| ObjectiveTo explore the interaction between gigantol/syringie acid(SA) and AR/iNOS. so as to further clarify caulis dendrobii mechanism of resistance to sugar cataract, lay a foundation for its development and utilization.Methods1. Fluorescently labeled gigantol and SA. Label the drug molecules with dansyl chloride and biotin at the basis of without affecting the the active group through chemical synthesis methods.2. Fluorescently labeled AR and iNOS. Application of recombinant DNA technology, we build gene expression vector of AR-GFP、AR-YFP and iNOS-DsRed, to transfected HLEC cells for expression. The drug as donor, and the protein as acceptor, through FRET to calculate energy transfer efficiency.3. The interaction between gigantol/SA and AR/iNOS at different temperatures are investigated with fluorescence spectrum, the binding constants, the binding sites and the thermodynamic parameter were obtained. The effect of gigantol/SA on the conformation of AR/iNOS was also analyzed syn-chronous fluorescence spectroscopy, the binding site area were obtained.4. A recombined vector pcDNA3.1-AR/pcDNA3.1-iNOS was constructedby gene engineering technique. The recombined vector was transfected into HLEC cells using lipidosome. Then detect the Raman spectra of a single living cells by using confocal microscopic Raman spectroscopy to analysis the influence of gigantol/SA on AR/iNOS secondary structure5. Research the change of surface morphology and three-dimensional conformation of AR/iNOS before and after adding gigantol/SA at single molecule by using AFM.Results1. Dansyl chloride labeled gigantol, SA and biotin labeled SA into HLEC cells and then405nm excitation wavelength of the fluorescence emission can be excited; receptor EGFP, YFP and DsRed were labeled AR, iNOS plasmid emit fluorescence at488nm,514nm and543nm excitation wavelength. Through explore different combinations of overlap regional for the emission spectrum of the donor and absorption spectra of acceptor, found dansyl chloride, biotin and EGFP, YFP and DsRed can not meet the experimental requirements for donor-recipient pairs in FRET, therefore can not be obtained by calculating fluorescence energy transfer efficiency and the role of the interaction distance.2. Results of the fluorescence spectra show that gigantol spontaneously by van der Waals forces and hydrogen bonding in combination with AR and make the protein fluorescence intensity increases. The interaction binding constant decreases with increasing temperature. Synchronous fluorescence spectra showed that gigantol acts on AR protein make fluorescence intensity of side chains of amino acid residues (tyrosine and tryptophan) increased and its emission wavelength blue shift. Raman spectroscopy showed that compared with normal HLEC cells, raman peaks of cells over expressing AR at1239cm-1,1450cm-1,1095cm-1,-1127cm-1,1034cm-1,1173cm-1, and1656cm-1have a significant differences, and compared with normal cells group, acting on cells of over express AR protein, characteristic peaks as the above-described attributable to the structure of α-helix or β-folding line, and phenylalanine, tryptophan and tyrosine residues occur in different degrees changes. Atomic force microscopy showed that the combine of gigantol and AR lead to AR molecular aggregation, the height increases, the surface morphology and three-dimensional conformational change.3. Results of the fluorescence spectra show that SA spontaneously by van der Waals forces and hydrogen bonding in combination with AR and make the protein fluorescence intensity increases. The interaction binding constant decreases with increasing temperature. Synchronous fluorescence spectra showed that SA acts on AR protein make fluorescence intensity of side chains of amino acid residues (tyrosine and tryptophan) increased and its emission wavelength were separately occur slightly red-shifted and blue shift. Raman spectroscopy showed that compared with normal HLEC cells, acting on cells of over express AR protein, characteristic peaks as the above-described attributable to the structure of a-helix and phenylalanine, tryptophan and tyrosine residues occur in different degrees changes. Atomic force microscopy showed that the combine of SA and AR lead to AR molecular aggregation, the number of nodes increases and the height increases, but the surface morphology and three-dimensional conformational Did not change significantly.4. Results of the fluorescence spectra show that gigantol spontaneously by van der Waals forces and hydrogen bonding in combination with iNOS and make the protein fluorescence intensity increases. The interaction binding constant decreases with increasing temperature. Synchronous fluorescence spectra showed that gigantol acts on iNOS protein make fluorescence intensity of side chains of amino acid residues (tyrosine and tryptophan) increased and the emission wavelength of tryptophan occur blue shift. Raman spectroscopy showed that compared with normal HLEC cells, raman peaks of cells aver expressing AR at1034cm-1、1095cm-1、1206cm-1、1239cm-1、1334cm-1、1450cm-1and1656cm-1have a significant differences, and compared with normal cells group, acting on cells of over express iNOS protein, characteristic peaks as the above-described attributable to the structure of α-helix and phenylalanine, tryptophan and tyrosine residues occur in different degrees changes. Atomic force microscopy showed that the combine of gigantol and iNOS lead to iNOS molecular aggregation and the height increases.ConclusionGigantol, SA with AR, iNOS interact to form complexes, effect on protein structure and amino acid side chain conformations environment exert its inhibitory effect AR, iNOS activity... |
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