Separation of amino acid phenylthiohydantoin derivatives by high-pressure liquid chromatography

Separation of the phenylthiohydantoin (PTH) derivatives of all 20 common amino acids is accomplished in approximately 11 min with excellent resolution by using high-pressure liquid chromatography. The chromatography is achieved at 50 degrees C on an Altex reversed-phase PTH-C18 column in an ammonium acetate-buffered acetonitrile, pH 4.5, mobile phase. Simple isocratic and linear gradient steps are used. Retention times for the various PTH-amino acids are very reproducible. Because the baseline is flat and free of background noise, PTH-amino acids can be detected in the low picomole range. The simplicity of this chromatographic system allows it to be easily automated.     

Isocratic separation of PTH-amino acids at picomole level by reverse-phase HPLC in the presence of sodium dodecylsulfate

The phenylthiohydantoin (PTH) derivatives of protein amino acids have been separated by reverse-phase high performance liquid chromatography (HPLC) on a fully end-capped C18 column using an isocratic solvent system. The developing solvent was 0.01 M sodium acetate buffer (pH 4.5) containing 39.5% acetonitrile and 0.02% sodium dodecylsulfate (SDS). With an automated liquid chromatography equipped with a dual-channel detector, operating at 254 and 313 nm, the present isocratic separation system was quite useful for routine microanalysis of PTH-amino acids released with a "gas-phase" sequencer. The time for one run was approximately 23 min and the limit of analysis approximately 2.5 pmol of a PTH-amino acid.     

High-pressure liquid chromatographic identification of phenylthiohydantoin derivatives of all twenty common amino acids

Phenylthiohydantoin (PTH) derivatives of all 20 common amino acids can be separated by high-pressure liquid chromatography. By using a Waters reversed-phase C₁₈ column eluted with a concave ethanol gradient in ammonium acetate, pH 5.1, all PTH derivatives were eluted in less than 30 min. The NH₂-terminal amino acid sequence of the human retinolbinding protein could unambiguosly be established for the first 40 residues. Likewise, HLA-DR antigens biosynthetically labeled with [³H]tyrosine and [³H]phenylalanine were subjected to automatic sequential degradation. Labeled PTH-amino acids were easily identified by the described chromatographic procedure.     

An optimized procedure for the separation of amino acid phenylthiohydantoins by reversed-phase HPLC

An optimization procedure for the separation of 24 PTH-amino acids by high-performance liquid chromatography on an inexpensive Merck Superspher Si 60 RP-8, (4.0 x 250 mm) column with PTH-Nle as an internal standard is described. The effects of pH, ionic strength, temperature and gradient were investigated. Using conventional HPLC equipment, the practical detection limit is about 5 pmol.     

Protein and lipid composition of a vitellin isolated from eggs of Sparus aurata

The protein and lipid composition of a vitellin isolated from eggs of Sparus aurata were characterized by SDS PAGE, N-terminal sequence analysis and lipid analysis by thin layer chromatography and gas chromatography. The lipoprotein complex contains proteins with apparent molecular weights of 69, 59, 23, 21 and 12 kDa and were characterized as vitellinogenin fragments by N-terminal sequencing. Lipid extraction and analysis indicate an association of cholesterol and phospholipids with the protein subunits. The phospholipids contain fatty acids with 14, 16 and 18 carbon atoms as determined by GC/MS.     

Manual solid phase sequence analysis of polypeptides using 4-N-N,-dimethylaminoazobenzene 4'-isothiocyanate.

A manual solid-phase method for sequence analysis of polypeptides is described. The immobilized polypeptide was subjected to stepwise degradation by Edman-type reagent, using the 4-N,N-dimethylaminoazobenzene 4'-isothiocyanate phenyllisothiocyanate double coupling method. The N-terminal amino acids were released (after conversion reaction) as 4-N,N-dimethylaminoazobenzene 4'-thiohydantoin (identified by thin layer chromatography) and phenylthiohydantoin derivatives. The method required 2--10 nmol polypeptide.     

Solid-phase sequencing on the gas-phase sequencer

Automated Edman degradation has been successfully used for determining the primary structure of numerous peptides and proteins. Quantitative solid-phase Edman degradation has great potential use for amino acid sequence analysis of synthetic peptides assembled on resin support by the Merrifield procedure. We report here the combined use of a modified gas-phase sequencer program and our improved reversed-phase HPLC analysis for PTH-amino acids to carry out the sequence analysis on synthesized peptide resins. This approach is far more sensitive than using glass beads on the conventional solid-phase sequencer. The peptide was assembled on copoly (styrene-1% divinylbenzene) resin beads at an initial substitution of 0.54 mmol/g. On a routine basis, 10-15 resin beads are used, and a repetitive yield of 94% is obtained: as few as 4 beads can be successfully sequenced. The HPLC PTH-amino acid analysis is sensitive down to subpicomole quantities. This procedure offers a sensitive and rapid analytical tool for checking the purity of peptides as they are being assembled on solid support.     

Fatty acid distribution of the normal sciatic nerve lipids in the rat

We isolated and purified the main lipids of the rat sciatic nerve. After methanolysis, fatty acids were isolated and purified by thin layer chromatography, and studied by gas chromatography. C 16 and C 18 fatty acids are the most abundant. Among the long-chain fatty acids, arachidonic acid (20:4) is present in some lipids; highly desaturated fatty acids in C 22 and C 24 are also present. In general, the fatty acids are highly saturated; cholesterol esters and ethanolamine phosphoglyceride fatty acids are highly unsaturated.     

Composition of phospholipids and phospholipid fatty acids in RAT mast cells

Summary The composition of phospholipids and phospholipid fatty acids in isolated rat serous fluid mast cells was analyzed by thin layer chromatography, gas-liquid chromatography and mass spectrometry. The phospholipids constituted about 50% of the mast cell lipids and phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylcholine, sphingomyelin and lysophosphatidylcholine were identified. The phosphatidylethanolamine fraction contained aldehydes and the highest proportion of unsaturated fatty acids. Sphingomyelin contained predominantly saturated fatty acids whereas the ratio unsaturated fatty acids: saturated fatty acids for the other phospholipids was more close to 1.     

Complete amino acid sequence of arachin subunit of molecular weight 21,000

A subunit of molecular weight 21,000 from arachin, the major peanut protein, was isolated in pure form and primary structure was determined. The subunit was fragmented with CNBr, trypsin, and NBS; the fragments were separated and isolated by PAGE, gel filtration, Dowex treatment, and paper electrophoresis, and Edman degradation on each fragment, including the intact subunit, was performed. The PTH-amino acids thus obtained were identified by UV spectroscopy and TLC. The complete sequence of 176 residues was established by overlapping technique.     

Free-radical oxidation of biological membrane lipids. V. Fluorescence of fatty acids and phospholipids]

The nature of fluorescence in UV and visible apectrum region of autooxidizing unsaturated fatty acids and phospholipids was partially determined by UV-adsorption, polarography in the system of organic solvents and thin layer chromatography.     

Hemolymph of Anopheles stephensi from uninfected and Plasmodium berghei-infected mosquitoes. 2. Free amino acids.

Determinations were made of free amino acids in hemolymph collected from adult female Anopheles stephensi mosquitoes. The hemolymph first was fractionated by extraction and precipitation procedures, after which qualitative determinations of free amino acids were made by high voltage thin layer electrophoresis, and thin layer chromatography. Subsequent quantitative determinations were made with an automatic amino acid analyzer. The concentration of total free amino acids in the hemolymph rose 60--70% after the mosquito took a blood meal, and remained relatively constant thereafter. When mosquitoes took a blood meal infected with the rodent malaria parasite Plasmodium berghei, the rise in total free amino acids was only 15--25%. The chief differences that occurred with individual free amino acids was that infected mosquitoes had greater increases in arginine, greater decreases in valine and histidine, and a total loss of detectable methionine.     

Cloning and expression of the Momordica charantia trypsin inhibitor II gene in silkworm by using a baculovirus vector

MCTI-II (Momordica charantia trypsin inhibitor II) isolated from bitter gourd (Momordica charantia LINN.) seeds is one of the serine protease inhibitors of the squash family. We cloned cDNA that encodes MCTI-II and constructed an expression system for MCTI-II by using a baculovirus vector. The recombinant baculovirus was inoculated to early fifth-instar larvae of the silkworm (strain: Shunrei x Shougetsu). Four days after infection, the hemolymph of silkworm larvae was collected and the recombinant protein was purified. Two kinds of expressed MCTI-II protein were obtained. An amino acid sequence analysis of the two proteins indicates that both were similar to the authentic inhibitor, except for the addition of a tripeptide derived from the vector at the N-terminus. One of the two inhibitors (MCTI-II A) resulted in a single PTH-amino acid in each Edman degradation cycle, while the other (MCTI-II B) resulted in two PTH-amino acids, suggesting the occurrence of cleavage of the reactive site. The inhibitory activities of MCTI-II expressed toward trypsin are examined in terms of the Ki value, these being 6.4 x 10(-10)M for MCTI-II A and 5.2 x 10(-10) M for MCTI-II B.     

Zur Bestimmung der Parameter der Bateman-Funktion bei der Auswertung von Feulgen-Hydrolysekurven

Zusammenfassung Die durch verschiedene Fixierungsmittel extrahierten Gewebsbestandteile — Glykogen, Lipide, Aminosäuren und Nucleinsäuren — wurden mit Hilfe der Dünnschichtchromatographie aufgetrennt und untersucht. Die Ergebnisse zeigen, daß die Dünnschichtchromatographie eine für histochemische Untersuchungen geeignete Methode ist, um die verschiedenen am Gewebe vorgenommenen Eingriffe zu kontrollieren.

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Summary Tissue components — glykogen, lipids, amino acids, nucleic acids — extracted with various fixation media, are separated by means of thin layer chromatography and tested. The results obtained show, that thin layer chromatography is a suitable method for histochemical investigations to control the various steps in tissue treatment.

     

Synthesis of a fully active snake venom cardiotoxin by fragment condensation on solid polymer.

A polypeptide cardiotoxin containing 60 amino acids with 4 disulfide bonds has been synthesized by the "fragment solid-phase" method. The identity of the synthetic product with native cardiotoxin was established by chromatography on Sephadex G-50, carboxymethyl-cellulose column chromatography, thin layer chromatography, disc gel electrophoresis, amino acid analysis, end group analysis, peptide mapping, circular dichroism spectra, and four biological tests.     

气相色谱法测定啤酒中的游离脂肪酸

气相色谱法测定啤酒中辛酸到二十二碳酸共１１种游离脂肪酸,采用多级溶剂萃取及薄层色谱纯化技术进行样品制备,并采充氮措施抑制脂肪酸的氧化产生,此方法有较好的重复性和回收率。     

Analysis of hepatotoxicity in maternal and fetal rats after glucocorticoid administration by lipid histochemistry and thin layer chromatography

Summary Changes in lipid metabolism of fetal and maternal rat livers were investigated on day 20 of pregnancy after administration of either 3 mg/kg or 24 mg/kg triamcinclone-acetonide or 124 mg/kg hydrocortisone in crystalline suspension to the mothers on day 15 of pregnancy. Sudan black B and Nile red as well as the UV-Schiff reaction and thin layer chromatography were used to study qualitatively the response of lipids to these glucocorticoids. Generally, after application of triamcinolone-acetonide fetal livers accumulated more lipids as toxic response to this glucocorticoid than the maternal organ; the degree of lipid accumulation was clearly dose-dependent in the fetuses. After hydrocortisone treatment, lipids in maternal livers were slightly, those in the fetuses were not affected. Histochemistry and thin layer chromatography revealed an accumulation of neutral lipids, especially of triglycerides and fatty acids which both contained increased amounts of ethylene bonds after treatment with triamcinolone-acetonide. The results also show that using combined histochemistry and thin layer chromatography, the analysis of hepatic lipids is a promising tool for the assessment of toxic effects of glucocorticoids on fetal and maternal hepatocytes in rats.Supported by the Deutsche Forschungsgemeinschaft (Sfb 174)     

Analysis of hepatotoxicity in maternal and fetal rats after glucocorticoid administration by lipid histochemistry and thin layer chromatography

Changes in lipid metabolism of fetal and maternal rat livers were investigated on day 20 of pregnancy after administration of either 3 mg/kg or 24 mg/kg triamcinolone-acetonide or 124 mg/kg hydrocortisone in crystalline suspension to the mothers on day 15 of pregnancy. Sudan black B and Nile red as well as the UV-Schiff reaction and thin layer chromatography were used to study qualitatively the response of lipids to these glucocorticoids. Generally, after application of triamcinolone-acetonide fetal livers accumulated more lipids as toxic response to this glucocorticoid than the maternal organ; the degree of lipid accumulation was clearly dose-dependent in the fetuses. After hydrocortisone treatment, lipids in maternal livers were slightly, those in the fetuses were not affected. Histochemistry and thin layer chromatography revealed an accumulation of neutral lipids, especially of triglycerides and fatty acids which both contained increased amounts of ethylene bonds after treatment with triamcinolone-acetonide. The results also show that using combined histochemistry and thin layer chromatography, the analysis of hepatic lipids is a promising tool for the assessment of toxic effects of glucocorticoids on fetal and maternal hepatocytes in rats.     

Lipid A from endotoxin: antigenic activities of purified fractions in liposomes.

Isolation of lipid A by acid hydrolysis of Shigella flexneri lipopolysaccharide resulted in a product that consisted of a heterogeneous mixture of bands when visualized by thin layer chromatography. Differential extraction with ethyl acetate and chloroform, or extraction with EDTA, followed by chloroform-methanol-water (Bligh-Dyer extraction), or a combination of both extraction schemes, resulted in partial purification of immunologically active lipid A. Eight fractions were purified further by preparative thin layer chromatography, and each of the fractions had phosphate, carbohydrate, and esterified fatty acids. Upon incorporation into liposomes, five of the eight purified fractions reacted with anti-lipid A serum, but the three fractions with the most number of esterified fatty acids failed to react with anti-lipid A serum. At least one fraction that originally was unreactive with anti-lipid A serum became reactive as a hapten inhibitor upon removal of esterified fatty acids by alkaline hydrolysis. Alkali-treated fractions from "unreactive" and "reactive" lipid A had similar activities as hapten inhibitors. Our data suggest that lipid A can exist in multiple forms that differ by the number and placement, and possibly by the type, of fatty acids linked to the carbohydrate of lipid A. Highly acylated forms of lipid A do not react with antiserum against the unpurified lipid A mixture, but removal of fatty acids does expose immunoreactive groups.