Home About us Contact
|
Home About us Contact | ||
|
|
||
Ultracentrifugation
Kinds of Ultracentrifugation Selected AbstractsNative and subunit molecular mass and quarternary structure of the hemoglobin from the primitive branchiopod crustacean Triops cancriformisFEBS JOURNAL, Issue 17 2006Morgane Rousselot Many branchiopod crustaceans are endowed with extracellular, high-molecular-weight hemoglobins whose exact structural characteristics have remained a matter of conjecture. By using a broad spectrum of techniques, we provide precise and coherent information on the hemoglobin of one of the phylogenetically ,oldest' extant branchiopods, the tadpole shrimp Triops cancriformis. The hemoglobin dissociated under reducing conditions into two subunits, designated TcHbA and TcHbB, with masses of 35 775 ± 4 and 36 055 ± 4 Da, respectively, determined by ESI-MS. Nonreducing conditions showed only two disulfide-bridged dimers, a homodimer of TcHbA, designated D1 (71 548 ± 5 Da), and the heterodimer D2 (71 828 ± 5 Da). Carbamidomethylation of free SH groups revealed the presence of three cysteines per subunit and indicated one intrasubunit and one intersubunit disulfide bridge. Ultracentrifugation and light-scattering experiments under nondenaturating conditions yielded mass estimates that suggested an uneven number of 17 subunits forming the native hemoglobin. This unrealistic number resulted from the presence of two size classes (16-mer and 18-mer), which were recognized by native PAGE and Ferguson plot analysis. ESI-MS revealed three hemoglobin isoforms with masses of 588.1 kDa, 662.0 kDa, and 665.0 kDa. The 16-mer and the smaller 18-mer species are supposed to be composed of TcHbA only, given the dominance of this subunit type in SDS/PAGE. Transmission electron microscopy of negatively stained specimens showed a population of compact molecules with geometrical extensions of 14, 16 and 9 nm. The proposed stoichiometric model of quarternary structure provides the missing link to achieve a mechanistic understanding of the structure,function relationships among the multimeric arthropodan hemoglobins. [source] Transmission Electron Microscopy and UV,vis,IR Spectroscopy Analysis of the Diameter Sorting of Carbon Nanotubes by Gradient Density UltracentrifugationADVANCED FUNCTIONAL MATERIALS, Issue 14 2009Romain Fleurier Abstract Diameter separation of single-walled carbon nanotubes is achieved via the density gradient ultracentrifugation process. Statistical analysis of the separated samples is performed using high-resolution transmission electron microscopy (HRTEM). The evolution of the diameter distribution with respect to the gradient density is extracted by analyzing hundreds of HRTEM images, and the results are found to be consistent with those estimated by UV,vis,IR spectroscopy. The efficiency of the separation process can be quantitatively characterized by the standard deviation of the diameter distribution, which is determined from the TEM analyses. This particular study indicated that for electric arc nanotubes dispersed in sodium cholate, diameter sorting is more efficient in the upper part of the gradient. [source] Length Fractionation of Carbon Nanotubes Using Centrifugation,ADVANCED MATERIALS, Issue 9 2008Jeffrey A. Fagan Scalable separation of single-walled carbon nanotubes (SWCNTs)by length and chirality is critical to the adaptation of these materials for applications. Ultracentrifugation of SWCNTs within a density gradient produces chiral separation of the NTs, and it is shown here that ultracentrifugation can also be used to produce length fractionated SWCNTs by exploiting their transient motion in response to applied centripetal acceleration. [source] Thrombin generation in vascular tissueJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 1 2006A. PATHAK Summary.,Background: Classically, it is thought that the vast majority of thrombin is generated on the surface of platelets, however, thrombotic events occur in patients despite treatment with potent antiplatelet agents. Methods and results: In freshly harvested left internal mammary artery (IMA) sections, addition of CaCl2 and platelet-poor plasma (PPP) were sufficient to stimulate a profound burst of thrombin and this effect was inhibited by antitissue factor antibodies. Ultracentrifugation of PPP to remove platelet microparticles had no effect on thrombin generation. Both the extrinsic and factor VIII-dependent pathways were necessary for IMA-supported thrombin generation as PPP derived from individuals deficient in factors V, VII, VIII or X did not support thrombin production. Small amounts of thrombin were generated utilizing factor IX (FIX)-deficient plasma, however, thrombin was not generated by aorta from FIX-deficient mice when FIX-deficient plasma was used. The addition of non-lipidated tissue factor (0.6 pm) and CaCl2 to actively proliferating cultured human aortic smooth muscle cells (SMC) resulted in a pronounced burst of thrombin generation occurring between 3 and 15 min after treatment. In the absence of tissue factor, thrombin was generated but at a slower rate and with a peak value 26% of that observed in the presence of tissue factor. Conclusion: Significant thrombin generation can occur on vascular tissue in the absence of platelets or platelet microparticles and on the surface of non-apoptotic SMC. [source] Analytical Ultracentrifugation in the Former USSR: The MOM UltracentrifugesMACROMOLECULAR BIOSCIENCE, Issue 7 2010Georges M. Pavlov No abstract is available for this article. [source] Design and Characterisation of an Artificial DNA-Binding CytochromeCHEMBIOCHEM, Issue 7 2004D. Dafydd Jones Dr. Abstract We aim to design novel proteins that link specific biochemical binding events, such as DNA recognition, with electron transfer functionality. We want these proteins to form the basis of new molecules that can be used for templated assembly of conducting cofactors or for thermodynamically linking DNA binding with cofactor chemistry for nanodevice applications. The first examples of our new proteins recruit the DNA-binding basic helix region of the leucine zipper protein GCN4. This basic helix region was attached to the N and C termini of cytochrome b562(cyt b562) to produce new, monomeric, multifunctional polypeptides. We have fully characterised the DNA and haem-binding properties of these proteins, which is a prerequisite for future application of the new molecules. Attachment of a single basic helix of GCN4 to either the N or C terminus of the cytochrome does not result in specific DNA binding but the presence of DNA-binding domains at both termini converts the cytochrome into a specific DNA-binding protein. Upon binding haem, this chimeric protein attains the spectral characteristics of wild-type cyt b562. The three forms of the protein, apo, oxidised holo and reduced holo, all bind the designed (ATGAcgATGA) target DNA sequence with a dissociation constant, KD, of approximately 90 nM. The protein has a lower affinity (KDca. 370 nM) for the wild-type GCN4 recognition sequence (ATGAcTCAT). The presence of only half the consensus DNA sequence (ATGAcgGGCC) shifts the KDvalue to more than 2500 nM and the chimera does not bind specifically to DNA sequences with no target recognition sites. Ultracentrifugation revealed that the holoprotein,DNA complex is formed with a 1:1 stoichiometry, which indicates that a higher-order protein aggregate is not responsible for DNA binding. Mutagenesis of a loop linking helices 2 and 3 of the cytochrome results in a chimera with a haem-dependent DNA binding affinity. This is the first demonstration that binding of a haem group to a designed monomeric protein can allosterically modulate the DNA binding affinity. [source] Mass spectrometrical analysis of the mitochondrial carrier Aralar1 from mouse hippocampusELECTROPHORESIS, Issue 11 2010Seok Heo Abstract Aralar1 is a mitochondrial aspartate/glutamate carrier and a key component of the malate,aspartate NADH shuttle system. An analytical approach to obtain high sequence coverage is important to predict conformation, identify splice variants and binding partners or generate specific antibodies. Moreover, a method allowing determination of Aralar1 from brain samples is a prerequisite for evaluating a biological role. Sucrose gradient ultracentrifugation was applied to enrich native membrane protein fractions and these were run on blue-native PAGE, followed by multidimensional gel electrophoresis. Spots from the third-dimensional gel electrophoresis were in-gel digested with trypsin, chymotrypsin and subtilisin. Subsequently, peptides were analyzed by nano-ESI-LC-MS/MS using collision-induced dissociation and electron transfer dissociation modes. ModiroÔ v1.1 along with Mascot v2.2 software was used for data handling. Aralar1 could be clearly separated, unambiguously identified and characterized from protein extracts of mouse hippocampus by the use of the multidimensional gel electrophoretic steps. The combined sequence coverage of Aralar1 from trypsin, chymotrypsin and subtilisin digestions was 99.85%. The results provide the basis for future studies of Aralar1 at the protein chemical rather than at the immunochemical level in the brain and thus challenge and enable determination of Aralar1 levels required for understanding biological functions in health and disease. [source] Effective elimination of nucleic acids from bacterial protein samples for optimized blue native polyacrylamide gel electrophoresisELECTROPHORESIS, Issue 14 2009Jingdan Liang Abstract Nucleic acids remaining within bacterial protein samples from Streptomyces lividans and Escherichia coli were found to interfere significantly with blue native polyacrylamide gel electrophoresis (BN-PAGE), a technique used frequently for analyzing bacterial protein complexes in proteomics studies. We have used ultracentrifugation and/or precipitation of cell lysates with streptomycin sulfate to eliminate nucleic acids from total and/or membrane protein samples. Nucleic acid-binding proteins were first enriched by precipitation with streptomycin sulfate, and contaminating nucleic acids were then eliminated by precipitation by adding polyethyleneimine. The performance of BN-PAGE was found to be dramatically improved by these sample preparation steps. [source] An effective skeletal muscle prefractionation method to remove abundant structural proteins for optimized two-dimensional gel electrophoresisELECTROPHORESIS, Issue 11 2005Bradley Jarrold Abstract Proteomic analysis of biological samples in disease models or therapeutic intervention studies requires the ability to detect and identify biologically relevant proteins present in relatively low concentrations. The detection and analysis of these low-level proteins is hindered by the presence of a few proteins that are expressed in relatively high concentrations. In the case of muscle tissue, highly abundant structural proteins, such as actin, myosin, and tropomyosin, compromise the detection and analysis of more biologically relevant proteins. We have developed a practical protocol which exploits high-pH extraction to reduce or remove abundant structural proteins from skeletal muscle crude membrane preparations in a manner suitable for two dimensional gel electrophoresis. An initial whole-cell muscle lysate is generated by homogenization of powdered tissue in Tris-base. This lysate is subsequently partitioned into a supernatant and pellet containing the majority of structural proteins. Treatment of the pellet with high-pH conditions effectively releases structural proteins from membrane compartments which are then removed through ultracentrifugation. Mass spectrometric identification shows that the majority of protein spots reduced or removed by high-pH treatment were contractile proteins or contractile-related proteins. Removal of these proteins enabled successful detection and identification of minor proteins. Structural protein removal also results in significant improvement of gel quality and the ability to load higher amounts of total protein for the detection of lower abundant protein classes. [source] Impact of postprandial lipaemia on low-density lipoprotein (LDL) size and oxidized LDL in patients with coronary artery diseaseEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 11 2006M. Granér Abstract Background, Remnant lipoprotein particles (RLPs) and oxidative stress are components of postprandial state. We investigated the concentrations of triglyceride-rich lipoproteins (TRLs), RLPs, low-density lipoprotein (LDL) size, and oxidized LDL (oxLDL) during alimentary lipaemia, and evaluated whether changes among these variables could be associated with the severity and extent of coronary artery disease (CAD). Materials and methods, Eighty men and 27 women with clinically suspected CAD underwent quantitative coronary angiography (QCA). TRLs were isolated by density gradient ultracentrifugation before and 6 h after an oral fat load. RLPs were measured by an immunoseparation method, oxLDL by ELISA, and LDL size by gradient gel electrophoresis. Results, Triglycerides, apolipoprotein (apo) B-48, and apoB-100 concentration in Swedberg flotation units (Sf) > 400 and in Sf 12,400 fractions were markedly increased at 6 h. Postprandial cholesterol content of RLPs (RLP-C) correlated with respective triglycerides in Sf > 400 (r = 0·737) and Sf 12,400 (r = 0·857), apoB-48 in Sf > 400 (r = 0·710) and Sf 12,400 (r = 0·664), apoB-100 in Sf > 400 (r = 0·812) and Sf 12,400 (r = 0·533). RLP-C correlated with oxLDL both in fasting and in fed state (r = 0·482 and r = 0·543, respectively) and inversely with LDL size (r = ,0·459 and r = ,0·442, respectively). (P < 0·001 for all). OxLDL was elevated postprandially (P < 0·001). In multivariate analysis, oxLDL was a determinant of severity and extent of CAD. Conclusion, Postprandial state is associated with oxidative stress. The magnitude of oxLDL increases during alimentary lipaemia and is associated with coronary atherosclerosis. [source] Human brain carboxypeptidase B, which cleaves ,-amyloid peptides in vitro, is expressed in the endoplasmic reticulum of neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 9 2001Akira Matsumoto Abstract Intracellular localization of novel human brain carboxypeptidase B (HBCPB) was investigated in human hippocampus, using immunohistochemistry by confocal laser microscopy and biochemical purification of the homogenate by density gradient ultracentrifugation. The former revealed that the majority of HBCPB was expressed in the endoplasmic reticulum, in which the HBCPB-specific C14-module immunoreactivity was colocalized with GRP78 immunoreactivity, a stress 70 heat shock protein specifically expressed in the endoplasmic reticulum. The latter showed that anti-C14-module immunoreactivity and prepro-HBCPB immunoreactivity were both enriched in the microsome fraction, especially in that of the endoplasmic reticulum-density fraction of normal human hippocampal homogenates from various sources. However, HBCPB prepared from human hippocampus showed exopeptidase activity for synthetic ,-amyloid 1,42 peptide, in which A, X-42 C-terminus immunoreactivity was decreased in a fashion dose-dependent of the amount of the protease added. These findings indicate that HBCPB, which is expressed in the endoplasmic reticulum of a group of neuronal perikarya, may play an important physiological role in degradation of ,-amyloid 1,42, which is specifically generated in the endoplasmic reticulum of human and rodent neurons and is also regarded as the most pathogenic and aggregatable species among all ,-amyloid peptides. [source] Structure,activity relationships of wheat flavone O -methyltransferase , a homodimer of convenienceFEBS JOURNAL, Issue 9 2008Jack A. Kornblatt Wheat flavone O -methyltransferase catalyzes three sequential methylations of the flavone tricetin. Like other flavonoid O -methyltransferases, the protein is a homodimer. We demonstrate, using analytical ultracentrifugation, that perchlorate dissociates the dimer into monomers. The resulting monomers retain all their catalytic capacity, including the ability to catalyze the three successive methylations. We show, using isothermal titration calorimetry, that the binding constant for S -adenosyl- l -methionine does not change significantly as the protein dissociates. The second substrate, tricetin, binds to the dimers but could not be tested with the monomers. CD, UV and fluorescence spectroscopy show that there are substantial changes in the structure of the protein as it dissociates. The fact that there are differences between the monomers and dimers even as the monomers maintain activity may be the result of the very low catalytic capacity of this enzyme. Maximal turnover numbers for the dimers and monomers are only about 6,7 per minute. Even though the binding pockets for S -adenosyl- l -methionine, tricetin, selgin and tricin are intact, selection of a catalytically competent structure may be a very slow step during catalysis. [source] pyr RNA binding to the Bacillus caldolyticus PyrR attenuation protein , characterization and regulation by uridine and guanosine nucleotidesFEBS JOURNAL, Issue 4 2008Casper M. Jørgensen The PyrR protein regulates expression of pyrimidine biosynthetic (pyr) genes in many bacteria. PyrR binds to specific sites in the 5, leader RNA of target operons and favors attenuation of transcription. Filter binding and gel mobility assays were used to characterize the binding of PyrR from Bacillus caldolyticus to RNA sequences (binding loops) from the three attenuation regions of the B. caldolyticus pyr operon. Binding of PyrR to the three binding loops and modulation of RNA binding by nucleotides was similar for all three RNAs. The apparent dissociation constants at 0 °C were in the range 0.13,0.87 nm in the absence of effectors; dissociation constants were decreased by three- to 12-fold by uridine nucleotides and increased by 40- to 200-fold by guanosine nucleotides. The binding data suggest that pyr operon expression is regulated by the ratio of intracellular uridine nucleotides to guanosine nucleotides; the effects of nucleoside addition to the growth medium on aspartate transcarbamylase (pyrB) levels in B. subtilis cells in vivo supported this conclusion. Analytical ultracentrifugation established that RNA binds to dimeric PyrR, even though the tetrameric form of unbound PyrR predominates in solution at the concentrations studied. [source] Calcite-specific coupling protein in barnacle underwater cementFEBS JOURNAL, Issue 24 2007Youichi Mori The barnacle relies for its attachment to underwater foreign substrata on the formation of a multiprotein complex called cement. The 20 kDa cement protein is a component of Megabalanus rosa cement, although its specific function in underwater attachment has not, until now, been known. The recombinant form of the protein expressed in bacteria was purified in soluble form under physiological conditions, and confirmed to retain almost the same structure as that of the native protein. Both the protein from the adhesive layer of the barnacle and the recombinant protein were characterized. This revealed that abundant Cys residues, which accounted for 17% of the total residues, were in the intramolecular disulfide form, and were essential for the proper folding of the monomeric protein structure. The recombinant protein was adsorbed to calcite and metal oxides in seawater, but not to glass and synthetic polymers. The adsorption isotherm for adsorption to calcite fitted the Langmuir model well, indicating that the protein is a calcite-specific adsorbent. An evaluation of the distribution of the molecular size in solution by analytical ultracentrifugation indicated that the recombinant protein exists as a monomer in 100 mm to 1 m NaCl solution; thus, the protein acts as a monomer when interacting with the calcite surface. cDNA encoding a homologous protein was isolated from Balanus albicostatus, and its derived amino acid sequence was compared with that from M. rosa. Calcite is the major constituent in both the shell of barnacle base and the periphery, which is also a possible target for the cement, due to the gregarious nature of the organisms. The specificity of the protein for calcite may be related to the fact that calcite is the most frequent material attached by the cement. [source] Small heat shock protein Hsp27 prevents heat-induced aggregation of F-actin by forming soluble complexes with denatured actinFEBS JOURNAL, Issue 22 2007Anastasia V. Pivovarova Previously, we have shown that the small heat shock protein with apparent molecular mass 27 kDa (Hsp27) does not affect the thermal unfolding of F-actin, but effectively prevents aggregation of thermally denatured F-actin [Pivovarova AV, Mikhailova VV, Chernik IS, Chebotareva NA, Levitsky DI & Gusev NB (2005) Biochem Biophys Res Commun331, 1548,1553], and supposed that Hsp27 prevents heat-induced aggregation of F-actin by forming soluble complexes with denatured actin. In the present work, we applied dynamic light scattering, analytical ultracentrifugation and size exclusion chromatography to examine the properties of complexes formed by denatured actin with a recombinant human Hsp27 mutant (Hsp27,3D) mimicking the naturally occurring phosphorylation of this protein at Ser15, Ser78, and Ser82. Our results show that formation of these complexes occurs upon heating and accompanies the F-actin thermal denaturation. All the methods show that the size of actin,Hsp27-3D complexes decreases with increasing Hsp27-3D concentration in the incubation mixture and that saturation occurs at approximately equimolar concentrations of Hsp27-3D and actin. Under these conditions, the complexes exhibit a hydrodynamic radius of ,,16 nm, a sedimentation coefficient of 17,20 S, and a molecular mass of about 2 MDa. It is supposed that Hsp27-3D binds to denatured actin monomers or short oligomers dissociated from actin filaments upon heating and protects them from aggregation by forming relatively small and highly soluble complexes. This mechanism might explain how small heat shock proteins prevent aggregation of denatured actin and by this means protect the cytoskeleton and the whole cell from damage caused by accumulation of large insoluble aggregates under heat shock conditions. [source] NEMO oligomerization in the dynamic assembly of the I,B kinase core complexFEBS JOURNAL, Issue 10 2007Elisabeth Fontan NF-,B essential modulator (NEMO) plays an essential role in the nuclear factor ,B (NF-,B) pathway as a modulator of the two other subunits of the I,B kinase (IKK) complex, i.e. the protein kinases, IKK, and IKK,. Previous reports all envision the IKK complex to be a static entity. Using glycerol-gradient ultracentrifugation, we observed stimulus-dependent dynamic IKK complex assembly. In wild-type fibroblasts, the kinases and a portion of cellular NEMO associate in a 350-kDa high-molecular-mass complex. In response to constitutive NF-,B stimulation by Tax, we observed NEMO recruitment and oligomerization to a shifted high-molecular-mass complex of 440 kDa which displayed increased IKK activity. This stimulus-dependent oligomerization of NEMO was also observed using fluorescence resonance energy transfer after a transient pulse with interleukin-1,. In addition, fully activated, dimeric kinases not bound to NEMO were detected in these Tax-activated fibroblasts. By glycerol gradient ultracentrifugation, we also showed that: (a) in fibroblasts deficient in IKK, and IKK,, NEMO predominantly exists as a monomer; (b) in NEMO-deficient fibroblasts, IKK, dimers are present that are less stable than IKK, dimers. Intriguingly, in resting Rat-1 fibroblasts, 160-kDa IKK,,NEMO and IKK,,NEMO heterocomplexes were observed as well as a significant proportion of NEMO monomer. These results suggest that most NEMO molecules do not form a tripartite IKK complex with an IKK,,IKK, heterodimer as previously reported in the literature but, instead, NEMO is able to form a complex with the monomeric forms of IKK, and IKK,. [source] Molecular mass of macromolecules and subunits and the quaternary structure of hemoglobin from the microcrustacean Daphnia magnaFEBS JOURNAL, Issue 14 2006Tobias Lamkemeyer The molecular masses of macromolecules and subunits of the extracellular hemoglobin from the fresh-water crustacean Daphnia magna were determined by analytical ultracentrifugation, multiangle laser light scattering and electrospray ionization mass spectrometry. The hemoglobins from hypoxia-incubated, hemoglobin-rich and normoxia-incubated, hemoglobin-poor Daphnia magna were analyzed separately. The sedimentation coefficient of the macromolecule was 17.4 ± 0.1 S, and its molecular mass was 583 kDa (hemoglobin-rich animals) determined by AUC and 590.4 ± 11.1 kDa (hemoglobin-rich animals) and 597.5 ± 49 kDa (hemoglobin-poor animals), respectively, determined by multiangle laser light scattering. Measurements of the hemoglobin subunit mass of hemoglobin-rich animals by electrospray ionization mass spectrometry revealed a significant peak at 36.482 ± 0.0015 kDa, i.e. 37.715 kDa including two heme groups. The hemoglobin subunits are modified by O-linked glycosylation in the pre-A segments of domains 1. No evidence for phosphorylation of hemoglobin subunits was found. The subunit migration behavior during SDS/PAGE was shown to be influenced by the buffer system used (Tris versus phosphate). The subunit mass heterogeneity found using Tris buffering can be explained by glycosylation of hemoglobin subunits. Based on molecular mass information, Daphnia magna hemoglobin is demonstrated to consist of 16 subunits. The quaternary structure of the Daphnia magna hemoglobin macromolecule was assessed by three-dimensional reconstructions via single-particle analysis based on negatively stained electron microscopic specimens. It turned out to be much more complex than hitherto proposed: it displays D4 symmetry with a diameter of approximately 12 nm and a height of about 8 nm. [source] Physical characterization of plakophilin 1 reconstituted with and without zincFEBS JOURNAL, Issue 14 2000Ilse Hofmann Plakophilin 1 (PKP1) belongs to the arm -repeat protein family which is characterized by the presence of a conserved 42-amino-acid motif. Despite individual members of the family containing a similar type of structural domain, they exhibit diverse cellular functions. PKP1 is ubiquitously expressed in human tissues and, depending on the type of cell, found prominently in the karyoplasm and/or in desmosomes. In surface plasmon resonance detection experiments, we noticed that PKP1 specifically bound zinc but not calcium or magnesium. Therefore we have used circular dichroism spectroscopy, limited proteolysis, analytical ultracentrifugation, electron microscopy and dynamic light scattering to establish the physical properties of recombinant PKP1 depending on the presence or absence of zinc. The , helix content of PKP1 was considerably higher when reconstituted with zinc than without. By atomic absorption spectroscopy 7.3 atoms zinc were shown to be tightly associated with one molecule of wild-type PKP1. The zinc-reconstituted protein formed globular particles of 21.9 ± 8.4 nm diameter, as measured by electron microscopy after glycerol spraying/rotary metal shadowing. In parallel, the average sedimentation coefficient (s20,w) for zinc-containing PKP1 was 41S and its diffusion coefficient, as obtained by dynamic light scattering, 1.48 × 10,7 cm2·s,1. The molecular mass of 2.44 × 106 obtained from s and D yields an average stoichiometry of 30 for the PKP1 oligomer. In contrast, PKP1, reconstituted without zinc, contained no significant amount of zinc, sedimented with 4.6S, and was present in monomeric form as determined by sedimentation equilibrium centrifugation. [source] Length Fractionation of Carbon Nanotubes Using Centrifugation,ADVANCED MATERIALS, Issue 9 2008Jeffrey A. Fagan Scalable separation of single-walled carbon nanotubes (SWCNTs)by length and chirality is critical to the adaptation of these materials for applications. Ultracentrifugation of SWCNTs within a density gradient produces chiral separation of the NTs, and it is shown here that ultracentrifugation can also be used to produce length fractionated SWCNTs by exploiting their transient motion in response to applied centripetal acceleration. [source] Determination of phthalate esters in cosmetics by gas chromatography with flame ionization detection and mass spectrometric detectionINTERNATIONAL JOURNAL OF COSMETIC SCIENCE, Issue 4 2005Huiming Chen GC-FID; GC-MS; Produits cosmétiques; Esters Phtaliques Synopsis A gas chromatography coupled with flame ionization detection (GC-FID) and mass spectrometric detection (MSD) method was developed to determine the six kinds of phthalate esters [dimethyl phthalate (DMP), diethyl phthalate (DEP), di- n -butyl phthalate (DBP), benzyl butyl phthalate (BBP), di(2-ethylhexyl) phthalate (DEHP) and di- n -octyl phthalate (DOP)] in cosmetics (solid, cream and liquid cosmetics). The cosmetics were extracted with methanol by ultrasonic and then separated with high-speed centrifugation. The upper clear layer was dried and filtered through a 0.45 ,m pore diameter filter. The filtrate was injected into GC-FID/GC-MS for detection. GC-FID chromatogram was applied for qualitative analysis, external standard method was used for quantitative analysis. Confirmation of phthalate presence was undertaken by GC-EI-MS. The recovery range of all phthalates were between 92.0 and 110.0% with relative standard deviations between 1.95 and 5.92%. The low detection limits of the method were: 0.1 ng for DMP, DEP, DBP and BBP, 0.5 ng for DEHP and DOP. The method had advantages of high precision and sensitivity, simplicity of pretreatment. The method can be used to test the six kinds of phthalate esters in cosmetics. Resume Une méthode d'analyse par chromatographie gazeuse couplée à une détection par ionization de flamme (GC - FID) et une détection spectrométrique de masse (MSD) a été développée pour analyser 6 sortes d'esters phtaliques (phtalate de diméthyle (DMP), phtalate de diéthyle (DEP), phtalate de di- n -butyle (DBP), phtalate de benzylbutyle (BBP), phtalate de di-2-éthylhexyle (DEHP) et phtalate de di- n -octyle (DOP)) dans des produits cosmétiques (solides, crèmes et liquides). Les produits cosmétiques sont extraits au méthanol sous ultrason, puis séparés par ultracentrifugation. La phase supérieure limpide est déshydratée et filtrée sur un filtre de diamètre de pore moyen égal à 0,45 ,m. Le filtrat est injecté dans le système GC - FID/GC-MS pour analyse. Les chromatogrammes GC-FID sont utilisés pour l'analyse qualitative, des standards externes ont été utilisés pour l'analyse quantitative. La GC-EI-MS permet de confirmer la présence des esters phtaliques. Le taux de récupération de tous les esters est compris entre 92 et 110% avec une déviation standard allant de 1,95%à 5,92%. La limite de détection par cette méthode est de 0,1 ng pour DMP, DEP, DBP et BBP, 0,5 ng pour DEHP et DOP. Les avantages de cette méthode sont sa haute précision, sa sensibilité et la simplicité du prétraitement. Cette méthode peut être utilisée pour doser la présence des six sortes d'esters phtaliques dans des produits cosmétiques. [source] Advances in membrane receptor screening and analysisJOURNAL OF MOLECULAR RECOGNITION, Issue 4 2004Matthew A. Cooper Abstract During the last decade there has been significant progress in the development of analytical techniques for the screening of ligand binding to membranes and membrane receptors. This review focuses on developments using label-free assays that facilitate ligand,membrane,receptor screening without the need for chemical-, biological- or radiological-labelled reagents. These assays include acoustic, optical surface plasmon resonance biosensing, sedimentation (analytical ultracentrifugation), chromatographic assays, isothermal titration calorimetry and differential scanning calorimetry. The merits and applications of cell-based screening systems and of different model membrane systems, including planar supported lipid layers, bead-supported membranes and lipid micro-arrays, are discussed. Recent advances involving more established techniques including intrinsic fluorescence, FRET spectroscopy, scintillation proximity assays and automated patch clamping are presented along with applications to peripheral membrane proteins, ion channels and G protein-coupled receptors. Novel high-throughput assays for determination of drug- and protein-partitioning in membranes are also highlighted. To aid the experimenter, a brief synopsis of the techniques commonly employed to purify and reconstitute membranes and membrane receptors is included. Copyright © 2004 John Wiley & Sons, Ltd [source] Expression of multiple AQP4 pools in the plasma membrane and their association with the dystrophin complexJOURNAL OF NEUROCHEMISTRY, Issue 6 2008Grazia Paola Nicchia Abstract Altered aquaporin-4 (AQP4) expression has been reported in brain edema, tumors, muscular dystrophy, and neuromyelitis optica. However, the plasma membrane organization of AQP4 and its interaction with proteins such as the dystrophin-associated protein complex are not well understood. In this study, we used sucrose density gradient ultracentrifugation and 2D blue native/sodium dodecyl sulfate,polyacrylamide gel electrophoresis and showed the expression of several AQP4 multi-subunit complexes (pools) of different sizes, ranging from , 1 MDa to ,500 kDa and containing different ratios of the 30/32 kDa AQP4 isoforms, indicative of orthogonal arrays of particles of various sizes. A high molecular weight pool co-purified with dystrophin and ,-dystroglycan and was drastically reduced in the skeletal muscle of mdx3cv mice, which have no dystrophin. The number and size of the AQP4 pools were the same in the kidney where dystrophin is not expressed, suggesting the presence of dystrophin-like proteins for their expression. We found that AQP2 is expressed only in one major pool of ,500 kDa, indicating that the presence of different pools is a peculiarity of AQP4 rather than a widespread feature in the AQP family. Finally, in skeletal muscle caveolin-3 did not co-purify with any AQP4 pool, indicating the absence of interaction of the two proteins and confirming that caveolae and orthogonal arrays of particles are two independent plasma membrane microdomains. These results contribute to a better understanding of AQP4 membrane organization and raise the possibility that abnormal expression of specific AQP4 pools may be found in pathological states. [source] Determination of particle heterogeneity and stability of recombinant adenovirus by analytical ultracentrifugation in CsCl gradientsJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 2 2008Xiaoyu Yang Abstract Recombinant adenoviruses (rAd), widely used as vectors for gene therapy, are generally purified by column chromatography and frequently contain empty capsids and other aberrant forms of virus particles. To determine particle heterogeneity we utilized analytical ultracentrifugation (AUC) in CsCl density gradients. Preparations of three different rAd vectors were assessed. AUC was able to resolve multiple density forms including two empty capsid types in various virus preparations. One unusual density form (form V), was noninfectious and lacked protein VI. AUC was able to quantify empty capsids and monitor their removal during process development. Their relative concentrations were reduced by either addition of an immobilized zinc affinity chromatography (IZAC) step or by extension of the infection time. The Adenovirus Reference Material (ARM), a wild-type Ad5, had 2.2% empty capsids and no other detectable minor particle forms. Finally, AUC was utilized to monitor the thermal instability of the three rAd vectors via the transformations of different density forms. The vector and empty capsids containing protein IX were more stable than those without IX. Together, these results exemplify AUC in CsCl density gradients as a valuable technique for evaluating product particle heterogeneity and stability. © 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:746,763, 2008 [source] Sedimentation behaviour and turbidity of carrot juices in relation to the characteristics of their cloud particlesJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 8 2003Martin Reiter Abstract The cloud stability of carrot juices was investigated using physical methods. In contrast to cloudy juices from fruits or other vegetables described so far, complete clarification of juice samples could not be achieved even after ultracentrifugation. Since the density of one particle fraction was almost equal to that of the continuous phase, this fraction was resistant to sedimentation by centrifugal forces up to 60 600 × g in an 8° Brix carrot juice. Cloud stability problems of carrot juices, therefore, are usually associated with bottom sediment formation, but not with visible loss of turbidity. Particle size and density were shown to be decisive for suspension stability, whilst both particle charge and serum viscosity did not show any effect on cloud stability. The reasons for the exceptional stability of the suspended particles are discussed. Based on three particle fractions, a new physical concept could be deduced according to particle size and density explaining the extraordinary suspension stability of carrot juices. Copyright © 2003 Society of Chemical Industry [source] Presence of HCV-RNA after ultracentrifugation of serum samples during the follow-up of chronic hepatitis C patients with a sustained virological response may predict reactivation of hepatitis C virus infectionALIMENTARY PHARMACOLOGY & THERAPEUTICS, Issue 5 2009I. CASTILLO Summary Background, Concentration of viral particles by ultracentrifugation of serum prior to PCR allows detection of hepatitis C virus (HCV) RNA in patients with undetectable viral RNA by conventional PCR assays. Aim, To analyse if HCV-RNA is detected after serum ultracentrifugation in chronic hepatitis C patients with a sustained virological response to antiviral therapy (defined as serum HCV-RNA negativity by conventional assays 6 months after the end of therapy). Methods, HCV-RNA was tested using real-time PCR in ultracentrifuged sera collected during the post-treatment follow-up (mean: 42 ± 27 months) in 57 sustained virological responders (SVR). Results, After serum ultracentrifugation, HCV-RNA was detected on at least one occasion during the follow-up in 29/57 (51%) SVR. Thirteen (23%) of these 57 SVR suffered a reactivation 18 ± 8 months after the end of therapy (reappearance of serum HCV-RNA detectable by conventional assays). Among reactivated patients, 11/13 (85%) had HCV-RNA in ultracentrifuged serum samples (detectable 10 ± 5 months before reactivation), while HCV-RNA was positive after ultracentrifugation in 18/44 (41%) long-term SVR (P = 0.01). Persistence of detectable HCV-RNA after serum ultracentrifugation was associated with reactivation (P = 0.001). Conclusions, Serum ultracentrifugation prior to PCR allows detection of HCV-RNA in SVR and its persistence may predict late reactivation. [source] ,-Conjugated Donor and Donor,Acceptor Metallo-PolymersMACROMOLECULAR RAPID COMMUNICATIONS, Issue 9-10 2010Andreas Wild Abstract Two zinc(II)- and two ruthenium(II) containing ,-conjugated metallo-polymers were synthesized and characterized in detail. We could prove by SEC, analytical ultracentrifugation (AUC) and viscosimetry the ruthenium(II) metallo-polymers to be high molar mass materials (Mfs,=,20,000,g,·,mol,1Ru1-2; Mfs,=,34,000,g,·,mol,1Ru1) exhibiting intrinsic viscosities of up to [,],=,192,·,cm3,·,g,1. Applying spin-coating we produced homogeneous films of the polymers and could, subsequently, investigate the photophysical properties in the solid state. Introducing the Ru(II) metallo-polymers mixed with PCBM[60] as photoactive layer in bulk-heterojunction solar cells resulted in very low efficiencies due to morphology problems. [source] Advances in the Synthesis and Characterization of Polypeptide-Based Hybrid Block CopolymersMACROMOLECULAR SYMPOSIA, Issue 1 2004Ivaylo Dimitrov Abstract Linear polystyrene- block -poly(Z-L-lysine) copolymers with a very narrow molecular weight distribution (polydispersity index < 1.03) could be obtained via the ring-opening polymerization of Z-L-lysine- N -carboxyanhydride using ,-(primary amino hydrochloride)-polystyrenes as macroinitiators in N,N -dimethylformamide as the solvent at 40-80 °C. The block copolymer samples were analyzed by means of NMR, size exclusion chromatography, and analytical ultracentrifugation. [source] A characteristic finite element method with local mesh refinements for the Lamm equation in analytical ultracentrifugationNUMERICAL METHODS FOR PARTIAL DIFFERENTIAL EQUATIONS, Issue 2 2009Jiangguo Liu Abstract The Lamm equation is a fundamental differential equation in analytical ultracentrifugation, for describing the transport of solutes in an ultracentrifuge cell. In this article, we present a characteristic finite element method with local mesh refinements for solving the Lamm equation. The numerical method is mass-conservative by design and allows relatively large time steps to be used. Numerical experiments indicate that the numerical solutions are oscillation-free in the region near the cell bottom, where mass build up and large concentration gradients occur. Positivity of solutions is also well kept. © 2008 Wiley Periodicals, Inc. Numer Methods Partial Differential Eq, 2009 [source] Increased aggregation propensity of IgG2 subclass over IgG1: Role of conformational changes and covalent character in isolated aggregatesPROTEIN SCIENCE, Issue 9 2010Heather Franey Abstract Aggregation of human therapeutic antibodies represents a significant hurdle to product development. In a test across multiple antibodies, it was observed that IgG1 antibodies aggregated less, on average, than IgG2 antibodies under physiological pH and mildly elevated temperature. This phenomenon was also observed for IgG1 and IgG2 subclasses of anti-streptavidin, which shared 95% sequence identity but varied in interchain disulfide connectivity. To investigate the structural and covalent changes associated with greater aggregation in IgG2 subclasses, soluble aggregates from the two forms of anti-streptavidin were isolated and characterized. Sedimentation velocity analytical ultracentrifugation (SV-AUC) measurements confirmed that the aggregates were present in solution, and revealed that the IgG1 aggregate was composed of a predominant species, whereas the IgG2 aggregate was heterogeneous. Tertiary structural changes accompanied antibody aggregation as evidenced by greater ANS (8-Anilino-1-naphthalene sulfonic acid) binding to the aggregates over monomer, and differences in disulfide character and tryptophan environments between monomer, oligomer and aggregate species, as observed by near-UV circular dichroism (CD). Differences between subclasses were observed in the secondary structural changes that accompanied aggregation, particularly in the intermolecular ,-sheet and turn structures between the monomer and aggregate species. Free thiol determination showed ,2.4-fold lower quantity of free cysteines in the IgG1 subclass, consistent with the 2.4-fold reduction in aggregation of the IgG1 form when compared with IgG2 under these conditions. These observations suggested an important role for disulfide bond formation, as well as secondary and tertiary structural transitions, during antibody aggregation. Such degradations may be minimized using appropriate formulation conditions. [source] Probing mechanisms of resistance to the tuberculosis drug isoniazid: Conformational changes caused by inhibition of InhA, the enoyl reductase from Mycobacterium tuberculosisPROTEIN SCIENCE, Issue 8 2007Nicole A. Kruh Abstract The frontline tuberculosis drug isoniazid (INH) inhibits InhA, the NADH-dependent fatty acid biosynthesis (FAS-II) enoyl reductase from Mycobacterium tuberculosis (MTB), via formation of a covalent adduct with NAD+ (the INH-NAD adduct). Resistance to INH can be correlated with many mutations in MTB, some of which are localized in the InhA cofactor binding site. While the InhA mutations cause a substantial decrease in the affinity of InhA for NADH, surprisingly the same mutations result in only a small impact on binding of the INH-NAD adduct. Based on the knowledge that InhA interacts in vivo with other components of the FAS-II pathway, we have initiated experiments to determine whether enzyme inhibition results in structural changes that could affect protein,protein interactions involving InhA and how these ligand-induced conformational changes are modulated in the InhA mutants. Significantly, while NADH binding to wild-type InhA is hyperbolic, the InhA mutants bind the cofactor with positive cooperativity, suggesting that the mutations permit access to a second conformational state of the protein. While cross-linking studies indicate that enzyme inhibition causes dissociation of the InhA tetramer into dimers, analytical ultracentrifugation and size exclusion chromatography reveal that ligand binding causes a conformational change in the protein that prevents cross-linking across one of the dimer,dimer interfaces in the InhA tetramer. Interestingly, a similar ligand-induced conformational change is also observed for the InhA mutants, indicating that the mutations modulate communication between the subunits without affecting the two conformational states of the protein that are present. [source] | ||||||||||||||||||||||