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71.
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Supramolecular interaction of 18‐crown‐6 ether with mesalazine and spectrofluorimetric determination of mesalazine in pharmaceutical formulations 下载免费PDF全文
Abdalla A. Elbashir Fatima Altayib Alasha Abdalla Hassan Y. Aboul‐Enein 《Luminescence》2015,30(8):1250-1256
The supramolecular interaction of protonated mesalazine (MSZ) and 18‐crown‐6 ether (18C6) has been examined by Ultraviolet–visible, FT‐IR and fluorescence spectroscopy. The formation of the inclusion complex has been confirmed based on the changes of the spectral properties. The MSZ–18C6 host–guest complex formed in (1:1) stoichiometry and the inclusion constant (K = 1.411 × 102 L mol–1) was ascertained by the typical double reciprocal plots. Furthermore, the thermodynamic parameters (ΔG°, ΔH° and ΔS°) of (MSZ‐18C6) were obtained. Based on the remarkable enhancement of the fluorescence intensity of MSZ produced through complex formation, a simple, accurate, rapid and highly sensitive spectrofluorometric method for the determination of MSZ in aqueous solution in the presence of 18C6 was developed. The measurement of relative fluorescence intensity was carried with excitation at 298 nm, emission 410 nm. All variables affecting the reactions were studied and optimized. Beer's law was obeyed in the concentration range of 0.1–0.9 µg/mL. The absorbance was found to increase linearly with increasing concentration of MSZ. The molar absorptivity, Sandell sensitivity, limit of detection (LOD) and limit of quantification (LOQ) were calculated. The validity of the described method was assessed, and the method was successfully applied to the determination of MSZ in its pharmaceutical formulation. In addition, a solid inclusion complex was synthesized by the coprecipitation method. Copyright © 2015 John Wiley & Sons, Ltd. 相似文献
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Bernd Anselment Danae Baerend Elisabeth Mey Johannes Buchner Dirk Weuster-Botz Martin Haslbeck 《Protein science : a publication of the Protein Society》2010,19(11):2085-2095
Refolding of proteins from solubilized inclusion bodies still represents a major challenge for many recombinantly expressed proteins and often constitutes a major bottleneck. As in vitro refolding is a complex reaction with a variety of critical parameters, suitable refolding conditions are typically derived empirically in extensive screening experiments. Here, we introduce a new strategy that combines screening and optimization of refolding yields with a genetic algorithm (GA). The experimental setup was designed to achieve a robust and universal method that should allow optimizing the folding of a variety of proteins with the same routine procedure guided by the GA. In the screen, we incorporated a large number of common refolding additives and conditions. Using this design, the refolding of four structurally and functionally different model proteins was optimized experimentally, achieving 74–100% refolding yield for all of them. Interestingly, our results show that this new strategy provides optimum conditions not only for refolding but also for the activity of the native enzyme. It is designed to be generally applicable and seems to be eligible for all enzymes. 相似文献
76.
Krastyu G. Ugrinov 《Biophysical journal》2010,98(7):1312-1320
Protein sequences evolved to fold in cells, including cotranslational folding of nascent polypeptide chains during their synthesis by the ribosome. The vectorial (N- to C-terminal) nature of cotranslational folding constrains the conformations of the nascent polypeptide chain in a manner not experienced by full-length chains diluted out of denaturant. We are still discovering to what extent these constraints affect later, posttranslational folding events. Here we directly address whether conformational constraints imposed by cotranslational folding affect the partitioning between productive folding to the native structure versus aggregation. We isolated polyribosomes from Escherichia coli cells expressing GFP, analyzed the nascent chain length distribution to determine the number of nascent chains that were long enough to fold to the native fluorescent structure, and calculated the folding yield for these nascent chains upon ribosome release versus the folding yield of an equivalent concentration of full-length, chemically denatured GFP polypeptide chains. We find that the yield of native fluorescent GFP is dramatically higher upon ribosome release of nascent chains versus dilution of full-length chains from denaturant. For kinetically trapped native structures such as GFP, folding correctly the first time, immediately after release from the ribosome, can lead to lifelong population of the native structure, as opposed to aggregation. 相似文献
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Using the whole-vacuolar mode of the patch clamp technique, we studied the effect of the chaotropic anions thiocyanate and nitrate on the electric currents generated by the proton pumping tonoplast ATPase and pyrophosphatase (PPiase), respectively, in vacuoles from suspension cells of Chenopodium rubrum L. Addition of KNO3 (150–250 m M ) or KSCN (70–150 m M ), and ATP (5 m M , obligatory) irreversibly inhibited the subsequent electric current through the tonoplast ATPase driven by 1 m M ATP, whereas PPiase-activity by 50 μ M PPi remained unaffected. The kinetics of inhibition, indicative of ATPase disintegration by the chaotropic anions, follows a single exponential (τ= 3.44 min). However, apparent ATPase disintegration did not measurably increase the tonoplast conductance. We conclude that, by contrast to organellar F-ATPases, upon disintegration the transmembrane proteolipid of the V-ATPase does not act as a proton conductor which, in the presence of chaotropic anions, like chloride or nitrate, would severely perturb solute compartmentation in the plant cell. 相似文献
78.
Localization of vacuolar transport receptors and cargo proteins in the Golgi apparatus of developing Arabidopsis embryos 总被引:5,自引:1,他引:4
Hinz G Colanesi S Hillmer S Rogers JC Robinson DG 《Traffic (Copenhagen, Denmark)》2007,8(10):1452-1464
Using immunogold electron microscopy, we have investigated the relative distribution of two types of vacuolar sorting receptors (VSR) and two different types of lumenal cargo proteins, which are potential ligands for these receptors in the secretory pathway of developing Arabidopsis embryos. Interestingly, both cargo proteins are deposited in the protein storage vacuole, which is the only vacuole present during the bent-cotyledon stage of embryo development. Cruciferin and aleurain do not share the same pattern of distribution in the Golgi apparatus. Cruciferin is mainly detected in the cis and medial cisternae, especially at the rims where storage proteins aggregate into dense vesicles (DVs). Aleurain is found throughout the Golgi stack, particularly in the trans cisternae and trans Golgi network where clathrin-coated vesicles (CCVs) are formed. Nevertheless, aleurain was detected in both DV and CCV. VSR-At1, a VSR that recognizes N-terminal vacuolar sorting determinants (VSDs) of the NPIR type, localizes mainly to the trans Golgi and is hardly detectable in DV. Receptor homology-transmembrane-RING H2 domain (RMR), a VSR that recognizes C-terminal VSDs, has a distribution that is very similar to that of cruciferin and is found in DV. Our results do not support a role for VSR-At1 in storage protein sorting, instead RMR proteins because of their distribution similar to that of cruciferin in the Golgi apparatus and their presence in DV are more likely candidates. Aleurain, which has an NPIR motif and seems to be primarily sorted via VSR-At1 into CCV, also possesses putative hydrophobic sorting determinants at its C-terminus that could allow the additional incorporation of this protein into DV. 相似文献
79.
Sergio Couoh-Cardel Elena Milgrom Stephan Wilkens 《The Journal of biological chemistry》2015,290(46):27959-27971
The membrane sector (Vo) of the proton pumping vacuolar ATPase (V-ATPase, V1Vo-ATPase) from Saccharomyces cerevisiae was purified to homogeneity, and its structure was characterized by EM of single molecules and two-dimensional crystals. Projection images of negatively stained Vo two-dimensional crystals showed a ring-like structure with a large asymmetric mass at the periphery of the ring. A cryo-EM reconstruction of Vo from single-particle images showed subunits a and d in close contact on the cytoplasmic side of the proton channel. A comparison of three-dimensional reconstructions of free Vo and Vo as part of holo V1Vo revealed that the cytoplasmic N-terminal domain of subunit a (aNT) must undergo a large conformational change upon enzyme disassembly or (re)assembly from Vo, V1, and subunit C. Isothermal titration calorimetry using recombinant subunit d and aNT revealed that the two proteins bind each other with a Kd of ∼5 μm. Treatment of the purified Vo sector with 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] resulted in selective release of subunit d, allowing purification of a VoΔd complex. Passive proton translocation assays revealed that both Vo and VoΔd are impermeable to protons. We speculate that the structural change in subunit a upon release of V1 from Vo during reversible enzyme dissociation plays a role in blocking passive proton translocation across free Vo and that the interaction between aNT and d seen in free Vo functions to stabilize the Vo sector for efficient reassembly of V1Vo. 相似文献
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