Vol. 72, No. 3, 1976

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RECONSTITUTION OF IRON-SUPEROXIDE DISMUTASE

Fumiyuki Yamakura

and

Koji Suzuki

Department of Chemistry, School of Medicine Juntendo University, Narashino, Chiba 275, JAPAN Received August 4,1976 Summary: Iron-free superoxide dismutase has been prepared by incubation with sodium carbonate buffer (pH ii.0) containing i0 mM dithiothreitol and 2 mM EDTA under anaerobic conditions and subsequent dialysis to return the pH to 7.8. Reconstituted enzyme was obtained from the apoenzyme by incubation under the same conditions, with addition of 12 mM ferrous ammonium sulfate instead of EDTA. The apoenzyme has no visible absorption and no significant amount of activity. The reconstituted enzyme had the same visible absorption and activity as the native enzyme. Four sulfhydryl groups per molecule of apoenzyme reacted rapidly with 5,5'-dithiobis-(2-nitrobenzoic acid) while holoenzyme reacted with the reagent very slowly.

Superoxide dismutase reaction

0~ + O~ + 2 H +

(EC 1.15.1.i), which catalyzes the > H202 + 02, is widely distributed

among oxygen-metabolizing organisms obes (2).

With a few exceptions

(i) and some obligate anaer-

(3,4), SOD was obtained as the

Mn or Fe-containing enzyme from procaryotes and mitochondria, whereas SOD from eytosoles of eukaryotes was found to contain Cu and Zn (i).

Since McCord and Fridovich (5) reported the prep-

aration of the apo- and reconstituted enzymes of Cu, Zn-SOD, many studies have been carried out on the properties of apo- and reconstituted

SOD (1,6,7).

Recently,

Ose and Fridovieh (8) have

extended the studies of the reversible removal of Mn atom from Mn-SOD of Escherichia eoli and the replacement of Mn with Co,Zn,

Abbreviation:

SOD, superoxide dismutase

Copyright © 1976 by Academic Press, Inc. All rights o] reproduction in any ]orm reserved.

1108

Vol. 72, No. 3, 1976

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

or Ni,

but they failed

Te-SOD

of E s c h e r i c h i a

succeeded

and

from P s e u d o m o n a s is composed

crystalline hydryl

Fe-SOD clude

enzyme

groups

describe

coli using

the

removal

of Fe atom from

same technique

which

had

with the Mn-SOD.

Fe-SOD 40,000

in the r e v e r s i b l e

has a m o l e c u l a r

of two subunits

contains

of equal

i.i g atoms

per mol of enzyme

the p r e p a r a t i o n

(9).

ovalis.

Fe is e s s e n t i a l

Furthermore,

the results

4 sulfhydryl

groups

cule and/or

chelated

The results

of q u a n t i t a t i v e

of h o l o e n z y m e

might

of

(9).

of Fe and 4 mol

In this

for enzymatic

weight

size

The

of sulf-

eommunieation,

of apo- and r e c o n s t i t u t e d

from P s e u d o m o n a s that

ovalis

have

activity studies

enzyme

of

led us to conof Fe-SOD. indicate

be buried

we

that

in enzyme

mole-

to Fe atoms. MATERIALS

AND METHODS

Fe-SOD was i s o l a t e d from P s e u d o m o n a s ovalis as d e s c r i b e d p r e v i o u s l y (9). E n z y m a t i c a c t i v i t y was m e a s u r e d by the m e t h o d of McCord and F r i d o v i c h (5) with a few m o d i f i c a t i o n s (9). For the studies of the enzyme stability , the enzymatic a c t i v i t y was estimated by the m e t h o d of N i s h i k i m i et al. (10). Protein was determined by the m i c r o - b i u r e t method of Itzhaki and Gill (ii), using bovine serum a l b u m i n as standard. Iron was d e t e r m i n e d by the m e t h o d Of M a s s e y (12) except that b a t h o - p h e n a n t h r o l i n e was used as a colo r i m e t r i c reagent. P o l y a c r y l a m i d e gel e l e e t r o p h o r e s i s was p e r f o r m e d at 5 ° C in a c c o r d a n c e with the method of Davis (13). Mobilities of the apo- and r e c o n s t i t u t e d SOD on p o l y a c r y l a m i d e gels (6, 7.5, 9, and 10.5 %) were a n a l y z e d a c c o r d i n g to the method of H e d r i c k and Smith (14). SOD a c t i v i t y on p o l y a c r y l a m i d e gel was located by the p h o t o c h e m i c a l m e t h o d of B e a u c h a m p and F r i d o v i c h (15). Sulfhydryl groups were d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y using 5,5'dithiobis-(2-nitrobenzoic acid) as d e s c r i b e d by Ellman (16). Optical a b s o r p t i o n spectra were m e a s u r e d with a Hitachi Model 124 Spectrophotometer.

RESULTS Purified

Fe-SOD

min and the a c t i v i t y Fig.

i, the enzyme 30 mg of the

and c o n t a i n e d

1.12

AND DISCUSSION

was treated

at various

of the enzyme

was

completely

pH's

was measured. inactivated

Fe-SOD,

which

g atoms

of Fe per mol

As

shown

5

in

at pH 10.9.

had a specific

1109

at 30 ° C for

activity

of enzyme,

of 3,200

was dialyzed

Vol. 72, No. 3, 1976

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I

I

I

>,o

-~ loo > u _J

25o if) w

I

o

I

9

I0

l

I

pH

Fig. i Effect of pH on the stability of SOD. The enzyme (34 ~g/ml) was i n c u b a t e d for 5 min at 30 ° C w i t h 0.i M s o d i u m c a r b o n a t e buffer, at various pH's as indicated. Residual a c t i v i t i e s were m e a s u r e d by the assay method of N i s h i k i m i et al. (i0).

against

distilled

water

carried

out under

anaerobic

The

enzyme

carbonate which

was

buffer

containing

against

(Whatman

DE-32)

eluted

by 50 mM of this

preparation

was

The a p o - S O D

lyzed

buffer instead

against

was

column

employed

of i N NaOH.

0.2 M sodium

for

buffer

carbonate

1110

The a p o - S O D to a DEAE-

(pH 7.8),

equili-

and then

i mM dithiothreitol. study.

25 min at 30 ° C with the

with a d d i t i o n Then

(pH 7.8)

previously

in the r e c o n s t i t u t i o n for

this

so-

(pH g.2)

buffer

and applied

buffer

This

5- 12 hr at 4 ° C,

phosphate

containing

incubated

of 2 mM EDTA.

substitution:

0.2 M s o d i u m

carbonate

water

were

i0 mM d i t h i o t h r e i t o l ,

(0,9 X 4 cm),

phosphate

buffer

as above,

N 2 gas

for 18 hr at 4 ° C.

ten fold with d i s t i l l e d

by 2.5 mM p o t a s s i u m

sulfate

0.2 M sodium

50 mM p o t a s s i u m

brated

alkaline

2 mM EDTA and

to pH ii by a d d i t i o n

against

procedures

25 min at 30 ° C with

0.5 mM d i t h i o t h r e i t o l

was diluted

This

using

i mM E D T A and i mM d i t h i o t h r e i t o l

subsequently

cellulose

The f o l l o w i n g

conditions

for

containing

was d i a l y z e d

containing and

incubated

pH was a d j u s t e d

lution

overnight.

of 12 mM ferrous enzyme

buffer

solution

(pH 9.2)

same

ammonium was dia-

containing

Vol. 72, No. 3, 1976

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Fig. 2 P o l y a c r y l a m i d e gel e ! e c t r o p h o r e s e s of native, apo-, and r e c o n s t i t u t e d SOD. Native SOD (a): 44 ~ g , a p o - S O D (b): 4 0 ~ g , r e c o n s t i t u t e d SOD (c): 3g ~ g , and r e c o n s t i t u t e d SOD (d): 4 ~ g were each subjected to p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s at pH 8.9 The gels (a,b,c) were stained for protein by Amido black i0 B; the achromatic zone indicates the enzymatic activity (d).

i mM d i t h i o t h r e i t o l at 4 ° C.

and I mM ferrous a m m o n i u m

A f t e r dialysis

of the r e c o n s t i t u t e d

changes

of 50 mM p o t a s s i u m phosphate

covered

by using a D E A E - c e l l u l o s e

buffer

sulfate for 5-12 hr enzyme against

(pH 7.8),

column as described

1111

two

it was reabove.

Vol. 72, No. 3, 1976

Fig.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

2 shows

the e l e c t r o p h o r e t i c

reconstituted

SOD on p o l y a e r y l a m i d e

and one m i n o r

were

SOD.

The p r o t e i n

the zones

of the apo-

method

bands

isomers,

(Fig.

gels.

of the r e c o n s t i t u t e d activity,

described

2).

which

in the text. SOD have

It appeared

the

to the a n a l y t i c a l

method

and

protein

band

and r e c o n s t i t u t e d SOD c o i n c i d e d

were

located

Major

with

by the

protein

same m o b i l i t y

that the major

of the apo- and r e c o n s t i t u t e d

according

of the apo-

One major

in both the apo-

and r e c o n s t i t u t e d

enzyme

protein

bands

of the enzymatic

photochemical

native

observed

patterns

bands

as the

and minor

SOD were

"charge"

of H e d r i c k

and

Smith

(15).

0.8

LLI 0 Z

0.6 0.5

03 n- 0 . 4 0 (/') I:]O

Reconstitution of iron-superoxide dismutase.

Vol. 72, No. 3, 1976 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS RECONSTITUTION OF IRON-SUPEROXIDE DISMUTASE Fumiyuki Yamakura and Koji S...
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