Study On The Effect Of Plant Active Ingredients On The Spectral Properties And Structure Of Proteins

The biological activity of active ingredients extracted from plants such as flavonoid, coumarins, saponins, alkaloids, polysaccharides, phenolic acids and etc.receives more and more attentions. β-Lactoglobulin(BLG), is the major whey protein in bovine milk. It is classified as a member of the lipocalin-protein family-capable of binding and transporting small hydrophobic molecules. Trypsin is a class of serine protease, trypsin could cleave the peptide bonds on the carboxyl side of lysine and arginine, It is an important protein in digestive system which was activated by enterokinase and also activate other zymogen.The study has constructed a method system, which combing multiple spectroscopy methods, containting fluorescence, ultraviolet-visible(UV–vis)absorption, circular dichroism(CD) and fourier transform infrared spectroscopy(FT-IR) with chemometrics and molecular docking. Investigating the binding properties and the effects of several plant active ingredients like osthole, etc, onβ-lactoglobulin and trypsin from the molecular level, which contributes to explore the pharmacological actions of drug molecules such as absorption, distribution and metabolism in vivo. These investigations will provide valuable information for designing highly active drugs and developing drugs. Also, The present work will help to understand the change of protein structure and biological function.The main contents were summarized as follows:1. A brief introduction concerning the structure, function and biological properties of β-lactoglobulin and trypsin. Also, the investigating methods and current situation about the interaction between small molecules and protein were summarized in this chapter, it will laid the theoretical basis for this paper.2. Under simulative physiological conditions, a method system was constructed by integrating multivariate curve resolution–alternating least squares(MCR–ALS)with UV–vis absorption, fluorescence and molecular docking, and applied to explore the binding properties of daphnetin(DAPH) or osthole to BLG. The result of fluorescence experiment suggested that the fluorescence quenching of BLG by DAPH and osthole were considered as a static quenching procedure, and there is an binding site between the trypsin and DAPH or osthole. The results of ΔH° and ΔS°were all positive, which meant that the binding of DAPH to BLG was driven mainly by hydrophobic interaction. The negative enthalpy change(ΔH°) and entropy change(ΔS°) indicated that the binding of osthole to BLG was driven mainly by hydrogen bond and van der Waals forces. The binding distance between osthole and BLG was determined to be 4.05 nm. Molecular docking to select the most optimal docking results found that DAPH and osthole bind to the central calyx of the BLG molecule,visually exhibited the stereo binding modes.3. A method system was created by synchronous fluorescence, circular dichroism(CD) and Fourier transform infrared spectroscopy, applied to investigate the interaction of DAPH or osthole with BLG and both effects on BLG conformation.Analysis of synchronous fluorescence revealed that the addition of DAPH to BLG change the microenvironment of tryptophan residues of BLG and decreased the hydrophobicity tryptophan, osthole affected on the microenvironment of tyrosine rather than that of tryptophan in BLG, and more tyrosine exposed to hydrophilic and polar microenvironment. CD and FT–IR spectra results showed that the binding interaction between BLG and DAPH or osthole changed the secondary structure of BLG. Furthermore, the increased in a-helix of BLG induced by DAPH was more remarkable than osthole, the structure of BLG became more closely.4. The effects of psoralen(PSO) and 8-Methoxypsoralen(8-MOP) on the properties of trypsin were investigated using multivariate curve resolution–alternating least squares(MCR–ALS) with UV–vis absorption, fluorescence and molecular docking. The results suggested that the the quenching mechanism of PSO and 8-MOP binding with trypsin were static quenching through the formation of binary complex,the binding of PSO and 8-MOP to trypsin was driven mainly by hydrophobic interaction, and there was a single class of binding sites on trypsin for both PSO and8-MOP. The molecular docking and trypsin activity assay showed that both PSO and8-MOP primarily interacted with the catalytic triad of trypsin(His57,Asp102 and Ser195) and led to the inhibition of trypsin activity. Results showed stability of PSO and 8-MOP were comparative largely destructed in the artificial simulation gastric juices.However,it was comparatively stable in the artificial simulation intestinal juice.5. Under simulative physiological conditions, The interaction between trypsin and PSO or 8-MOP and trypsin conformational change were detected by three dimensional fluorescence, circular dichroism(CD), and Fourier transform infrared(FT-IR) spectroscopy, The results found that the addition of PSO and 8-MOP to trypsin both led to the second structure alteration of trypsin and resulted in the rearrangement of the polypeptides of protein with the reduction of α-helix andβ-sheet content and increases in β-turn and random coil structures.