美洛昔康与溶菌酶作用机制的光谱分析及理论模建研究 -

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美洛昔康与溶菌酶作用机制的光谱分析及理论模建研究

doi:10.3788/CO.20201302.0354

程旭^{1, 2, 3,},
刘保生^{1, 2, 3,,},
张红彩^{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21375032

详细信息

作者简介:
程旭(1993—), 男, 山东潍坊人, 硕士研究生, 2017年于济宁学院获得学士学位, 主要从事分子发光学理论与应用研究。E-mail:2547034720@qq.com
刘保生(1963—), 男, 河北省保定人, 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学位、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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- 被引次数:0
出版历程
- 收稿日期:2019-03-29
- 修回日期:2019-04-30
- 刊出日期:2020-04-01

Spectral analysis and theoretical modeling of the working mechanism of meloxicam and lysozyme

CHENG Xu^{1, 2, 3,},
LIU Bao-sheng^{1, 2, 3,,},
ZHANG Hong-cai^{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, Baoding 071002, China

Funds:

National Natural Science Foundation of China21375032

More Information

Corresponding author:LIU Bao-sheng, E-mail:lbs@hbu.edu.cn

摘要

摘要:为了探究美洛昔康与溶菌酶的作用机制，在pH=7.40的实验条件下，采用荧光光谱、同步荧光光谱和理论模建分析技术研究了类风湿性关节炎药物美洛昔康与溶菌酶分子之间的相互作用。结果表明，美洛昔康能够以静态猝灭形式有效地猝灭溶菌酶的内源荧光，形成1：1的复合物，并使溶菌酶的构象发生改变。热力学结果表明，美洛昔康-溶菌酶体系的主要作用力类型为疏水作用力。理论模建结果表明，该体系除疏水作用外还存在氢键作用，且美洛昔康被溶菌酶的活性氨基酸残基Glu35和Asp52包围，结合作用改变了溶菌酶催化活性中心处氨基酸残基的微环境。当患者服用15 mg美洛昔康时，美洛昔康与溶菌酶的蛋白结合率 W（ B）为3.71%~8.79%，说明美洛昔康与溶菌酶的结合对溶菌酶自身抗炎、抗菌功能的影响不大，体系药物结合率 W（ Q）为1.08%~1.14%，说明溶菌酶与美洛昔康结合不会影响美洛昔康的药效。该研究从理论上证明了溶菌酶在血浆环境中与药物美洛昔康结合后，对溶菌酶本身功能和美洛昔康的药效不会产生严重影响。
- 美洛昔康/
- 溶菌酶/
- 荧光分析/
- 理论模建/
- 结合率
Abstract:In order to explore the active mechanisms of meloxicam and lysozyme, the interaction between the molecules of rheumatoid arthritis drugs, meloxicam and lysozyme was studied using fluorescence spectroscopy, synchronous fluorescence spectroscopy and theoretical modeling analysis under the pH=7.40 experimental conditions. The results showed that meloxicam was able to effectively quench the endogenous fluorescence of lysozyme, forming a 1:1 complex and changing the conformation of lysozyme. Thermodynamic results indicated that the main type of meloxicam-lysozyme system was a hydrophobic interaction. The results of theoretical modeling indicated that the system had hydrogen bonds in addition to hydrophobic interactions and that meloxicam was surrounded by the active amino acid residues Glu35 and Asp52 from the lysozyme, which changed the microenvironment of amino acid residues at the active center of the lysozyme. When a patient took meloxicam 15 mg, the protein binding rate of meloxicam to lysozyme W( B) was 3.71%~8.79%, indicating that the combination of meloxicam and lysozyme has little effect on anti-inflammatory and antibacterial function of lysozyme itself, and the system drug binding rate W( Q) was 1.08%~1.14%, indicating that the combination of lysozyme and meloxicam does not affect the efficacy of meloxicam. This study theoretically proved that lysozyme does not have a serious effect on its own function or the efficacy of meloxicam after it is combined with meloxicam in plasma.
- meloxicam/
- lysozyme/
- fluorescence analysis/
- theoretical modeling/
- binding rate

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图 1MEL-LYSO的荧光发射光谱(T=310 K,λ_ex=280 nm)

Figure 1.Fluorescence emission spectra of MEL-LYSO system (T=310 K,λ_ex=280 nm).C_LYSO=5.0×10^-7mol/L, 1~9C_MEL=(0, 0.05, 0.2, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0)×10^-5mol/L

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图 2MEL与LYSO作用的相对荧光曲线(T=310 K)

Figure 2.Relative fluorescence curves of the interaction between MEL and LYSO(T=310 K). Concentration of LYSOC_LYSO=5.0×10^-7mol/L, Concentration of MELC_MEL=(0.05, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0)×10^-5mol/L

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图 3MEL-LYSO体系的荧光强度随NaCl浓度的变化(T=310 K)

Figure 3.Fluorescence intensity of MEL-LYSO system as a function of NaCl concentration (T=298 K).C_LYSO=5.0×10^-7mol/L,C_MEL=1.0×10^-5mol/L,C_Nacl=(0, 0.1, 0.2, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0) ×10^-1mol/L

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图 4MEL-LYSO体系的同步荧光光谱(T=310 K) (a) Δλ=60 nm; (b) Δλ=15 nm

Figure 4.Synchronous fluorescence spectra of MEL-LYSO system (T=310 K). (a) Δλ=60 nm; (b) Δλ=15 nm.C_LYSO=5.0×10^-7mol/L, 1~9C_MEL=(0, 0.05, 0.2, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0)×10^-5mol/L

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图 5MEL与LYSO相互作用的理论模建图。(a) MEL与LYSO发生作用区域及MEL与LYSO的氨基酸残基之间的氢键; (b) MEL与LYSO结合部位的氨基酸残基分布情况

Figure 5.Theoretical modeling of the interaction between MEL and LYSO. (a) Regions where MEL interacts with LYSO and hydrogen bonds between docked MEL and amino acids residues of LYSO; (b) Distribution of amino and residues at the bonding site of MEL and LYSO

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表 1不同温度下MEL-LYSO体系的猝灭反应参数

Table 1.Quenching reactive parameters of MEL-LYSO at different temperatures

λ_ex/(nm)	T/(K)	K_q/(L·mol^-1·s^-1)	K_sv/(L·mol^-1)	r₁	K_a/(L·mol^-1)	n	r₂
280	293	2.08×10¹²	2.08×10⁴	0.993 2	2.34×10⁴	1.08	0.992 2
	308	1.59×10¹²	1.59×10⁴	0.993 9	1.71×10⁴	1.06	0.996 9
	318	1.28×10¹²	1.28×10⁴	0.995 7	1.31×10⁴	1.22	0.995 6
295	293	1.53×10¹²	1.53×10⁴	0.993 8	1.62×10⁴	1.17	0.994 2
	308	1.18×10¹²	1.18×10⁴	0.996 5	1.23×10⁴	1.12	0.996 9
	318	0.89×10¹²	0.89×10⁴	0.997 4	0.93×10⁴	1.03	0.991 5
注：r₁为方程F₀/F~[L]的线性相关系数;r₂为方程lg[(F₀-F)/F]~lg{[L]-n[B_t](F₀-F)/F₀}的线性相关系数；[B_t]=5.0×10^-7mol/L

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表 2不同温度下MEL-LYSO体系的热力学参数(λ_ex=280 nm)

Table 2.The thermodynamic parameters of MEL-LYSO at different temperatures(λ_ex=280 nm)

T/K	K_a/L·mol^-1	ΔH/kJ·mol^-1	ΔS/J·mol^-1·K^-1	ΔG/kJ·mol^-1
298	2.34×10⁴		7.82	-24.93
310	1.71×10⁴	-22.59	8.15	-25.12
318	1.31×10⁴		7.76	-25.06

下载: 导出CSV

表 3MEL-LYSO体系的对接能量(单位:kJ/mol)

Table 3.Docking energy of MEL-LYSO system (unit: kJ/mol)

Protein PDB ID	ΔG₀	ΔE₁	ΔE₂	ΔE₃
2LYZ	-27.81	-31.53	-31.11	-0.42

下载: 导出CSV

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