Binding of chemicals to melanins re-examined: adsorption of some drugs to the surface of melanin particles.

This work presents a first attempt to study the interaction of some drugs with melanins, realistically considered as solid aggregates of primary particles. This situation appears similar to the adsorption of organic molecules onto the surface of colloidal absorbers, as active carbon, zeolites or titanium dioxide. We have applied some of the most popular theoretical models used in technological applications with the aim to give a more realistic picture of the melanin-drug interaction responsible for some observed side effects in vivo. Moreover, this approach can simplify the problem of the search of the physical parameters dominating the binding processes, by reducing the phenomenon to a simple physisorption/chemisorption, at least in a first approximation. We have studied the binding to melanin of gentamicin, methotrexate and chlorpromazine, molecules with different physico-chemical and structural characteristics. Our study demonstrates the possibility to fit experimental adsorption data with Langmuir, Freundlich, Tempkin and Dubinin-Radushkevich equations. In such a way we obtain binding parameters useful to characterize the drug-surface interaction in terms of energy and of mean affinity.

Magnetic investigations of human mesencephalic neuromelanin.

Pigmentation of neurons in substantia nigra is due to neuromelanin, a pigment that stores large amounts of iron. Human mesencephalic neuromelanin has been investigated by means of magnetic susceptibility measurements as a function of temperature. Magnetic measurements provide a physico-chemical characterization of the iron cluster buried in the organic melanin matrix and support the view that iron is not simply chelated, but rather is organized in a three-dimensional network. The paramagnetism of isolated iron ions is observed, in agreement with electron paramagnetic resonance spectroscopy. Furthermore, antiferromagnetic grains with a large size distribution function are present. These grains contain N spins coupled antiferromagnetically; however, N(1/2) spins are decoupled from the grain bulk and parallelly aligned. The latter subgrains are superparamagnetic with a blocking temperature ranging between 5 K and room temperature. This behavior has not been observed in synthetic melanin, where the paramagnetic contribution is strongly enhanced. Preliminary results on pigment isolated from patients affected by Parkinson's disease, a neurodegenerative pathology involving primarily pigmented neurons in substantia nigra pars compacta, show a lower total magnetization compared to control neuromelanin. The temperature behavior of zero field cooling and field cooling magnetizations is similar for both. The significant depletion of iron content in Parkinson's disease neuromelanin could indicate a progressive Fe migration from its storage environment to the cytosol.

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).

X-ray absorption fine-structure spectroscopy studies of Fe sites in natural human neuromelanin and synthetic analogues.

X-ray absorption fine-structure spectroscopy is used to study the local environment of the iron site in natural (human) neuromelanin extracted from substantia nigra tissue and in various synthetic neuromelanins. All the materials show Fe centered in a nearest neighbor sixfold (distorted) oxygen octahedron; the Fe-O distances, while slightly different in the natural and synthetic neuromelanin, are both approximately 2.0 A. Appreciable differences arise, however, in the second (and higher) coordination shells. In this case the synthetic melanin has the four planar oxygens bound to carbon rings with Fe-C distances of approximately 2.82 and 4.13 A; the human sample does not show the 2.82 A link but instead indicates a double shell at approximately 3.45 and 3.78 A.

Anomalous temperature fluorescence quenching of N-Trp terminal peptides.

The photophysics of Trp-containing peptides is extremely affected by the position of the indole ring with respect to substituents. In this work an unusual temperature fluorescence quenching behavior is presented. The N-tryptophan terminal peptides (N-Trp) show an increase of the static emission intensity as rising the temperature from 10 to about 40 degrees C. the anomaly is typical of the N-Trp terminal peptides since neither tryptophan (Trp) nor glycyl-tryptophan (Gly-Trp) and alanyl-tryptophan (Ala-Trp) show the same trend; a similar behavior is not detected in the C-tryptophan terminals. The other important features are the wavelength and pH dependence of the effect. The anomaly is in fact detected only at neutral pH and for excitation wavelength near the red edge of the UVB absorption band of indole. An interpretation of the anomaly is suggested, though more sophisticated techniques are needed to better focus the problem; the model proposed involves the superimposition of a ground state effect (the temperature-induced equilibrium shift from the zwitterionic to the anionic form of the peptides) and an excited state mechanism. At present no unique interpretation can be provided about the excited state mechanism that favors the anomaly ans some suggestions are discussed.

The interaction of melanin with 8-methoxypsoralen: effect on radiative and nonradiative transitions. A fluorescence and pulsed photoacoustic study.

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.

Application of photoacoustic phase angle spectroscopy (phi AS) to eumelanins and pheomelanins.

A new technique, based on the measurement of the phase angle of the photoacoustic signal, was used for investigating the absorption spectra of melanins in the dry state. The main advantages of such a method are the insensitivity to scattering of the light and the applicability to nontransparent or highly adsorbing and thermally thick samples. The spectra obtained on different kinds of natural and synthetic melanins show much more details than the corresponding ones obtained spectrophotometrically on aqueous suspensions of the pigment. Some relevant features of the spectra still cannot be interpreted, yet we were able to show the presence in eumelanins of dopachrome at 475 nm and melanochrome at 540 nm, and in pheomelanins of compounds previously isolated from red chicken feathers. Moreover, the well known trend of the optical absorption, i.e., its decrease with increasing wavelength, was unambiguously confirmed.

Quenching of the excited states of 8-methoxypsoralen by synthetic Eumelanin.

The interaction of 8-methoxypsoralen (8-MOP) with synthetic eumelanin was investigated using static and time-resolved fluoresence and pulsed photoacoustic calorimetry. Spectroscopic data indicate the absence of interaction in the ground state, 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 mechanism. 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.

X-ray diffraction studies on melanins in lyophylized melanosomes.

A series of experiments was performed on lyophylized melanosomes in order to analyze the melanin in the natural state as polymerized into these organelles and to verify in such biological amorphous material the possibility of obtaining intensity scattering curves from which Bragg distances can be calculated. The results confirm the feasibility of the method and show that melanins in melanosomes maintain many structural features of the purified form and that the biochemical composition of the organelles can be responsible for the observed differences in the diffractograms. The presence in melanosomes of supramolecular paracrystalline aggregates was also clearly demonstrated.

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.

13C solid-state cross polarization/magic-angle-spinning nuclear magnetic resonance spectra of natural and synthetic melanins.

The availability of NMR spectrometers operating in cross polarization/magic angle spinning (CP-MAS) has provided a powerful tool for the structural elucidation of insoluble materials. In this 13C NMR study of eumelanins we report the first direct evidence of the presence of different chemical functionalities in synthetic and natural eumelanins. These spectra contain useful information for the characterization of melanins from different sources.

Fluorescence depolarization studies of melanosomal membranes from different sources.

In the present paper we report a comparative study of physical properties and biochemical composition of isolated melanosomal membranes extracted from bovine eyes and from an equine spleen melanoma. Some biophysical characteristics of such membranes were obtained by steady-state and time resolved fluorescence spectroscopy using DPH as fluorescent probe. By these methods we have measured both static fluorescence polarization and fluorescence lifetimes and from the experimental data we have calculated the rotational correlation times by Perrin's equation. Since dynamic and static parameters, such as fluidity and molecular order, can be determined by these methods, the results are discussed on the basis of the recent theories of the role of the biochemical composition in the molecular structure and properties of membranes.

Effect of U.V. irradiation and of various chemical conditions on the oxidation of NADH by pheomelanins.

The oxidation reaction of NADH with synthetic and natural pheomelanins was studied under various physicochemical conditions in order to obtain information about the molecular steps involved in the electron transfer process. The comparison with the previous data on eumelanins suggests that the reaction pathways are the same and that the greater photosensitivity of pheomelanins, observed in other experiments, is manifested in the redox reactions with NADH as an increased formation of quinone groups and of superoxide anions.

Involvement of superoxide ions in the oxidation of NADH by melanins.

The reactions of oxidation of NADH by Dopa and Dopamine-melanins show inhibition with low quantities of Superoxide dismutase, the enzyme which catalyzes the reaction: O2- + O2- + 2H+ leads to H2O2 + O2 and which has been used as an indicator of the involvement of superoxide ions in many biochemical systems. The reaction was also investigated in the presence of metal ions (Cu2, Zn2+ and Co2+) that are able to increase the extent of oxidation, and also in this case the inhibition effect of superoxide dismutase was observed. The results are interpreted in terms of a single electron pathway involving the superoxide ion and NAD.

Oxidation of NADH by melanin: effect of UV light and copper ions.

The oxidation reaction of NADH with synthetic melanin from L-dopa was investigated under various physicochemical conditions both by spectrophotometric measurements and ESR spectroscopy. The different amounts of NADH oxidized and the effects on the formation and decay of melanin free radicals were compared. Some hypotheses on a common physical mechanism involved in electron transfer reaction and in electron excitation through a liquid-solid interface are presented.

Reversible and irreversible changes in hydrogen ion titration curves of melanins.

Hydrogen ion titration curves obtained on melanins prepared and modified differently show recognizable differences. Melanin prepared with NaOH exhibits irreversible loss of functional groups in the base range once titrated acidic. Melanin prepared with NH4OH shows two main branches selected by incubation at an extreme pH for 24 h. Longer exposure reveals other curves but they disappear once a 24-h schedule of titration is resumed. The existence of two major pH branches appears consistent with the oxidation-reduction properties of melanins. These results demonstrate the ability to obtain a reproducible feature in the hydrogen ion titration curves. Thus it is finally possible to quantitate CO2 adsorbed to melanin in solution at basic pH.

Isoelectric focusing study of radiation damage to dry conalbumin.

The effect of gamma-rays on iron-free conalbumin was studied by isoelectric focusing technique. The results obtained can be interpreted on the basis of radiation-induced conformational changes of the macromolecules through a cleavage of disulphide bridges. Treatments with reagents acting on disulphide bridges lead to isoelectric focusing patterns that confirm this hypothesis.