biophysical and biochemical processes which originate from the same “elementary” event: the absorption of a photon by the photoreceptor molecule.