Isolation and characterization of polypeptide at the picomole level. Pre-column formation of peptide derivatives with dimethylaminoazobenzene isothiocyanate.
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ABSTRACT:
SUBMITTER: Chang JY
PROVIDER: S-EPMC1163408 | biostudies-other | 1981 Dec
REPOSITORIES: biostudies-other
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Project description:This paper describes a manual method for N-terminal sequence analysis of polypeptides at subnanomole sensitivity. The polypeptide is degraded stepwise by using the dimethylaminoazobenzene isothiocyanate/phenyl isothiocyanate double-coupling method, and the released dimethylaminoazobenzenethiohydantoins of amino acids were identified by reversed-phase high-pressure liquid chromatography. The dimethylaminoazobenzenethiohydantoins are coloured compounds and can be detected in the visible region with the sensitivity limit of 1 pmol (signal-to-baseline noise ratio 5). A high-pressure liquid-chromatographic method was developed for complete analysis of all amino acid dimethylaminoazobenzenethiohydantoin derivatives, including the by-products of serine and threonine. Thus, without use of an automatic sequenator or radioactive materials, it is possible to determine the complete sequence of peptides and N-terminal sequence of proteins with less than 1 nmol of material.
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Project description:4-NN-Dimethylaminoazobenzene 4'-isothiocyanate was synthesized for the purpose of improving the ease and sensitivity of peptide sequence analysis. The method of 4-NN-dimethylaminoazobenzene 4'-isothiocyanate synthesis, the preparation of 24 4-NN-dimethylaminoazobenzene-4'-thiohydantoins of amino acids and their t.l.c. separation are described. All the thiohydantoins, except those of leucine and isoleucine, could be satisfactorily separated by chromatography on a two-dimensional polyamide sheet. The sensitive azo group permits the detection of 4-NN-dimethylaminoazobenzene-4'-thiohydantoins of amino acids as red spots down to pmol amounts directly on the sheet. A simple sensitive method for sequencing dipeptides and the first two or three N-terminal amino acids of proteins is also reported. The colour change of the spots from purple to blue to red after being exposed to HCl vapour, corresponding to the chemical change from 4-NN-dimethylaminoazobenzene-4' isothiocyanate to the 4-NN-dimethylaminoazobenzene-4'-thiocarbamoyl amino acid derivative to the 4-NN-dimethylaminoazobenzene-4'-thiohydantoin amino acid derivative, reveals a very interesting and valuable feature of this reagent.
| S-EPMC1172628 | biostudies-other
Project description:A manual high-sensitivity sequencing method is described, in which 4-NN-dimethylaminoazobenzene 4'-isothiocyanate is used for the stepwise degradation of amino acid residues from the peptides. The 4-NN-dimethylaminoazobenzene 4'-thiazolinones of amino acids that were released, after conversion into their thiohydantoin derivatives, were identified by t.l.c. on polyamide sheets. This new method is simple and sensitive, and requires only 2-10nmol of peptides or proteins for extended sequence analysis. The method was tested on the sequence analysis of a hexapeptide (Leu-Trp-Met-Arg-Phe-Ala), bradykinin, glucagon and native lysozyme. Results show that the proposed procedure is a sensitive method for the sequence determination of short peptides as well as for the partial sequence determination of intact proteins.
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Project description:(1) Background: Sodium taurocholate cotransporting polypeptide (NTCP) functions as a key receptor for the hepatitis B virus (HBV) infection. Analyzing HBV and NTCP interaction is an important issue not only for basic research but also for the development of anti-HBV therapeutics. We developed here a novel model system to analyze the interaction of NTCP with liposomes instead of HBV. (2) Methods: Liposomal binding and endocytosis through NTCP in HEK293T cells were achieved by serial treatments of HEL293T cells transiently expressing NTCP-green fluorescence protein (GFP) fusion protein with a synthetic biotinylated pre-S1 peptide (Myr47-Bio) and streptavidin (SA) complex (i.e., Myr47-Bio+SA) followed by biotinylated liposomes. By this procedure, binding of [biotinylated liposomes]-[Myr47-Bio+SA]-[NTCP-GFP] was formed. (3) Results: Using this model system, we found that liposomal binding to NTCP on the cell surface via Myr47-Bio+SA was far more efficient than that to scavenger receptor class B type 1 (SR-B1). Furthermore, liposomes bound to cell surface NTCP via Myr47-Bio+SA were endocytosed into cells after cells were cultured at 37 °C. However, this endocytosis was suppressed by 4 °C or cytochalasin B treatment. (4) Conclusions: This model system will be useful for not only analyzing HBV entry mechanisms but also screening substances to prevent HBV infection.
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Project description:The potential of exosomes as biomarker resources for diagnostics, prognostics and even for therapeutics is an area of intense research. Despite the various approaches available, there is no consensus with respect to the best methodology for isolating exosomes and to provide substantial yields with reliable quality. Differential centrifugation is the most commonly used method but it is time-consuming and requires large sample volumes, thus alternative methods are urgently needed. In this study two precipitation-based methods and one column-based approach were compared for exosome isolation from distinct biofluids (serum, plasma and cerebrospinal fluid). Exosome characterization included morphological analyses, determination of particle concentration, stability and exosome preparations' purity, using different complementary approaches such as Nanoparticle Tracking Analysis, Electrophoretic Light Scattering, Transmission Electron Microscopy, EXOCET colorimetric assay, protein quantification methods and western blotting. The three commercial kits tested successfully isolated exosomes from the biofluids under study, although ExoS showed the best performance in terms of exosome yield and purity. Data shows that methods other than differential centrifugation can be applied to quickly and efficiently isolate exosomes from reduced biofluid volumes. The possibility to use small volumes is fundamental in the context of translational and clinical research, thus the results here presented contribute significantly in this respect.
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Project description:Staphylococcus epidermidis releases factors that activate the HIV-1 long terminal repeat, induce cytokine release, and activate nuclear factor B in cells of macrophage lineage. The active material had a mass of 34,500 daltons, was inactivated by proteases and partitioned into the phenol layer on hot aqueous phenol extraction, and thus was termed phenol-soluble modulin (PSM). High performance liquid chromatography (HPLC) of crude PSM yielded two peaks of activity designated PSM peak 1 and peak 2. MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) mass spectroscopy indicated the presence of two components in peak 1, which were designated PSM and PSM. Peak 2 contained a single component, designated PSM. Separation of PSM and PSM in peak 1 could be achieved by a second HPLC procedure. The structure of each component was determined by amino acid sequence analysis and identification and sequencing of their genes. PSM, PSM, and PSM were 22-, 44-, and 25-amino acid, respectively, strongly hydrophobic polypeptides. PSM was identified as Staphylococcus epidermidis delta toxin, whereas PSM and PSM exhibited more distant homology to previously described staphylococcal toxins. They appeared to exist as a complex or aggregate with activity greater than the component parts. The properties of the S. epidermidis PSMs suggest that they may contribute to the systemic manifestations of Gram-positive sepsis.
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Project description:Solvent-based on-column focusing is a powerful and well known approach for reducing the impact of pre-column dispersion in liquid chromatography. Here we describe an orthogonal temperature-based approach to focusing called temperature-assisted on-column solute focusing (TASF). TASF is founded on the same principles as the more commonly used solvent-based method wherein transient conditions are created that lead to high solute retention at the column inlet. Combining the low thermal mass of capillary columns and the temperature dependence of solute retention TASF is used effectively to compress injection bands at the head of the column through the transient reduction in column temperature to 5°C for a defined 7mm segment of a 6cm long 150?m I.D. column. Following the 30s focusing time, the column temperature is increased rapidly to the separation temperature of 60°C releasing the focused band of analytes. We developed a model to simulate TASF separations based on solute retention enthalpies, focusing temperature, focusing time, and column parameters. This model guides the systematic study of the influence of sample injection volume on column performance. All samples have solvent compositions matching the mobile phase. Over the 45-1050nL injection volume range evaluated, TASF reduces the peak width for all solutes with k' greater than or equal to 2.5, relative to controls. Peak widths resulting from injection volumes up to 1.3 times the column fluid volume with TASF are less than 5% larger than peak widths from a 45nL injection without TASF (0.07 times the column liquid volume). The TASF approach reduced concentration detection limits by a factor of 12.5 relative to a small volume injection for low concentration samples. TASF is orthogonal to the solvent focusing method. Thus, it can be used where on-column focusing is required, but where implementation of solvent-based focusing is difficult.
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Project description:Pre-column fluorescent derivatization has been used for the fast quantification of amino acids using high-performance liquid chromatography (HPLC) systems. However, it generally requires an offline in-vial derivatization process with multiple derivatization reagents. The offline derivatization requires the same number of reaction vials as the number of sample vials for use as a reaction chamber for the derivatization reaction in an autosampler. Therefore, the number of samples analyzed per batch using the pre-column derivatization method is halved. To benefit from the pre-column derivatization method, we transformed the derivatization process from an offline chamber process to an online in-needle process (in-needle Pre-column Derivatization for Amino acids Quantification; iPDAQ). Fluorescent derivatization in the injection needle obviated the need for vacant vials as reaction chambers. Consequently, the throughput per batch improved up to two times, and the consumption of derivatization reagents was reduced to less than one-tenth of that in the conventional vial method. We demonstrated to separate and quantify the amino acids in various biological samples. Herein, we presented a novel HPLC-based amino acid quantification method that enables the continuous analysis of a large number of samples. The iPDAQ facilitates accurate amino acid quantification due to the automation of derivatization and achieves improvement in the throughput and reduction of analysis labor.
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Project description:1. Three azo-dye-binding proteins were identified in the soluble cell supernatant fraction from livers of rats that had received 4-dimethylaminoazobenzene by intraperitoneal injection. 2. One is basic and was highly purified. It has an isoelectric point of pH8.4 in barbital-sodium chloride buffer, I0.1, an S(20,w) value of 3.5s and a molecular weight determined by Sephadex chromatography of 45000. 3. It does not have N-terminal amino acids with free alpha-amino groups. 4. Digestion with Pronase gives rise to a single azo-dye-bound peptide, which on hydrolysis is shown to contain glycine, alanine, serine, threonine, glutamic acid and aspartic acid. The amino acid that binds the azo-dye was not identified. 5. On starch-gel electrophoresis the basic protein separates into a double band, indicating microheterogeneity. 6. The other two proteins were partially purified and occur in a fraction together. They have isoelectric points near neutrality and a molecular weight as determined by Sephadex chromatography of 13800. 7. The absorption spectra in formic acid of both the basic and the low-molecular-weight proteins are similar. The azonium ion has an absorption maximum at 518mmu and another adsorbed chromogen is present with an absorption maximum at 395mmu.
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Project description:Assembly of amyloid-beta peptide (A?) into cytotoxic oligomeric and fibrillar aggregates is believed to be a major pathologic event in Alzheimer's disease (AD) and interfering with A? aggregation is an important strategy in the development of novel therapeutic approaches. Prior studies have shown that the double N-methylated analogue of islet amyloid polypeptide (IAPP) IAPP-GI, which is a conformationally constrained IAPP analogue mimicking a non-amyloidogenic IAPP conformation, is capable of blocking cytotoxic self-assembly of A?. Here we investigate the interaction of IAPP-GI with A?40 and A?42 using NMR spectroscopy. The most pronounced NMR chemical shift changes were observed for residues 13-20, while residues 7-9, 15-16 as well as the C-terminal half of A?--that is both regions of the A? sequence that are converted into ?-strands in amyloid fibrils--were less accessible to solvent in the presence of IAPP-GI. At the same time, interaction of IAPP-GI with A? resulted in a concentration-dependent co-aggregation of A? and IAPP-GI that was enhanced for the more aggregation prone A?42 peptide. On the basis of the reduced toxicity of the A? peptide in the presence of IAPP-GI, our data are consistent with the suggestion that IAPP-GI redirects A? into nontoxic "off-pathway" aggregates.
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