Vol. 17
No. 1
Chemical Research in Chinese Universities
2001
6~ 13
Binding Constants for Terbium( ) with
Chicken Apoovotransferrin*
YANG Bin sheng
* *
and L I Ying qi
( I nst itute of Molecular Science, Shanx i University , T aiy uan 030006, P . R . Chi na)
( Received N ov. 17, 1999)
3+
T he binding of T b to chicken apoovot ransferrin w as st udied by monitoring t he fluorescent
intensity of T b3+ at 549 nm. T he conditional equilibrium const ant s for t he complexat ion of T b3+
by chicken apoovotransferrin in 0 1 mol/ L hepes, at pH 7 4 and room temperature w ere mea
sured. T he successive m acroscopic binding const ants are lg K 1 = 9 08
0 22. T he molar f luorescence enhancement of T b
4
3+
0 12 and lg K 2 = 7 36
apoovot ransf errin com plex is ( 2 06
0 14)
- 1
! 10 mol ∀L. T he f luorescence quenching experiment and the t itration of N term inal monofer
ric ovotransferrin show ed t hat T b
sit e of apoovot ransf errin.
3+
has a preference for being bound to the N term inal binding
Keywords T erbium; T ransferrin; Binding const ant; Fluorescence
Article ID 1005 9040( 2001) 01 006 08
Introduction
T he transferrins are a superfamily of metal binding proteins, w hich are single chains, 80
kDa glycoproteins, characterized by their ability to bind iron tightly( an effective binding con
20. 7
[ 1, 2]
stant is about 10 ) , but reversibly
. Serum transferrin, found in blood and ex tracellular
fluids, functions in iron transport and homeostasis, maint aining the essent ial element in a non
toxic form and shuttling it betw een peripheral sites of absorpt ion, storage, release and utiliza
t ion. Ovotransferrin exists in avian egg w hite. Ovotransferrin acts as bacteriost ats by scaveng
ing iron and keeping it from infect ious organisms, w hich is probably inferred from the fact that
in v iv o the protein is largely present in their apo ( iron free) forms and is then able to bind
iron so tightly that it is unavailable for bacterial grow th[ 3] .
T he t hree dim ensional structure of ovotransferrin, as revealed by X ray cryst allo
graphy [ 4] , is sim ilar to those of hum an lactoferrin and rabbit serum transferrin, which are
folded into tw o homologous lobes, each containing two dissim ilar domains w ith one F e3+ and
2-
one CO3 bound to a specific site in each interdomain cleft. T he coordination spheres of iron
are identical in the tw o lobes. Each iron atom is linked to one aspartyl carboxylate oxygen,
two tyrosyl phenolate oxygens, and one histidyl nitrogen. T he remaining tw o coordination re
* Supported by the N ational Natur al Science Foundation of China ( No. 200710220) , Natural Science Foundation of
Shanx i Prov ince( N o. 991013) and Shanxi Returned scholar Foundation.
* * T o whom corr espondence should be addressed.
No. 1
YANG Bin sheng and L I Ying qi et al .
7
quirem ents of iron are satisfied by the tw o oxygens of the carbonate co anion which is secured
to the protein by a netw ork of hydrogen bonding to an arginine nitrogen, a threonine oxygen,
and the N terminus of a helix protruding into the binding cleft. Although similar, the sites are
not identical, differing in t heir chemical, their spectroscopic, and possibly their physiological
properties[ 5] .
3+
T here have been a num ber of studies on T b binding to transferrin, mostly aim ing at us
[ 6]
ing the m etal ion as spectroscopic probes . In t his work the fluorescence spectra have been
used to m easure the binding constants for sequentially binding tw o T b3+ ions to apoovotrans
ferrin, w ith particular em phasis on the difference betw een apoovotransferrin and apotransfer
3+
rin. It w as found that T b w as bound to the N terminal binding site of apoovotransferrin
preferably, w hereas it w as bound to C terminal binding site of apotransferrin preferably.
Experiment
Materials
Nitrilotriacetic acid ( NT A ) , N 2 hydroxyethyl piperazine N # 2 ethane sulfonic acid
( hepes) , disodium ethylenediaminetetraacet ic acid( EDT A) , ferrous ammonium sulfate and
1
sodium perchlorate w ere all analytical grade reagents and w ere used as received. Chicken egg
w hite apoovotransferrin and human serum apotransferrin w are purchased from Sigma.
Apoovotransferrin or apotransferrin ( 100 ∃ 150 mg ) w as dissolved into 8 mL of 0 1
m ol/ L hepes buffer, pH 7 4, containing 0 1 mol/ L sodium perchloride. T he volum e of the
solut ion was reduced at room temperature to 1 m L by using an Am icon Model 8010 ultrafiltra
t ion cell fitted w it h an XM 50 membrane at 4 13 ! 105 Pa of nitrogen gas to remove the
chelating agents. T he dilution and concentration step w as repeated four times. T hen the
buffer was changed into the buffer noncontaining sodium perchlorate and the dilut ion and con
centration step w as repeated tw ice. T he concentration of protein w as determined from the ab
sorbance at 278 nm by using an extinct ion coefficient of 93 000 cm - 1 ∀m ol- 1∀L [ 7] for apo
1%
[ 8]
transferrin and at 280 nm of E 1cm = 11 3 for apoovotransferrin , respectively.
Diferric ovotransferrin solution w as prepared by adding 2 m ol of ferrous ammonium sul
fate to apoovotransferrin and was used w ithout further purification.
N terminal m onoferric transferrin w as prepared from apoovotransferrin by adding 1 mol
of ferrous ammonium sulfate and w as used w ithout further purificat ion.
T erbium chloride hexahydrate w as 99 9% . A stock solution w as prepared by dissolving a
w eighed sample of the chloride in a sm all volume of dilute hydrochloric acid. T he solution w as
diluted to the m ark w ith distilled w ater. T he terbium stock solution was standardized by com
plexometric titration w ith ethylenediam inetetraacetic acid and Xylenol orange as the metal in
dicator in acet ic acid sodium acetate buffer at pH 5 5.
Since apoovotransferrin has a high affinity towards metal ions, precautions should be tak
en to avoid contam ination by extraneous metal ions. All the glassw ares including cuvet te w ere
rout inely soaked in 1 mol/ L HNO 3 and then rinsed w ith distilled water.
Chemical Research in Chinese Universities
8
2
Vol. 17
Method
Fluorescence spectra w ere recorded w ith a Perkin Elm er LS 50B Fluorescence Spec
trophotom eter. A solution of apoovotransferrin in 0 1 mol/ L hepes, pH 7 4, w as added to a
dried, 1 cm fluorescent cuvette. Apoovotransferrin w as titrated w ith an acidic solution of
T b3+ ions at am bient bicarbonate concentration. T he fluorescent spectrum w as recorded from
470 nm to 630 nm after each addition. In the determ ination of binding constants the titrations
3+
w ith T b w ere done under the same instrument conditions. Diferric ovotransferrin and N ter
m inal monoferric ovotransferrin were also titrated. T o correct the dilution during each titration
and norm alize the results from run to run, the fluorescent intensity data w ere converted to flu
orescence enhancement, dividing the fluorescent intensity by the analytical concentrat ion of
protein.
Results and Discussion
Terbium Fluorescent Spectra
For apoovotransferrin being titrated by the addition of an acidic T b3+ solution in 0 1 mol/
L hepes, pH 7 4, at room temperature, the
fluorescent em ission spectra from 470 nm to
1
630 nm are show n in Fig. 1. It can be seen
that t here is a double peak at 491 nm, the
peak at 549 nm is the strongest am ong the
peaks. T he intensity at 549 nm is gradually in
3+
creased w ith the addit ion of T b . After an
amount of T b3+ has been added, the fluores
cent intensity at 549 nm approached a max i Fig. 1
m um and does not increase cont inuously.
In an aqueous solution T b3+ is only w eak
ly lum inescent principally because it is an ex
tremely w eak absorber of electromagnetic radi
at ion, orbital forbidden f f transition. In gen
eral, there are four characteristic fluorescent
peaks, near 490, 545, 590 and 623 nm,
w hich are attributed to the transitions from
5
7
7
7
7
D 4 to F 6 , F 5 , F 4 and F 3, respect ively.
5
The fluorescence emission spectra pro
duced by the addition of Tb3+ ( 0 15
mmol/ L) to 1 5 mL apoovotransferrin
( 9 4 mol/ L) in 0 1 mol/ L Hepes, pH
7 4 at room temperature with ambient
bicarbonate.
M easured conditions were slit 5 nm, fliter 390
nm,
ex
295 nm. Cur ve a is baseline. Other
curv es, V ( T b 3+ ) / L: b . 10; c. 20; d . 40;
e. 70; f . 100; g. 140; h. 200; i . 260; j .
320, respectively .
7
As the energy gap betw een the D 4 and F 0
3+
levels of T b approx im ately m atches the energy of t he five vibrat ional quanta of the O ∃ H
group, the displacement of the coordinate w ater m olecule by another ligand w ill be revealed
experim entally by a large increase in the fluorescence intensity of T b3+ . T he fluorescent en
hancement of T b3+ is determined by the ligands. T he fluorescent enhancem ent effect is small
er for the ligands, w hich are non containing aromatic, low m olecular w eight ligands. In the
presence of aromat ic containing ligands there is no contribution of free T b
3+
to the total mea
No. 1
YANG Bin sheng and L I Ying qi et al .
9
sured fluorescent intensity. T b3+ fluorescence can also be neglected[ 6] for the complexes,
w hich are formed from T b3+ w ith non containing arom atic, low molecular weight ligands. So
3+
the increase of the fluorescent intensit y at 549 nm is the origin of the binding of T b to
apoovotransferrin in Fig. 1. T o correct for dilution effects and to norm alize the dat a from run
to run, the fluorescent intensity is converted to fluorescence enhancement, T b3+ fluorescence
enhanced by apoovotransferrin, the fluorescent intensity at 549 nm is divided by the analytical
concentration of apoovotransferrin. T he resulted fluorescence enhancement is plotted v ersus
the ratio of total T b3+ to the analytical apoovotransferrin concentration as in Fig. 2.
In Fig. 2 the initial part of the titration is linear. T he linear behavior indicates that in the
3+
early stages of the titration, apoovotransferrin binds essent ially 100% of T b in each aliquot
of titration. U nder these conditions the initial slope of the t itration curve is equal to the molar
fluorescence enhancem ent of T b3+ apoovotransferrin com plex ( E apo ) , fluorescence intensity
3+
3+
per m ole bound T b in the T b apoovotransferrin complex . T he molar fluorescence en
hancement calculated by this method is ( 2 06 0 14) ! 104 mol- 1∀L . Based on the value it
follow s that the complete saturation of the two binding sites of apoovotransferrin should yield a
final fluorescence enhancem ent of 4 12 ! 104 mol- 1∀L . As more T b3+ was added, the in
3+
3+
creasing saturation of T b binding sites led to the less effective binding of added T b , and
the plot of fluorescence enhancement v ersus equiv T b3+ began to curve dow nw ard. T he t itra
t ion curve eventually leveled off at about 3 75 ! 104 mol- 1 ∀L after the addition of alm ost
4 m ol of T b3+ , w hich indicated t hat the tw o binding sites were not saturated w ith T b3+ .
Since the ferric ion was bound so m uch m ore t ightly than the lanthanide, the presence of
ferric ions at both the binding sites blocked T b3+ binding to the m etal binding sites. So diferric
ovotransferrin and N terminal m onoferric ovotransferrin w ere also titrated w ith T b3+ . T he
3+
titrat ion curves are show n in F ig. 2. T here w as no measureable T b fluorescence during the
titrat ion of diferric ovotransferrin. It means that under the experim ental condition the T b3+
fluorescence completely com es from t he binding of T b3+ on the m etal binding sites of apoovo
Fig. 2
Titration curves for the addition of Tb3+
to approximately 9 4
mol/ L apoovo
transferrin, N terminal monoferric and
diferric ovotransferrin in 0 1 mol/ L hep
Fig. 3
Plot of n, the number of Tb3+ bound per
apoovotransferrin, N terminal monoferric
transferrin vs. the Tb3+ ion added to the
es buffer at pH 7 4 and room tempera
proteins, based on the titration of aproxi
mately 9 4 mol/ L proteins in 0 1 mol/ L
ture.
hepes buffer at pH 7 4 and room tempera
ture.
Chemical Research in Chinese Universities
10
Vol. 17
transferrin w hen apoovotransferrin is titrated by the addition of T b3+ . Com paring t he titration
curve of the vacant C terminal site w ith the titration curve of apoovotransferrin, the titration
of the vacant C term inal site began w ith a much low er slope and reached a max imum of only
1 58 ! 104 mol- 1∀L . T hus the C terminal site appears to have a low er binding const ant and
Tb
3+
is bound to the N term inal binding site of apoovotransferrin preferably.
T he number of T b3+ bound per apoovotransferrin molecule, n , at any point in protein
titrat ion curves is defined as follow s.
n = E obs/ E apo
( 1)
w here E obs is the observed fluorescence enhancement at any titration point, E apo is the molar
fluorescence enhancem ent of the T b3+ apoovotransferrin complex. T itration curves in Fig. 2
are converted to the plot of n v ersus equivalent of T b3+ as show n in F ig. 3. It can be seen that
each apoovotransferrin can bind 1 76 of T b
2 Terbium Binding Constants
T he successive binding of T b
equilibria:
3+
3+
. For F eN ovotransferrin n is 0 75.
to apoovotransferrin can be described by tw o sequent ial
Apoovotransferrin+ T b
T b apoovotransferrin
( 2)
T b apoovotransferrin + T b
T b apoovotransferrin T b
( 3)
T he t hermodynam ic binding constants for these equations are written as t he follow ing
equat ions
[ T b apoovotransferrin]
[ T b] [ apoovotransferrin]
[ T b apoovotransferrin T b]
K2 =
[ T b apoovotransferrin] [ T b]
K1=
( 4)
( 5)
w here K 1 and K 2 are apparent binding constants, w hich are limited to the experimental condi
t ions. [ T b] and [ apoovotransferrin] refer to the molarities of free T b3+ and free apoovotrans
ferrin, [ T b apoovotransferrin] and [ T b apoovotransferrin T b] refer to the molarities of pro
teins, w hich bind one and tw o T b3+ ions, respectively.
It is presum ed that there is no difference in the m olar fluorescence enhancement for the
two binding sites of apoovotransferrin. T hen the fluorescence enhancem ent at any point in
titrat ion curve can be calculated by first using the init ial guesses of K 1 and K 2 to solve the ap
propriate mass balance equation for [ apoovotransferrin] and [ T b] and then t hose values w ere
subst ituted into equation 6
E apo K 1 [ T b] [ apoovotransferrin] + 2E apo K 1 K 2[ T b] 2 [ apoovotransferrin]
E calcd =
( 6)
[ apoovotransferrin] t ot
w here [ apoovotransferrin] t ot refers to t he analytical concentrat ion of apoovotransferrin. T he
m olar fluorescence enhancement of the T b apoovotransferrin complex , E apo , is obtained from
the initial slope of t he apoovotransferrin t itration curve. T hus K 1 and K 2 are left as the only
unknowns needed to calculate E calcd. In principle the titration curve in Fig. 2 can be fit by us
ing non linear least squares and K 1 and K 2 as the only adjustable parameters to obtain K 1 and
K 2 . In practice using a chelating agent as the com petition ligand can decrease the correlation
No. 1
YANG Bin sheng and L I Ying qi et al .
11
of K 1 and K 2 to obtain more accurate K 1 and
K 2 as descried previously
[ 6]
. So t he titrations
w ith T b3+ w ere repeatedly by using some m etal
titrant solut ions that contained a range of con
centrations of NT A. T he chelating agent com
peted w ith apoovotransferrin for T b3+ and thus
at any point in the titration there w as a distri
3+
bution of T b between apoovotransferrin and
the chelating agent, NT A, w hich systemat ical Fig. 4
ly reduced the observed fluorescence enhance
3+
m ent as the NT A%T b molar rat ios increased.
3+
A series of titrat ion curves for T b NT A solu
t ions is show n in Fig. 4. For NT A part icipat ion
in the reaction the m ass balance equations
3+
Titrations of approximate 9 4
mol/ L
apoovotransferrin in 0 1 mol/ L hepes at
room temperature and pH 7 4 wi th 0 15
mmol/ L solutions of Tb3+ that contained
variational NTA%Tb3+ molar ratios.
n( NT A) %n ( T b3+ ) : a. 0; b . 0 24; c. 0 49;
d . 0 73; e. 0 98; f . 1 22; g. 1 47.
should include apoovotransferrin, T b
and
NT A. T he titrat ion data in Fig. 4 can be fitted by using equation 6 yet, in w hich K 1 and K 2
are only the adjustable param eters.
Using difference UV titrations Harris [ 7] demonstrated that w ith both binding sites apo
transferrin binding tw o lanthanide ions, either sm aller or larger lanthanide ions and the
amount of saturation w ere affected by the concentrat ion of bicarbonate. For instance, an am
bient bicarbonate concentration is about 0 14 m mol∀L - 1 at pH 7 4, it w ill interfere w ith
T b3+ to be bound to apoovotransferrin. T he solubilit y product of RE 2( CO 3) 3 falls in the range
of 10- 31 to 10- 33 4[ 9] . T his gives an upper limit on the concentration of free T b3+ in the range
of 10- 6 to 10- 7 mol/ L. Since the T b3+ carbonate species is not included in the mass balance
equat ions, the nonlinear least squares fitting is restricted to the points for w hich the calculated
3+
- 6
concentration of free T b is not allow ed to exceed 10 mol/ L . From Fig. 4 the effective
binding constants for the com plexes of T b3+ w it h apoovotransferrin are lg K 1 = 9 08 0 12
and lgK 2= 7 36 0 22.
Preference for Metal binding Site
In different molar rat ios of T b3+ to
apoovotransferrin or apotransferrin, T b3+
apoovotransferrin or T b3+ apotransferrin com
3
plexes can be prepared. T he addition of ferrous
ammonium sulfate to a solution of T b3+
apoovotransferrin or T b3+ apotransferrin leads
the fluorescent intensity at 549 nm to being
3+
Fig. 5 Quenching curveas of Tb fluorescence by
quenched. Fluorescence quenching curves are
ferrous ions.
obtained by plotting t he relative fluorescence
c [ Tb (
) ] / c ( Apoov otransferrin ) : a. 1 6;
intensity v ersus c [ F e ( & ) ] / c ( Protein ) ,
b . 1 6; c. 0 7; d. 0 7.
Chemical Research in Chinese Universities
12
Vol. 17
w hich is the m olar rat io of ferrous to analytical apoovotransferrin or apotransferrin concentra
t ion. T he titration curves of T b3+ apoovotransferrin and T b3+ apotransferrin are show n in
F ig. 5. Since the ferric ion is bound to apoovotransferrin or apotransferrin so m uch more tight
3+
ly than the lanthanide, T b ions occupying both N term inal and C term inal binding sites can
be displaced by added ferric or ferrous ions. It can be seen t hat t he titration curves are biphasic
and the relative fluorescence intensities are near zero w hen c [ Fe( &) ] / c ( Protein) approaches
2. It means that T b3+ ions bound to N terminal and C terminal binding sites can be displaced
completely by ferric ions. How ever, the quenching curves are different for T b3+ apoovotrans
3+
3+
ferrin and T b apotransferrin. For T b apotransferrin the quenching rate is larger in c [ F e
( &) ] / c( Protein) > 1 than in c [ Fe( &) ] / c ( Protein) < 1( see curves a and c in F ig. 5) . For
T b3+ apoovotransferrin the quenching rate is larger in c [ Fe( &) ] / c ( Protein) < 1 than in c
[ Fe( &) ] / c ( Protein) > 1( see curves b and d in F igure 5) .
4 Discussions
Ow ing to the forbidden f f transition and nonradiative energy loss to O ∃ H oscillators in
3+
3+
the first coordinat ion sphere of the T b ion, T b fluorescence is very w eak in an aqueous so
lution. On account of displacem ent of coordinating w ater molecules and the tw o tyrosyl pheno
late oxygens of apoovotransferrin coordinating to T b3+ directly, apoovotransferrin m akes the
fluorescence of T b3+ increased largely after T b3+ is bound to apoovotransferrin. T he natural
3+
substrate of apoovotransferrin is Fe , for the metal difference U V spectrophotometeric t itra
t ions show a sharp break at the point of the saturation of the two m etal binding sites. Howev
er, a dist inct end point is seldom observed for other met al ions[ 10] . T hus in the T b3+ fluores
cence t itration curve there is no dist inct break at 2 m ol of T b3+ . In fact the final E obs value is
consistently less than tw ice the E apo value that w ould be expected for fully formed T b
3+
apoovotransferrin T b3+ , indicating that the two binding sites of apoovotransferrin are not sat
urated even in the presence of a relatively large excess of T b3+ .
At pH 7 4 iron ( ferric and ferrous salts) is bound specifically to the N term inal binding
[ 11]
[ 7]
site of apoovotransferrin
or apotransferrin and lanthanide ions are bound to C terminal
binding site of apotransferrin preferably [ 6, 10] . T hus it can be seen that T b3+ is bound to N ter
m inal binding site of apoovotransferrin preferably from F ig. 5[ 12] . T he sequent ial macroscopic
binding constants for the two T b3+ ions being bound to apoovotransferrin are lg K 1 = 9 08
3+
0 12 and lgK 2= 7 36 0 22. T he difference betw een lgK 1 and lgK 2 is 1 72. Since T b is
bound to the N term inal binding site of apoovotransferrin preferably( see Fig. 2 and Fig. 5) , it
is reasonable to assign K 1 correponding to the stronger N term inal site and K 2 corresponding
to the w eaker C term inal site.
T he crystal structure of diferric ovotransferrin shows t hat t he overall conformation is sim
ilar to those of hum an lactoferrin and rabbit serum transferrin, how ever, the relative orienta
t ion of the two lobes, w hich may be related to the class specificity of transferrin to receptors,
is different from those of either hum an lactoferrin or rabbit serum transferrin[ 4] . T he differ
ence between their detailed structures can be related to the difference betw een physiological or
funct ional characteristics of transferrins. T he circularly polarized lum inescence spectroscopy
No. 1
YANG Bin sheng and L I Ying qi et al .
13
from hum an lactoferrin is very sim ilar to that of hum an serum transferrin, however, ovotrans
ferrin shows differences in line shape and magnitude [ 13] . Fluorescence quenching experim ents
3+
showed that T b is bound to the N terminal binding site of apoovotransferrin preferably,
w hereas it is bound to the C terminal binding site of apotransferrin preferably. T his is in a
greement w it h the results of crystallography and circularly polarized lum inescence spec
troscopy.
Acknowledgements
T he authors are gratef ul to Dr . W . R . H arris, Dep artm ent of Chem istry , Univ ersi
ty of M issour i St Louis, f or the supp ly of ap oovotransf er rin.
References
[ 1]
A isen, P. , L iebman, A . and Zweier, J. , J. Biol. Chem. , 253, 1 930( 1978)
[ 2]
M art in, R. B. , Savory , J. , Brown, S. , et al . , Clin. Chem. , 33, 405( 1987)
[ 3]
Baker, E. N. , in Advances in Inorg anic Chemistry , 11, 389( 1994)
[ 4]
K uro kawa, H. , M ikami, B. and Hirose, M . , J. M ol. Biol. , 254, 196( 1995)
[ 5]
Baldw in, D. A . and Eg an, T . J. , S. A fr . J. Sci. , 83, 22( 1987)
[ 6]
Y ang Bin sheng and Harr is, W. R. , Chinese. J. Biochem. M ol. Biol. , 15, 462( 1999)
[ 7]
Harris, W . R. and Chen, Y . , Inor g. Chem. , 31, 5 001( 1992)
[ 8]
Chasteen, N . D. and W illiams J. , Biochem. J. , 193, 717( 1981)
[ 9]
M artell, A. E. and Smith, R . M . , Cr itical Stability Constants, Plenum, N ew Yor k, 1974
[ 10]
Y ang Bin sheng and Harr is W. R. , Acta Chimica Sinica, 57, 503( 1999)
[ 11]
W illiams, J. and Evans, R . W . , P roteins of I ron M etabolism, Gr une and Stratton, New Y ork, 169( 1977)
[ 12]
Y ANG Bin sheng and Har ris, W. R . , Chem. J. Chinese U niversit ies, 19, 1 057( 1998)
[ 13]
Abdollahi, S. and Harr is, W . R. , J. P hys. Chem. , 100, 1 950( 1996)