JOURNAL OF
Inorganic
Biochemistry
Journal of Inorganic Biochemistry 100 (2006) 1462–1469
www.elsevier.com/locate/jinorgbio
Synthesis, characterization and properties of chromium(III)
complex [Cr(SA)(en)2]Cl Æ 2H2O
Bin Liu a, Ying-Qi Li a, Bin-Sheng Yang
a
a,*
, Shu-Ping Huang
b
Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education,
Institute of Molecular Science, Shanxi University, Taiyuan 030006, China
b
Institute of Modern Chemistry, Shanxi University, Taiyuan 030006, China
Received 26 October 2005; received in revised form 5 April 2006; accepted 14 April 2006
Available online 9 June 2006
Abstract
The reaction of chromium(III) chloride, salicylic acid (SA) and ethylenediamine (en) led to the formation of chromium complex
[Cr(SA)(en)2]Cl Æ 2H2O(1). The crystal structure belongs to monoclinic system with the space group P2(1), R1 = 0.0358. In this compound,
Cr(III) atom is six-coordinated in octahedral coordination geometry by one phenolic hydroxyl oxygen, one carboxylate oxygen from the
salicylic acid and four nitrogen atoms from two ethylenediamine molecules, respectively. The transfer manners of Cr(III) from the title compound to the low-molecular-mass chelator, ethylenediamine-N,N,N 0 ,N 0 -tetraacetic acid (EDTA) and the iron-binding protein apoovotransferrin (apoOTf) were followed by a combination of UV–visible (UV–Vis) and fluorescence spectra in 0.01 M Hepes at pH 7.4. The results
show that Cr(III) can be transferred from the complex to apoovotransferrin with the retention of the salicylate acted as a synergistic anion.
2006 Elsevier Inc. All rights reserved.
Keywords: Chromium(III); Salicylic acid; Apoovotransferrin; Transfer
1. Introduction
In the last 15 years, nutritional studies have suggested
that chromium(III) may have an essential role in mammals.
Chromium is known to activate enzymes, maintain protein
stability and enhance carbohydrate metabolism [1–5].
Chromium(III) picolinate, Cr(pic)3, has been the most
thoroughly studied of these synthetic products and has
become a very popular nutritional supplement [6]. However, the effect of Cr(pic)3 has been extremely contentious.
Of particular concern are recent reports that the complex
can efficiently cleave DNA in the presence of biological
reducing agents and can induce strand breaks in chromosomes of intact cells [7–10]. Hence, a search has been
underway to identify the biologically active form of chromium, that is, the biomolecule that binds chromium(III)
*
Corresponding author. Tel.: +86 351 7016358.
E-mail address: yangbs@sxu.edu.cn (B.-S. Yang).
0162-0134/$ - see front matter 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2006.04.004
and possesses an intrinsic function associated with insulin
action in mammals [11,12].
Up to date, the mechanisms of absorption of chromic
ions are uncertain. Little is known of the fate of Cr(III)
intaken orally. Essentially no data exist on the forms of
chromium(III) in food as a result of its very low concentration [13]. The fate of chromium(III) once it enters the
bloodstream is better elucidated. The iron-transport protein transferrin has been proposed to serve as the major
chromium transport agent [6]. Vincent has studied the
interaction of Cr(pic)3 with transferrin by UV–Vis spectrum [14,15]. In order to explore the mechanism of chromium(III) absorption, one novel chromium(III) complex
[Cr(SA)(en)2]Cl Æ 2H2O was synthesized, and then the manner, in which chromium is transferred from the complex to
apoovotransferrin (apoOTf), was followed by a combination of UV–visible (UV–Vis) and fluorescence spectra. As
a control experiment, EDTA was employed first as a simple competition agent at different temperature. The ligand
B. Liu et al. / Journal of Inorganic Biochemistry 100 (2006) 1462–1469
salicylic acid was chosen originally because of its low toxicity and the similar structure with that of aspirin.
2. Experiments
2.1. Materials and physical measurements
All manipulations were performed under aerobic conditions, and all chemicals were all analytical grade reagents
and were used without further purification. Apoovotransferrin was obtained from Sigma (Lot 115H7080). Deionized water was used throughout. All glassware, including
absorption and fluorescence cuvettes (1 · 1 cm), were routinely soaked in 1 M HNO3 and then rinsed with deionized
water.
UV–visible (UV–Vis) spectra were measured with a
HP8453 UV–Vis spectrophotometer. Fluorescence spectra
were measured with a Hitachi 850 fluorescence spectrophotometer. The temperature of the solutions was maintained
at 15 or 37 C by a jacketed cell holder connected to an
external circulating water bath (Shimadzu TB-85 or
Huber). Chemical analyses for carbon, hydrogen, and
nitrogen were determined by microanalysis using a Perkin–Elmer 240B elemental analyzer. The X-ray data were
collected on a Smart Apex CCDX diffractometer.
2.2. Preparations of protein samples
Apoovotransferrin was purified according to the literature [16]. The final concentration was determined from
the absorbance at 278 nm using an extinction coefficient
of 91 200 M1 cm1.
2.3. Synthesis of the complex
CrCl3 Æ 6H2O (0.67 g, 2.5 mM), salicylic acid (SA)
(0.34 g, 2.5 mM) and granular (Mesh size 20) zinc (0.10 g,
1.5 mM) was added into methanol (30 mL) and then was
refluxed for 1 h. To the mixture ethylenediamine (en) was
added dropwise and allowed to refluxed for 15 min as a
pink precipitate formed in >70% yield. The precipitate
was filtered, washed with methanol and dried. The dried
powder was dissolved in water and an X-ray quality crystal
was obtained after two days at room temperature.
2.3.1. Analysis
Calc. for C11H24O5N4ClCr (%): C, 34.78; H, 6.32; N,
14.76. Found (%): C, 34.57; H, 6.23; N, 14.83.
2.4. X-ray structure determination of CrC11H24O5N4Cl
A single crystal of dimensions 0.40 · 0.20 · 0.20 mm
was mounted on a glass fiber and used for date collection.
Cell constants and an orientation matrix for date collection
were obtained by least-squares refinement of diffraction
date from reflections with 2.44–26.81 using a Bruker
SMART APEX CCD automatic diffractometer. Date were
1463
collected at 293 K using Mo Ka radiation (k = 0.71073 Å)
and the x-scan technique and corrected for Lorentz and
polarization effect (SADABS).
The structure was solved by direct methods (SHELX97)
[17] and subsequent differences Fourier map and then
refined on F2 by a full-matrix least-squares procedure using
anisotropic displacement parameters [18]. After several
cycles of refinement, the hydrogen atoms of ethylenediamine and salicylic acid legends were located at their
calculated positions and were refined using a riding model,
with Csp3–H = 0.97 Å, Csp2–H = 0.93 Å and Nsp2–H =
0.90 Å, and with Uiso(H) = 1.5 Ueq(parent atom). The
hydrogen atoms of the water solvate molecules were
located in a difference Fourier map and constrained to ride
on their parent atoms, with O–H = 0.85 Å and Uiso(H) =
1.2 Ueq(O). Atomic scattering factors are from International Tables for X-ray Crystallography [19] and molecular
graphics from SHELXTL [20]. A summary of the crystal
date, experiment details and refinement results is given in
Table 1.
3. Results and discussion
3.1. Description of the structure of [Cr(SA)(en)2]ClÆ2H2O
The structure of [Cr(SA)(en)2]Cl Æ 2H2O was determined
by X-ray crystallography. The perspective structure and
the atomic numbering schemes for the chromium complex
are shown in Fig. 1. Selected bond lengths and angles are
given in Table 2.
Table 1
Crystal data and structure refinement for [Cr(SA)(en)2]Cl Æ 2H2O
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal color
Crystal system
Space group
Unit cell dimensions
a (Å)
b (Å)
c (Å)
b ()
Vol (Å3)
Z
Crystal density (g/cm3)
Crystal size (mm3)
Index ranges
h Range for data collection ()
Final R indices [I 2r]
R indices (all data)
F(0 0 0)
Absorption coefficient (mm1)
Reflections collected
Unique reflections [Rint]
Largest difference in peak and hole (e Å3)
CrC11H24N4O5Cl
379.79
293 (2)
0.71073 (Mo Ka)
red
monoclinic
P2(1)
8.354(2)
9.856(3)
10.120(3)
94.045(3)
831.2(4)
2
1.517
0.40 · 0.20 · 0.20
9 6 h 6 7,
9 6 k 6 11,
11 6 l 6 12
2.44–26.81
R1 = 0.0358, Rw2 = 0.0834
R1 = 0.0368, Rw2 = 0.0841
398
0.876
3430
2654 [0.0220]
0.46 and 0.23
1464
B. Liu et al. / Journal of Inorganic Biochemistry 100 (2006) 1462–1469
3.2. Fluorescence and UV–Vis spectra of the complex
In Fig. 2, the characteristic absorption peak of the salicylic acid occurs at 298 nm (curve a), while in the complex
this peak exhibits red shift to 326 nm (curve b). The electronic spectrum in the visible region of the complex (curve
c, attributed to d–d transitions) is altered in intensity and
shifts in position of the adsorption bands relative to the
corresponding Cr(III) aquo ions.
Salicylic acid contains carboxylic group and phenolic
hydroxyl (HOOCC6H4OH, the pK values of SA are 2.97
and 13.40 for the carboxylic and phenol groups) [21].
Fig. 3 displays the fluorescence spectrum of salicylic acid
in 0.01 M Hepes buffer at pH 7.4, with a maximum emission speak near 410 nm (curve b). With the coordination
235 nm
2
Absorbance
Fig. 1. The structure of the title compound, with the atom-numbering
scheme. Displacement ellipsoids are drawn at the 30% probability level for
non-H atoms.
Table 2
Selected bond lengths (Å), angles ()
O1–Cr1–O2
O2–Cr1–N1
O2–Cr1–N4
O1–Cr1–N3
N1–Cr1–N3
O1–Cr1–N2
N1–Cr1–N2
N3–Cr1–N2
1.903(2)
2.064(3)
2.090(3)
91.15(10)
90.01(10)
95.46(11)
89.53(11)
92.93(11)
176.17(10)
82.23(11)
91.87(11)
Cr1–O2
Cr1–N2
Cr1–N4
O1–Cr1–N1
O1–Cr1–N4
N1–Cr1–N4
O2–Cr1–N3
N4–Cr1–N3
O2–Cr1–N2
N4–Cr1–N2
b 326 nm
1.941(2)
2.095(3)
2.082(3)
508 nm
c
94.14(11)
91.69(11)
171.93(11)
176.92(11)
81.52(12)
87.64(11)
92.04(11)
The main structure of the title compound is made up of
one [Cr(SA)(en)2]+ complex cation, one Cl and two water
solvate molecules. The chromium(III) center is a octahedron with four nitrogen atoms (Cr–N, 2.064–2.095 Å) from
two en molecules, one phenolic hydroxyl oxygen (Cr–O,
1.903 Å) and carboxylate oxygen (Cr–O, 1.941 Å) from
SA ligand. These two oxygen atoms and two nitrogen
atoms occupy the equatorial position and the other nitrogen atoms occupy the axial position. The complex unit consists of one six-member ring (C–C–O–Cr–O–C) and two
five-member rings (C–N–Cr–N–C) in the molecule.
[Cr(SA)(en)2]Cl Æ 2H2O belongs to monoclinic system with
the space group P2(1), a = 8.354(2) Å, b = 9.856(3) Å,
c = 10.120(3) Å, b = 94.045(3), V = 831.2(4) Å3 and Z =
2. There are four kinds of intermolecular hydrogen bonds
formed which stabilize the conformation with the distance
in the ranges of 3.068–3.114 Å for O–H O, 3.165 Å for
O–H Cl, 2.907–3.012 Å for N–H O and 3.268–
3.600 Å for N–H Cl, respectively.
0
300
400
500
600
Wavelength/nm
Fig. 2. Absorption spectra of [Cr(SA)(en)2]+ and SA (a) [SA] = 4.0 ·
104 M. (b) [Cr(SA)(en)2]+ = 3.7 · 104 M. (c) [Cr(SA)(en)2]+ = 2.0 · 102.
400
Fluorescence intensity/a.u.
Cr1–O1
Cr1–N1
Cr1–N3
a 298 nm
1
b
300
200
100
a
0
360
400
440
Wavelength/nm
Fig. 3. Fluorescence spectra of the [Cr(SA)(en)2]+ and SA, 0.05 M Hepes,
pH 7.4, room temperature, slit, 10 nm; filer, 350 nm; kex, 280 nm; (a)
[Cr(SA)(en)2]+ = 1.0 · 106 M. (b) [SA] = 1.0 · 106.
B. Liu et al. / Journal of Inorganic Biochemistry 100 (2006) 1462–1469
of salicylic acid to Cr(III) the fluorescence intensity at
410 nm is quenched and the title complex hardly has any
fluorescence in the same condition, as shown in Fig. 3
(curve a).
3.3. Transfer of chromium
3.3.1. The competition with EDTA
Chromium levels in tissues and biological fluids are
extremely low, generally within an order of magnitude of
the detection limits of current analytical techniques. The
common bioinorganic probes for Cr(III) are of limited utility [6]. As described above, the spectra are sensitive to the
changes of the coordination sphere Cr(III) center for
[Cr(SA)(en)2]+, especially such as the dissociation of SA,
give rise to significant changes in the complex’s absorption
and fluorescence spectra. Therefore it enables us to follow
the reactivity of [Cr(SA)(en)2]+ towards biomolecule in a
low concentration by those spectra. In order to research
the interaction of the complex with transferrin, EDTA
was employed as a simple competitive ligand first.
To examine the transfer of Cr(III) from [Cr(SA)(en)2]+
to EDTA, 10 equiv. of EDTA was added to 10 lM
[Cr(SA)(en)2]+ in 0.01 M Hepes, pH 7.4. The reaction mixture was stored at 37 C. Both the UV–Vis spectra and
fluorescence spectra were monitored as a function of time
until the spectra became constant with time. The UV–Vis
spectra are shown in Fig. 4. It can be seen that the absorption peaks at 326 nm for the coordinated SA decrease and
the peaks at 298 nm for free SA increase gradually. There
are two isosbestic points at 310 nm and 280 nm, respectively. Curves of absorbance at 326 nm and 298 nm, A326
vs. time and A298 vs. time for the reaction are shown in
Fig. 4 inset. It is obvious that the complex [Cr(SA)(en)2]Cl
1465
is decompounded by EDTA, meanwhile, salicylic acid
ligand is free from Cr(III) gradually. The rate constant is
calculated to be (2.20 ± 0.05) · 102 M1 h1.
The fluorescence spectra are shown in Fig. 5. Sample
was excited at 280 nm and the emission was monitored
from 350 nm to 480 nm. The characteristic fluorescence
intensity of SA at 410 nm increase gradually. Curve of
F410 vs. time for the reactions at different temperature are
displayed in Fig. 5 inset, the rate constant at 37 C is
(1.90 ± 0.03) · 102 M1 h1. It shows that SA ligand is
released from the complex gradually. In contrast, the
absorbance spectra of the complex [Cr(SA)(en)2]Cl is stable
in Hepes buffer at 37 C. Furthermore, the rate constants
obtained from the two methods are in good agreement,
so it well indicates that Cr(III) are combined to EDTA,
and SA or en ligands are competitively replaced. This procedure can be briefly illustrated by the following sketch:
þ EDTA
½CrðSAÞðenÞ2 ƒƒ! CrðEDTAÞ þ SA þ 2en
3.3.2. The competition with apoovotransferrin (apoOTf)
The manner in which chromium is transported in organism is unknown. The iron-transport protein transferrin, the
second most abundant protein in blood serum, has been
proposed to serve as the major chromium transport agent.
A molecule of transferrin (80 kDa) is made up of two
similar halves, the N- and C-lobes, joined by a bridging
peptide. Each lobe is comprised of two domains, the N I
and N II for the N-lobe and C I and C II for C-lobe, which
form a cleft containing a high-affinity binding site for ferric
ion and other ions. The ferric ion is bound in a distorted
coordination to four protein ligands (in human transferrin
by Asp63, Tyr95, Tyr188 and His249 for the N-lobe, or by
A 298
.2
Absorbance
0
100
Time/h
200
1
.4
2
3
.2
d
.4
A 326
.6
1
1200
Fluorescence intensity/a.u.
A
.6
F410 /a.u.
300
.8
2
600
0
200
0
200 400 600
Time/h
c
100
4
b
a
0
0. 0
280
320
Wavelength/nm
360
Fig. 4. Absorption spectra at different time for the mixture of 10 lM
[Cr(SA)(en)2]+ with 10 equiv. of EDTA, 37 C, 0.01 M Hepes, pH 7.4,
time (h) 1, 0; 2, 50; 3, 100; 4, 150 h. Inset: curves of absorbance at 296 nm
and 326 nm with time.
400
440
Wavelength/nm
Fig. 5. Fluorescence changes with time (a ! d) for the mixture of 10 lM
[Cr(SA)(en)2]+ with 10 equiv. of EDTA at 15 C in 0.01 M Hepes, pH 7.4,
time (h): a, 0; b, 4; c, 30; d, 50 h. Inset: curves of fluorescence at 410 nm
with time at different temperature, 1, 37 C; 2, 15 C.
B. Liu et al. / Journal of Inorganic Biochemistry 100 (2006) 1462–1469
Asp392, Tyr426, Tyr517 and His585 for the C-lobe) and
two ligands from a synergistically bound, bidentate anion
[22–24]. Binding constants of chromium to transferrin were
reported to be K1 = 1.42 · 1010 M1 and K2 = 2.06 ·
105 M1. In this paper, apoovotransferrin (apoOTf) was
utilized in place of serum transferrin because of its ready
availability in quantity and its cost; the binding properties
of apoOTf are nearly identical to serum transferrin [14].
ApoOTf can bind Fe(III) and other metal ions tightly
[25,26] in the presence of synergistically bound anion that
is usually carbonate. The UV–Vis spectra of Cr(III)-OTf
reported previously are similar to that of Cr(III)-saturated
transferrin [15].The addition of Cr(III) to apoOTf results in
enhancement of the intensity of the UV absorption bands
at ca. 240 and 291 nm (arising in part from tyrosine residues which serve as metal ligands) as a result of Cr(III)
binding [15].
Furthermore, fluorescence quenching measurements can
also be used to monitor metal binding [23]. The fluorescence is dominated by emission from Trp residues, but
the fluorescence may be quenched when a cation, such as
a transition metal or a lanthanide ion, is bound nearby.
In the paper, titration of CrCl3 to apoOTf was monitored
by fluorescence spectra at 20 C in 0.01 M Hepes buffer at
pH 7.4. When the metal–protein binding had been equilibrated after 30 min, a fluorescence spectrum was recorded
and a new aliquot of titration was added. To correct for
dilution during each titration and normalize the results
from different titration, the fluorescence intensity was converted to molar fluorescence intensity (FM) by dividing the
fluorescence intensity by the analytical concentration of
apoOTf, as shown in Fig. 6. Highly effective quenching
occurred at 336 nm in this titration, fluorescence intensity
of apoOTf decreased to about 50%. From Fig. 6, it can
be seen that one apoOTf molecule binds approximately
2 equiv. of Cr(III). Such behavior arises from the interac-
tion of the Cr(III) with the hydrophobic pockets in the protein structure (harboring the tryptophan residues), thus
leading to the energy transfer quenching of tryptophan
fluorescence. Whereas quenching was scarcely observed in
the titration of salicylic acid to apoOTf in the same condition, meanwhile, the fluorescence intensity at 410 nm
increased regularly with the increasing of concentration
of salicylic acid. Thus, for apoOTf, both changes in fluorescence spectrum (336 nm) and the UV–Vis spectrum at ca.
240 and 291 nm can be used to investigate the transfer of
chromium from the complex [Cr(SA)(en)2]Cl to protein.
For convenience, some characteristic fluorescence peaks
or UV–Vis absorption peaks appeared in this paper are
listed in Table 3.
First, solution of 18 lM [Cr(SA)(en)2]+ with 1 equiv. of
apoOTf in 0.01 M Hepes, pH 7.4 was excited at 280 nm at
37 C. The fluorescence of apoOTf at 336 nm (characteristic of tryptophan residue) is quenched with time slowly
(a ! c), as shown in Fig. 7. Curve of F336 vs. time for the
reaction is given in Fig. 7 inset. The quenching phenomenon at 336 nm results from the decomposition of the
complex and combination of Cr(III) to the protein. The
rate constant at 37 C is calculated to be (5.02 ± 0.05) ·
102 M1 h1. The peak at 410 nm does not appear as
Table 3
Characteristic fluorescence peaks or UV–Vis absorption peaks
Free-salicylate
Coordinated-salicylate
apoOTf
Cr-OTf
Fluorescence intensity/a.u.
600
7
4
F 336 /1 0 M
-1
8
6
Fluorescence
peak kmax (nm)
UV–Vis absorption
peak (nm)
410 (strong)
410 (very weak)
336
336 (weak)
298
326
280
240, 291
a
a
b
F 336 /a.u.
1466
400
200
c
0
400
200
400
Time/ h
200
5
320
0
1
2
3
4
[Cr] / [apoOTf]
Fig. 6. Titration curve for the addition of CrCl3 to apoOTf at 20 C in
0.01 M Hepes, pH 7.4.
360
Wavelength/nm
400
Fig. 7. Fluorescence spectra at different time (a ! c) for the reaction of
18 lM [Cr(SA)(en)2]+ with 1 equiv. of apoOTf at 37 C in 0.01 M Hepes at
pH 7.4. Inset: the changes of fluorescence at 336 nm with time.
B. Liu et al. / Journal of Inorganic Biochemistry 100 (2006) 1462–1469
expectation, although the same concentration of SA with
apoOTf exhibits a strong fluorescence peak at 410 nm
(shown in Fig. 8).
Fig. 9 shows the difference UV spectra of the mixture of
18 lM [Cr(SA)(en)2]+ with 1 equiv. of apoOTf at 37 C
blanked as protein solution. With time going it reveals a
significant enhancement in the absorbance at 291 and
240 nm. To be surprising, the peak at 326 nm nearly does
not decrease. Curve of A291 vs. time for the reaction is
shown in Fig. 9 inset. The increase may be attributed to
the binding of Cr(III) to residues of the protein. The rate
Fluorescence intensity/a.u.
600
400
3
2
200
1
330
360
390
Wavelength/nm
420
Fig. 8. Fluorescence spectra for the addition of 2.3 · 104 M SA to
2.3 · 106 M apoOTf 2 mL at 37 C in 0.01 M Hepes buffer, pH 5.0. V
(SA)/lL: 1, 0 lL; 2, 20 lL; 3, 50 lL.
.6
1467
constant at 37 C is calculated to be (5.02 ± 0.05) ·
102 M1 h1. It is well consistent with the result from fluorescence spectra approximately, meaning that Cr(III) has
transferred from the title complex to apoOTf and the Cr–
apoOTf bonds are formed.
However, neither concomitant appearance of free salicylate fluorescence at 410 nm nor any decrease in the intensity of the Cr(III)-salicylate charge transfer band at 326 nm
are observed throughout this competition reaction. These
interesting results allow us to infer that SA ligand is not
replaced by apoOTf in the transfer of Cr(III) to the protein. This may bring about some questions, such as the fate
of en and SA ligands. To identify it, some control experiments were carried out.
It is impossible to observe directly the free en ligands in
the reaction mixture only by UV–Vis spectra or fluorescence spectra. In this paper [Cr(en)3]Cl3 was employed to
react with apoOTf in an attempt to uncover the fate of
en ligands. Two equivalents of [Cr(en)3]Cl3 was added to
14 lM apoOTf in 0.01 M Hepes, pH 7.4. Sample was
allowed to incubate at 37 C. Both the UV–Vis spectra
and fluorescence spectra were monitored as a function of
time. The UV–Vis spectra changes are depicted in
Fig. 10. Curve of A291 vs. time for the reaction is shown
in Fig. 10 inset. The spectra also show enhancement of
the intensity of the UV–Vis absorption bands at ca. 240
and 291 nm. The rate constant at 37 C is calculated to
be (2.15 ± 0.03) · 103 M1 h1. When the chromium complexes (CrCl3 [15], [Cr(en)3]Cl3 and [Cr(SA)(en)2]Cl) react
with apoOTf, they all exhibit very similar UV–Vis spectra
in the range of 240–300 nm, and either of the fluorescence
intensity of apoOTf at 336 nm in those three reactions is
quenched gradually. Based on the coordination demands
or binding abilities of the apoOTf site, it can be proposed
.45
e
.16
.4
A 291
a
A298
.30
.15
.08
.2
0.00
0.00
0
Time/h
.2
40
80 120
Time/ h
ΔA
ΔA
0 100 200 300
.1
d
a
0.0
250
300
Wavelength/nm
350
Fig. 9. Difference UV spectra for the reaction of 18 lM [Cr(SA)(en)2]+
with 1 equiv. of apoOTf at different time, 37 C, 0.01 M Hepes, pH 7.4,
time (h): a, 0; b, 25; c, 75; d, 96; e, 120 h. Inset: curves of absorbance at
291 nm with time.
0.0
250
300
Wavelength/nm
350
Fig. 10. Difference UV spectra for the reaction of 2 equiv. of [Cr(en)3]Cl3
with 14 lM apoOTf at different time, 37 C, 0.01 M Hepes, pH 7.4, time
(h): a, 6; b, 12; c,24; d, 60. Inset: curves of absorbance at 291 nm with time.
1468
B. Liu et al. / Journal of Inorganic Biochemistry 100 (2006) 1462–1469
that two en ligands are dissociated during the transfer of
Cr(III) from [Cr(SA)(en)2]Cl to apoOTf.
Then, 1 equiv. of [Cr(SA)(en)2]Cl was pre-incubated
with 18 lM apoOTf in 0.01 M Hepes, pH 7.4 at 37 C
for 8 days (period I). Then excessive EDTA was added
to the mixture and was stored at 37 C again (period
II). Both the UV–Vis spectra and fluorescence spectra
were monitored as a function of time through the course,
as shown in Figs. 11 and 12, respectively. Sample is
referred for simplicity as [Cr(SA)-apoOTf] + EDTA. In
period II with the addition of 250–2500 equiv. of EDTA,
.6
ΔA
.4
a
.2
c
0.0
250
300
350
Wavelength/nm
400
Fig. 11. Difference UV spectra for the reaction of excessive EDTA with
18 lM [Cr(SA)-apoOTf], EDTA (equiv./apoOTf) a, 0; b, 250; c, 2500.
Fluorescence intensity/a.u.
300
c
200
a
100
0
320
360
Wavelength/nm
400
Fig. 12. Fluorescence spectra for the reaction of excessive EDTA with
18 lM [Cr(SA)-apoOTf], EDTA (equiv./apoOTf) a, 0; b, 250; c, 2500.
Scheme 1.
the Cr(III)-salicylate charge transfer band (kmax =
326 nm) and the UV–Vis absorption bands at ca. 240
and 291 nm all decrease; the peaks at 298 nm for the free
SA appear (Fig. 11). Meanwhile, the quenched fluorescence intensity of apoOTf at 336 nm is restored, with
the appearance of fluorescence of free SA at 410 nm
(Fig. 12). All these phenomena clearly show the coordinated SA to Cr(III) bound by apoOTf is replaced by
excessive EDTA in period II and apoOTf or SA molecules are free from Cr(III). In other words, a very stable
complex [Cr(SA)–apoOTf] is formed in period I and then
destroyed by excessive EDTA in period II.
A number of literatures have indicated that dozens of
organic anions (including salicylate) could substitute for
carbonate as the synergistically bound anions in iron–
transferrin complex [27,28]. A carboxylate group and a
proximal polar group (hydroxy, keto, amino, or carboxylate) within 6.3–7.0 Å of the carboxylate are required for
synergistic binding. Dubach and his coworkers used EPR
spectra to examine the mode of synergistic anion binding.
It is proposed that these anions behave as bidentate
ligands, with coordination to the iron through both the carbonate and proximal polar group [29]. Salicylate–Fe(III)–
transferrin complex shows characteristic change transfer
band in the visible spectrum with kmax = 450 nm, implying
direct metal–anion bonding. The salicylate anion readily
forms a chelate ring and is proposed to have the same function as the synergistically bound anion. This theory is
favored by the results obtained in this paper. However,
en molecular does not fit for the demands of synergistically
bound, and it is easily replaced by apoOTf. Based on the
discussion above, it can be inferred in reason that salicylate
serves as the role of synergistically bound anion when
Cr(III) is transferred from the title complex to the protein
and the salicylate–Cr(III)–transferrin ternary complex is
formed. Perhaps the summing-up can be illustrated in
Scheme 1.
Vincent and co-workers [14] has studied the interaction
of Cr(pic)3 with apoOTf in 5% DMSO and 95% Tris
(0.05 M, pH 7.5) by UV–Vis spectrum. It showed that
the Cr(III) could not be transferred from Cr(pic)3 to apoOTf unless the metal was reduced to the Cr(II) level. In
contrast, [Cr(SA)(en)2]+ is relatively easy to release
[Cr(SA)]+ to apoOTf and forms a very stable complex
salicylate–Cr(III)–transferrin.
B. Liu et al. / Journal of Inorganic Biochemistry 100 (2006) 1462–1469
4. Conclusions
This paper describes the synthesis and characterization
of the complex [Cr(SA)(en)2]Cl Æ 2H2O and the reaction
with apoovotransferrin. The mechanisms of transfer of
chromium from the complex to EDTA and apoOTf are different. Competition studies show that Cr(III) can be transferred from the complex to apoovotransferrin, with the
retention of the salicylate, it is inferred first by the author
that a very stable ternary complex salicylate–Cr(III)–transferrin was formed. The use of salicylic acid is desirable for
further studies to test the role of chromium metabolism.
References
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
5. Abbreviations
Hepes
EDTA
apoOTf
SA
en
N-2-hydroxyethyl-piperazine-N 0 -2-ethansulfonic
acid
ethylenediamine-N,N,N 0 ,N 0 -tetraacetic acid
apoovotransferrin
salicylic acid
ethylenediamine
[11]
[12]
[13]
[14]
[15]
[16]
[17]
6. Supplementary materials
[18]
[19]
Crystallographic data for the compound has been
deposited with the Cambridge Crystallographic Data Centre, CCDC Nos. 289525. Copies of this information may be
obtained free of charge from The Director, CCDC, 12
Union Road, Cambridge, CB2 1EZ, UK, fax: +44 1223
336 033, e-mail: deposit@ccdc.cam.ac.uk or http://www.
ccdc.cam.ac.uk.
Acknowledgements
This work was supported by the National Natural Science Foundation of PR China (No. 20371031) and the
Natural Science Foundation of Shanxi Province (No.
20031017).
1469
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
J.B. Vincent, Acc. Chem. Res. 33 (2000) 503–510.
R.A. Anderson, Clin. Physiol. Biochem. 4 (1986) 31–41.
C.M. Davis, J.B. Vincent, J. Biol. Inorg. Chem. 2 (1997) 675–679.
A. Levina, R. Codd, C.T. Dillon, P.A. Lay, Prog. Inorg. Chem. 51
(2002) 145–250.
W. Mertz, Nutr. Rev. 56 (1998) 174–177.
J.B. Vincent, Polyhedron 20 (2001) 1–26.
T.O. Berner, M.M. Murphy, R. Slesinski, Food Chem. Toxicol. 42
(2004) 1029–1042.
D.D.D. Hepburn, J.B. Vincent, J. Inorg. Biochem. 94 (2003) 86–93.
K.R. Manygoats, M. Yazzie, D.M. Stearns, J. Biol. Inorg. Chem. 7
(2002) 791–798.
S. Chaudhary, J. Pinkston, M.M. Rabile, J.D.V. Horn, J. Inorg.
Biochem. 99 (2005) 787–794.
J.B. Vincent, J. Nutr. 130 (2000) 715–718.
J.B. Vincent, Nutr. Rev. 58 (2000) 67–72.
B.J. Clodfelder, R.G. Upchurch, J.B. Vincent, J. Inorg. Biochem. 98
(2004) 522–533.
N.E. Chakov, R.A. Collins, J.B. Vincent, Polyhedron 18 (1999) 2891–
2897.
Y. Sun, J. Ramirez, S.A. Woski, J.B. Vincent, J. Biol. Inorg. Chem. 5
(2000) 129–136.
W.R. Harris, B.-S. Yang, S. Abdollahi, Y. Hamada, J. Inorg.
Biochem. 76 (1999) 231–242.
G.M. Sheldrick, Program for the Solution of Crystal Structure,
University of Gottingen, Germany, 1997.
G.M. Sheldrick, Program for the Refinement of Crystal Structure,
University of Gottingen, Germany, 1997.
International tables for X-ray Crystallography, vol. C, Kluwer
Academic Publishers, Doordrecht, The Netherlands, 1995.
G.M. Sheldrick, SHELXTL/PC. Version 5.1, Bruker AXS Inc.,
Madison, WI, USA, 1999.
N. Arnaud, J. Georges, Analyst 124 (1999) 1075–1078.
E.N. Baker, Adv. Inorg. Chem. 41 (1994) 389–463.
B.-S. Yang, J.-Y. Feng, Y.-Q. Li, F. Gao, Y.-Q. Zhao, J.-L. Wang, J.
Inorg. Biochem. 96 (2003) 416–424.
] B.J. Clodfelder, J. Emamaullee, D.D. Hepburn, N.E. Chakov, H.S.
Nettles, J.B. Vincent, J. Biol. Inorg. Chem. 6 (2001) 608–617.
W.R. Harris, P.K. Bali, Inorg. Chem. 27 (1988) 2687–2691.
B.-S. Yang, W.R. Harris, Acta Chim. Sinca 57 (1999) 503–509.
M.S. Shongwe, C.A. Smith, E.W. Ainscough, H.M. Baker, A.M.
Brodie, E.N. Baker, Biochemistry 31 (1992) 4451–4458.
M.R. Schlabach, G.W. Bates, J. Biol. Chem. 250 (1975) 2182–
2188.
J. Dubach, B.J. Gaffney, K. More, G.R. Eaton, S.S. Eaton, Biophys.
J. 59 (1991) 1091–1100.