Bovine serum albumin–vanadium complex hybrid: Synthesis, crystal structure, SOD activities and asymmetric catalytic oxidation of sulfides

Article abstract of DOI:10.1016/j.poly.2019.01.050

Two symmetrical double-sided Schiff base ligands, H₄L1 and H₄L2, were synthesized by condensation of salicylaldehyde and 2-hydroxy-1-naphthaldehyde with oxalodihydrazine, respectively. The crystal structures of their vanadium complex

Full text of DOI:10.1016/j.poly.2019.01.050

Accepted Manuscript
Bovine serum albumin–vanadium complex hybrid: Synthesis, crystal structure,
SOD activities and asymmetric catalytic oxidation of sulfides
Jingnan Shi, Yi Wei, Yuzhen Zhang, Jie Tang, Hedong Bian, Qing Yu, Fuping
Huang
PII:
S0277-5387(19)30072-5
https://doi.org/10.1016/j.poly.2019.01.050
POLY 13723
DOI:
Reference:
To appear in:
Polyhedron
Received Date:
Revised Date:
Accepted Date:
24 October 2018
18 January 2019
22 January 2019
Please cite this article as: J. Shi, Y. Wei, Y. Zhang, J. Tang, H. Bian, Q. Yu, F. Huang, Bovine serum albumin–
vanadium complex hybrid: Synthesis, crystal structure, SOD activities and asymmetric catalytic oxidation of
sulfides, Polyhedron (2019), doi: https://doi.org/10.1016/j.poly.2019.01.050
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Bovine serum albumin–vanadium complex hybrid: Synthesis,
crystal structure, SOD activities and asymmetric catalytic
oxidation of sulfides
a
b
a
b
a, b
Qing Yu, b
Jingnan Shi, Yi Wei, Yuzhen Zhang, Jie Tang, Hedong Bian,*
b
Fuping Huang
a
Guangxi Key Laboratory of Chemistry and Engineering of Forest Products, School of Chemistry and Chemical
Engineering, Guangxi University for Nationalities, Nanning, Guangxi, 530008, PR China
b
Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources (Ministry of Education of
China), School of Chemistry and Pharmacy, Guangxi Normal University, Guilin, Guangxi, 541004, PR China
Abstract：Two symmetrical double-sided Schiff base ligands, H
4
L1 and H L2, were
4
synthesized by condensation of salicylaldehyde and 2-hydroxy-1-naphthaldehyde
with oxalodihydrazine, respectively. The crystal structures of their vanadium
complexes were analyzed by single crystal X-ray diffraction. The interaction between
the synthesized complexes and BSA was studied by various spectral methods. The
binding parameters including quenching constant KSV, binding constant K, and
binding mode were obtained using fluorescence quenching method. The study shows
that the quenching type was a static quenching process caused by formation of
complex. The SOD activities of the complexes were determined by xanthine
oxidase-NBT reduction method. The antioxidant capacity of the complexes was
determined by the ABTS method. The results indicate that the activity of hybrid
protein was more pronounced than that of the corresponding complex. At the same
time, the hybrid protein can catalyze asymmetric oxidation of methyl phenyl sulfide.
Keywords： Dioxalyl dihydrazone; Vanadium; Bovine serum albumin; Catalytic
activity

1. Introduction
[
1]
Serum albumin (SA) is the most abundant protein in vertebrate plasma . One of
the most outstanding properties of serum albumin is their capacity to bind small
[
2]
molecules reversibly, such as amino acids, inorganic ions, drugs, and fatty acids . In
recent years, bovine serum albumin (BSA) is widely used in biochemical
[
3]
experiments . Moreover, BSA is selected as the protein model for its easy
[4]
purification , stability, water solubility, abundance, low cost, medical importance,
[5]
and unusual ligand-binding properties . Research shows that BSA is a globulin in
bovine serum, contains 583 amino acid residues including tryptophan 212 and
tryptophan 134. Trp-212 is located in the hydrophobic binding bag of BSA, while
[6]
Trp-134 is located on the molecular surface .
Free radicals are a kind of negatively charged ions with oxidative activity
[7]
produced in the metabolic process of an organism . The dynamic balance of free
radical production and scavenging in human body is strongly linked to the growth and
development of human body, pathology, physiology, disease and aging. Under
different physiological conditions, free radical homeostasis dynamics in aerobic
[8]
organisms must be maintained in order to sustain life . Superoxide dismutase (SOD)
was first discovered by Irwin Fridovich and Joe Mccord. It is an active substance
from living organisms and can remove harmful substances in the body. Superoxide is
[9]
one of the most common free radicals in the body . Superoxide dismutase can
transform molecular oxygen and hydrogen peroxide in all oxygen-metabolizing
organisms and some anaerobic bacteria by catalyzing superoxide anions[10]. It
constitutes the main defense mechanism of cells against oxidative stress and can resist
the harmful effects of superoxide. Studies have shown that SOD is essential in
oxygen-hyperactive diseases such as various cardiovascular diseases, inflammatory
diseases, diabetes and cancer[11]. The vanadium(V) complexes with salen type ligands
were a promising class of compounds in different SOD mimics. Furthermore, salen
complexes have been shown to play an important role in treatment of diseases such as

Parkinson's disease, alzheimer's disease and motor neuron disease to oxidative stress
from multiple sclerosis and excitotoxic neuronal damage[10]
.
Chiral sulfoxide is widely used as chiral ligands and chiral auxiliaries of
metals[12]. Therefore, the preparation of enantiomers for the synthesis of pure
sulfoxide is of great significance[13]. The most effective way to synthesize chiral
sulfoxides is the asymmetric oxidation of thioether catalyzed by chiral metal
complexes[14]. The rational design of various chiral metal complex catalysts to
catalyze sulfur ether has attracted the attention of many chemical researchers in recent
years. As catalysts, Schiff base metal complexes are often used in catalytic
asymmetric cyclopropanation, polymerization, oxidation of olefins and
electrocatalysis[15]. Particularly, using transition metal complexes as a catalyst, mainly
based on titanium, vanadium, manganese, and iron is an area of current interest[16]
.
2. Experimental
2
.1. Materials and Instrumental
All chemicals were of analytical reagent (A.R.) and were used without any further
purification. BSA was purchased from Sigma-Aldrich (Sweden). Fourier transform
infrared spectra of the samples with a KBr disc were obtained on a Perkin Elmer
−
1
Spectrum One FT-IR spectrometer (4000-400 cm ). Elemental analysis was
performed on a Perkin Elmer 2400 analyser. Single crystal date collected on a Bruker
Smart 1000 CCD diffractometer using Mo-Kα radiation (λ = 0.71073 Å). CD spectra
recorded on jasco-810 Round dichroism spectrometer. Fluorescence spectra measured
in RF-5301 fluorescence spectrophotometer (Japan Shimadzu Company). Raman
spectroscopy obtained on a Laser Micro-Raman Spectrometer (Renishaw Invia +
Plus). HPLC experiments were carried out using LC-20AB High performance liquid
1
chromatograph (Japan Shimadzu Company). H-NMR spectra of the compounds were
recorded on a Bruker NMR spectrometer (300 MHz).
2
.2. Synthesis of the ligands

The ligands were prepared following the procedure previously reported[17,18]
.
1
2
N' ,N' -bis(2-hydroxy-1-naphthylmethylene)oxalohydrazide (H
oxalyl dihydrazide (1.50 g, 12.7 mmol) and 2-hydroxy-1-naphthaldehyde (4.80 g,
5.4 mmol) in anhydrous ethanol solution (600 mL) was refluxed for 2 h under
4
L1). A mixture of
2
constant stirring using magnetic stirrer. The product was filtered and washed with
ethanol, then dried in vacuo. The yellow powder product was obtained. Yield: 48%.
-
1
Anal. Calc. (%) for C24
H
18
N
4
O
4
(M = 426.13 g·mol ): C, 67.60; H, 4.25; N, 13.14.
-
Found: C, 68.10; H, 4.15; N, 13.04. EIS-MS (m/z): [H
3
L1] , 425.12. FT-IR (KBr
-1
phase, cm ): 3454(s, O-H), 3293(s, N-H), 1660(s, C=O), 1622(s, C=N), 1283(m,
1
C-O), 1041(w, N-N). H NMR (300 MHz, DMSO-d
6
): δ = 7.23-8.21(m, 12H,
naphthyl), 9.72(s, 2H, CH=N), 12.58(s, 2H, OH), 12.79(s, 2H, NH).
1
2
N' , N' -bis(2-hydroxybenzylidene)oxalohydrazide (H
the same procedure as described for H L1 with salicylaldehyde (9.76 g, 79.8 mmol)
and oxalyl dihydrazide (4.72 g, 39.9 mmol). A yellow product was obtained. Yield:
4
L2). It was prepared using
4
-
1
6
1
5%. Anal. Calc. (%) for C16
H
14
N
4
O
4
(M = 326.30 g·mol ): C, 58.89; H, 4.32; N,
-
7.17. Found: C, 58.70; H, 4.52; N, 17.20. EIS-MS(m/z): [H
3
L2] , 325.09. FT-IR
-1
(
KBr phase, cm ): 3275(s, C-H), 3205(w, N-H)， 1668(s, C=O), 1608(s, C=N),
1
1
265(w, C-O), 1034(w, N-N). H NMR (300 MHz, DMSO-d
6
): δ = 6.91-7.54(m, 8H,
aryl), 8.79(s, 2H, CH=N), 10.98(s, 2H, OH), 12.63(s, 2H, NH).
O
O
O
O
N
NH
NH
N
N
NH
NH
N
OH
HO
OH
OH
Scheme 1. Structure of the ligand H
4
L1 (left) and H
4
L2 (right).
2
.3. Synthesis of complexes

The ligands were obtained with sufficient purity and used without further
purification in the synthesis of the complexes. (H NMe [(VO (L1)] (1). A mixture
L1 (0.12 g, 0.3 mmol), V (0.05 g, 0.3 mmol), triethylamine (5 drops), dimethyl
formamide (10 mL) and anhydrous ethanol (7 mL) was sealed in a Teflon-lined
2
2
)
2
)
2 2
H
4
2
O
5
o
o
stainless steel vessel, heated at 80 C for 72 h and then cooled to 40 C at a rate of 5
oC/h. Yellow-green lumpy crystals of 1 were obtained and picked out, washed with
ethanol and dried in air. Yield: 73%. Anal. Calc. (%) for C28
H
28
N
6
O
8
V
2
(M = 678.44
-1
g·mol ): C, 48.83; H, 2.73; N, 9.49. Found: C, 49.24; H, 3.13; N, 8.76. FT-IR (KBr
-1
phase, cm ): 1617(s, C=N), 1243(s, C-O), 1105(w, N-N), 899(s, V=O).
NMe [(VO (L2)]·2(H O) (2). A mixture of NH VO (0.3 mmol), H
0.3 mmol), dimethyl formamide (10 mL), anhydrous ethanol (7 mL), and
(H
2
2
)
2
2
)
2
2
4
3
4
L2
(
triethylamine (5 drops) was placed in a 20 mL Teflon-lined stainless steel vessel. The
rest of the synthesis process is the same as complex 1. The resulting crystalline
product was washed with ethanol and dried in air. Color: Reddish brown. Yield: 62%.
-
1
Anal. Calc. (%) for C20
H
28
N
6
O
10
V (M = 614.37 g·mol ): C, 39.20; H, 2.47; N, 11.43.
2
-1
Found: C, 38.12; H, 3.54; N, 10.31. FT-IR (KBr phase, cm ): 1603(s, C=N), 1267(s,
C-O), 1145(w, N-N), 904(s, V=O).
2
.4. X-ray crystallography
The crystal of suitable size was selected and placed on the single crystal
diffractometer. X-ray single-crystal diffraction data for 1 and 2 were collected. The
graphite monochromatic Mo Kα (λ = 0.71073 Å) at 293(2) K. The structures were
solved by direct methods and further refinements were carried out by full-matrix least
squares on F using SHELXTL Program package[19,20]. Mixed hydrogenation,
hydrogen atoms were refined with isotropic thermal parameters and non-hydrogen
atoms were refined with anisotropic thermal parameters. The crystallographic and
structural modification data are shown in Table 1.
2
Table 1
Crystal data and structure refinement for 1 and 2.
Compound
1
2

Formula
Formula weight
Crystal system
Space group
a (Å)
C
28
H
28
N
6
O
8
V
2
C
20
H
28
N
6
O
10
V
2
678.44
Triclinic
P1
614.37
Monoclinic
P2 /n
1
8.069 (3)
9.670 (3)
9.695 (3)
7.987 (2)
19.296 (6)
8.555 (3)
90
b (Å)
c (Å)
α (°)
91.466 (6)
99.390 (6)
93.354 (6)
744.6 (4)
1
β (°)
97.781 (5)
90
γ (°)
V/(Å3)
1306.2 (7)
2
Z
-
3
D
c
(g cm )
1.518
1.567
F (000)
350
636
θ range for data collection (°)
Reflections collected
2.5 to 26.3°
8651
2.1 to 26.3°
14781
2666
3
018
R(int) = 0.042]
1.08
= 0.0404
ωR = 0.1029
= 0.0626
ωR = 0.1120
Independent reflections
Goodness-of-fit on F2
Final R indices [I > 2σ (I)]
[
[R(int) = 0.031]
1.109
R
1
R
1
= 0.0379
ωR = 0.1115
= 0.0447
ωR = 0.1166
2
2
R
1
R
1
R indices (all data)
2
2
2
.5. Synthesis of the hybrid proteins
The Schiff base complexes and BSA were dissolved in the Tris-HCl buffer
-6
-1
solution (pH = 8.0) to prepare the stock solution (1.0 × 10 mol·L ). The mixture
containing 2400 μL complex stock solution and 300 μL BSA stock solution was kept
o
under stirring at 4 C for 10 hours, then transferred to a treated dialysis bag and
-1
dialyzed for 3 times in a phosphate buffer (PB) solution (0.1 mol·L , pH = 7.0) to
remove the non-reacted reactants. The resulting hybrid proteins were purified by
Sephadex G75 gel chromatography and the concentration of the hybrid proteins were
determined by Coomassie brilliant blue method after concentration.

2
.6. BSA binding experiments
The interaction mechanism was tested by UV absorption spectra, fluorescence
spectra, circular dichroism spectra and Raman spectra. The pH = 7.43 phosphate
buffer saline (PBS) solution was used to dilute BSA, and the concentration was 1.0 ×
1 -
6
0 mol·L . The 3 mL diluted BSA solution was added to the colorimetric dish and
-1
mixed proportionally with the complex solution with a concentration of 1.0 × 10-3
-1
mol·L .The fixed excitation wavelength was at 280 nm, slit widths was 5/5 nm. At
the temperature of 293, 299 and 306 K, the fluorescence quenching spectrum within
the range of 290-450 nm was scanned. Measurement of circular dichroism spectra and
UV and Raman spectrum is resemblance to fluorescence quenching.
2
2
.7. Antioxidant activity measurements
.7.1. SOD activity experiment
Rate constants for the reactions of complexes and hybridized proteins with the
superoxide anion, were decided by competition kinetics using NBT as the detector
molecule[21]. The reaction solution was prepared by dissolving 100 μL xanthine
-2
-1
-4
-1
solution (5.0 × 10 mol·L ) and 200 μL NBT solution (2.5 × 10 mol·L ) in 1.7 mL
PB solution at pH = 7.4. The xanthine oxidase was mixed to the solution with a total
-
1
volume of 2 mL produced a ΔA550 nm of 0.024 min . The detection of complex and
SOD activity of adduct was basically consistent with the above operation. The
difference was that complex or complex-BSA hybrid was added before adding
xanthine oxidase. The metal complex-BSA adduct was synthesized by incubating
equimolar amounts of BSA and the metal complex for 10 minutes in the buffer
solution at room temperature. Each sample test took 6 concentrations and three
parallel determinations for each solution were carried out. The value of IC50 was
determined by NBT analysis[22]
.
+
2
.7.2. ABTS· scavenging ability experiment
The free radical scavenging activities were investigated by ABTS method

[
23]
according to the modified literature study . Briefly, an equal amount of ABTS
-3
-1
-3
-1
solution (5 × 10 mol·L ) and potassium persulfate solution (5 × 10 mol·L ) were
+
mixed and stored in the dark for 12-16 hours to give a dark green ABTS· stock
solution. The working fluid was obtained by diluting the stock solution with PBS
solution before use. A certain amount of complexes and hybridized protein solutions
at different concentrations were added to the working solution and mixed. Kinetics
was used to determine the wavelength of 734 nm and to test the absorbance value of
the mixture at 734 nm. Take 5 concentrations for each sample and perform 3 parallel
experiments.
2
.8. Catalytic oxidation of methyl phenyl thioether experiment
The reaction mixture was composed of a solution containing 0.05 mol·L-1
phosphate buffer，methyl phenyl thioether (8 mmol), the catalyst complex-BSA
adduct (0.25 μmol), tert-butyl hydroperoxide (TBHP) as an oxidant (12 μmol), the
o
total volume of 30 mL, under 25 C constant stirring using magnetic stirrer. The
reaction was quenched by adding an appropriate amount of saturated anhydrous
sodium sulfite solution, extracted with dichloromethane, dried over anhydrous sodium
sulfate. The methylene chloride was removed by vacuum spinning on a rotary
evaporator to give a pale yellow oily liquid. It was tested by HPLC at room
temperature with a column of Chiralcel OB-H, Φ = 4.6 mm × 250 mm. The
enantiomeric excess (ee) was analyzed using the formulas[24]
:
ee% = [peakarea(S - R)/(S + R)] × 100%
(2.1)
3. Results and discussion
3
.1. IR spectral studies
The characteristic ligand bands of C=O and N-H display at about 1660/1668 and
-1
3
293/3205 cm for H
4
L1/H L2, respectively, which conforms the presence of the free
4
ligands in the keto form. The disappearance of those bands in the spectra of the both

complexes indicates the enolisation of the amide functionality and forming a N=C-O
-1
bond. The ligands show bands at 1622/1608 cm for H
4
L1/H L2. These bands shift
4
-1
toward lower wavenumbers (1617/1603 cm for 1/2), indicating complexation of the
nitrogen atom to the vanadium center[25]. In addition, the strong vibrations centered
around 899/904 cm for 1/2 indicate the presence of cis-VO
[25, 26].
-
1
2
3
.2. Crystal structures of complexes
Fig. 1. X-ray crystal structure of 1 (left) and 2 (right). Hydrogen atoms are omitted for clarity.
Fig. 1 represents the perspective view of the molecular structure of 1 and 2,
respectively. The complexes 1 and 2 have the similar crystal structures. The structure
of 2 is also similar to other vanadium(V) complexes with dioxalyl dihydrazone
[
27]
published previously . Therefore, the crystal structure of 1 is discussed in detail here.
Complex 1 consists of two dimethylammonium and one centrosymmetric dianion
2
-
[
V(O)
2
(µ-L1)V(O)
2
] . Each vanadium(V) atom is coordinated with one N atom and two
O atoms from the ligand L1 and two oxido groups. The Reedijk distortion index τ[28]
τ = 0 for a square pyramid, and τ = 1 for a trigaonal bipyramid) value is 0.075,
(
indicating slightly distorted square pyramidal configuration for the vanadium(V)
atoms. The equatorial plane of V1 contains of phenolate oxygen O4, imine nitrogen
N2, the oxygen O1 from a deprotonated alkoxide, and O2 from oxido group. V1
deviates by 0.5151 Å from the equatorial plane. Another oxido group O3 occupies the
apical position of the coordination sphere. The V=O bonds [V1–O2: 1.644 (2) Å and
V1–O3: 1.611 (2) Å] are characteristic of oxido-type O atoms with strong π bonding
[29].
Table 2
Selected bond lengths (Å) and angles (º) for the complexes 1 and 2.

1
V1—O1
1.9732 (19)
1.644 (2)
V1—O4
V1—N2
1.904 (2)
2.141 (2)
V1—O2
V1—O3
1.611 (2)
O1—V1—N2
O2—V1—O1
O2—V1—O4
O2—V1—N2
O3—V1—O1
73.54 (8)
O3—V1—O2
O3—V1—O4
O3—V1—N2
O4—V1—O1
O4—V1—N2
108.68 (11)
105.32 (11)
103.08 (10)
143.53 (10)
81.06 (9)
92.79 (9)
95.20 (9)
147.82 (10)
105.62 (10)
2
V1—O4
1.900 (2)
V1—O3
V1—N2
1.634 (2)
2.143 (2)
V1—O1
1.9894 (17)
1.623 (2)
V1—O2
O4—V1—O1
O4—V1—N2
O1—V1—N2
O2—V1—O4
O2—V1—O1
152.58 (8)
82.35 (8)
O2—V1—O3
O2—V1—N2
O3—V1—O4
O3—V1—O1
O3—V1—N2
108.87 (13)
114.52 (10)
95.50 (10)
92.34 (9)
73.82 (7)
103.68 (11)
98.50 (10)
135.82 (11)
3
3
.3. Spectroscopic characterization of the hybrid proteins
.3.1. UV–Vis analysis
The hybrid proteins were determined using a UV–Vis spectroscopic method. The
response signal of UV absorption comes from amino acid residues such as Trp, Try
and Phe in proteins[30]. As shown in Fig. S1 the peaks at about 200 – 300 nm in the
absorption spectra of two complexes are assigned to the n-π* transition of the benzene
ring. In addition, the peaks at about 330 – 380 nm are attributed to the π-π* transition
of C=N; the peaks at about 400 – 500 nm are corresponding to the d-d transition of a
high spin[30,31]. As shown in Fig. S1, the peaks of the hybrid proteins not only show
the characteristic peak of BSA, but also show the corresponding complexes of
characteristic peak, which lead us to conclude that stable hybrid proteins were formed.
3
3
.4. BSA Binding Properties
.4.1. UV–Vis absorption spectra

UV–Vis absorption spectra are feasible and effective method to study the effect
of complexes on protein structure changes. Conformational changes of protein
[
32]
molecules can be inferred by UV–Vis absorption spectrum . From UV spectra (Fig.
S2), the peak value was around 203 and 279 nm, respectively. The absorption peak at
2
03 nm was related to the change of protein skeleton[33]. The absorption band at 203
nm showed a low chromium effect with a small red shift in the presence of vanadium
compounds. This proves that the microenvironment of the amino acid residues of
BSA has changed. The change in peak strength near 279 nm is induced by the π-π*
and n-π* transformation of aromatic amino acid residues such as Trp, Tyr and Phe[34]
.
Its peak changes were small, indicating that changes of the microenvironment of the
amino acid residues had little impact on Trp, Tyr and Phe. The results show that the
interaction between vanadium complexes and BSA is obvious.
3
.4.2. Fluorescence quenching spectra
Fluorescence spectroscopy is one of the important methods to study the
interaction between small molecular compounds and biological macromolecule[34]
.
The fluorescence quenching mechanism, binding constants and number of binding
sites can be obtained by analyzing the fluorescence spectra of biomolecules and small
molecule compounds. Phenylalanine, tyrosine and tryptophan are inherent amino acid
residues capable of fluorescence in BSA molecules. However, the intrinsic
fluorescence of BSA is produced by tryptophan[35]. When the excitation wavelength is
at 280 nm, the fluorescence of BSA is induced by Trp-212 located in the hydrophobic
pocket of BSA. Fluorescence quenching of Trp-212 occurs when small molecular
compounds interact with proteins. In nature, fluorescence quenching is due to the
[
36]
decrease of fluorescence quantum yield from fluorophore . The interaction between
vanadium complexes and BSA was tested by fluorescence quenching experiment at
three temperatures (293, 299, and 306 K). From Fig. S3, at 293 K, when λex = 280 nm,
BSA was found a strong fluorescence at about 343 nm. When the concentration of
BSA were fixed, increases the concentration of the complexes regularly, the peak

shape unchanged, and the fluorescence intensity of BSA gradually decreases, which
proves that the interaction between the complexes and BSA.
3
.4.3. Fluorescence quenching mechanism
The fluorescence quenching can be better explained by the Stern-Volmer
[
37]
relation :
F
0
/F =1+ Ksv[Q]=1+ k
qτ
0[Q]
(3.1)
Where F is the fluorescence intensity in the presence of a quencher in BSA, F
fluorescence intensity in the absence of quencher in BSA, k is the bimolecular
quenching rate constant, KSV is the dynamic quenching constant, τ is the average
0
is the
q
0
-8
fluorescence lifetime of the biomolecule in the absence of quencher (about 10 s). [Q]
is the initial concentration of the complexes.
3.0
2.5
2.0
1.5
1.0
293 K
1.5
293 K
299 K
306 K
2
3
99 K
06 K
1.4
1.3
1.2
1.1
1.0
2
4
6
8
10
2
4
6
8
10
-
6
-6
[V-1]10 mol/L
[V-2]10 mol/L
Fig. 2. The Stern-Volmer plots for the binding of the complexes with BSA.
According to the Eq. (3.1), the Stern-Volmer curve can be obtained by taking [Q]
as the abscissa and F /F as the ordinate (Fig. 2). The value of Ksv and k can be
obtained by using this curve, and are listed in Table 3. The values of k are much
bigger than the maximum scattering collision quenching constant (2 × 1010
L·mol s )
-1 -1 [38]. And as the temperature increases, the quenching constant decreases. So
it can be determined that the quenching type of complexes and protein is a static
0
q
q
quenching process caused by intermolecular complexes[39]
Table 3
.
The Stern−Volmer quenching constants of complexes with BSA.

4
−1
k
(×10 L∙mol ∙s ) Ra
12
−1 −1
S.D.b
Complexes
T(K)
K
SV(×10 L∙mol )
q
2
93
23.7
17.0
14.3
5.7
23.7
17.0
14.3
5.7
0.96487
0.9905
0.01597
0.00589
0.00415
0.00174
0.00173
0.00082
BSA-1
299
3
2
06
93
0.99338
0.99278
0.99124
0.99744
BSA-2
299
06
5.2
5.2
3
4.5
4.5
a
R is the correlation coefficient
b
S.D. is the standard deviation for the KSV values
3
.4.4. The binding constant (K ) and the number of binding site (n) of the complexes
b
with BSA
The information can be obtained by the following formula Eq. (3.2)[40]
log [(F0 − F ) / F ] = log K + n log[Q] (3.2)
, F and [Q] are the same as in Eq. 3.1, n is the number of binding sites, and
.
b
where F
0
K
b
b
is the binding constant. From a plot of log[(F0 − F)/F] versus log[Q] (Fig. 3), n and
can be obtained. The relevant parameters are listed in Table 4.
K
0
.0
0.2
-0.4
0.6
0
0
0
.4
.2
.0
2
2
3
93 K
99 K
06 K
-
293 K
99 K
306 K
2
-
-
-
-
-
-
-
-
0.2
0.4
0.6
0.8
1.0
1.2
1.4
-0.8
-1.0
-
-
-
1.2
1.4
1.6
-
5.0
-4.8
-4.6
-4.4
-4.2
-4.0
-5.0
-4.8
-4.6
-4.4
-4.2
-4.0
log [V-1]
log [V-2]
Fig. 3. The Logarithmic plots for the binding of complexes with BSA.
From Fig. 3 and Table 4, the number of binding sites obtain is about 1, that is,

the complexes bind to BSA at a molar ratio of 1: 1 in a certain concentration range.
Table 4
The static binding constants K and the number of binding sites n of complexes with BSA at three
b
temperatures.
K
b
ΔG0
ΔH0
ΔS0
Complexes
T(K)
n
-1
(kJ·mol-1)
-1
-1
4
−1
(
kJ·mol )
(J·mol ·K )
(
×10 L∙mol )
2
93
5.80
3.49
0.53
1.78
1.76
0.84
1.405
1.344
0.969
1.323
1.135
1.098
-132.48
-130.89
-130.85
-168.90
-166.80
-166.75
BSA-1
299
-132.45
-168.86
-5.22
3
2
06
93
BSA-2
299
06
-6.88
3
3
.4.5. Combination type determination
According to the related thermodynamic parameters, the acting force between
the complexes and BSA can be determined. The temperature has no obvious change,
the enthalpy change can be ΔH as a constant[41]. According to van' t Hoff equation
0
0
0
can be inferred that the reaction enthalpy change ΔH and entropy change ΔS .
lnK = - ΔH / RT + R ΔS0
0
(3.3)
-1
-1
where T is the absolute temperature and R is the gas constant (8.3151 J·mol ·K ).
0
0
Using the Eq. (3.3) to map, the curve is obtained. The values of ΔH and ΔS are
calculated and are listed in Table 4.
12
11
10
9
8
1
2
0
.00325
0.00330
0.00335
/ T
0.00340
1
Fig. 4. On the basis of van’t Hoff plot for the binding of complex with BSA in 0.05 mol∙L-1

phosphate buffer of pH 7.43.
0
0
0
ΔG =ΔH / TΔS
(3.4)
0
Then the free energy change ΔG can be estimated from the Eq. (3.4). The Gibbs
free energy is negative at the three experimental temperatures in Table. 4, proves that
0
interaction can occur spontaneously. According to the law of thermodynamics, ΔH >
0
0
0
0
and ΔS > 0 is mainly for the hydrophobic force, ΔH < 0 and ΔS < 0 is mainly for
0
0
hydrogen bonding and van der Waals forces, ΔH < 0 and ΔS > 0 is mainly for the
electrostatic forces [41]. Therefore, it can be concluded that the type of complexes and
BSA is hydrogen bonding and van der Waals forces.
3
.4.6. Circular dichroism spectra
Circular Dichroism (CD) spectrum is an extremely important method to study
secondary structure changes of proteins. It is simpler, faster and more accurate than
other methods[42]. Proteins are biological macromolecules made up of many different
kinds of amino acids arranged in different order. However, proteins do not exist in the
form of long chains, but bend and fold to form a three-dimensional structure.
Common protein secondary structure is α−helix and β−sheet[43]. The circular
dichroism of protein mainly consists of the sum of circular dichroism of active
chromophore and folding structure. The peptide bond has circular dichroic absorption
peaks in the range of 190 – 250 nm, mainly caused by n-π electron transition. The
strongest round dichroic signal is the α−helix part[44]. It can be seen from Fig. 5, with
the addition of complexes of different concentrations, the BSA peak shape remains
basically unchanged, and the position of the absorption peak moves slightly upward.
This indicates that the complexes interact with the peptide bond with not change the
main structure of BSA. The protein remains α−helix is the major structure, but it
reduces the amount of protein in the helix, leading to changes in the
microenvironment of BSA. The structural content of the helical structure in protein
molecules is determined by Eq. (3.5) [37]
.
MRE = obs (m deg)/ (10  n  l  Cp )
(3.5)

where MRE is the molar ellipticity, C
p
is the molar concentration of the protein, θobs is
ellipticity, n is the number of amino acid residues, and l is the path length. The α-helix
[37]
structure of BSA was analyzed by the Eq. (3.6) .
α-helix% =[(MRE - 4000) / (33000- 4000)]×100
(3.6)
2
08
where MRE208 is the observed MRE value at 208 nm. From the Fig. 5, Eqs. (3.5) and
3.6), the results of the α-helix content of BSA were obtained and listed in Table 5.
(
2
0
20
0
BSA-2
BSA-1
0
-
-
-
-
20
40
60
80
-20
-40
-60
-80
-100
-120
c
a
-
-
-
-
100
120
140
160
c
a
-
140
160
-
2
00
210
220
230
240
250
200
210
220
230
240
250
Wavelength (nm)
Wavelengh (nm)
–
6
–1
Fig. 5. The CD spectra of complex-BSA system. [BSA] = 1.0 × 10 mol∙L . From a to c, the
ratio of [BSA]: [complex] were 1:0, 1:1and 1:2.
Table 5
The content of α−helix of BSA in the absence of and presence of complexes.
[
Complex]:[BSA]
0:1
1:1
2:1
BSA-1(%)
BSA-2(%)
52.76
52.76
43.06
35.28
26.82
20.21
The helix content of the BSA molecule is gradually reduced due to the increased
complexes concentration. This indicates that the complexes bind to the amino acid
residue in the BSA molecule, destroying the network of hydrogen bond skeleton
shaped between the amino acid residues. For this reason, the α−helix structure content
of the protein is lowered, and the secondary structure of BSA is changed. In the
interaction systems, the α−helix structure changes caused by 2 are relatively larger.
3
.4.7. Raman spectroscopic studies
Raman spectroscopy is also used in this work to investigate the interaction

between the complexes and BSA. In the Raman spectrum of BSA, its main chain
conformation is mainly reflected by the absorption peak of the peptide chain[45]
Therefore, its conformational changes can be displayed by the change of the band[46]
Fig. 6 is a general Raman spectrum of BSA interaction with the complexes.
.
.
In the Raman spectrum, the characteristic absorption peak due to the C=O
stretching vibration of the peptide bond of the protein appears in the range of 1610 –
-1
1
700 cm in the amide I band and the characteristic peak in the amide II band in the
-1
range of 1600 – 1500 cm . These absorption peaks are caused by the stretching
vibration of protein CN and accompanying NH bending vibration[47]. The
-1
characteristic absorption peaks in the 1651 – 1663 cm range belong to the α-helix
structure of the BSA molecule. Fig. S6 is a curve fit plot of the BSA amide I band and
the fit results are listed in Table 6. According to the fitting results, the decrease of the
spiral content of BSA was significantly reduced from 65.13% to 33.50% after the
interaction of complex 2 with BSA. This is consistent with the results obtained from
the circular dichroism spectrum, which alters the change in the secondary structure of
the protein.
56000
52000
48000
44000
40000
36000
32000
28000
24000
BSA
BSA-1
BSA-2
800
1000
1200
1400
1600
1800
2000
Wavelength (nm)
Fig. 6. Raman spectra of free BSA and of BSA after addition of complex. [BSA] = [complex]
−5
−1
=
1×10 mol∙L .
Table 6
The curve fitting results of Raman amide I of BSA.
α−helix
short segment
β−turn
System
BSA
Peak
Peak
(cm-1)
Peak
(cm-1)
Content (%)
65.13
Content (%)
20.66
Content (%)
14.21
-
1
(
cm )
1653
1618
1680

BSA-1
BSA-2
1665
1658
56.58
33.50
1612,1623 11.92
1645,1678 57.90
1695,1705 10.50
1697 8.60
3
3
.5. Superoxide dismutase activity
.5.1. SOD activity
The degree of NBT reduction was followed by spectrophotometry, and the rate
of reaction of the complexes and its hybrid protein to O
·- were determined by its
competitive kinetic method[11]. In this system, XO is catalyzed by XOD to produce
·-. When O ·- encounters NBT, it produces blue formazan, which has its maximum
2
O
2
2
[
48]
absorption peak at 550 nm . When SOD is added to the system, the reduction
reaction of NBT is inhibited so that the reaction rate is slowed down. The SOD
activity was indirectly tested by comparing the rate at which blue formazan was
produced in the absence of the SOD enzyme. Record the absorbance value at 550 nm,
and use this data as the ordinate[49]. The time is plotted on the abscissa and a straight
line can be fitted. The SOD activity was calculated in this way. When natural SOD or
artificially simulated SOD enzymes are added to the reaction system, they can
disproportionate superoxide anion radicals, generate hydrogen peroxide and oxygen,
compete with superoxide anion radicals reflected by NBT, and hinder the formation
of formazan. The slope of the line is reduced. The degree of slope reduction is a
manifestation of the magnitude of SOD activity. The greater the degrees of slope
reduce, the stronger the activity is.
Experimental studies showed that 1 did not show SOD activity, and the addition
of BSA could not effectively improve its activity. Fig. S7 shows the NBT reduction
O
·- reaction rate of 2 and its protein adduct at various concentrations. As shown in
2
the figure, as the concentration of the complex and the protein adduct gradually
increases, the slope of the corresponding curve gradually decreases. This indicates
that the rate at which the complex and its corresponding hybrid protein catalyze the

decomposition of O
simulated enzyme. The experimental results show that the method can catalyze the
decomposition rate of O
·- better than the decomposition rate of complex alone. The
reaction rate constant was obtained by the Eq. (3.7).
2
·- accelerates with the increase of the concentration of the
2
V0
Kcat
-
=
[cat]
1
Vcat
Kdetector[detector]
(3.7)
Where V
0
is the uninhibited reduction rate of the detector molecule and Vcat is the
reduction rate of the detector molecule inhibited by the complexes. Kdetector is the
constant for the reaction and its value is 5.88 × 10 M ∙s-1[50]. Kcat is the apparent rate
4
-1
constant of the inhibitor. The abscissa represents concentration of complexes and their
hybrids and (V
0
/Vcat) -1 is plotted as ordinate. It can be seen from Fig. 7. From the Eq.
(
3.8), the IC50 and Kcat values could be determined in Table 7[51]
.
K = K
[detector]/ IC50
detector
(3.8)
cat
0
0
0
0
0
0
0
0
0
0
0
.50
2
.45
BSA-2
.40
.35
.30
.25
.20
.15
.10
.05
.00
1
2
3
4
5
6
7
8
9
10
concentration (10-6 M)
Fig. 7. The SOD-like activity of complex (■) and complex-BSA adduct (●) in the Fridovich
assay (NBT = 250 μM).
Table 7
IC50 and Kcat values of the complexes and the corresponding hybrid proteins.
-6
−1
6
-1 −1
Substance
IC50 (×10 mol∙L )
K
cat (×10 L∙mol ∙s )
R
Native SOD
0.0158
-
0.9942

2
25.65
13.92
5.73
0.9689
0.9877
BSA-2
10.56
The IC50 value of the natural SOD enzyme was 0.0158 μmol·L-1[52]. For 2, the IC50
value was markedly reduced, and the Kcat value was increased when hybridized with
protein. In the system, the activity of 2 was improved by BSA, because the
hydrophobic cavities and amino acid residues of BSA provide appropriate catalytic
environment after forming hybrid proteins.
+
3
.5.2. ABTS· scavenging ability
ABTS can be oxidized by an oxidizing agent such as K
2
S
2
O
8
and MnO to react
2
+
electronically to form green ABTS· radicals. However, when both the antioxidant
+
+
and the ABTS· radical are present, the production of the ABTS· radical is
suppressed, and the absorbance at the maximum absorption of 734 nm is also lowered,
and the color changes from green to colorless[53]. For this reason, the change in
absorbance of ABTS at 734 nm before and after the addition of antioxidants can
demonstrate the antioxidant capacity of the sample[54]. The inhibition rate is analyzed
by Eq. (3.9). Using inhibition rate and sample concentration to plot Fig. 8, the
antioxidant concentration can be determined.
Activity = [(A
is absorbance of the blank and A is absorbance of the sample. The sample
concentration with inhibition rate of 50% is IC50 value.
0
- A) / A] × 100%
(3.9)
where A
0
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
90
80
70
60
50
40
30
VC
1
BSA-1
BSA
VC
2
BSA-2
BSA
20
10
0
10
20
30
40
50
10
20
30
40
50
Concentration (μM)
Concentration (μM)

Fig. 8. Trends in the inhibition of ABTS radicals by complexes with standard Vitamin C (VC) at
various concentrations.
4
0.25
40
35
30
25
20
15
10
5
0
1
4.28
7
.42
5
.02
5.18
3.47
VC
BSA
1
BSA-1
2
BSA-2
Compounds
Fig. 9. The radical scavenging activity of the compounds and VC.
From Fig. 8, the change in VC concentration has no effect on the ability to clear
+
+
ABTS· . This proves that it quickly reaches equilibrium with ABTS· . The
scavenging ability of BSA and its hybrid protein with metal complexes increased with
the increasing concentration. After the formation of hybrid protein, the IC50 value of
+
the complexes decreased significantly. The ABTS· radical scavenging ability is
reflected by the IC50 value and listed in Fig. 9. After the formation of the hybrid
protein, the IC50 value of the complexes is reduced, and the order of activity is
BSA-2 > VC > BSA-1 > 1 > 2 > BSA. Although 1 has no SOD activity, it exhibits
+
·-, 1 has
some ABTS· scavenging activity. It is indicated that in addition to O
2
antioxidant capacity against other free radicals in biological fluids. The IC50 value of
+
the complexes for ABTS· scavenging activity clearly show that the present
+
complexes can be used as ABTS· radical scavengers.
3
.6. Catalytic oxidation of methyl phenyl thioether
Methyl phenyl sulfide was used as substrate to study the catalytic oxidation
activity with the complexes and their corresponding hybridized proteins as enzyme[55]
.
The reaction product was further purified by column chromatography and then was
tested by NMR, as shown in Fig. S8. The results show that the chemical shift δH1 =
2
.74 (t, 3H) is hydrogen on the methyl group, and δH2 = 7.26 - 7.66 (m, 5H) is

hydrogen on the benzene ring, and their integral ratio is δH2: δH1 = 5: 3. These
indicate the product synthesized is methylphenyl sulfoxide.
Yield (%)
ee (%)
BSA-1
︷
1
2
BSA-2
89
︷
83.8
︷ ︷84.5
80.6
80.6
80
60
40
20
0
76.1
77.5
76.7
78.6
65.5
55.7
5
4.5
2
2.51
19.48
15.77
1
2.94
.01 3.02 5.19
2
2.5 1.22 0.45
20 30
0.93
0 20 30
1.
0
20 30
0
20 30
0
o
Temperature ( C)
Fig. 10. Rate and Enantiomeric excess of methyl phenyl sulfoxide catalyzed by complex
and their corresponding hybrid proteins.
The catalytic activity of enzyme catalysts is very sensitive to temperature. The
influence of temperature on catalytic reaction is mainly reflected in reaction speed and
catalyst activity. Generally speaking, the decrease of temperature slows down the
reaction speed, which is conducive to the orderly progress of asymmetric reactions,
thus improving enantioselectivity. However, when the temperature is too low, the
activity of catalyst decreases, which is not conducive to the high conversion rate and
high yield of sulfoxide products. When the temperature is too high, the reaction speed
is accelerated, which is conducive to the improvement of the conversion rate and
sulfoxide yield, but the high temperature tends to deactivate the artificial enzyme
catalyst and change the enantiomer activation energy, resulting in the reduction of
enantiomer selectivity. Therefore, determining the appropriate reaction temperature is
one of the important factors to obtain the products with high efficiency and high
enantiomers selectivity. The effects of temperature on the catalytic activity of
asymmetric catalytic oxidation of benzothioether were studied. The results are shown
o
in Fig. 10. The results showed that when the reaction temperature is 20 C, the
optimum conversion and sulfoxide yield of the hybrid proteins were obtained. Under
this condition, the ee values were catalyzed by BSA-1 and BSA-2 reached 22.51%
[
56]
and 15.77%, with the yield of 77.5 and 84.5%, respectively. In our previous work ,

the
vanadium
complex
with
Schiff
base
(2,
2’
-[(1,
2
-ethanediyl)bis(nitrilopropylidyne)]bisphenol) catalyze methyl phenyl sulfoxide
under the similar reaction condition with the ee value of 27%, which is slightly higher
than 1 and 2.
4. Conclusion
In this paper, two vanadium metal complexes with Schiff base ligands were
synthesized. The interactions between the complexes and BSA were studied by UV
spectroscopy, circular dichroism spectroscopy, fluorescence quenching and Raman
spectroscopy. It was found by fluorescence quenching spectroscopy that the
interaction mechanism between the two complexes and BSA is static quenching.
Calculating the binding constants at different temperatures, binding sites and
thermodynamic parameters, it was found that hydrogen bonding and van der Waals
forces are the main forces for maintaining the stability of hybrid proteins. Both
circular dichroism and Raman spectroscopy indicated that the complexes changed the
α-helix structure of BSA to different degrees, and the change trend was consistent.
Using xanthine oxidase-NBT reduction and ABTS·+ free radical scavenging
experiments, the antioxidant activity of the complex and its hybrid protein was
determined. It was found that 1 had a weak ability to eliminate O
·-, but a certain
2
+
+
activity of ABTS· . Both SOD activity and ABTS· activity of 2 were better than that
of 1. The results of asymmetric catalytic oxidation of phenylmethyl sulfide show that
the complex and its corresponding hybrid proteins have certain catalytic activity.
BSA-1 possesses a higher stereoselectivity than BSA-2. The addition of serum
albumin can enhance the antioxidant activity and catalytic activity. These complexes
are effective SOD mimics with catalytic activities. The application of such metal
complexes might be as pharmaceutical agents and catalysts.
Acknowledgements

We gratefully acknowledge the National Natural Science Foundation of China
21361003, 21101035), the Guangxi Natural Science Foundation of China
016GXNSFDA380005, 2016GXNSFFA380010, 2016GXNSFBA380237)，Specific
(
2
research project of Guangxi for research bases and talents (2017AD19029), New
Century Ten, Hundred, Thousand Talents Project in Guangxi, the National Natural
Science Foundation of Guangxi University for Nationalities and the Guangxi
University for Nationalities Graduate Scientific Research Innovation Project
(
gxun-chxzs2017120), Specific research project of Guangxi for research bases and
talents（AD18126005).
Appendix A. Supplementary data
CCDC (#1868577 and #1868578) contains the supplementary crystallographic data
for
http://www.ccdc.cam.ac.uk/conts/retrieving.html,
Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44)
223-336-033; or e-mail: deposit@ccdc.cam.ac.uk.
1
and 2. These data can be obtained free of charge via
or from the Cambridge
1
References
[
[
1] H. Cao, Y. Yi, Biometals 30 (2017) 1-11.
2] L. Buzoglu, E. Maltas, M. Ozmen, S. Yildiz, Colloids Surf., A 442 (2014)
1
39-145.
[
[
[
[
[
3] J. Chen, G. Song, Y. He, Q. Yan, Microchim. Acta 159 (2007) 79-85.
4] Y. Z. Zhang, J. Zhang, F. F. Li, Mol. Biol. Rep. 38 (2011) 2445-2453.
5] J. Tong, Zhang H , Yang H , Mol. Biol. Rep. 37 (2010) 1741-1747.
6] Z. Chen, J. Zhang, C. Liu, Biometals 26 (2013) 827-838.
7] Ç. S, M. Köse, F. Tümer, A. Gölcü, M. Tümer, Spectrochim. Acta, Part A 151
(
2015) 821-838.
8] J. Yu, L. Ge, S. Liu, P. Dai, S. Ge, W. Zheng, J. Inorg. Organomet Polym. 21
2011) 809-815.
9] M. N. Islam, A. A. Kumbhar, A. S. Kumbhar, M. Zeller, R. J. Butcher, M. B.
Dusane, B. N. Joshi, Inorg. Chem. 49 (2010) 8237-8246.
[
[
(
[
[
10] I. N. Zelko, T. J. Mariani, R. J. Folz. Free Radical Biol. Med. 33 (2002) 337-349.
11] E. F. Zhang, Y. Wei, F. P. Huang, Q. Yu, H. D. Bian, H. Liang, F. H. Lei, J. Mol.
Struct. 1155 (2018) 320-329.

[
[
12] C. R. Johnson, R. A. Kirchhoff, H. G. Corkins, J. Org. Chem. 39 (1974) 23-42
13] J. Han, V. A. Soloshonok, K. D. Klika, J. Drabowicz, A. Wzorek, Chem. Soc.
Rev. 47 (2018) 1307-1350.
[
14] P. Patel, B. Parmar, R. I. Kureshy, N. H. Khan and E. Suresh, Dalton Trans. 47
(
2018) 8041-8051.
[
[
[
15] A. M. Abu-Dief, I. M. A. Mohamed, J.Basic Appl. Sci. 4 (2015) 119-133.
16] S. Mohebbi, S. Bahrami, A. Shangaie, Transition Met. Chem. 36 (2011) 425-431.
17] H. Hosseini-Monfared, N. Asghari-Lalami, A. Pazio, K. Wozniak, C. Janiak,
Inorg. Chim. Acta 406 (2013) 241-250.
[
18] R. A. Lal, L. M. Mukherjee, A. N. Siva, A. Pal, S. Adhikari, Polyhedron 12 (1993)
2
351-2354.
[
[
[
19] G. M. Sheldrick, Acta Crystallogr, Sect. A: Found. Adv. 71 (2015) 3-8.
20] G. M. Sheldrick, Acta Crystallogr. Sect. C: Struct. Chem. 71 (2015) 3-8.
21] V. A. Kostyuk, A. I. Potapovich, E. N. Strigunova, T. V. Kostyuk, I. B. Afanas'Ev,
Arch. Biochem. Biophys. 428 (2004) 204-208.
[
[
[
[
[
[
[
22] K. Parschat, C. Canne, J. Hüttermann, R. Kappl, S. Fetzner, Biochim. Biophys.
Acta 1544 (2001) 151-165.
23] Y. Song, J. Jiang, J. Ma, S. Y. Pang, Y. Z. Liu, Y. Yang, C. W. Luo, J. Q. Zhang, J.
Gu, W. Qin, Environ. Sci. Technol. 49 (2015) 11764-11771.
24] J. Tang, P. F. Yao, X. L. Xu, H. Y. Li, F. P. Huang, Q. Q. Nie, M. Y. Luo, Q. Yu, H.
D. Bian, RSC Adv. 6 (2016) 44154-44162.
25] M. R. Maurya, C. Haldar, A. Kumar, M. L. Kuznetsov, F. Avecilla, J. C. Pessoa,
Dalton Trans. 42 (2013) 11941-11968.
26] M. R. Maurya, N. Chaudhary, F. Avecilla, P. Adao, J.C. Pessoa, Dalton Trans. 44
(
2015) 1211-1232.
27] S. Gupta, M. V. Kirillova , G. D. S. Mf, A. J. Pombeiro, A. M. Kirillov, Inorg.
Chem. 52 (2013) 8601-8611.
28] A. W. Addison, T. N. Rao, J. Reedijk, J. V. Rijn, G. C. Verschoor, J. Chem. Soc.
Dalton Trans. 7 (1984) 1349-1356.
[
[
[
[
29] Z. Chi, L. Zhu, X. Lu, J. Mol. Struct. 1001 (2011) 111-117.
30] E. M. Clérico, M. R. Ermácora, Arch. Biochem. Biophys. 395 (2001) 215-224.
31] Y. Hu, G. Zhang, J. Yan, Molecular Biology Reports 41 (2014) 1693-1702.
32] K. Tishchenko, E. Beloglazkina, M. Proskurnin, V. Malinnikov, D. Guk, M.
Muratova, O. Krasnovskaya, A. Udina, D. Skvortsov, R. R. Shafikov, J. Inorg.
Biochem. 175 (2017) 190-197.
[
[
33] Z. Liu, X. Guo, Z. Feng, L. Jia, J. Solution Chem. 44 (2015) 293-306.
34] S. Rudra, S. Dasmandal, C. Patra, A. Kundu, A. Mahapatra, Spectrochim. Acta,
Part A 166 (2016) 84-94.
[
[
35] D. U. Chuan-Rong, X. Luo, J. R. Wei, H. E. Ting-Ting, X. Y. Zheng, C. W. Lin,
Chem. Res. Chin. Univ. 29 (2013) 854-860.
36] F. U. Jiaxin, K. W. Wang, G. E. Yushu, F. L. Jiang, X. H. Sun, Sci. China. Chem.
5
4 (2011) 788-796.
[
[
37] A. Abedi, Z. M. Lighvan, S. N. Ostad, Monatsh Chem 147 (2016) 1-8.
38] Q. L. Gong, X. G. Hu, G. Y. Fang, X. H. Li, J. Mol. Model. 18 (2012) 493-500.

[
39] X. J. Guo, A. J. Hao, X. W. Han, P. L. Kang, Y. C. Jiang, X. J. Zhang, Mol. Biol.
Rep. 38 (2011) 4185-4192.
[
[
40] X. Pan, P. Qin, R. Liu, J. Wang, J Agric Food Chem 59 (2011) 6650-6656.
41] İ. D, R. Aydın, Ö. Vatan, T. Sevgi, D. Yılmaz, Y. Zorlu, Y. Yerli, Ç. B, E.
Demirkan, Ç. N, J. Biol. Inorg. Chem. 22 (2017) 1-25.
[
42] M. Mahanthappa, M. A. Savanur, B. Puthusseri, S. Yellappa, J. Mater. Sci. 53
(
2018) 202-214.
[
[
[
[
43] S. Basak, K. K. Rajak, Inorg. Chem. 47 (2008) 8813-8822.
44] N. J. Greenfield, Nat. Protoc. 1 (2006) 2527-2535.
45] T. R. Weikl, K. A. Dill, J. Mol. Biol. 332 (2003) 953-963.
46] M. A. Pimenta, E. D. Corro, B. R. Carvalho, C. Fantini, L. M. Malard, Acc.
Chem. Res. 48 (2015) 41-47.
[
47] M. S. Dresselhaus, A. Jorio, M. Hofmann, G. Dresselhaus, R. Saito, N. Lett. 10
(
2010) 751-758.
[
[
[
[
[
48] A. Yoshida, Y. Kaburagi, Y. Hishiyama, Carbon 44 (2006) 2333-2335.
49] X. Bai, Y. Gong, H. Yang, D. Gong, Heritage Sci. 2 (2014) 1-5.
50] F. A. Abdlseed, M. M. El-Ajaily, Int. J. PharmTech Res. 1 (2009) 1097-1103.
51] V. Oliveri, G. Vecchio,Eur. J. Med. Chem. 46 (2011) 961-965.
52] O. Vajragupta, L. J. Boonchoong P. Berliner, Free Radical Res. Commun. 38
(
2004) 303-314.
[
53] V. Oliveri, A. Puglisi, G. Vecchio, Dalton Trans. 40 (2011) 2913-2919.
[
[
54] L. Zheng, M. Zhao, C. Xiao, Q. Zhao, G. Su, Food Chem. 192 (2016) 288-294.
55] T. Anna, K. Olga, K. Ekaterina, F. Tatyana, T. Mikhail, Int. J. Mol. Sci. 16 (2015)
2
5353-25376.
56] J. Tang, P. Yao, F. Huang, M. Luo, W. Yi, H. Bian, Tetrahedron Asymmetry, 28
2017) 1700-1707.
[
(
Title: Bovine serum albumin–vanadium complex hybrid: Synthesis, crystal
structure, SOD activities and asymmetric catalytic oxidation of sulfides
Authors: Jingnan Shi, Yi Wei, Yuzhen Zhang, Jie Tang, Hedong Bian,* Qing Yu,
Fuping Huang
Two symmetrical double-sided Schiff base ligands and their vanadium
complexes has been synthesized and fully characterized. The interaction of the two
vanadium complexes with bovine serum albumin (BSA) was investigated by
spectroscopic techniques. SOD-like activitand ABTS free radical scavenging ability
were also studied. The results showed that the microenvironment of BSA can enhance
the activity of the complexes. The complexes can catalyze asymmetric catalytic
oxidation of Methyl phenyl sulfide.

Title: Bovine serum albumin–vanadium complex hybrid: Synthesis, crystal
structure, SOD activities and asymmetric catalytic oxidation of sulfides
Authors: Jingnan Shi, Yi Wei, Yuzhen Zhang, Jie Tang, Hedong Bian,* Qing Yu,
Fuping Huang