| Qualitative and quantitative analysis about Cytochrome c and its noncovalent complexes by electrospary ionization mass spectrometry are reported in this paper. As ESI-MS has an ability of forming sample with multiple charges, so it has a unique advantage in the analysis of macromolecules. In addition, ESI-MS has features of speed, specific, sensitive which led it is widely used in many fields and disciplines. As a soft ionization method, ESI-MS has an irreplaceable position in the study of noncovalent complexes. It can not only maintain complexes'stability in relatively mild conditions, but also get molecular weight, stoichiometry and other specific information of noncovalent complexes.In ESI-MS, instrument parameters play a significant impact on the sample's signal intensity. The appropriate parameters must meet that the signal intensity of samples are strong enough and conditions should be selected as mild as possible, which helps maintain the natural configuration of protein and noncovalent complexes. The impact of various instrument parameters on the signal intensity is studied in the subject, and the optimal experimental conditions for Cytochrome c noncovalent complexes are explored. In addition, by changing the collision voltage, we verify the heme of cytochrome c bind to the amino acid residues in a covalent and stable form by using tandem mass spectrometry.Three different types of small molecules (saponins, oligosaccharides , triterpene) are selected to mix with Cytochrome c and the formations of noncovalent complexes whether or not are detected by ESI-MS in this paper. The results show that Cytochrome c can form noncovalent complexes with the saponins (Saikosaponin A, Saikosaponin C and Ginsenoside Re). As the structure of saponins is composed of oligosaccharides and triterpene, we choose three oligosaccharides (Raffinose, Stachyose, Maltotriose) and three triterpenes (Glycyrrhetinic acid, Ursolic acid, Oleanic acid) to mix with Cytochrome c. The mass spectrum results show that the three oligosaccharides can bind to Cytochrome c, while the three triterpenes can not. This study shows that in the process of forming noncovalent complexes with Cytochrome c, the effective binding part of saponin is the oligosaccharides part rather than the triterpene part. Oligosaccharide is selected as ligand to form noncovalent complexes with Cytochrome c is first reported at present. The unique advantages of ESI are proved by studying noncovalent complexes of Cytochrome c, and this subject gives a new horizon about the choice of ligand.In this subject, saponins and oligosaccharides could form noncovalent complexes with Cytochrome c are proved through qualitative research, and quantitative calculation of noncovalent complexes'stability is finished by specified direct calculation method. Using the speicified direct calculation method, we get dissociation constants Kd1 and Kd2 of these noncovalent complexes between Cytochrome c and saponins/oligosaccharides. And the corresponding stoichiometric ratios are 1:1, 1:2. Comparisons of dissociation constants in these noncovalent complexes can refelect the relative stability.When these ligands form noncovalent complexes with Cytochrome c, we study on their binding site and mode of action through direct experimental method. In this subject, we investigate the formation mechanism of Cytochrome c through experiments rather than theoretical or computer simulation. According to the calculated dissociation constants, the structure of ligands and the known literature, we give a detailed and clear explanation about the differences in stability for these noncovalent complexes with different ligands. After knowing the ligands bind to Cytochrome c in the formation of hydrogen bond, we find that the amounts of hydroxyl in ligands play a positive role in the stabiliy of noncovalent complexes no matter the bind formation is H-H or H-T. In addition, the structure and steric hindrance of ligands can not be neglected in forming process of noncovalent complexes.Unlike the theoretical calculation reported in the literature, we select Cytochrome c as model molecule and investigate it by ESI-MS, then we choose ligands to form noncovalent complexes with Cytochrome c. This subject analyzes the bondig manner of Cytochrome c noncovalent complexes and the factors which affect the noncovalent complexes'stability. This subject not only provides a new horizon for the selection of new ligand binding to cytochrome C, but also gives a further validate that the ESI-MS is an effective analysis means for protein noncovalent complexes. |
