The preparation, properties, and metabolism of 14C-bicarbonate-labelled transferrin

Vol.53,No.l,

BIOCHEMICAL

1973

THE PREPARATION,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

PROPERTIES,

14C-BICARBONATE-LABELLED

AND METABOLISM

OF

TRANSFERRIN*

J. Martinez-Medellin** and H.M. Schulman+ Lady Davis Institute for Medical Research Jewish General Hospital, Montreal, Canada. Received

May

7,

1973

Summary: 14C-bicarbonate-labelled transferrin was prepared in order to study the role of bicarbonate in the cell-mediated release of iron from transferrin. "C-bicarbonate bound to transferrin only in the presence of iron and with a ratio of bound bicarbonate to bound iron of one. The transferrin"C-bicarbonate complex was very stable in Tris-HCl buffered at pH 7.59.0 even in the presence of excess non-radioactive bicarbonate. However, oxalate, citrate, and phosphate promoted a rapid exchange of transferrinbound 14C-bicarbonate with bicarbonate present in the medium. Rabbit reticulocytes effected a temperature-dependent release of 14C-bicarbonate from transferrin at the same rate at which they incorporated the existence of a coordinated mechanism "Fe from transferrin -- suggesting in the cells for the release of both iron and bicarbonate from transferrin. Introduction: Transferrin erythroid

cells

has been well from

for

transferrin

ferric

iron

and for forming

broad

absorption

Permanent

chemically, is

peak with

by a grant address:

t To whom correspondence

synthesis.

apotransferrin

bound with

one molecule

a characteristic

a maxiumum around

stability from

constant

the Medical

Facultad Autonoma should

Cop.vrighr 0 I9 73 by Academic Press. Im. All rights of reprohtctiorl iti arz.y form reserved.

iron

to developing

Although whereby

the protein

iron

is

released

unknown.

Fe ut

a complex

transports

the mechanism

conditions

each

has an apparent

which

in hemoglobin

physiological

(1),

* Supported

serum protein

utilization

by cells

bound

complex

the

characterized

Under

*

is

can bind

two atoms of

of bicarbonate salmon-pink

465 mu. greater

Research

The iron

than Council

lO24M-1,

32

color

having

in this which

of Canada.

de Ciencias, Universidad National de Mexico, Mexico 20, D.F. be addressed.

is also

would

a

BIOCHEMICAL

Vol. 53, No. 1,1973

appear

to exclude

transferrin

the

in controlling mechanism

of release

there

anions;

for

‘transferrin, the

role

is

Materials

paper

the

release

of iron

from

transferrin

for

would

in the

studying

(4)

binding

be an ideal release

a variety

tool

of iron

on the stability

under

of the

in

bicarbonate

(1))

in the cell-mediated

transferrin

an understanding

role

red

states.

or oxalate

data

a significant

may be important

requirement

citrate

may play

(3),

and pathological

as studied

from

iron

transferrin

we present

complex

of iron

from

labelled

of the anion

Fet*-H14C03-

of the

incubated

certain

of iron

to

for

from

studytrans-

transferrin-

of conditions,

by reticulocytes

(or

including

the

in vitro.

and Methods: NaH14C03 (42.4

HCl were

previously

mCi/m mole)

purchased

Transferrin described

gradients

(6)

from

from

anemic

and incubated

as ferric

chloride

in

Corp.

rabbit

serum

with

Chicago

low background

Searle)

and counted

rabbits

were

as previously in a Nuclear

14Gbicarbonate

pretreated

from

mCi/mg)

and charged

with

iron

as

(5).

5gFe was measured containing

and 5gFe (3-30

Amersham/Searle

was purified

Reticulocytes

were

for

synthesis

an absolute

example,

In this

release

hemoglobin

normal

H14C03--

ferrin.

O.lN

as a mechanism

of intracellular

of iron

in both

Because

ing

availability

the rate.of

formation

other

diffusion

(2). Since

cell

simple

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

were

Triton

described Chicago

either

X-100

washed

counter

or dissolved

in a Nuclear

Chicago

gamma counter.

to aluminum

Chem. Co.)

planchettes

and counted

in NCS Solubilizer Unilux

on dextran

(5).

automatic

applied

(Sigma

and purified

II-A

liquid

Samples which

in a Nuclear (hersham/

scintillation

counter. It

had been determined

are dried

on planchettes

lost

the

the

into

planchettes.

transferrin-bound

atmosphere,

that

when samples

at room temperature, while

On the other

containing free

transferrin-bound hand,

NCS dissolves

14C-bi carbonate. 33

14C-bicarbonate

14Lbicarbonate

is

14C-bicarbonate and fixes

rapidly

remains both

free

and

on

Vol.53,No.1,1973

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE I

Transferrin* (mu Moles)

Hl4CO - Bound* (mp Mofes)

% Saturation* with iron

-iron

157

1.4

< 0.5

tiron

473

867

92

*After

dialysis

for

24 hours against

Molar Ratio Hl4C03-/Transferrin

0 1.8

0.05 M tris-HCl

buffer,

pH 7.5.

Values used in the calculations: Transferrin

mol. wt.

Apotransferrin: Transferrin:

= 76,000 (7)

E 430 m,, = 11.1 (8) E Ifsmu = 0.6

To form 14C-bicarbonate-labelled of 50 mM Tris-HCl ature with

buffer,

a six-fold

amount of iron

against

transferrin,

pH 8.3, was incubated

molar excess

two 4 liter

apotransferrin for

contained

The solution

saturating

120 mg of sodium citrate

was then dialyzed

changes of 5 mM Tris-HCl

in 1.0 ml

one hour at room temper-

of NaHl4C03 and a two-fold

-- the iron solution

5.6 mg of FeC13 per ml.

(7)

buffered

and

for 20 hours at 4'C at pH 7.5.

Results: Table I shows that we obtained H14C03- and Fe*' shows that

to apotransferrin

Tris-HCl

buffered

at pH 7.5.

appreciable

loss

stored

at -20°C the radioactivity

Hl4C03-

of radioactivity, was lost

atmospheric

conditions.

complex was very dialyzed

stable against

of

It also in

in 0.05M this

and when the complex was

at a rate

COP).

34

of binding

is not bound to apotransferrin

It could be extensively

without

by exchange with

stoichiometery

our incubation

bicarbonate

The transferrin-

buffer

(probably

(1) with

under these conditions

the absence of iron.

the expected

of about 4% per month

Less than 10% of the radiIactivity

BIOCHEMICAL

Vol.53,No.1,1973

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

60

120

180

240

300

Minutes

Figure

1:

The effect of various anions on the stability of the transferrin Hl4CO3- complex. Transferrin - Hl4C03- solutions, at a concentration of 3.3 mg/ml in 50 mM?ris-HCl, pH 7.5, were incubated at room temperature, At the indicated times samples were removed, dried on planchettes, and assayed for radioactivity as described in Methods. A: control, B: NaHC03 (16.8 mg/ml), C: 10 mM sodium citrate, D: 10 mM potassium oxalate or 100 mM sodium phosphate.

was lost ing

in two hours

a lOO-fold

excess

On the the

other

radioactivity

order

hand,

loss there

either

citrate,

oxalate,

than

the complex

with

experiment, It

that

in Tris-HCl

being

buffer

contain-

than

iron

we conclude

competing

anions

anions from

35

With

the (there

all

three

in the A465m,, during with

extinction that were

a rapid

Fe*

5 and

coefficient

appreciable

transferrin

promoted

1);

> phosphate

of apotransferrin

have a lower

was not removed

the competing

(Figure

> citrate

a 6% decrease

(1), the

appreciably

oxalate

the complexes

bicarbonate

or 100 mM phosphate

in 10 mM phosphate).

or phosphate

with

10 mM oxalate,

decreased

anions

Since

complexes

and that

seems likely

10 mM citrate,

was less

of incubation.

of the

in

of radioactivity

hours

trations

was incubated

of NaHC03.

of these

was no measurable anions

complex

of the transferrin

of activity

competing

when the

not

formed

concenduring

by these

agents.

exchange

between

the

BIOCHEMICAL

Vol.53,No.1,1973

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Figure 2: Incorporation of 5gFe from transferrin and removal of Hl4COq' bys 25% from transferrin cell suspensions, were incubated in medium (5) containing 0.51 mg/ml of iron-saturated transferrin labelled with either 5gFe or Hl4C03-. Curve A: The stability of Hl4CO3?-transferrin at 0°C. Reticulocytes were incubated at 0°C in mediumcontaining H"CO - transferrin. At the indicated times portions of the cell suspension iere removed and the radioactivity determined as described in Methods. Curve B: Incorporation of 5gFe from transferrin by reticulocytes at 37'C. Reticulocytes were incubated at 37°C in mediumcontaining 5YF transferrin. At the indicated times portions of the cell suspension we:; removed and cooled in an ice bath. The cells were washed five times in normal saline by centrifugation and resuspension at 4"C, after which they were assayed for 5gFe. Curve C: The removal of Hl4CO3- from transferrin by reticulocytes at 37' i: The procedure was the sameas for Curve A except that the suspension was incubated at 37Y. transferrin-bound

bicarbonate and bicarbonate present in the mediumwhich was.

formed from dissolved atmospheric CO2. To see whether a relationship of iron from transferrin

exists between the cell-mediated

and the transferrin-bound

were independently incubated with transferrin or H14C03-. In both cases iron-saturated

bicarbonate,

reticulocytes

labelled with either

transferrin

at a final

removal

5gFe+++ concentration

of 0.51 mg/ml was present in the medium. As can be seen in Figure 2 there was an excellent cells

correlation

between the rate of incorporation

of 5gFe into the

and the rate of disappearance of H14C03', which was presumably lost to

36

Vol.53,No.1,1973

the atmosphere.

There

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

was no loss

of HT4CO3-

medium

at 37OC in the

absence

medium

at O°C in the presence

from

of reticulocytes,

the transferrin

or from

in the

transferrin

in the

of reticulocytes.

Discussion: Our finding of H14C0 3 - is Like

them,

confirms

we also

found

that

can displace authors

of transferrin were

likely

bicarbonate

the

conditions into

in which

hemoglobin.

of iron

from

transferrin ing

can actively

reticulocytes that

between

complex, behaviour

even

of the

the

and the

release

events

are

in our transferrin-

in the

presence

complex

iron

between

that

medium.

incorporate

these

suggests

anions

reaction

our

under

from

kinetics

transferrin

of incorporation

of H14C03coordinated

from in develop-

cells. bicarbonate

in the

removal

to result

in the

the physiologically (2),

of suitable of bicarbonate

release

metabolize

We would

is complex

absence

enzymatic

further

the

reaction

amounts

because

competing

in the

unlike

of appreciable

This

(1).

citrate,

However,

CO2.

by the

correlation

into

case oxalate,

probably

of atmospheric

us to examine

seem to indicate

transferrin-iron

iron

anions,

of an exchange

reticulocytes

transferrin would

Since the

competing

dissolved

The excellent

erythroid

in this

of the transferrin-H14C03-

NaHC03, allowed

of Young and Perkins

the formation

bicarbonate

result

one molecule

from transferrin.

presence

and bicarbonate

The stability of excess

the

of radioactive

was most

anions,

not detect

with

to apotransferrin

observations

bicarbonate

done in the

displacement

the

certain

we could

complexed

experiments

bound

each Fe +++ bound

bound

the previous

system

for

also

and phosphate,

the

that

of iron

and since anions

significant transferrin

(4),

it

from

transferrin

from

the protein,

is

anion

involved

in

does not complex conceivable

by cells thereby

that

would

with

the

be sufficient

enabling

the

cells

the iron.

like

to acknowledge

the excellent

Rose Sidloi.

37

technical

assistance

of

to

Vol. 53, No. 1,1973

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

References: 1. ::

4. 5. 6. 7. 8.

J.W. Young and D.J. Perkins, Eur. J. Biochem 4, 385 (1968) P. Aisen and A. Leibman, Biochem. Biophys. Res. Commun 32, 220 (1968) H.M. Schulman, Biochim. Biophys. Acta, 155, 253 (1968) E.M. Price and J.F. Gibson, Biochim. Bi=ys. Res. Commun 46-, 646 (1972) J. Martinez-Medellin and H.M. Schulman, Biochim. Biophys Acta 264, 272 (1972 H.M. Schulman, Biochim. Biophys. Acta 148, 251 (1967) R.M. Palmour and H.E. Sutton, Biochem.-T& 4026 (1971) J.H. Katz, J. Clin. Invest. 40, 2143 (1961

38