BIOCHEMICAL

Vol. 71, No. 4, 1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

THE EFl?!ECTOPOXYGENOE ALKALINE SOLUTIONS OF SUF'EROXIDE DISMUTASE Y.A.Symonyan and R.M.Ralbandyan Institute

of Biochemistry,

Academy of

Sciences Armenian SSR, Yerevan,375044 (USSR) Received

June

21,1976

SUMMARY Tne blowing of oxygen through strongly alkaline solutions of SOD leads to the drop of pH by more than 3 units. The rate of the process depends linearly on the concentration of SOD. The effect of oxygen on the modification of ttle shape and the decrease of the intensity of EF!R signal of SOD were observed. The incubation of strongly alkaline solutions of SOD under vacuum leads to the reduction of the protein copper. The data obtained suggest, that the reduced copper may be at least partially reoxidized by oxygen. It is suggested that at pH 12.5 and higher in the presence of SOD the reaction of electron transfer from hydroxyl anion to the oxygen takes place: .OH + O;OH- + 02INTRODUCTION Superoxide reaction

of dismutation

the reverse hydrogen

dismutases

process

peroxide

In the dismutation is important

of oxygen anion

of generation t

in the media of different has been shown that

modifications

as well radicals

as from

02 are involved

the properties

acidity.

and thus

of the enzyme

In the recent

work (3)

it

in weakly acid media the enzyme undergoes

a change in the environment tional

t

two protons

to investigate

the

oxygen (1,2):

2HteH202

process

radicals

of superoxide

and molecular

20;' it

(SOD) are known to catalyze

of the copper and some coaformatake place. On the other hand, the data

Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

1131

Vol. 71, No. 4, 1976

available perties

BIOCHEMICAL

(4,5)

indicate

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

that no essential

of the enzyme occur in alkaline

Moreover,

the obsarved

stability

solutions

up to pH 12.0.

changes in the EPR and optical

of SOD at pH 12.5 are still the remarkable

changes in the pro-

reversible.

stability

These facts

of SOD in the alkaline

of the protein

spectra

must have a certain

suggest

media.

Such

biological

sig-

nificance. In the present

communication

and vacuum on the properties pay attention strongly

we report

effects

of SOD alkaline

to a reaction

possibly

of oxygen

solutions

catalyzed

and

by SOD in

basic media.

MATERIAL AND METHODS es was obThe preparation of SOD from bOViP8 erythroc tained by the method of &Cord and Fridovich (6 3" and it was further purified as described in the work (7).The final preparation was electrophoretically homogeneous and had spectral purity index (A26dA6S0 > of 26. Concentrations of the protein were estimated by using molar extinction of 300 M-lcrn -' for the band at 680 nm. The apoprotein was prepared by the method of &Cord and Fridovich (6). The pH was adjusted by adding 0.5 M KOH to the protein solution in 0,005 Y phosphate buffer, pH 7.4. EPR spectra were recorded on a Varian E-4 instrument at -16OOC. Optical spectra were obtained at 2209 in 10 mm cells. The incubation of the protein under vacuum (10 mm of Hg) was lasted for several hoursRESULTS The aeration gen was found is clearly

of strongly

solutions

to lead the drop of their

observed

12.5 or higher.

basic

if

initial

In various

control

or in the presence

of the apoprotein,

tion

Also,

solutions

the following

oqgen.

containing

no proteins

no decrease

was bubbled

From obtained

conclusions

kinetic

may be drawn. 1132

was

when oxygen

no changes of pH of alkaline

were noted when the solution

but not with

solution

experiments,

alkaline

oxy-

PH. This phenomenon

pH of the protein

was blowed through was observed.

of SOD with

of their protein

pH solu-

with nitrogen,

curves

(Fig.

1)

Vol. 71, No. 4,1976

Fig.

BIOCHEMICAL

AND BIOPHYSICAL

1. Time course of pH drop at different concentrations of SOD at blowing of oxygen, The inserted part shows the dependence of the initial rate of pH drop on concentration of SOD. Oxygen pressure in all cases was 20 mm of Hg. x - data in the absence of the protein.

The final

decrease

in pH ( ApHN3.5)

does not depend on

the concentration

of SOD. Similar

results

pH of the protein

solution

aeration

adjusted

before

to 13.2. Whereas, after

no remarkable the solution gen leads

was 11.2.

In the second line

were incubated

solutions

under vacuum for

pH of SOD solutions decrease

observed

of the integral

in the course of incubation

the reduction

of the protein

copper.

1133

oxg-

the effect

was studied. initial

4 hours.

was 12.7 or higher intensity

that

as the cri-

and EPR spectra with

pH of

only when their

of experiments

as in optical

served when the protein

oxygen was

when the initial

of SOD solutions

when

hours of aeration

high with pH of 12.5 being

changes in pH as well

sive

with

to the drop of pH in SOD solutions

point.

initial

were obtained

Thus it has been established

vacuum on the properties

11.7

several

drop of pH was observed

pH were sufficiently tical

RESEARCH COMMUNICATIONS

of

lo were ob-

pH of 7.4 or However,

when

the progres-

of EPR spectra

was

under vacuum, indicating Thus, "self-reduction"

Vol. 71, No. 4, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of the copper took place when strongly SOD were subjected that

this

to vacuum-storation.

reduced protein

dized by the blowing was decreased The results

It has been found

might be, at least,

of oxygen through

of

partially

the solution

reoxiwhich pH

of these experiments

are shown in Fig.

2.

*oo

2. Changes in EPR (A) and optical (B) spectra of SOD (4.10-4&) in the course of oxygen blowing. 1- (-1 SOD was aerated at pH 7.4 during 4 hours. 2- (---I SOD immediately after the alkalysation to pH 12.70 sample 2 after 20 min aeration by oxygen. sample 2 after 90 min aeration by oxygen. EPR spectra were taken under the followconditions: frequency 9.38 GHs, power 10 mW, modulation amplitude 6.3 gauss, time constant 0.3 seem

Fig.

3 represents

before

and after

cluded

the short-time

of integral tial

solutions

from 12.7 to 9.3 in the course of the blowing.

a0

Fig.

basic

intensity

the optical

the aeration

intensity increase

blowing

and EPR spectra

of SOD

of the solutions.

As may be con-

of oxygen results

in 30% decrease

of the EPR spectrum and in the essenof the nitrogen

1134

superhyperfine

struc-

Vol.71,No.4,

Fig.

1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

3. Effect of vacuum on EPR spectra of SOD. l- SOD alkalyzetd to pH 12.7, 2- SOD 8lkRlyzed to pH 13.5, 3- sam18s 1 or 2 after incubation uILd8r vacuum for 4 hours PpH was not changed). 4- SOD at pH 7.4 after incubation under vacuum for x) hours (the shape and intensity were not changed). 5- sample 3 after aeration by oxygen for 5 hours, (pH became 9.3). Conditions of EPR spectroscopy were similar to those of Pig. 2.

ture.Although

mor8 prolonged

aeration

of th8 solution

results

in the EPR spectrum which have the shape characteristic

for

neutral

re-

and basic media, however

mains 3C% lower be

noted that

relate

than that

basic

changes in optical observed. SOD copper

of the initial

In control

solutions

and EPR spectra

spectra

experiments

basic

hand, th8 incubation

(pH

completely

12.5)

of strongly

by oxygen no were

reduction

solutions basic

cor-

when the neutral

in pH of such solutions.

1135

It should

of these solutions

Thus, oxygen leads to the partial in strongly

intensity

spectrum0

of SOD were aerated

drop by mor8 than 3 units other

integral

the changes in optical

with EPR data.

or mildly

its

of the

and to the On the

solution8

of SOD

Vol. 71, No. 4, 1976

BIOCHEMICAL

under vacuum results

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in partial

reduction

changes in the pH of the solutions

of copper,

but no

were noted.

DISCUSSION The phenomenon OP the reduction proteins (8,9).

in weakly basic media was observed It has been suggested

a reducing

group which is

the properties plasmin).

The optical

in alkaline

proteins

(e.g.

of some functional of the protein

takes place

coordinated

The effect

here should be connected

concentration

with

in the medium. Therefore

transfer

from hydrcql

+ 02 w.OH In the absence of the protein this (12) proposed

that

of superoxide

some low-aolecular

weight

for

+ O;reaction

a reaction

radicals COmpleXeS

of carbohydrates. to explain

solutions

the loss it

of

the rise

of

may be sug-

of SOD the reaction (1) does not proceed

the effectivity

such

anion

anion to oxygen takes place:

OH-

essential

pH the

and the substitution

than with

of electron

the copper being

At this

of oxygen on pH of alkaline

media in the presence

possible

or cerulo-

to the copper by hydroxyl

in basic

oxidation

Prom those of

groups (in particular,

groups from the medium rather

the formation

PH. However

lactase

(4).

gested that

patiadi

alkaline

different

changes were observed

of SOD described proton

at

is

However at pH 12.5 and

of water molecule,

hydroxyl

dissociated

there

media with pH about 12.0.

(10)) (11).

in these proteins

works

of SOD were not changed

deprotonisation

occurs

in several

and EPR spectra

certain

imidaeol

that

of SOD are essentially

the blue copper-containing

higher,

of blue copper-containing

of this is responsible

in alkaline generally

process.

solutions

for of

used Per the

By means of the reaction (1) it is the partial reduction of the protein copper

1136

BIOCHEMICAL

Vol. 71, No. 4,1976

in the course

of the aeration

cause generated

superoxide cu+2

On the other

radicals

+ o;--

in contrast

not accompanied that

basic

cu+l

t

properties:

o2 of the copper under

to the reduction reaction

solutions,be-

possess reducing

by aeration,

by the drop of pH, gives

in the last

RESEARCH COMMUNICATIONS

of strongly

hand, the "self-reduction"

vacuum which, imagine

AND BIOPHYSICAL

there

was

us the possibility

to

is the equilibrium:

cu+2

+ o*-2- cu+l + 0 2 in accordance with this scheme the removal

Actually,

under v8cuu.m should there

were superoxide It

media,

with

spectrum

results

is similar

tose oxidase

are stabilized

or in some organic

of the so-called

of the copper,

solvents

it

the nitrogen

(13).

pink copper-containing to that

in rather In connec-

should be noted that

enzyme, galac-

dismutase

the superoxide

radical

Galac-

activity

enzyme the copper atom is also linked

atoms. Besides,

the EPR

of SOD at pH 12.5 (14).

was shown to have superoxide

In this

if

in the solution.

these radicals

the obtained

tose oxidase, (15,161.

in the reduction

radicals

is known th8t

alkaline tion

result

of oxygen

with

is coor-

dinated to the copper atom of the enzyme. It is known (17,18) th8t in SOD the copper atom is linked with 4 atoms of imida201 nitrogens.

In the first

of SOD the water molecule in strongly (11).

alkaline

Therefore

in the coordinetion

media,

coordination

sphere of the copper

which is involved is substituted

it may be suggested

that

sphere the electron

such as:

1137

in the process by hydroxyl

at

ion

at pH 12.5 and higher tranefer

takes place

BIOCHEMICAL

Vol. 71, No. 4, 1976

Thus, from the above data it similarities

exist

the low-molecular hydrates

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

is possible

to think

that

between the mechanisms of galectose weight

copper complexes,

and copper-containing

oxidizing

certain oxidase, of carbo-

SOD.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. il. 12. 13, 14. 15. 16. 17. 18.

Fridovich, I. (1974) Adv. in Enzymol 9, 35-97. Hodgson, E.K. and Fridovich, I. (1973) Biochim. Biophys. Res. Comms. 2, 270-274. Fee, J.A. and Phillips, W.D. (1975) Biochim. Biophys. Acta u, 25-38. Rotilio, G. Finazzi-Agro, A., Calabrese, L. Bossa, Fe, h. and Mondovi, B. (1371) Biochem!stry &Q Guerrieri, 616-622. Symonyan, Y.A. and Nalbandyau R.Y. (1972) Doklady Academyi Nauk (Russ.) a, 1131-1173. &Cord, J.M. and Fridovich, I. (1969) J. Biol. Chem. a, 6049-6055. Symonyan M.A. and Nalbandyan, R.M. (1975) Biochimiya (Russ.) k& 726-732. Brill, A.%, Bryce, G.P. and Maria, H.Y. (1368) Biochim. Biophys. Acta 154, 342-350. Fee, J.A. MalmstrGm, B.G. and V'&ngKrd, T. (1970) Biochim. &ioph se Acta 1 136-143. Fee, J.A. and 5icorleto, 3 JE. (1973) Biochemistry 12, 4893-4899. Terenzi M. Rigo, A., Franconi C., Mondovi, Be, Calai. a&d Rotilio, G. (19743 Biochim Biophys. Acta zeh30-236. Fatiadi, A.J. (1970) J. Res. Natl. Bureau of Stand. a, 723-731. Maricle, D.L. and Hodgson, W.G. (1965) Anal. Chem. 2, 1562-1565. Cleveland, Le, Coffman, R.E., Coon, P. and Davis, L. (1975) Biochemistry 14, 1108-1115. Hamilton, G.H., Libby, R.D. and Hartzell, C.R. (1973) Biochem. Biophys. Res. Comms. a, 333-340. Kwiatkowski, L.D. and Kosman, D.J. (1973) Biochem. Biophys. Res. Comms. 53, 715-721. Forman, H.J., Eivans, H.J., Hill, R.L. and Fridovich, I. (1973) Biochemistry & 823-827. Richardson J. Thomas, K.A., Rubin, B.H. and Richardson, D. (1475)'Proc. Natle Acad. Sci. USA 72, 1349-1359.

1138

The effects of oxygen on alkaline solutions of superoxide dismutase.

BIOCHEMICAL Vol. 71, No. 4, 1976 AND BIOPHYSICAL RESEARCH COMMUNICATIONS THE EFl?!ECTOPOXYGENOE ALKALINE SOLUTIONS OF SUF'EROXIDE DISMUTASE Y.A.Sym...
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