采用电喷雾质谱法研究柴胡皂苷与细胞色素 c 的非共价相互作用。

Study on the noncovalent interactions of saikosaponins and cytochrome c by electrospray ionization mass spectrometry.

机构信息

Key Laboratory of Analytical Chemistry of the State Ethnic Affairs Commission, College of Chemistry and Materials Science, South-Central University for Nationalities, Wuhan, Hubei, 430074, PR China.

出版信息

Rapid Commun Mass Spectrom. 2012 Apr 15;26(7):719-27. doi: 10.1002/rcm.6151.

DOI:10.1002/rcm.6151

PMID:22368050

Abstract

RATIONALE

The study of interactions between protein and pharmaceutical molecules including natural extracts has become of increasing interest in biological and biomedical research. An investigation of the interaction between saikosaponins and cytochrome c (Cyt c) by electrospray ionization mass spectrometry (ESI-MS) is described in this study. Saikosaponins are found in Bupleurum falcatum (a flowering plant), and they are glycosides that consist of saccharides and the sapogenins of triterpenoids.

METHODS

Seven model molecules of saccharides and triterpenes, namely maltose (Mal II), maltotriose (Mal III), raffinose (Raf), and stachyose (Sta), glycyrrhetinic acid (Gly), ursolic acid (Urs) and oleanic acid (Ole), were chosen to perform a series of ESI-MS control experiments for the exploration of the interaction groups in saikosaponins with Cyt c. The dissociation constants of detected noncovalent complexes were determined by using a direct ESI-MS assay.

RESULTS

We have observed in the ESI mass spectra the formation of Cyt c complexes with saikosaponins a and c, and these saccharides, with 1:1 and 1:2 stoichiometry. Our results showed that no complex ions of triterpenes and Cyt c were detected in the ESI-MS and similar Kd values were obtained for the Cyt c complexes of saikosaponins and saccharides. This demonstrates that the glycosyl moiety in the saikosaponins is the effective interaction group with Cyt c. We propose that saikosaponins and saccharides interact with Cyt c by hydrogen bonds. The binding affinity of these six ligands with Cyt c is shown to be in the order Ssa > Ssc > Raf, Mal III > Sta ≥ Mal II.

CONCLUSIONS

The ESI-MS methodology presented in this study enables us to investigate the interactions of saikosaponins with Cyt c, and allows the direct determination of binding constants. These results could guide further research for providing insights into the structure-binding relationship of ligands with Cyt c.

摘要

研究蛋白质与包括天然提取物在内的药物分子之间的相互作用已经成为生物和生物医学研究中日益关注的课题。本研究通过电喷雾电离质谱（ESI-MS）研究了柴胡皂苷与细胞色素 c（Cyt c）之间的相互作用。柴胡皂苷存在于柴胡（一种开花植物）中，是由糖和三萜类皂甙元组成的糖苷。

方法

选择 7 种糖和三萜类模型分子，即麦芽糖（Mal II）、麦芽三糖（Mal III）、棉子糖（Raf）和水苏糖（Sta）、甘草次酸（Gly）、熊果酸（Urs）和齐墩果酸（Ole），进行一系列 ESI-MS 对照实验，以探索柴胡皂苷与 Cyt c 的相互作用基团。通过直接 ESI-MS 测定法测定检测到的非共价复合物的离解常数。

结果

我们在 ESI 质谱中观察到柴胡皂苷 a 和 c 与 Cyt c 形成复合物，这些糖以 1:1 和 1:2 的化学计量比形成复合物。我们的结果表明，在 ESI-MS 中未检测到三萜类化合物和 Cyt c 的复合物离子，并且获得了柴胡皂苷和糖与 Cyt c 的复合物的相似 Kd 值。这表明柴胡皂苷中的糖基部分是与 Cyt c 有效相互作用的基团。我们提出，柴胡皂苷和糖通过氢键与 Cyt c 相互作用。这 6 种配体与 Cyt c 的结合亲和力顺序为 Ssa > Ssc > Raf、Mal III > Sta ≥ Mal II。