Tài liệu Báo cáo khoa học: Application of a fluorescent cobalamin analogue for analysis of the binding kinetics A study employing recombinant human transcobalamin and intrinsic factor pdf

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Application of a uorescent cobalamin analogue
for analysis of the binding kinetics
A study employing recombinant human transcobalamin
and intrinsic factor
Sergey N. Fedosov
1
, Charles B. Grissom
2
, Natalya U. Fedosova
3
, Sứren K. Moestrup
4
, Ebba Nexứ
5
and Torben E. Petersen
1
1 Protein Chemistry Laboratory, Department of Molecular Biology, University of Aarhus, Denmark
2 Department of Chemistry, University of Utah, Salt Lake City, UT, USA
3 Department of Physiology and Biophysics, University of Aarhus, Denmark
4 Department of Medical Biochemistry, University of Aarhus, Denmark
5 Department of Clinical Biochemistry, AS Aarhus University Hospital, Denmark
Cobalamin (Cbl, vitamin B
12
) is a cofactor for two
crucial enzymes in mammals [1]. Therefore, an
enhanced inux of the vitamin is required during cell
growth to satisfy high synthetic and energetic
demands. Intensive uptake of Cbl was suggested to be
a good marker of the fast growing tissues including
malignant cells [2]. However, declining application of
radioactive
57
Co-labeled Cbl prompts investigation of
alternative ligands. Imaging of tumours with the help
of Cbl derivatives, as well as targeted delivery of
Keywords
cobalamin; uorescence; intrinsic factor;
transcobalamin
Correspondence
S. N. Fedosov, Protein Chemistry
Laboratory, Department of Molecular
Biology, University of Aarhus, Science Park,
Gustav Wieds Vej 10, 8000 Aarhus C,
Denmark
Fax: +45 86 13 65 97
Tel: +45 89 42 50 92
E-mail: snf@mb.au.dk
(Received 9 June 2006, revised 31 July
2006, accepted 18 August 2006)
doi:10.1111/j.1742-4658.2006.05478.x
Fluorescent probe rhodamine was appended to 5Â OH-ribose of cobalamin
(Cbl). The prepared conjugate, CBC, bound to the transporting proteins,
intrinsic factor (IF) and transcobalamin (TC), responsible for the uptake of
Cbl in an organism. Pronounced increase in uorescence upon CBC attach-
ment facilitated detailed kinetic analysis of Cbl binding. We found that TC
had the same afnity for CBC and Cbl (K
d
ẳ 5 ã 10
)15
m), whereas inter-
action of CBC with the highly specic protein IF was more complex. For
instance, CBC behaved normally in the partial reactions CBC + IF
30
and
CBC + IF
20
when binding to the isolated IF fragments (domains). The lig-
and could also assemble them into a stable complex IF
30
CBCIF
20
with
higher uorescent signal. However, dissociation of IF
30
CBCIF
20
and IF
CBC was accelerated by factors of 3 and 20, respectively, when compared
to the corresponding Cbl complexes. We suggest that the correct domain
domain interactions are the most important factor during recognition and
xation of the ligands by IF. Dissociation of IFCBC was biphasic, and
existence of multiple proteinanalogue complexes with normal and partially
corrupted structure may explain this behaviour. The most stable compo-
nent had K
d
ẳ 1.5 ã 10
)13
m, which guarantees the binding of CBC to IF
under physiological conditions. The specic intestinal receptor cubilin
bound both IFCBC and IFCbl with equal afnity. In conclusion, the
uorescent analogue CBC can be used as a reporting agent in the kinetic
studies, moreover, it seems to be applicable for imaging purposes in vivo.
Abbreviations
Cbl, cobalamin (vitamin B
12
); CBC, uorescent derivative of Cbl; CNCbl, cyano-cobalamin; GdnHCl, guanidine hydrochloride; HC, haptocorrin;
IF, intrinsic factor; TC, transcobalamin; RU, response units.
4742 FEBS Journal 273 (2006) 47424753 ê 2006 The Authors Journal compilation ê 2006 FEBS
conjugated drugs, is rapidly becoming a perspective
direction of Cbl-related research [3,4]. Yet, there is a
gap between the number of new derivatives and the
detailed knowledge about their interaction with the
specic protein carriers, which are the key players in
targeted delivery.
Uptake of dietary Cbl is a complex process because
only a limited amount of the vitamin is available from
natural sources. Three specic proteins, intrinsic factor
(IF), transcobalamin (TC) and haptocorrin (HC), are
involved in transportation (reviewed in [58]). IF is
responsible for gastrointestinal uptake of vitamin B
12
,
and this protein is particularly sensitive to any changes
introduced into the structure of the ligand. After-
wards, Cbl is transferred to TC, which delivers the
vitamin to different tissues via the blood circulation.
TC is also quite specic for the true cobalamins. The
third carrier HC is present in many body uids and
has low substrate specicity. It is assumed to be a
storage, protective or scavenging protein. HC even-
tually binds all Cbl-resembling molecules and trans-
ports them to the liver, where they are either stored
or disposed. Yet, the exact function of HC remains
unknown.
Afnity of the transporting proteins for Cbl still
remains a controversial issue with an extraordinary
dispersion of the reported equilibrium dissociation
constants K
d
ẳ 10
)9
)10
)15
m [5,7,1015]. However,
the major reasons of this discrepancy are rather arti-
cial. Thus, insufcient equilibration of two binding
species at the point of equivalence, e.g., E + S , ES
at E
0
% S
0
, leads to severe overestimation of K
d
as dis-
cussed previously [10]. Inapplicability of the equilib-
rium methods for a near-irreversible binding was also
pointed out by other authors [12]. It was concluded
that the separate kinetic determination of k
+
and k

gives a much more adequate estimation of K
d
.
Attempts to follow the association and dissociation
kinetics were made using radioactive
57
Co-labeled Cbl
by the charcoal method [5,7,12,13], change in absorb-
ance of Cbl [10,14], and plasmon resonance signal [15].
However, all the above methods were not completely
adequate for the task, because partial protein precipi-
tation in the rst protocol or low signal to noise ratio
in the two latter procedures could compromise the
accuracy of measurements. In this respect, application
of a highly sensitive uorescent probe seems to be
advantageous in terms of the protein concentrations,
time scale and amplitude of response.
Molecular mechanisms of Cbl recognition by the
transporting proteins are not completely understood. A
probable structural basis of the IFligand interactions
was recently inferred from the properties of its two pro-
teolytic fragments [9,10]. Thus, the small C-terminal
fragment IF
20
(13 kDa peptide with % 7 kDa of carbo-
hydrates) had a relatively high afnity for Cbl and was
suggested to be the primary subject of substrate binding.
The larger N-terminal fragment IF
30
(30 kDa peptide)
bound the ligand with low afnity. However, interaction
between IF
30
and the saturated IF
20
Cbl complex was
necessary to stabilize the bound ligand within a rm
sandwich-like complex IF
30
CblIF
20
. In addition, only
two assembled fragments could bind to the specic
receptor cubilin [10]. Based on these facts, the sequential
interaction of Cbl with the two domains of the full
length IF was suggested.
The structure of the kindred protein TC (human
and bovine) in complex with H
2
OCbl was recently
solved on the atomic level [16]. The found architecture
of the TCligand complex was very similar to the one
suggested for IF [9,10]. TC consists of two domains
with Cbl placed in-between. The ligand was essentially
enwrapped, and its solvent accessible surface decreased
to % 7% with only the ribose moiety exposed. In total,
34 hydrogen and hydrophobic contacts between TC
and the ligand ensured a very strong retention of Cbl.
Additionally, a His residue substituted for water of
H
2
OCbl, which added to protection of the ligand
against reduction and coordination of other com-
pounds. The structure of TCCbl complex directly
indicated that a foreign label (e.g., a uorescent probe)
should be conjugated to 5Â OH ribosyl group of Cbl to
minimize loss of afnity.
The present work describes the binding of a uores-
cent Cbl analogue CBC-244 to the Cbl-transporting
proteins IF and TC. In the interpretation of our results
we emphasize the following issues: (i) kinetic character-
ization of the new ligand; (ii) its applicability in the
binding studies of other corrinoids; and (iii) potential
pertinence to the physiological studies.
Results
Preparation of the proteins
The experiments were performed on the recombinant
human proteins IF and TC puried from plants [17]
and yeast [18], respectively. Both proteins were origin-
ally obtained as Cbl-saturated holo-forms, and prepar-
ation of the unsaturated apo-forms required their
denaturing. Unfolding of TC with 5 m guanidine
hydrochloride (GdnHCl) was earlier found to be the
best in terms of the protein recovery [14,18]. However,
similar approach to IF gave some variation in its Cbl
binding properties, as discussed elsewhere [10]. In the
present study, we have found that denaturing in 8 m
S. N. Fedosov et al. Application of a uorescent Cbl analogue
FEBS Journal 273 (2006) 47424753 ê 2006 The Authors Journal compilation ê 2006 FEBS 4743
urea followed by a renaturing dilution (see below)
provided better recovery of IF and improved its ligand
binding properties, as will be demonstrated below.
Synthesis of the uorescent Cbl analogue
CBC-244
The uorescent conjugate of Cbl (Fig. 1A) was pre-
pared by coupling of 5- (and 6-) carboxyrhodamine
succinimidil ester (5 6 mixed isomers) to an amino
derivative of Cbl modied at 5ÂOH-ribose [19,20]; see
below for details. Two isomers of CBC-244 were
then separated by reverse phase HPLC and examined
for their binding to IF and TC. Both derivatives
behaved in most respects quite similarly (data not
shown), yet, the binding of 5Â CBC-244 to the tested
proteins was 1.5-fold faster. The experiments des-
cribed in the present article were performed with
5Â form, and below we will refer to 5Â CBC-244 as
CBC.
Spectral properties of CBC
The coefcient of molar absorbance for rhodamine moi-
ety of CBC was estimated as e
527
ẳ 90 000 m
)1
ặcm
)1
.
In the below experiments we used concentrations of
CBC Ê 1 lm, where no self-quenching was observed,
and the intensity of CBC uorescence linearly depended
on CBC concentration (data not shown). The excitation
and emission spectra of CBC, either free or bound to
the Cbl-specic proteins, are presented in Fig. 1B.
Attachment to the transporting proteins, especially to
IF, clearly induced increase in the quantum yield of the
uorescent ligand, allowing direct monitoring of the
binding-dissociation reactions. Presence of 2 lm Cbl
(cyano-, aquo-, adenosyl-forms) in the solutions
together with CBC (both free and protein bound)
caused approximately 6% quenching of the uorescent
signal immediately after mixing as demonstrated in
Fig. 1C. This effect was insignicant at the Cbl concen-
trations below 1 lm, but required correction when con-
centrations increased to 2 lm and above.
Binding of CBC to IF or TC
As a pilot experiment, an isotope dilution assay was
conducted, where increasing concentrations of the
cold ligand (Cbl or CBC) competed with the radio-
active ligand
57
Co-labeled Cbl for the binding to IF
(or TC). It appeared that both the analogue and Cbl
efciently displaced
57
Co-labeled Cbl according to the
ratio of their half-saturation points Cbl
0.5
CBC
0.5
ẳ
A
BC
Fig. 1. Fluorescent conjugate 5Â CBC-244. (A) Chemical structural of CBC (M
r
ẳ 2042). (B) Excitation and emission spectra of CBC in solution
or bound to the Cbl specic proteins, [CBC] ẳ 0.5 l
M, [TC] ẳ 1 lM, [IF] ẳ 1 lM, pH 7.5, 20 C. (C) Fluorescence quenching (F
q
ẳ 0.94ặF
0
)
induced by 2 l
M Cbl in the solution of 0.5 lM CBC (free or bound to TC or IF), incubation time 0.51 min.
Application of a uorescent Cbl analogue S. N. Fedosov et al.
4744 FEBS Journal 273 (2006) 47424753 ê 2006 The Authors Journal compilation ê 2006 FEBS
0.2 and 0.4 for IF and TC, respectively. Therefore, the
uorescent probe was subjected to further kinetic
analysis.
Interaction of CBC with the specic binders was
monitored over time, where increasing amplitude of
the uorescent signal reected binding process (Fig. 2).
The experiments were performed with varying protein
concentrations keeping the initial concentration of
CBC constant. The same nal amplitude of uorescent
response was reached after 30 s of incubation, there-
fore the reactions obeyed an irreversible bimolecular
mechanism E + S ES in the time scale of the
experiment. The data were tted by the corresponding
equation [10]. Both IF and TC demonstrated the
same rate constant of CBC binding k
+CBC
ẳ 64
5 lm
)1
ặs
)1
. The amplitude of relative response for IF
was, however, three-fold higher (Table 1).
Binding of CBC to IF fragments IF
20
or IF
30
The binding reactions were conducted at constant
CBC and variable concentrations of the peptides IF
20
and IF
30
(Fig. 3). The preliminary equilibrium analysis
in Fig. 3A indicated that the ligandpeptide interaction
was reversible for IF
20
+ CBC and IF
30
+ CBC, but
nearly irreversible for the three component mixture
A B
Fig. 2. Binding of CBC to IF and TC. (A) CBC + IF IFCBC. (B) CBC + TC TCCBC. Both reactions were followed in 0.2 M P
i
buffer,
pH 7.5, 20 C. Final concentrations in the cuvette: [CBC] ẳ 0.5 l
M, [protein] ẳ 0.5, 1.0, 2.5 lM. See text and Table 1.
Table 1. Interactions between IF, TC and the ligands CBC, cyano-cobalamin (CNCbl). All reactions were carried out at 20 C and pH 7.5. The
results are presented as mean SD. Bold type indicates the rate constant for CBC differing from the corresponding coefcients for Cbl.
*Data for H
2
OCbl and
57
Co-labeled CNCbl from references [9,10,14,18]. RU, response units.
Reaction
DFluor.
(RUặl
M
)1
)
k
+
ã 10
)6
(M
)1
ặs
)1
) k

(s
)1
) K
d
(M)
IF
20
+L, IF
20
L
L ẳ CBC 0.75 0.05 61 8 9 2 1.5 0.3 ã 10
)7
L ẳ Cbl % 60 % 9 % 1.5 ã 10
)7
L ẳ Cbl* 14 3 4 3 3 2 ã 10
)7
IF
30
+L, IF
30
L
L ẳ CBC 0.82 0.08 2 1 160 30 84ã 10
)5
L ẳ Cbl* 3.5 0.6 140 40 4.0 2 ã 10
)5
IF
20
L + IF
30
, IF
20
LIF
30
L ẳ CBC 2.0 0.1 4.2 0.4 1.2 0.3 ã 10
)3
2.9 0.7 ã 10
)10
L ẳ Cbl % 4 5.0 1.5 ã 10
)4
% 10
)10
L ẳ Cbl* 4.0 0.5 % 10
)4
% 10
)11
IF + L , IFL
L ẳ CBC 2.7 0.1 64 6 (65%) 8 ã 10
)6
(25%) 2 ã 10
)4
1.2 0.2 ã 10
)13
3.1 0.4 ã 10
)12
L ẳ Cbl 74 10 4 1 ã 10
)7
51ã 10
)15
L ẳ Cbl* 2060 10
)5
)10
)6
10
)13
)10
)14
TC + L , TCL
L ẳ CBC 1.0 0.1 64 5 4 1 ã 10
)7
61ã 10
)15
L ẳ Cbl 68 2 3.2 0.6 ã 10
)7
51ã 10
)15
L ẳ Cbl* 30100 10
)7
10
)14
)10
)15
S. N. Fedosov et al. Application of a uorescent Cbl analogue
FEBS Journal 273 (2006) 47424753 ê 2006 The Authors Journal compilation ê 2006 FEBS 4745
IF
20
+IF
30
+ CBC at the concentrations used. The
curves were tted by the square-root equation [10] to
estimate the maximal amplitude of response DF and
the equilibrium dissociation constants. The small
glyco-peptide IF
20
had relatively high afnity for the
uorescent ligand with K
CBC,20
ẳ 0.13 0.04 lm.On
the contrary, the binding of CBC to the larger frag-
ment IF
30
was much weaker, K
CBC,30
ẳ 83 14 lm.
Similar results were found earlier for Cbl as well [10].
The maximal amplitude of uorescent response for the
isolated peptides was relatively low when compared to
the three component mixture IF
30
+IF
20
+ CBC and
the full length IF (Fig. 3A and Table 1).
The time course of the binding between CBC and
peptides is presented in Fig. 3B,C. The corresponding
rate constants k
+CBC
and k
CBC
for IF
20
and IF
30
were calculated as described earlier [10], and the results
are presented in Table 1. The obtained values were
comparable with those known for H
2
OCbl [10].
Association of the fragments IF
20
CBC + IF
30
When the preformed complex IF
20
CBC was mixed
with the low afnity unit IF
30
a noticeable increase in
the uorescence was observed over time (Fig. 3D). It
was ascribed to association of two IF fragments into a
complex IF
20
CBCIF
30
as was observed earlier for the
true substrate Cbl [9,10]. The main phase [DF ẳ 2.0
response units (RU)ặlm
)1
] presumably reected the bi-
molecular reaction IF
20
CBC + IF
30
IF
20
CBC
IF
30
with k
F20+30
ẳ 4.2 0.4 lm
)1
ặs
)1
. An additional
mono-molecular transition A B with k ẳ 1.2
0.2 s
)1
was observed at the end of the reaction.
This slow exponential phase accounted for a relatively
small increase in the uorescent signal (DF ẳ 0.15
RUặlm
)1
). Possible explanation of this effect is
presented below.
Competitive binding of CBC and Cbl, calculation
of k
+
We have tested the application of the uorescent ana-
logue CBC as a tool for investigation of the binding
kinetics of nonuorescent ligands. Cyano-cobalamin
(CNCbl) was examined in the present setup. Simul-
taneous injection of CBC and Cbl to the specic
binding protein (either IF or TC) led to a competitive
binding of the two ligands (Fig. 4). The reaction
A
C D
B
Fig. 3. Binding of CBC to the fragments IF
20
and IF
30
. (A) Equilibrium binding of 0.5 lM CBC to IF
20
,IF
30
and IF
20
+IF
30
. The amplitude of
the uorescent response in equilibrium was measured at 15 s from the reaction start. The uorescence level did not change during this
time interval. (B) Time-dependent change in uorescence induced by binding of [CBC] ẳ 0.5 l
M to [IF
20
] ẳ 0.5, 0.75, 1.0, 2.5 lM . (C) Time-
dependent binding of [CBC] ẳ 0.5 l
M to [IF
30
] ẳ 1, 10, 20, 40 lM. (D) Time-dependent binding of [IF
20
CBC] ẳ 0.5 lM to [IF
30
] ẳ 0.4, 0.8, 2,
4 l
M. See text and Table 1.
Application of a uorescent Cbl analogue S. N. Fedosov et al.
4746 FEBS Journal 273 (2006) 47424753 ê 2006 The Authors Journal compilation ê 2006 FEBS
obeyed a bidirectional irreversible mechanism, e.g.,
IFCbl Cbl + IF + CBC IFCBC, at least in
the shown time scale. The corresponding rate constants
k
+Cbl
and k
+CBC
were calculated by computer simula-
tions (see below), and their values appeared to be quite
similar, k
+
ẳ 6070 lm
)1
ặs
)1
(Table 1). The obtained
results demonstrated good correlation with earlier data
for H
2
OCbl and CNCbl [14,15].
Dissociation of IFCBC and IFCbl in chase
experiments
When measuring CBC dissociation, the binding pro-
teins were rst loaded with the uorescent probe and
then exposed to a four-fold excess of Cbl. Presence of
Cbl caused gradual decrease in the total uorescence
ascribed to dissociation of CBC. Detachment of Cbl
was monitored in the opposite manner. The binding
protein was initially saturated with Cbl, and then the
uorescent probe was added. The latter displaced Cbl
in the binding site, and an increase of uorescence was
registered. Dissociation of the initially bound ligand
was expected to be the rate limiting step in all above
cases. Control samples (CBC + Cbl and IFCBC
without additives) were also monitored throughout the
experiment, see below.
The charts for dissociation of IFCBC and IFCbl
versus time are shown in Fig. 5A. Already a rough
comparison of the dissociation velocities indicated at
least a 10-fold faster liberation of the uorescent ana-
logue when compared with Cbl. The CBC dissociation
spanned at least 90% of the total amplitude, which
allows one to describe the reaction as a unidirectional
process and t it by exponential approximation. Sur-
prisingly, the mono-exponential t was quite inadequate
(dotted line, Fig. 5A), and the data were analysed by
a double-exponential function instead. Approximately
25% of CBC was liberated with k
)1
% 2 ã 10
)4
s
)1
,
whereas dissociation of the following 6575% was char-
acterized by k
)2
% 8 ã 10
)6
s
)1
. Possible explanation of
the multiphasic kinetics is presented below.
Dissociation of IFCbl in the presence of CBC was
hardly noticeable (Fig. 5A, bottom curve). An approxi-
mate value of k
Cbl
was estimated from the initial slope
equal to v
0
ẳ k
Cbl
ặ[IFCbl] (Fig. 5A, dashed line). We
have veried the dissociation process by simulating its
behaviour with help of the below scheme:
IF ỵ CBC () IF CBC;
k
ỵCBC
ẳ 70 lM
1
S
1
; k
CBC
ẳ 1 10
5
s
1
IF ỵ Cbl () IF Cbl; k
ỵCbl
ẳ 70 lM
1
S
1
;
k
Cbl
is the fitting parameter.
The unknown rate constant, obtained from the best t,
corresponded to k
Cbl
ẳ 4 ã 10
)7
s
)1
.
Dissociation of TCligand complexes
In contrast to IF, dissociation of two TCligand com-
plexes occurred equally slowly (Fig. 5B). The corres-
ponding rate constants (Table 1) were calculated from
the initial slopes: v
0, CBC
ẳ k
CBC
ặ[TCCBC]
0
and
v
0, Cbl
ẳ k
Cbl
ặ[TCCbl]
0
.
Dissociation of the cleaved IFligand complexes
The assembled peptideligand complexes IF
30
CBC
IF
20
and IF
30
CblIF
20
were exposed to the external
substitutes, Cbl or CBC, respectively. This caused dis-
sociation of the original structures and recombination
of the peptides with the added ligand. Considering
the already known rate constants, the rate-limiting
step of the whole process was expected to be
detachment of IF
30
from the assembled complex, e.g.,
IF
30
CBCIF
20
IF
30
+ CBCIF
20
.
A B
Fig. 4. Competition between CBC and CNCbl for the binding to the transport proteins. (A) Binding of [CBC] ẳ 0.5 lM to [IF] ẳ 0.5 lM in the
presence of different Cbl concentrations (0, 0.2, 0.5, 1.0 l
M). (B) Binding of [CBC] ẳ 0.5 lM to [TC] ẳ 0.5 lM at different Cbl concentrations
(0, 0.25, 0.5, 1.0 l
M). See text and Table 1 for details.
S. N. Fedosov et al. Application of a uorescent Cbl analogue
FEBS Journal 273 (2006) 47424753 ê 2006 The Authors Journal compilation ê 2006 FEBS 4747
As seen from the data in Fig. 5C, stability of both
IF
30
CblIF
20
and IF
30
CBCIF
20
was lower than that
of the full length protein (Fig. 5A), and the original
structures dissociated in one hour. Rough evaluation
revealed a three-fold faster disassembly of IF
30
CBC
IF
20
(curve at the top) when compared with IF
30
Cbl
IF
20
(curve at the bottom). All other interactions seemed
to be the same for both ligands, considering the nal
equilibrium levels at time Ơand the concentrations
of the reagents used. The whole process was computer
simulated according to the below scheme:
IF
20
ỵ CBC () IF
20
CBC;
k
ỵCBC
ẳ 61lM
1
S
1
; k
CBC
ẳ 9s
1
IF
30
ỵ IF
20
CBC () IF
30
CBCIF
20
;
k
F20ỵ30
ẳ 4lM
1
s
1
; k
F2030
is the fitting parameter
IF
20
ỵ Cbl () IF
20
Cbl;
k
ỵCbl
ẳ 61lM
1
S
1
; k
Cbl
ẳ 9s
1
IF
30
ỵ IF
20
Cbl () IF
30
CblIF
20
;
k
20ỵ30
ẳ 4lM
1
s
1
; k
2030
is the fitting parameter.
Binding of the free ligands to IF
30
was ignored as insig-
nicant under conditions of the experiment. Optimal
values of the tting parameters k
F20)30
and k
20)30
were
found for each curve: 1.2 ã 10
)3
s
)1
and 3.6 ã 10
)4
s
)1
(top dashed curve, Fig. 5C); 9.0 ã 10
)4
s
)1
and 5.0 ã
10
)4
s
)1
(bottom dashed curve, Fig. 5C). Then, the
obtained parameters were corrected to get the general
t of the whole system with the same set of coefcients.
The solid curves in Fig. 5C show the simulations for
k
20)30
values presented in Table 1.
Reliability of CBC-uorescence method
The data of CBC-based measurements (Table 1)
showed a good correlation with the results obtained
earlier for Cbls by different methods [10,14,18]. Only
the rate constant of IFCbl dissociation deviated from
our previous data and pointed to better retention of
the ligand by the current protein preparation (Table 1).
The difference could be caused by either changed rena-
turing procedure for IF or inaccuracy of one of the
kinetic methods. In order to verify the current data of
A

B

D C
Fig. 5. Dissociation of the protein-ligand complexes. (A) IFligand dissociation followed by uorescence method: [IFCBC] ẳ 0.5 lM,
[Cbl] ẳ 2 l
M (top curve); and [IFCbl] ẳ 0.5 lM, [CBC] ẳ 0.55 lM (bottom curve). (B) TCligand dissociation followed by uorescence method:
[TCCBC] ẳ 0.5 l
M, [Cbl] ẳ 2 lM (top curve); and [TCCbl] ẳ 0.5 lM, [CBC] ẳ 1 lM (bottom curve). (C) Dissociation of IF fragments followed
by uorescence method: IF
30
CBCIF
20
ẳ (0.6 lM IF
30
+ 0.5 l M CBC + 0.5 lM IF
20
), [Cbl] ẳ 2 lM (top curve); and IF
30
CblIF
20
ẳ (0.6 lM IF
30
+0.5 lM Cbl + 0.5 lM IF
20
), [CBC] ẳ 1 lM (bottom curve). (D) Dissociation of IFligand followed by absorbance method: [IFH
2
OCbl] ẳ 15 lM,
[CNCbl] ẳ 50 l
M; inset presents transition in the absorbance spectra of the protein-associated ligands IFH
2
OCbl IFCNCbl.
Application of a uorescent Cbl analogue S. N. Fedosov et al.
4748 FEBS Journal 273 (2006) 47424753 ê 2006 The Authors Journal compilation ê 2006 FEBS
uorescent measurements we repeated the dissociation
experiment with IF according to the previously des-
cribed method [10], where change in the absorbance
spectrum of IFCbl was measured upon displacement
of H
2
OCbl by CNCbl, Fig. 5D. The estimated value of
k
H
2
OCbl
ẳ 5 ã 10
)7
s
)1
corroborated higher stability
of IFCbl from the current protein preparation.
Binding of IFCBC and IFCbl to the specic
receptor
Binding of two proteinligand complexes IFCBC and
IFCbl to the receptor cubilin was tested by surface
plasmon resonance. Identical pattern of records
(Fig. 6) implied that both complexes were recognized
by the receptor equally well. The experiment suggests
that the tertiary structure of the receptor recognition
site in IFCBC is indistinguishable from that of
IFCbl.
Discussion
In the present article we demonstrate that the uores-
cent Cbl analogue CBC (Fig. 1A) binds to the trans-
porting proteins TC and IF. Interaction of CBC with
the Cbl specic proteins was accompanied by signi-
cant change in its uorescence (Fig. 1B). Therefore,
the binding-dissociation reactions could be monitored
directly in time making this uorescent conjugate par-
ticularly suitable for rened analysis of the Cbl binding
kinetics.
Interaction between CBC and TC was not affected
by presence of the 5ÂO-ribosyl conjugated uorophore,
as was expected from the crystallographic data for
TCCbl complex [16], and the binding-dissociation
curves of CBC and Cbl were identical (Figs 2B,4B
and 5B, Table 1). Using a new and more sensitive
approach we conrm correctness of the lowest equilib-
rium dissociation constants for TCCbl and TCCBC
complexes (K
d
ẳ 5 ã 10
)15
m
)1
). Impressive dissoci-
ation stability of TCCBC implies its essential resem-
blance to TCCbl, and therefore, suggests normal
transportation of the uorescent probe in the organ-
ism, especially taking into account moderate variation
of the receptor afnity for apo- holo-TC [21,22].
Attachment of CBC to the most Cbl-specic protein
IF was fast and matched the binding velocity of Cbl,
k
+CBC
% k
+Cbl
% 70 ã 10
6
m
)1
ặs
)1
(Table 1). Detach-
ment of CBC from IF was, however, accelerated by a
factor of 20 (Fig. 5A, main phase). Regardless the lat-
ter fact, retention of CBC by IF was still formidable
with K
d
ẳ 120 fm for 6575% of the protein. This
seems to be quite enough to bind the ligand under
physiological conditions (IF % 50 nm).
Another interesting observation concerns biphasic
dissociation of IFCBC with k
)1CBC
ẳ 2 ã 10
)4
s
)1
for
the fast phase (25%) and k
)2CBC
ẳ 8 ã 10
)5
s
)1
for
the slow one (6575%), (Fig. 5A, upper curve). We do
not think that the effect is caused by the original het-
erogeneity of IF preparation because the protein was
homogeneous in all other respects. An alternative
explanation seems to be more probable. Thus, distor-
ted shape of the analogue causes partial corruption of
its bonds with IF. As a consequence, the ligand and
the protein form several complexes with different dis-
sociation stability being in equilibrium, e.g., (IF
CBC)
1
, (IFCBC)
2
. If transition between these
conformations is sufciently slow, dissociation of the
ligand would be described by two to three rate coef-
cients (which was, indeed, observed). No such effect
was found for dissociation of TCCBC which was in
all respects indistinguishable from that of TCCbl
(Fig. 5B). We can therefore surmise that the suf-
ciently wide opening at 5Â OH-ribosyl group found in
TCCbl complex [16] might be quite narrow in IF
Cbl. Consequently, the bonding of CBC at its conju-
gated 5Â O-ribosyl group is partially unaccomplished in
IF. Presence of a slow equilibrium at this site (e.g.,
bound ô unbound) may account for the discussed
biphasic dissociation of IFCBC. The general structure
of the obtained IFCBC complex was, however, close
to IFCbl, because both of them bound to the specic
receptor cubilin in a uniform manner (Fig. 6).
It is known that IF is the most Cbl-specic binder
among three transporting proteins [5,7]. This feature
makes the mechanism of interaction between IF and
the ligand especially interesting as a kinetic example of
the utmost substrate selectivity. We have earlier sug-
gested a two domain organization of IF, where the
Fig. 6. Interaction of IF with the receptor-coated BIACore chip in
the presence or absence of the ligand. At time 120 s IF was added
to the receptor-coated chip either alone (bottom curve) or in com-
plex with Cbl or CBC (top curves). Washing out procedure was
started at t ẳ 600 s. Free ligands (Cbl, CBC) did not affect the
baseline (bottom curves).
S. N. Fedosov et al. Application of a uorescent Cbl analogue
FEBS Journal 273 (2006) 47424753 ê 2006 The Authors Journal compilation ê 2006 FEBS 4749
distant units IF
30
and IF
20
are assembled by the sub-
strate into a rm complex [9,10]. This architecture of
the Cbl-transporting proteins was directly demonstra-
ted by crystallographic studies of TC [16], another
member of this family. Highly sensitive uorescent
analogue provided an opportunity to investigate indi-
vidual contributions of different domains to the pro-
cess of substrate recognition, using the fragments IF
30
and IF
20
as a model.
Binding of CBC to the isolated fragments IF
20
and
IF
30
closely resembled that for Cbl (Fig. 5C, Table 1).
In other words, two domains were not very specic if
taken separately, at least in the example shown. Lack-
ing specicity for ligands seems to be caused by insuf-
cient contact area in each domain. Indeed, the
maximal uorescent signal in the two-component mix-
tures IF
20
+ CBC and IF
30
+ CBC (30% and 30%)
was lower than that in the complete three-component
mixture IF
20
+ CBC + IF
30
(100%). This observation
points to a reduced number of potential proteinligand
bonds when the two domains are taken apart. On the
other hand, simultaneous interaction of the two frag-
ments domains with the sandwiched ligand had a
cooperative character. It leads to higher uorescent
response and better xation of CBC. Final stabiliza-
tion of IF
30
CBCIF
20
can occur after series of transi-
tions at the domaindomain interface, which may be
the reason for the slow exponential phase during inter-
action of IF
20
CBC with IF
30
(Fig. 3D).
The discussed interdomain adjustments are expected
to be dependent on the geometry of ligands placed
in-between. Presence of a substrate with inappropriate
shape would disturb IF
30
IF
20
interface and decrease
stability of the nal proteinligand complex, possibly
creating several erroneous or alternative conforma-
tions. The weaker ligand retention and biphasic disso-
ciation kinetics of IFCBC (Fig. 5A) are in agreement
with the presented speculations. The peptide link,
which connects the two domains in the full length pro-
tein, is not just a spectator of proteinligand interac-
tions. Thus, it adds to both ligand afnity and
specicity of IF. This statement is based on the follow-
ing observations: (a) the uncleaved IF retained
Cbl CBC better than the separated fragments glued
by the ligand (Fig. 5A and C, respectively); (b) dis-
crimination between CBC and Cbl was better
expressed for the full length protein (20-fold difference)
than for the peptides (three-fold difference). It is poss-
ible that the right or wrong positioning of the
domains by the link prior to the substrate binding par-
tially accounts for different specicity of IF, TC and
HC for Cbl. The probable scheme of interaction
between IF, the ligand and the receptor is presented in
Fig. 7. The step(s) responsible for discrimination
between CBC and Cbl is specied.
It is generally accepted that IF serves as a reliable
shield, protecting organisms against uptake of corri-
noids with deviating structure. Yet, calculations show
that IF would be partially saturated under physiologi-
cal concentrations of this protein (% 50 nm) even if the
afnity for a ligand is decreased by a factor of 10
6
(e.g., to K
d
ẳ 110 nm). Additional observation indi-
cates that the reduced afnity for the analogue CBC
had no effect on the recognition of IFCBC complex
by the specic receptor cubilin immobilized on the
detecting chip (Fig. 6). All the above facts mean that
the intestinal uptake of analogues can be quite feasible.
In this regard we plan to examine a group of ana-
logues concerning details of their binding to the speci-
c proteins and receptors.
In conclusion, the binding of a uorescent Cbl ana-
logue (CBC) to two Cbl-transporting proteins TC and
IF was found to be normal and close to normal,
respectively. Applicability of CBC as a tool for analysis
of the binding kinetics was established and allowed to
make several inferences concerning the proteinligand
and proteinreceptor interactions. Furthermore, our
results provide strong arguments that the transportation
routes of CBC and Cbl would be identical in the human
body. CBC appears to be useful for tracing accumula-
tion of vitamin B
12
in cancer cells and other tissues.
Experimental procedures
Materials
All standard chemicals were purchased from Merck (White-
house Station, NJ, USA), Roche Molecular Biochemicals
(Mannheim, Germany), Sigma-Aldrich (Cambridge, MA,
USA). H
2
OCbl CNCbl and
57
Co-labeled Cbl were obtained
from Sigma-Aldrich and ICN Pharmaceutical Ltd (Costa
Mesa, CA, USA), respectively.
Fig. 7. Schematic presentation of IF interaction with the ligands
and the receptor. Both CBC and Cbl (lled circles) bind preferen-
tially to IF
20
domain, thus inducing assembly of IF
20
S and IF
30
units into a composite structure recognized by the receptor. The lig-
and binding step, which seems to be responsible for reduced afn-
ity for the analogue, is indicated with ! sign.
Application of a uorescent Cbl analogue S. N. Fedosov et al.
4750 FEBS Journal 273 (2006) 47424753 ê 2006 The Authors Journal compilation ê 2006 FEBS
Methods
Expression and purication of human recombinant
IF and TC
The recombinant Cbl binding proteins and their fragments
were isolated from plants and yeast as described earlier
[9,17]. Preparation of the unsaturated apo-form of IF was
although modied. Thus, the Cbl-saturated holo-IF
(1 mgặmL
)1
) was dialysed against 20 volumes of 8 m urea
(30 C) instead of 5 m GdnHCl. The incubation was con-
tinued for 46 days with three changes of the urea solution.
Renaturation was achieved by 1 : 10 dilution with 0.2 m
phosphate buffer pH 7.5 at 20 C. The protein was after-
wards concentrated 50 : 1 by ultraltration and dialysed
against excess of 0.2 m phosphate buffer pH 7.5.
Synthesis of the uorescent Cbl analogue CBC-244
Activation of the 5Â hydroxyl group in the a-ribofuranoside
moiety of CNCbl was performed with help of 1,1Â-dicarbo-
nyl-di-(1,2,4-triazole) as described elsewhere [19,20], where-
upon 4,7,10-trioxa-1,13-tridecanediamine was conjugated as
a spacer [19,20]. Amino group of the spacer was used for
the attachment of the uorophore, 5 6-carboxyrhodamine
6G, succinimidyl ester (5 6 mixed isomers) from Molecular
Probes (Eugene, OR, USA), according to recommendations
of the manufacturer. The product was a mixture of 5Â and
6Â forms in the ratio 44 : 53. The above isomers were separ-
ated by reverse phase HPLC on C-18 column.
Measurement of uorescence spectra
Excitation spectra of 5Â C-CBC-244 were recorded in the
range 400550 nm (excitation bandpass 3 nm), using emis-
sion wavelength 600 nm (bandpass 5 nm). Emission spectra
were recorded in the range 500600 nm (bandpass 5 nm),
excitation wavelength 480 nm (bandpass 3 nm).
Measurement of the binding kinetics with uorescent
probe CBC
Increase in uorescence upon binding of CBC to the Cbl
specic proteins was recorded on DX.17 MV stopped-ow
spectrouorometer (Applied Photophysics, Leatherhead,
UK), using excitation wavelength 525 nm (bandpass
7 nm) with 550 nm cut-off lter on the emission side.
The binding was carried out in 0.2 m phosphate buffer
pH 7.5, 20 C, at 0.5 lm CBC and varying concentrations
of the binding protein or peptide (0.52.5 lm). All experi-
ments were performed in triplicate, and the average
records are presented.
Experiments on competitive binding of CBC and Cbl to
the specic proteins (IF or TC) were conducted as des-
cribed above. Final concentrations of the reagents in the
cuvette were 0.5 lm binding protein, 0.5 lm CBC, 0.25
1 lm Cbl.
Measurement of the dissociation kinetics with the
uorescent probe CBC
A ligand exchange method was used in the below chase
experiments, e.g., IFCBC + Cbl IFCbl + CBC.
Changes of the emission spectra were recorded over time in
the mixtures proteinCBC (0.5 lm) + Cbl (2 lm) or pro-
teinCbl (0.5 lm) + CBC (0.551 lm) when measuring dis-
sociation of CBC or Cbl, respectively. Two control samples
for each binding protein contained (i) proteinCBC (0.5 lm)
and (ii) CBC (0.5 lm) + Cbl (2 lm) or Cbl (0.5 lm) + CBC
(0.551 lm). The concentration of proteinCBC complex
(e.g., for IF) at time t was calculated according to the equa-
tion:
IF CBC
t
ẳ
F
sample
F
min
q F
max
F
min
IF
0
where F
sample
is uorescence of the experimental sample
(e.g., IFCBC + Cbl or IFCbl + CBC) at time t; q is a
quenching coefcient determined separately for the corres-
ponding mixture (example in Fig. 2C); parameters F
max
and
F
min
correspond to the control probes (e.g., IFCBC and
CBC + Cbl) and indicate the maximal and minimal poss-
ible uorescence for the experimental sample; IF
0
corres-
ponds to the total concentration of the binding sites.
Measurement of the dissociation kinetics by absorbance
method
This procedure was described earlier [10]. Briey, the mix-
ture of IFH
2
OCbl (15 lm) and CNCbl (50 lm)inP
i
buf-
fer, pH 7.5, 20 C was incubated over time. Free ligands
were adsorbed on charcoal, and the absorbance spectra
were recorded. Concentration of appearing IFCNCbl was
calculated by comparison with the standards IFH
2
OCbl
and IFCNCbl according to the equation:
IF CNCbl
t
ẳ
DA
352
ỵ DA
361
ị
DA
max
352
ỵ DA
max
361
ị
IF
0
where, e.g., DA
352
corresponds to change of absorbance at
wavelength 352 nm in the reaction sample after incubation
time t; DA
max
352
ẳjA
CNCbl
A
H
2
OCbl
j stands for maximal poss-
ible change in the amplitude at wavelength, e.g. 352 nm; IF
0
represents total concentration of the binding sites.
Binding of IF to the receptor
IF, with or without ligands, interacted with the specic
receptor cubilin immobilized on the surface of the detect-
ing chip in BIACore 2000 instrument (Biacore Interna-
tional AB, Uppsala, Sweden) [24].
S. N. Fedosov et al. Application of a uorescent Cbl analogue
FEBS Journal 273 (2006) 47424753 ê 2006 The Authors Journal compilation ê 2006 FEBS 4751
Data processing
The data for irreversible and reversible bimolecular reac-
tions E + S ES and E + S , ES (Figs 3 and 4) were
subjected to nonlinear regression analysis using the appro-
priate equations [10]. The rate constants k
+S
and k
S
were
calculated by a tting program kyplot 4 (Kyence Lab Inc.,
Tokyo, Japan). Complex reactions without algebraic solu-
tion were simulated and tted using program gepasi 3.2
(http://www.gepasi.org) [23] supplied by kinetic schemes
presented in the main text.
Acknowledgements
This work was supported by Lundbeck Foundation
and Cobento Biotech A S.
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