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Low Salt Concentrations (low + salt_concentration)
Selected AbstractsStepwise proteolytic removal of the , subdomain in ,-lactalbuminFEBS JOURNAL, Issue 15 2001The protein remains folded, can form the molten globule in acid solution Bovine ,-lactalbumin (,-LA) is an ,/, protein which adopts partly folded states when dissolved at low pH (A-state), by removal of the protein-bound calcium at neutral pH and low salt concentration (apo-state), as well as in aqueous trifluoroethanol. Previous spectroscopic studies have indicated that the A-state of ,-LA at pH 2.0, considered a prototype molten globule, has a native-like fold in which the helical core is mostly retained, while the , subdomain is less structured. Here, we investigate the conformational features of three derivatives of ,-LA characterized by a single peptide bond fission or a deletion of 12 or 19/22 amino-acid residues of the , subdomain of the native protein (approximately from residue 34 to 57). These ,-LA derivatives were obtained by limited proteolysis of the protein in its partly folded state(s). A nicked ,-LA species consisting of fragments 1-,3,40 and 41,123 (nicked-LA) was prepared by thermolytic digestion of the 123-residue chain of ,-LA in 50% (v/v) aqueous trifluoroethanol. Two truncated or gapped protein species given by fragments 1,40 and 53,123 (des,1-LA) or fragments 1,34 and 54-,57,123 (des,2-LA) were obtained by digestion of ,-LA with pepsin in acid or with proteinase K at neutral pH in its apo-state, respectively. The two protein fragments of nicked or gapped ,-LA are covalently linked by the four disulfide bridges of the native protein. CD measurements revealed that, in aqueous solution at neutral pH and in the presence of calcium, the three protein species maintain the helical secondary structure of intact ,-LA, while the tertiary structure is strongly affected by the proteolytic cleavages of the chain. Temperature effects of CD signals in the far- and near-UV region reveal a much more labile tertiary structure in the ,-LA derivatives, while the secondary structure is mostly retained even upon heating. In acid solution at pH 2.0, the three ,-LA variants adopt a conformational state essentially identical to the molten globule displayed by intact ,-LA, as demonstrated by CD measurements. Moreover, they bind strongly the fluorescent dye 8-anilinonaphthalene-1-sulfonate, which is considered a diagnostic feature of the molten globule of proteins. Therefore, the , subdomain can be removed from the ,-LA molecule without impairing the capability of the rest of the chain to adopt a molten globule state. The results of this protein dissection study provide direct experimental evidence that in the ,-LA molten globule only the , domain is structured. [source] Differential interactions of plasmid DNA, RNA and genomic DNA with amino acid-based affinity matricesJOURNAL OF SEPARATION SCIENCE, JSS, Issue 17-18 2010Angela Sousa Abstract The development of a strategy to plasmid DNA (pDNA) purification has become necessary for the development of gene therapy and DNA vaccine production processes in recent years, since this nucleic acid and most of contaminants, such as RNA, genomic DNA and endotoxins, are negatively charged. An ideal separation methodology may be achieved with the use of affinity interactions between immobilized amino acids and nucleic acids. In this study, the binding behaviour of nucleic acids under the influence of different environmental conditions, such as the composition and ionic strength of elution buffer, and the temperature, is compared with various amino acids immobilized on chromatography resins. Supercoiled (sc) plasmid isoform was isolated with all matrices used, but in some cases preferential interactions with other nucleic acids were found. Particularly, lysine chromatography showed to be an ideal technology mainly on RNA purification using low salt concentration. On the other hand, arginine ligands have shown a greater ability to retain the sc isoform comparatively to the other nucleic acids retention, becoming this support more adequate to sc pDNA purification. The temperature variation, competitive elution and oligonucleotides affinity studies also allowed to recognize the dominant interactions inherent to biorecognition of pDNA molecule and the affinity matrices. [source] Effects of ionic strength on lysozyme uptake rates in cation exchangers.BIOTECHNOLOGY & BIOENGINEERING, Issue 2 2005I: Uptake in SP Sepharose FF Fluorescence scanning confocal microscopy was used in parallel with batch uptake and breakthrough measurements of transport rates to study the effect of ionic strength on the uptake of lysozyme into SP Sepharose FF. In all cases the adsorption isotherms were near-rectangular. As described previously, the intraparticle profiles changed from slow-moving self-sharpening fronts at low salt concentration, to fast-moving diffuse profiles at high salt concentration, and batch uptake rates correspondingly increased with increasing salt concentration. Shrinking core and homogeneous diffusion frameworks were used successfully to obtain effective diffusivities for the low salt and high salt conditions, respectively. The prediction of column breakthrough was generally good using these frameworks, except for low-salt uptake results. In those cases, the compressibility of the stationary phase coupled with the shrinking core behavior appears to reduce the mass transfer rates at particle-particle contacts, leading to shallower breakthrough curves. In contrast, the fast uptake rates at high ionic strength appear to reduce the importance of mass transfer limitations at the particle contacts, but the confocal results do show a flow rate dependence on the uptake profiles, suggesting that external mass transfer becomes more limiting at high ionic strength. These results show that the complexity of behavior observable at the microscopic scale is directly manifested at the column scale and provides a phenomenological basis to interpret and predict column breakthrough. In addition, the results provide heuristics for the optimization of chromatographic conditions. © 2005 Wiley Periodicals, Inc. [source] Physicochemical interactions between drugs and superdisintegrantsJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 12 2008Nelly Fransén We have evaluated the interactions between superdisintegrants and drugs with different physicochemical characteristics, which may affect the in-vivo absorption e.g. after mucosal administration. The binding of sodium salicylate, naproxen, methyl hydroxybenzoate (methylparaben), ethyl hydroxybenzoate (ethylparaben), propyl hydroxybenzoate (propylparaben), atenolol, alprenolol, diphenhydramine, verapamil, amitriptyline and cetylpyridinium chloride monohydrate (CPC) to different superdisintegrants (sodium starch glycolate (SSG), croscarmellose sodium (CCS) and crospovidone) and one unsubstituted comparator (starch) was studied spectrophotometrically. An indication of the in-vivo effect was obtained by measuring the interactions at physiological salt concentrations. SSG was investigated more thoroughly to obtain release profiles and correlation between binding and ionic strength. The results showed that the main interactions with the anionic hydrogels formed by SSG and CCS were caused by ion exchange, whereas the neutral crospovidone exhibited lipophilic interactions with the non-ionic substances. The effect of increased ionic strength was most pronounced at low salt concentrations and the ion exchange interactions were almost completely eradicated at physiological conditions. The release profile of diphenhydramine was significantly affected by the addition of salt. It was thus concluded that the choice of buffer was of great importance for in-vitro experiments with ionic drugs. At physiological salt concentrations the interactions did not appear to be strong enough to influence the in-vivo bioavailability of any of the drug molecules. [source] Expression, purification and preliminary crystallization of amaranth 11S proglobulin seed storage protein from Amaranthus hypochondriacus L.ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2010Mary Rose Tandang-Silvas 11S globulin is one of the major seed storage proteins in amaranth. Recombinant protein was produced as up to ,80% of the total bacterial protein using Escherichia coli Rosetta-gami (DE3) containing pET21d with amaranth 11S globulin cDNA. The best expression condition was at 302,K for 20,h using LB medium containing 0.5,M NaCl. The recombinant protein was easily separated from most of the Escherichia coli proteins by precipitation with 0,40% ammonium sulfate solution. It formed aggregates at low temperature and at low salt concentrations. This behaviour may imply that it has a more hydrophobic nature than other 11S seed globulins. The crystals diffracted to 6,Å resolution and belonged to space group P63, with unit-cell parameters a = b = 97.6, c = 74.8,Å, , = 120.0°. One subunit of a trimer was estimated to be present in the asymmetric unit, assuming a Vsol of 41%. To obtain the complete structure solution, experiments to improve crystallization and flash-cooling conditions are in progress. [source] Titration of poly(dA-dT) · poly(dA-dT) in solution at variable NaCl concentrationBIOPOLYMERS, Issue 2 2004Marta Airoldi Abstract CD and uv absorption data showed that high molecular weight poly(dA-dT) · poly(dA-dT), at 298 K, undergoes an acid-induced transition from B-double helix to random coil in NaCl solutions of different concentrations, ranging from 0.005 to 0.600M. Similarly, titration of the polynucleotide with a strong base causes duplex-to-single strands transition. The base- and acid-induced transitions were both reversible by back-titration (with an acid or, respectively, with a base): the apparent pKa were the same in both directions. However, the number of protons per titratable site (adenine N1) required to reach half-denaturation was in great excess over the stoichiometric value; to a much larger extent, the same effect was observed also for the deprotonation of the N3H sites of thymine. Moreover, in the basic denaturation experiments, at low salt concentrations ([NaCl],0.300M) less acid than calculated was needed to back-titrate the base excess to half-denaturation. Both effects could be qualitatively justified on the basis of the counterion condensation theory of polyelectrolytes and considering the energy barrier created by the negatively charged phosphodiester groups to the penetration of the OH, ions inside the double helix and the screening effect of the Na+ ions on such charges, in the deprotonation experiments. © 2004 Wiley Periodicals, Inc. Biopolymers, 2004 [source] A novel purification strategy for retrovirus gene therapy vectors using heparin affinity chromatographyBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2005María de las Mercedes Segura Abstract Membrane separation and chromatographic technologies are regarded as an attractive alternative to conventional academic small-scale ultracentrifugation procedures used for retrovirus purification. However, despite the increasing demands for purified retroviral vector preparations, new chromatography adsorbents with high specificity for the virus have not been reported. Heparin affinity chromatography is presented here as a novel convenient tool for retrovirus purification. The ability of bioactive retroviral particles to specifically bind to heparin ligands immobilized on a chromatographic gel is shown. A purification factor of 63 with a recovery of 61% of functional retroparticles was achieved using this single step. Tentacle heparin affinity supports captured retroviral particles more efficiently than conventional heparin affinity chromatography supports with which a lower recovery was obtained (18%). Intact, infective retroviral particles were recovered by elution with low salt concentrations (350 mM NaCl). Mild conditions for retrovirus elution from chromatographic columns are required to preserve virus infectivity. VSV-G pseudotyped retroviruses have shown to be very sensitive to high ionic strength, losing 50% of their activity and showing membrane damage after a short exposure to 1M NaCl. We also report a complete scaleable downstream processing scheme for the purification of MoMLV-derived vectors that involves sequential microfiltration and ultra/diafiltration steps for virus clarification and concentration respectively, followed by fractionation by heparin affinity chromatography and final polishing by size-exclusion chromatography. Overall, by using this strategy, a 38% yield of infective particles can be achieved with a final purification factor of 2,000. © 2005 Wiley Periodicals, Inc. [source] | ||||||||||