Browsing by Subject "Fluorescence"
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- PublicationOpen AccessA Fast and Accurate Method for the Quantification of Doxycycline in Goat Plasma and Milk by HPLC Using a Fluorescence Detector(2024-12-17) Martínez, José; Hernandis, Verónica; Badillo, Elena; Escudero, Elisa; Yuste Pérez, María Teresa; Galecio, Juan Sebastián; Marín, Pedro; FarmacologíaDoxycycline is an antimicrobial agent used in veterinary medicine to treat a variety of bacterial infections. To date, no analytical technique utilising HPLC with fl uorescence detection has been documented for the quantifi cation of doxycycline concentrations in goat plasma or milk. Consequently, the objective of the present study was to propose a rapid HPLC assay with fl uorescence detection for the quantifi cation of doxycycline in the aforementioned samples, thereby facilitating the conduct of pharmacokinetic studies and the detection of residues in diverse goat tissues. Proteins were precipitated with methanol and trifl uoroacetic acid in a single step. Doxycycline was separated on a XBRIDGE C18 column using an isocratic method. Sample volume injected into the HPLC system was 50 µl. Fluorescence detection was conducted with an excitation wavelength of 380 nm and an emission wavelength of 520 nm. The retention times of doxycycline and danofl oxacin (internal standard) were determined to be 8.0 and 5.5 minutes, respectively. The calibration curves for plasma and milk exhibited linearity over the concentration range of 0.1 to 2 μg/mL. The limit of detection was 0.065 μg/ mL, while the limit of quantifi cation was 0.1 μg/mL in both matrices. The accuracy and precision of the method were consistently within the limits of 10.9% for plasma and 10.5% for milk. The fi ndings of this study may be employed in the quantifi cation of doxycycline in goat plasma and milk, thus facilitating the conduct of pharmacokinetic studies.
- PublicationOpen AccessA way to increase the bioaccesibility and photostability of roflumilast, a COPD treatment, by cyclodextrin monomers(MDPI, 2019-05-04) Matencio Durán, Adrián; Hernández-García, Samanta; López Nicolás, José Manuel; García Carmona, Francisco; Bioquímica y Biología Molecular A; Facultad de BiologíaRoflumilast is an orally available inhibitor of phosphodiesterase (PDE) type 4, which is widely used in chronic obstructive pulmonary diseases. However, it has low solubility and adverse effects include diarrhea and nausea. Since its solubilization may improve treatment and, dismissing any adverse effects, its interaction with cyclodextrins (CDs) was studied. The Higuchi-Connors method was used to determine the complexation constant with different CDs, pH values and temperatures. Molecular docking was used to predict interaction between the complexes. An in vitro digestion experiment was carried out to test roflumilast protection. Finally, the photostability of the complex was evaluated. The complex formed with β-CD had the highest K11 value (646 ± 34 M−1), although this value decreased with increasing temperature. Similarly, K11 decreased as the pH increased. In vitro digestion showed that CDs protect the drug during digestion and even improve its bioaccessibility. Finally, CDs reduced the drug’s extreme photosensitivity, originating a fluorescence signal, which is described for first time. The kinetic parameters of the reaction were obtained. This study not only completes the complexation study of roflumilast-CD, but also points to the need to protect roflumilast from light, suggesting that tablets containing the drug might be reformulated.
- PublicationRestrictedBiosynthesis of a novel polymeric chitosan-betaxanthin and characterization of the first sugar-derived betalains and their effects in the in vivo model Caenorhabditis elegans(Elsevier, 2020-09-28) Guerrero Rubio, María Alejandra; Hernández García, Samanta; Gandía Herrero, Fernando; García Carmona, Francisco; Bioquímica y Biología Molecular "A"Betaxanthins are nitrogenous plant pigments belonging to the family of betalains and they are known for their health-promoting effects and fluorescent properties. A novel biotechnological approach in the synthesis of these compounds has allowed the synthesis of high amounts of known betalains and of novel, tailor-made betalains through the condensation of the structural unit - betalamic acid - with amine groups of different compounds. Here we describe the synthesis and characterization of chitosan-betaxanthin, the first fluorescent polymeric betaxanthin which forms nanoparticles and that might combine the fluorescent properties of betalains and the properties of chitosan, a sugar polymer widely used with medical purposes. In addition, glucosamine, the structural unit of chitosan, and its stereoisomer galactosamine were shown to condense in solution with betalamic acid. This produced novel molecules with spectral and in vivo antioxidant and anti-aging properties similar to those of biological betaxanthins, which are the first sugar-derived betaxanthins described.
- PublicationOpen AccessCharacterization of betalains, saponins and antioxidant power in differently colored quinoa (Chenopodium quinoa) varieties(Elsevier, 2017-05-02) Jiménez-Atiénzar, Mercedes; Ibáñez-Tremolada, Martha; Gómez-Pando, Luz Rayda; Cabanes Cos, Juana; Escribano Cebrián, Josefa; Gandía Herrero, Fernando; García Carmona, Francisco; Bioquímica y Biología Molecular "A"Quinoa was the traditional grain crop used by the prehispanic civilizations in America. Grains are white, black, yellow, and red-violet and plants are cultivated in vast areas of Peru, Bolivia and Ecuador. The recent description of the betacyanin pigment betanin in red-violet varieties is here further analyzed detecting the presence of amaranthin not previously identified in quinoa grains. Yellow-orange grains are characterized for the first time and up to four different betaxanthins are found to be responsible for this coloration. The native fluorescence of the identified betaxanthins makes the surface of the yellow quinoa grains glow with green fluorescent light. The presence of betalains is correlated with high antioxidant and free radical scavenging activities measured under the FRAP, ABTS and ORAC assays in grain extracts of 29 Peruvian varieties. TEAC equivalence is as high as 44.1 and 47.4 mmol Trolox/kg for the yellow and red-violet varieties analyzed respectively.
- PublicationOpen AccessFluorescent Bioinspired Protein Labeling with Betalamic Acid. Derivatization and Characterization of Novel Protein-Betaxanthins(2016-10) Francisco, García-Carmona; Mercedes, Jiménez-Atiénzar; Cabanes Cos, Juana; Escribano Cebrián, Josefa; Gandía Herrero, Fernando; Bioquímica y Biología Molecular ABetaxanthins are the water-soluble pigments that bestow yellow coloration to fruits, flowers and roots of plants of the Caryophyllales order and present autofluorescence after excitation with blue light. In this work, the semi-synthesis of betaxanthins derived from macromolecules is achieved for the first time by exploiting the reactivity of amine groups belonging to proteins. The synthesis of protein-betaxanthins is demonstrated by spectrophotometry and HPLC-ESI-TOF-MS mass analysis. The derivatization with betalamic acid was in a ratio 1:1 and yielded protein-betaxanthins yellow in color that exhibited fluorescent properties with a maximum excitation wavelength of 476 nm and a maximum emission wavelength of 551 nm. Moreover, staining can be started from purified betalamic acid or directly from raw red beet root extracts. The novel bioinspired labeling reaction allowed protein detection in conventional fluorescence scanning and imaging systems and opens a new perspective for betalamic acid derived molecules as fluorescent probes with multiple biological applications.
- PublicationOpen AccessLight emission in betalains: from fluorescent flowers to biotechnological applications(Cell Press, 2020-02) Guerrero Rubio, María Alejandra; Escribano Cebrián, Josefa; Gandía Herrero, Fernando; García Carmona, Francisco; Bioquímica y Biología Molecular AThe discovery of visible fluorescence in the plant pigments betalains revealed the existence of fluorescent patterns in flowers of plants of the order Caryophyllales, where betalains substitute anthocyanins. The serendipitous initial discovery led to a systemized characterization of the role of different substructures on the photophysical phenomenon. Strong fluorescence is general to all members of the family of betaxanthins linked to the structural property that the betalamic acid moiety is connected to an amine group. This property has led to bioinspired tailor-made probes and to the development of novel biotechnological applications in screening techniques or microscopy labeling. Here, we comprehensively review the photophysics, photochemistry, and photobiology of betalain fluorescence and describe all current applications.
- PublicationRestrictedNanoparticles of betalamic acid derivatives with cyclodextrins. Physicochemistry, production characterization and stability(Elsevier, 2020-07-14) Guerrero Rubio, Alejandra; Matencio Durán, Adrián; Gandía Herrero, Fernando; López Nicolás, José Manuel; García Carmona, Francisco; Bioquímica y Biología Molecular AThe use of betalains as bioactive compounds has been widely investigated. However, their use as a functional food requires a more exhaustive study because of their photosensitivity and poor stability. For this reason, this works studies the stabilization of two highly bioactive betalain derivatives, phenylethylamine-betaxanthin (Ph- Bx) and indoline-betacyanin (In-Bc) by nanoencapsulation in natural a modified cyclodextrins (CDs). Methyl- β-CD (Mβ-CD) showed the best results for Ph-Bx while Hydroxypropyl-β-CD (HPβ-CD) was better in the case of In- Bc. In addition, the effect of pH and temperature on encapsulation was studied finding that the encapsulation constant (KF) value increased as the pH decreased in the case of Ph-Bx but not in the case of In-Bc. The KF decreased as the temperatures increased. The thermodynamic parameters, ΔH◦, ΔS◦ and ΔG◦, were also calculated. Molecular docking provided further insight into how the different interactions influenced the encapsulation constant. The results show that the presence of CDs during the biotechnological synthesis of Ph-Bx and In- Bc increased production by 51 and 26%, respectively, and also increased the stability of both derivatives. This study represents a first step towards increasing the production of this type of compound for introduction into functional foods.
- PublicationRestrictedReversible bleaching of betalains induced by metals and application to the fluorescent determination of anthrax biomarker(Elsevier, 2020-05-01) Guerrero Rubio, M. Alejandra; Martínez Zapata, Joaquín; Henarejos Escudero, Paula; Gandía Herrero, Fernando; García Carmona, Francisco; Bioquímica y Biología Molecular ABetalains are antioxidant pigments of plant origin that are found in nature in the order Caryophyllales. They are classified into two groups: betacyanins, which present a violet coloration, and betaxanthins, fluorescent betalains with yellow coloration. In this work, the effect of metals, such as copper and europium, on the spectroscopic properties of betalains is studied. For the first time, the reversibility of spectral changes in fluorescence and absorbance is considered after the addition of chelating substances. These results explain the general bleaching of betalains by metals. The reversibility of the betalain-metal complexes is applied to the fluorescent detection of the presence of spores from Bacillus by the interaction of the complexes with the chelator dipicolinic acid, which is the main and characteristic component of endospores. This fast, economic method allows the detection of spores below the infective dose for anthrax by using a flower pigment present in Mirabilis jalapa.
- PublicationEmbargoStructural implications on color, fluorescence, and antiradical activity in betalains(2010-05-14) Escribano Cebrián, Josefa; Gandía Herrero, Fernando; García Carmona, Francisco; Departamento de Bioquímica y Biología Molecular A; Facultad de VeterinariaBetalains are water-soluble pigments with high antiradical capacity which bestow bright colors on Xowers and fruits of most plants of the order Caryophyllales. They are classiWed as betacyanins, exhibiting a violet coloration, and betaxanthins, which exhibit yellow coloration. Traditionally, betalains have been defined as condensation products of betalamic acid with different amines and amino acids, but the implication of the pigment structure for their properties has not been investigated. This paper explores different structural features of the betalains, revealing the clues for the switch from yellow to violet color, and the loss of fluorescence. A relevant series of 15 betalain-related compounds (both natural and novel semisynthetic ones) is obtained and characterized by chromatography, UV-vis spectrophotometry, fluorescence, and electrospray ionization mass spectroscopy. Antiradical properties of individual pure compounds in a broad pH range are studied under the ABTS•+ radical assay. Relevance of specific bonds is studied, and diferences between betaxanthins and betacyanins are used to explore in depth the structure–antiradical activity relationships in betalains.
- PublicationOpen AccessThe histidine phosphocarrier kinase/phosphorylase from Bacillus Subtilis is an oligomer in solution with a high thermal stability(MDPI, 2021-03-22) Neira, José L.; Cámara Artigas, Ana; Hernández Cifre, José G.; Ortore, María Grazia; Química FísicaThe histidine phosphocarrier protein (HPr) kinase/phosphorylase (HPrK/P) modulates the phosphorylation state of the HPr protein, and it is involved in the use of carbon sources by Grampositive bacteria. Its X-ray structure, as concluded from crystals of proteins from several species, is a hexamer; however, there are no studies about its conformational stability, and how its structure is modified by the pH. We have embarked on the conformational characterization of HPrK/P of Bacillus subtilis (bsHPrK/P) in solution by using several spectroscopic (namely, fluorescence and circular dichroism (CD)) and biophysical techniques (namely, small-angle X-ray-scattering (SAXS) and dynamic light-scattering (DLS)). bsHPrK/P was mainly a hexamer in solution at pH 7.0, in the presence of phosphate. The protein had a high conformational stability, with an apparent thermal denaturation midpoint of ~70 C, at pH 7.0, as monitored by fluorescence and CD. The protein was very pH-sensitive, precipitated between pH 3.5 and 6.5; below pH 3.5, it had a molten-globule-like conformation; and it acquired a native-like structure in a narrow pH range (between pH 7.0 and 8.0). Guanidinium hydrochloride (GdmCl) denaturation occurred through an oligomeric intermediate. On the other hand, urea denaturation occurred as a single transition, in the range of concentrations between 1.8 and 18 M, as detected by far-UV CD and fluorescence.
- PublicationOpen AccessThe inclusion complex of oxyresveratrol in modified cyclodextrins: a thermodynamic, structural, physicochemical, fluorescent and computational study(Elsevier, 2017-04-06) Matencio Durán, Adrián; López Nicolás, José Manuel; García Carmona, Francisco; Bioquímica y Biología Molecular A; Facultad de BiologíaThe interaction between oxyresveratrol (a type of stilbene with high biological activity) and modified cyclodextrins (CDs) was studied. Using HPLC-RP, was seen to form a 1:1 complex with all the CDs tested. The best CD in this respect was MβCD (KF = 606.65 ± 30.18 M−1), the complexation showing a strong dependence on pH and temperature: The complexation constant (KF) decreased as the pH and temperature increased. The thermodynamic parameters studied (ΔH°, ΔS° and ΔG°) showed negative entropy, enthalpy and Gibbs free energy change at 25 °C. In addition, fluorescence signal of oxyresveratrol increased when MβCD was added. The oxyresveratrol emission and excitation spectra were obtained for first time. A 1H NMR was carried out to study the structure of the complex and, DSC studied demonstrated the complexation. A computational study by molecular docking was made to complement the structural study.
- PublicationRestrictedUnfolding and refolding in vitro of a tetrameric, a-helical membrane protein: the prokaryotic potassium channel KcsA(American Chemical Society, 2005-10-06) Barrera Olivares, Francisco Nicolás; Renart Pérez, María Lourdes; Molina Gallego, María Luisa; Poveda Larrosa, José Antonio; Encinar Hidalgo, José Antonio; Fernández Carvajal, Asia María; Neira Faleiro, José Luis; González Ros, José Manuel; Bioquímica y Biología Molecular B e Inmunología2,2,2-Trifluoroethanol (TFE) effectively destabilizes the otherwise highly stable tetrameric structure of the potassium channel KcsA, a predominantly α-helical membrane protein [Valiyaveetil, F. I., Zhou, Y., and MacKinnon, R. (2002) Biochemistry 41, 10771−10777]. Here, we report that the effects on the protein structure of increasing concentrations of TFE in detergent solution include two successive protein concentration-dependent, cooperative transitions. In the first of such transitions, occurring at lower TFE concentrations, the tetrameric KcsA simultaneously increases the exposure of tryptophan residues to the solvent, partly loses its secondary structure, and dissociates into its constituent subunits. Under these conditions, simple dilution of the TFE permits a highly efficient refolding and tetramerization of the protein in the detergent solution. Moreover, following reconstitution into asolectin giant liposomes, the refolded protein exhibits nativelike potassium channel activity, as assessed by patch-clamp methods. Conversely, the second cooperative transition occurring at higher TFE concentrations results in the irreversible denaturation of the protein. These results are interpreted in terms of a protein and TFE concentration-dependent reversible equilibrium between the folded tetrameric protein and partly unfolded monomeric subunits, in which folding and oligomerization (or unfolding and dissociation in the other direction of the equilibrium process) are seemingly coupled processes. At higher TFE concentrations this is followed by the irreversible conversion of the unfolded monomers into a denatured protein form.