The interaction of
6-amino-4-aryl-3-methyl-1-phenyl-1H-pyrazolo[3,4-b] pyridine-5-carbonitrile
with two kinds of snake venom: A fluorescence quenching study
Lian
Shuqin, Wu Yunming, Yin Xiaoxing
(School of Pharmacy, Xuzhou Medical College, Xuzhou, Jiangsu 221004)
Abstract The interactions between the
two kinds of snake venom (cobra venom and viper venom) and
6-amino-4-aryl-3-methyl-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile
at pH 7.6 were investigated by fluorescence quenching and
6-amino-4-(2-hydroxyphenyl)-3-methyl-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-
carbonitrile (I) was studied in detail. The quenching constants KSV,
binding constants K and sites n of I with venom were determined. The
results indicated that both of interactions were static quenching procedures because of
the formation of new compounds and there had a strong binding between them.
Keywords cobra venom, viper venom, 6-amino-4-(2-hydroxylphenyl)-3-methyl-1-phenyl-1H-pyrazolo
[3,4-b]pyridine-5-carbonitrile, interaction
1. INTRODUCTION
Fluorescence quenching is the decrease of the quantum yield of fluorescence from a
fluorophore induced by a variety of molecular interactions with quencher molecule.
Fluorescence quenching can be dynamic, resulting from collisional encounters between the
fluorophore and quencher, or static, resulting from the formation of a ground-state
complex between the fluorophore and quencher [1]. In both cases, molecular
contact is required between the fluorophore and the quencher for fluorescence quenching to
occur [2]. Application of the fluorescence quenching technique can also reveal
the accessibility of the fluorophores to quenchers.
The combination of drug and protein is an importantly pharmic and
kinetic character. Studying the fluorescence quenching of pharmaceutical molecular to
proteins, we can get many relative information about proteins molecular, so the study is
widely much accounted of.
There have been no studies on fluorescence quenching of snake venom
induced by drugs or other small molecules. In this paper, we synthesized
6-amino-4-aryl-3-methyl-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5- carbonitrile
(Fig. 1) and their interactions with the two kinds of snake venom were subsequently
studied. And the interactions of I with cobra venom and viper venom were researched
respectively in detail. The quenching constants KSV, binding constants K
and binding sites n based on the fluorescence quenching were calculated.
Fig. 1 Molecular structure
R = H, 2-OH, 4-NO2, 4-CN, 4-Br, 4-Cl, 4-CH3, 2-Cl, 4-OCH3,
3-NO2, 4-N(CH3)2
2. EXPERIMENTAL
2.1 Reagent and tnstrumentation
Cobra venom and viper venom were purchased from the of in Wuyi Mountain Snake Research
Institute (Fujian, China).
6-amino-4-aryl-3-methyl-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile were
synthesized from aromatic aldehyde, malononitrile and 1-phenyl-3-methyl-1H-pyrazol-5-amine
(recrystallized 2 times with 95% ethanol, normalized purity (HPLC) 99.50 - 99.86%). 0.9%
(w/v) NaCl aqueous solution was used to keep biologically ionic strength. The Tris
(hydroxymethylaminomethane) buffer (> 99.5%) and other agents were of analytical
purity. Redistilled water was used throughout.
An F-4500 spectrophotometer (Hitachi, Japan) was used and the cell
dimension is 1×1×4 cm3, the slit width was 3 nm. The pH values were measured
with a Aiwang (Shanghai, China) pH meter.
2.2 Experimental process
The following reagents were added to a 10 mL tube in the order indicated: 1.0 mL of venom
(1.0 mg/mL), adequate I (100 ug/mL) and 2.0 mL of Tris-HCl buffer solution (0.05 mol/L, pH
= 7.6). After adding 0.9% NaCl aqueous solution to the tube, the absorption spectra and
fluorescence spectra were measured at room temperature. Fluorescence measurements were
taken at excitation and emission wavelengths of 282 and 300~500 nm, respectively, with a
resolution of 3 nm.
3. RESULTS AND DISCUSSION
3.1 Fluorescence spectra
Fig. 2 were fluorescence spectra of which cobra venom and viper venom respectively mixed
(v: v = 1: 1).
a
b
Fig. 2 The emission spectrum of cobra venom (a)
and viper venom (b) at room temperature and 282 nm.
1. cobra venom or viper venom (0.1 mg/mL)
2. I (10 ug/mL)
3. cobra venom-I or viper venom-I (0.1 mg/mL) + I (10 ug/mL)
From Fig. 2, we can
observe an obvious decrease in the relative fluorescence intensity (RFI) of cobra
venom and viper venom, but the intensity of I was not foundamental changed. That
was to say there existed mutual action and stabilizing complex which is on ground state
and no fluorescence was formed. The fluorescence of two kinds of venom may be due to the
tryptophane in molecular chain [3]. So the content of tryptophane in corbric
venom may be more than that one in viper venom according to Fig. 2.
3.2 The binding properties and quenching mechanism
The fluorescence quenching of cobra venom and viper venom with varying concentrations
of I is shown in Fig. 3.
a
b
Fig. 3 a. Fluorescence spectra
of 0.1 mg/mL cobra venom at l ex = 282 nm showing the quenching effect of
increasing concentrations of I (0, 3, 5, 7, 10, 12, 15, 18, 20 mg/mL). Spectra were
recorded at pH 7.6.
b. Fluorescence spectra of 0.1 mg/mL viper venom at l ex = 282 nm
showing the quenching effect of increasing concentrations of I (0, 3, 5, 7, 10, 12,
15, 18 mg/mL). Spectra were recorded at pH 7.6.
Fluorescence quenching is
described by the well-known Stern–Volmer equation:
F0/F=1+KSV[Q]
where F0 and F denote the steady-state fluorescence
intensities in the absence and in the presence of quencher I, respectively, KSV
is the Stern–Volmer quenching constant, and [Q]
is the concentration of the quencher. Hence, the equation was applied to determine KSV
by linear regression of a plot of F0/F against [Q].
The fluorescence of two
kinds of venom were quenched by I. Respectively, the quenching Stern-Volmer figures
of cobra venom and viper venom with varying concentrations of I are shown in
Fig. 4. It showed that both of curves have linear relation (r= 0.992, 0.995). The
constants KSV can be caculated (Table 1).
Fig. 4 The Stern-Volmer plot of cobra venom and viper venom quenching spectrum
1. cobra venom-I 2. viper venom-I
Table 1 quenching
constants KSV、binding constants
K and binding sites n
|
10-4KSV
/L.mol-1 |
10-4K
/L.mol-1 |
n |
cobra venom-I |
1.73 |
0.89 |
0.38 |
viper venom-I |
2.32 |
1.05 |
0.44 |
To dynamic
quenching style, Ksv= Kd= Kqt 0. To biomacromolecule, t 0=
108 s, qsv,max= 2.0×1010 L× mol- 1 × s-1
[5]. If the quenching phenomenon of venom and I were dynamic, rate constant kq
should far more than 2.0×1010 L× mol-1× s-1. The
result in the paper is opposite, so the quenching reason was that two styles formed new
compounds and they were static quenching proceedings. When considering the effect of I on
the fluorescence spectra of two kinds of venom, there was no apparent l em
shift. This suggests no other change in the immediate environment of the fluorophores
unless I is close to the fluorophores to occur the quenching effect. This means that the
molecular conformation of the two kinds of venom is affected.
So the quenching reason was that a new steady complex which has no
fluorescence was formed and it was a static quenching proceeding with the equation:
Log (F0-F)/F = Log K + n Log [Q]
The binding constants K and binding sites n could be calculated from
Fig. 4 (r = 0.991, 0.992), and the calculated results (Table 1) indicated that it had
strong binding between I and two kinds of snake venom.
3.3 Effect of substituted group of different aromatic aldehyde
Other 11 aromatic aldehydes (R=-H, 4-NO2,
4-CN, 4-Br, 4-Cl, 2-OH, 4-CH3, 2-Cl, 4-OCH3, 3-NO2,
4-N(CH3)2) were also researched. The result indicated that only I
(R=2-OH) can do so. We supposed that this activity was mainly come from –OH which can bind with two kinds of venom through hydrogen bond.
4. SUMMARY
It was demonstrated the two proceedings are static. According to the maximum
excitation and the maximum emission, we can infer that tryptophan may be the component of
cobra venom and viper venom from Fujian Province and the content of tryptophan in viper
venom is less than in cobra venom.
I can interact with cobra venom
and viper venom. Whether I can reduce the toxicity of snake that needs to be
further investigated in future work.
ACKNOWLEDGEMENTS We are
grateful to the foundation of the "Natural Science Research Project of University in
Jiangsu Province" (No. JH03-038) for financial
support.
REFERENCES
[1] Lakowicz, J R. Principles of Fluorescence Spectros copy, 2nded. Kluwer Academic/Plenum
Publishers: New York, 1999, 698.
[2] Athina P, Rebecca J G, Richa R. Journal Agricultrual Food Chemistry, 2005, 53: 158
[3] Chen G Z, Huang X Z. Fluorescence Analytical Method, Beijing: Science Press, 1990,
115.
[4] Feng X Z, Lin Z, Yang L J et al. Talant, 1998, 47: 1223.
[5] Lakowicz J R, Weber G. Biochemistry, 1973, 12: 4161.
6-氨基-4-芳基-5-氰基-3-甲基-1-苯基吡啶并[2,3-c]吡唑与两种蛇毒的相互作用:有关荧光猝灭的研究
廉淑芹,吴云明,印晓星
(徐州医学院药学院,徐州,江苏 221004)
摘要 运用荧光猝灭的原理在pH7.6的生理条件下,眼镜蛇毒与蝮蛇蛇毒与系列6-氨基-4-芳基-5-氰基-3-甲基-1-苯基吡啶并[2,3-c]吡唑化合物之间的相互作用被研究,并且筛选出6-氨基-4-对羟基苯基-5-氰基-3-甲基-1-苯基吡啶并[2,3-c]吡唑(I)进行详细研究。猝灭常数KSV、结合常数K以及结合位点n被获得。该实验证明眼镜蛇毒-I或蝮蛇蛇毒-I之间的相互作用均是由于新化合物的生成,而这一过程是静态猝灭的过程,并且它们之间均存在很强的结合。
关键词 眼镜蛇毒;蝮蛇蛇毒;6-氨基-4-对羟基苯基-5-氰基-3-甲基-1-苯基吡啶并[2,3-c]吡唑;相互作用
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