Eur. J. Biochem.

21 (1971) 55-59

The Binding of Scandium Ions to Transferrin in wiwo and in witro

Anthony W. FORD-HUTCHINSON
and Deryck J. PERKINS
Chemical Pathology Department, St. George's Hospital Medical School, London
(Received March 11/April 29, 1971)

1. Studies on the binding of scandium t o human apotransferrin showed that two Sc3+were
bound specifically a t the two iron-binding sites.
2. The binding of each Sc3f involves the ionisation of two phenolic tyrosyl residues.
3. The scandium * protein complex has similar resistance to denaturants and chemical reagents
as the iron complex.
4. The only protein complex observed, when quantities ofSc3f below the free iron binding capa-
city of serum were added t o whole serum in vitro, was the Sc3+* transferrin complex.
5. The injection of Sc3+,as a 1:2 citrate complex, into rabbits led after 48 h to all the plasma
Sc3+being present as the transferrin complex.

Previous studies on the metabolism of scandium
were initiated through possible hazards arising from
its presence in nuclear explosion sites [l], because of
its chemical similarity to yttrium and the lanthanides,
whose biological properties have been extensively
studied [2] and since in vivo it is produced from the
decay of 47Ca,which is used for studies of calcium
metabolism in man [3]. The distribution and ex-
cretion of scandium in mice [4] and man [5,6] were
studied and electrophoresis was used to show Sc3+
binding to the p-globulin fraction of plasma [7].
The &globulin, transferrin, in its pure form was
shown to bind a number of trivalent metal ions,
including scandium [8]. Transferrin was also impli-
cated in the transport of various metals in the plasma,
apart from iron, including chromium [9], gallium [lo],
indium [ll]and manganese [121. This report describes
the specific binding of Sc3+ to the iron binding sites
of transferrin and shows that in vivo transferrin is
implicated in the transport of Sc3+in Serum.
MATERIALS AND METHODS
Materials
Apotransferrin was obtained from Hoechst Ltd.
(Hounslow, Middlesex, England). 46Scin 0.1 M HC1
(0.5-3 mCi/mgSc ) and 69Fe as ferric chloride in
0.1 M HC1 (3.3 mCi/mg Fe) were obtained from the
Radiochemical Centre (Amersham, Bucks, England).
Sephadex G-25 and 6-150 beads and DEAE-A50
resin were obtained from Pharmacia (G.B.) Ltd.,
(Wembley, Middlesex, England). Chelex 100 resin,
200-400 mesh, was made by Biorad Laboratories
(Richmond, California, U.S.A.). Scandium chloride
(high purity) was obtained from Johnson, Matthey &
Co. (Hatton Garden, London E.C.l., England).
Cyanogum 41 was obtained from BDH Chemicals
Ltd. (Poole, Dorset, England). All solutions were
made with deionised, glass-distilled water.
Methods
Difference spectroscopy readings were taken on a
UnicamSPSOOrecording spectrophotometerfittedwith
a scale expansion unit. All other spectrophotometric
readings were taken with a Gilford 2000 spectrophoto-
meter fitted with a Unicam SP500 monochromator.
Radioactivity measurements of solutions containing
46Sc or S9Fewere made using a y scintillation spectro-
meter. All protein concentrations were determined
using an E~~~ = 9.23 x lo4 11131 (the on binding
Sc3f to tranferrin is less than lo/, as shown below).
For binding measurements by gel filtration, apotrans-
ferrin (5 ml, 50/, w/v) in 0.1 M Tris-C1 buffer a t the
appropriate p H was equilibrated, for either 1 h a t
23 "C or one week a t 4 "C, with a ten-fold excess of
-
Sc3f as a 1 :2 Sc3f citrate complex containing 10 pCi
of 4 % ~ .Excess metal was removed on a 35 ~2 cm
column of Sephadex G-25 equilibrated with 0.1 M
Tris-C1 buffer. Fractions (3 ml) were collected and
the protein eluates localised and metal concentra-
tions determined by radioactivity measurements.
-
The scandium transferrin samples thus obtained
(from the longer incubation period only) were passed
down a 2 0 x 2 cm column of Chelex 100. Fractions
were treated as for the Sephadex 6-25 column.
Ultraviolet difference spectra upon metal binding
were recorded using dual compartment cuvettes.
I n one compartment was placed protein (5O/, w/v)
in p H 8.0 0.2 M Tris-C1 buffer; in the other 2.0 mM
scandium and 4.0 mM trisodium citrate in the same
buffer. For the titration of scandium and apotrans-
56 Scandium Binding to Transferrin
ferrin measurements of the absorbance a t 246nm
were taken of apotransferrin (0.5O/,) in 0.1 M Tris-C1
pH 8.0 before and 48 h after the addition of small
amounts of 1.0 mM scandium and 2.0 mM trisodium
citrate. Urea denaturation difference spectra were
obtained using single compartment cells. Apotransfer-
rin (2 ml, 4O/,, w/v) in p H 8.0 0.1 M Tris-C1 buffer
was added to a 5 ml volumetric flask and where ap-
propriate 0.4 ml of 1.0 mM iron, or 0.4 ml 1.0 mM
scandium and 2.0 mM trisodium citrate were added
and equilibrated. Solid urea (to make the solutions
8 M in urea) was added to the appropriate flasks and
the volumes made up to the mark and difference spec-
tra recorded between solutions with and without urea.
Periodate oxidations were carried out similarly
to the method of Azari and Phillips [14]. 10 ml of
0.1 M NaHC0,-K,CO, buffer, p H 8.5, containing
50 mg of apotransferrin and where appropriate 0.2 ml
of 0.01 M Fe3+ or Sc3+plus 0.02 M trisodium citrate
was added to 1 0 m l of the above buffer containing
0.01 M sodium periodate. The tubes were then in-
cubated a t 32 "C. Aliquots (4 ml) were withdrawn for
analysis and added t o 0.1 ml of 0.3 M sodium arsenite
+
and 0.1 ml of 0.01 M Fe3+ 0.02 M trisodium citrate
(0.5 ml in the case of scandium transferrin). (This
allows for the fact that Fe3+displaces Sc3+from trans-
ferrin.) Incubation was for 1 h a t 23 "C and then activ-
ity was measured by measuring iron binding from the
absorbance a t 465 nm.
For binding studies to serum proteins in vitro,
pooled normal human serum was used throughout. To-
tal iron and free iron-binding capacity were deter-
mined [15]. Transferrin was separated from serum
according t o the method of Killander [16] except that
a 2 5 x 3 5 cm column of Sephadex G-I50 (flow rate
20 ml/h) was usedinstead of a Sephadex G-200 column.
Serum (1.5 ml) was preincubated with either 0.3 pCi
of 59Fe or 0.5 pCi of 46Sc(containing enough carrier
and citrate to half saturate the free iron binding capa-
city of the serum). Fractions (3 ml) were collected
and the protein content determined by light absorb-
ance a t 254 nm and the metal content by their radio-
activity. The transferrin peak obtained from the ion-
exchange resin was homogenous on cellulose acetate
electrophoresis.
Serum proteins were also separated on gradient
polyacrylamide gels (5x 120mm) consisting of 20 mm
of 6O/,gel (cyanogum4l),30mm of 7.5O/,gel and 70mm
of go/, gel made up in buffer (Tris 45.75 g/l, 60 ml
1 N HC1/1, pH 8.9). The electrode buffer contained
0.6 g/1 Tris and 28.8 g/l glycine, p H 8.3. Gels were
run a t 2 mA per gel for 10 min and then 4 mA per gel
for 2-2.5 h. 25 pl (50 pl for experiments in vivo) of
serum, with a n equal volume of loo/, (w/v) sucrose
in gel buffer Containing one drop of 50/, bromophenol
blue as marker, was applied to the gels which, after
electrophoresis, were sliced into 24 equal segments for
radioactivity measurements. Protein bands were
Eur. J. Biochem.
located by staining with amido black. It was found
that the position of transferrin could be accurately
predicted from the position of the start line and the al-
bumin band which were made visible by the bromo-
phenol blue marker dye.
For experiments in vivo rabbits (New Zealand
Whites) were injected intravenously in groups of
three with 0.5 mg (1mCi) or 0.005 mg (0.01 mCi) of
scandium, containing *Y3c as a 1:2 scandium citrate -
complex ; injection solutions were brought to neutral
p H with 2 M Tris. Blood samples (2 ml) were removed
a t intervals, the first sample being taken after 10 min,
and the radioactivity in the plasma was measured.
For the larger dose, plasma samples were run on gra-
dient acrylamide gels which were segmented and
counted. Samples were frozen quickly for storage
prior t o electrophoresis. Control experiments showed
that no detectable redistribution of the metal occurs
when samples were stored frozen.
RESULTS
Sc3+ Binding to Pure Transferrin
Gel filtration studies of the binding of Sc3+(10-fold
excess) to apotransferrin were performed with two
different incubation times, 1h a t 23 "C and 1 week
a t 4 "C (Fig. 1).The binding is slower a t the lower p H
values and is incomplete after 1h. After 1 week at
4 "C excess metal binding is observed, over and above
the two atoms per mole expected for specific binding
to the two iron binding sites. Passage of this metal
protein solution through a column of Chelex 100 re-
moves the excess metal and 2 atoms/mole remain
bound a t p H 8. Further passage of scandium trans- -
ferrin through Chelex 100 columns results in no sig-
nificant loss of metal (< 5O/,).
I I I 1 I
7.5 8.0 a5 9.0 9.5
PH
Fig. 1. T h e effect of pH on the binding of Sc3+ to apotransferrin.
Apotransferrin [5 ml, 50/4 (w/v)] in 0.1 M Tris-C1 buffer was
incubated with Sc3+ (10-fold excess; 1 :2 citrate complex).
Excess metal was removed on Sephadex G-25 column, non-
specifically bound metal on a Chelex 100 column. 0,1 week
incubation, 4 "C after Sephadex G-25 column; 0, 1 week in-
cubation, 4 "C after Sephadex G-25 column followed by Chelex
100 column; 0 , 1 h incubation, 23 "C, after Sephadex 6 - 2 5
column
V01.21, NO.1, 1971 A. W. FORD-HUTCHINSON
and D. J. PERIUNS
The difference spectrum obtained on binding Sc3+
to apotransferrin is very similar to the phenolic
tyrosine ionisation difference spectra found for the
colourless metal conalbumin complexes [17]. Two
maxima occur a t 246 nm and 297 nm. The A E~~~
of 4.1i & 0 . 1 8 ~ 1 0(S.D.)
~ (14 readings) for this
reaction indicates that four tyrosyl residues are ionised
per protein molecule on binding two Sc3+(for phenolic
tyrosyl ionisation in conalbumin, A E~~~ is about
I x lo4 [17]). No further spectral changes were ob-
served 2 h after mixing with the concentrations of
reactants used. The bindings was followed spectro-
photometrically a t 246 nm on the Gilford 2000 re-
cording spectrophotometer. The optimum citrate to
scandium ratio was determined by this technique,
and found to be 2 :1. Excess citrate inhibited the
reaction and with ratios of citrate to scandium of
5 : I, or over, binding was very slow. The titration
experiment (Fig. 2) shows that no further increase in
absorbance is observed after two atoms of Sc3+ are
bound per mole of transferrin.
The binding of iron to conalbumin or transferrin
results in much greater stability towards physical,
chemical and enzymatic treatments [IS]. A con-
venient method of following the urea denaturation of
transferrin is by ultraviolet difference spectroscopy
[19]. Iron * transferrin and scandium transferrin - those found for conalbumin [19]. All difference spectra
show very little change in absorption in 8 M urea.
Apotransferrin, however, shows large spectral changes.
These difference spectra do not allow for possible
errors due to solvent effects but if apotransferrin
versus apotransferrin in 8 M urea was plotted against
-
(a)iron .transferrin versw iron transferrin in 8 M urea
sc3+ (moIes/mole transferrin)
Fig.2. The titrution of apotransferrin with Sc3+.The absorbance
at 246 nm was measured before and 48 h after the addition
of small amounts of 1.0 Mm scandium incorporating 2.0 mM
trisodium citratc to apotransferrin (0.5O/,) ;n 0.1 M Tris-C1
pH 8.0 a t 23 O C
57
Table. The periodate oxidation of transfewin and its metal
complexes
Apotransferrin (50 mg in 10 mlO.1 M NaHC0,-K,CO, buffer,
pH 8.5) and where appropriate 0.2 ml 0.01 M Fe3+ in 0.02 M
trisodium citrate was added to the 10 ml of the buffer contain-
ing 0.01 M sodium periodate. Tubes were incubated a t 32 "C
and aliquots (4ml)withdrawn for analysis and added to 0.1 ml,
0.3 M sodium arsenite and 0.1 ml of 0.01 Fe3+, 0.02 M tri-
sodium citrate (0.5 ml for the scandium * transferrin complex).
Incubation was for 1h a t 23 "C and activity was then deter-
mined by measuring iron binding by the absorbance a t
465 nm
Chromogenic activity
-.
'rime Iron Scandium
Apotransferrin . ,,.ransferrin .Transferrin
min "0 "lo "0
0 100.0 100.0 100.0
5 43.7 100.0 100.0
I0 33.2 100.0 100.0
15 32.3 100.0 99.0
25 31.3 100.0 95.2
and (b) scandium transferrin versus scandium
-transferrin in 8 M urea, two almost identical difference
spectra were obtained. Negative peaks are seen a t
284 and 292 nm. These spectra are very similar to
were formed with I0 min of mixing with no further
changes apparent for up t o 3 days. Resistance to
periodate oxidation also shows the stabilising effect
of iron on conalbumin [14], Transferrin behaves in a
very similar way to conalbumin (Table). Both iron
and scandium confer resistance to periodate oxidation
and as for conalbumin, no evidence was found for tyro-
syl ionisation difference spectra upon oxidation.
Xca+ Binding to Xerum in vitro
Gel filtration of serum on Sephadex G-I50 and
chromatography on DEAE-Sephadex A50 show that
all the protein-bound Sc3f is in the form of scan-
-
dium transferrin when amounts of Sc3+ were added
below the quantity necessary t o saturate the free
iron-binding capacity of the serum. Similar results
were obtained using 6BFe.
Human serum was also separated on gradient poly-
acrylamide gels. The method used separated albumin
and transferrin, the two principal metal binding pro-
teins, distinctly from the rest of the serum proteins.
This method has the advantage of showing the amount
of free metal associated with small molecular weight
constituents of the serum. Experiments in vitro with
pooled human serum showed that all the protein-
bound Sc3f was bound to transferrin with concentra-
tions of Sc3+ less than the free iron-binding capacity
of the serum. I n addition to this, about 30°/, of the
metal ion was associated with the small molecular
weight, or "free" band on the gels. With concentra-
58 Scandium Binding to Transferrin
3o0 2 4
Fig. 3. The decay of Sc3+ in vivo in plasma. Rabbits were injected in groups of three with ( 0 )0.5 mg (1 mCi) and (0)0.005 mg
(0.01 mCi) of 46Sc (1:2 citrate complex). Samples of blood (2 ml) were taken at intervals and the radioactivity in the
plasma was measured. (A) represents in detail the first 6 h shown in (B)
tions of Sc3+ greater than the free iron-binding capa-
city general binding to all protein fractions was ob-
served.
Sc3+ Binding to Plasma in vivo
Gradient polyacrylamide gels were also used t o
follow the fate of Sc3+ in vivo. 10 min after the injec-
tion of the rabbits, 75O/, of the metal in the plasma
is associated with the small molecular weight band and
2501, with the transferrin. After 1 h these figures are
reversed and after 24 h 95O1, of the metal is bound to
the transferrin. The quantity of metal bound t o the
transferrin is maximal after 20 min while the quantity
on the small molecular weight band decreased from
the initial (10 min) sample. After 48 h all detectable
radioactivity is associated with the transferrin band.
The decay of Sc3+in whole plasma for two different
doses (3 rabbits each) is shown in Fig.3. From the gel
results it is concluded that all values after 24 h will
probably only represent radioactivity present as
-
scandium transferrin. A linear section of the loga-
rithmic plot for both doses can be seen giving a half-
life of about 4.5 h. This probably represents the half-
life of the free Sc3+. However, over the time period
studied no simple logarithmic decay of the scandium
- transferrin complex was observed.
DISCUSSION
Scandium although it has many of the chemical
properties of yttrium and the lanthanides also shows
similarities to the first transition series [20-221.
This is probably due t o the smaller atomic radius of
Sc3+ (68 pm, cf. 69 pm for Fea+)compared with those
of yttrium and the lanthanides (85-106 pm) [all.
Preliminary experiments in this laboratory show that
yttrium and the lanthanides are not bound specifically
Eur. J. Biochem.
6
o.,
Time (h)
0
- 50 100 150 200 250
to the iron binding sites of transferrin thus providing
further evidence for the disimilarity of scandium and
the rest of the group IIIa elements.
Due to the hydrolysis of the Sc3+ion a t alkaline
pH, the binding experiments were performed using
the metal as its citrate complex. The scandium ci- -
trate complexes are very slow to equilibrate across
dialysis membranes and thus the gel filtration tech-
nique was used for studying metal binding, rather
than equilibrium dialysis, although this is not en-
tirely satisfactory as the protein and metal do not
remain in equilibrium. I n this case, the binding t o
the specific iron binding sites is strong enough t o
allow its use. The method could not be used for quan-
titative studies of weaker non-specific interactions.
The results show that Scs+ is bound specifically
onto the iron-binding sites of transferrin, conferring
on the protein similar stability t o denaturants and
chemical reagents as iron. Two tyrosyl residues are
ionised per Sc3f bound. The binding of iron results in
the release of three H+ per Fe3+bound [23], but spec-
trophotometry suggested that these were all produced
from phenolic tyrosyl ionisations [24]. More recent
work on conalbumin suggests that the number of
tyrosyl residues involved in binding is two for each
trivalent metal ion and one for each divalent metal ion.
The third proton released, they suggest, may be de-
rived from a tryptophanyl residue, as has been pro-
posed for the coloured metal * conalbumin complexes
[25]. It is possible that a comparative study of the
colourless complexes (e. g. scandium * transferrin)
versuS the coloured complexes may explain some of
these discrepancies.
This work also shows that scandium, like chro-
mium, gallium, indium and manganese, is transported
in plasma as the transferrin complex. The mechanism
of removal of scandium remains obscure and the pro-
Vol. 21, No. 1, 1971 and D. J. PERKINS
A. W. FORD-HUTCHINSON
cess is not a simple logarithmic decay over the time
studied, as would occur for the simple degradation or
removal of the transferrin complex from the serum.
We wish to thank the Medical Research Council and the
Governors of St. George’s Hospital, London, S.W.l. for finan-
cial support. We are also indebted to Dr. G. Franglen for help
with polyacrylamide gel electrophoresis.
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A. W. Ford-Hutchinson and D. J. Perkins
Department of Chemical Pathology
St. George’s Hospital Medical School
9 Knightsbridge, London, S.W.l, Great Britain