Induced Chirality in Sulfasalazine by Complexation With Albumins: Theoretical and Experimental Study.

Through molecular recognition, drugs can interact and complex with macromolecules circulating in the body. The serum albumin transport protein, found in several mammals, has several interaction sites where these molecules can be located. The drug sulfasalazine (SSZ) is known in the literature to complex at drug site 1 (DS1) in human serum (HSA) and bovine serum (BSA) proteins. This complexation can be studied using various spectroscopic techniques. With the techniques used in this work, absorption in the ultraviolet and visible regions (UV-Vis) and electronic circular dichroism (ECD), a significant difference was observed in the results involving HSA and BSA. The application of theoretical methodologies, such as TD-DFT and molecular docking, suggests that the conformation that SSZ assumes in DS1 of the two proteins is different, which exposes it to different amino acid residues and different hydrophobicities. This difference in conformation may be related to the location of DS1 where the drug interacts or to the possibility of SSZ moving in the BSA site, due to its larger size, and moving less freely in HSA.

In vivo modification of the enamel pellicle and saliva resveratrol levels after use of resveratrol-containing orodispersible capsules.

OBJECTIVES: To evaluate in vivo 1) the bioavailability of trans-resveratrol when administered through sublingual capsules; 2) the effect of resveratrol on the protein composition of the acquired enamel pellicle (AEP). DESIGN: Ten volunteers received a sublingual capsule containing 50 mg of trans-resveratrol. Unstimulated saliva was then collected after 0, 30, 60, and 120 min and AEP was collected after 120 min following administration of the capsule. In the next week, the volunteers received a placebo sublingual capsule, and saliva and AEP were collected again. Saliva samples were analyzed for free trans-resveratrol using high-performance liquid chromatopgraphy (HPLC), and AEP samples were subjected to proteomic analysis (nLC-ESI-MS/MS). RESULTS: Trans-resveratrol was detected in saliva at all the time points evaluated, with the peak at 30 min. A total of 242 proteins were identified in both groups. Ninety-six proteins were increased and 23 proteins were decreased in the Resveratrol group. Among the up-regulated proteins, isoforms of cystatins, PRPs, Mucin-7, Histatin-1, Lactotrasnferrin and Lysozyme-C were increased and the isoforms of Protein S100, Neutrophil defensins, Albumin, PRPs, and, Statherin were decreased in Resveratrol group. CONCLUSION: The sublingual capsule is effective at increasing the bioavailability of trans-resveratrol in saliva. Several proteins involved in important processes to maintain systemic and oral health homeostasis were identified. These proteins differently expressed due to the presence of trans-resveratrol deserve attention for future studies, since they have important functions, mainly related to antimicrobial action.

Circular dichroism spectrum of (R)-(+)-3,3'-dibromo-1,1'-bi-2-naphthol in albumin: Alterations caused by complexation-Experimental and in silico investigation.

This study describes the interaction of human serum albumin (HSA) with the binol derivative (R)-(+)-3,3'-dibromo-1,1'-bi-2-naphthol (R-BrB), which has its optical activity based on the prohibitive energetic barrier for conversion into the enantiomer (S)-(+)-3,3'-dibromo-1,1'-bi-2-naphthol (S-BrB). The objective was to assess the ability of HSA to differentiate axial enantiomers based on their binding efficiency and their impact on the CD spectra. We discovered that both enantiomers were effective ligands, and the CD signal disappeared when equimolar amounts of R-BrB and S-BrB were simultaneously added, indicating no preference for either enantiomer. The complexation resulted in a significant signal increase at 250 nm and a bathochromic effect at 370 nm. Molecular docking simulations were performed, and the lower energy pose of R-BrB was selected for DFT calculations. The theoretical CD spectra of free and complexed R-BrB were obtained and showed alterations corroborating the experimental results. By comparing the difference spectrum (HSA:R-BrB minus HSA) with the spectrum of free RBrB in water or ethyl alcohol, we concluded that the CD signal intensification was due to the increased solubilization of R-BrB upon binding to HSA.

Role for Carboxylic Acid Moiety in NSAIDs: Favoring the Binding at Site II of Bovine Serum Albumin.

The molecular structures of nonsteroidal anti-inflammatory drugs (NSAIDs) vary, but most contain a carboxylic acid functional group (RCOOH). This functional group is known to be related to the mechanism of cyclooxygenase inhibition and also causes side effects, such as gastrointestinal bleeding. This study proposes a new role for RCOOH in NSAIDs: facilitating the interaction at the binding site II of serum albumins. We used bovine serum albumin (BSA) as a model to investigate the interactions with ligands at site II. Using dansyl-proline (DP) as a fluorescent site II marker, we demonstrated that only negatively charged NSAIDs such as ibuprofen (IBP), naproxen (NPX), diflunisal (DFS), and ketoprofen (KTP) can efficiently displace DP from the albumin binding site. We confirmed the importance of RCOO by neutralizing IBP and NPX through esterification, which reduced the displacement of DP. The competition was also monitored by stopped-flow experiments. While IBP and NPX displaced DP in less than 1 s, the ester derivatives were ineffective. We also observed a higher affinity of negatively charged NSAIDs using DFS as a probe and ultrafiltration experiments. Molecular docking simulations showed an essential salt bridge between the positively charged residues Arg409 and Lys413 with RCOO-, consistent with the experimental findings. We performed a ligand dissociation pathway and corresponding energy analysis by applying molecular dynamics. The dissociation of NPX showed a higher free energy barrier than its ester. Apart from BSA, we conducted some experimental studies with human serum albumin, and similar results were obtained, suggesting a general effect for other mammalian serum albumins. Our findings support that the RCOOH moiety affects not only the mechanism of action and side effects but also the pharmacokinetics of NSAIDs.

Increase in plasma resveratrol levels and in acid-resistant proteins in the acquired enamel pellicle after use of resveratrol-containing orodispersible tablets.

OBJECTIVE: This study evaluated the effect of administration of trans-resveratrol-containing orodispersible tablets on the protein composition of the AEP and on blood plasma trans-resveratrol concentrations. METHODS: Ten volunteers participated in two crossover double-blind phases. In each phase, after dental prophylaxis, they received a trans-resveratrol (15 mg) orodispersible tablet, or a placebo tablet (without actives). The AEP formed after 120 min was collected with electrode filter papers soaked in 3 % citric acid. Blood samples were collected 30, 45, 60 and 120 min after the use of the tablet. After protein extraction, AEP samples were analyzed by shotgun labelfree quantitative proteomics and plasma samples were analyzed by high-performance liquid chromatography (HPLC). RESULTS: Eight hundred and two proteins were identified in the AEP. Among them, 336 and 213 were unique to the trans-resveratrol and control groups, respectively, while 253 were common to both groups. Proteins with important functions in the AEP had increased expression in the trans-resveratroltreated group, such as neutrophil defensins, S100 protein isoforms, lysozyme C, cystatin-D, mucin-7, alphaamylase, albumin, haptoglobin and statherin. Trans-resveratrol was detected in the plasma at all the times evaluated, with the peak at 30 min. CONCLUSIONS: The administration of trans-resveratrol in sublingual orodispersible tablets was effective both to increase the bioavailability of the polyphenol and the expression of antibacterial and acid-resistant proteins in the AEP, which might benefit oral and general health.

Menadione and protocatechuic acid: A drug combination with antitumor effects in murine osteosarcoma cells.

Osteosarcoma (OS) is a primary malignant bone tumor that has an abnormal expression of oncogenesis and tumor suppressors and causes dysregulation of various signaling pathways. Thus, novel therapeutic strategies for OS are needed to overcome the resistance of traditional treatments. This study evaluated the cytotoxic and anticancer effects of the association between menadione (MEN) and protocatechuic acid (PCA) in murine OS cells (UMR-106). The concentrations were 3.12 µM of isolated MEN, 500 µM of isolated PCA, and their associations. We performed cell viability assays, morphology modification analysis, cell migration by the wound-healing method, apoptosis by flow cytometry, reactive oxygen species (ROS) production, gene expression of NOX by RT-qPCR, and degradation of MMP-2 and 9 by zymography. Our results showed that the association of MEN+PCA was more effective in OS cells than the compounds alone. The association decreased cell viability, delayed cell migration, and decreased the expression of NOX-2 and ROS. In addition, the MEN+PCA association induced a slight increase in the apoptotic process. In summary, the association can enhance the compound's antitumor effects and establish a higher selectivity for tumor cells, possibly caused by significant mitochondrial damage and antioxidant properties.

Toxicological Profile and Anti-Inflammatory Effect of Mucoadhesive Gel from Residues of Agave sisalana and Punica granatum.

Inflammation is a natural protective reaction of the body against endogenous and exogenous damage, such as tissue injuries, trauma, and infections. Thus, when the response is adequate, inflammation becomes a defense mechanism to repair damaged tissue, whereas when the response is inadequate and persistent, the increase in inflammatory cells, cytosines, and chymosins impair tissue regeneration and promote a response harmful to the organism. One example is chronic tissue inflammation, in which a simple lesion can progress to ulcers and even necrosis. In this situation, the anti-inflammatory medications available in therapy are not always effective. For this reason, the search for new treatments, developed from medicinal plants, has increased. In this direction, the plants Agave sisalana (sisal) and Punica granatum (pomegranate) are rich in saponins, which are secondary metabolites known for their therapeutic properties, including anti-inflammatory effects. Although Brazil is the world's leading sisal producer, approximately 95% of the leaves are discarded after fiber extraction. Similarly, pomegranate peel waste is abundant in Brazil. To address the need for safe and effective anti-inflammatory treatments, this study aimed to create a topical mucoadhesive gel containing a combination of sisal (RS) and pomegranate residue (PR) extracts. In vitro experiments examined isolated and combined extracts, as well as the resulting formulation, focusing on (1) a phytochemical analysis (total saponin content); (2) cytotoxicity (MTT assay); and (3) a pharmacological assessment of anti-inflammatory activity (phagocytosis, macrophage spreading, and membrane stability). The results revealed saponin concentrations in grams per 100 g of dry extract as follows: SR-29.91 ± 0.33, PR-15.83 ± 0.93, association (A)-22.99 ± 0.01, base gel (G1)-0.00 ± 0.00, and association gel (G2)-0.52 ± 0.05. In MTT tests for isolated extracts, cytotoxicity values (µg/mL) were 3757.00 for SR and 2064.91 for PR. Conversely, A and G2 exhibited no cytotoxicity, with increased cell viability over time. All three anti-inflammatory tests confirmed the presence of this activity in SR, PR, and A. Notably, G2 demonstrated an anti-inflammatory effect comparable to dexamethasone. In conclusion, the gel containing SR and PR (i.e., A) holds promise as a novel herbal anti-inflammatory treatment. Its development could yield economic, social, and environmental benefits by utilizing discarded materials in Brazil.

Solvent-Induced Lag Phase during the Formation of Lysozyme Amyloid Fibrils Triggered by Sodium Dodecyl Sulfate: Biophysical Experimental and In Silico Study of Solvent Effects.

Amyloid aggregates arise from either the partial or complete loss of the native protein structure or the inability of proteins to attain their native conformation. These aggregates have been linked to several diseases, including Alzheimer's, Parkinson's, and lysozyme amyloidosis. A comprehensive dataset was recently reported, demonstrating the critical role of the protein's surrounding environment in amyloid formation. In this study, we investigated the formation of lysozyme amyloid fibrils induced by sodium dodecyl sulfate (SDS) and the effect of solvents in the medium. Experimental data obtained through fluorescence spectroscopy revealed a notable lag phase in amyloid formation when acetone solution was present. This finding suggested that the presence of acetone in the reaction medium created an unfavorable microenvironment for amyloid fibril formation and impeded the organization of the denatured protein into the fibril form. The in silico data provided insights into the molecular mechanism of the interaction between acetone molecules and the lysozyme protofibril, once acetone presented the best experimental results. It was observed that the lysozyme protofibril became highly unstable in the presence of acetone, leading to the complete loss of its ß-sheet conformation and resulting in an open structure. Furthermore, the solvation layer of the protofibril in acetone solution was significantly reduced compared to that in other solvents, resulting in fewer hydrogen bonds. Consequently, the presence of acetone facilitated the exposure of the hydrophobic portion of the protofibril, precluding the amyloid fibril formation. In summary, our study underscores the pivotal role the surrounding environment plays in influencing amyloid formation.

A flaw in applying the FRET technique to evaluate the distance between ligands and tryptophan residues in human serum albumin: Proposal of correction.

Due to its primordial function as a drug carrier, human serum albumin (HSA) is extensively studied regarding its binding affinity with developing drugs. Förster resonance energy transfer (FRET) is frequently applied as a spectroscopic molecular ruler to measure the distance between the binding site and the ligand. In this work, we have shown that most of the published results that use the FRET technique to estimate the distance from ligands to the binding sites do not corroborate the crystallography data. By comparing the binding affinity of dansyl-proline with HSA and ovotransferrin, we demonstrated that FRET explains the quenching provoked by the interaction of ligands in albumin. So, why does the distance calculation via FRET not corroborate the crystallography data? We have shown that this inconsistency is related to the fact that a one-to-one relationship between donor and acceptor is not present in most experiments. Hence, the quenching efficiency used for calculating energy transfer depends on distance and binding constant, which is inconsistent with the correct application of FRET as a molecular ruler. We have also shown that the indiscriminate attribution of 2/3 to the relative orientation of transition dipoles of the acceptor and donor (κ2) generates inconsistencies. We proposed corrections based on the experimental equilibrium constant and theoretical orientation of transition dipoles to correct the FRET results.

Antiglycation, antioxidant and cytotoxicity activities of crude extract of Turnera ulmifolia L. before and after microencapsulation process.

Turnera ulmifolia L. is used in folk medicine and it is known to have anti-hyperglycemic effect on the organism in order to reduce complications in diabetic patients. Glycation process is directly related to oxidative stress, acting as an important endogenous source, inducing the production of free radicals, and thus increasing the production of reactive oxygen species. The encapsulation technology on natural compounds can minimize and even mitigate the risk of loss of biological activity in order to maintain their activities against oxidative stress and glycation. The present study aimed to evaluate the antiglycation and antioxidant activities of T. ulmifolia extract before and after encapsulation and cytotoxicity of the crude extract. This study presents important information about the biological activities, highlighting antioxidant and antiglycation potential and no cytotoxicity of Turnera ulmifolia crude extract, a species of genus Turnera that has been poorly studied. T. ulmifolia crude extract presented flavonoids as main active compounds. The results showed a promising activity in scavenging free and peroxyl radicals, chelating iron ions and inhibiting BSA glycation. In addition, this study showed the possible encapsulation of bioactive compounds using maltodextrin as wall material.

The dimerization of methyl vanillate improves its effect against breast cancer cells via pro-oxidant effect.

Phenolic phytochemicals are a group of organic compounds with potent antioxidant features but can also act as powerful pro-oxidants. These characteristics are effective in reducing metastatic potential in cancer cells, and this effect has been associated with reactive oxygen species (ROS). Methyl vanillate (MV) and its dimer, methyl divanillate (DMV), are potent antioxidants. In the present study, we investigated the effects of MV and DMV on breast cancer cell lines MCF-7 and MDA-MB-231 and compared the results using the non-tumor cell line HB4a. Our results indicated that the compounds performed a pro-oxidant action, increasing the generation of ROS. DMV decreased the viability cell, showing a higher apoptotic effect and inhibition of proliferation than MV on both cell lines, with significant differences between groups (p < 0.05). Some modulation of NOX4, NOX5, and DUOX were observed, but the results did not correlate with the intracellular production of ROS. The dimer showed more effectivity and pro-oxidant effect than MV, impacting cell line MCF-7 in higher extension than MDA-MB-231. In conclusion, and corroborating with reported works, the dimerization of natural phenolic compounds was associated with improved beneficial biological effects as a potential cytotoxic agent to tumor cells.

Antiglycation and antioxidant activities of the crude extract and saponin fraction of Tribulus terrestris before and after microcapsule release.

OBJECTIVE: The present study investigated antiglycation and antioxidant activities of crude dry extract and saponin fraction of Tribulus terrestris. It also developed a method of microencapsulation and evaluated antiglycation and antioxidant activities of crude dry extract and saponin fraction before and after microcapsule release. METHODS: Antiglycation activity was determined by relative electrophoretic mobility (REM), free amino groups and inhibition of advanced glycation end-product (AGE) formation. Antioxidant activity was determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric ion-reducing antioxidant power (FRAP), nitric oxide (NO) and thiobarbituric acid reactive species (TBARS) tests. Microcapsules were prepared using maltodextrin as wall material and freeze-drying as encapsulation technique. Morphological characterization of microcapsules was evaluated by scanning electron microscopy, and encapsulation efficiency and microcapsule release were determined by total saponins released. Antiglycation and antioxidant assays were performed using crude dry extract and saponin fraction of T. terrestris before and after release. RESULTS: Saponin fraction showed an increase of 32.8% total saponins. High-performance liquid chromatography-mass spectrometry analysis showed the presence of saponins in the obtained fraction. Antiglycation evaluation by REM demonstrated that samples before and after release presented antiglycation activity similar to bovine serum albumin treated with aminoguanidine. Additionally, samples inhibited AGE formation, highlighting treatment with saponin fraction after release (89.89%). Antioxidant tests demonstrated antioxidant activity of the samples. Crude dry extract before encapsulation presented the highest activities in DPPH (92.00%) and TBARS (32.49%) assays. Saponin fraction before encapsulation in FRAP test (499 µmol Trolox equivalent per gram of dry sample) and NO test (15.13 µmol nitrite formed per gram of extract) presented the highest activities. CONCLUSION: This study presented antiglycation activity of crude dry extract and saponin fraction of T. terrestris, besides it demonstrated promising antioxidant properties. It also showed that the encapsulation method was efficient and maintained biological activity of bioactive compounds after microcapsule release. These results provide information for further studies on antidiabetic and antiaging potential, and data for new herbal medicine and food supplement formulations containing microcapsules with crude extract and/or saponin fraction of T. terrestris.

Anti-tumour potential and selectivity of caffeic acid phenethyl ester in osteosarcoma cells.

Osteosarcoma is the most common type of bone cancer, and metastasis is widespread decreasing the survival rate. The search for new therapeutic strategies has increased for phytochemicals due to their potential as antioxidants and anticancer properties. Thus, we evaluated the caffeic acid phenethyl ester (CAPE) and caffeic acid's (CA) anticancer properties on UMR-106 murine osteosarcoma cells. The IC25 and IC50 were 1.3 and 2.7 µM for CAPE and 91.0 and 120.0 µM for CA, respectively. This study shows the potential anticancer properties of CAPE and highlights how a phenethyl ester component addition can improve the pharmacological potency in relation to its precursor CA. Our results showed that CAPE was more efficient and selective in reducing the viability of tumor cells compared to the control osteoblasts (MC3T3-E1) (p < 0.05). In addition, CAPE was 44-fold (IC25) and 70-fold (IC50) more cytotoxic than CA. CAPE also decreased ROS generation and cell migration. In summary, CAPE was more selective for tumor cells, preserving normal ones, suggesting its potential role as an anticancer drug.

2-acetylphenothiazine protects L929 fibroblasts against UVB-induced oxidative damage.

Ultraviolet B (UVB) light corresponds to 5% of ultraviolet radiation. It is more genotoxic and mutagenic than UVA and causes direct and indirect cellular damage through the generation of reactive oxygen species (ROS). Even after radiation, ROS generation may continue through activation of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) enzyme. Long-term exposure can progress to premature skin aging and photocarcinogenesis. To prevent damage that is caused by UVB radiation, several studies have focused on the topical administration of compounds that have antioxidant properties. 2-Acetylphenothiazine (ML171) is a potent and selective inhibitor of NOX1. The present study investigated the antioxidant potential and photoprotective ability of ML171 in UVB-irradiated L929 fibroblasts. ML171 had considerable antioxidant activity in both the DPPH• and xanthine/luminol/xanthine oxidase assays. ML171 did not induce cytotoxicity in L929 fibroblasts and increased the viability of UVB-irradiated cells. ML171 also inhibited ROS production, the enzymatic activity of NOX, depolarization of the mitochondrial membrane, and DNA damage. Additionally, ML171 protected cell membrane integrity and induced fibroblast migration. These results suggest that the incorporation of ML171 in topical administration systems may be a promising strategy to mitigate UVB-induced oxidative damage in L929 fibroblasts.

MOF-Based Erodible System for On-Demand Release of Bioactive Flavonoid at the Polymer-Tissue Interface.

Plant-derived compounds incite applications virtually on every biomedical field due to the expedient antioxidant, anti-inflammatory and antimicrobial properties in conjunction with a natural character. Here, quercetin (QCT), a flavonoid with therapeutic potentials relevant to the oral environment, was encapsulated within metal-organic frameworks (MOFs) to address the concept of on-demand release of phytochemicals at the biointerface. We verified the applicability of a microporous MOF (ZIF-8) as a controlled-release system for QCT, as well as investigated the incorporation of QCT@ZIF-8 microparticles into a dental adhesive resin for desirable therapeutic capabilities at the tooth-restoration interface. QCT was encapsulated within the frameworks through a water-based, one-step synthetic process. The resulting QCT@ZIF-8 microparticles were characterized with respect to chemical composition, crystal structure, thermal behavior, micromorphology, and release profile under acidic and physiological conditions. A model dental adhesive formulation was enriched with the bioactive microparticles; both the degree of conversion (DC) of methacrylic double bonds and the polymer thermal behavior were accounted for. The results confirm that crystalline QCT@ZIF-8 microparticles with attractive loading capacities, submicron sizes, high thermal stability and responsiveness to environmental pH change were successfully manufactured. The concentration of QCT@ZIF-8 in the resin system was a key factor to maintain an optimal DC plateau and rate of polymerization. Essentially, one-step encapsulation of QCT in biocompatible ZIF-8 matrices can be easily achieved, and QCT@ZIF-8 microparticles proved as smart platforms to carry bioactive compounds with potential use to prevent microbial and enzymatic degradation of hard tissues and extracellular matrix components.

Paracoccidioides brasiliensis Releases a DNase-Like Protein That Degrades NETs and Allows for Fungal Escape.

Paracoccidioidomycosis is a systemic fungal disease, considered endemic in Latin America. Its etiological agents, fungi of the Paracoccidioides complex, have restricted geographic habitat, conidia as infecting form, and thermo-dimorphic characteristics. Polymorphonuclear neutrophils (PMNs) are responsible for an important defense response against fungus, releasing Neutrophil Extracellular Traps (NETs), which can wrap and destroy the yeasts. However, it has been described that some pathogens are able to evade from these DNA structures by releasing DNase as an escape mechanism. As different NETs patterns have been identified in PMNs cultures challenged with different isolates of Paracoccidioides brasiliensis, the general objective of this study was to identify if different patterns of NETs released by human PMNs challenged with Pb18 (virulent) and Pb265 (avirulent) isolates would be correlated with fungal ability to produce a DNase-like protein. To this end, PMNs from healthy subjects were isolated and challenged in vitro with both fungal isolates. The production, release, and conformation of NETs in response to the fungi were evaluated by Confocal Microscopy, Scanning Microscopy, and NETs Quantification. The identification of fungal DNase production was assessed by DNase TEST Agar, and the relative gene expression for hypothetical proteins was investigated by RT-qPCR, whose genes had been identified in the fungal genome in the GenBank (PADG_11161 and PADG_08285). It was possible to verify the NETs release by PMNs, showing different NETs formation when in contact with different isolates of the fungus. The Pb18 isolate induced the release of looser, larger, and more looking like degraded NETs compared to the Pb265 isolate, which induced the release of denser and more compact NETs. DNase TEST Agar identified the production of a DNase-like protein, showing that only Pb18 showed the capacity to degrade DNA in these plates. Besides that, we were able to identify that both PADG_08528 and PADG_11161 genes were more expressed during interaction with neutrophil by the virulent isolate, being PADG_08528 highly expressed in these cultures, demonstrating that this gene could have a greater contribution to the production of the protein. Thus, we identified that the virulent isolate is inducing more scattered and loose NETs, probably by releasing a DNase-like protein. This factor could be an important escape mechanism used by the fungus to escape the NETs action.

Interaction between 1-pyrenesulfonic acid and albumin: Moving inside the protein.

Due to the high sensitivity to alterations in microenvironment polarity of macromolecules, pyrene and its derivatives have long been applied in biosciences. Human serum albumin (HSA), besides its numerous physiological functions, is the main responsible by transport of endogenous and exogenous compounds in the circulatory system. Here, a comprehensive study was carry out to understand the interaction between HSA and the pyrene derivative 1-pyrenesulfonic acid (PMS), which showed a singular behaviour when bound to this protein. The complexation of PMS with HSA was studied by steady state, time-resolved and anisotropy fluorescence, induction of circular dichroism (ICD) and molecular docking. The fluorescence quenching of PMS by HSA was abnormal, being stronger at lower concentration of the quencher. Similar behaviour was obtained by measuring the ICD signal and fluorescence lifetime of PMS complexed in HSA. The displacement of PMS by site-specific drugs showed that this probe occupied both sites, but with higher affinity for site II. The movement of PMS between these main binding sites was responsible by the abnormal effect. Using the holo (PDB: ID 1A06) and apo (PDB: ID 1E7A) HSA structures, the experimental results were corroborated by molecular docking simulation. The abnormal spectroscopic behaviour of PMS is related to its binding in different regions in the protein. The movement of PMS into the protein can be traced by alteration in the spectroscopic signals. These findings bring a new point of view about the use of fluorescence quenching to characterize the interaction between albumin and ligands.

Oxidative Alteration of Trp-214 and Lys-199 in Human Serum Albumin Increases Binding Affinity with Phenylbutazone: A Combined Experimental and Computational Investigation.

Human serum albumin (HSA) is a target for reactive oxygen species (ROS), and alterations of its physiological functions caused by oxidation is a current issue. In this work, the amino-acid residues Trp-214 and Lys-199, which are located at site I of HSA, were experimentally and computationally oxidized, and the effect on the binding constant with phenylbutazone was measured. HSA was submitted to two mild oxidizing reagents, taurine monochloramine (Tau-NHCl) and taurine dibromamine (Tau-NBr2). The oxidation of Trp-214 provoked spectroscopic alterations in the protein which were consistent with the formation of N'-formylkynurenine. It was found that the oxidation of HSA by Tau-NBr2, but not by Tau-NHCl, provoked a significant increase in the association constant with phenylbutazone. The alterations of Trp-214 and Lys-199 were modeled and simulated by changing these residues using the putative oxidation products. Based on the Amber score function, the interaction energy was measured, and it showed that, while native HSA presented an interaction energy of -21.3 kJ/mol, HSA with Trp-214 altered to N'-formylkynurenine resulted in an energy of -28.4 kJ/mol, and HSA with Lys-199 altered to its carbonylated form resulted in an energy of -33.9 kJ/mol. In summary, these experimental and theoretical findings show that oxidative alterations of amino-acid residues at site I of HSA affect its binding efficacy.

Solvent-dependent inversion of circular dichroism signal in naproxen: An unusual effect!

The electronic circular dichroism (ECD) spectra of naproxen enantiomers were studied as a function of solvents using experimental (circular dichroism) and theoretical (time-dependent density functional theory) approaches. The (R)- and (S)-naproxen enantiomers presented an unusual inversion in their ECD signals in the presence of ethanol and water when compared with polar aprotic solvents such as acetonitrile. From a practical point of view, these findings deserve great attention because these solvents are widely used for high-performance liquid chromatography analysis in quality control of chiral pharmaceutical drugs. This is particularly relevant to naproxen because the (S)-naproxen has anti-inflammatory properties, whereas (R)-naproxen is hepatotoxic. A time-dependent density functional theory computer simulation was conducted to investigate the signal inversion using the solvation model based on density, a reparameterization of polarized continuum model. Electronic circular dichroism signals of conformers were calculated by computer simulation and their contribution to the combined spectra obtained according to Boltzmann weighting. It was found that the experimentally observed ECD signal inversion can be associated with the minor or major contribution of different conformers of naproxen.

Dansylglycine, a fluorescent probe for specific determination of halogenating activity of myeloperoxidase and eosinophil peroxidase.

Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are enzymes present in neutrophil and eosinophil leukocytes, respectively. Here, we present the development of a sensitive and specific assay for determination of the halogenating enzymatic activity of MPO and EPO based on the electrophilic attack of HOCl and HOBr on aromatic ring of dansylglycine (DG). We found that the intrinsic fluorescence of DG was promptly depleted by the action of these acids. In the presence of the enzymes, the fluorescence bleaching was dependent of chloride (Cl-) and bromide (Br-), which makes the assay able to distinguish the halogenating from the peroxidase activity. A linear correlation was obtained between the hydrogen peroxide (H2O2) concentration and the fluorescent decay. Similarly, the enzyme activity was measured by keeping constant H2O2. The method was applied for studding MPO/EPO specific inhibitors as 5-fluortryptamine (reversible inhibitor) and 4-hydroxybenzhydrazide (irreversible inhibitor). Differently of the taurine chloramine/3,3',5,5'-tetramethylbenzidine assay, which is among the most used technique, the dansylglycine assay was able to differentiate these inhibitors based on their kinetic behavior. In conclusion, this assay can differentiate the peroxidase and halogenating activity of MPO and EPO. Moreover, the method is adequate for real-time measurement of the production of HOCl and HOBr.