Preparation and properties of clostridial apoferredoxins

Vol. 29, No. 2, 1967

BIOCHEMICAL AND BIOPHYSICALRESEARCH

PREPARATION AND PROPERTIES OF CLOSTRIDIAL Jen-shiang Department

Received

iron

September

A procedure

has previously

and inorganic

or "acid-labile"

isolation

for

the reconstitution

doxin

on Chelex

agent.

the mercurial,

of ionic These

could

if

reconstitution

two alternate

compare

requirements

for

preparations

employ

ferredoxin,

rather

mixture.

(Malkin

reconstitution

components

of these

treatment

with

were

and

incorporated omitted

from

we wish

the

to

of apoferredoxins,

apoferredoxins, from

the iron

sulfide,

any one of the

communication

of ferredoxin

to remove

sodium if

of

reducing

or sulfide

the preparation

of ferre-

the addition

and a mercaptide

other

and

by chromatography

required

In this

for

ferredoxin,

by treatment

was obtained

of the

and the conditions

protein

nor was any iron

properties

than

clostridial

by Fe(SOs)(NHs)nSOs,

or both

acid

the removal

and was purified

sulfide,

methods

the chemical

this

Reconstitution

be supplied

incubation

from

from

inorganic

either

for

was prepared

columns.

was omitted,

the protein

Berkeley

apoferredoxin,

No reconstitution

components

describe

protein,

mersalyl,

iron,

2-mercaptoethanol. three

sulfide

The apoprotein

and Sephadex

a source

of California,

been described

of ferredoxin

1966b).

with

C. Rabinowitz

University

of the resulting

Rabinowitz,

APOFERREDOXINS

26, 1967

the

into

Hong and Jesse

of Biochemistry,

COMMUNICATIONS

and describe

these

proteins.

and inorganic

a mercurial,

sulfide

the These

from

as previously

described. M&?&s modification

and Mater&G--Protein of the phenol

and have been corrected

concentrations

method

so that

as described all

values

246

were

determined

by Lovenberg,

are expressed

by a

et al.

as dry weight

(1963), of

Vol. 29, No. 2, 1967

The radioactivities

protein. were

BIOCHEMICAL AND BIOPHYSICALRESEARCH

determined

with

10 ml of Bray's as described mined

groups

acid

Iron

acid-l-

protein

as previously

by the procedure Apoferredoxin-I:

described

by Malkin

4000-fold

by Lovenberg,

et aZ.

et al.

was perpH 8.5,

excess

by

iodoacetic

NaOH before G-25.

use.

The

The enzymic reaction

(1963). from CZostridiwn

acidi-

(1963).

was prepared (1966b)

used had a

the phosphoroclastic

used was prepared

and Rabinowitz

acid

in 0.1 p1 Tris.HCl,

on Sephadex

The protein

The free

by carboxymethylation

concentrated

of Buchanan,

was deter-

Carboxymethylation

using

ferredoxin

1963).

iodoacetic

was determined

in

bathophenanthroline

determined

with with

Counter

sulfide

et at.,

15 minutes

by chromatography

described

Crystalline urici

for

had been neutralized

of ferredoxins

with

Acid-labile

were

anaerobically

was isolated

activity

(1962).

cpm per umole.

at room temperature

which

I Scintillation

The radioactive

of 63,500

apoferredoxin

Mark

(Lovenberg,

"C.

and "C-apoferredoxins

was determined

of apoferredoxins

activity

treating

Chicago

described

iodoacetic

formed

'$Fe-ferredoxins

by Doeg and Ziegler

sulfhydryl

specific

a Nuclear

solution.

as previously

using

of

COMMUNICATIONS

for

according

to the method

the preparation

of apo-

ferredoxin-(mersalyl-Chelex-Sephadex). Apoferredoxin-II:

Ferredoxin,

pH 7.4,

was made 4% in TCA with

allowed

to stand

throughout bleached

this

HCl,

period

rpm for

The precipitate

peated

1 hour

for

pH 9.0,

and the with

colorless

supernatant

solution

material

supernatant

taken

247

y formic

was

was centrifuged was discarded. acid

The washing

and allowed was re-

up in 4 ml of 0.1 E Tris.

was removed was then

mixture

was completely

suspension

3 ml of 0.023

E Tris.HCl,

of H2S gas was noted

The whi,te

was finally

and the insoluble

This

of ferredoxin

at 0“ C and centrifuged.

The precipitate

The almost

color

period.

was washed

once.

Evolution

and the brown

10 minutes

30 minutes

a 15% TCA solution.

at 0" C.

at the end of this

at 17,000

to stand

for

14 mg, in 11 ml of 0.015

passed

by centrifugation. over

a Sephadex

G-25

Vol. 29, No. 2, 1967

column

(1.2

Tris.HCl,

BIOCHEMICAL

x 38 cm) which pH 8.5,

apoferredoxin

had been previously

and the protein

prepared

in this

as for

respect

to P-mercaptoethanol Reconstitution

II,

330,000

mercaptoethanol tion

mixture

8.5,

and incubated were

3 umoles

were

8 cm) equilibrated water

prior

washed 0.05

to use.

successively M Tris.HCl,

The ferredoxin ing

0.58

with

with

0.05

applying

containing

was finally

Mwith

of 4 ml.

(specific

with

activity

NP gas.

The reac-

The reconstituted

the reaction

mixture,

!j Tris.HCl,

ferre(0.8

and washed

x

with

the column

pH 7.4,

M Tris.HCl,

pH

column

M NaCl to remove 0.005

=

of 2-

0.1 E Tris*HCl,

pH 6.5,

at

At the

and 140 umoles

phosphate,

with

4 hours

M 2-mercapto-

on DEAE-cellulose

0.23

eluted

0.07

volume

deaerated

at 37" C.

10 ml of 0.15

pH 7.4,

was

and 20 ml of excess pH 7.4,

reagents. contain-

M NaCl. of Ferredoxin

ferredoxin

and 3 umoles

was reisolated

ResuZts--The for

are shown in Table

iron,

I.

for

and 0.05

15 minutes

Apoferredoxin-

c 2-mercaptoethanol at 37" C with

of Na2S when present.

in

3 umoles

of

The reconstituted

as above.

analyses

sulfide,

from Apoferredoxin-III: pH 8.5,

of 1.6 ml was incubated

5gFeS04(NHs)2S04

and III

flushing

3 fold

1.8 mg, in 0.1 MTris.HCl,

a volume

still

1 M potassium

After

Reconstitution

III,

the same

NP gas for

8 M urea,

in a final

by chromatography with

The

Apoferredoxin-

of 5gFeS04(NHs)2S04

15 minutes

reisolated

using

were

of Na2S when present,

diluted

for

solutions

pH 8.5,

added while

was then

E

apoferredoxin-II.

by flushing

of antifoam

3 umoles

cpm/umole),

doxins

anaerobically

amount

end of the incubation,

the same buffer.

from Apoferredoxin-II:

in 0.1 M Tris.HCl,

and a small

0.02

(P-MET).

1.8 mg, was incubated

ethanol

with

was prepared

but all

of Ferredoxin

room temperature

with

way is designated

apoferredoxin-II,

COMMUNICATIONS

equilibrated

was eluted

Apoferredoxin-III

Apoferredoxin-III: procedure

AND BIOPHYSICALRESEARCH

of native free

ferredoxin

sulfhydryl

The apoproteins

groups were

248

and apoferredoxins and enzymic

essentially

free

II

activity of iron

and

Vol. 29, No. 2, 1967

Table

I.

BIOCHEMICAL

Properties

of Apoferredoxins

Sample

1. Native

AND BIOPHYSICALRESEARCH

Iron

ferredoxin

COMMUNICATIONS

and Reconstituted

Acidlabile sulfide

Free -SH

Aag0

motes/mote protein

A&l/ ml/cm

6.5

3.94

6.0

0.2al

Ferredoxins

Enzymic activity

Yield

% 100

% --

2 . Apoferredoxin-Iu

0.06

0.00

0.0

0.11

0

75

3. Apoferredoxin-II

0.16

0.01

0.7

0.00

0

67

4. Apoferredoxin-III

0.11

0.01

7.5

0.00

0

70

6.0 6.6 6.4

5.9 6.8 6.0

3.74 2.65 4.02

108 114 119

6. Apoferredoxin-II t S= t Fe'+ t 2-MET@

0.58

0.52

0.00

7

55

7. Apyf;;zdoxin-II + urea + 2-METg/

0.95

0.13

0.00

0

77

8. Apoferredoxin-II t urea t s= f E-METE.

0.05

0.10

0.00

0

75

5.8 6.8 5.8 6.2

5.8 7.1 5.1 5.6

3.24 3.27 3.94 4.25

108 106

72

2.1

0.1

0.49

3

79

0.00

0.26

0.00

3

86

5. Apoferredoxin-II + urea + S= + Fe++ t 2-MET

9. Apoferredoxin-III + Fe"

t S=

+ 2-MET

10. Apoferredoxin-III t Fe* + Z-MET"-/ 11. Apoferredoxin-III + P-MET
70 75 89c-/

:: 8sC/

1;:

t S=

Q/ Previously

described

(Lovenberg,

et ail.,

1963).

!?I Apoferredoxin-I contained 4.3 moles of mercury per mole protein as determined by calorimeter analysis (Tyuma, et aZ., 1966); Malkip, R. observations. and Rabinowitz, J. C., unpublished d Double procedure were then

the amounts added.

of the

iron

g Protein was reisolated by gel chromatographed on OEAE-cellulose

d Protein was re' isolated by gel of by chromatography on DEAE-cellulose.

249

salt

and Na2S used

in the standard

filtration column.

on Sephadex

G-25 and

filtration

on Sephadex

G-25

instead

Vol. 29, No. 2, 1967

acid-labile

and show no enzymic

sulfhydryl

sulfhydryl free

groups

ethanol free

per mole groups

per mole

groups

The properties Ferredoxin was reduced

be reconstituted

with

2-mercaptoethanol

for

4 hours

salt

and sodium

sulfide.

captoethanol

in the absence

from

in 8 y urea

if

the

separately.

protein

mixtures

could

reconstitution.

ferredoxin-II tained

from either'apoferredoxins

In contrast

treated

with

iron

with

2-mercaptoethanol

of iron spectra. ias

to the behavior

The yield from

and 2-merbe reconsti-

was added from

sulfide

to

incubation and the other

however

it

did

not

sulfide.

apoferredoxin-III

of sulfide.

by the Prior

and urea

showdd full

apo-

P-mercaptoethanol

sulfide

ferredoxins

and sulfide

from

sulfide

or acid-labile

from

pH

of an iron

ferredoxin

or sodium

and a source

and apoferredoxin-III

absorption

Nor could

salt

The reconstituted

the same amounts

had identical

of either

the apoprotein

not be reconstituted sodium

I.

shown in Table if

was recovered,

be reconstituted

an iron

are

could

salt

reconstitution

of

to the addition

or the sodium

iron

The yield

70%.

75% of the apoprotein

the

amounts

'of the awfeyredoxin-III for

salt

About

for

significant

of both

iron

the

and 0.1 E TrissHCl,

salt,

7

prevents

prior

previously

either

required

Ferredoxin addition

the

containing

components contain

only

about

in 8 E urea

of urea.

the apoferredoxin

oxidation.

apoferredoxin-II

of an iron

of free

of P-mercapto-

apparently

is

of

contains

The presence

from

from

Ferredoxin

by the addition

0.7 moles

ferredoxins

at room temperature

ferredoxin-II

tuted

of protein.

of reconstituted

content

apoferredoxin-III

ferredoxin

could

8.5,

while

in their

in their

contains

of the apoprotein from

are similar

differ

of apoferredoxin-III

II and III

apoferredoxins

but

of protein

in the preparation sulfhydryl

activity

Apoferredoxin-II

groups.

sulfhydryl

II and III

Apoferredoxins

sulfide.

properties free

BlOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS

formed

treatment

was not from

enzymic activity

as the native of ferredoxin

required

both

apo-

and con-

ferredoxin, reconstituted

70-908.

of apoferredoxins-I

and II,

apoferre-

and

Vol. 29, No.2,

1967

BIOCHEMICAL

doxin-III

binds

absence

of Na2S.

lower

than

protein

some iron

that

nor

its

content.

isolated

does it

after

groups,

not

In previous

(Malkin

they

for

1966a)

ferredoxin

could

be reconstituted

if

groups

had been

the mercury-containing

mercaptoethanol described they

are

free

differ

supported

report

reconstitution the addition

with

2-mer-

from

E-mercapto-

(Lovenberg,

these

et aZ., ferredoxin

sulfhydryl

apoproteins

treated

with

salt

the preparation

However, in which

as the S-mersalyl

were

groups.

apoferredoxins.

isolated

of an iron

of

by Suzuki

"acid-labile"

by the findings

and Rabinowitz,

teins

apoprotein

from

Prior

derivatives

an excess

and NazS.

previously

the

of 2-

The apoproteins

described

in

that

of mercury.

The recent as the source

from

protected

in the presence

here

prepared

no detectable

from

free

in the case of apoferre-

contained

not be reconstituted

can

possesses

from a',a'-dipyridyl-treated

could

sulfhydryl

salt.

ferredoxin

in 8 E urea

derivatives

Ferredoxin

free

sulfhydryl

apoferredoxin-mersalyl

and Rabinowitz,

of an iron that

to

in the protein

to disulfides.

apoferredoxin

prepared

equivalent

the apoferredoxin

are oxidized

is

iron-containing

be detected

absence

reconstitution

studies,

and apoferredoxin

could

in the

protein

test

experiments

if

salt

of acid-labile

the enzymic

these

to free

is necessary

ethanol-treated

in

NazS in the

if

the

amounts

sulfide

from

an iron

However

significant

apoferredoxin

but

with

to the recovered

ferredoxin.

with

of the disulfides

doxin-11:

bound

activity

appears

from

captoethanol

1963)

exhibit

It

incubated

of iron

in native

incubation

be reconstituted

reduction

is

No acid-labile

Discussion--

sulfhydryl

it

does not contain

sulfide, iron

The amount found

isolated

when

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1966a),

sulfide reported

sulfide

(1967)

in non-heme

in our

previous

nor by the observation

of ferredoxins of sodium

and Kimura

acid-labile

as well

as of iron

251

iron

of cysteine

proteins

is not

investigations

reported

containing

described.

in support

here, sulfide

to the

three

(Malkin since requires apopro-

the

Vol. 29, No.2,

1967

BIOCHEMICAL

Supported, Institute

in part,

of Arthritis

Health

AND BIOPHYSICALRESEARCH

by Research

and Metabolic

Grant

A-2109

Diseases,

from

United

COMMUNICATIONS

the

National

States

Public

Service. REFERENCES

Buchanan,

B. B.,

Lovenberg,

W., and Rabinowitz,

J.

C., Proc.

NatZ. Acad.

sci. u. s., 49, 345 (1963). _m Doeg,

K. A.,

Lovenberg, 238, s-m Malkin, Malkin, Suzuki, Tyuma,

R.,

D. M., Arch.

and Ziegler, W., Buchanan, 3899 (1963).

B. B.,

Biochem.

and Rabinowitz,

Biuphys., J.

97, 37 (1962).

..,5

C., J. BtioZ. Chem.,

and Rabinowitz,

J. C.,

Biochemistry,

R., and Rabinowitz, 822 (1966b).

J. C.,

Biochwn. Biophys. Res. Conmun., 23, -w

K., I.,

and Kimura, Benesch,

T.,

R. E.,

5, 1262 (1966a).

Biochem. Biophys. Res. Commun.,22, 514 (1967). and Benesch,

252

R., Biochemistry,

5, 2957

(1966).