Vol. 84, No. 2, 1978 September

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

29, 1978

Pages

MELANIN PHOTOREACTIONS IN AERATED MEDIA:

335-341

ELECTRON SPIN RESONANCE

EVIDENCE FOR PRODUCTION OF SUPEROXIDE AND HYDROGEN PEROXIDE C. C. Felix,

J. S. Hyde,

T. Sarna+

and R. C. Sealy

National Biomedical ESR Center, Department of Radiology, Wisconsin, 8700 W. Wisconsin Avenue, Milwaukee, Wisconsin

August

Received

Medical 53226

College

of

3,1978

Summary. Electron spin resonance measurements on aerated melanin suspensions during photoirradiation show changes in the microwave saturation of melanin free radicals and formation of adducts in the presence of spin traps. These observations indicate that oxygen is reduced to superoxide and hydrogen peroxide. Introduction.

Melanins

in man is believed their

containing

evidence

for

in vivo

free

their

systems,

for

oxygen

both

-tPermanent Department

resonance

are produced of these

a triplet

changes

intrinsic

free

light-induced)

dark

radicals obtained

We here

in the presence

which

2

However,

is

essential

a sound if

with

of light.

amplitude

and the mixture irradiation,

evidence

of light,

for

causing

(i)

light;

we have

and adduced understanding

one is

to model

melanins

is

poorly

A slow

uptake

of

3 and Copeet with

oxygen

of radicals but rapid

an increase

address: Institute of Molecular Biology, Jagiellonian of Biophysics, Grodzka 53, Rrakgw, Poland.

ESR - Electron Spin Resonance, Abbreviations: alanine, DMPO - 5,5'-dimethyl-1-pyrroline-l-oxide,

Addi-

with media

by serve

investigations.

has been reported,

during

provide

g-semiquinones)

of oxygen

of ESR signal

function

in part,

in deaerated

the presence

primary

characterized,

(ESR)

radicals

systems

in the

whose

upon irradiation

precursor.

and in

suspensions

are unclear. by melanin

spin

on irradiated

reversible

plus

involved

electron

in the dark

by melanin

have described

radicals'(probably

radicals

having

pigments

They are

and the mode of interaction

both

intrinsic

for

of oxygen

understood,

tration

free

the ESR spectra

of the effect

oxygen

probe

transient

characterized

of biological

to be photoprotection. persistent

as a convenient tional,

form a class

the

al, 4

concen(i.e.

processes

scavenging

of

in the microUniversity,

Dopa - B-dihydroxyphenyl SOD - Superoxide dismutase. 0006-291X/78/0842-0335$01.00/0

335

Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 84, No. 2, 1978

wave saturation reduction

BIOCHEMICAL

AND BIOPHYSICAL

of the ESR resonances

of the

scavenged

oxygen

of the free

to hydrogen

way of an initial

one-electron

findings

important

implications

may'

an oxygen

may have

pigment that

darkening

hydrogen

peroxide

reduction

have is

involved

RESEARCH COMMUNICATIONS

radicals

peroxide,

to yield in melanin requirement,

and for at least

(ii) in part

superoxide.

These

biology:

immediate

and there

is

by

a possibility6

in melanogenesis.

Methods. Experiments described were carried out with samples of synthetic melanin from DL-$-dihydroxyphenyl alanine (DL-Dopa), prepared as previously described.' However, experiments with natural melanin from bovine eye8 gave similar results. Melanin aqueous suspensions (ca. 10 mg/ml) at ambient temperature were irradiated with filtered light of 320-600 nm from an Eimac VIX-3OOUV 300 W Xenon lamp in the cavity of a Varian E-109 spectrometer. Deoxygenation was achieved by freeze-pump-thaw cycles on a vacuum line. Results

and Discussion.

on the

extent

presence

This 1, where

absence

of saturation,

of the microwave all

readily

samples. than

absence iment oxygen samples

samples these

resulted, signals

deoxygenated,

concentration.)

signal

amplitudes

dissolved

ESR signals were

observed

We believe

in the medium,

within

that since

in microwave

radicals

to the same levels

were over

rapid

the

time

light-induced

whereas

photolysis

irradiation

few minutes, found

336

for

behavior

saturates

changes

observed,

these

saturation

or the a very

time

square is

found

much more

oxygen.

samples,

with

In the to the

0.1 mW, this sample

state

microwave

be proportional

containing

of oxygen-containing

a ground

and

of oxygen

below

the

air-saturated

independent

and at powers

upon

radicals

were

should

depend

melanin

of incident

samples

gave no change

intrinsic

as a function

no changes

free

the

the deoxygenated

(up to 1 hour).

the intrinsic

for for

dependent

such as oxygen,

powers

of the melanin

depletion

in turn

At higher

those

of light

is

amplitudes

shown

power,

Upon photolysis ration

demonstrated

are

ESR signal

species

amplitudes

suspensions

(ESR lineshapes

for

is

that

which

paramagnetic

ESR signal

power.

known

saturation,

phenomenon

oxygen-saturated

root

is well

of microwave

of fast-relaxing

triplet. in Fig.

It

whereas period

exper-

reflect

of deoxygenated

photolysis

in an increase deoxygenated

in the

of the

changes

of the ESR signals mixture,

in satu-

in

samples.

from

either

of aerated saturation (Data

of

BIOCHEMICAL

Vol. 84, No. 2, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(Microwave

Power fh”*

amplitude of the intrinsic free radical in: Fig. 1. ESR signal (a) oxygensaturated (m); (b) air-saturated (0); and (c) deoxygenated (A) aqueous suspensions of synthetic melanin as a function of microwave power.

obtained

at low microwave

radical

concentration

mixture

microwave

power)

(see Table) saturated all MS

rate -1

>.

diated

the change

recorded

have measured

under

the

time

conditions

this

time than

of oxygen

removal,

rate, (ca.

removal

of transient

M s-l).

The Table

of

required

solutions

portion

that

in amplitude

as a function

that

This

established

no detectable

change

in

of the

irradia-

was occurring.)

By monitoring tion

power

which

after

time

approximately

A[02]/At, may be limited

radicals shows

melanin times

solutions, is

sample

microwave

the onset

five

air-saturated

50%) of the

also

of extreme

to deoxygenate

is

for

of the ESR spectrum

approximately by oxygen

volume,

by self-reaction the effect

337

saturation

(200 mW

of irradiation,

we

suspensions

and find

greater

oxygen-

for

indicating

thatthe

2 x 10 -6

the same (ea. from

the unirra-

is much lower

than

the

(from

> 2 x 10

of catalase

diffusion

over-

Ref.

2,

and superoxide

rate -4

dismutase

of

vol. 84, No. 2, i 978

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE Nelanin

Suspension

Deoxygenation

Air saturated Air saturated + superoxide dismutase Air saturated + catalase Air saturated + catalase + superoxide dismutase Oxygen saturated

time/mina

106

1.9 t 0.1

2.2

+ 0.1

2.1 i: 0.1

2.0

f 0.1

3.1

2 0.1

1.34

f 0.05

3.2 2 0.1 11.4 2 0.5

1.30 2.0

? 0.05 ? 0.1

aTime required for 95% change of the difference between tp original ESR signal amplitude and that for the deoxygenated sample. Average rates of oxygen removal calculated from deoxygenation times assuming that airand oxygen-saturated suspensions have oxygen concentrations close to those for water, viz. 0.25 and 1.4 mM respectively.

on the rate

of oxygen

hydrogen

peroxide

Catalase

(360 ug/ml)

oxide

has a much smaller

is

also

its

the rate

Van Woert') affect.

enzymes

disproportionation

20' 2 To demonstrate have used novel

evidence

method

is

scavenger persistent the

the method

based

upon

2H+ r

production trapping'

superoxide

nitroxide

spin-adduct,

The reaction

between

hydrogen

which

(120

ug/ml)

with

superoxide

the absence

whose

338

(2)

+ '2 melanin

irradiation,

has recently

provided

biological free

ESR spectrum

radical

is

we 10,ll

systems.

a relatively

characteristic

HO; 12)

The

to a radical

(DMPO) to give

DMPO and 0; (&d/or

(3).

of enzyme

(1)

in other

of a transient

per-

peroxide.

'2'2

during

generation

the addition

with

consistent in

of

and (2).

H20 + 5i02

such as 5,5'-dimethylpyrroline-l-oxide

addend.

equation

for

of spin

is

and hydrogen

superoxide dismutase,

superoxide

(1)

dismutase

mode of disappearance

catalase H2°2 -

destruction

consistent

superoxide

the latter

to oxygen

the

reactions

by 40X,

whereas

However,

predominant

catalyze

respectively,

decreases

(cf.

if

These

and superoxide

formation

production

removal.

is

of

shown in

Vol. 84, No. 2, 1978

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

.

..

;;

a

Fig. 2. ESR Spectra of free radicals detected during photoirradiation of an aqueous suspension of synthetic melanin in the presence of the spin trap DMF'O. Lines marked A are attributed to the adduct of superoxide with DMPO. The melanin resonance (W) is off-scale in this display. Owing to loss of spin adduct following oxygen depletion (rapid under these conditions) the spectra are composites from several samples scanned over adjoining magnetic field intervals. (a) without superoxide dismutase; (b) with 120 rig/ml superoxide dismutase (only part of this spectrum is shown).

Fig. tions

2(a)

0;+ >rar+ H+_ d&,H I o- obtained from irradiated O* shows spectra

of melanins

and an impurity superoxide

containing

M DMPO.

in the DMPO (O),

spin-adduct

are clearly

of an authentic

01 adduct

spectrum

(Fig.

2(b))

when superoxide

dismutase

is

reduction

concluded to superoxide

superoxide

that

adduct

and thence

339

from melanin attributable that

their

positions

11 and they in

the reaction

of oxygen to hydrogen

are not

solu(m) to the

confident

since

included

reduction

(4) - (5)).

(A)

We are

those

electron

the

visible.

match

therefore

from

lines

resonances

A

is

indeed

other

Besides

marked

It

are

0.15

(3) oxygen-saturated

the lines exactly present medium.

proceeds

by a one-

peroxide

(reactions

Vol. 84, No. 2, 1978

BIOCHEMICAL

e O2

AND BIOPHYSICAL

l

of the one-electron

However,

although

ESR-detectable electron

donation

on the polymer semiquinones reflect That

low

ities.

Despite

melanins

of these

important

consequences

scavenging

bleach15 reductions

are

does free

such a dependence

raises

intriguing

may prove

modify are

although

16

also

of may well

reduction

damaging 14 species

by melanin

that

formed

reaction

experimental

biology

of

seem likely

radicals

our

peroxide

melanin

of the concentration

of one-electron

under

established.

reduction.

peroxide

yield

in melanin

chemically

the

capable

overall

of hydrogen

by irradiated

for

been firmly

reversible

to detect

and hydrogen

potentially

or otherwise

Acknowledgements. Grant 5-4PI-RR01008

the well-documented

yield

production

slow

photoinduced

overallquantum

yet

a dependence

transient

(cf.13

low

has not

concentratioqit

Our failure

their

(5)

on oxygen the

superoxide

o2 + H202

found

oxygen).

irradiated

to give

from

surface

(4)

reductant

not yet

radicals is

with

the

oxygen

we have free

0;

+

20; 2H The identity

RESEARCH COMMUNICATIONS

there (high

is

possibilconditions, to have

evidence

concentrations

the pigment).

of

Other

for

a

can

one-electron

to be expected.

This work was supported by National and by National Science Foundation

Institutes of Health Grant PCM 76-14831.

References. 1. Commoner, B., Townsend, J., and Pake, G. W. (1954) Nature, 176, 689-691. 2. Felix, C. C., Hyde, J. S., and Sealy, R. C. submitted for publication. 3. Van Woert, M. H. (1968) Proc. Sot. Exp. Biol. Med., 2, 165-171. 4. Cope, F. W., Sever, R. J., and Polis, B. D. (1963) Arch. Biochem. Biophys., 100, 171-177. Pathak, M. A. (1968).Advanees in Biology of Skin, 8, 397-420. 5. E.g., R. P., Okun, M. R., Edelstein, L. M., and Cariglia, N. (1974) 6. Patel, J. Invest. Dermatol., 63, 374-377. 7. Felix, C. C., Hyde, J. S., Sarna, T., and Sealy, R. C. (1978) J. Am. Chem. Sot., 100, 3922-3926. 8. Plumer, J. I., and Kopac, M. J., in Pigment Cell Growth (edit. by Gordon, M.) (Academic Press, New York, 1953), p. 305. 9. Janzen, E. G. (1971) Accounts Chem. Res., 5, 31-40. J. R. (1974) Can. J. Chem., 52, 10. Harbour, J. R., Chew, V., and Bolton 3549-3553. Sealy, R. C., Swartz, H. M., and Olive, P. L. (1978) Biochem. Biophys. 11. Res. Comm., 82, 680-684. J. R. (1975) Biochem. Biophys. Res. Comm., 12. Harbour, J. R., and Bolton, 64, 803-807. R. L. (1973) J. C. S. Faraday I, 69, 13. E.g., Patel, K. B., and Willson, 814-825.

340

Vol. 84, No. 2, 1978

14. 15. 16.

Fridovich, 239-277 Sacchi, Skin, 2, Chauffe, 565-572.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in Biology (edit. by Pryor, W. A.), A, I., in Free Radicals (Academic Press, New York, 1976). S., Lanzi, G., and Zannotti, L. (1969) Advances in Biology of 169-175. L., Windle, J. J., and Friedman, M. (1975) Biophys. J., 15,

341