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Reverse-phase High-performance Liquid Chromatography (reverse-phase + high-performance_liquid_chromatography)
Selected AbstractsIsolation and Identification of Bitter Peptides of Tryptic Hydrolysate of Soybean 11S Glycinin by Reverse-phase High-performance Liquid ChromatographyJOURNAL OF FOOD SCIENCE, Issue 8 2003I M.-R. ABSTRACT: The 21 peptides purified from the bitter fraction of tryptic hydrolysates of soybean 11S glycinin by using gel-permeation high-performance liquid chromatography (HPLC) and a series of 3 C18 reverse phase (RP)-HPLC were in the molecular weight range of 200-1400 Da and showed mostly the hydrophobicity of less than 1400 cal/mol. Although the primary structures of the bitter peptides from 11S glycinin were not exactly the same as those of the proglycinin, many bitter peptides were basic mimics of the common structure, indicating the significance of the primary structure of a peptide playing a role in the bitter taste perception. [source] How do enamelysin and kallikrein 4 process the 32-kDa enamelin?EUROPEAN JOURNAL OF ORAL SCIENCES, Issue 2006Yasuo Yamakoshi The activities of two proteases , enamelysin (MMP-20) and kallikrein 4 (KLK4) , are necessary for dental enamel to achieve its high degree of mineralization. We hypothesize that the selected enamel protein cleavage products which accumulate in the secretory-stage enamel matrix do so because they are resistant to further cleavage by MMP-20. Later, they are degraded by KLK4. The 32-kDa enamelin is the only domain of the parent protein that accumulates in the deeper enamel. Our objective was to identify the cleavage sites of 32-kDa enamelin that are generated by proteolysis with MMP-20 and KLK4. Enamelysin, KLK4, the major amelogenin isoform (P173), and the 32-kDa enamelin were isolated from developing porcine enamel. P173 and the 32-kDa enamelin were incubated with MMP-20 or KLK4 for up to 48 h. Then, the 32-kDa enamelin digestion products were fractionated by reverse-phase high-performance liquid chromatography (RP-HPLC) and characterized by Edman sequencing, amino acid analysis, and mass spectrometry. Enamelysin cleaved the 32-kDa enamelin only after it was deglycosylated. Kallikrein 4 digestion of the 32-kDa enamelin generated nine major cleavage products, six of which were successfully characterized. After 12 h of digestion with KLK4, all of the 32-kDa enamelin had been cleaved, but some cleavage products persisted after 48 h of digestion. [source] Peptides of human gingival crevicular fluid determined by HPLC-ESI-MSEUROPEAN JOURNAL OF ORAL SCIENCES, Issue 6 2005Elisabetta Pisano The acidic-soluble protein content of human gingival crevicular fluid was analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC), and the eluent deriving from the chromatography separation was directly introduced into an ion-trap mass spectrometer through electrospray ionization (ESI-IT MS). By this technique the molecular weight of peptides/proteins was determined with a precision of ,,1/10,000 amu. On the basis of the chromatographic behavior and the knowledge of the molecular mass value, some peptides and proteins soluble in acidic solution were unambiguously recognized. Besides high quantities of human serum albumin, , -defensins 1,4 and minor amounts of cystatin A, statherin, basic PB salivary peptide and other unidentified components were detected. The presence of , -defensins in gingival crevicular fluid is in agreement with their relevant contribution to protein composition deriving from granulocyte secretions. Other peptides and proteins abundant in human saliva, such as proline-rich proteins (PRPs) and histatins, were not detected in gingival crevicular fluid. Further investigations will be necessary to establish the origin of statherin and PB salivary peptide in gingival crevicular fluid. [source] Isolation and characterization of antimicrobial proteins and peptide from chicken liverJOURNAL OF PEPTIDE SCIENCE, Issue 6 2007Guan-Hong Li Abstract Endogenous antimicrobial peptides and proteins are crucial components of the innate immune system and play an essential role in the defense against infection. Antimicrobial activity was detected in the acid extract of livers harvested from healthy adult White Leghorn hens, Gallus gallus. Two antimicrobial proteins and one antimicrobial polypeptide were isolated from the liver extract by cation-exchange and gel filtration chromatography, followed by two-step reverse-phase high-performance liquid chromatography (RP-HPLC). These antimicrobial components were identified as histones H2A and H2B.V, and histone H2B C -terminal fragment using peptide mass fingerprinting and partial sequencing by tandem nanoelectrospray mass spectrometry. The proteins and the peptide identified in the present study, which exhibited antimicrobial activity against both Gram-positive and Gram-negative bacteria, were thermostable and showed salt-resistant activity. The antimicrobial properties of histones and histone fragment in chicken provide further evidence that histones, in addition to their role in nucleosome formation, may play an important role in innate host defense against intracellular or extracellular microbe invasion in a wide range of animal species. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd. [source] Heparin-binding proteins of human seminal plasma: purification and characterizationMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 12 2008Vijay Kumar Abstract Human seminal plasma (HuSP) contains several proteins that bind heparin and related glycosaminoglycans. Heparin binding proteins (HBPs) from seminal plasma have been shown to participate in modulation of capacitation or acrosome reaction and thus have been correlated with fertility in some species. However, these have not been studied in detail in human. The objective of this study was to purify major HBPs from HuSP in order to characterize these proteins. HBPs were isolated by affinity,chromatography on Heparin,Sepharose column, purified by reverse-phase high-performance liquid chromatography (RP-HPLC) and Size-exclusion chromatography and checked for purity on sodium-dodecyl PAGE (SDS,PAGE). Identification of HBPs was done by matrix-assisted laser desorption-ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS). Here we report the purification and identification of seven HBPs in seminal fluid. The major HBPs are lactoferrin and its fragments, semenogelin I fragments, semenogelin II, prostate specific antigen, homolog of bovine seminal plasma-proteins (BSP), zinc finger protein (Znf 169) and fibronectin fragments. In this study we are reporting for the first time the purification and identification of BSP-homolog and Znf 169 from HuSP and classified them as HBPs. Here we report the purification of seven clinically important proteins from human seminal fluid through heparin affinity chromatography and RP-HPLC, in limited steps with higher yield. Mol. Reprod. Dev. 75: 1767,1774, 2008. © 2008 Wiley-Liss, Inc. [source] A survey of sesamin and composition of tocopherol variability from seeds of eleven diverse sesame (Sesamum indicum L.) genotypes using HPLC-PAD-ECDPHYTOCHEMICAL ANALYSIS, Issue 4 2008Kelly S. Williamson Abstract The objective of this study was to determine the composition and content of sesamin and desmethyl tocopherols such as , -tocopherol (,T), , -tocopherol (,T) and , -tocopherol (,T) in seeds of sesame (Sesamum indicum L.) for 11 genotypes conserved in the United States Department of Agriculture (USDA), Agricultural Research Service (ARS) and Plant Genetic Resources Conservation Unit (PGRCU) in Griffin, Georgia, USA. Seed accessions studied were collections from eight countries worldwide, including one landrace from Thailand and two cultivars from Texas, USA. Novel methodologies and analytical techniques described herein consisted of reverse-phase high-performance liquid chromatography (HPLC) connected in series with two detection systems specific for each analyte class. Photodiode array detection was employed for sesamin analysis and electrochemical array detection was used in the determination of tocopherols. A preliminary study was conducted to assess sesamin levels in 2003 and tocopherol levels in 2004 from sesame seed samples conserved at the USDA, ARS and PGRCU. In 2005, sesame seed samples were grown, harvested and evaluated for sesamin as well as tocopherol levels. The overall results (n = 3) showed that sesamin, ,T, ,T and ,T levels were 0.67,6.35 mg/g, 0.034,0.175 µg/g, 0.44,3.05 µg/g and 56.9,99.3 µg/g respectively, indicating that the sesame seed accessions contained higher levels of sesamin and ,T compared with ,T and ,T. Statistical analysis was conducted and significant differences were observed among the 11 different sesame genotypes. This suggests that genetic, environmental and geographical factors influence sesamin and desmethyl tocopherol content. Copyright © 2007 John Wiley & Sons, Ltd. [source] Quantitative determination of anti-fungal and insecticide amides in adult plants, plantlets and callus from Piper tuberculatum by reverse-phase high-performance liquid chromatographyPHYTOCHEMICAL ANALYSIS, Issue 5 2003Hosana M. Debonsi Navickiene Abstract A rapid, sensitive and reliable reverse-phase HPLC method was used for the quantitative determination of the anti-fungal and insecticide amides, dihydropiplartine (1), piplartine (2), ,,,, -dihydropiperine (3) and pellitorine (4) in plants in natura, in plantlets in vitro and ex vitro, and in callus of Piper tuberculatum. Well-resolved peaks were obtained with good detection response and linearity in the range of 15.0,3000 µg/mL. The plants in natura contained compounds 1,4, the plantlets ex vitro and in vitro accumulated compounds 1,2 and 1,4, respectively, while only amide 4 was found in callus. Copyright © 2003 John Wiley & Sons, Ltd. [source] Quantitative determination of cytotoxicFriedo-nor -oleanane derivatives from five morphological types of Maytenus ilicifolia (celastraceae) by reverse-phase high-performance liquid chromatographyPHYTOCHEMICAL ANALYSIS, Issue 2 2002Waldemar Buffa Filho Abstract Five different morphological types of Maytenus ilicifolia of the same age and harvested under the same conditions showed distinct accumulations of some friedo-nor -oleananes. A rapid, sensitive and reliable reverse-phase HPLC method (employing an external standard) was used for the determination of the cytotoxic triterpenoids, 20,-hydroxymaytenin, 22,-hydroxymaytenin, maytenin, celastrol and pristimerin in each of the five types. Well resolved peaks with good detection response and linearity in the range1.0,100,µg/mL were obtained. Copyright © 2002 John Wiley & Sons, Ltd. [source] Development and validation of a HPLC method for determination of levonorgestrel and quinestrol in rat plasmaBIOMEDICAL CHROMATOGRAPHY, Issue 7 2010Tao Tang Abstract Levonorgestrel and quinestrol, commonly known as EP-1, has long been used in the control of wild rodents. Up to the present time, however, no method for simultaneous quantification of levonorgestrel and quinestrol in rat plasma has been reported. In the present study, a sensitive reverse-phase high-performance liquid chromatography with ultraviolet detection (RP-HPLC-UV) method for quantification of levonorgestrel and quinestrol in rat plasma has been developed. It uses a Kromasil ODS C18 column and acetonitrile-0.1% formic acid (85,:,15, v/v) mobile phase at ambient temperature. The plasma sample was prepared by hexane,isoamyl alcohol extraction (90,:,10, v/v). The flow rate and detection wavelength were 1.0,mL/min and 230,nm. The correlation coefficients were greater than 0.9995 within 0.08,50,,g/mL for levonorgestrel and 0.12,50,,g/mL for quinestrol, and the limits of detection were 0.02 and 0.05,,g/mL for levonorgestrel and quinestrol, respectively. Average recovery ranged from 92.5 to 96.3% and inter-day RSDs were less than 7.56%. This method can be applied to the further pharmacokinetic study of levonorgestrel and quinestrol in rat plasma. Copyright © 2009 John Wiley & Sons, Ltd. [source] High-performance liquid chromatography and LC-ESI-MS method for identification and quantification of two isomeric polyisoprenylated benzophenones isoxanthochymol and camboginol in different extracts of Garcinia speciesBIOMEDICAL CHROMATOGRAPHY, Issue 8 2009Satyanshu Kumar Abstract A rapid, sensitive and simple reverse-phase high-performance liquid chromatography,electrospray ionization mass spectrometric method has been developed for the identification and quantification of two isomeric polyisoprenylated benzophenones, isoxanthochymol and camboginol, in the extracts of the stem bark, seeds and seed pericarps of Garcinia indica and in the fruit rinds of Garcinia cambogia. The separation of isoxanthochymol and camboginol was achieved on a Perkin Elmer RP8 column (10 × 2.1 mm with 5.0 µm particle size) using a solvent system consisting of a mixture of acetonitrile,water (80:20, v/v) and methanol,acetic acid (99.0:1.0, v/v) as a mobile phase in a gradient elution mode. The limits of detection and quantification were 5 and 10 µg/mL for isoxanthochymol and 50 and 100 µg/mL for camboginol, respectively. The intra- and inter-day precisions were 2.34 and 3.41% for isoxanthochymol and 3.35 and 3.66% for camboginol. The identity of the two isomeric compounds in the samples was determined on a triple quadrupole mass spectrometer with ESI interface operating in the negative ion mode. The method was used to identify and quantify isoxanthochymol and camboginol in the different extracts of two Garcinia species, Garcinia indica and Garcinia cambogia. Copyright © 2009 John Wiley & Sons, Ltd. [source] Simultaneous quantification of acylated and desacylated ghrelin in biological fluidsBIOMEDICAL CHROMATOGRAPHY, Issue 12 2008Suleyman Aydin Abstract This study reports simultaneous quantification of both acylated and desacylated forms of ghrelin in biological samples, utilizing a reverse-phase high-performance liquid chromatography (HPLC) system. The HPLC assay was also compared with RIA assays in use. Biological samples (serum, saliva, urine, milk) known for the presence of ghrelin were collected from a total of eight post-partum women and eight male volunteers. Analysis of ghrelin with HPLC was also validated for linearity, precision, detection limit and accuracy. An elution time of 6 min was observed for pure (commercial) desacylated human ghrelin and for the same form of the hormone from all body fluids studied. The elution time for acylated pure human ghrelin and that in body fluids, however, was around 16 min. The mean recovery rate was over 90% for both forms with no significant interference. The lowest detectable levels for acylated and desacylated ghrelin with the method used here were 11 (±2) and 14 (±3) pg mL,1, respectively. Given its simplicity, accuracy, time and cost-effectiveness, the HPLC method described here for determination of two forms of ghrelin (active and inactive) might prove useful for certain diagnostic purposes. Copyright © 2008 John Wiley & Sons, Ltd. [source] Analysis of amino acids in nectar from Silene colorata Poiret (Caryophyllaceae)BOTANICAL JOURNAL OF THE LINNEAN SOCIETY, Issue 1 2007ANASS TERRAB Nectar samples were collected from Silene colorata Poiret (Caryophyllaceae), in three different populations from south-western Spain: Zahara de la Sierra (Cádiz), Bornos (Cádiz) and Bormujos (Seville). Samples were analysed for amino acids by reverse-phase high-performance liquid chromatography with precolumn phenylisotiocyanate (PITC) derivatization. The method has the advantage of being highly sensitive, capable of detecting nanogram (ng) quantities of amino acids. Eighteen amino acids were identified and quantified. The mean number of amino acids in a nectar sample was 14 (SD = 2.8). Six amino acids (threonine, alanine, arginine, proline, tyrosine and methionine) were detected in all samples, accounting for 83% of the total amino acids content; proline and arginine were the most abundant amino acids, accounting for 40% and 20% of the total amino acids, respectively. The mean amounts of amino acids in nectar samples per population were 824, 782 and 356 µm in Zahara de la Sierra, Bornos and Bormujos, respectively. Environmental variations such as temperature and sunlight are factors influencing the metabolic processes of nectar production. Our results may contradict the theory that the chemical constituents of floral nectar vary according to the kinds of pollinators. © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society, 2007, 155, 49,56. [source] Natural Antigenic Peptides from Squamous Cell Carcinoma Recognized by Autologous HLA-DR8,restricted CD4+ T CellsCANCER SCIENCE, Issue 8 2002Hiroaki Kondo A large number of human tumor antigens recognized by CD8+ cytotoxic T lymphocytes (CTL) have been identified. Some of them have been employed in clinical trials and have achieved some objective responses. However, little is known about those that are recognized by CD4+ T cells, except for a very few that were identified from melanomas. Previously, we reported that an oral squamous cell carcinoma (SCC) cell line, OSC,20, was effectively lysed by HLA-DRB1·08032 (HLA-DRS)-restricted autologous CD4+ T cell line, TcOSC,20. In this study, we performed two steps of chromatographic purification of the tumor cell lysate in combination with mass spectrometry. We found one reverse-phase high-performance liquid chromatography (RP-HPLC) fraction that was effectively recognized by the T cells. We analyzed the fraction by nano-liquid chromatography/electrospray ionization ion trap mass spectrometry (LC/MS/MS) and found six representative ions. We could determine the primary amino acid sequence of each of the six ions. Three of them contained a potential HLA-DR8 binding motif, and TcOSC,20 showed a rather strong cytotoxic response to one of the synthetic pep tides, namely, amino acid residues 321,336 of human a-enolase. Thus, several gene products of squamous cancer cells are endogenously processed and may be presented on HLA class II molecules, so that they could constitute target molecules for autologous CD4+ T cells. [source] Synthesis and biological activity of GnRH antagonists modified at position 3 with 3-(2-methoxy-5-pyridyl)-alanine,CHEMICAL BIOLOGY & DRUG DESIGN, Issue 2 2005M.P. Samant Abstract:, Degarelix is a potent very long-acting GnRH antagonist after subcutaneous administration. In this paper, we describe the synthesis of two analogs of degarelix incorporating racemic 3-(2-methoxy-5-pyridyl)-alanine (2-OMe-5Pal, 5) at position 3. The two diastereomers were separated by reverse-phase high-performance liquid chromatography (RP-HPLC) and the absolute stereochemistry at position 3 in the peptides was determined by enzymatic digestion with proteinase K. These analogs were tested in vitro for their ability to antagonize the GnRH receptor and in vivo for duration of action in a castrated male rat assay. Analog 7 with D2-OMe-5Pal was potent in vitro (IC50 = 5.22 nm); however, analog 8 with L2-OMe-5Pal at position 3 in degarelix lost potency as an antagonist of the human GnRH receptor (IC50 = 36.95 nm). Both the analogs were found to be short-acting in vivo. [source] Studies on the conformational properties of CP-1042,55, the hinge region of CP-10, using circular dichroism and RP-HPLCCHEMICAL BIOLOGY & DRUG DESIGN, Issue 6 2000E. Lazoura Abstract: The conformational properties of CP-1042,55, a peptide corresponding to the hinge region of CP-10, were investigated using circular dichroism spectroscopy and reverse-phase high-performance liquid chromatography (RP-HPLC). The circular dichroism studies indicated that CP-1042,55 formed considerable secondary structure in the presence of hydrophobic solution environments including 50% acetonitrile, 50% trifluoroethanol and 200 mm sodium dodecyl sulfate, which comprised a mixture of ,-helix and ,-sheet. The effect of temperature on the conformation of CP-1042,55 was investigated between 5 and 40°C, with very small changes in the spectra being observed.RP-HPLC was then used to investigate the effect of temperature on the conformation of CP-1042,55 in the presence of a hydrophobic surface. Using a C18 -adsorbent, CP-1042,55 exhibited a conformational transition at 25°C, which was associated with an increase in the chromatographic contact area and the binding affinity of the peptide for the stationary phase. In addition, near-planar bandbroadening behaviour indicated that conformational species interconverted with rapid rate constants compared with the chromatographic time scale. These results indicated that the conformational change at 25°C in theRP-HPLC system most likely corresponds to the unfolding of an ,-helical and/or ,-sheet structure to an extended coil structure. Therefore, the strong chemotactic properties of this peptide may be attributed to its ability to form considerable secondary structure in the presence of a hydrophobic environment. [source] Cytotoxic Polyketides Containing Tetramic Acid Moieties Isolated from the Fungus Myceliophthora Thermophila: Elucidation of the Relationship between Cytotoxicity and StereoconfigurationCHEMISTRY - A EUROPEAN JOURNAL, Issue 24 2007Yu-Liang Yang Dr. Abstract Five new polyketides that contain tetramic acids, myceliothermophins A,E, were isolated from the thermophilic fungus Myceliophthora thermophila. Two sets of 5-alkyl-5-hydroxyl (or 5-methoxyl)-1H -pyrrol-2(5H)-one diastereomers, myceliothermophins A/B and C/D, were separated as pure compounds by using silica-gel column chromatography and recycling reverse-phase high-performance liquid chromatography (RP-HPLC). The relative configurations of the chiral centers in 5-alkyl-5-hydroxyl (or 5-methoxyl)-1H -pyrrol-2(5H)-one moieties were deduced from NOESY correlations. In the cytotoxic assay, the 5-(2-methylpropyldiene)-1H -pyrrol-2(5H)-one analogue (myceliothermophin E) exhibited inhibition against four cancer cell lines. In addition, the significant inhibitory effect of myceliothermophins A and C and the inactivity of myceliothermophins B and D revealed the importance of the relative configurations of 5-alkyl-5-hydroxyl (or 5-methoxyl)-1H -pyrrol-2(5H)-one moieties on their cytotoxicity potency against cancer cells. [source] | ||||||||||||||||