Study On The Interaction Mechanism Between Several Protein And Phenylpropanoid Glycoside From Kudingcha

The protein is essential for life process and plays an important role in the movement and development of life. Serum albumin is the most abundant protein transporting a great number of endogenous and exogenous compounds in the body as carriers. Trypsin and lipase exist in the human pancreas, and the activity of trypsin was found very high in the body of patients with severe pancreatitis. Lipase is responsible for the metabolism of fat in the human diet. The interaction of the important transport protein and digestible protein with small molecule drugs may be helpful for the treatment of obesity and pancreatitis, and can help us better understand the biological process of the compound in its metabolism, and these experimental results could provide useful information on the compatibleness and the use of natural plant glycosides at molecule level and would be of biological significance in pharmacology and clinical medicine. In this thesis, the interactions of phenylpropanoid glycosides(Ligupurpuroside A and Ligupurpuroside B) with BSA, trypsin and lipase were studied using UV-visible spectrophotometry, fluorescence spectroscopy, circular dichroism spectroscopy and molecular modeling technique under imitated physiological conditions. Detailed studies on the quenching type, binding site, binding constant, binding force, protein conformational change were performed to explore the mechanism of interaction between them. The main contents are summarized as follows:The mechanism of the interactions of Ligupurpuroside A and Ligupurpuroside B with BSA respectively has been investigated by spectroscopic and docking methods. Experimental results show that the two small molecules can quench the intrinsic fluorescence of BSA through a combined quenching way, both the two drugs can bind to BSA to form ground state complexs, the BSA intrinsic fluorescence quenching ability of Ligupurpuroside A is greater than that of Ligupurpuroside B. The values of binding site(n) for the two small molecules binding to BSA are approximately equal to 1, and both can bind to BSA avidly, suggesting that Ligupurpuroside A and Ligupurpuroside B can be stored and transported to the target site in human body. Binding constants obtained further show that Ligupurpuroside A binds to BSA more strongly than Ligupurpuroside B does. Thermodynamic studies show that it is an enthalpy-driven and exothermic process for Ligupurpuroside A binding to BSA, and van der Waals interactions and hydrogen bonds are the main forces between them, while the interaction of Ligupurpuroside B with BSA is a weak endothermic process with an obvious increase of entropy, and dominated by hydrophobic interactions. Based on F?rster’s theory of non-radiation energy transfer, the distance of Ligupurpuroside A to BSA fluorophore is 2.73 nm, which is shorter than that for Ligupurpuroside B. From UV-visible spectra, synchronous fluorescence spectroscopy and circular dichroism, both Ligupurpuroside A and Ligupurpuroside B can induce the loosening and unfolding of BSA backbone, then decrease the hydrophobicity of the microenvironment of Trp, and increase the hydrophobicity of the microenvironment of Tyr. Comparing to Ligupurpuroside B, Ligupurpuroside A more deeply insert into the hydrophobic cavity of the BSA. Molecular simulation results show that both the two drugs can insert into the hydrophobic cavity of BSA, resulting in reduction of the fluorescence intensity of BSA. In addition, the calculated free energy change and binding distance between Trp 213 and small molecule are concordant with the experimental resultsThe enzyme activity experiments show that two small molecules(Ligupurpuroside A and Ligupurpuroside B) can inhibit trypsin activity through a competitive manner, and they have the potential to treat severe pancreatitis. Fluorescence spectroscopy experiments indicate that Ligupurpuroside A can reduce the intrinsic fluorescence of trypsin through a combined quenching way, while Ligupurpuroside B quenchs the intrinsic fluorescence of trypsin by a static way. Thermodynamic experiments show that Ligupurpuroside A binding to trypsin is mainly an entropy-driven spontaneous process, hydrophobic interaction is the main driving force. On the contrary, the binding process between Ligupurpuroside B and trypsin is enthalpy driven and the electrostatic forces play the dominant role. Conformational experiments suggest that the hydrophobicity around the tryptophan residues decreases, and hydrophobicity of the micro-environment of the tyrosine residues increases after binding to Ligupurpuroside A. Binding to Ligupurpuroside B leads to an increase of the hydrophobicity around the tyrosine residues of trypsin. Molecular docking shows that both Ligupurpuroside A and Ligupurpuroside B can enter the hydrophobic cavity of trypsin, and locate near Trp215 and Tyr228 of trypsin.The interactions between the two drugs(Ligupurpuroside A, Ligupurpuroside B) and lipase have been studied using a variety of spectroscopic techniques and molecular docking. The enzyme activity experiments show that phenylpropanoid glycosides can inhibit the activity of lipase by a competitive way, and the inhibition ability of Ligupurpuroside A is greater than that of Ligupurpuroside B. The fluorescence spectroscopy results show that both the two small molecules quench the fluorescence of lipase via static quenching mechanisms. Binding experiments show that the binding constant of Ligupurpuroside A-lipase is greater than Ligupurpuroside B-lipase. The thermodynamic analysis suggests that the binding of Ligupurpuroside A to trypsin is mainly entropy driven, and the hydrophobic interactions play a major role in the binding reactions. Ligupurpuroside B and lipase is an enthalpy-entropy driven process, and the interaction is largely driven by hydrophobic interactions, meanwhile the electrostatic interactions can also not be excluded. The synchronous fluorescence spectroscopy and circular dichroism experiments shows that the conformation of lipase did not change when interacting with small moleculues. The molecular docking results displayed the specific binding site and the detailed information about hydrogen bonds between the Ligupurpuroside A and trypsin. Concretely, both the two small molecules bind to the active site of hydrophobic cavity and interact with nearby tryptophan residues and tyrosine residues of lipase, thus weaken the intrinsic fluorescence of lipase.