Study on the Interactions Between Caffeoylquinic Acids With Bovine Serum Albumin: Spectroscopy, Antioxidant Activity, LC-MSn, and Molecular Docking Approach.
ABSTRACT: Clarified the binding mechanism of drugs with plasma proteins could provide fresh insights into the drug development. Caffeoylquinic acids (CQAs) are a kind of phenolic acid compounds which has extensive biological effects. This study investigated the binding mechanism of three CQAs, including chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid, with bovine serum albumin (BSA) by using multi-spectroscopic techniques, including fluorescence, UV-Vis, Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopy, LC-MSn, molecular docking and antioxidant activity assessment. In addition, the influences of PBS buffer, Tris-HCl buffer and water as solvents on the characteristics of CQAs and BSA interaction were also investigated. The results showed that intrinsic fluorescence of BSA was quenched by CQAs and the interaction was static quenching with the formation of a non-fluorescent complex. The binding of CQAs and BSA was spontaneous, and Van der Waals forces and hydrogen-bond interaction occupied crucial roles in the binding. All the three CQAs could bind to Site I in Domain IIA. The weakest interaction between neochlorogenic acid and BSA may due to its larger polarity. The results also indicated that the binding affinity of CQAs had a descending order of Tris-HCl > H2O > PBS. This study firstly clarified the binding mechanism of CQAs with BSA and changes of the binding in different solvents, and provided fresh insights into this drug transportation and metabolism.
Project description:In order to induce tumours on dicotyledonous plants, the bacterium Agrobacterium tumefaciens needs to be able to sense signal molecules, i.e. phenolic compounds. In order to identify putative chemoreceptors or environmental sensors involved in vir gene induction, we undertook the purification of a phenol-binding protein by affinity chromatography on a syringamide Ultrogel A4 column equilibrated at pH 5.6. A mild extraction of bacterial proteins with a Tris/HCl buffer at pH 9.0 led to the purification of a 39 kDa protein (Pbp39) with a pl of 4.3 after specific elution of the affinity matrix with sodium syringate. When the affinity chromatography was performed at neutral pH, barely any protein was isolated, indicating the importance of an acidic pH for optimal affinity. A microplate binding experiment revealed that both syringlyl biotinylated-BSA and sinapyl-biotinylated-BSA bound at pH 5.6 to the plate coated with Pbp39.
Project description:Identification and characterization of HP1BP3 (a human histone H1 homologue) as a novel chromatin retention factor essential for the co-transcriptional processing of pri-miRNA. We generated BAC transgenic cells at 80% confluency (~1x107) were cross-linked with 1% formaldehyde for 10 minutes at 37Â°C, and quenched with 125 mM glycine at room temperature for 5 minutes. The fixed cells were washed twice with cold PBS, scraped, and transferred into 1 ml PBS containing protease inhibitors (Roche). After centrifugation at 700 g for 4 minutes at 4Â°C, the cell pellets were resuspended in 100 Î¼l ChIP lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl [pH 8.1] with protease inhibitors) and sonicated at 4Â°C with a Bioruptor (Diagenode) (30 seconds ON and 30 seconds OFF at highest power for 15 minutes). The sheared chromatin with a fragment length of ~200 â 600 bp) was centrifuged at 20,000 g for 15 minutes at 4Â°C). 100 Î¼l of the supernatant was used for ChIP or as input. A 1:10 dilution of the solubilized chromatin in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl 16.7 mM Tris-HCl [pH 8.1]) was incubated at 4Â°C overnight with 6 Î¼g/ml of a goat anti-GFP (raised against His-tagged full-length eGFP and affinity-purified with GST-tagged full-length eGFP). Immunoprecipitation was carried out by incubating with 40 Î¼l pre-cleared Protein G Sepharose beads (Amersham Bioscience) for 1 hour at 4Â°C, followed by five washes for 10 minutes with 1ml of the following buffers: Buffer I: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 150 mM NaCl; Buffer II: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 500 mM NaCl; Buffer III: 0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl [pH 8.1]; twice with TE buffer [pH 8.0]. Elution from the beads was performed twice with 100 Î¼l ChIP elution buffer (1% SDS, 0.1 M NaHCO3) at room temperature (RT) for 15 minutes. Protein-DNA complexes were de-crosslinked by heating at 65Â°C in 192 mM NaCl for 16 hours. DNA fragments were purified using QiaQuick PCR Purification kit (QIAGEN) and eluted into 30 Î¼l H2O according to the manufacturerâs protocol after treatment with RNase A and Proteinase K.
Project description:The nature and mechanisms of interaction between two selected methyl benzoate derivatives (methyl <i>o</i>-methoxy <i>p</i>-methylaminobenzoate-<b>I</b> and methyl <i>o</i>-hydroxy <i>p</i>-methylaminobenzoate-<b>II</b>) and model transport protein bovine serum albumin (BSA) was studied using steady-state and time-resolved spectroscopic techniques. In order to understand the role of Trp residue of BSA in the <b>I</b>-BSA and <b>II</b>-BSA interaction, the effect of free Trp amino acid on the both emission modes (LE-locally excited (<b>I</b> and <b>II</b>) and ESIPT-excited state intramolecular proton transfer (<b>II</b>)) was investigated as well. Experimental results show that the investigated interactions (with both BSA and Trp) are mostly conditioned by the ground and excited state complex formation processes. Both molecules form stable complexes with BSA and Trp (with 1:1 stoichiometry) in the ground and excited states. The binding constants were in the order of 10<sup>4</sup> M<sup>-1</sup>. The absorption- and fluorescence-titration experiments along with the time-resolved fluorescence measurements show that the binding of the <b>I</b> and <b>II</b> causes fluorescence quenching of BSA through the static mechanism, revealing a 1:1 interaction. The magnitude and the sign of the thermodynamic parameters, Δ<i>H</i>, Δ<i>S</i>, and Δ<i>G</i>, determined from van't Hoff relationship, confirm the predominance of the hydrogen-bonding interactions for the binding phenomenon. To improve and complete knowledge of methyl benzoate derivative-protein interactions in relation to supramolecular solvation dynamics, the time-dependent fluorescence Stokes' shifts, represented by the normalized spectral response function <i>c(t)</i>, was studied. Our studies reveal that the solvation dynamics that occurs in subpicosecond time scale in neat solvents of different polarities is slowed down significantly when the organic molecule is transferred to BSA cavity.
Project description:Octacalcium phosphate (OCP) has been shown to enhance new bone formation, coupled with its own biodegradation, through osteoblasts and osteoclast-like cell activities concomitant with de novo hydroxyapatite (HA) formation and serum protein accumulation on its surface. However, the nature of the chemical environment surrounding OCP and how it affects its metabolism and regulates protein accumulation is unknown. The present study examined how the degree of supersaturation (DS) affects the bovine serum albumin (BSA) adsorption onto OCP in 150 mM Tris-HCl buffer at 37 °C and pH 7.4, by changing the Ca2+ ion concentration. The amount of BSA adsorbed onto OCP increased as the DS increased. In addition, the amount of newly formed calcium phosphate, which could be OCP, was increased, not only by increases in DS, but also at lower equilibrium concentrations of BSA. The increased adsorption capacity of BSA was likely related to the formation of calcium phosphate on the adsorbed OCP. Together the results suggested that the formation of new calcium phosphate crystals is dependent on both the DS value and the adsorbate protein concentration, which may control serum protein accumulation on the OCP surface in vivo.
Project description:While the regulation of metabolic enzymes by oncogenic drivers or tumor suppressors has been intensively studied over recent years, our understanding of how metabolic processes directly regulate cell proliferation has remained fragmentary. Here we show how the alteration of metabolism directly affects cell cycle progression in cancer cells. We found that activation of the nuclear receptor peroxisome-proliferation activated receptor gamma (PPARM-NM-3), a transcriptional master regulator of lipid metabolism, inhibits the growth of lung adenocarcinoma cells by triggering a metabolic switch that inhibits pyruvate oxidation and reduces glutathione levels. These PPARM-NM-3-induced metabolic changes result in a marked increase of reactive oxygen species (ROS) levels that lead to rapid hypophosphorylation of retinoblastoma protein (RB) and cell cycle arrest. Both of these changes can be prevented by suppressing pyruvate dehydrogenase kinase 4 (PDK4) or M-NM-2-oxidation of fatty acids. Thus, we provide a mechanism that directly links metabolic changes to inhibition of cancer cell cycle progression by altering ROS levels. We generated PPARG-LAP BAC transgenic NCI-H2347 and NCI-H1993 cell lines using the BAC-transgenesis approach. Cells at 80% confluency (~1-1.5x107) were cross-linked with 1% formaldehyde for 10 minutes at 37M-BM-0C, and quenched with 125 mM glycine at room temperature for 5 minutes. The fixed cells were washed twice with cold PBS, scraped, and transferred into 1 ml PBS containing protease inhibitors (Roche). After centrifugation at 700 g for 4 minutes at 4M-BM-0C, the cell pellets were resuspended in 100 M-NM-<l ChIP lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl [pH 8.1] with protease inhibitors) and sonicated at 4M-BM-0C with a Bioruptor (Diagenode) (30 seconds ON and 30 seconds OFF at highest power for 12 minutes). The sheared chromatin with a fragment length of ~200 M-bM-^@M-^S 600 bp) was centrifuged at 10,000 g for 10 minutes at 4M-BM-0C). 100 M-NM-<l of the supernatant was used for ChIP or as input. A 1:10 dilution of the solubilized chromatin in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl 16.7 mM Tris-HCl [pH 8.1]) was incubated at 4M-BM-0C overnight with 6 M-NM-<g/ml of a goat anti-GFP (raised against His-tagged full-length eGFP and affinity-purified with GST-tagged full-length eGFP). Immunoprecipitations were carried out by incubating with 40 M-NM-<l pre-cleared Protein G Sepharose beads (Amersham Bioscience) for 1 hour at 4M-BM-0C, followed by five washes for 10 minutes with 1ml of the following buffers: Buffer I: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 150 mM NaCl; Buffer II: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 500 mM NaCl; Buffer III: 0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl [pH 8.1]; twice with TE buffer [pH 8.0]. Elution from the beads was performed twice with 100 M-NM-<l ChIP elution buffer (1% SDS, 0.1 M NaHCO3) at room temperature (RT) for 15 minutes. Protein-DNA complexes were de-crosslinked by heating at 65M-BM-0C in 192 mM NaCl for 16 hours. DNA fragments were purified using QiaQuick PCR Purification kit (Qiagen) and eluted into 30 M-NM-<l H2O according to the manufacturerM-bM-^@M-^Ys protocol after treatment with RNase A and Proteinase K.
Project description:Cells were washed with cold PBS twice and scraped from the plates, then centrifuged at 1000 rpm for 5 min to pellet cells. Washed cells were lysed with 400 L of cell lysis buffer (10 mM Tris-HCl pH7.5, 150 mM NaCl, 0.15% NP-40 and proteinase inhibitor cocktail) and incubated on ice for 10 min. The suspension was carefully add at the top of sucrose buffer (10 mM Tris-HCl pH7.5, 150 mM NaCl, 24% sucrose), and centrifuged at 3200g for 10 min to collect nuclear pellets. Pellets were resuspended in 250 L of Glycerol buffer (20 mM Tris-HCl pH7.5, 75 mM NaCl, 0.5 mM EDTA pH8.0, 50% glycerol), then then immediately add 250 μl Nuclear lysis buffer (10 mM Tris-HCl pH7.5, 300 mM NaCl, 7.5 mM MgCl2, 0.2 mM EDTA pH8.0, 1% NP-40, 1M urea). Mix by vortexing for 4 s and incubate on ice for 2 min. Centrifuge the lysate at 13,000 × g for 2 min to precipitate the chromatin–RNA complex. Brieﬂy rinse the chromatin pellets with PBS-EDTA. RNA was extracted with Trizol reagent. RNA libraries were prepared according to manual of SMARTer smRNA-Seq Kit for Illumina (Clontech, 635031). Overall design: Examination of m6A levels in zebrafish embryo.
Project description:Biogenic polyamines are found to modulate protein synthesis at different levels, while polyamine analogues have shown major antitumor activity in multiple experimental models, including breast cancer. The aim of this study was to examine the interaction of bovine serum albumin (BSA) with biogenic polyamines, spermine and spermidine, and polyamine analogues 3,7,11,15-tetrazaheptadecane x 4 HCl (BE-333) and 3,7,11,15,19-pentazahenicosane x 5 HCl (BE-3333) in aqueous solution at physiological conditions. FTIR, UV-visible, CD, and fluorescence spectroscopic methods were used to determine the polyamine binding mode and the effects of polyamine complexation on protein stability and secondary structure. Structural analysis showed that polyamines bind BSA via both hydrophilic and hydrophobic interactions. Stronger polyamine-protein complexes formed with biogenic than synthetic polyamines with overall binding constants of K(spm) = 3.56 (+/-0.5) x 10(5) M(-1), K(spmd) = 1.77 (+/-0.4) x 10(5) M(-1), K(BE-333) = 1.11 (+/-0.3) x 10(4) M(-1) and K(BE-3333) = 3.90 (+/-0.7) x 10(4) M(-1) that correlate with their positively charged amino group contents. Major alterations of protein conformation were observed with reduction of alpha-helix from 63% (free protein) to 55-33% and increase of turn 12% (free protein) to 28-16% and random coil from 6% (free protein) to 24-17% in the polyamine-BSA complexes, indicating a partial protein unfolding. These data suggest that serum albumins might act as polyamine carrier proteins in delivering polyamine analogues to target tissues.
Project description:Blood paper cards provide an effective DNA storage method. In this study, we used three DNA dissolving reagents (Tris-EDTA [TE] buffer, Tris-HCl buffer, and water) and one common commercially available kit (DN131 from MRC Inc) to elute DNA from 105 human blood paper cards collected up to 10 years ago. These DNA samples were used as templates for amplification of a single nucleotide polymorphism (SNP, C125T) region of human caspase-12 by PCR and a specific Taqman genotyping assay using the same amount of DNA. We show that DNA isolated by Tris-HCl buffer has higher yield and quality in comparison to DN131 solution. PCR success rate to amplify caspase-12 C125T SNP using Tris-HCl is comparable to the method using DN131 (89.5% vs 87.6%). The Taqman genotyping success rate using Tris-HCl is higher than using DN131 (81.9% vs 70.5%). Using TE or water, PCR success rates are lower than using DN131 (73.3% [TE]; 72.4% [H2O]), but Taqman genotyping success rates are comparable to the method using DN131 (70.5% [TE]; 79.1% [H2O]). We concluded that using Tris-HCl is a reliable and effective method to elute DNA from old human blood paper cards. The crude DNA isolated by Tris-HCl can be used to study genetic polymorphisms in human populations.
Project description:The goal of this study is to test ionic strength and buffering capacity in polysome extraction buffer on ribosome footprints in Arabidopsis root and shoot. Overall design: Ribo-seq generated from Arabidopsis root and shoot prepared with four different polysome extraction buffers. All four buffers contain 2% polyoxyethylene 10 tridecyl ether, 1% deoxycholic acid, 1 mM DTT, 100 µg/mL cycloheximide, and 10 unit/mL DNase I, but with different ionic strength or buffering capacity listed below. Buffer A: 200 mM Tris-HCl (pH 8), 50 mM KCl, 25 mM MgCl2; buffer B: 200 mM Tris-HCl (pH 8), 40 mM KCl, 20 mM MgCl2; buffer C: 200 mM Tris-HCl (pH 8), 30 mM KCl, 15 mM MgCl2; buffer D: 100 mM Tris-HCl (pH 8), 40 mM KCl, 20 mM MgCl2. 8 libraries in total.
Project description:Two specimens of human articulage were successively extracted with solutions of phosphate-buffered saline (PBS), 7 M-urea and 4 M-guanidine hydrochloride (Gdn-HCl). Proteoglycans from individual extracts were fractionated by DEAE-Sephacel chromatography and gel chromatography on Sephacryl S-400. The presence of three populations of large proteoglycans was demonstrated in all three extracts by composite agarose/polyacrylamide-gel electrophoresis (CAPAGE). The population corresponding to the fastest CAPAGE band of aggregating proteoglycans was shown to be extremely polydisperse, having Mr (as estimated by SDS/PAGE) decreasing continuously from more than 300,000 to the size corresponding to 'free' hyaluronic acid-binding region (HABR) (about 70,000). A rather polydisperse set of HABR-containing fragments which spanned a broad range of sizes, and also differed in their keratan sulphate contents, was isolated from both 7 M-urea and 4 M-Gdn-HCl extracts. PBS and 7 M-urea extracts, but not the Gdn-HCl extract, further contained small proteoglycans, identified as fast-migrating bands on CAPAGE electrophoretograms. One of those small species was recognized with an antibody against the small proteoglycan PG II; the other two remain to be positively identified. However, the glycosaminoglycan of the small species which was present exclusively in the PBS extract was identified as keratan sulphate; this species may thus belong to the family of small keratan sulphate-containing proteolygans.