荧光光谱法结合分子对接研究人血清白蛋白对齐墩果酸与熊果酸的异构体识别作用
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摘要
五环三萜化合物齐墩果酸(OA)与熊果酸(UA)为同分异构体,具有相似的理化性质和稍有差异的药理活性。目前人们主要采用各种色谱、质谱类方法实现对OA与UA的异构体识别,未见使用荧光光谱法的报道。提出了一种使用荧光猝灭法实现对OA与UA异构体识别的方法。首先考察了两种常见的血清蛋白—牛血清白蛋白(BSA)与人血清白蛋白(HSA)同OA与UA的作用情况,结果表明OA与UA均可有效地猝灭BSA与HSA的荧光发射。对所得荧光猝灭数据计算可知OA, UA与BSA, HSA作用的双分子猝灭速率常数(K_q)均远大于生物大分子荧光猝灭所观察到的最大散射碰撞速率常数2.0×10~(10) L·(mol·s)~(-1),说明猝灭类型均为静态猝灭,即OA与UA均是通过与BSA及HSA形成稳定复合物方式实现荧光猝灭的。应用双对数方程对所得荧光猝灭数据计算可知OA, UA与BSA, HSA所形成的复合物中结合位点数在0.90～1.26之间,说明所形成的复合物为1∶1型。BSA与OA, UA所形成复合物的表观结合常数(K_A)为同一数量级,相差不大,但是HSA与OA, UA所形成复合物的K_A差别很大, HSA-UA复合物的KA比HSA-OA复合物高124.91倍,表明HSA-UA复合物的稳定性更强。同步荧光实验结果显示, OA与UA的加入对于HSA波长差(Δλ)为60 nm同步荧光光谱的影响大于Δλ为15 nm的同步荧光光谱,由此可以说明OA与UA在HSA上的结合位点可能位于Trp残基附近。分子对接模拟计算结果表明OA与UA均对接在HSA结构中一个疏水性空腔中,主客体之间存在强烈的氢键与疏水作用。OA同Arg218, His242, Pro447等残基间存在氢键作用,键长分别为2.95, 2.97与3.17?,此外还与Lys195, Lys199, Trp214, Arg222, Leu238, Asp451和Tyr452等七个氨基酸残基间存在疏水作用。UA同Trp214, Arg218和Lys444等残基存在氢键作用,键长分别为3.01, 2.88与2.65?,此外还与Leu198, Gln221, Arg222, Asn295, Val343, Pro447, Cys448, Asp451和Val455等9个氨基酸残基间存在疏水作用。由于UA同HSA作用位点数目多于OA,说明UA与HSA疏水性空腔的空间匹配程度更高。因此,认为HSA-UA与HSA-OA复合物间稳定性差异是HSA实现对OA与UA异构体识别的原因。
Oleanolic acid(OA) and ursolic acid(UA) are isomeric pentacyclic triterpenoid compounds, which share the similar physical and chemical properties but a little bit different pharmacological activity. At present, various chromatography and mass spectrometry methods have been used to discriminate OA and UA isomerically, but there seems no fluorescence spectroscopy-based method reported. This paper presents a method for the identification of OA and UA isomers using fluorescence quenching. Firstly, two common serum albumins, bovine serum albumin(BSA) and human serum albumin(HSA), interacting with OA and UA are examined, respectively. Experimental result shows that OA and UA can effectively quench fluorescence emission of BSA and HSA. The result of obtained fluorescence quenching data shows the bi-molecular quenching constants of BSA(or HSA) and OA(or UA) are much higher than the maximum scatter collision-quenching rate constant generally observed for any quencher against biopolymer(2.0×10~(10) L·(mol·s)~(-1)), indicating the type of quenching is static quenching, namely, OA and UA can form stable complexes with BSA and HSA. Double logarithm equation was also applied to the fluorescence quenching data and binding site numbers of the complexes formed by OA or UA and BSA or HSA are obtained between 0.90 and 1.26, indicating all complexes are 1∶1 type. The apparent binding constants(KA) of BSA-OA and BSA-UA complexes are at the same order of magnitude, but those of HSA-OA and HSA-UA are very different. The KA of HSA-UA is 124.91 times higher than that of HSA-OA, indicating HSA-UA has much higher stability. Synchronous fluorescence experimental result shows that the spectrum at 60 nm wavelength difference(Δλ) can be quenched more effectively than that of 15 nm Δλ alongwith the addition of OA and UA, showing the binding sites of OA and UA at HSA may beclose toTrp residues. The results of molecular docking simulation show that both OA and UA are docked at a hydrophobic cavity in HSA, and there is strong hydrogen bonding and hydrophobic effect between the host and the guest. In addition, three hydrogen bonding interaction are found between OA and Arg218, His242 and Pro447 residues of HSA with bond lengths of 2.95, 2.97 and 3.17 ?, respectively. There are seven amino acid residues of HSA that has hydrophobic effect with OA including Lys195, Lys199, Trp214, Arg222, Leu238, Asp451 and Tyr452. Whereas UA forms three hydrogen bonds with Trp214, Arg218 and Lys444 residues of HSA with bond length of 3.01, 2.88 and 2.65 ?, respectively. Nine amino acid residues including Leu198, Gln221, Arg222, Asn295, Val343, Pro447, Cys448, Asp451 and Val455 have hydrophobic effect with UA. Since UA has stronger hydrogen bonding and more hydrophobic effect with HSA, it is more spatially fitted to the HSA's hydrophobic cavity. Thus, according to the fluorescence spectral data and molecular docking results, the stability difference of HSA-OA and HSA-UA may be responsible for discrimination of OA and UA isomers.
Oleanolic acid(OA) and ursolic acid(UA) are isomeric pentacyclic triterpenoid compounds, which share the similar physical and chemical properties but a little bit different pharmacological activity. At present, various chromatography and mass spectrometry methods have been used to discriminate OA and UA isomerically, but there seems no fluorescence spectroscopy-based method reported. This paper presents a method for the identification of OA and UA isomers using fluorescence quenching. Firstly, two common serum albumins, bovine serum albumin(BSA) and human serum albumin(HSA), interacting with OA and UA are examined, respectively. Experimental result shows that OA and UA can effectively quench fluorescence emission of BSA and HSA. The result of obtained fluorescence quenching data shows the bi-molecular quenching constants of BSA(or HSA) and OA(or UA) are much higher than the maximum scatter collision-quenching rate constant generally observed for any quencher against biopolymer(2.0×10~(10) L·(mol·s)~(-1)), indicating the type of quenching is static quenching, namely, OA and UA can form stable complexes with BSA and HSA. Double logarithm equation was also applied to the fluorescence quenching data and binding site numbers of the complexes formed by OA or UA and BSA or HSA are obtained between 0.90 and 1.26, indicating all complexes are 1∶1 type. The apparent binding constants(KA) of BSA-OA and BSA-UA complexes are at the same order of magnitude, but those of HSA-OA and HSA-UA are very different. The KA of HSA-UA is 124.91 times higher than that of HSA-OA, indicating HSA-UA has much higher stability. Synchronous fluorescence experimental result shows that the spectrum at 60 nm wavelength difference(Δλ) can be quenched more effectively than that of 15 nm Δλ alongwith the addition of OA and UA, showing the binding sites of OA and UA at HSA may beclose toTrp residues. The results of molecular docking simulation show that both OA and UA are docked at a hydrophobic cavity in HSA, and there is strong hydrogen bonding and hydrophobic effect between the host and the guest. In addition, three hydrogen bonding interaction are found between OA and Arg218, His242 and Pro447 residues of HSA with bond lengths of 2.95, 2.97 and 3.17 ?, respectively. There are seven amino acid residues of HSA that has hydrophobic effect with OA including Lys195, Lys199, Trp214, Arg222, Leu238, Asp451 and Tyr452. Whereas UA forms three hydrogen bonds with Trp214, Arg218 and Lys444 residues of HSA with bond length of 3.01, 2.88 and 2.65 ?, respectively. Nine amino acid residues including Leu198, Gln221, Arg222, Asn295, Val343, Pro447, Cys448, Asp451 and Val455 have hydrophobic effect with UA. Since UA has stronger hydrogen bonding and more hydrophobic effect with HSA, it is more spatially fitted to the HSA's hydrophobic cavity. Thus, according to the fluorescence spectral data and molecular docking results, the stability difference of HSA-OA and HSA-UA may be responsible for discrimination of OA and UA isomers.
引文
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[2] Kong L,Li S,Liao Q,et al.Antiviral Research,2013,98(1):44.
[3] Fontanay S,Grare M,Mayer J,et al.Journal of Ethnopharmacology,2008,120(2):272.
[4] Liu H,Shi Y,Wang D,et al.Journal of Pharmaceutical & Biomedical Analysis,2003,32(3):479.
[5] Gao R,Wang L,Yang Y,et al.Biomedical Chromatography,2015,29(3):402.
[6] Janicsák G,Veres K,Kállai M,et al.Chromatographia,2003,58(5-6):295.
[7] Kamatou G P P,Viljoen A M,Sleenkamp P.Food Chemistry,2010,119(2):684.
[8] Xu X H,Su Q,Zang Z H.Journal of Pharmaceutical Analysis,2012,2(3):238.
[9] Chen Q,Zhang Y,Zhang W,et al.Biomedical Chromatography:BMC,2011,25(12):1381.
[10] Huang L,Chen T,Ye Z,et al.Journal of Mass Spectrometry,2007,42(7):910.
[11] Caligiani A,Malavasi G,Palla G,et al.Food Chemistry,2013,136(2):735.
[12] Yu Z,Cui M,Yan C,et al.Rapid Communications in Mass Spectrometry,2007,21(5):683.
[13] Hanwell M D,Curtis D E,Lonie D C,et al.Journal of Cheminformatics,2012,4(1):17.
[14] Morris G M,Huey R,Lindstrom W,et al.Journal of Computational Chemistry,2009,30(16):2785.
[15] WU Yu-hang,HAN Zhong-bao,MA Jia-ze,et al(吴雨杭,韩忠保,马嘉泽,等).Spectroscopy and Spectral Analysis(光谱学与光谱分析),2016,36(3):765.
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[17] Kandagal P B,Shaikh S M T,Manjunatha D H,et al.Journal of Photochemistry & Photobiology A Chemistry,2007,189(1):121.
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