The aim of this work was to compare free radical properties of some model neuromelanins obtained from dopamine and its mixture with 5-S-cysteinyldopamine in various molar ratios. The EPR method was used in these investigations; the samples were detected at X and K bands. The parameters of EPR spectra, free radicals concentrations in air and in vacuum, the influence of microwave power and temperature on intensity of resonance signals were measured

Dzierżęga-Lęcznar, A.

Skrzypek, Danuta

Ziółkowska, J.

Acta Physica Polonica A

English

Skrzypek, D.

Biblioteka Nauki - repozytorium artykuÅÃ³w

EPR Investigations of Model Neuromelanins

Repozytorium Uniwersytetu Śląskiego RE-BUŚ

             Title: EPR investigations of model neuromelanins  Author: Danuta Skrzypek, A. Dzierżęga-Lęcznar, J. Ziółkowska  Citation style: Skrzypek Danuta, Dzierżęga-Lęcznar A., Ziółkowska J. (2000). EPR investigations of model neuromelanins. "Acta Physica Polonica A" (Vol. 98, nr 5 (2000), s. 561-566).  Vol. 98 (2000)	 ACTA PHYSICA POLONICA A 	 No. 5Proceedings of the International Conference "Condensed Matter Physics", Jaszowiec 2000EPR INVESTIGATIONS OF MODELNEUROMELANINSD. SKRZYPEK „,* , A. DZIERŻĘGA-LĘCZNAR b AND J. ZIÓLKOWSKA „„ Institute of Physics, University of Silesia, Uniwersytecka 4, 40-007 Katowice, Polandb Faculty of Pharmacy, Silesian Medical AcademyNarcyzów 1, 41-200 Sosnowiec, PolandThe aim of this work was to compare free radical properties of somemodel neuromelanins obtained from dopamine and its mixture with 5-S-cys-teinyldopamine in various molar ratios. The EPR method was used in theseinvestigations; the samples were detected at X and K bands. The parametersof EPR spectra, free radicals concentrations in air and in vacuum, the influ-ence of microwave power and temperature on intensity of resonance signalswere measured.PACS numbers: 76.30.Rn, 87.15.—v1. IntroductionMost dopaminergic neurons of human brain substantia nigra are pigmentedwith neuromelanin. It seems that this biopolimer may be actively involved in neu-rodegenerative processes that occur during natural ageing and Parkinson's dis-ease [1]. Numerous data indicate that the nigral pigment consists of indole-type(eumelanin) and benzothiazine-type (pheomelanin) subunits, derived oxidativelyfrom dopamine and 5-S-cysteinyldopamine, respectively [2, 3]. EPR studies evi-denced that both structural constituents of natural neuromelanin are the sourcesof distinct kind of stable organic radicals [4].It has been found previously that pure eumelanin synthesized from dopamineis characterized by o-semiquinone oxygen radicals, whereas 5-S-cysteinyldopamine--derived pheomelanin contains nitrogen-centered semiquinoneimine radicals [5].The aim of this work was to compare free radical properties of some model neu-romelanins obtained from dopamine and its mixture with 5-S-cysteinyldopaminein various molar ratios.*corresponding author; e-mail: dskrzyp@us.edu.pl(561)562 	 D. Skrzypek et al.2. Materials and methods5-S-cysteinyldopamine was obtained by tyrosinase-catalyzed oxidation of dop-amine in the presence of cysteine and purified by ion-exchange chromatography [6].Model neuromelanins were synthesized by autooxidative polymerization ofdopamine (DA) and 5-S-cysteinyldopamine (5-S-CDA), mixed in various molar ra-tios, in 50 mM sodium phosphate buffer (pH 7.0) at 310 K. with vigorous stirringand light protection. Pure DA-melanin and 5-S-cysDA-melanin were also synthe-sized using the same procedure.Spectra EPR at X band (-„ 9 GHz) were recorded with a standard Radiopanspectrometer system. At K band (— 22 GHz) a MB 257 microwave bridge wasused with a spectrometer.3. Results and discussionFree radical properties of synthetic pheomelanins obtained from various cys-teinyl adducts of dopamine, as well as those of eu-/pheo-copolymer from dopamineand 5- S-cysteinyldopamine were compared previously at X band [5]. For all thestudied samples, complex EPR spectra with a poorly resolved hyperfine structurewere recorded. According to Sealy et al. [7], the EPR signals were characterized ad-ditionally by "R"-factor, defined as the height ratio of central high-field to low-fieldmaximum. Such a parameter seems to be helpful for determination of the relativecontent of eu- and pheo-type components in the melanin pigments of mixed char-acter.The presence of nitrogen centered free radicals with g-values of 2.0052 wasproved for pure pheomelanin from 5-S-cysteinyldopamine. For eu-/pheo-copolymer,derived oxidatively from an equimolar mixture of dopamine and 5-S-cysteinyldop-amine, lowering g-value to 2.0046, as well as a significant increase in R-factorcould be observed. Hence, it was concluded that oxygen free radicals of eumelanincomponent were predominant in this kind of biopolymer.It was evidenced by degradative studies [3] that human substantia nigraneuromelanin could contain variable amounts of pheomelanin component. Thenatural pigment should be modeled then by synthetic copolymers derived fromdopamine and 5-S-cysteinyldopamine rather than by pure dopamine melanin.It can be assumed that, depending on its chemical composition, human neu-romelanin may be involved in some patho-physiological processes with variousefficiency. The ratio of pheomelanin to eumelanin subunits implies not only intrin-sic structure and aggregation mode of the biopolymer, but also its free radical typeand concentration. The EPR spectra of free radicals in synthetic dopamine-melaninand 5-S-cysteinyldopamine-melanin are distinguishable [5]. The DA-mel has a sin-gle line, whereas the 5-S-CDA-mel, in which the adduct is sulfur, has a spectrumcharacteristic of an immobilized radical that is partly nitrogen centered, mostprobably a semiquinonimine. In all copolymers (Fig. 1) EPR signals similar tothose found for 5-S-CDA melanin are present.The parameters of the EPR spectra for the investigated model neurome-lanins are given in the Table. The samples of pheomelanin-type have gapp valuesEPR Investigations of Model Neuromelanins	 563Fig. 1. EPR spectra of DA/5-S-CDA-mel: molar ratio — (2.5 : 7.5) (a), (1 : 1) (b),(7.5 : 2.5) (c).564 	 D. Skrzypek et al.characteristic of free radicals nitrogen-centered. The mixed type melanin exhibitrelatively higher R-factor compared to pure pheomelanin (sample A).3.1. EPR measurement at X (9 GHz) and K (22 GHz) bandsWe also recorded EPR spectra of synthetic neuromelanins at K band. Fig-ure 2 shows the spectra obtained at various microwave frequencies for 5-S-CDA-mel(for example).Fig. 2. EPR spectra of model 5-S-CDA-mel at X (a) and K (b) bands. The smoothlines mean the fitting to Eq. (2).14N hyperfine coupling in π radicals is generally axial and anisotropic(Az > A1), g-factor is also axial. The Zeeman splitting is proportional to themicrowave frequency because the EPR resonance condition derived from the first--order perturbation theory for the hyperfine term while neglecting the fine struc-ture, quadrupolar and nuclear Zeeman terms is as follows:whereThe critical points of the powder pattern arising from Eq. (1) have been calculatedfor the case of axial symmetry. When the magnetic field is swept and the frequencyheld constant, the critical points occur at the following fields:The comparison of EPR measurements at X and K bands shows that the complexspectra for 5-S-CDA-mel and copolymers DA/5-S-CDA-mel are the case of axialsymmetry. The estimation is based on the comparison of experimental spectrawith the spectra calculated from Eq. (2). The results obtained by this procedureare shown in the Table. We concluded that the same kind of free radicals in allinvestigated model pheomelanins is present, as those found for 5-S-CDA-melanin.EPR Investigations of Model Neuromelanins	 5652.2. Microwave power saturationEPR studies were performed for model neuromelanins in the presence ornot of atmospheric oxygen in the environment of samples. Progressive microwavepower saturation of EPR signal was used to obtain information about the partici-pation of oxygen molecules in the changes occurring in the free radicals populationand spin relaxation processes.Fig. 3. The influeuce of microwave power on the intensities of the EPR signals ofDA/5-S-CDA-mel: sample C. P0 and P — total microwave power produced by klystronand used microwave power, respectively. The intensities were normalized.Figure 3 shows that the intensity of EPR signal (I) saturates with the mi-crowave power (P 1 / 2 ) leading to deviations of linearity between I and P 1 / 2 . Sim-ilar figures were obtained for all model neuromelanins. The higher values of P1/2(that are inversaly proportional to T1 T2 — the product of transversal and longi-tudinal relaxation times) are obtained when oxygen is present. The concentrationof paramagnetic centers in DA-mel is not dependent on the presence of molecu-lar oxygen, whereas air removing from other model neuromelanins leads to thedecrease in paramagnetic centers concentrations. However, it seems that in thesecases the accessibility of oxygen to paramagnetic centers is low.3.3. Temperature investigations of model neuromelaninsThe experiments were performed at a series of temperatures from 300 K to150 K. Figure 4 shows the product IT as a function of temperature T (I was ob-tained by a double integration of the first derivative spectra — it was necessary torelate signal amplitudes to paramagnetic susceptibility). The data were normalizedto T = TR. The susceptibility of normal paramagnetics is universally proportional566 	 D. Skrzypek et al.Fig. 4. Temperature dependences IT vs. T for model neuromelanin: (a) samples E,A,C;(b) samples D,B.to the temperature T. Thus the product IT is constant vs. T. We believe that thedistinct changes might be interpreted in terms of thermal induced radicals.Our experiments (data not shown) also indicate that temperature depen-dences of IT were not affected by the presence of atmospheric oxygen in thesample environment.4. ConclusionsIt was evidenced [3] that human substantia nigra neuromelanin could containvariable amounts of pheomelanin component. The natural pigment should be mod-eled then by synthetic copolymers derived from dopamine and 5-S-cysteinyldop-amine rather, than by pure dopamine melanin.We investigated free radical properties of these copolymers. By monitoringthe signal intensities and progressive microwave power saturation it was ascer-tained that model neuromelanins interact with molecular oxygen. The estimationfor experimental results at X and K bands shows that complex EPR spectra forsynthetic neuromelanin are the case of axial symmetry. The measurements per-formed at a series of temperatures may suggest the presence of thermal inducedradicals.References[1] M.B.H. Youdim, D. BenShachar, P. Riederer, J. Neur. Transm. 43, 113 (1994).[2] R. Carstam, C. Brinck, A. Hindemith-Augustsson, H. Rorsman, E. Rosengren,Biochim. Biophys. Acta 1097, 152 (1991).[3] G. Odh, R. Carstam, J. Paulson, A. Wittbjer, E. Rosengren, H. Rorsman, J.Neurochem. 62, 2030 (1994).[4] W.S. Enochs, M.J. Nilges, H.M. Swartz, J. Neurochem. 61, 68 (1993).[5] A. Dzierżęga-Lęcznar, B. Pilawa, K. Stępień, T. Wilczok, Nucleonika 42, 343(1997).[6] S. Ito, K. Fujita, M. Yoshioka, D. Sienko, T. Nagatsu, J. Chromatograph. 375,134 (1986).[7] R.C. Sealy, J.S. Hyde, C.C. Felix, I.A. Menon, G. Prota, Science 217, 545 (1982).

EPR investigations of model neuromelanins

https://rebus.us.edu.pl/bitstream/20.500.12128/8470/1/Skrzypek_EPR_investigations_of_model_neuromelanins.pdf

EPR investigations of model neuromelanins

Abstract

Similar works

Full text

Available Versions

Biblioteka Nauki - repozytorium artykuÅÃ³w

Repozytorium Uniwersytetu Śląskiego RE-BUŚ

EPR investigations of model neuromelanins

Abstract

Similar works

Full text

Available Versions

Biblioteka Nauki - repozytorium artykuÅÃ³w

Repozytorium Uniwersytetu Śląskiego RE-BUŚ

Biblioteka Nauki - repozytorium artykuÅÃ³w