同步荧光光谱探究头孢西丁钠与溶菌酶的结合机制

张红彩; 刘保生; 程旭

doi:10.3788/CO.2019-0112

同步荧光光谱探究头孢西丁钠与溶菌酶的结合机制

doi: 10.3788/CO.2019-0112

张红彩^{1, 2, 3,},
刘保生^{1, 2, 3, ,},
程旭^{1, 2, 3}

1.
河北大学化学与环境科学学院，河北保定 071002
2.
河北省分析科学技术重点实验室，河北保定 071002
3.
国家级化学实验教学示范中心，河北保定 071002

基金项目: 国家自然科学基金资助项目（No.21375032）

详细信息

作者简介:
张红彩(1993—)，女，河北石家庄人，硕士研究生，2017年于保定学院获得学士学位，主要从事分子发光学理论与应用研究。E-mail：1179497976@qq.com

刘保生(1963—)，男，河北保定人，硕士，研究员，1986年、1992年于河北大学分别获得分析化学专业学士、硕士学位，主要从事分子发光学理论与应用研究。E-mail：lbs@hbu.edu.cn

通讯作者:
刘保生(1963—)，男，河北保定人，硕士，研究员，1986年、1992年于河北大学分别获得分析化学专业学士、硕士学位，主要从事分子发光学理论与应用研究。E-mail：lbs@hbu.edu.cn

中图分类号: O657.3
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出版历程
- 收稿日期: 2019-07-23
- 修回日期: 2019-08-20
- 刊出日期: 2020-06-01

Study on the binding mechanism of cefoxitin sodium to lysozyme by synchronous fluorescence spectroscopy

ZHANG Hong-cai^{1, 2, 3
,},
LIU Bao-sheng^{1, 2, 3
, ,},
CHENG Xu^{1, 2, 3}

1.
College of Chemistry & Environmental Science, Hebei University, Baoding 071002, China
2.
Key Laboratory of Analytical Science and Technology of Hebei Province, Baoding 071002, China
3.
National Chemistry Experimental Teaching Demonstration Center, Hebei University, Baoding 071002, China

Funds: Supported by National Natural Science Foundation of China (No. 21375032)

More Information

Corresponding author: lbs@hbu.edu.cn

摘要

摘要: 在模拟生理学条件下(pH=7.40)，采用同步荧光法研究了头孢西丁钠(CFXS)和溶菌酶(LYSO)中的荧光基团酪氨酸(Tyr)残基、色氨酸(Trp)残基之间的相互作用。结果表明：CFXS以静态猝灭的方式猝灭LYSO中的Tyr、Trp残基的荧光，结合位点数n ≈1。310 K时，Tyr与Trp残基反应的荧光猝灭比率分数N_SFQR(Trp)(60.25%)>N_SFQR(Tyr)(39.75%)，结合位置更靠近Trp残基。Hill系数n_H约为1，表明CFXS与LYSO中Tyr与Trp残基的结合不会影响后继配体与蛋白质的结合。CFXS与LYSO中Tyr残基的药物结合率W(Q)为0.19%~0.13%，Trp残基的药物结合率W(Q)为0.23%~0.14%，游离的药物含量几乎不变，这表明CFXS与LYSO中Tyr与Trp残基的结合基本不影响药物的疗效。Tyr残基的蛋白结合率W(B)为52.69%~54.67%，Trp残基的蛋白结合率W(B)为67.67%~69.39%，因此，蛋白中游离的氨基酸残基数目会明显降低。CFXS-LYSO结合体系的主要作用力类型是疏水作用，分子对接结果表明CFXS与LYSO之间还存在氢键作用，且两者的最佳结合位置在LYSO的活性中心附近，两者的结合改变了活性中心处氨基酸残基的微环境。
- 同步荧光法 /
- 头孢西丁钠 /
- 溶菌酶 /
- 荧光基团 /
- 分子对接
Abstract: Under simulated physiological conditions (pH=7.40), the interaction between tyrosine (Tyr) residue and tryptophan (Trp) residue in lysozyme (LYSO) and cefoxitin sodium (CFXS) was studied using synchronous fluorescence spectroscopy. The results showed that CFXS quenched the fluorescence of Tyr and Trp residue in LYSO by static quenching, and that the number of binding sites n was nearly 1. At 310 K, the fluorescence quenching ratio of CFXS with Trp residue N_SFQR(Trp)(60.25%) was higher than that of N_SFQR(Tyr)(39.75%), indicating that the binding position was closer to the Trp residue. The Hill coefficient n_H was about 1, indicating that the binding of CFXS to the Tyr and Trp residues in LYSO did not affect the binding of subsequent ligands to proteins. The drug binding rate of CFXS to Tyr residue in LYSO was 0.19% to 0.13%, and the drug′s binding rate to Trp residue was 0.23% to 0.14%, respectively. The content of the free drug was almost unchanged. The results showed that the combination of Tyr and Trp residue in LYSO and CFXS did not affect the efficacy of the drug. The protein binding rate of Tyr residue was 52.69% to 54.67%, and the protein binding rate of Trp residue was 67.67% to 69.39%, implying the amount of free amino acid residue in the protein decreased significantly. The main force of the CFXS-LYSO binding system was a hydrophobic interaction. The results of molecular docking showed that there was still a hydrogen bond between the CFXS and LYSO, and the best binding position was near to the active center of the LYSO. The combination of the two substances changed the microenvironment for the amino acid residue at the active center.
- synchronous fluorescence /
- cefoxitin sodium /
- lysozyme /
- fluorescent group /
- molecular docking

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图 1 CFXS-LYSO体系的同步荧光光谱 (T=310 K) (a) Δλ=15 nm；(b) Δλ=60 nm

Figure 1. Synchronous fluorescence spectra of CFXS-LYSO system (T=310 K) (a) Δλ=15 nm; (b) Δλ=60 nm

下载: 全尺寸图片幻灯片

图 2 CFXS-LYSO体系荧光强度随NaCl浓度的变化 (T=310 K)

Figure 2. Fluorescence intensity of CFXS-LYSO system as a function of NaCl concentration C_Nacl (T=310 K)

下载: 全尺寸图片幻灯片

图 3 CFXS与LYSO相互作用的分子对接图

Figure 3. Molecular docking model of the interaction between CFXS and LYSO

下载: 全尺寸图片幻灯片

表 1 CFXS-LYSO体系的同步荧光猝灭反应参数

Table 1. Reactive parameters of synchronous fluorescence quenching for CFXS-LYSO system

Δλ/nm	T/K	K_sv/L·mol⁻¹·s⁻¹	k_q/L·mol⁻¹	r₁	K_a/L·mol⁻¹	n	r₂
Δλ=60	298	2.18×10⁴	2.18×10¹²	0.990 5	1.73×10⁴	1.18	0.992 2
	310	1.61×10⁴	1.61×10¹²	0.992 4	1.58×10⁴	1.03	0.991 3
	318	1.26×10⁴	1.26×10¹²	0.994 1	1.39×10⁴	0.96	0.993 5
Δλ=15	298	1.33×10⁴	1.33×10¹²	0.993 8	0.96×10⁴	1.07	0.995 7
	310	1.17×10⁴	1.17×10¹²	0.991 4	0.84×10⁴	1.17	0.992 2
	318	0.83×10⁴	0.83×10¹²	0.995 2	0.68×10⁴	1.12	0.993 4
r₁为方程I₀ /I~[L]的线性相关系数；r₂为方程lg[(I₀-I)/I]~lg{[L]-n[B_t](I₀-I)/I₀}的线性相关系数；[B_t]=5.0×10⁻⁷ mol/L。

下载: 导出CSV

表 2 不同温度下CFXS-LYSO体系的热力学参数

Table 2. Thermodynamic parameters of CFXS-LYSO system at different temperatures

System	T/K	K_a/L·mol⁻¹	ΔH/kJ·mol⁻¹	ΔS/J·mol⁻¹ K⁻¹	ΔG/kJ·mol⁻¹
Δλ=15 nm	298	0.96×10⁴	−13.15	32.10	−22.71
	310	0.84×10⁴		32.70	−23.28
	318	0.68×10⁴		32.01	−23.33
Δλ=60 nm	298	1.73×10⁴	−11.68	41.93	−24.17
	310	1.58×10⁴		42.70	−24.91
	318	1.39×10⁴		42.58	−25.22

下载: 导出CSV

表 3 不同温度下CFXS和LYSO中Tyr、Trp残基的Hill系数

Table 3. Hill coefficients of Tyr and Trp residues in CFXS and LYSO at different temperatures

T/K	Δλ=60 nm		Δλ=15 nm
T/K	n_H	r₃	n_H	r₃
298	0.94	0.993 7	0.99	0.997 1
310	1.15	0.994 8	1.07	0.993 3
318	0.92	0.996 7	0.94	0.995 2
n_H为体系的Hill系数；r₃为方程lg[Y/(1-Y)]~lg[L]的线性相关系数。

下载: 导出CSV

表 4 CFXS-LYSO体系的对接能量(单位：kJ/mol)

Table 4. Docking energy of CFXS-LYSO system (unit: kJ/mol)

Protein PDB ID	ΔG₀	ΔE₁	ΔE₂	ΔE₃
2LYZ	−24.81	−39.78	−36.44	−3.34

下载: 导出CSV

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同步荧光光谱探究头孢西丁钠与溶菌酶的结合机制

doi: 10.3788/CO.2019-0112

通讯作者:
刘保生(1963—)，男，河北保定人，硕士，研究员，1986年、1992年于河北大学分别获得分析化学专业学士、硕士学位，主要从事分子发光学理论与应用研究。E-mail：lbs@hbu.edu.cn

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Study on the binding mechanism of cefoxitin sodium to lysozyme by synchronous fluorescence spectroscopy

Corresponding author: lbs@hbu.edu.cn

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《中国光学（中英文）》跻身光学领域期刊Q3区

留言板

同步荧光光谱探究头孢西丁钠与溶菌酶的结合机制

doi: 10.3788/CO.2019-0112

通讯作者: 刘保生(1963—)，男，河北保定人，硕士，研究员，1986年、1992年于河北大学分别获得分析化学专业学士、硕士学位，主要从事分子发光学理论与应用研究。E-mail：lbs@hbu.edu.cn

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Study on the binding mechanism of cefoxitin sodium to lysozyme by synchronous fluorescence spectroscopy

Corresponding author: lbs@hbu.edu.cn

计量

出版历程

目录

《中国光学（中英文）》跻身光学领域期刊Q3区

通讯作者:
刘保生(1963—)，男，河北保定人，硕士，研究员，1986年、1992年于河北大学分别获得分析化学专业学士、硕士学位，主要从事分子发光学理论与应用研究。E-mail：lbs@hbu.edu.cn