Effect of carbamylation on the molecular recognition action of amino benzothiazole by carrier protein

Article abstract of DOI:10.1016/j.saa.2019.117595

Increasing benzothiazole derivatives containing amino or N-acyl structures in position 2 have been largely developed as pesticides and medicines. However, the structure-function relationship of 2-substituted benzothiazole derivatives has seldom been illustrated from the perspective of their albumin-binding nature. Herein, to probe the influence of carbamylation on the albumin-binding nature of benzothiazole derivatives, formyl group was introduced to the amine group of 2-amino benzothiazole (ABT) to yield a novel modified ABT (MABT). Their protein-binding properties were systematically deciphered by spectroscopy, molecular modeling and density functional theory (DFT) calculations. The interaction mechanisms, recognition thermodynamics and binding geometry were investigated and compared. The structural alteration of human serum albumin was explored using synchronous fluorescence emission and circular dichroism spectrum technologies. Based on experimental results, the structures of protein complex with MABT and ABT were revealed by molecular docking method. The differences in energy transfer efficiency and molecular orientation of ABT and MABT in new complexes were tentatively explained by DFT calculations. The work was expected to help to understand the impact of different substituents on the bioactivity of benzothiazole derivatives and guide for structural designs of new compounds.

Full text of DOI:10.1016/j.saa.2019.117595

Journal Pre-proof
Effect of carbamylation on the molecular recognition action of amino benzothiazole by
carrier protein
Hua-xin Zhang, Qing-hua Xia, Dan Zhou
PII:
S1386-1425(19)30985-0
https://doi.org/10.1016/j.saa.2019.117595
SAA 117595
DOI:
Reference:
To appear in: Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
Received Date: 24 July 2019
Revised Date: 12 September 2019
Accepted Date: 2 October 2019
Please cite this article as: H.-x. Zhang, Q.-h. Xia, D. Zhou, Effect of carbamylation on the molecular
recognition action of amino benzothiazole by carrier protein, Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy (2019), doi: https://doi.org/10.1016/j.saa.2019.117595.
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2019 Published by Elsevier B.V.

Graphical abstract
The hydrophobic heterocyclic scaffold of benzothiazole guided ABT and MABT to Sudlow’s
site I in the subdomain IIA of HSA, while the carbamylation of ABT on position 2 resulted in the
opposite molecular orientation and significantly increased energy reception efficiency, which were
reasonably explained by the alterations of dipole moment and energy gap between HOMO and
LUMO.

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Effect of carbamylation on the molecular recognition action of amino
benzothiazole by carrier protein
a, b, *
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Hua-xin Zhang
, Qing-hua Xia , Dan Zhou
a
Ministry-of-Education Key Laboratory for the Synthesis and Application of Organic Functional
Molecules, Hubei University, Wuhan 430062, P. R. China
b
Hubei Key Laboratory of Drug Synthesis and Optimization, Jingchu University of Technology,
Jingmen 448000, P. R. China
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*Corresponding authors.
Tel.: +8618986973258
E-mail address: h.x.zhang@yeah.net (H. X. Zhang), d.zhou@hubu.edu.cn (D. Zhou)
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Abstract:
Increasing benzothiazole derivatives containing amino or N-acyl structures in position 2 have
been largely developed as pesticides and medicines. However, the structure-function relationship of
2-substituted benzothiazole derivatives has seldom been illustrated from the perspective of their
albumin-binding nature. Herein, to probe the influence of carbamylation on the albumin-binding
nature of benzothiazole derivatives, formyl group was introduced to the amine group of 2-amino
benzothiazole (ABT) to yield a novel modified ABT (MABT). Their protein-binding properties
were systematically deciphered by spectroscopy, molecular modeling and density functional theory
(DFT) calculations. The interaction mechanisms, recognition thermodynamics and binding
geometry were investigated and compared. The structural alteration of human serum albumin was
explored using synchronous fluorescence emission and circular dichroism spectrum technologies.
Based on experimental results, the structures of protein complex with MABT and ABT were
revealed by molecular docking method. The differences in energy transfer efficiency and molecular
orientation of ABT and MABT in new complexes were tentatively explained by DFT calculations.
The work was expected to help to understand the impact of different substituents on the bioactivity
of benzothiazole derivatives and guide for structural designs of new compounds.
Keywords: Benzothiazole derivative; Fluorescence probe; Molecular modeling; Thermodynamics;
DFT
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1. Introduction
The biological action of an active compound is highly dependent on its binding tendency to
plasma proteins since many key physiological processes of exogenous molecules are closely related
to their protein-binding ratio, such as transportation, distribution and clearance [1-3]. The nature of
plasma-binding has become one of the essential items when evaluating a new compound which is
going to be applied in vivo [4].
Benzothiazole, chemically referred to as 1,3-benzothiazole, is an aromatic heterocyclic
compound, whose activity is obviously superior to other heterocyclic because of the introduction of
sulfur atoms. The nine atoms in this heterocyclic scaffold are coplanar, which provides its derivative
a steady chemical structure that allows more applications. When substituents are attached, this
molecule becomes especially active in biology, which makes benzothiazole derivatives gained great
attention and widely used in pesticide, medicine and other fields [5].
The type and the position of substituent group are two main factors that influence the
physiological and biological activities of benzothiazole derivatives. Since this heterocyclic stem
nucleus is electron-withdrawing group, electron-donating substituent should be favorable for
increasing biological activity. On the other hand, a large number of experiments show that
2-substituted benzothiazole has higher activity. Considering these two points, 2-amino
benzothiazole (ABT) was selected as a target ligand. To inspect the influence of the
electron-donating ability of substituent group on the protein-binding properties of benzothiazole
derivatives, a novel modified ABT, N-1, 3-benzothiazol-2-ylformamide (MABT) was synthesized
for comparison.
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The scaffolds of ABT and MABT exist in plenty of commercial products such as riluzole (an
amyotrophic lateral sclerosis drug), frentizole (an antiviral drug and immunosuppressant),
benzthiazuron and methabenzthiazuron (pesticides) (Fig. 1) and there are still a large number of
substituted active compounds being researched as candidates for pesticides and drugs [6]. So, the
present study is of considerable value.
The task of present paper was: (1) to synthesize MABT from ABT by formylation reaction of
amine group; (2) to investigate the interaction of HSA with ABT and MABT respectively, including
interaction mechanisms, recognition thermodynamics and binding geometry, binding power, and the
structure of protein-ligand complex; (3) to analyze the difference in serum-binding characteristics
between ABT and MABT, and to explain the reasons. This study was expected to help to understand
the action mechanism of benzothiazole derivatives with different substituents and guide for their
structural designs.
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2. Experimental
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2.1. Chemicals
HSA，warfarin and tris(hydroxymethyl)aminomethane (Tris) were supplied by Sigma-Aldrich
Chemical Co. (Milwaukee, USA). Ibuprofen and KCl were purchased from Roche Diagnostics
(Mannheim, Germany). 2-amino benzothiazole (ABT) was purchased from Macklin Inc. (Shanghai,
China) and N-1, 3-benzothiazol-2-ylformamide (MABT) was synthesized in our laboratory.
The buffer of pH 7.40 was prepared by mixing 0.05 M Tris aqueous and 0.15 M HCl aqueous at
proper ratio and KCl (0.5 M) was added to simulate physiological condition. The working solution
of ABT (0.5 mM), MABT (0.5 mM), ibuprofen (10 mM) and warfarin (10 mM) were dissolved by
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ethanol. HSA solution (50 µM) was dissolved by buffer. 2 mL of 50 µM HSA solution and different
volumes of ABT or MABT were mixed in a battery of 10 mL colorimetric tubes and diluted to scale
lines with buffer. The mixtures were kept at specific temperature for 0.5 h before spectroscopic
determination.
2.2. Instruments
All fluorescence was measured on a Hitachi F-4600 fluorescence spectrophotometer. The
temperature was maintained by a thermostatic circulator bath. A Jasco J-810 Spectropolarimeter, a
Purkinje TU-1901 spectrophotometer, a Testo 206-pH2 pH-meter and a Mettler-Toledo Excellence
plus electronic analytic balance were employed to record CD spectra, UV-vis spectra, pH values
and weights, respectively.
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2.3. Inner filter effect correction
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The data were corrected by equation 1, as described in literatures [7, 8]:
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(
Aex+ Aem)
F = F 10
(1)
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obs
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2.4. Molecular docking
Molecular modeling for ABT-HSA and MABT-HSA complexes was performed by the famous
docking software AutoDock 4 [9]. The concrete procedures were based on literature [10] as
experiments had revealed the definite binding site of present ligands.
2.5. DFT study
All computation results were given by ORCA (Version 4.0.1) [11]. The researches were done
in the gas phase, and M06-L functional with 6-311G(d) basis set was employed [12], which was
thought to be one of the functionals with small errors [13]. The electrostatic potential was drawn on
ABT and MABT molecule and visualized by VMD (Version 1.9.3) program [14].
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2.6. Synthesis of MABT
MABT was synthesized from ABT by formylation reaction of amine group (Fig. 2). Formic
acid (88%) was dried by anhydrous magnesium sulfate and filtered. 2.5 g of dried formic acid (54
mmol) and 7.1 g of diacetyl oxide (69 mmol) was mixed and refluxed for 3h. Then, the temperature
was kept lower than 40 ℃ 1 and 3 g of ABT (8.7mmol) were gradually added to the mixed acid
anhydride. Afterwards, 15 mL of H C OC H was added and stirred for another 22 h. The residues
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were filtered and washed by diethyl ether for several times, affording the white solid product (in a
yield of 42.0%).
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3. Results and Discussions
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3.1. The emission spectra
Aromatic amino acid residues in HSA make this protein a fluorophore, which can give
intensive fluorescence near 345 nm when excited by ultraviolet radiation. Fig. 3 recorded the
fluorescence emission spectra of ABT, MABT, HSA and their mixed systems between 280 nm and
480 nm.
As we could see in Fig. 3, even at their highest concentration in systems, both ABT and MABT
gave little fluorescence at 345nm when excited by 285 nm. It suggested ABT or MABT made no
contribution to the emission intensity of HSA, which ensured the reliability of the data. When HSA
was fixed at 10 µM, the emission intensity decreased with increasing amount of ABT (Fig.3 A) and
MABT (Fig.3 B), which was usually referred as fluorescence quenching [15, 16]. MABT showed
obviously stronger quenching ability than ABT in the same concentrations, which implied that the
energy transfer efficiency between protein and ligand significantly enhanced when the amine group
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was replaced by the acylated amine group.
3.2. The quenching mechanism
Fluorescence quenching contains dynamic and static mechanism [17]. These two types can be
judged by the fluorescence lifetime of phosphor at excited state, the relationship between quenching
constant and temperature or the ultraviolet spectra of interaction systems [18, 19]. In our
experiments, the quenching spectra were recorded at 310K, 304K, 298K and 292K, respectively.
The corrected emission intensity data were plotted by Stern-Volmer equation [20]:
F0
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=
1+ K [Q]
(2)
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Assuming their quenching belonged to dynamic mechanism, the slopes of regression equation
(i.e. Stern-Volmer constant, K ) should represent dynamic quenching constants and it should
SV
increase when temperature rises [21]. But the Stern-Volmer constants of ABT-HSA systems (Fig.4A)
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decreased from 5.381×10 L·mol at 292 K to 4.437×10 L·mol at 310 K, and MABT-HSA
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systems (Fig.4B) from 5.186×10 L·mol at 292 K to 4.436×10 L·mol at 310 K. The dynamic
quenching constants we detected were inversely correlated with temperature. It was untenable
because higher temperature would always promote diffusion. Therefore, our previous assumption of
dynamic quenching could be ruled out.
To confirm the quenching mechanism, ultraviolet absorption of ABT, MABT, HSA and their
mixed systems were recorded (Fig. 5). From Fig. 5 we could see, the absorbance of HSA-ligand
systems around 220 nm (blue curves) was much lower than the superposition (light violet curves) of
the separated absorbance of HSA (black curves) and ligands (red curves), which indicated that HSA
had formed new complex with both ABT and MABT at ground state [22]. Considering the results of
quenching constants and ultraviolet spectra, the quenching process between ABT/MABT and HSA
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could be identified as static mechanism.
3.3. Binding thermodynamics
Static quenching means new complex formation. The observed binding constant (K ) and
b
binding site number (n) could be calculated by the logarithm equation described in literature [23]:
F0 − F
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log(
) = log K + nlog[Q]
(3)
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The plots and regression equations were presented in Fig. 6. Thermodynamic parameters are
θ
θ
important in evaluating the affinity between ABT/MABT and HSA [24]. The
∆H and ∆S of an
isobaric process can be calculated by the indefinite integral form of Van’t Hoff equation (Fig. 7):
θ
θ
−
∆H
∆S
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ln K =
+
(4)
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And ∆G can be calculated by its definition:
θ
θ
θ
∆
G = ∆H －T·∆S
(5)
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The considerable value of binding constants in Table 1 means both ABT and MABT can bind
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effectively with HSA. The order of binding constants between HSA and MABT (10 ) is one
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thousand times bigger than ABT (10 ), indicating the stability of MABT-HSA complex is higher
θ
θ
than ABT-HSA. The negative ∆G and positive ∆S suggests the interactions between ABT/MABT
and HSA are spontaneous and entropy-driven process. But the protein-binding tendency of MABT
is much greater. The most obvious distinction between ABT and MABT is their enthalpy changes
θ
(∆
H ) when binding to HSA. ABT-HSA interaction is an exothermic process while MABT-HSA
interaction is an endothermic one, which should be considered when designing pesticide or drug
molecules based on benzothiazole scaffold.
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3.4. The binding powers
According to the thermodynamic rules reported by Ross [25], it could be speculated that the
main powers that stabilized the structures of ABT-HSA and MABT-HSA complex was electrostatic
forces and hydrophobic interactions, respectively.
3.5. The conformation investigation
Synchronous fluorescence emission (SFE) techniques has been widely applied to detect the
nature of amino acid residues in protein [26]. The shift of maximum emission wavelength in
synchronous spectra reflects the changes in polarity around the chromophore: a red shift means an
increase in the polarity while a blue shift denotes an increase in hydrophobicity [23]. Based on these
rules, we could infer that the binding of ABT had led to increases in polarity around both tryptophan
residue and tyrosine residue in HSA, as the maximum emission wavelength of both tryptophan and
tyrosine moved to long wavelength direction (Fig. 8 A and B). The binding of MABT to HSA made
no obvious impact on the microenvironment of tryptophan (Fig. 8 C) but the polarity around
tyrosine strikingly increased (Fig. 8 D).
To quantitatively detect the influence of ABT/MABT binding, circular dichroism (CD)
spectroscopy is employed [27]. The data of CD spectra were converted to mean residue ellipticity
(Fig. 9) and used to calculate the fine structures (Table 2).
It could be found in Table 2 that ABT and MABT had altered the contents of fine structures.
The total
ABT and MABT were added, respectively. The total
7.0%) and 4.2% (5.8% to 10.0%), respectively. Under the same conditions, MABT brought more
significant changes in total -helix and -strand structures. Similar cases could also be found in the
α-helix structure reduced by 2.9% (59.9% to 57.0%) and 8.5% (59.9% to 51.4%) after
β
-strand structure increased by 1.2% (5.8% to
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content of β-turns and irregular structure. These data demonstrated MABT would bring greater
influence on the secondary structure of proteins than ABT. It implied that pesticides or drugs
containing ABT and MABT scaffolds might present different activity or side effect.
3.6. Site-selective binding on HSA
To find out the binding site of ABT and MABT, warfarin and ibuprofen were used as
molecular probes for Sudlow’s site I and site II [28, 29], respectively. The spectra data (Fig. 10)
were fitted by equation 3 (Fig. 11) to calculate the new binding constants. As expected, the value of
K decreased after molecular probes were added, but in strikingly different levels. For both ABT
b
and MABT, the influence of warfarin was obviously stronger than ibuprofen (insets of Fig. 11),
which implied the warfarin had occupied the site of ABT and MABT. So, the primary site for ABT
and MABT was Site I. It might be the heterocyclic scaffold of benzothiazole that guided the
compound to specified binding site.
3.7. Molecular docking
The 3D crystallographic structure reported by He and Cater [30, 31] shows that HSA is a
heart-shaped molecule consisting of 585 amino acid residues. Each of its three domain
compromises ten helices which are ascribed to two subdomains. The first six helices are regarded as
subdomain A and the last four as subdomain B.
Since the site markers experiments have revealed the binding site of ABT and MABT locates
in Sudlow’s site I, the grid centers were set at 32.895, 13.107 and 7.466. The optimal docking
results showed that ABT and MABT bound almost the same location in site I in the subdomain IIA
(Fig. 12), based on which we thought it was amine group and heterocyclic scaffold of benzothiazole
that guided ABT and MABT into the binding location . Though binding in the same location, the
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molecular orientations of ABT and MABT were almost precisely opposite in new complexes, as
shown in Fig. 13.
In Fig. 14, the residues close to ABT and MABT (within 3.5Å) were displayed, which showed
ABT and MABT molecule were surrounded by different residues in subdomain IIA. The residues
that directly interact with ABT mainly distributed on the amino terminal, leaving the left
semi-sphere (benzene terminal) uncovered, as shown in Fig. 14A. It looked like some attractive
force had pulled ABT molecule to the right side and we speculated two gravitations might be
responsible for it. Firstly, the only one hydrophilic residue (SER287) within 3.5Å was located in the
amino terminal of ABT molecule. Secondly, the carboxyl oxygen of ARG257 formed two hydrogen
bonds (H-bonds) with the amine group of ABT. The existence of H-bonds was complied with the
conclusion on the binding power between ABT and HSA in Section 3.4.
On the other hand, MABT was entirely enclosed by a closed pocket made up of LUE219,
ARG222, PHE223, LEU238, VAL241, ARG257, LEU260, ALA261, ILE264, SER287, ILE290
and ALA291 (Fig. 14B). Most of these residues are hydrophobic, indicating the primary power
fixing MABT was hydrophobic interactions, which was also in accordance with the result of
thermodynamic discussion.
3.8. DFT calculations
In order to understand the difference in protein-binding characteristics of benzothiazole
derivatives when amine group on position 2 was substituted by formamide group, DFT calculations
were performed according to the method mentioned in experimental section. The electrostatic
potential and the dipole moments of ABT and MABT molecules were presented in Fig. 15, where
we could find that the dipole moments of two ligands were almost in opposite direction (blue
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arrows in Fig. 15). It implied that electrostatic force existed not only in ABT-HSA complex as the
primary power, but also in MABT-HSA complex as an auxiliary power that could not be ignored.
Then the opposite orientation of ABT and MABT in protein-ligand complexes described in
molecular docking study could be easily understood.
Another significant difference between HSA-ABT and HSA-MABT systems revealed by the
results of fluorescence detection was the energy transfer efficiency. To analyze why HSA possessed
obviously higher energy transfer efficiency to MABT than to ABT, the frontier molecular orbits of
ABT and MABT were calculated and compared. According to the frontier molecular orbital theory,
the electron transition from HOMO to LUMO is most easily to happen under external stimulus. The
energy gap between HOMO and LUMO (∆E= ELUMO-EHOMO) of the ABT and MABT molecules
were employed to analyze their efficiency of energy reception. The HOMO and the LUMO of ABT
and MABT molecules in combination with their energy levels were listed contrastively in Table 3.
We could find in Table 3 that the energy levels of both HOMO and LUMO reduced when the
amine group of ABT was substituted by formamide group, which suggested that the stability of
MABT was superior to that of ABT. The energy gap (∆E) of the ABT and MABT molecules were
calculated to be 3.89 eV and 3.53 eV, respectively. The maximum fluorescence emission
wavelength of HSA (energy donor) was 345 nm (Fig. 3), whose energy corresponded to 3.59 eV. It
was obvious that the energy gap (ELUMO-EHOMO) of the ABT matched better with the energy of
donor, which meant, compared to ABT, the valence electron of MABT was more easily to accept
the energy and jump to unoccupied molecular orbital with higher energy levels. The theoretical
reasoning based on structural calculation reasonably accounted for the experimental facts that
MABT quenched the characteristic fluorescence of HSA much more effectively than ABT in the
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same concentrations
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4. Conclusions
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In present paper, MABT was synthesized from ABT by formylation reaction of amine group.
Then the protein-binding characteristics of ABT and MABT were systematically determined and
compared. The results showed that both ABT and MABT could bind with HSA in vitro. The orders
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of equilibrium constants for MABT and ABT were 10 and 10 L·mol , respectively, indicating a
higher energy transfer efficiency in HSA-MABT systems than HSA-ABT systems. Their
θ
θ
interactions with HSA were entropy-driven (∆S <0) as well as spontaneous (∆G <0) processes, but
with different heat effects (exothermic for ABT but endothermic for MABT). The primary power
that stabilized the structures of ABT-HSA and MABT-HSA complex was electrostatic force and
hydrophobic interaction, respectively. Compared to ABT, MABT caused greater influence on HSA’s
fine structure under the same conditions. Site marker competitive experiments suggested both ABT
and MABT bound to Sudlow’s site I. The optimal conformation presented the interaction details,
which also rationally interpreted the conclusions drawn from spectral and thermodynamic studies.
The energy gap between HOMO and LUMO of ABT and MABT molecules explained their
different energy reception efficiency from HSA, and dipole moments were responsible for their
opposite molecular orientation in new complex. Therefore, the hydrophobic heterocyclic scaffold of
benzothiazole derivatives may determine their binding sites in protein, while the substitutes in
position 2 will greatly affect the energy transfer efficiency, binding affinity, binding power and
thermodynamics through altering the charge distribution of whole molecule.
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Acknowledgements
We acknowledged the support of National Natural Science Foundation of China (21571055,
21673069) and Research Foundation of Education Bureau of Hubei Province (B2018240).
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3
61
Table 1 Physical and chemical parameters for ABT-HSA and MABT-HSA interaction
θ
θ
θ
Kb
L·mol )
∆H
∆S
∆G
Ligands T(K)
n
-
1
-1
-1
-1
-1
(
(kJ·mol ) (J·mol ·K )
(kJ·mol )
-23.88
-24.19
-24.49
-24.80
-40.62
-41.52
-42.41
-43.30
4
2
2
3
3
2
2
3
3
92
98
04
10
92
98
04
10
1.873×10
1.743×10
1.587×10
1.518×10
1.840×10
1.895×10
1.929×10
1.976×10
1.139
1.142
1.139
1.138
1.671
1.681
1.694
1.698
4
4
4
7
7
7
7
ABT
-9.102
2.899
50.62
149.0
MABT
3
3
62
63
-
18 -

3
64
Table 2 Alterations of fine structures of HSA
System
Regular
Distorted
Regular
-strand
%)
Distorted
β
-turns (%)
Unordered
α-helix (%)
α-helix (%)
β
β
-strand (%)
structure (%)
(
HSA
40.2
37.5
32.1
19.7
19.5
19.3
2.8
3.4
5.0
3.0
3.6
5.0
12.8
14.5
19.7
20.9
21.3
25.8
HSA-ABT
HSA-MABT
3
3
65
66
-
19 -

3
67
Table 3 Frontier molecular orbital of ABT and MABT molecules.
Molecule
ABT
HOMO
EHOMO/eV
LUMO
ELUMO/eV
∆E/eV
-5.24
-1.35
3.89
MABT
-5.82
-2.29
3.53
3
68
69
3
-
20 -

3
70
3
3
71
72
Fig. 1. Some commercial products containing the scaffold of ABT or MABT.
-
21 -

3
73
3
3
74
75
Fig. 2. Synthetic route to MABT.
-
22 -

(
A)
-
5
-1
(B)
600
-
5
-1
-1
T=310K
c(HSA)=1.0*10 mol L
T=310K
c(HSA)=1.0*10 mol L
-
4
-1
-4
6
00
50
00
50
0
c(ABT) /10 mol L
c(MABT) /10 mol L
0
0
0
0
0
1
1
1
.25
.5
.75
0.25
0.5
0.75
1
1.25
1.5
1.75
1.75
4
3
1
450
300
150
0
.25
.5
1.75
.75
1
ABT only
350
MABT only
350 400
Wavelength /nm
3
00
400
Wavelength /nm
450
300
450
376
377
378
Fig. 3. Influence of ABT (A) and MABT (B) on the emission of HSA.
-
23 -

(A
)
2
3
(
B)
F /F=1+5.381×10 [ABT]
5
.0
.8
.6
.4
.2
.0
0
F /F=1+5.186×10 [MABT]
0
3
F /F=1+4.963×10 [ABT]
12
5
0
F /F=1+4.947×10 [MABT]
0
3
1
1
1
1
1
F /F=1+4.624×10 [ABT]
5
0
F /F=1+4.496×10 [MABT]
0
3
F /F=1+4.437×10 [ABT]
5
0
9
6
3
0
F /F=1+4.436×10 [MABT]
0
2
2
3
3
92K; R=0.9998
98K; R=0.9995
04K; R=0.9994
10K; R=0.9997
292K; R=0.9191
298K; R=0.9138
304K; R=0.9139
310K; R=0.9145
0
4
8
12
16
20
0
4
8
12
16
20
-5
-1
-5
-1
[
ABT] /10 mol
·L
[MABT] /10 mol·L
3
79
80
3
Fig. 4. Stern-Volmer plots of ABT-HSA systems (A) and MABT-HSA systems (B).
3
81
-
24 -

(
A) 2.0
(
B)
-
5
-1
-5
-1
c(HSA)=10 mol L
c(HSA)=10 mol L
2
.0
.5
.0
.5
-
4
-1
-4
-1
c(ABT)=1.75*10 mol L
c(MABT)=1.75*10 mol L
1
1
0
0
.5
.0
.5
.0
HSA
MABT
HSA-MABT
HSA+MABT
1
1
0
HSA
ABT
HSA-ABT
HSA+ABT
0.0
2
20
240
260
280
300
220
240
260
280
300
Wavelength /nm
Wavelength /nm
3
82
83
3
Fig. 5. Ultraviolet spectra of ABT-HSA systems (A) and MABT-HSA systems (B).
3
84
-
25 -

(
A) 0.2
(B)1.5
log(F /F-1)=4.273+1.139*log[ABT]
log(F /F-1)=7.265+1.671*log[MABT]
0
0
log(F /F-1)=4.241+1.142*log[ABT]
log(F /F-1)=7.278+1.681*log[MABT]
0
.0
0
0
log(F /F-1)=4.201+1.139*log[ABT]
log(F /F-1)=7.285+1.694*log[MABT]
0
1.0
0
-
-
-
-
-
-
0.2
0.4
0.6
0.8
1.0
1.2
log(F /F-1)=4.181+1.138*log[ABT]
log(F /F-1)=7.296+1.698*log[MABT]
0
0
0
.5
292K; R=0.9971
98K; R=0.9985
304K; R=0.9988
10K; R=0.9993
0.0
0.5
2
2
3
3
92K; R=0.9744
2
98K; R=0.9704
04K; R=0.9722
10K; R=0.9728
3
-
-
4.6
-4.4
-4.2
log[ABT]
-4.0
-3.8
-4.6
-4.4
-4.2
log[ABT]
-4.0
-3.8
385
386
387
Fig. 6. Binding equilibriums of ABT-HSA systems (A) and MABT-HSA systems (B).
-
26 -

(
A)9.9
(B)
lnK=1095/T+6.089
R=0.9790
lnK=-348.7/T+17.93
R=0.9887
1
6.80
6.78
6.76
6.74
6.72
9
9
9
.8
.7
.6
1
1
1
1
0.00320
0.00328
-10.00336
0.00344
0.00320
0.00328
-10.00336
0.00344
-1
-1
T
/K
T /K
388
389
390
Fig. 7. The plots of logK versus 1/T for ABT-HSA (A) and MABT-HSA (B) interactions.
-
27 -

(
A)
-
5
-1
delta lamda: 60nm
(B)
c(HSA)=1.0*10 mol L
5
-1
1
20 c(HSA)=1.0*10 mol L
delta lamda: 15nm
7
50
00
50
00
50
0
-4
-1
c(ABT) /10 mol L
-4
-1
c(ABT) /10 mol L
6
4
3
1
0
9
6
3
0
0
0
0
0
0
0
0
1
1
1
.25
.5
.75
0.25
0.5
0.75
1
.25
.5
1
.25
1.5
.75
1.75
1
2
20
240
260
280
300
320
260
280
Wavelength /nm
300
320
Wavelength /nm
3
91
(
C)
-
5
-1
(D)
delta lamda: 60nm
5
-1
c(HSA)=1.0*10 mol L
1
20 c(HSA)=1.0*10 mol L
delta lamda: 15nm
7
50
00
50
00
50
0
-4
-1
c(MABT) /10 mol L
-4
-1
c(MABT) /10 mol L
0
6
4
3
1
9
6
3
0
0
0
0
0
0
0
1
1.25
1
1
.25
.5
.75
0
0.25
0.5
0.75
1
1.25
1.5
.5
.75
1.75
2
20
240
260
280
300
320
2
60
280
Wavelength /nm
300
320
Wavelength /nm
3
92
93
3
Fig. 8. SFE spectra of ABT-HSA and MABT-HSA systems.
3
94
-
28 -