A radioimmunoassay for copper containing superoxide dismutase

Vol. 88, No. 2, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

May 28, 1979

Pages 337-345

A RADIOIMMUNOASSAY

FOR

COPPER

SU PEROXIDE

A.

Baret,

P. Michel,

CERB,

HIA

CONTAINING

DISMUTA

M. R.

Imbert

St Anne,

SE

and J. L.

83800

Toulon,

Morcellet

Naval

and A. M. Institut

de Biologie et Marie

Received

January

Michelson

Physico-Chimique, Curie,

75005

13, rue

Pierre

Paris

30,1979

SUMMARY.

- Highly radioactive rat and bovine copper containing superoxide up to 1700 Ci/mmole of protein were prepared by iodination ses (Cu-SOD) $yYL% I and chloramine T. Labelled human Cu-SO 92’5’;; -;?E;y$ c,o,u,p,l:,n,ate the enzyme with N-succinimidyl 3-(4-hydroxy, 5 - ( These tracer molecules can be used with anti-bovine or anti-human Cu-SOD antisera to estimate various Cu-SODS as protein (rat, bovine, human) with a sensitivity of 20-80 picogrammes, using standard radio-immunochemical techniques. Control ion

superoxide

(SOD)

in vivo

02-

present

(1).

among

all

aerobic

nature

of the metal

strongly There

of the enzymes,

cuprozinc these problems

state

depends

the quantity

are

species,

upon

several

types

but differ

at the active

It is thus assay

of the steady

of interest

in their

site

concentration

ubiquitous and in the

a sensitive,

specific,

and easy

(2).

to develop

Radioimmunological

and can contribute

between

of molecular AND

mangano

techniques

to the solution

the metabolism MATERIALS

are

location

SODS can be achieved.

with

which

dismutase

subcellular

if discrimination

connected

of superoxide

of enzyme,

particularly

requirements

of the radical

of a wide

and

answer variety

of

oxygen.

METHODS

Superoxide dismutases. Rat, human and bovine cuprozinc superoxide dismutases were prepared according to the technique of McCord and Fridovich (3). After purification, the specific activity of the bovine enzyme was 3300 NBT-riboflavin units/mg (4). In addition, a commercial bovine blood SOD (Sigma) has been used (SA : 2700 McCord and Fridovich units/mg). Before use, the purity of these preparations was tested by polyacrylamide gel electrofo’calisation (Multiphor LKB). 0006-291 337

X/79/1 00337-09$01.00/O

Copyright @ 1979 by Academic Press, Inc. All rights ofreproduction in anyform reserved.

Vol. 88, No. 2, 1979

BIOCHEMICAL

AND EIOPHYSICAL RESEARCH COMMUNICATIONS

Antibovine SOD and antihuman SOD antibodies. Antibovine and antihuman SOD antisera were obtained by immunisation of rabbits, with, for the first injection, 1 mg of protein followed by three further injections of 0. 5 mg at monthly intervals. The Cu-SOD preparation was emulsified with complete Freund adjuvant and administered subcutaneously. 125

Iodine labelling of cuprozinc SOD. The labelling of bovine anqgt enzymes was effected with 2 pg of protein in 2 ~1 of buffer, 200 PCi of Na I (IMS 30, of chloramine T in 0. 5 M phosphate Amersham) in 2 ~1, and small quantiq$$ I incorporation into the protein was buffer pH 7. 5 (5). The percentage of measured by precipitation of the iodinated enzyme by 10 ‘$ trichloracetic acid in presence of 1 mg/ml of bovine serum albumin (BSA) as carrier. For each labelling, the quantity of chloramine T (generally about 1. 5 pg) giving a yield of iodination between 40 ‘$ and 50 ‘$ of the iodine used is determined. When this percentage is reached, the reaction is stopped by addition of sodium metabisulfite, the quantity of which is twice that of chloramine T, and the labelling medium is diluted with 1 ml of diluent buffer (0. 1 M phosphate pH 7. 6, containing 2 % BSA). The product is purified on Sephadex G 100 with the samebuffer as eluent (Fig. 1). Labelling of the human enzyme wasle&ected by coupling human I) iodophenyl) propionate Cu-SOD with N-succinimidyl 3-(4-hydroxy, 5-( using the technique described by Bolton and Hunter (6). The tracer is purified on Ultrogel Aca 44 (Industrie Biologique FranGaise) with 0. 1 M phosphate buffer pH 7. 6 containing 0. 5 % gelatin as eluent buffer, Two peaks of radioactivity are observed. The first corresponds to the immunoreactive fractions used as tracer, whereas the second corresponds to the coupling product of introduced before the chromatographic the excess of reagent with glycocol, step to terminate the reaction (6). This technique was used since treatment of human Cu-SOD with chloramine T gives rise to aggregates. Radio-immunoassay. The incubation mixture for radio-immunoassay contains purified or unpurified preparations of SOD appropriately diluted (0. 1 ml), labelled enzyme (0. 3 ml, 5000 cpm), and diluted antiserum (0. 1 ml) in a final volume of 0. 5 ml. All dilutions are made with 2 ‘$I BSA in 0. 1 M sodium phosphate pH 7. 6. The duration of the incubation at ambient temperature is. 18 to 24 hours. The separation of the bound fraction from free enzyme is effected by addition of 1 ml of a solution of 30 % polyethylene glycol 6000 in 0. 1 M phosphate pH 7. 6 after addition of 100 ~1 of normal bovine serum in each tube. After centrifugation, the supernatant is discarded and the radioactivity of the precipitate is counted in a Packard 5360 scintillation spectrometer. RESULTS

High

specific

pattern Cu-SOD elution The

which

activity

iodinated

is obtained

on a column volume

immunoreactivity

of 300 pl of tracer

Figure

on gel filtration

of Sephadex

at peak

SOD.

of the different (about

10 000 cpm)

the chromatographic

of the iodination

G 100.

no II is identical

1 shows

Three to that

fractions

mixture

of rat

are

observed.

The

of unlabelled

purified

peaks

was determined

in the presence

338

SOD.

by incubation

of an antibody

excess

Vol. 88, No. 2, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Fractions Fig. 1 - Chromatography G 100 -e*-, cpm ;++@

(100

ul of a 10-j

corresponding peaks

SOD has been

1700

least

Fractions

to constitute

ponding

The

the tracer.

to 0. 8 atom Ci per

4 days

of iodine

mmole

of protein.

Assays

of stability

at 4” C, with

degradation

of the iodinated

Figure

2 shows

the anti-bovine Under

Two

these

homologous

antiserum

radioactivity

from

of peak

radioactivity

that the tracer

for

no II are iodinated

is 51 uCi/ug,

corres.

activity

is stable

in immunoreactivity. at -20”

value

of the bovine

Its value

21 days

and one heterologous obtained

it is possible

the antibody Cu-SOD

with

bovine

(R, S. 5), at a final

as the quantity

of bovine

no II and a zero

of SOD and a specific

shown

value

for

of

at

Moreover,

C shows

only

chro-

slight

protein.

Cu-SOD

is defined

molecule

G 100 after

curves

conditions;

specific

have

standard

sensitivity

femtomoles

per

a maximal

to the maximum

no decrease

on Sephadex

curves.

The

of peak

by radio-immunoassay.

matography

Standard

presents

radioactivity

corresponding

determined

rat Cu-SOD on Sephadex bound to antibody.

immunoreactivity

to the maximum

I and III.

diluted

dilution).

125 I - iodinated % radioactivity

of

sites,

to measurea of enzyme Hence

can be estimated

339

were

iodinated dilution

displaces

homologous with

precision

used.

Cu-SOD

using

of k.

pg of bovine which

with

system

_

SOD.

The

5 ‘$I of the sera,

about

as protein.

2.4 The

BIOCHEMICAL

Vol. 88, No. 2, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

vi3, 1.0.

.8-

.6.

4 .

\

.2.

5

10

.018

,156

5>p

pl ok% hemolysate

1 .312

.625

1.25

2.5

diluted or homogenate

5

10 ng

*

pure bovine Cu SOD

125 Fip. 2 - Dosage curves using bovine I Cu-SOD and antiantibodies (RS 5) with purified bovine C-SOD (N bovine Cu- SOD bovine liver homogenate () and bovine hemolysate (e+-) ; lack of cross reactions with human (-*-*) and rat (-II ) hemolysate B/Be is the ratio of radioactivity precipitated in presence of the different cold SODS compared with that precipitated in their absence.

technique oxidase which give

is thus

about

and luminol

twice

as sensitive

(7) which

is valuable

gives

an enzymic

activity

valuable

information

since

extremely biopsy

small

samples.

plus

dilution

of the antiserum

system

antibovine

to detect

both

of 3-6

Conjunction

protein

double

using

are

can thus

xanthine

femtomoles,

of the two

and activity

approach

antisera

enzyme,

but

techniques

can

measured,

be used

using

for human

the heterologous

system

are

shown

in Fig.

3. The

000.

Under

these

conditions,

20 pg (0. 6 femtomoles)

rat

125I

final it is

of bovine

enzyme.

4 presents I Cu-SOD

with

(R. S. 5) is l/50

80 pg of rat

Figure 125 human

(2.4

obtained

Cu-SOD

and 800 pg of human

of the antiserum enzyme

of SOD.

This

curves

Cu-SOD

enzyme

in the region

assay

s.

samples. Standard

possible

as the enzymic

),

standard plus

(R. S. 6) is l/25

curves

antihuman 000.

obtained

Cu-SOD

It is possible

femtomoles).

340

with

antisera. to detect

the homologous The 80

final

dilution

pg of human

BIOCHEMICAL

Vol. 88, No. 2, 1979

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

.8 -

.6-

.78

1.56 312

6.25

12.5

25

100 pl of

Fig. 3 - Cross reactions of purified rat (-*-*-) (-*-+) bovinel#--Y Cu-SODS and rat liver homogenate using I rat Cu-SOD as tracer and anti-bovine B/Be as in fig. 2.

homogenate

, human (m ) and ( -1 in the system Cu-SOD antibodies (RS 5).

.6

:

f$$l2P Purified reaction

I

6.25 . .078

.

25

,

.0156

.3l2

.625

l&l 1 1.25

of 100 fofd dilutecl?&olysate 25

5

ose response curves in the homologous I-human Cu-SOD and anti human Cu-SOD human Cu-SOD (Ct), human hemolysate is shown with rat hemolysate (W).

341

M ng

pure human Cu SOD

w

human Cu-SOD system antibody diluted 25 000 fold. (W). Lack of cross

BIOCHEMICAL

Vol. 88, No. 2, 1979

Interspecies species

specificity. specificity

SOD inhibit

With

is very

interaction

A similar cross

result

reactions

enzymes,

same

is obtained

with

are

are

observed

when

dilution

curves

appear

to parallel

those

reaction

occured

between

nor

(Table

I),

Cross

reactions

a cross

which

with

reaction

G 100 showed material

proteins.

(2 %) with

bovine

SOD.

of the enzymatic

by radio with

activities

(Table

II).

The

by the riboflavin-NBT -immunoassay.

the specific

determined

The

activity

by protein

value

Moreover,

of different SOD (Fig.

catalase

the

rat 3).

organs

No cross

in any

system

identical

been

presented on Sephadex

of human

results

are

method

(4) per

of SOD.

(8).

erythrocyte

erythrocyte

SOD.

SOD were

techniques

expressed

pg of protein

enzyme

determined

using

in enzymatic

3. 08 units/pg,

human

by a

to those

described

and enzymatic

obtained,

preparations

of the catalase

are

of human

of the purified

and rat

is due to contamination

activity

of the radioimmunological

of 40 donors

determined

reaction

specific

No

or rat

human

and Cu-SODS

Purification

by SOD has already

specific

bovine,

rat

4).

and bovine

is studied.

Only

characteristics

Determination

blood

cross

(Fig.

Cu-SOD.

bovine

of catalase

by conjunction

between

leiognathi)

erythrocyte

homogenate.

system

I- Cu-SOD

Mn-SOD

other

enzymatic

human

the purified

human

apparent

rat liver

the homogenates

with

(P.

immunological

with

system

with

purified

Contamination

Individual

human

the inter-

human

SOD and the antibovine

is obtained 125

used,

rat nor

iodinated

reactions

obtained obtained

this

systems

Neither

the homologous

cross

bacterial

that

whose

I).

the heterologous

are

with

result

between

hand,

evident

(Table

the bovine

The

On the other

Cu-SODS

the two homologous

marked

between

SODantibodies(Fig.2).

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

units

SOD determined may

(2925

be compared

NBT

units/mg)

assay. DISCUSSION

The erythrocuprein assay

preparation

of stable

is an essential

of this

enzyme.

iodinated

bovine

difficulty

in labelling

of the SOD with

condition

Previously,

SOD has been the enzyme

complete

lack

and high for

a low used

for

which

specific

radioactivity

the elaboration

specific

studies

has been

encountered

of immunoreactivity

342

of the radio-immuno-

radioactivity

metabolic

(action

iodinated

(40 Ci/mmole) (9).

The

greatest

is aggregation of chloramine

T).

as

Vol. 88, No. 2, 1979

BIOCHEMICAL

AND BIOPHYSICAL

TABLE Cross

reactions

of SODS

studied

Mn - SOD

heterologous

Heterologous system

(rat tracer plus antibovine SOD antiserum)

Bovine

I

100

systems

system

Human

Human

&-SOD

and

Homologous

Species

Pnzyme

I

in homologous

I

RESEARCH COMMUNICATIONS

q0

25 %

0%

Rat

0%

Bovine

0%

Swordfish

0%

Bacterial

0%

0 %

0.01 72

Human

0%

0%

0%

0% 100

%

0%

I

100

%

190

% 0.5

I

%

-

TABLE

Specific

activity

of normal

Enzyme units/ml number

of subjects

standard

This

which

to a dimerisation two

cysteine

39

enzyme

proportion

enzyme

enzyme,

provides

be strictly which

controlled retains

use of N-succinimidyl

a tracer

suitable

with

for

343

correspond

bridges

between

of the retention

the rat

weight

volume two

and bovine

in order

all its

0.069

could

of disulfide

to a molecular

Hence,

3.08

6. 37

Cu-SOD,

the radio-immunoassay.

fold

enzymes,

to obtain

immunological

3-(4-hydroxy,

ug

39

407.71

by the value

corresponds

enzyme.

must

of iodinated

the human

propionate

iodinated

of oxidant

is suggested

which

40

the human

due to formation

units/

vg/ gHb

1. 02

for

Specific activity

of protein

59. 75

34. 9

This

Cu-SOD

40

1223

is complete

residues.

of the intact

the quantity

For

pg/ ml blood

of the protein,

of the aggregated that

units/gHb

2. 88

aggregation,

erythrocyte Immunoassay

183 deviation

human

activity

39

mean

II

a high properties.

5-(

125

I) iodophenyl)

Vol. 88, No. 2, 1979

BIOCHEMICAL

Studies are

highly

SODS.

specific

(This

sequences system

of the cross for

is also

different

RS 5 anti-bovine

percentage

differences

differences

of the analogous The without

reaction.

Moreover

will

concentration

erythrocyte

closely

to that

no significant

for

the mammalian

both

enzyme

between

system).

The

amino

Mn-SOD

variation value),

rat

Cu-SOD.

The

does

is needed.

detection

not show

of choice

with

of enzyme

of application,

any cross

since

Comparison

activity

the specific

by protein

among

subjects

normal

we are levels

with

activity

presently

should

(standard

of the

and shows deviation

work

(contract pour

was

supported

no 77. 4. 0842),

la Recherche

Medicale

by grants

from

D. G. R. S. T. FranGaise

CNRS

(Contract

(E. R.

estimation

little

difficulty.

103),

INSERM

no 77. 7. 0280)

the Fondation

and the D. R. M. E.

REFERENCES 1.

2. 3. 4. 5.

Michelson, A.M., Puget, K., Durosay, P. and Bonneau, J. C. (1977) in “Superoxide and Superoxide Dismutases”. ed. A. M. Michelson, J. M. McCord and I. Fridovich, p. 467-499, Academic Press, London. Fridovich, I. (1975) Ann. Rev. Biochem. 44, 147-149. McCord, J. M. and Fridovich, I. (1969) J.%ol. Chem. -’244 6049-6055. Beauchamp, C. and Fridovich, I. (1971) Anal. Biochem. -44, 276-287. Roth, J. (1975) in “Methods in Enzymology” Vol. XXVI B. ed. B. W. O’Malley and J. G. Hardman, p. 223-233, Academic Press, New York.

344

less

radio-immunoassay

developing,

present

other

corresponds

estimation

a similar

no prior

or enzyme

by radio-immunoassay,

as determined

and assay

ACKNOWLEDGEMENTS This

acid

or sequence

allows

which

In conjunction

low

systems

by the heterologous

conformational

modifications

which

at very

common

revealed

technique

as determined

Mn-SODS

proteins

or bovine

indicate

to be tested

enzyme

the homologous

and iodinated

be the routine

Cu-SOD,

2. 5 $I of the mean

of the human

of erythrocuprein

As an example

individual

sequence

only

that

of the enzymes.

from

it should

of purified

acid

are

reactions

parts

distinguish

human

than

of cross

interference

(10).

shown

SOD antiserum

of the material

techniques

species

radio-immunoassay

in tissues

purification

amino

RESEARCH COMMUNICATIONS

have

of the rat homologous

of SODS from with

reactions

an antigenic

true

AND BIOPHYSICAL

of

Vol. 88, No. 2, 1979

6. 7. 8. 9.

10.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Bolton, A. E. .and Hunter, W. M. (1972) J. Endoct. 55,~ XXX-XxX1. Puget, K. and Michelson, A.M. (1974) Biochimie 56, 1255-1267. Halliwell, B. (1973) Biochem. J. -J135 379-381. Huber, W. and Saifer, M. G. P. in “Superoxide and Superoxide Dismutases” ed. A. M. Michelson, J. M. McCord and I. Fridovich, p. 11-17, Academic Press, London. Crapo, J.D. and McCord, J. M. (1976) Am. J. Physiol. -J231 1196-1203.

345