The role of ferroxidase-II and a ferroxidase inhibitor in iron mobilization from tissue stores

Vol.

96, No.

October

4, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages

31, 1980

THE

ROLE

OF FERROXIDASE IN IRON

-11 AND

MOBILIZATION

FROM

Richard W. Topham, G. Peyton Neatrour, Richard B. Russo,

September

TISSUE

INHIBITOR

STORES

James H. Woodruff, Melissa P. Calisch, and Mark R. Jackson

Department University Virginia Received

A FERROXIDASE

1532-1539

of Chemistry of Richmond 23173

17,198O SUMMARY

Injection of ferroxidase-II into copper-deficient rabbits resulted in a rapid, large, increase in the serum iron concentration which was equivalent to the increase observed when ceruloplasmin was injected into the same A recently discovered serum inhibitor of ferroxidase-II, was also animals. shown to potently inhibit ceruloplasmin. Acceleration of iron mobilization from storage tissues by dietary manipulation or repetitive bleeding of rabbits leads to a large decrease in the serum content of the inhibitor and a corresponding increase in the total serum ferroxidase activity. These studies suggest that ferroxidase-II could serve as a viable, alternative mobilizer of iron from tissue stores and that the recently discovered serum ferroxidase inhibitor could participate in the regulation of the efflux of iron from tissue stores. INTRODUCTION Numerous have

--in vivo

established

of iron

from

that tissue

ceruloplasmin stores.

the mobilization

of iron

into

(1,2.

transferrin

shown

to be essential

provides, iron

at least

metabolism

and in vitro

studies

It has been

for in part,

The

its ferroxidase the much

tightly

sought

Inc. reserved.

1532

(6,8,

ceruloplasmin

“missing

to ceruloplasmin 9). link”

(l-7)

the mobilization

and incorporation

bound

activity

(10).

Z 1980 b! .4cademic Press, Q/‘ reprodrrcfior~ in anx ,/brtn

that

the oxidation

copper

laboratories

facilitates

postulated

0006-291X/80/201532-08$01.00/0 Copwght .A11 righrs

several

(ferroxidase-I)

by promoting

4, 6).

from

Thus, between

facilitates of iron has

been

ceruloplasmin copper

and

Vol.

96, No.

4, 1980

BIOCHEMICAL

An objection that

anemia

by very

to this

is rarely

low

proposed

observed

plasma

AND

in Wilson’s

ceruloplasmin

human

IV-I

disease

serum

account

for

has

the fact

that

can be accounted

may

account

To date, ceruloplasmin

ferroxidase-II

play

plasmin

been

communication

its purification

Mature,

studies

discovered

the iron

disease.

reported

iron

from

alternative

which

serum

mobilization

for

rabbit

it has stores

ceruloinhibitor

serum

indicate

(13).

that

of iron

ferroxidase facilitated

for

tissue

serum

from

and

Thus,

the --in vivo mobilization

from

inhibitor by both

cerulo-

ferroxidase-II.

MATERIALS Animals.

to those

of a naturally-occurring,

of promoting

in regulating

(10,12)

ferroxidase-II.

as a viable

serum

in Wilson’s

can mobilize

describes

the recently

(ferroxidase-1)and

with

would

with

ceruloplasmin

similar

during

Wilson’s

is associated

studies,

serve

from

from

of ferroxidase-II

observed

performed

thereby,

and

existence

is rarely

ferroxidase-II

obtained

and that

a role

anemia

and purification

(ferroxidase-II)

serum,

activity

is

characterized

identification

of plasma

the existence

is capable

stores

could

have

for was

present

The

ferroxidase

mobilization

and,

Evidence

The

that

whether

system

of ferroxidase-II

tissue

iron

ascertained

plasmin.

(11).

for by the content

(3, 5,7),

in an --in vivo

reported

a disorder

The

of human

COMMUNICATIONS

of ceruloplasmin

ferroxidase

fraction

that more

for the fact

no in -- vivo

not been

been

role

disease,

(3,6,10).

copper-containing,

the Cohn

RESEARCH

physiological

of a non-ceruloplasmin, serum,

BIOPHYSICAL

female

AND New

METHODS

Zealand

White

rabbits

were

used

in these

studies. Diets. The copper-deficient purchased from ICN Nutritional ceiving copper-deficient or low

The

Assay enzymic

and low iron diets used in these studies were Biochemicals, Cleveland, OH. Animals reiron diets also received doubly deionized water.

of Ferroxidase Activity and the Inhibition oxidation and incorporation of iron into

1533

of Ferroxidase Activity. transferrin was measured

Vol.

96, No.

BIOCHEMICAL

4, 1980

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

exhibits maximal spectrophotometrically at 460 nm where Fe 3+-transferrin absorbance. This assay for enzymic Fe3+ -transferrin formation has been described in detail and validated in numerous previous reports (1,2,4,6,7,8-15). In assays containing no inhibitor, each cuvette (1.8 ml capacity) contained 0. 3 50 ml of a 0. 6 M acetate buffer, pH 6. 0; 0.250 ml of a 2% (w/v) apotransferrin solution: 0.300 ml of a 4 x 10e4 ferrous ammonium sulfate solution; 0.100 ml of a solution of either whole serum, ferroxidase-II, or ceruloplasmin; and 0. 100 ml of 0.05 M acetate buffer, 0. 15 M NaCl, pH 5. 5. In assays containing inhibitor, the 0. 100 ml of 0. 05 M acetate buffer was replaced by 0. 100 ml of a solution of the inhibitor which was prepared in the same buffer. The amount of inhibition was determined by obtaining the difference in the initial velocities of the ferroxidase reaction of assays with and without inhibitor and was reported either as % inhibition or as A( AA46Ofmin). Preparation and Injection of Ceruloplasmin and Ferroxidase-II. Purified ceruloplasmin and ferroxidase-II were obtained as previously described (13, 16). Prior to injection, purified ceruloplasmin and ferroxidase-II preparations were dialyzed with mammalian Ringer’s solution, In the case of ceruloplasmin, each rabbit received intravenously 1. 36 ml possessing a total ferroxidase activity content of 32. 5 mg. of 6.85 AA460 /min and a total protein In the case of ferroxidase-II, each rabbit received intravenously 2.00 ml possessing a total ferroxidase activity of 6. 48 A A460 /min and a total protein content of 37.0 mg. Determination of Serum Iron Concentrations. The total iron concentration of serum samples was determined utilizing Sigma Diagnostic Kit No. 565 MO) which is based on the quantitative color(Sigma Chemical Co. , St. Louis, metric determination for serum iron reported by Persijn et al. (17). Determination of Total Ferroxidase Activity and the Total Inhibitor ConSerum samples were immediately assayed for tained in Whole Rabbit Sera. ferroxidase activity and the total ferroxidase activity per ml of whole serum calculated. To analyze the total content of ferroxidase inhibitor present in these serum samples, it was necessary to separate the inhibitor from the This separation was effected by gel-filtration on ferroxidase enzymes. Sephadex G-200 as previously described (13). Three ml samples of serum were applied to a column (1.5 x 45 cm) of Sephadex G-200 and protein was eluted from the column with 0. 05 M acetate buffer, 0. 15 M NaCl, pH 5. 5. Three major bands of protein were eluted from the column. The first of these bands contained the serum ferroxidases and the third band contained the ferroxidase The fractions comprising the third band were combined and coninhibitor. centrated by lyophilization. The resulting residue was redissolved in 2 ml of 0.05 M acetate buffer, 0. 15 M NaCl, pH 5. 5. The inhibitory power of this reconstituted sample was analyzed against ferroxidase-II as previously described and the total inhibition per ml of original serum calculated.

RESULTS To ascertain an --in vivo

system;

whether the effects

ferroxidase-II

was

of ceruloplasmin

1534

capable

of mobilizing

and ferroxidase-II

iron injection

in on

Vol.

96, No.

4, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

TABLE

EFFECT ON

OF

THE

THE

SERUM

INJECTION IRON

OF

RESEARCH

I

FERROXIDASE-II

CONCENTRATION

AND

IN

Time

Serum Concentration

Ferroxidase-II Fe/100

ml

serum)

54.8

70.1

1 hr.

110

204

1 day

151

281

iron

tained

on copper-deficient

concentration

iron

concentration

were

achieved.

with

an amount

dase

activity

were

were The

monitored

copper

same

copper

which

had a total This

The

ferroxidase ferroxidase-II

iron

concentration

recently

discovered

to see if it might and also

inhibit

with

(3, ‘7).

activity injection (Table (13) be a more

were

White

injected

then

also

values

serum

experiments

an amount

for both

intravenously

in serum

an additional

thirty

ferroxi-

iron

con-

performed days,

these

of ferroxidase

-II

to the ceruloplasmin resulted

main-

and serum

injected

increase

with

equivalent

activity

minimal

similar

After

rabbits

20% of the total

resulting

favorably

ceruloplasmin.

until

equalled

The

were

Zealand

ferroxidase

animals

which

animals animals

serum

-deficient

I ) compared

-deficient

in New

periodically

animals.

copper-deficient

in the serum

The

of ceruloplasmin

(Table

injected.

compared

diets.

of normal

centration

activity

Iron

0

serum

vestigated

RABBITS

Geruloplasmin (ug

in other

CERULOPLASMIN

COPPER-DEFICIENT

After

Injection

COMMUNICATIONS

in a rapid,

previously large,

increase

I). serum

inhibitor

general When

1535

of ferroxidase-II

inhibitor

of serum

samples

of purified

was

in-

ferroxidase ceruloplasmin

Vol.

96, No.

4, 1980

BIOCHEMICAL

EFFECT

OF

DIET

ON

ACTIVITY

Rabbit

AND

THE

AND

BIOPHYSICAL

TABLE

II

SERUM

CONTENT

THE

OF

FERROXIDASE

Diet

Total

I

Diet,

Low

Iron

Refed

2

Low

Ferroxidase Activity

Refed

were with

tested

with

White

for

weeks,

ferroxidase

1.17

1.14

2.13

0.437

1.12

1.28

Diet,

the total

amount values

comparable amounts

compared

rabbits then

the possible

physiological

maintained

first

on a diet

greatly

of ferroxidase

of low

iron

iron

were

for

the animals

were

from

74% inhibition

was

of this

ferroxidase

on a diet

four

for

on a low

1536

a diet

tissues;

of normal four

weeks.

and the total refed

serum

storage

and the total

maintained inhibitor

role

content

content

decreased

activity, observed

ferroxidase-II.

of iron

were

of ferroxidase

of the inhibitor,

of the efflux

the animals

when

amounts

to 53% with

of normal

inhibitor after

4 weeks

equivalent

to a diet

increased

to normal

0.924

in the regulation

Zealand

a return

0.913

4weeks

4weeks

To investigate

four

0.298

possessing

ceruloplasmin

inhibitor

2.80

4weeks

and ferroxidase-II,

A(AA460/min/mlserum)

4weeks

Diet,

Normal

serum)

Ferroxidase Inhibition

0.977

Diet,

Diet,

Total

0.925

Normal

Iron

FERROXIDASE

4 weeks

Diet,

Normal

COMMUNICATIONS

INHIBITOR

(AA46g/min/ml Normal

RESEARCH

iron

weeks, The

ferroxidase iron

two New

diet

followed

total

amount activity

(Table

ferroxidase of normal

content

of greatly

II).

activity iron

by

content.

Both returned

Vol.

96, No.

BIOCHEMICAL

4, 1980

AND

Tome

Fig.

serum

rabbit,

this

corroborate

content

that

of inhibitor

maintained

accelerate animal

on alternate decline

the first ferroxidase

activity

from

for

a period

weeks,

both

hibitor

in the serum

amount

of bleeding (Fig.

the total

iron

of iron

in the total

five weeks

an inverse

of normal

days

1).

ferroxidase returned

COMMUNICATIONS

lweeksl

serum

was

a corresponding

When

the repetitive

observed

was

amount

to from

A steady, during

increase was

a third

removed

occurred

steady

the

bled

weeks.

inhibitor

and the total

to the levels

repetitively Blood

bleeding

between

activity;

consecutive

of ferroxidase

with

Ferroxidase of repetitive

existed

stores.

of nine

of

ferroxidase

content,

tissue

activity

Content 1 = period

relationship

and the total

on a diet

the mobilization

dramatic

RESEARCH

Effect of Repetitive Bleeding on the Serum Activity and the Ferroxidase Inhibition. bleeding; 2= period of no bleeding.

1.

To further total

BIOPHYSICAL

in the total

ceased

for

four

of ferroxidase

at the beginning

in-

of the study.

DISC uSSION These mobilization could

serve

studies of iron

demonstrate from

as a viable

tissue alternative

that stores for

ferroxidase-II

can promote

as effectively

as ceruloplasmin

ceruloplasmin

1537

in Wilson’s

the --in vivo and thus disease

and

Vol.

96, No.

other

sera

differ

markedly

The

only

of low

that

ceruloplasmin

tissue

This

the iron-mobilizing

ferroxidase

bilization

of iron

receiving

low

fed diets

iron

composition

proteins

evidence

of these

serum

(11, 14,15).

possess,

to promote

additional

in the serum

diets

of normal

iron

with

this

activity

are

the copper-

the mobilization

of iron

to support

proteins

the proposal

is a result

be more

iron

of their

lower

levels

Hence,

bled.

discovered,

Thus,

of its mechanism

in the

by the changes observed

inhibitor tissue of this

of action

are

when

observed

and bleeding

from

and characterization

inhibitor

animals

activity

ferroxidase of iron

of animals

content

corroborated

the dietary

pool

the serum was

ferroxidase

serum

of the mobilization

between

was

and the total

the serum

of the same

of the serum

the moiron

of the ferroxidase

the serum

correlation

upon

of the plasma

content.

activity

This

dependent

the maintenance

than

ferroxidase

the purification

and the elucidation

should

Such a correlation

content.

repetitively

recently

for

contain

of inhibitor

in the regulation concerning

should

rabbits.

content

diets

stores

ferroxidase

that was that

iron

tissue

and the total studies

the rabbit

low

diets

of normal

dietary

inhibitor

from

total

of inhibitor

tions

serum

and the ability

capability

receiving

and greater

volved

these

COMMUNICATIONS

and ferroxidase-II

and chemical

provides

receiving

animals

suggest

RESEARCH

activity. Animals

than

which

activity

stores.

BIOPHYSICAL

Ceruloplasmin

properties

features,

ferroxidase

AND

content.

in physical

common

dependent from

BIOCHEMICAL

4, 1980

studies could

stores. serum

currently

in

be inInvestiga-

ferroxidase in progress.

ACKNOWLEDGEMENTS The author wishes to thank Mrs. tion of the typescript. This work was Grant 1 ROI AM 26383-01 and a faculty Richmond. 1.

Osaki, S., 2746-2751.

Johnson,

D.A.,

Dorothy supported research

REFERENCES and Frieden,

1538

Ogden for her help in the preparaby National Institutes of Health grant from the University of

E.

(1966)

J. Biol.

Chem.

-’241

Vol.

2. 3. 4. 5. 6. 7. 8. 9. 10.

11. 12. 13. 14. 15. 16. 17.

96, No.

4, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Osaki, S., and Johnson, D.A. (1969) J. Biol. Chem. 244, 5557-5758. Ragan, A.A., Nacht, S., Lee, G. R., Bishop, C. R., and Cartwright, G.E. (1969) Am. J. Physiol. 217, 1320-1323. Osaki, S., Johnson, D.A., Topham, R. W., and Frieden, E. (1970) Fed. Proc. 2, 695. Roeser, H. P. , Lee, G. R. , Nacht, S. , and Cartwright, G. E. (1970) J. Clin. Invest. 49, 2408-2417. and Frieden, E. (1971) J. Biol. Chem. 246, Osaki, S., Johnson, D.A., 3018-3023. Williams, D. M. , Lee, G. R. , and Cartwright, G. E. (1974) Am. J. Physiol. 227, 1094-1097. Gki, S. (1966) J. Biol. Chem. 241, 5053-5059. 0. (1967) J. Biol. Chem. 242,2653-2657. Osaki, S., and Walaas, Frieden, E., and Osaki, S. (1970) The effects of Metals on Cells, Subcellular Elements, and Macromolecules (edited by Maniloff, J. , Coleman, M. W. ) Thomas, Springfield, IL. 39-72. J. R. , and Miller, Topham, R. W. , and Frieden, E. (1970) J. Biol. Chem. 245, 6698-6705. E. (1967) ClinChem. Johnson, D.A., Osaki, S., and Frieden, -’13 142-150 Chin, J. L., Calisch, M. P. , and Topham, R. W. (1978) Int. J. Biochem. 2, 775-783. D.A. (1974) Arch. Biochem. Biophys. 160, Topham, R. W., and Johnson, 647 -654. Topham, R. W., Sung, S.M., Morgan, F. G., Prince, W. D., and Jones, S.H. (1975) Arch. Biochem. Biophys. 167, 129-137. Deutsch, H. F. (1960) Arch. Biochem. Biophys. 89, 225-229. (1971) Clin. Chem. Persijn, J. P., Van Der Silk, W. , and Riethorst,. Acta 35, 91-98.

1539