Vol. 90, No. 4, 1979

AND BIOPHYSICAL

BIOCHEMICAL

RESEARCH COMMUNICATIONS Pages

29, 1979

October

1207-1213

'MJ3 EFFECT OF X-RAYS oi\l PHOYER~WLS OF SUPEROXIDE DISLIUTASE Li.A.Symonyan, Institute Sciences, Received

July

R.LNalbandyan

of Biochemistry,

Academy of

Armenian SSR. Yerevan

375044,

USSR

11,1979

Summary Cuprozinc superoxide dismutase was isolated from liver of rats irradiated witn lethal doses of X-rays. In contrast to tne enzyme isolated from normal animals, superoxide dismutase from irradiated rats was found to be in reduced inactive state. Under aerobic conditions the reduced enzyme is oxidized spontaneously during a week resulting in preparations with properties of the normal enzyme. The irradiation by X-rays of superoxide dismutase isolated from normal animals also led to reduction of tne enzyme and decrease in its activity. The enzyme reduced by irradiation is again oxidized during storage under aerobic conditions. Introduction Recent investigations tne injecting into

cuprozinc

animals

lethal

before

effects

ministration

of Petkau et al. superoxide

exposing

of ionizing of additional

radiation.

nary studies

that

than before the effect we report of rats

the irradiation. of the irradiation on some properties

irradiated

(SOD) intravenously protects

X-irradiation

of the lethal

These results

effect

of cuprozinc

questions

about

In the present

study

as tne effect 0006-291X/79/201

1207

of SOD was ratner

SOD isolated

Copyright All rights

ad-

after

raised

on SOD itself.

that

In prelimi-

were made immediately

with X-rays , as well

them from the of the ani-

effect.

the radioprotective

when injections

have shown that

It was also stated

enzyme after

decrease

more pronounced

dismutase

triem to X-rays

mals leads to further we found

/1,2/

from liver of X-rays

on

207-07$01.00/O

@ I979 by Academic Press, Inc. of reproduction in anyform reserved.

Vol. 90, No. 4, 1979

SOD isolated sults

from normal

obtained

of SOD in vitro connected was

with

at least

Materials

BIOCHEMICAL

clearly brings

animals indicate

(in vitro that

RESEARCH COMMUNICATIONS

experizients).

the irradiation

about an inactivation

a reduction partially

AND BIOPHYSICAL

of enzpic

copper.

The re-

of animals

or

of the enzyme tnat

is

This inactivation

reversible.

and methods

SOD frown bovine and rat erythrocytes was isolated essentially according to I&Cord and Bridovich /3/. The preparation was subsequently cilroma-tograpned twice on DEAE-Sephadex A-53. Central fractions obtained after the second chroaatoF;raphy had a spectral inwas observed on heating dex, A263/A6831 of 25-26. iJo turbidity this preparation at GOOCfor 10 min. The enzyme activity was deterinined according to Misra and kkidovich /4/ or Risnikixi et al. /5/. SOD from liver of normal or irradia-ted rats was prepared by the metnod of Reiss and Gersnon /6/ rnodifiea by introduction of a DEAE-Sephadex A-50 chromatography step to obtain preparations with the above mentioned criteria of purity. Tne isolation procedure was carried out within 4%~ The irradiation of Wistar line rats was performed using a therapeutic X-rays instrument "RUM-11" (USSR). Tne X-ray beam was filtered with 3.5 mm Cu +l m;ll Al. The total dosage was 850 roentgens, the dose power being 30 roentgens/min. Tne lethality 5 days after irradiation by this dosage was 4%506. After irradiation the rats were killed and their olood anu livers were collected to isolate SOD by the same method that was used for normal blood and liver. X-ray irradiation of SOD dissolved in 0.01 id1phosphate buffer was "HJP-203/20" (USSR) at a dose power of conducted in an instrument 20.103 radians/min for 30 min. During irradiation the protein solution was maintained at 18'C. Analytical electropnoresis in 7.5$ polyacrylamide gels was carried out according to Davis /7/. Electrophoretograins were coloured with Amide Black to localize protein zones; positions of SOD active zones were determined in gels of parallel runs according to Nishikini et al. /5/. Molecular weigilt was determined by the lnethod of Weber and Osborn /8/. The metal content was deterrained by atoinic absorption("AAS-1: GDR).

1208

Vol. 90, No. 4, 1979

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Optical spectra were obtained on a "Specord" instrument (GDR) EPR spectra were recorded temperature in 10 rm~~cells. at room on a 'Yarian E-4" instrument operating at -16OOC; modulation amplitude, 6.3 gauss; microwave frequency, 9.12 Ghz and microwave power, 10 mW. Results Preparations by the tra

of

same method

preparations.

these

tee-ted

for

normal

ted

from animals

storage rats

or optical

SOD from

preparation

similar

weights.

no EPR spectrum

after

have

and molecular

between

SOD isolated

became

and

are

In

contrast

for to

0

Fig-l.

Changes in properties spectra, B-optical ted from normal rat to Nishikimi et al. of rats irradiated 48h after irradiation. storage at 6OC for

SOD from

the

region activity

than

properties

2 4 (SOD).

that

animals, was deof the

of

however, SOD from

SOD. Fig.1

10 -“9 M

however,

normal

visible

We found,

of normal

UV-spec-

differences,

was lower

a week the

rats

contents,

The specific

of magnitude*

those

zinc

to in

irradiated

and

certain

rats. animals

by 2 orders

similar

copper

There

irradiated

5-10°C

normal

absorption

irradiated

at

from

SOD from that irradia-

shows diffe-

8

of SOD after irradiation of rats. A-EPR spectra and C-activities. I-Enzyme isolalivers; activity was measured according /4/s II-Enzyme isolated from the liver by 850 roentgens. Livers were collected III-The preceding preparation after 6 days.

1209

Vol. 90, No. 4, 1979

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH CO!vVWNlCATlONS

,

p.0 Ld’

I)

l

-0-o :O /O -/O

0.0

0

2

2

4

Fig.2.

2

-0

8

w

Jd

4

6

10

8

12

mm). lo-%

intensity of the protein from irradiated animals during storage at 6'C. ( )-data for irradiated rats, ( )-data for unirradiated rats (controls).

Changes in EF’R signal

Pig.3. Changes in properties of SODof bovine erythrocytes after irradiation of the enzyme in vitro and during storage of the irradiated SOD. The enzyme was irradiated at 20.103 radians/miz and stored at 6OC. A-SOD activity. (A)-unirradiated enzyme, (Al-after irradiation, (0 j-storage of the irradiated preparation at 6% for 48h. B-EPR si@;nal intensity (arbitrary units). rences in the EPR and optical SOD between

irradiated

spectra

and non-irradiated

so the loss of these differences preparation.

The kinetics

SOD in the course in Pig.2.

of storage

and irradiated

animals

liver

just

tely

after

irradiation,

not only during The in vitro rats

or bovine

in the activity

rats.

after

storage

of the appearance

No differences

extracts

as well

of the EPR signal preparation

in electrophoretograms

before

irradiation erythrocytes

The decrease that

in activity

it

as

of SOD isolated was found 1210

of

is shown

to bring

of crude

than immedia-

the inactivation

but after

al-

of SOD from normal

death was more pronounced

indicating irradiation

demonstrates

of the irradiated

of tne irradiated

were found.

It

of

proceeds

well.

from livers

of normal

about the bleaching

Vol. 90, No. 4, 1979

BIOCHEMICAL

of the protein as well

and a decrease

as a decrease

such as the content unchanged. before

in its

addition

The

RESEARCH COMMUNICATIONS

in the intensity activity

of metals

irradiation

spectra

AND BIOPHYSICAL

of its

(r'ig.3A).

and molecular

of 10'3~J mannitol

did not prevent

EPR spectrum

Other properties

wei@t

were,

however,

to the enzyme solution

changes in the optical

and EPR

or in activity.

X-ray

irradiation

of SOD in vitro

ges in SOD as irradiation mals, like at least

of the animals.

the enzyme irradiated partially

clearly

SOD from irradiated

.in vitro,

in the course

causes tne same chanregained

its

under

aerobic

of storage

ani-

properties conditions.

These data are shown in Pig.313. Discussion The data presented at the irradiation the SOD rather sent it

here indicate

is probably

connected

than the destruction

is difficult

sible,

however,

vitro

and in vivo

peroxide tissues ties.

similar.

are involved

/lo/.

/9/.

by X-rays

There is evidence

that

scavenger

brain,

zing radiation

than bone marrow or spleen, low /ll/.

1211

proper-

superoxide

of radiation

of superoxide

letharadicals,

high levels

are known to be more resistant

It may be concluded

reductive

formed when

reductive

with

in

hydrogen

peroxides

Animal tissues

of SOD (liver, is relatively

plauof water

with

among these intermediates,

effect.

kidney)

irradiation

atomic hydrogen,

in the oxygen enhancement

radioprotective

and in vitro

of the enzyme irradiated

Organic

of

At pre-

seems quite

also have certain

SOD, which is a potent

has a clear

molecule.

in viva

The X-ray

electrons,

of SOD reduction

of many intermediates

radicals

are irradiated

radicals lity

and superoxide

chemical

This suggestion

is known to cause the formation such as solvated

with

reduction

because the properties were very

the inactivation

of the protein

to claim that

is caused by the same reductant.

properties

tnat

to ioni-

in which the SOD level from tnis

fact

and from

Vol. 90, No. 4, 1979

BIOCHEMICAL

the data obtained

here that

pends,

extent,

to a great

tive

intermediates

tion

of SOD that

interaction

formed

the radiation

resistance

on the catalytic

activity

during

irradiation.

and after

occurs when it

is irradiated

of the enzyme with

might be considered dismutation

superoxide

as the first

catalyzed

0;‘

t

to the generally oxidized

--c

accepted

by another

de-

of SOD on reducIf

is a result radicals,

the reducof the

then this

process

of the

i.e: cut1

o2 .

t

scheme of enzymatic

in the second stage of tne catalytic be again

of tissues

stage of the normal cycle

by SOD /12/, cu+*

According

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

cycle,

dismitation

the enzyme copper should

superoxide

radical

2H+ -b

CU+~ t

in the presence

of

protons: cut1

o;-

t

t

In terms of this

scheme tne reduction

reflect

that

the fact

protons,

with

be mainly

H202 is one of the possible

was found

by irreversible The thermal

the reduced

protein

oxidized

SOD was inactive

SOD without We suggest reduction

that

of SOD during

peroxidative

should

/14/.

aging.

in reduction,

Neverthless,

the aut-

spectra

The partial

differing

inactivation

properties

may be connected We believe

of the chemical

lead to a decrease

damage.

1212

expo-

of the en-

also that

reduction

of

was observed

the enzyme of young animals

such inactivation

of SOD as the result radiation

of SOD also results

macromolecular

as compared with

of

to cause enzyme inactivation

is autoxidizable.

protein

changes in its

in old animals

of SOD. Prolonged

and had EPR and optical

from tnose of the native

to

state.

changes in the FJ?R spectrum treatment

although

appears

and a deficiency

in the reduced

reductants

sure of SOD to H202, however, zyme /13/.

of SOD irradiated

excess of substrate

the enzyme should

accompanied

H202 .

wita

/15/.

the chemical

the inactivation

in the course of ir-

of the resistance

of cells

to

Vol. 90, No. 4, 1979

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

REFERENCES

1. Petkau, A., Chelack, W.S., Pleskach, S.D., Meeker, B.E. and Brady, C.M. (1975) Biochem.BiophysrRes.Commun. a, 886-893. 2. Petkau, A., Kelly, K., Chelack, W.S., Pleskach, S.D., Barefoot, C. and Meeker, B.E. (1975) Biochem.Biophys.Res.Com.mun. a, 1167-1174. (1969) J.Biol.Cnem. e, 6045-6055. 3. XcCord, J.M. and Fridovich,I. 4. &!isra, H.P. and Fridovicn, I. (1972) J.Biol.Cnem.x, 3170-3175. 5. Nishikimi, &, Rae, X.A. and Jagi, K. (1372) Biochem.Biophys.Res. Comnun. 46, 839-854. 6. Reiss, U. and Gershon, D. (1976) Europ. J. Biochem. 63, G17-623. 7. Davis, B.J. (1964) Ann. N.-Y. Acad.Sci. 121, 404-427. 8. Weber, I(. and Osborn, Id. (1369) J.Biol.Chem. s, 4406-4412. 9. Errera, X1. and Forssberg, A. eds. (1960) in $jIechanisms in Radiobiology, ~01.2, Academic press New York. 10. Misra, H.P. and Fridovich, I. (1976) Arch.Biochem.Biophys. a 577-581 and references therein. 11. Harts, J.M., Deutscn, H.F. and Funakosi, S. (19'73) Clin.Chem. Acta. 46, 125-132. 12. Fielden, E.M., Roberts, P.B., Bray, R.C., Lowe, D.J., Maunter, G.N., Rotilio, G. and Calabrese, L. (1974) Biochem. J. 13q, 49-60. 13. Bray, R.C., Cockle, S.A., Fielden, E.X., Roberts, P.B., Rotilio, G. and Calabrese, L. (1374) Biochem. J. 139, 43-43. and Balbandyan R.&l. (1375) Biochimia (Russ.) 4C, Symonyan, M.A. 14. 726-732.

15. Reiss,

U. and Gershon,

D. (1976)

255-262.

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