317 - Simultaneous Electrochemical-Electron Spin Resonance (SEESR) studies on natural and synthetic melanins

BioelecfrocLenzisfry J.

EZecfroanaZ

Elsevier

and

Ciiem.

Sequoia

Bioenergefics

116 (rgSo)

S.A..

7 (rgSo)

r53-r65

153-165

Lausanne

-

Printed

in

Italy

317 - Simultaneous EIectrochemica&EIeetron Spin Resonance (SEESR) Studies on Natural and Synthetic Melanins* by STANISLAW EUKIEWICZ, and ZENOW NATUSZAK Department

of

Institute

Biophysics.

University.

3r-ooL

Xanuscript

received

Krakbw,

KRZYSZTOF of

Molecular

RESZKA Biology

of

the

Japellonian

Polancl

September

gth

rg7g

Simultaneous Electrochemical-Electron Spin Resonance (SEESR) measurements have not previously been used to study the redox properties of melanins despite chemical evidence for the oxidation-reduction capacity of these polymers_ This paper describes experiments in which natural and synthetic melanins were electrochemically reduced or oxidized with simultaneous esamination of their ESR absorption_ These measurements demo&rated that : active ; I. melanins are electrochemically and electroosidation may strongly affect ESR 3. their electroreduction signal intensity ; induced changes are both pH-dependent and char3- electrochemicahy acteristic of melanin type ; 4- distinct phases in the increase and decrease in ESR absorption can be distinguished correspondin g to the acceptance or donation of single electrons by melanins. The data provide direct experimental evidence that electroreduction and electroosidation of melanins proceed vin discrete one-electron steps. In the light of these findings it appears moreover that some natural melanins possess an extremely high donor-acceptor capacity_ Intmduction The technique of Simultaneous Electrochemical-Electron Spin Resonance studies (SEESR) was introduced nearly 20 years ago by JhKI and GESKE [I]_ This methodical approach made it possible to measure

3-S

* Presented at September 1979.

o3oz--_1g$3 /So/orj3-or6g

tha 5th \\-eimar

@

International (D_D_R)_

Elsevier

Sequoia

Symposium

S.A.

on

Bioelectrochemistry,

154

-Enkiewicz, Resrh-

and

Matuszak

changes in the content of unpaired electrons (k in the paramagnetic properties) of sampks undergoing electrochemical alterations in the resonant cavity of ESR spectrometers. It proved to- be particularily useful for identifying transient free radical intermediates generated ekctrochemicahy during the ESR examination of the material. However. it has not been applied to the study of the redox properties of biological polymersFor example. the o-xidizing and reducing capacity of natural and synthetic melanins has been a subject of numerous investigations for at ieast 40 years [zJ_ FspeciaIIy in the last decade one can notice a growing interest in chemical, physical and biologica! features of these pigments [3-6$ Accordingly, some new data concerning the redox prop-

erties of melanins have also been published [pro]. The progress made in this field indicates that all types of mehmins should probably be re-

garded as classical redox poly_mers as defined by CASS~DY and KUX [II]It is therefore surprising that SEESR has not yet been applied in the analysis of the interesting redox and paramagnetic properties of melanins and related polymers since the method itself and the problem to be investigated are both well established_ The present paper describes an attempt to fill this gap by arialysing with the use of SEESR changes in the ESR spectra of natural and synthetic melanins induced electrcchemicaIly, i-e_ by electroreduction and/or electrooxidation of these poIymers.

Experimental Se\-era1 types of natural and synthetic melanins were chosen for Among natural pigments, aspergillin was studied the SEESR analvsk in detail_ This material was extracted from the myceha of a fungus rfsp~gz-ks n&r by a mild akahne digestion as described elsewhere[rn.131Some of the pigments used were of animal origin, isolated either from bovine eyes or from hamster melanotic turnours For these materiak, the same technique of preparation and purification was used as in the previous studies on melanins done in this Laboratory [I+ x3]. Synthetic DOPAand catecholmeIanins were prepared by- con\-entional methods [16, 175 ESR measurements were carried out using a VAKIAS X-band spectrometer with IOO kHz field modulation and a TE,,. rectangular cavity_ The design of the flat ekctrochemical cell is shown in Fig_ I. The process of electroreduction was performed in most cases at a mercury cathode except for some electroo_xidation and/or eiectroreduction experiments where a platinum electrode was used_ l-he saturated caIomei electrode (S-C-E.) served as counter and reference electrodesUelanin samples were dissolved or suspended in \-erona1 buffer at

Ekctrochemical

and ESR

Studies

on Melanins

r5.5

pH’s within the range zA5-g.S. The buffer contained 0-1 M KC1 as a basic electrolyteIn some instances dissolved KOH or HCI had to be added if pH-values exceeding the range of veronal buffer were necessary_

Fig_

r_

The flat electrochemicai ESR cell for the measurements in the rectangular TE,,, resonant cavity I F - the fiat part of the measuring cell ; WE - the working electrode (mercury or platinum) ; T - the tungsten wire electrode ; CE - the counter electrode (SCE) : RE - the reference electrode (‘XX) ; X2 - the inlet for gseous nitrogen_

The voltage from a potentiostat was changed stepwise (in IOO mV steps) from o to -z-o V or toward positive values_ It was necessary to wait several minutes following each change of the applied potential difference (U) in order both the current and the ESR signal amplitude to reach constant values_ Only then were ESR spectra recorded_ -411 measurements were carried out at room temperature (22 OC f 10). Reagents were of anaIytical ,gade.

W5

I)

Eukiewicz,

Reszka

Changes in the intensify of the ESR

and

Xatuszak

absoq5tian of n a t u I a I melanins

Since one would expect the electrochemically induced changes to be pH-dependent, SEESR analysis was carried out at several hydrogen ion concentrations_ The diagram shown in Fig 2 is representative of data recorded It was found that only the amplitude of the from natural pigmentsESR signals becomes modified during the electrochemical treatment of the sample ; both the shape and width of the ESR absorption line remain Thus, the relative values of ESR absorption intensity serve constant. as indicators of quantitative changes in the paramagnetic properties of the meIanin occurring under the influence of an EBIF acting at the merThe rise in the ESR signal amplitude corresponds to an cury cathode. increase in the concentration of unpaired electrons associated with intermediate free radical products_ The drop of the signal intensity is an expression of an opposite change, Ce_ of a decrease in the concentration of substances at a one-electron state of oxidation or reduction_ (Provided of course that saturation effects brought about by excessive microwave power are not involved)_ As can be seen from Fig_ 2, four phases can be clearly distinguished -280 -240 -200 - 160

Fig_ 2_ The ekctrochemicaliy induced changes in the paramagnetic properties and ESR spectrum of aspergiflin (0-S m&m?_ The SEESR analysis was carried out in veronal buffer (pH 5.6) with 0.1 M KC1 using mercury cathode. The amplitude of the ESR signal (A) is given in arbitrary units and the applied voltage in volts (V)_ The measurements were performed at room temperature (2z* r°C)_

Electrochemical

and

ESR

Studies

on

Melanins

157

in the course of a full electrochemical cycle (which comprises an increase of the applied voltage followed by its decrease) when looking at the behaviour of paramagnetic properties I Phase I_ - Consists of a moderate increase in ESR signal amplitude around -1.0 V which becomes very strong and increases rapidly at about -1-4 V, reaching a maximum near -1.8 V. The above values of potential difference at which the inflections of the curve occur pertain to a particular type of natural melanin (aspergillin) at a particular pH (8.6) and vary for other melanins or for other acidities of the solution_ Phase II. - This starts upon further change of the applied voltage up to about -z_o. and is characterized by a steep drop of the ESR absorption intensity_ Phase III. - This takes place when the applied voltage is changed A distinct rise in the ESR in the reverse direction, i_e_ toward zero_ signal is again evident although less strong than that seen in phase I. .;\Ioreover, its maximum point was at about -023 V_ This implies that the process does not follow the same pathway as in phase II when the voltage is returned stepwise to its original value at the start of experimentPhase IV. - This is characterized by a rapid drop of the ESR signal amplitude between -0-S and -0-5 V, the starting and end points having slightly different values_ The curve shown in Fig. 2 reveals a kind of hysteresis. The possible nature of this will be briefly discussed in the next part of this paper_ The course of each electrochemical cycle is much the same when Thus, four analodealing with melanins of similar origin and structure_ gous phases consisting of successive increase and decrease in the intensity of ESR absorption can be observed in the case of native bovine eyes and hamster melanoma pigments_ The curves differ only in the intensity of the electrochemically induced changes of the ESR signals and in the value of the applied voltage at which the maxima and minima occurThis is evident upon comparing the diagrams shown in Figs 2 and 3_ Both curves illustrate the behaviour of the same type of melanin (aspergillin one of the native and most comples forms of this pi,ment). The other hinds of examined melanins derived from bovine eyes or hamster melanoma do not differ significantly in their response to SEESR treatment except that the electrochemically induced changes in their ESR spectra are much less pronounced than for aspergiilin.

The electrochemical and ESR data concerning two types of synthetic melanins I catechol-melanin (at pH 7-o). and DOPA-melanin (at pH S-6) are presented in Fig_ 4 and s_ There is a close resemblance in the behaviour of their ESR absorption when the applied voltage is changed from o to -2.0 V and back again. However, one also notices that the relative changes in ESR signal intensity are not so high as for

Lukiewicz.

Reszka

and Hatuszak

240

A

- 200 - 160 P

+I

- 120

‘e

- a0

it I \ O\

-40

\

O‘Q Q %+-O-Q--_-o Uvs. S.c.E_
-2.0

_0

I 0

Fig_ 3_ The changes in the param agnetic properties and ESR spectrum of aspergillin (0-S m&m? The sample was dissolved in veronal induced electrochemicaily at the mercury cathode_ measurements buffer (pH - 7-o) with 0-1 M KCI_ Otherwise the conditions of the SEESR and the adopted denotations were the same as in the diagram shown in Fig z.

aspergillin, and that the parts of the curve corresponding to phases II and III are present whiie phases I and IV, mentioned in the preceding section, are missing_ The electrochemically induced alterations are enhanced in strongly alkaline solutions (cf. Fig_ 6) but the bi-phasic course of the process remains unchanged-40 -32 P

0P-+y-Q/

‘“b

i

-6 -o-o-o

d e-o-

24

-

.i;

we_

0

:q-,z;

- ‘6 -3

-8

,5-w-u Its_ SCECV)

-2_O

-LO

-1.5

The eketrochemically

-OS

0

0

induced changes in the content of unpaired dectrons and amplitude catechol-melanin sample (1.0 m&m‘) in veronal buffer (pH - 7-o) with 0.1 M KCL The e_xperimentrrl conditions and denotations are otherwise identical as in the case of SEESR data presented in Fig_ 2. of the ESR

siD4

of

a

Electrochemical

The diagram (1.1 mg[cmx) 0-r

_I1 KCI.

and

ESR

Studies

illustrating the results of the SEESR at the mercuccathode carried out For denotations see Fig. z_

on

Jtelanins

159

analysis of a DOPA-melanin sample \-ero~~al buffer (pH - S.6) with

using

Attention should be drawn at this point to a feature characteristic In all these examples (and at to all the curves presented in Fig. +-6_ the other pH values not included because of the limited space) one does not observe any increase of ESR absorption intensity when the applied The significance of this voltage is char&g from -1.5 toward -2-o V. finding will be discussed below_

at the p L a t i

IL IL 41s

eLectrode

The measurement of the ESR spectra and SEESR experiments with the use of a platinum electrode were carried out in a similar way to those described in Sections I and 2 and were done with the same types of melanins over a wide range of pH valuesThe results of these SEESR study were aualogous to the data previously established with mercury cathode. These data therefore also demonstrate that electrochemical

treatment brings about distinct changes in the ESR spectra and that

these changes depend on the type of -melanin aud pH of the solution_ However, in contrast to the use of the mercury electrode, the platinum electrode made it possible to electrochemically induce alterations in the paramagnetism of samples at much lower values of the voltage (ranging between -0.6 to fo.6 V)_

Eu?siewicz.

160

Reszka

and

Matuszak

-80 -64 oA++-.+

i rs

-32

8-“” fl

H-f

d-9

- 1s (/vs. -15

-20J Fig_

-1.0I

scE.w -0s

0,

O

6_

The diagram showing the dependence of the paramagnetic properties and ESR signal intensity of the DOPA-melanin sample (1-1 m&m?) on the E3IIF at the mercury electrode_ The SEESR analysis was carried out at room temperature using KOH (023 31). The denotations are the same as in Fig_ z_

The examples of SEESR analysis performed in a strongly alkaline environment (0-S M KOH) are especially interesting because such a high pH tends to shift the redos equilibrium of melanin oxidizab!e groups toward an increased amount of forms at the one-electron state of reduction- Thus, the changes in the param agnetic properties of the polymer may be quite different under these conditions depending on the type and structure of the melanin. For instance aspergillin demonstrates a rise of ESR absorption intensity not only during electroreduction but also upon electrooxidation (cf_ Fig. 7). which is not the case with synthetic melanins. As one can see from Fig_ S and g. both the catecholmeknin and DOPA-melanin reveal a drop of their ESR signal amplitude This would indicate that the same type of during eIectrooxidation_ melanin may behave either as an electron donor or electron acceptor in different environmentai conditions (camp_ also Fig. 4. 5 and 6).

The changes found in the paramagnetic propertk of melanins upon electrochemical oxidation/reduction are well pronounced. especially One may question whether they resulted from in the case of aspergillin. direct interactions of melanin with the electrode or from the chemical In parreactions of melanin with some secondary electrolytic products_ ticular, the latter possibility shouid be considered in those iristauces where the applied voltage was high (-1-5 to -LO V), and could cause

Electrochemical

and

ESR

Studies

on

161

BIelanins

-48

-06 Fig_ The

I -02

-04

7_ changes

t 0.2

0

in the

ESR

spectrum

t

r

0.4

0.6

and

content

of

unpaired

electrons

in the

aspergillin

sample (0-S mgjcms). induced electrochemically at a platinum electrode in 0-S N The esperimcntal conditions and denotations are given in the description of Fig

KOHo.

electrolytic decomposition of water. For example a local increase of pH at the surface of the mercury cathode could account for the increase in the ESR signal amplitude of melanins. the paramagnetic properties of which have been shown to be pH-dependent [IS, rg]_ Thus, an eventual rise of the DH-value near the electrode should be associated with a corresponding &crease in the intensity of ESR signals of melanin.

-0.6

-04

-02

0

02

04

0.6

Fig. 5. The electrochemically induced changes in the paramagnetic properties and ESR spectrum observed at the platinum electrode using of a catechol-melanin sample (LO mg/cm=). The adopted o.S M KOH. The measurements were carried out at room temperaturedenotations are the same as in Fig. z.

16-z

3Lukiewicz. Reszka and Natuszak

Fig_ Q_ The dependence of the panmagnetism and ESR signal intensit>(-4-1) of a DOPX-mel;min ~amplc (I.? m&m=) dissolved in 0-S -III KOH OII the E_lLF at the pl;rtir;um electrode_ The conditions of the SEESrZ analysis and denotations as in Fig. =_

However, this interpretation does not seem satisfactory for the following reason.5 Frrst, such an assumption is not consistent with data for ali the The above-mentioned electrochemically induced svstems investigated. &crease in ESR signals was found for natural melanins (in particular aspergillin) but not for synthetic melanins. Esamination of the latter materials revealed. within the same range of voltages. a decrease in the ESR absorption, (cf_ Fig_ +6)_ This behaviour would not be consistent with an explanation attributing the observed changes in the paramagnetic properties to the interaction of melanin with secondary products at the ions), since an increase in ESR mercury cathode (e.g., with hydroxide signal amplitude with rising pH is one of the features characteristic of all types of mehtnins [3_oi_ Thus, buffering of the solution was probably sufficient to suppress local deviations of the pH near the electrode, since a distinct drop in the ESR signal intensity was observed with synthetic melanins_ The second reason for rejecting the above interpretation is that electrochemicahy-induced alterations of paramagnetic properties could ako be seen at much lower values of the apphed voltage (between -0.6 and to-6 V) if platinum electrodes were used. These values are outside the range of potential differences at which water is electrolytically decomposing-

Electrochemical

and

ESR

Studies

on ~Ieianins

1‘53

On these grounds one must conclude that melanins are eIectrochemically active and that the observed paramagnetic effects are due to their direct interactions with the electrodes_ The efficiency of electroreduction and electroo-xidation of melanin This finding is easily understandable was shown to be pH-dependent_ considering that at least some part of the redox and paramagnetic properties of these polymers is determined by quinones and hydroquinones. It is known that the redo-u equilibria of quinone-hydroquinone solutions are modified by pH changes [zI]. Thus, the four phases in the course of the electrochemical-ESR cycle, defined in the preceding part of this paper, are likely to correspond to successive steps of reduction and o_xidation of an organic substance, according to the ~fICHAELIS concepts [zz]_ In particular I Phase I. - One-electron reduction of an acceptor (R), i.e. the transfer of a first electron from the electrode, leading to the formation of an anion-radical (R’) : R+e-+R’ (I) Such a process should be associated with an increase in free radical concentration and ESR signal intensity: Phase -11. - Transfer of the second electron leading to reduction of the anion-radical (RT) to the fully reduced product (R”-) I R’

fe-+R’-

(11)

This interaction is associated with a decrease in both free radical concentration and ESR absorption_ Phase III. - _A reversal of the previous phase, i.e. a one-electron oxidation of the reduced acceptor (R”-) : R2--e-_tR-

(III)

Here again a rise in anion-radical content and in ESR signal should occur_ Phase IV. - A second oxidation step restoring a fully o_xidized diamagnetic form of the substance (R) I R--e--+R

(IV)

The above reaction will result in a drop of both free radical intermediates and paramagnetism of the sample. Phases I and IV may be absent if the substance is chemically reduced prior to SEESR analysis as in the case of melanins in strongly alkaline solutions (cf. e-g_ Fig_ @)_ The electrochemically induced ESR changes discussed are most probably determined not only by the quinoid and hydroquinoid components of melanin structure but also by other double-bonded components. In the iight of some recent findings [23] this seems to be especially likely when platinum electrodes are used_ However, it would be quite

Eukiewicz. Reszka and Xatuszak

164

difiicdt at present to estimate, on the basis of the hitherto accumulated data,_ the contribution of different oxidizable sites in melanin to the overall observed effect. To answer this sort of question, separate series of SEESR studies using melauins with a chemically modified structure would be needed, It is evident from the above discussion that the experimental data presented in this paper are in good agreement with the MICHAJZLISconcept of reversibIe two-step oxidation and reduction of organic substances_ Thus, these findings appear to represent a direct experimental proof of a single-electron mechanism of electroreduction and electrooxidation of meianius. As was pointed out in the introduction, it is surprising that SEESR measurements have not previously been appIied in the field of melanin research_ One of the possible reasons for this is the insolubility of melauins iu almost all solvents_ The discovery of an easily water-soluble aspergillin [IZ, 241 and identification of its paramagnetism and relationship with melanin pigments [12,34.25] led to the SEESR study of Nevertheless, technical difficulties associated this group of polymers_ with this kind of measurements remain_ They are mainly due to au inconvenient geometry of commercially avaiiable conventional, flat eIectrochemical cells adapted for use with YlX& rectangular cavities_ The main problem with such a system is the very limited area of contact between the Sample under examination and the surface of the working electrode_ The observations are tim-onsuming since it takes a comparatively In future investigations long time for the system to come to equilibrium_ of this kind it would therefore be desirable to design electrochemical cells with a more favourable geometry_ However, in spite of these limitations the method of SEESR analysis has proved to be a useful approach to the study of redox properties of melanins and has provided some new evidence of a high electronexchange capacity for these polymers.

Acknowhxigements One of the authors (S-L.) is greatly indebted to Professor JAMES S_ HYDE (National Biomedical ESR Center, Medical College of Wisconsin) for suggesting ways of improving the geometry of electrochemical cells in future investigations, and is very grateful to Professor ROGER C_ SEALY for a stimulating discussion and linguistic correction of the manuscript_

References [I]

-AH_

MAKI

and

[z] F_H_J_ FICGE. 131 R_C_

SEALI-.

D-H_ GESKE. PYOC_ Sot. Exp.

C.C.

FELIS.

J_ Chenr. Phys_ BioZ. iWed_. 41

J_S_ HYDE

and

30

HAL

1356

(195~)

(1939)

127

SWVARTZ.

in

&ee

RadicaZs

Eiectrochemical in Biology. (in

WA_

PRYOR

and

ESR

(Editor).

Studies

Academic

165

BIeIanins

Press, New York (1979)

TATHACHARL

and

BLS_

BLOIS.

BiopJzys_

[s] S_ E~KIEWICZ. FoZia Hislochem_ Cyfochern_ Unique Properties of EsJ V. RILEY (Editor),

(1976)

[7] E-V_

VolGAS.

(1976)

H-F_

HXBERMIAN

666

E-V_ G-AN. K-31_ Lax,

[g]

96 (1977) 15 IA_ MESOS. S-L. LEU

J_

9 (1969)

77

19 (x972) g3 MeZanocytes. S.

h-arger.

and

A.

JLENox.

H-F_ H_A~~ERM_AX and and

H-F.

HABERMAX,

&C/J_ I__&

Biocherw_

Xew- York (1965) [I=] R-A_ XICOL_AUS. MeZanins. [14]

J-I_

RESZKA.

Pia.

D.

PL~MER

and

M.J.

Academic

Base1

XESOS.

Biophys_

Br_

Can_ J_ Biochewr_

J_

Thesis.

Press, Xew York

[IS] 2. X*T~SZ_AK. M_ SC_ [16] CC. FELIX. J.S. HYDE.

in Pigment

Cell Growth.

M.

753

Iog

John

Herman. Paris (rg6S) p_ 130 Ja~ellonian University. Krak&v

KOPAC.

173

DermatoL

55 (1977)

I.A. MENOS and H-F_ HABERMAN. BY_ J_ DernzafoZ. 97 (Ig77j H-G_ CASSID~ and K.X. Kus. Oxidation Reduction Poiynzers.

K.

IV

3

[S]

[13]

Vol

print)

[4] Y-T.

[IO] [II]

on

G~RDOS

\Viley.

(1979) (Editor),

(1933) p_ 305

T/zesis. Jaf$ellonian University. Krak6w (1979) T. SARSA and R.C. SEAL\-, /_ Am. Chem. SOG.

100

[I?] [IS] [zg]

('97s) 3921 M. PRZECALISSKI. F_ J_ GRADY and MS. BLOIS. A.B.

Fzo] [ZI] FE] [z3]

T. SARKA. Ph. D. Thesis. Jagie1lonia.n University, Krak&v (1974) G_ TOLLIS and C. STEELISK. Biochinz. Biophys. Ada 112 (1966) 377 L. ~tICHAELIS. Cl,em. Rev_ 16 (1935) 450 AitR (Pubi_) Jfoscow (1977) p_ +I L-I_ ALTROPOV, TheoreticaZ Chemistry,

[z_r] S-S_ (1970)

[q]

ZDASOVA 53

K. RESZI~A and Ramis 75 j75

UJLCS University of Lublin (1976) Ph. D. Thesis. D-C. BORG. J_ Am. Chenr_ SOL 90 (196s) zgag ZAHLAS and J-E_ I\Lurxc. BiophysJ_ 4 (1964) 471

and

1V.D.

POCHODESKO.

S_ P_.J_AP. conf_

Izc.

Radio-Microwu-ze

Akad.

Xazrk

Specie_.

SSR Poznai

Biol.

1

(1975)