On the Stabilizing Effect of Aspartate and Glutamate and Its Counteraction by Common Denaturants.

By performing differential scanning calorimetry(DSC) measurements on RNase A, we studied the stabilization provided by the addition of potassium aspartate(KAsp) or potassium glutamate (KGlu) and found that it leads to a significant increase in the denaturation temperature of the protein. The stabilization proves to be mainly entropic in origin. A counteraction of the stabilization provided by KAsp or KGlu is obtained by adding common denaturants such as urea, guanidinium chloride, or guanidinium thiocyanate. A rationalization of the experimental data is devised on the basis of a theoretical approach developed by one of the authors. The main contribution to the conformational stability of globular proteins comes from the gain in translational entropy of water and co-solute ions and/or molecules for the decrease in solvent-excluded volume associated with polypeptide folding (i.e., there is a large decrease in solvent-accessible surface area). The magnitude of this entropic contribution increases with the number density and volume packing density of the solution. The two destabilizing contributions come from the conformational entropy of the chain, which should not depend significantly on the presence of co-solutes, and from the direct energetic interactions between co-solutes and the protein surface in both the native and denatured states. It is the magnitude of the latter that discriminates between stabilizing and destabilizing agents.

Structural Characterization of Ectodomain G Protein of Respiratory Syncytial Virus and Its Interaction with Heparan Sulfate: Multi-Spectroscopic and In Silico Studies Elucidating Host-Pathogen Interactions.

The global burden of disease caused by a respiratory syncytial virus (RSV) is becoming more widely recognized in young children and adults. Heparan sulfate helps in attaching the virion through G protein with the host cell membrane. In this study, we examined the structural changes of ectodomain G protein (edG) in a wide pH range. The absorbance results revealed that protein maintains its tertiary structure at physiological and highly acidic and alkaline pH. However, visible aggregation of protein was observed in mild acidic pH. The intrinsic fluorescence study shows no significant change in the λmax except at pH 12.0. The ANS fluorescence of edG at pH 2.0 and 3.0 forms an acid-induced molten globule-like state. The denaturation transition curve monitored by fluorescence spectroscopy revealed that urea and GdmCl induced denaturation native (N) ↔ denatured (D) state follows a two-state process. The fluorescence quenching, molecular docking, and 50 ns simulation measurements suggested that heparan sulfate showed excellent binding affinity to edG. Our binding study provides a preliminary insight into the interaction of edG to the host cell membrane via heparan sulfate. This binding can be inhibited using experimental approaches at the molecular level leading to the prevention of effective host-pathogen interaction.

A novel sulfamethoxazole derivative as an inhibitory agent against HSP70: A combination of computational with in vitro studies.

In the current study, a novel derivative of sulfamethoxazole (a sulfonamide containing anti-biotic) named ZM-093 (IUPAC name: (E)-4-((4-(bis(2-hydroxyethyl)amino)phenyl)diazenyl)-N-(5-methylisoxazole-3-yl)benzenesulfonamide) was synthesized via common diazotization-coupling reactions from sulfamethoxazole and subsequently characterized through NMR/FT-IR spectroscopy. After evaluation, the compound was geometrically optimized at the DFT level of theory with BL3YP method and 6/31++G (d,p) basis set and from the optimized structure, several molecular descriptors important in the biological reactivity of the compound, such as global reactivity parameters, molecular electrostatic potential, average local ionization energy, and drug-likeness features of the compound were computationally analyzed. The experimental in vitro investigations of the interaction between ZM-093 and heat shock protein 70 (HSP70), a protein that is highly expressed in several types of cancers, exhibited a significant inhibitory effect against the chaperone activity of HSP70 for the titled compound (P-value < 0.01) and the comparison between the experimental studies with the mentioned computational analysis, as well as molecular docking, illustrated that ZM-093 may inhibit HSP70 through binding to its substrate-binding domain. Finally, by taking all the previous results into account, a new method for assessing the inhibitory activity of ligand to HSP70 is introduced based on protonography, a recently developed method that is dependent on the catalytic activity of carbonic anhydrase on polyacrylamide gel electrophoresis.

The inhibition mechanism of the texture deterioration of tilapia fillets during partial freezing after treatment with polyphenols.

The inhibition mechanism of the texture deterioration of tilapia fillets after treatment with polyphenols during partial freezing for 49 days was studied. Carnosic acid (CA), procyanidin (PA), quercetin (QE), and resveratrol (RSV) treatments had significantly higher hardness values (over 230 g) than the control group (183 g) on day 49 (P < 0.05). Polyphenol treatments were effective in delaying the protein degradation, lipid oxidation and spoilage microbe growth. Moreover, the kinetic model showed that the predicted shelf life of tilapia fillets treated with PA (102 d) was extended by 25 d compared to the control group (77 d). It was the proposed possible mechanism that polyphenols comprehensively maintained the protein conformation (increased hydrogen bonds and decreased disulfide bonds) and retarded protein denaturation and degradation, protecting the texture of the fillets. Therefore, polyphenols can be used to maintain texture and extend the shelf life of tilapia fillets during partial freezing.

Essential Oils of Foeniculum vulgare subsp. piperitum and Their in Vitro Anti-Arthritic Potential.

Wild Foeniculum vulgare subsp. piperitum (C.Presl) Bég. flowers, fruits and leaves were extracted with steam distillation and obtained essential oils (EOs) were characterized using GC/MS. The study was designed to verify the potential effectiveness of fennel EOs in the treatment of inflammation and arthritis. Since tissue proteins denaturation is a major cause of arthritic diseases, fennel EOs and their main constituents were evaluated for their ability to inhibit the heat-induced proteins degradation using bovine serum albumin as a protein model. Moreover, the in vitro inhibitory effects of the three EOs on the pro-inflammatory mediator nitric oxide (NO) production were verified in LPS-stimulated RAW 264.7 cells. Estragole (28.81-33.40 %), anethole (24.16-27.40 %), fenchone (9.76-18.48 %), α-phellandrene (1.63-8.37 %) and limonene (5.54-6.05 %) were the major constituents. All the EOs showed a concentration-dependent biological activity, being the flower EO the most effective in inhibiting NO production (IC50 =232.2±11.3 µg/mL). The leaf EO showed a very good bovine serum albumin (BSA) anti-denaturation activity (IC50 =95.9±2.4 µg/mL). Moreover, four components were proved to be effective in protecting protein from heat-induced degradation, being α-phellandrene the most active compound (IC50 =73.2±1.9 µg/mL).

Mechanistic insights into the urea-induced denaturation of human sphingosine kinase 1.

Sphingosine kinase 1 (SphK1) plays a significant role in various cellular processes, including cell proliferation, apoptosis, and angiogenesis. SphK1 is considered as an attractive target for drug development owing to its connection with several diseases, including cancer. In the current work, the urea-induced unfolding of SphK1 was performed at pH 8.0 and 25 °C using CD and fluorescence spectroscopy. SphK1 follows a biphasic unfolding transition (N â‡Œ I â‡Œ D) with an intermediate (I) state populated around 4.0 M urea concentration. The circular dichroism ([θ]222) and fluorescence emission spectra (λmax) of SphK1 with increasing concentrations of urea were analyzed to calculate Gibbs free energy (ΔG0) for both the transitions (N â‡Œ I and I â‡Œ D). A significant overlap of both the transitions obtained by two spectroscopic properties ([θ]222 and λmax) was observed, indicating that both N â‡Œ I and I â‡Œ D transition follow two-step equilibrium unfolding pattern. Also, we performed 100 ns molecular dynamics (MD) simulations to get atomistic insights into the structural changes in SphK1 with increasing urea concentrations. Our results showed a consistent pattern of the SphK1 unfolding with increasing urea concentrations. Together, spectroscopic and MD simulation findings provide deep insights into the unfolding mechanism and conformational features of SphK1.

Effect of marinating ingredients on temperature-induced denaturation of hemoglobin and its relation to red blood spot formation in cooked chicken breast.

Red blood spot (RBS) commonly found in cooked chicken breast has caused severe economic loss as it is perceived as a sign of undercooked product. The objectives of this study were to investigate the cause of RBS as related to common ingredients used in marination, based on both chicken breast and isolated chicken hemoglobin (Hb) models. The effect of sodium chloride (NaCl), sodium tripolyphosphate (STPP), and glucose on thermal denaturation of Hb was investigated along with the extent of RBS formation in cooked marinated chicken breast. After vacuum tumbling for 65 min and subsequent storage at 4 °C for 20 hr, STPP and glucose were not absorbed into the center of chicken breast. However, Na+ was absorbed after 12 hr storage. The denaturation temperature (Td ) of isolated chicken Hb decreased to 65.8 °C in the presence of 1.5 M NaCl, while that of the control was 69.4 °C. STPP at pH 9 decreased Td of Hb to 61.4 °C. The alkaline pH induced by STPP destabilized the Hb structure. RBSs were observed at 100% incidence when cooked to core temperatures of 50 and 70 °C for 1 min. RBSs were completely eliminated at core temperature of 85 °C. The ingredients used during marination appeared to have a minimal effect on RBS formation due to their limited absorption into the chicken breast. The cooking temperature is a major factor governing RBSs, as it directly affects the denaturation of Hb.

Mechanistic insights into the urea-induced denaturation of a non-seleno thiol specific antioxidant human peroxiredoxin 6.

Peroxiredoxin 6 (Prdx6) is a unique enzyme among mammalian peroxiredoxins as it lacks resolving cysteine. It is found to be involved in number of different diseases including tumours and its expression level is highest in lungs as compared to other organs. It has been found that Prdx6 plays a significant role different metabolic diseases, ocular damage, neurodegeneration and male infertility. It is a bifunctional protein having phospholipase A2 and peroxidase (also has the ability to reduce phospholipid hydroperoxides) activities. In order to complete the peroxidise reaction cycle it requires glutathione catalyzed by glutathione S-transferase. Equilibrium unfolding and conformational stability of Prdx6 was studied by using urea as a chemical denaturant to understand the changes it goes under cellular stress conditions. Three different spectroscopic methods were employed to monitor urea-induced denaturation. From the results obtained, it was found that the urea denaturation of Prdx6 follows a variable two state process due to non-coincidence of the normalized transition curves obtained from different optical probes. The different denaturation curves were normalized and thermodynamic parameters, ΔGDo, Gibbs free energy change related to the urea-induced denaturation, midpoint of denaturation (Cm), and m = (δΔGD / [urea]) were obtained. The structural information of Prdx6 were further analysed by several parameters obtained by 100 ns MD simulation. The results of MD simulation clearly favour the outcome of spectroscopic studies.

Influence of crowding agents on the dynamics of a multidomain protein in its denatured state: a solvation approach.

It is now well appreciated that the crowded intracellular environment significantly modulates an array of physiological processes including protein folding-unfolding, aggregation, and dynamics to name a few. In this work we have studied the dynamics of domain I of the protein human serum albumin (HSA) in its urea-induced denatured states, in the presence of a series of commonly used macromolecular crowding agents. HSA was labeled at Cys-34 (a free cysteine) in domain I with the fluorophore 6-bromoacetyl-2-dimethylaminonaphthalene (BADAN) to act as a solvation probe. In partially denatured states (2-6 M urea), lower crowder concentrations (~ < 125 g/L) induced faster dynamics, while the dynamics became slower beyond 150 g/L of crowders. We propose that this apparent switch in dynamics is an evidence of a crossover from soft (enthalpic) to hard-core (entropic) interactions between the protein and crowder molecules. That soft interactions are also important for the crowders used here was further confirmed by the appreciable shift in the wavelength of the emission maximum of BADAN, in particular for PEG8000 and Ficoll 70 at concentrations where the excluded volume effect is not dominant.

The anti-oxidant enzyme, Prdx6 might have cis-acting regulatory sequence(s).

Peroxiredoxin 6 (Prdx6) is a ubiquitously expressed 1-cysteine Peroxiredoxin found throughout all phyla. In mammals, under different physiological conditions, it has evolved from a peroxidase to a multifunctional enzyme. Among the mammalian Prdx6's, human and rat Prdx6's are the most extensively studied. Our study revealed that human and rat Prdx6's exhibit differences in their peroxidase activity. These two Prdx6's have only 8% difference in their primary sequence (with 19 amino acids) with no apparent modification at any of the key conserved residues. In the present communication, we have investigated the roles of thermodynamics, structure and internal flexibility of Prdx6 to account for the difference in their peroxidase activity. We discovered that these amino acid variations have led to structural alterations in human Prdx6 so that it shows enhanced intrinsic dynamics (or flexibility) than the rat protein. We could also identify the gain of intrinsic dynamics of the catalytic site in human Prdx6 due to relocation of an important active site residue (R132) to the loop region as the most plausible reason for high catalytic activity in the human protein as compared to rat variant. Since it is the thioredoxin fold that upholds the peroxidase function, certain structural alteration in the Prdx6 structure might help to regulate the efficiency of thioredoxin folds. Our results hint that Prdx6 might have a cis-acting regulatory sequence(s).

Study of Anti-oxidant, Anti-inflammatory, Genotoxicity, and Antimicrobial Activities and Analysis of Different Constituents found in Rhizome Essential Oil of Curcuma caesia Roxb., Collected from North East India.

BACKGROUND: This investigation was designed to evaluate the chemical composition, antioxidant, anti-inflammatory, genotoxicity, and antimicrobial activities of Curcuma caesia Roxb rhizome essential oil. METHODS: Gas Chromatography/Mass Spectroscopy (GC/MS) analysis was performed to determine the chemical composition, standard antioxidative test DPPH assay, reducing power assay, in vitro antiinflammatory activity (egg albumin denaturation, protease inhibitory assay) by using standard methods. Similarly, antimicrobial activity was tested using the disc diffusion method, minimum inhibitory concentration ability (MIC); while to test genotoxicity, Allium cepa assay was used. RESULTS: GC/MS analysis revealed eucalyptol (28.55%), epicurzerenone (19.62%), and camphor (21.73%) as the major components of C. caesia rhizome essential oil. Potent antioxidant (IC50= 48.08±0.003 µg/mL), anti-inflammatory (IC50= 121.7±0.0013 µg/mL), and antimicrobial activities of the essential oil were recorded better than the standard drugs Fluconazole for fungus and Ciprofloxacin for bacteria. The essential oil also possessed a strong antibacterial effect against two tested bacterial strains B. subtilis and B. cereus with 7.5 µg/mL MIC value, while for fungal strains the essential oil was most effective against S. cereviaceae with an MIC value of 2.5 µg/mL. All the data were recorded in triplicates. Allium cepa assay revealed minor genotoxicity with mitotic index, MI= 27.70%; chromosome aberration, A= 1.1% of C. caesia rhizome essential oil. CONCLUSION: C. caesia rhizome essential oil possesses potent antioxidant, anti-inflammatory, and antimicrobial properties with negligible genotoxicity. Hence, the present study is highly significant for the utilization of rhizome of C. caesia, a high-value ethnopharmacological plant for advanced R & D and commercial application.

Investigating the interaction of porcine pancreatic elastase and propanol: A spectroscopy and molecular simulation study.

The response of porcine pancreatic elastase (PPE) to propanol was examined by various techniques including UV-vis spectrophotometry, spectrofluorometry and circular dichroism, as well as molecular docking and molecular simulation. These techniques were used to investigate the structural changes and elastase activity in the presence of propanol. This work was performed at three temperatures of 303, 313 and 323 K, with the pH value of 8.5 (Tris buffer). The results of the UV-vis spectrophotometry indicated the transfer of tryptophan to an environment with low hydrophobicity. Fluorescence measurements also revealed the quenching of fluorescence intensity was induced by propanol, and dynamic quenching was the proposed quenching mechanism. Kinetic studies also suggested the inhibitory effect (noncompetitive) of propanol on elastase. Further, Circular Dichroism (CD) spectra showed that propanol caused slight alterations in the secondary structures of PPE (0.3% increase for the α-helix and 0.5% decrease for the ß-sheet). Addition of propanol decreased the Tm (Melting Temperature) parameter from 332.8 K to 330.1 K.

The Effects of Storage on Quality and Nutritional Values of Ehrenberg's Snapper Muscles (Lutjanus Ehrenbergi): Evaluation of Natural Antioxidants Effect on the Denaturation of Proteins.

: Protein denaturation in frozen minced fillets (Ehrenberg's Snapper), stored at -25°C was studied; 50.0 mg biomass/50g mince fillets treated with cinnamon, cumin, turmeric, garlic, ginger and 25.0 mg of vitamin C were used to slow protein denaturation. FT-IR stretching vibration of Amide-A (νNH) at 3300 cm-1; Amide-I stretching (νC=O) between 1600-1690 cm-1 and Amide-II stretching (νCN) and bending (δNH) between 1480 and 1575cm-1 were used as marker peaks. Garlic was the most significant (P ≤0.01) in controlling the rate of protein denaturation when νNH was used as a marker peak. DSC analysis showed that turmeric presented the highest effect on delaying the denaturation of sarcoplasmic proteins with a ∆H0=73.7J/g followed by garlic-treated mince fillets ∆H0=70.1J/g. All spices used were efficient in stopping the denaturation of myosin with the highest ∆H0=769.3 J/g registered for cinnamon-treated mince fillets. Actin was less vulnerable to denaturation in comparison to myosin and sarcoplasmic proteins.

Indole-3-propionic acid has chemical chaperone activity and suppresses endoplasmic reticulum stress-induced neuronal cell death.

Insoluble aggregated proteins are often associated with neurodegenerative diseases. Previously, we investigated chemical chaperones that prevent the aggregation of denatured proteins. Among these, 4-phenyl butyric acid (4-PBA) has well-documented chemical chaperone activity, but is required at doses that have multiple effects on cells, warranting further optimization of treatment regimens. In this study, we demonstrate chemical chaperone activities of the novel compound indole-3-propionic acid (IPA). Although it has already been reported that IPA prevents ß-amyloid aggregation, herein we show that this compound suppresses aggregation of denatured proteins. Our experiments with a cell culture model of Parkinson's disease are the first to show that IPA prevents endoplasmic reticulum (ER) stress and thereby protects against neuronal cell death. We suggest that IPA has potential for the treatment of neurodegenerative diseases and other diseases for which ER stress has been implicated.

Cytotoxic and anti-inflammatory resorcinol and alkylbenzoquinone derivatives from the leaves of Ardisia sieboldii.

Medicinal plants belonging to the genus Ardisia are traditionally used to cure various human diseases including inflammation and cancer. This study aimed to purify and characterize cytotoxic and anti-inflammatory compounds from Ardisia sieboldii leaves. Bioassay-guided chromatographic analyses yielded three compounds, 2-methyl-5-(8Z-heptadecenyl) resorcinol (1), 5-(8Z-heptadecenyl) resorcinol (2), and ardisiaquinone A (3), whereas liquid chromatography-electrospray ionisation-mass spectrometry chemical profiling revealed the presence of diverse resorcinol and alkylbenzoquinone derivatives in cytotoxic 70% methanol extracts. Chemical structures of 1-3 were confirmed by spectroscopic methods including 1H NMR (nuclear magnetic resonance), 13C NMR, and electrospray ionisation-mass spectrometry. Compounds 1 and 2 were purified from A. sieboldii for the first time, and all three compounds showed cytotoxicity against a panel of cancer cell lines and brine shrimps in a dose-response manner. Among them, compound 2 exhibited the highest cytotoxicity on cancer cells (IC50 values of 8.8-25.7 µM) as well as on brine shrimps (IC50 value of 5.1 µM). Compounds 1-3 exhibited anti-inflammatory effects through inhibiting protein denaturation (IC50 values of 5.8-9.6 µM), cyclooxygenase-2 activity (IC50 values of 34.5-60.1 µM), and nitrite formation in RAW 264.7 cells. Cytotoxic and anti-inflammatory activities of 1-3 demonstrated in this study deserve further investigation for considering their suitability as candidates or leads to develop anticancer and anti-inflammatory drugs.

Garlic clove extract assisted silver nanoparticle - Antibacterial, antibiofilm, antihelminthic, anti-inflammatory, anticancer and ecotoxicity assessment.

Facile and low cost garlic clove extract based silver nanoparticles was synthesized and its broad spectrum of therapeutic activity including antibiofilm, antiparasitic and anti-breast cancer activity was evaluated. The synthesized garlic­silver nanoparticles (G-AgNPs) were characterized by various physico-chemical techniques. G-AgNPs showed good optical property, highly crystalline nature, spherical shape and uniformly dispersed with size measuring between 10 and 50 nm. G-AgNPs have shown greater anti-bacterial and antibiofilm activity on clinically important pathogens methicillin-resistant S. aureus and P. aerigunosa at 100 µg ml-1. The efficacy of G-AgNPs against earthworm evidenced its effectiveness as anti-helminthic agent in treating intestinal parasites. The significant inhibition of BSA protein denaturation proves its anti-inflammatory property. In addition, G-AgNPs have shown remarkable anticancer effect and significantly inhibited the human breast cancer cell (MCF-7) viability at 100 µg ml-1 after 24 h. A noticeable change in the morphology of MCF-7 cells was also noticed. G-AgNPs were non-toxic to human HEK293 embryonic cells. Also, the non-toxic nature of G-AgNPs to C. cornuta and no morphological, physiological changes proved its safety to the environment. It is concluded that G-AgNPs have a broad range of biological applications and it can be used as an eco-friendly material without having negative effects in the environment.

Effect of soluble soybean polysaccharides on freeze-denaturation and structure of myofibrillar protein of bighead carp surimi with liquid nitrogen freezing.

This paper investigated the synergistic effect of 3% soluble soybean polysaccharides (SSPS) and liquid nitrogen freezing (-80 °C) on the freezing process and protein denaturation of bighead carp surimi. Freezing curve showed that liquid nitrogen freezing could significantly minimize the elapsed time of maximum-ice-crystal formation zone. Both liquid nitrogen freezing and SSPS were useful in preventing protein denaturation of surimi during 12-week frozen storage. Protein denaturation results indicated that SSPS-LNfreezing surimi1 had the highest protein solubility, Ca2+-ATPase activity and total sulfhydryl content. SDS-PAGE indicated that SSPS and liquid nitrogen freezing could effectively inhibit the decrease of myosin heavy chain concentration after 12 weeks of frozen storage. Raman spectra showed that tryptophan and tyrosine were exposed to polar microenvironment, the ɑ-helix and ß-sheet turned into random coil and ß-turn, and the conformation of disulfide bond changed from trans-gauche-trans (t-g-t) to gauche-gauche-trans (g-g-t). Either SSPS or liquid nitrogen freezing could mitigate these changes during frozen storage and a synergistic effect emerged on preventing myofibrillar protein denaturation and protein structure change. The combination of SSPS with liquid nitrogen freezing could be applied to freeze bighead carp surimi.

Structural Stabilization of Human Transthyretin by Centella asiatica (L.) Urban Extract: Implications for TTR Amyloidosis.

Transthyretin is responsible for a series of highly progressive, degenerative, debilitating, and incurable protein misfolding disorders known as transthyretin (TTR) amyloidosis. Since dissociation of the homotetrameric protein to its monomers is crucial in its amyloidogenesis, stabilizing the native tetramer from dissociating using small-molecule ligands has proven a viable therapeutic strategy. The objective of this study was to determine the potential role of the medicinal herb Centella asiatica on human transthyretin (huTTR) amyloidogenesis. Thus, we investigated the stability of huTTR with or without a hydrophilic fraction of C. asiatica (CAB) against acid/urea-mediated denaturation. We also determined the influence of CAB on huTTR fibrillation using transmission electron microscopy. The potential binding interactions between CAB and huTTR was ascertained by nitroblue tetrazolium redox-cycling and 8-anilino-1-naphthalene sulfonic acid displacement assays. Additionally, the chemical profile of CAB was determined by liquid chromatography quadruple time-of-flight mass spectrometry (HPLC-QTOF-MS). Our results strongly suggest that CAB bound to and preserved the quaternary structure of huTTR in vitro. CAB also prevented transthyretin fibrillation, although aggregate formation was unmitigated. These effects could be attributable to the presence of phenolics and terpenoids in CAB. Our findings suggest that C. asiatica contains pharmaceutically relevant bioactive compounds which could be exploited for therapeutic development against TTR amyloidosis.

Unveiling novel diphenyl-1H-pyrazole based acrylates tethered to 1,2,3-triazole as promising apoptosis inducing cytotoxic and anti-inflammatory agents.

Meagre and suboptimal therapeutic response along with the side effect profile associated with the existing anticancer therapy have necessitated the development of new therapeutic modalities to curb this disease. Bearing in mind the current scenario, a series of 1,2,3-triazole linked 3-(1,3-diphenyl-1H-pyrazol-4-yl)acrylates was synthesized following a multi-step reaction scheme. Initial screening for anticancer potential was done by in vitro sulforhodamine B assay against four human cancer cell lines- MCF-7 (breast), A549 (Lung) and HCT-116 and HT-29 (Colon). On evaluation, several compounds showed promising growth inhibition against all the cell lines, particularly compounds 6e, 6f and 6n. Among them, compound 6f displayed IC50 values of 1.962, 3.597, 1.764 and 4.496 µM against A549, HCT-116, MCF-7 and HT-29 cell lines respectively. Furthermore, the apoptosis inducing potential of the compounds was determined by Hoechst staining and DNA fragmentation assay. Colony formation inhibition assay was also carried out to determine the long term cytotoxic potential of the molecules. Moreover, compounds 6e, 6f and 6n were also evaluated for anti-inflammatory activity by protein albumin denaturation assay and red blood cell membrane stabilizing assay.

Monitoring of lysozyme thermal denaturation by volumetric measurements and nanoDSF technique in the presence of N-butylurea.

The results of thermal studies of denaturation of hen egg white lysozyme (HEWL) in water and an aqueous solution of N-butylurea (BU) are presented. High-precision densimetric measurements were used to characterize and analyze the changes of the specific volume, v, during temperature elevation. The temperature of the midpoint of protein denaturation was also determined by nanoDSF technique (differential scanning fluorimetry). The densities of lysozyme solutions were measured at temperatures ranging from 298.15 to 353.15 K with an interval of 5 K at atmospheric pressure (0.1 MPa). The concentration of the protein covered the range from 2 to 20 mg per 1 ml of the solution. The optimal range of the concentration for the densimetric measurements was roughly estimated. In the transition region, the structural changes of the protein are accompanied by the biggest increase of ν values with temperature. Our measurements show that this effect can be monitored from volumetric data without precise determination of protein concentration. The results prove that a two-state model of denaturation could be used for data interpretation. Contrary to common misconception, the volumetric measurements suggest that the denatured protein does not necessarily need to be in a fully extended state. In this way, the 'protein volume paradox' could be explained. The surface area of the protein remains unchanged and thus the increase of the specific volume of the protein is relatively small. Additionally, the self-stabilizing effect of the protein in BU solution was reported. For the HEWL in pure water, this phenomenon was not observed.