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

doi:10.3964/j.issn.1000-0593(2019)07-2190-06

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Abstract 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 (2.0×10¹⁰ 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 Å, 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 Å, 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.

Key words：Human serum albumin; Oleanolic acid; Ursolic acid; Isomeric discrimination; Molecular docking

Received: 2018-04-19 Accepted: 2018-09-11

ZTFLH:

O657.3

Corresponding Authors: YU Zhan E-mail: yuzhan@synu.edu.cn

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https://www.gpxygpfx.com/EN/10.3964/j.issn.1000-0593(2019)07-2190-06 OR https://www.gpxygpfx.com/EN/Y2019/V39/I07/2190