THE JOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 242, No. 19, Issue of October 10, pp. 42864286,1967

Printedin u.s..~.

The Kinetics and Mechanism of Fe(II1) Exchange between

Chelates and Transferrin
III. THE AMOUNT OF IRON-ETHYLENEDIAMINETETRAACETATE INITIALLY BOUND TO PROTEIN*

(Received for publication, March 20, 1967)

CAROLYN BILLUPS, LEON PAPE,$ AND PAUL SALTMAN$

From the Graduate Program of Biochemistry, University of Southern California, Los Angeles, California 90007

SUMMARY approximately 5% of the iron-EDTA was bound to the protein.

Under quite similar conditions, Aisen el al. estimated that
With the use of Sephadex gel and collodion membrane
approximately 85% of the protein iron-binding sites w-ere filled
filtrations it has been possible to determine the fraction of
in the same time period.
iron-ethylenediaminetetraacetate which reacts rapidly with
This discrepancy is of particular significance in light of the
apotransferrin. The values obtained for ratios of iron-EDTA
mechanism of transfer we proposed. Our kinetic measurements
to protein binding site of 1: 1 (4 to 7% of binding sites oc-
were attributed to formation of a reactive species of iron-EDTA,
cupied) and 5 : 1 (37% of binding sites occupied) correspond
Fe-EDTA*, which is able to interact with the apoprotein.
with similar values obtained by spectrophotometry. The
That Fe-EDTA* constitutes but a small fraction of the total
suggested participation of a chemically active iron-EDTA
iron-EDTA was deduced from the spectral concentration of
species in the reaction is further strengthened.
the intermediate formed in the initial reaction. Upon learning
of the Swedish group’s results, we repeated our spectral experi-
ments several times and were able to confirm our initial findings.
We therefore felt it imperative to confirm or reject these meas-
urements with the use of independent techniques involving
We have recently reported on the mechanisms involved in neither optical spectrometry nor electron paramagnetic resonance.
the transfer of iron from low molecular weight chelates, including This paper presents results obtained by determining the amount
citrate, nitrilotriacetate, and ethylenediaminetetraacetate to of iron-EDTA-protein by means of the techniques of Sephadex
the specific /3i iron-binding protein, transferrin (1, 2). The gel filtration as w-e11as collodion membrane filtration to separate
kinetics of the transfer reactions was determined by visible high and low molecular weight species of the iron.
and ultraviolet spectrophotometric techniques. The results
EXPERIMENTAL PROCEDURE
of these studies led us to postulate the formation of an inter-
mediate complex between the iron chelate and the protein. Apotransferrin solutions were prepared with protein obtained
This intermediate then dissociated to yield the metalloprotein from Behringwerke (Marburg-Lahn, Germany). The protein
plus the free chelator. Similar experiments by Aisen et al. was extensively dialyzed against 5 x 10e3 M Tris-HCl buffer,
(3) extended these observations to include measurements of the pH 7.4, prior to use, in order to remove low molecular weight
chelating agent-iron-protein complex with the sensitive method chelating agents known to be present in the preparations of
of electron paramagnetic resonance. Although qualitatively this material. Final protein concentrations of both 1O-4 and
their results were quite similar to ours, there was a quantitative 10e3 metal binding eq per liter for the gel filtration experiments
discrepancy in the fraction of the iron chelate presented to the and 10e4 metal binding eq per liter in the membrane filtration
protein that was bound in the initial rapid reaction. When experiments were used. The determination of the values was
iron-EDTA was present in the same concentration as the carried out as described previously (4). All solutions were
iron-binding sites available on apotransferrin, we found that prepared in 5 x 10e3 M Tris-HCl buffer, pH 7.5, and equilibrated
* This research was supported by the John A. Hartford Founda- with the laboratory atmosphere by extensive aeration prior to use.
tion, Inc., and United States Public Health Service Award AMO5- Radioactive 59Fe-EDTA solutions were prepared with ferric
484. nitrate (Mallinckrodt) and disodium-EDTA (Mallinckrodt) at
$ Permanent address, Department of Physics, California State final concentrations of 10m3 M each in the stock solution. Isotope
College at Los Angeles. was obtained as 59FeC18 (New England Nuclear) and added
$ Research Career Development Awardee, United States Public
Health Service. Present address, University of California, San in sufficient amounts to the ferric nitrate solution prior to the
Diego, LaJolla, California. addition of chelating agent to provide approximately lo6 cpm

4284

This is an Open Access article under the CC BY license.

Issue of October 10, 1967 C. Billups, L. Pape, and P. Xaltman 4285

per ml. The disodium-EDTA was added, and the solution was TABLE 1
adjusted to pH 7.5 with 1OP M NaOH. Radioactivity was Estimation of 5sPe-EDTA bound to apotransferrin
with
determined in all of the solutions assayed with a y-well NaI membrane filtration
scintillation crystal and a pulse height analyzer peaked at The reaction mixture (volume 1.0 ml) was 5 X 10e3 M in Tris-
1.1 m.e.v. HCl, pH 7.5, and lo+ M in protein metal binding sites. SSFe-
Sephadex G-25 (coarse) u-as washed and equilibrated with EDTA was added to provide 1: 1 and 5: 1 ratios of iron chelate to
5 x 10V3 M Tris-HCl, H 7.5, and a 28.5 X 3.0 cm column wa.s binding site. The membranes were calibrated with 1OP M @Fe-
EDTA and 10e4 M 59Fe-transferrin. Results are averages of
prepared. The column was calibrated isotopically with 5sFe-
four experiments under each condition. Values for the per-
EDTA in the absence of protein and spectrophotometrically centage of the protein binding sites filled with 59Fe are compared
with Fe-transferrin prepared from Fe-nitrilotriacetate, a chelat- with values calculated from spectral measurements under similar
ing agent known to yield quantitatively all of its iron to the conditions (2).
protein within the first few seconds of the reaction (1). The
Binding sites filled
materials were eluted wit,h 5 x 10-Z M Tris-HCl buffer, pH 7.5, Radioactivity
With 69Fe
at a flow rate of 1 ml/100 sec. Fractions of 1 ml were collected, Conditions
and the radioactivity was determined sts described above. The Filtrate Membrane NOW Membrane Spectros-
filterable filtration COPY
absorbance was measured at 470 rnp to determine the concentra- I i

tion of Fe-transferrin. cflmx lo-3 % %

Collodion membrane filtration was also used. The membrane 59Fe-EDTA 78.1 l.B 0
filters were obtained from Carl Schleicher and Schiill Company 59Fe-transferrin. 0.5 0.9 49.1 97.2 100
and extensively washed with disodium-EDTA, then HzO, 59Fe-EDTA-apo-
and finally Tris buffer. They were mounted in the glass suction transferrin (1: 1). 53.5 1.5 4.2 7.1 5.5
apparatus also supplied by Carl Schleicher and Schiill. The 69Fe-EDTA-apo-
transferrin (5: 1) 247.7 3.7 20.0 3G.8 27.4
radioactive solution was placed in the collodion bag, a vacuum
was established in the outside chamber, and the total con-
tents of the bag were filtered. The collodion membrane was solutions were placed on the Sephadex column and the elution
then washed four times with Tris buffer, and the total amount commenced. The pattern of radioactivity in the 5sFe-EDTA
of radioactivity in the filterable fraction was determined. The
plus apotransferrin experiment is compared with that obtained
nonfilterable fraction was taken up in 1.0 ml of the Tris buffer with SSFe-EDTA (Fig. 1). It is clear that the low molecular
and counted. The radioactivity remaining in the membrane weight chelate and the protein-bound iron are resolved by this
was also determined. The membranes were calibrated against technique. When 5SFe-EDTA and apoprotein are mixed, only
SSFe-EDTA and 59Fe-protein. The total elapsed time for each a small fraction, approximately 4 y. of the total activity present,
experiment was less than 1 hour. moves with the high molecular weight fraction. The position
of the small amount of high molecular w-eight material corre-
RESULTS AND DISCUSSION
sponds to that observed with the Fe-protein as the reference
Filtration with Sephadex G-%-Solutions containing final substance. Similar results w-ere obtained with both 10e3 and
concentrations of either 10e3 or low4 M apotransferrin and 10e3 10m4 M apoprotein.
or lOA M 59Fe-EDTA were prepared in a total volume of 0.45 Filtration with Collodion Membranes-Experiments similar to
ml. Carrier Fe-transferrin, 5 x 1O-4 M, was added, and the those described above mere carried out with the membrane
technique. The final concentration of apotransferrin was 10-J
M. The final volume of reaction mixture uas 1.0 ml. The
results from a series of experiments in which ratios of 5sFe-EDTA
to protein binding sites were 1: 1 and 5 : 1 are shown in Table I.
These results confirm and extend those obtained by Sephadex
gel filtration and corroborate the initial spectrophotometric
results reported in our earlier communication (2). The values
for the fraction of complex were obtained from spectral data
=‘Fe - EDTA + ApoTrf
(see Fig. 4, Reference 2) with a molar absorptivity of 6520 = 1300
for each metal binding site.
We believe that the results presented in this paper confirm
our previous results that only a small fraction, approximately
5% of the iron-EDTA present in solution, is in a form which
can rapidly combine with the binding sites of the protein. As
we have proposed, it would appear that the formation of an
active species, Fe-EDTAX, precedes the binding of the iron
chelate to the protein to form the ternary complex. The
Volume (ml.) nature of this ligand exchange process is presently under in-
FIG. 1. Elution pattern of radioactivity of apotransferrin, vestigation.
lo-3 M binding sites per liter, mixed with 6sFe-EDTA, 1O-3 M We are unable to resolve the discrepancy between the results
(O-O), with a Sephadex G-25 column. The elution pattern obtained with electron paramagnetic resonance (3) and the
of iron transferrin (O----O) measured spectrophotometrically
and 6”Fe-EDTA (- - -) are superimposed for comparison. spectrophotometric (2) and filtration data presented here. It
4286 Kinetics of Fe Binding by Transferrin. II1 Vol. 242, No. 19

might be argued that the iron-EDTA forms both a loosely and REFERENCES
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rium between Fe-EDTA and anotransferrin is nerturbed bv 2. BATES,G. W., BILLUPS,C., ANDSALTMAN,P., J. Biol. Chem.,
filtration, then only the tightly &bound protein complex would 242, 2816 (1967).
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