Pier Raimondo Crippa

New melanic pigments in the human brain that accumulate in aging and block environmental toxic metals

Neuronal pigments of melanic type were identified in the putamen, cortex, cerebellum, and other major regions of human brain. These pigments consist of granules 30 nm in size, contained in organelles together with lipid droplets, and they accumulate in aging, reaching concentrations as high as 1.5–2.6 μg/mg tissue in major brain regions. These pigments, which we term neuromelanins, contain melanic, lipid, and peptide components. The melanic component is aromatic in structure, contains a stable free radical, and is synthesized from the precursor molecule cysteinyl-3,4-dihydroxyphenylalanine. This contrasts with neuromelanin of the substantia nigra, where the melanic precursor is cysteinyl-dopamine. These neuronal pigments have some structural similarities to the melanin found in skin. The precursors of lipid components of the neuromelanins are the polyunsaturated lipids present in the surrounding organelles. The synthesis of neuromelanins in the various regions of the human brain is an important protective process because the melanic component is generated through the removal of reactive/toxic quinones that would otherwise cause neurotoxicity. Furthermore, the resulting melanic component serves an additional protective role through its ability to chelate and accumulate metals, including environmentally toxic metals such as mercury and lead.

Time-resolved photoacoustic spectroscopy: New developments of an old idea

Abstract Acoustic waves generated by heat emission in radiationless transitions from photoexcited molecules can be detected by suitable transducers. Their study allows the investigation of thermal relaxations, thus providing thermodynamic and kinetic data on short-lived species produced by the absorption of pulses of light. In this field of research the best technique has proved to be the so-called pulsed-laser, time-resolved photoacoustic spectroscopy, which is based on piezoelectric detection of pressure waves in the time domain. Deconvolution processing of the transient signals gives both the lifetimes of excited states and the energy content of the transitions, provided that decay times are in the range 5 ns-5 μs. Moreover, when compared with proper theoretical models emphasizing the energy balance, the photoacoustic results can help to build a complete picture of the deactivation pathways, including photochemical events. The biophysical applications, although numerous and widespread both in basic and applied research, offer the real possibility of giving information on photobiological processes in conditions very close to the living state. Among the more significant contributions obtained in this area, the results on photosynthesis and photosensitivity of plants and photosynthetic micro-organisms, structural and functional dynamics of respiratory proteins, photocycles of rhodopsin and bacteriorhodopsin and photophysical properties of several natural pigments are particularly relevant, together with some medical and biotechnological applications. Another promising field of application of photoacoustics concerns photoactive drugs and the photophysics of fluorescent probes for conformational studies of proteins, nucleic acids and membranes. In general terms, time-resolved photoacoustic spectroscopy promises to become one of the most powerful techniques in photobiophysics, provided that some limitations in data analysis and time resolution are removed by technical improvements.

Photoacoustic studies of non-radiative relaxation of excited states in melanin

Photoacoustic measurements made at various chopping frequencies on dense acqueous melanin suspensions have revealed a significant dependence upon pH and redox state. It is shown that such behaviour, differing from the simple predictions of the Rosencwaig-Gersho theory, can be explained in terms of finite carrier diffusion and recombination times. The implications of these findings with respect to the amorphous semiconductor model proposed to describe the dynamic role of epidermal melanin are discussed. From the experimental data, values of physical parameters were calculated which allow a qualitative correlation between chemical states and electronic behaviour and, consequently, some aspects of the molecular biology of the melanosome, founded on a rigorous base.

Fungal melanins differ in planar stacking distances.

Melanins are notoriously difficult to study because they are amorphous, insoluble and often associated with other biological materials. Consequently, there is a dearth of structural techniques to study this enigmatic pigment. Current models of melanin structure envision the stacking of planar structures. X ray diffraction has historically been used to deduce stacking parameters. In this study we used X ray diffraction to analyze melanins derived from Cryptococcus neoformans, Aspergillus niger, Wangiella dermatitides and Coprinus comatus. Analysis of melanin in melanized C. neoformans encapsulated cells was precluded by the fortuitous finding that the capsular polysaccharide had a diffraction spectrum that was similar to that of isolated melanin. The capsular polysaccharide spectrum was dominated by a broad non-Bragg feature consistent with origin from a repeating structural motif that may arise from inter-molecular interactions and/or possibly gel organization. Hence, we isolated melanin from each fungal species and compared diffraction parameters. The results show that the inferred stacking distances of fungal melanins differ from that reported for synthetic melanin and neuromelanin, occupying intermediate position between these other melanins. These results suggest that all melanins have a fundamental diffracting unit composed of planar graphitic assemblies that can differ in stacking distance. The stacking peak appears to be a distinguishing universal feature of melanins that may be of use in characterizing these enigmatic pigments.

Oxygen adsorption and photoreduction on fractal melanin particles

The main putative functions of melanins in living cells, that is photoprotection and dark or light-dependent oxido-reductive activity, still requires an interpretation which takes into account the micro-mesoscopic structure of native melanin particles. It is indeed well established that a different chemical composition of melanins, even if derived from different biosynthetic pathways, has only a little influence on the biological and physical properties of the solid aggregates, the common form in which the pigment is found [P.R. Crippa et al., Chemistry of melanins, in: A. Brossi (Ed.) The Alkaloids, vol. 36, Academic Press, New York, 1989, pp. 253-323]. In the present work a model for interfacial electron transfer is proposed describing the process of light induced superoxide formation through a monoelectronic reduction of dioxygen adsorbed on melanin solid surface. This process is presumed to be dependent on the surface fractal characteristics, and its kinetics must be interpreted as a heterogeneous interfacial reaction involving light produced carriers and the adsorbed acceptor, like in colloidal inorganic semiconductors such as TiO(2).

THE INTERACTION OF MELANIN WITH 8-METHOXYPSORALEN: EFFECT ON RADIATIVE and NONRADIATIVE TRANSITIONS. A FLUORESCENCE and PULSED PHOTOACOUSTIC STUDY

Abstract –The interaction of 8‐methoxypsoralen (8‐MOP) with synthetic eumelanin was investigated using static and time‐resolved fluorescence and pulsed photoacoustic calorimetry. Due to the strong overlap of the absorption bands of melanin and 8‐MOP, a method is presented to account for the systematic errors introduced by the optical filter effect exerted by each absorbing species in the fluorescence and the photoacoustic measurements. As a preliminary step to the understanding of the nonradiative behavior of the psoralen‐melanin complexes, the photoacoustic parameters of 8‐MOP in various solvents were determined. Spectroscopic data indicate the absence of interaction at the ground‐state level, whereas the singlet excited state of 8‐MOP is quenched by the pigment; the average fluorescence lifetimes are independent of the melanin concentration, thus indicating a static quenching mechanism. The photoacoustic data show that the quenching process involves an increased intersystem crossing probability, which is almost unaffected by the presence of oxygen, as expected for a molecule essentially acting as a type I photosensitizing agent.

An XAS study of the sulfur environment in human neuromelanin and its synthetic analogs

Neuromelanin is a complex molecule accumulating in the catecholaminergic neurons that undergo a degenerative process in Parkinson’s disease. It has been shown to play either a protective or a toxic role depending on whether it is present in the intraneuronal or extraneuronal milieu. Understanding its structure and synthesis mechanisms is mandatory to clarify the reason for this remarkable dual behavior. In the present study, X-ray absorption spectroscopy is employed to investigate the sulfur binding mode in natural human neuromelanin, synthetic neuromelanins, and in certain structurally known model compounds, namely cysteine and decarboxytrichochrome C. Based on comparative fits of human and synthetic neuromelanin spectra in terms of those of model compounds, the occurrence of both cysteine- and trichochrome-like sulfur coordination modes is recognized, and the relative abundance of these two types of structural arrangement is determined. Data on the amount of cysteine- and trichochrome-like sulfur measured in this way indicate that among the synthetic neuromelanins those produced by enzymatic oxidation are the most similar ones to natural neuromelanin. The interest of the method described here lies in the fact that it allows the identification of different sulfur coordination environments in a physically nondestructive way.

Type I photosensitized reactions of oxopurines. Kinetics and thermodynamics of the reaction with triplet benzophenone by time-resolved photoacoustic spectroscopy

Abstract Benzophenone photosensitized reactions of caffeine, theophylline and theobromine were investigated in acetonitrile by time-resolved laser-induced photoacoustics. In the three cases global quenching rate constants of triplet benzophenone were measured as a function of temperature and it was observed that this is a non-activated process. Besides, for theobromine and theophylline heats for NH hydrogen abstraction reactions were determined. In agreement with semiempirical calculation predictions, hydrogen abstraction is thermodynamically more favorable and faster for theophylline (Δ H =−265 kJ mol −1 , k r =9.6×10 8 M −1 s −1 ) than for theobromine (Δ H =−168 kJ mol −1 , k r =3.7×10 8 M −1 s −1 ).

TIME-RESOLVED PHOTOACOUSTIC CALORIMETRY OF TRYPTOPHAN IN WATER and ORGANIC SOLVENTS

Abstract –Very little has been done in the direct study of nonradiative decays of Trp, indole and derivatives, in spite of their relatively low fluorescence quantum yields. Time‐resolved photoacoustic calorimetry is an ideal technique for this kind of study: it is very sensitive in the detection of small amounts of heat released and, in principle, allows a broad band of temporal resolution. The photoacoustic apparatus used for our measurements offers a temporal window between 10 ns and 10 μs. The analysis of the waveforms, based on a particular deconvolution method, simultaneously gives the fraction of energy released and the associated lifetime. In a broad sense, time‐resolved photoacoustic spectroscopy can be seen as a complementary method to traditional radiative techniques such as static, time‐resolved fluorescence and flash photolysis. The present work presents studies of Trp in different solvents in order to acquire new information about the interaction of this chromophore with solvents. From our measurements the high sensitivity of Trp to solvents is confirmed. The formation of complexes is evident at the excited state (exciplexes), between the chromophore and one or more molecules of solvent. These exciplexes are characterized by having an energy different from that of the singlet and triplet of Trp. Moreover, photoacoustic measurements detect, in water, the presence of another electronic state, which seems to have characteristics similar to a triplet‐like state reported in other work.

A model for interfacial electron transfer on colloidal melanin

Abstract Though melanins are involved in photochemical reactions (mainly of oxido-reductive type) in vitro and this activity is supposed to have biological implications, no satisfactory model of the reaction kinetics has so far been proposed. The main difficulty arises from the particulate structure of the insoluble melanins and the consequent necessity to describe their reactivity in the framework of heterogeneous chemistry, i.e., at the solid-liquid interface. Our paper presents a simplified model of the monoelectronic reduction reaction of dioxygen, based on well-established experimental facts and some reasonable assumptions: (1) surface adsorption of O2 on colloidal melanins can be described by a Langmuir isotherm; (2) the kinetics of superoxide formation is photodependent and includes an interfacial electron-transfer process; (3) the photochemical behaviour of the single melanin granule can be described in terms of the electronic properties of amorphous semiconductor particles. Some satisfactory comparisons with experimental data and calculated values of the kinetic constants for the process are presented and discussed.