Crystal structure and interaction with bovine serum albumin of the Cu(I/II) complex [C20H32Cu2I3N 4]n

Article abstract of DOI:10.1080/00958972.2012.706282

[C₂₀H₃₂Cu₂I₃N₄] _n was synthesized and characterized by elemental analysis, ESI-MS spectrometry, and IR spectra. The crystal structure was determined by X-ray single-crystal diffraction. The

Full text of DOI:10.1080/00958972.2012.706282

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Crystal structure and interaction with
bovine serum albumin of the Cu(I/II)
complex [C20H32Cu2I3N4] n
Yu-Fen Liu a , Hai-Tao Xia a & De-Fu Rong b
a School of Chemical Engineering, Huaihai Institute of Technology,
Lianyungang, Jiangsu, China
b Beilun Entry-Exit Inspection and Quarantine Bureau of China,
Ningbo, Zhejiang, China
Accepted author version posted online: 25 Jun 2012.Published
online: 10 Jul 2012.
To cite this article: Yu-Fen Liu , Hai-Tao Xia & De-Fu Rong (2012): Crystal structure and interaction
with bovine serum albumin of the Cu(I/II) complex [C20H32Cu2I3N4] n , Journal of Coordination
Chemistry, 65:16, 2919-2934
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Journal of Coordination Chemistry
Vol. 65, No. 16, 20 August 2012, 2919–2934
Crystal structure and interaction with bovine serum albumin
of the Cu(I/II) complex [C20H32Cu2I3N4]n
YU-FEN LIUy, HAI-TAO XIA*y and DE-FU RONGz
ySchool of Chemical Engineering, Huaihai Institute of Technology, Lianyungang,
Jiangsu, China
zBeilun Entry-Exit Inspection and Quarantine Bureau of China, Ningbo,
Zhejiang, China
(Received 19 January 2012; in final form 8 May 2012)
[C20H32Cu2I3N4]n was synthesized and characterized by elemental analysis, ESI-MS spectro-
metry, and IR spectra. The crystal structure was determined by X-ray single-crystal diffraction.
The binding of the complex with bovine serum albumin (BSA) was studied by fluorescence
spectroscopy under simulated physiological conditions. The binding constant (Kb), the number
of binding sites (n), and the corresponding thermodynamic parameters DH, DS, DG were
calculated based on the van’t Hoff equation. The complex had strong ability to quench the
fluorescence from BSA, and the quenching mechanism of this complex to BSA was static
quenching. Hydrogen bonds and van der Waals forces are the interactions between the Cu(I/II)
¨
complex and BSA. According to the Forster non-radiation energy transfer theory, the binding
average distance between the donor (BSA) and the acceptor (Cu(I/II) complex) was obtained.
The effect of the complex on the BSA conformation was also studied by using synchronous
fluorescence spectroscopy.
Keywords: Cu(I/II) Complex; Crystal structure; Bovine serum albumin; Fluorescence spectrum
1. Introduction
Copper is a physiologically important metal [1, 2], playing an important role in growth,
scavenging harmful free radicals, and preventing oxidative damage to cells, protein
synthesis, and the activity of metal enzymes [3], capable of participating in a variety of
redox cycling reactions [4]. Protein is the major target of many types of medicines.
Studying the structure and function of proteins are important in biochemistry,
chemistry, and medicine. Serum albumins are the most abundant proteins in the
circulatory system with many important physiological functions, contributing to the
osmotic blood pressure and mainly responsible for the maintenance of blood pH [5–7].
The most important physiological role of albumins is the binding, transport, and
deposition of a variety of endogenous and exogenous substances in blood [8], such as
fatty acids, drugs, metabolites, metal ions, and other biologically-active compounds
present in the blood [9]. Distribution, free concentration, and metabolism of various
*Corresponding author. Email: xht161006@hhit.edu.cn
Journal of Coordination Chemistry
ISSN 0095-8972 print/ISSN 1029-0389 online ß 2012 Taylor & Francis
http://dx.doi.org/10.1080/00958972.2012.706282

2920
Y.-F. Liu et al.
drugs can be significantly altered as a result of their binding to serum albumin [10].
Thus, investigating the interaction between serum albumins and drugs is important for
understanding the transportation and distribution of drugs in the body and clarifying
the action mechanism and pharmaceutical dynamics, important in chemistry, life
sciences, and clinical medicine. Among the serum albumins, bovine serum albumin
(BSA) is usually selected as the protein model because of its abundance, low cost, ease
of purification, stability, medical importance, and unusual ligand-binding properties.
Studies are consistent with the fact that human and BSAs are homologous proteins
[11, 12]. Techniques used to detect the interaction between drugs and serum albumin
include fluorescence spectroscopy [8, 13–15], UV-spectrophotometry [16, 17], Fourier
transform infrared (FT-IR) [18, 19], circular dichroism spectroscopy, and cyclic
voltammetry [20, 21]; fluorescence quenching is a useful method to study the reactivity
of chemical and biological systems since it allows non-intrusive measurements of
substances in low concentration under physiological conditions [22]. It can reveal
accessibility of quenchers to serum albumin’s fluorophores, help in understanding
binding mechanisms of serum albumin to compounds, and provide clues to the nature
of the binding phenomenon. Despite the numerous studies on copper complexes and
their interactions with bio-macromolecules [23–25], in this study, we report the crystal
structure of a new copper (I/II) complex containing N1-benzylpropane-1,2-diamine.
The binding properties of this complex with BSA have been carried out using
fluorescence spectroscopy, giving binding constants at different temperatures in
HCl-Tris (pH 7.4) buffer solution. The enthalpy changes (DH) and entropy changes
(DS) between BSA and the complex are calculated and the interaction between BSA and
the complex are discussed.
2. Experimental
2.1. Materials
BSA was purchased from Beijing Biosea Biotechnology Company; its molecular weight
is assumed to be 67000. All BSA solutions are prepared in a pH 7.4 buffer solution, and
BSA stock solution (1.0 ꢀ 10ꢁ4 mol Lꢁ1) was kept in the dark at 4ꢂC. NaCl (analytical
grade, 0.5 mol Lꢁ1) is used to maintain the ionic strength. Buffer solution consist of Tris
(0.05 mol Lꢁ1) and HCl (0.05 mol Lꢁ1), and the pH is adjusted to 7.4 by adding
0.1 mol Lꢁ1 NaOH at 298 K. The Cu(I/II) complex solutions (3.0 ꢀ 10ꢁ5 mol Lꢁ1) were
prepared in ethanol. All other materials were of analytical reagent grade; solutions are
prepared with doubly-distilled water.
2.2. Physical measurements
Elemental analyses were measured on a Perkin-Elmer 2400c Element analyzer. Infrared
spectra were recorded on a Nicolet 5DX FT-IR spectrophotometer using KBr discs
from 4000 to 400 cmꢁ1. Fluorescence spectra were measured with a Shimadzu RF-5301
fluorophotometer equipped with a 150 W Xenon lamp source and 1.0 cm quartz cell.
Absorption spectra were studied with a Shimadzu UV-2550 PC spectrophotometer.
MS spectra were recorded on a Shimadzu LCMS-2020 mass spectrometer using ethanol
as mobile phase.

Cu(I/II) Interaction with BSA
2921
2.3. Preparation of ligand
To a solution of benzaldehyde (0.1 mol) in ethanol (20 mL), propane-1,2-diamine
(0.1 mol) in ethanol (20 mL) was added. The mixed solution was stirred for 5 h at 70ꢂC,
allowed to stand, and the precipitate was collected by filtration. The precipitate was
dissolved in 40 mL ethanol : chloroform (1 : 2 v/v) and solid NaBH4 (0.4 mol) was
added. The mixture was stirred at 45ꢂC for 7 h and then water was added to 200 mL,
statically separated, the yellow viscous liquid was obtained by evaporating the organic
solvent. Anal. Calcd for C10H16N2 (%): C, 73.12; H, 9.82; N, 17.06. Found (%):
C, 73.45; H, 9.77; N, 17.09. IR (KBr, ꢀ (cmꢁ1)): 3306, 3034, 2968, 2812, 1603, 1545,
1451, 1154, 1025, 732, 693.
2.4. Preparation of complex
To a solution of the ligand (10 mmol) in methanol (30 mL), a mixture of CuAc2
(5 mmol) and NaI (5 mmol) in water (10 mL) was added. The mixture was stirred at
70ꢂC for 2 h and the color of the solution turned gradually green. The solvent was
cooled to room temperature, a green precipitate was collected by filtration, washed with
water and methanol successively, and finally dried to afford green powder in 67.9%
yield. Recrystallization of the complex from ethanol at room temperature gave crystals
suitable for X-ray single-crystal diffraction. Anal. Calcd for C20H32Cu2I3N4 (%):
C, 28.72; H, 3.86; N, 6.70. Found (%): C, 28.45; H, 3.73; N, 6.72. IR (KBr, ꢀ (cmꢁ1)):
3440, 2924, 2852, 1637, 1494, 1450, 1384, 1230, 1070, 765, 700, 617, 557. ESI-MS (m/z):
881.6, 838.5, 707.9, 673.9, 415.3, 372.9, 291.8, 279.1, 198.1.
2.5. X-ray crystal structure determination
A single-crystal of the complex suitable for X-ray crystallographic analysis was
mounted in sealed glass capillaries. Diffraction data were collected with a Bruker
SMART 1000 CCD diffractometer by the use of graphite monochromated Mo-Ka
˚
radiation (ꢁ ¼ 0.71073 A) at 298(2) K. The crystal structure was solved by direct
methods and all non-hydrogen atoms were located with successive difference Fourier
syntheses. The structure was refined by full-matrix least-squares on F2 with anisotropic
thermal parameters for all non-hydrogen atoms. Hydrogen atoms were added
according to theoretical models. All calculations were performed using the programs
contained in the SHELXL package [26, 27]. A summary of crystallographic data and
refinement parameters for the Cu(I/II) complex are given in table 1.
2.6. Fluorescence spectra
The fluorescence measurements of complex-BSA solutions were performed at different
temperatures by keeping the concentration of BSA fixed at 3 ꢀ 10ꢁ6 mol Lꢁ1 while
varying the Cu(I/II) complex concentration from 0 to 7 ꢀ 10ꢁ6 mol Lꢁ1. The widths of
both the excitation slit and the emission slit were set to 5.0 nm. Fluorescence spectra
were recorded from 300 to 500 nm at an excitation wavelength of 285 nm.

2922
Y.-F. Liu et al.
Table 1. Crystal data and structure refinement for complex.
Formula
Formula weight
Temperature (K)
C20H32N4Cu2 I3
836.28
293(2)
0.71073
Monoclinic
P2(1)
˚
Wavelength (A)
Crystal system
Space group
Unit cells and dimensions (A, )
ꢂ
˚
a
b
c
ꢂ
ꢃ
ꢄ
9.8764(15)
13.4861(19)
10.6808(18)
90
103.892(2)
90
1381.0(4), 2
2.011
3
˚
Volume (A ), Z
Calculated density (Mg mꢁ3
F(000)
)
794
0.31 ꢀ 0.30 ꢀ 0.29
Crystal size (mm3)
ꢅ Range for data collection (ꢂ)
Reflections collected
1.96–25.01
6877
Independent reflection
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I 4 2ꢆ(I)]
4781 [R(int) ¼ 0.0491]
Full-matrix least-squares on F2
4781/1/331
0.99
R1 ¼ 0.0579, wR2 ¼ 0.1306
ꢁ3
˚
Largest difference peak and hole (e A
)
0.810 and ꢁ0.694
2.7. Energy transfer between protein and Cu(I/II) complex
The absorption spectrum of the Cu(I/II) complex was recorded at 298 K from 300 to
500 nm (three replicates). The emission spectrum of BSA was also recorded at 298 K in
the same range (three replicates). Then, the overlap of the UV absorption spectrum of
complex with the fluorescence emission spectrum of BSA was used to calculate the
energy transfer.
2.8. Synchronous fluorescence spectra
Synchronous fluorescence spectra were collected with Dꢁ ¼ 15 and Dꢁ ¼ 60 nm,
respectively, under the same experimental conditions.
3. Results and discussion
3.1. Crystal structure of complex
The molecular structure of the complex is shown in figure 1 and selected bond distances,
bond angles, and hydrogen bonds are summarized in table 2. The Cu(I/II) complex
contains a dinuclear unit, an iodide bridges the pair of coppers, resulting in the
formation of a polymer. Copper(II) is six-coordinate with a distorted octahedral

Cu(I/II) Interaction with BSA
2923
Figure 1. Molecular crystal structure of the Cu(I/II) complex [symmetry code: A (1 ꢁ x, ꢁ1/2 + y, 1 ꢁ z)].
coordination geometry; the coordination atoms are four nitrogen atoms from two
ligands and two iodides. The distortion leads to I–Cu–I angles ranging from 163(3)ꢂ to
171.31(17)ꢂ. The ligand donors are in the equatorial position with axial positions
occupied by iodides. The Cu2–N distances are all different, ranging from 1.979(14) to
˚
2.054(17) A, shorter than that found for [Cu0.92Zn0.08I2(C5H5N)4] ꢃ 2C5H5N [28]
˚
(average Cu–N 2.037 A) and longer than that for Cu–N (Cu3L2(MeCN)2I2](MeCN)2,
˚
˚
average 2.008 A) [29]. The average Cu2–I distance is 3.277 A, slightly longer than that
˚
[29] of (Cu2–I1 3.211(1) A) and significantly shorter than that [28] of (Cu1–I1 3.460(5)
˚
A). The copper(I) is three-coordinate with three iodides and exhibits a triangle planar
arrangement with angles close to 120ꢂ (sum of the three angles ¼ 359.7ꢂ). The Cu1–I
˚
bond lengths range from 2.463(19) to 2.577(5) A, shorter than those reported for
copper(I) iodide complexes [30–34]. Hydrogen atoms attached to N, C1, C2, C3, C11,
C12, C13 and C1, C2, C11, C12 are disordered over two sites; the site occupancies are
refined to 0.56(15) and 0.44(15), respectively. The I1, I2, and I3 are also disordered over
two sites with site occupancies refined to 0.04(3) and 0.96(3), 0.985(5) and 0.015(5),
0.71(14) and 0.29(14), respectively.
There is one N–H ꢃ ꢃ ꢃ I and one C–H ꢃ ꢃ ꢃ Cg intramolecular hydrogen bond in the
structure of the Cu(I/II) complex. The Cu(I) and Cu(II) units are linked into 1-D
polymer chains parallel to the b-axis by I1, I2 atoms, and C10—H10 ꢃ ꢃ ꢃ Cg hydrogen
bonds (center of ring C15 to C20) shown in figure 2. Adjacent polymer chains are linked
into sheets parallel to the [001] plane by N4–H4B ꢃ ꢃ ꢃ I3iii hydrogen bonds shown in
figure 3, and neighboring [001] sheets are linked into a 3-D network structure through
van der Waals forces.

2924
Y.-F. Liu et al.
ꢂ
˚
Table 2. Selected bond lengths (A), hydrogen bond lengths (A) and bond angles ( ) for complex.
˚
Cu1–I30
Cu1–I10
Cu1–I20
Cu1–I2
Cu1–I3
Cu1–I1
Cu2–N2
2.463(19)
2.47(5)
2.53(9)
2.558(3)
2.56(3)
Cu2–N4
Cu2–N3
Cu2–N1
1.998(14)
2.018(17)
2.054(17)
3.18(9)
3.28(6)
3.299(4)
3.357(3)
i
Cu2–I20
Cu2–I10
Cu2–I1
Cu2–I2
2.577(5)
1.979(14)
i
i
I30–Cu1–I10
I30–Cu1–I20
I10–Cu1–I20
I30–Cu1–I2
I10–Cu1–I2
I20–Cu1–I2
I30–Cu1–I3
I10–Cu1–I3
I20–Cu1–I3
I2–Cu1–I3
I30–Cu1–I1
I10–Cu1–I1
I20–Cu1–I1
I2–Cu1–I1
I3–Cu1–I1
N2–Cu2–N4
N2–Cu2–N3
N4–Cu2–N3
N2–Cu2–N1
N4–Cu2–N1
N3–Cu2–N1
116.0(16)
120(4)
110(5)
122.7(12)
120.6(15)
29(3)
16(3)
116(3)
108(3)
117.9(8)
116(2)
19(7)
N3–Cu2–I20
N1–Cu2–I20
N2–Cu2–I10
N4–Cu2–I10
N3–Cu2–I10
N1–Cu2–I10
68(2)
109(2)
78(4)
101(4)
98(5)
85(5)
163(3)
87.5(5)
92.0(5)
85.5(6)
96.8(6)
154(2)
15(5)
85.8(5)
94.8(5)
90.0(6)
88.0(6)
22(2)
i
I20 –Cu2–I10
N2–Cu2–I1
N4–Cu2–I1
N3–Cu2–I1
N1–Cu2–I1
i
I20 –Cu2–I1
120(2)
I10–Cu2–I1
N2–Cu2–I2i
N4–Cu2–I2i
N3–Cu2–I2i
N1–Cu2–I2i
120.43(11)
121.4(6)
178.6(7)
97.8(6)
83.5(6)
83.9(6)
94.8(6)
177.2(8)
94.3(16)
86.7(16)
i
I20 –Cu2–I2i
I10–Cu2–I2i
163(5)
171.31(8)
109.70(17)
106.65(8)
I1–Cu2–I2i
Cu1–I1–Cu2
Cu1–I2–Cu2ii
i
N2–Cu2–I20
i
N4–Cu2–I20
D–H ꢃ ꢃ ꢃ A
D–H
H ꢃ ꢃ ꢃ A
D ꢃ ꢃ ꢃ A
ffD–H ꢃ ꢃ ꢃ A
N4–H4B ꢃ ꢃ ꢃ I3iii
0.9
0.93
2.76
2.92
3.62(2)
3.75(3)
161
149
C10–H10 ꢃ ꢃ ꢃ Cgii
i
iii
Symmetry codes: 1 ꢁ x, ꢁ1/2 þ y, 1 ꢁ z; ii1 ꢁ x, 1/2 þ y, 1 ꢁ z; ꢁ1 þ x, y, z. Cg (center of ring C15–C20).
Figure 2. CH ꢃ ꢃ ꢃ Cg hydrogen bond contacts of Cu(I/II) complex [symmetry code: A (1 ꢁ x, ꢁ1/2 + y,
1 ꢁ z), B(x, ꢁ1 + y, z), C(1 ꢁ x, 1/2 + y, 1 ꢁ z)].

Cu(I/II) Interaction with BSA
2925
Figure 3. The N–H ꢃ ꢃ ꢃ I hydrogen bond contacts of Cu(I/II) complex [symmetry code: A (1 ꢁ x, ꢁ1/2 + y,
1 ꢁ z), D(ꢁx, ꢁ1/2 + y, 1 ꢁ z), E(2 ꢁ x, ꢁ1/2+y, 1 ꢁ z), F(ꢁ1 + x, y, z), G(1 + x, y, z)].
3.2. ESI-MS and fluorescence spectroscopy
ESI-MS was used to verify the solution phase stability of the Cu(I/II) complex. The
0.5 mL sample ethanol solution (10ꢁ5 mol Lꢁ1) was injected into the electrospray source
at a flow rate of 0.5 mL minꢁ1, probe voltage 4.5 kV (positive mode), nebulizing gas
flow 1.5 L minꢁ1, drying gas pressure 0.1 MPa, temperatures of MS source block and
probe were set at 200ꢂC and 300ꢂC, respectively. ESI mass spectra and the structural
assignments for Cu(I/II) complex are shown in figure 4. The results show that the
Cu(I/II) complex is stable in ethanol.
Fluorescence measurements give information about the molecular environment in the
vicinity of the fluorophore molecules. The BSA molecule has three intrinsic
fluorophores, tryptophan, tyrosine, and phenylalanine residues. Generally, BSA
solutions excited at 285–290 nm emit fluorescence, attributable mainly to the trypto-
phan residues. A valuable feature of the intrinsic fluorescence of proteins is the high

2926
Y.-F. Liu et al.
[Cu(C10H16N2)3]2+
279.1
2.0x106
1.5x106
1.0x106
[M-2I+C H N ]2+
372.9
10 16
2
[M+2H]2+
415.3
[M-2I]2+
291.8
5.0x105
[M+H]+
838.5
[M-I]+
[M-C10H16N2+H]+
[Cu(C10H16N2)2]2+
198.1
[M+EtOH]+
881.6
707.9
673.9
0.0
100
200
300
400
500
m/z
600
700
800
900
1000
Figure 4. ESI-MS spectrum in the positive ion mode of a ethanol solution of [C20H32Cu2I3N4]n.
sensitivity of tryptophan to its local environment, changes in emission spectra of
tryptophan are common in response to protein conformational transitions, subunit
association, substrate binding, or denaturation [35]. Figure 5 shows the fluorescence
emission spectra of BSA with various amounts of Cu(I/II) complex. It is obvious that
BSA has a fluorescence emission peak at 347 nm. When different concentrations of the
Cu(I/II) complex solution was titrated into a fixed concentration of BSA, a decrease in
the fluorescence intensity of BSA was observed, indicating that there was an interaction
between the Cu(I/II) complex and BSA. Furthermore, the maximum wavelength of
BSA shifted from 347 to 345 nm after the addition of the Cu(I/II) complex, so a slight
blue shift of the maximum emission wavelength of BSA was observed, suggesting that
the microenvironment of tryptophan residues changed after the addition of the Cu(I/II)
complex. Quantitative analysis of the binding of the Cu(I/II) complex to BSA was
carried out using fluorescence quenching at 347 nm at different temperatures.
Fluorescence quenching is the decrease in the quantum yield for fluorescence from a
fluorophore induced by a variety of molecular interactions with quencher molecules.
Quenching can occur by different mechanisms, which are usually classified as dynamic
quenching and static quenching. Dynamic quenching depends upon diffusion, where
higher temperatures result in larger diffusion coefficients. As a result, the bimolecular
quenching constants are expected to increase with increasing temperature. Static
quenching refers to the formation of a ground state fluorophore–quencher complex,
where increased temperature is likely to result in decreasing stability of the complexes,
and thus lower values for the static quenching constants.
To clarify the fluorescence quenching mechanism of BSA by the Cu(I/II) complex,
the procedure of the fluorescence quenching was first assumed to be a dynamic

Cu(I/II) Interaction with BSA
2927
200
100
0
300
400
500
wavelength/nm
Figure 5. Emission spectra of BSA in the presence and absence of Cu(I/II) complex. [BSA]: 3ꢀ10ꢁ6mol Lꢁ1
;
[Cu(I/II) complex]: 0, 1, 2, 3, 4, 5, 6, 7ꢀ10ꢁ6mol Lꢁ1. ꢁex ¼ 285 nm; ꢁem ¼ 347 nm; pH ¼ 7.4; T ¼ 293 K.
quenching process; the fluorescence quenching data are usually analyzed by the
Stern–Volmer equation:
F 0=F ¼ 1 þ Kqꢇ0½QŠ ¼ 1 þ Ksv½QŠ
where F0 and F represent the fluorescence intensities in the absence and in the presence
of the quencher, respectively. Kq is the quenching rate constant, Ksv is the dynamic
quenching constant, ꢇ0 is the average lifetime of the biomolecule without quencher, and
[Q] is the concentration of quencher. Since the fluorescence lifetime of the biopolymer is
10ꢁ8 s [36], the quenching rate constant Kq can be calculated using the above equation.
Stern–Volmer curves of F0/F versus [Q] at different temperatures are shown in figure 6
and the corresponding Stern–Volmer quenching constants Ksv are listed in table 3. The
values of the Stern–Volmer quenching constants Ksv and Kq decrease with increase in
temperature, and the values of Kq are much greater than the maximum scatter collision-
quenching constant of the biomolecule (2.0 ꢀ 1010 mol Lꢁ1 sꢁ1) [8], indicating that the
probable quenching mechanism of fluorescence of BSA by the Cu(I/II) complex is static
quenching rather than dynamic collision.
3.3. Binding constants and the number of binding sites
For static quenching, the binding constant and the number of binding sites can be
determined by the following equation [12]:
ꢂꢀ
ꢁ
ꢃ
log F 0 ꢁ F =F ¼ log Kb þ n log½QŠ

2928
Y.-F. Liu et al.
293K
303K
313K
2.0
1.8
1.6
1.4
1.2
1.0
1
2
3
4
5
6
6
[Q] X 10 mol/L
Figure 6. The Stern-Volmer plots for the interaction of Cu(I/II) complex with BSA.
Table 3. Stern–Volmer quenching constants, binding constants, binding points, and thermodynamic
parameters of Cu(I/II)complex–BSA.
T (K) Ksv (L molꢁ1) Kq (L molꢁ1 sꢁ1) Kb (L molꢁ1
)
n
DH (kJ molꢁ1) DG (kJ molꢁ1) DS (J molꢁ1 Kꢁ1
)
293
303
313
1.84 ꢀ 105
1.15 ꢀ 105
7.59 ꢀ 104
1.84 ꢀ 1013
1.15 ꢀ 1013
7.59 ꢀ 1012
5.95 ꢀ 108 1.68
2.49 ꢀ 107 1.45
5.94 ꢀ 105 1.18
ꢁ263.42
ꢁ49.56
ꢁ42.26
ꢁ34.96
ꢁ729.54
where Kb and n are the binding constant and the number of binding sites, respectively.
According to this equation, the binding parameters can be obtained by a plot of the
double-logarithm curve log[(F0 ꢁ F)/F] versus log [Q] (figure 7). The values of Kb and n
were calculated from the values of the intercept and slope of the plots, respectively, with
results summarized in table 3. The Cu(I/II) complex binds to BSA and the binding
constant decreases with increasing temperature, indicating that the stability of the
Cu(I/II) complex–BSA system is reduced. The interactions are probably associated with
hydrogen bonding and weakening of the complex–BSA stability. Values of n are 1–2,
indicating that there are one or two classes of binding sites on BSA for the Cu(I/II)
complex.
3.4. Binding mode between the Cu(I/II) complex and BSA
The interaction between the Cu(I/II) complex and BSA include hydrophobic forces,
electrostatic forces, van der Waals interactions, and hydrogen bonds. The signs and
magnitudes of the thermodynamic parameters determine the nature of the forces

Cu(I/II) Interaction with BSA
2929
0.4
0.0
293K
303K
313K
–0.4
–0.8
–1.2
–1.6
–6.0
–5.8
–5.6
–5.4
–5.2
–5.0
log[Q]
Figure 7. Plots of log(F0ꢁF)/F vs. log[Q].
actually taking part in the complex–BSA interaction. According to the DH and DS, the
interaction between the Cu(I/II) complex and BSA can be concluded. If the temperature
changes little, the reaction enthalpy change is regarded as a constant. In order to
elucidate the interaction of Cu(I/II) complex with BSA, binding studies were carried out
at 293, 303, and 313 K. The thermodynamic parameters were calculated from the van’t
Hoff equation and corresponding thermodynamically functions based on the
temperature effect. The following two equations were used:
DH DS
ln K¼ ꢁ
þ
RT
R
DG¼ ꢁ RT ln K¼DH ꢁ TDS
where R is the gas constant, T is the experimental temperature, and K is the binding
constant at the corresponding T. The value of DH and DS were obtained from ln K
versus 1/T plot (figure 8). The thermodynamic parameters for the interaction of the
Cu(I/II) complex with BSA are shown in table 3. The binding constant of the Cu(I/II)
complex–BSA decreases with increase in temperature, suggesting that the binding
reaction of the Cu(I/II) complex with BSA is exothermic. According to the rules
summarized by Ross and Subramanian [37], negative enthalpy and entropy values
indicate that hydrogen bonds and van der Waals forces play a major role in the
interaction of the Cu(I/II) complex with BSA.
3.5. Synchronous fluorescence studies on Cu(I/II) complex binding to BSA
The synchronous fluorescence of BSA can be used to study the environment of amino
acid residues by measuring the possible shift in maximum emission wavelength, since
such shifts reflect the changes of polarity around the chromophore. When the value

2930
Y.-F. Liu et al.
20
18
16
14
12
3.20
3.25
3.30
3.35
3.40
3.45
1000/T(K)
Figure 8. Van’t Hoff plot for the interaction of BSA and Cu(I/II)complex.
(Dꢁ) of the difference between excitation and emission wavelengths is fixed at 15 nm,
synchronous fluorescence spectra only show information of the tyrosine residues,
whereas when Dꢁ is fixed at 60 nm, it provides the information of the tryptophan
residues. Figure 9 presents the synchronous fluorescence spectra of BSA in the presence
of the Cu(I/II) complex. When the Cu(I/II) complex was gradually added, the
maximum emission wavelength of the tryptophan residues did not undergo a significant
shift, suggesting that the interaction of the Cu(I/II) complex with BSA does not affect
the polarity and conformation of the tryptophan residue micro-region. In contrast, an
obvious blue shift was observed when Dꢁ ¼ 15 nm, showing that the Cu(I/II) complex
entered into the hydrophobic cavities and the conformation of BSA was changed. So we
conclude that tyrosine residues participated in the molecular interaction between the
Cu(I/II) complex and BSA.
3.6. Energy transfer between the Cu(I/II) complex and BSA
According to Forster’s non-radiative energy transfer theory, the efficiency of energy
¨
transfer mainly depends on the following factors: (1) the extent of overlap between the
fluorescence emission spectrum of the donor and the UV absorbance spectrum of the
acceptor, (2) the orientation of the transition dipole of donor and acceptor, and (3)
the distance between the donor and the acceptor is less than 8 nm. The overlap of
the UV-Vis absorption spectrum of the Cu(I/II) complex with the fluorescence emission
spectrum of BSA is shown in figure 10. The energy transfer efficiency is defined in the
following equation:
ꢂ
ꢃ
ꢀ
ꢁ
6
E ¼ 1= 1 þ ðr=R0Þ ¼ 1 ꢁ F=F 0

Cu(I/II) Interaction with BSA
2931
(a)
30
20
10
0
280
300
320
340
wavelength/nm
(b) 200
150
100
50
0
300
350
400
wavelength/nm
Figure 9. Synchronous fluorescence spectra of interaction between BSA and Cu(I/II) complex.
(a) Dꢁ ¼ 15 nm; (b) Dꢁ ¼ 60 nm. [BSA]: 3ꢀ10ꢁ6 mol Lꢁ1
; [Cu(I/II) complex]: 0, 1, 2, 3, 4, 5, 6,
7ꢀ10ꢁ6 mol Lꢁ1. pH ¼ 7.4; T ¼ 303 K.
where E is the efficiency of energy transfer between the donor and the acceptor, F and
F0 are the fluorescence intensities of BSA in the presence and absence of the Cu(I/II)
complex, r is the distance between the donor and the acceptor, and R0 is the critical

2932
Y.-F. Liu et al.
120
100
80
60
40
20
0
0.25
0.20
0.15
0.10
0.05
0.00
b
a
300
350
400
450
500
wavelength/nm
Figure 10. Spectral overlap of Cu(I/II) complex absorption (a) with BSA fluorescence (b) [BSA] ¼ [Cu(I/II)
complex]: 1ꢀ10ꢁ5 mol Lꢁ1 (T ¼ 293 K).
distance at which transfer efficiency equals 50%. The value of R0 can be calculated
using the following equation:
R60 ¼ 8:79 ꢀ 10ꢁ25ðK2 ꢃ Nꢁ4 ꢃ È ꢃ JÞ
where K2 is the spatial orientation factor of the dipole, N is the refractive index of the
medium, È is the fluorescence quantum yield of the donor, and J is the overlap integral
of the fluorescence emission spectrum of donor and absorption spectrum of the
acceptor, which can be calculated by the following equation:
R 1
FðꢁÞ"ðꢁÞꢁ4dꢁ
0
R
J ¼
1 FðꢁÞdꢁ
0
where F(ꢁ) is the fluorescence intensity of the donor in the wavelength range ꢁ to ꢁ þ Dꢁ
and "(ꢁ) is the molar absorption coefficient of the acceptor at wavelength ꢁ. In the
present case, K2 ¼ 2/3, N ¼ 1.336, È ¼ 0.15 [38]. From the overlapping spectrum, J can
be
evaluated
by
integrating
the
spectra
for
ꢁ ¼ 300–450 nm,
J ¼ 2.82 ꢀ 10ꢁ15 cm3 L molꢁ1, E ¼ 0.22, R0 ¼ 2.07 nm, and r ¼ 2.56 nm were calculated.
Obviously, the donor–acceptor distance is less than 8 nm, and 0.5 R0 5 r 5 1.5 R0,
which implies a high possibility of energy transfer from BSA to the Cu(I/II) complex [7].
4. Conclusions
The crystal structure of the Cu(I/II) complex [C20H32Cu2I3N4]n was determined by
single-crystal X-ray diffraction. The interactions of the complex with BSA in

Cu(I/II) Interaction with BSA
2933
physiological buffer solution were studied by fluorescence spectroscopic methods. The
results indicate that the Cu(I/II) complex is a strong quencher, and the decreasing
values of the binding constants with increasing temperature indicate interaction with
BSA through a static quenching procedure. The values of n revealed the presence of one
or two classes of binding sites on BSA. The thermodynamic parameters of the binding
interaction were determined and their values suggest that hydrogen bonds and van der
Waals forces play a major role in interactions of the Cu(I/II) complex with BSA. The
binding distance r between the Cu(I/II) complex and BSA indicates that energy transfer
from BSA to the Cu(I/II) complex occurs. Synchronous fluorescence spectroscopy
indicates that the conformation of BSA was changed in the presence of the Cu(I/II)
complex.
Supplementary material
Crystallographic data for the structures in this article have been deposited with the
Cambridge Crystallographic Data Center as supplementary publication CCDC No.
692706 for the title complex. Copies of the data can be obtained, free of charge, on
application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK.
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