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Query: UNIPROT:P01178 (oxytocin)
 15,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Experimental conditions and parameters involved in high performance liquid chromatography (HPLC) separations of the peptide hormone oxytocin and seven of its diastereoisomers, namely [1-hemi-D-cystine]-, [2-D-tyrosine]-, [4-D-glutamine]-, [5-D-asparagine]-, [6-hemi-D-cystine-], [7-D-proline]-, and [8-D-leucine]-oxytocin, on reverse phase columns were investigated. The effects of solvent, pH, and salt concentration were studied. Using the solvent systems 10% tetrahydrofuran-ammonium acetate buffer or 18% acetonitrile-ammonium acetate buffer and the muBondapak C18 support, oxytocin was separated from each of its diastereoisomers under all conditions studied, but the order of elution of diastereoisomers was highly dependent on solvent and to a lesser extent on pH. Separations of the hormone and its diastereoisomers on reverse phase HPLC and on classical partition chromatography on Sephadex G-25 were compared. The results are discussed in terms of the interactions of the solute with the reverse phase column and the solvent system. Implications of these findings in terms of the different solution conformations of the peptides are discussed.
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PMID:The separation of peptide hormone diastereoisomers by reverse phase high pressure liquid chromatography. Factors affecting separation of oxytocin and its diastereoisomers--structural implications. 3 28

A simple, isocratic, sensitive (1 ng), and specific high-performance liquid chromatographic (HPLC) method based on photodiode-array detection (PAD) is described for simultaneous quantitation of the bioactive peptides, lysine vasopressin (LVP), arginine vasopressin (AVP) and oxytocin (OXY). Acidified pig plasma and left ventricular (LV) tissue samples were first extracted with Sep-Pak C18 columns, and the bioactive peptides were eluted with methanol, then dried at 37 degrees C and reconstituted with HPLC mobile phase. The bioactive peptides were separated by HPLC on a Dynamax 3009-A C8 column with a mobile phase of 0.1% trichloroacetic acid-50 mM heptanesulfonic acid-30mM triethylamine-20% acetonitrile in water, pH 2.5 and identified with a Waters 990-PAD system (spectrum index plots in the range 200-400 nm). Standards of LVP, AVP and OXY and their mixtures showed a linear increase in the range 5 to 100 ng and were eluted at 6.1, 6.9 and 4.6 min, respectively. Spectrum analysis showed a distinct absorption peak at 280 nm, corresponding to peptide bonds. The reproducibility of the method coefficient of variation for standards is 6.9, 5.8 and 4.7% for LVP, AVP and OXY, respectively. In plasma and tissue it is much higher: 12.9% (LV tissue) and 18.6% (plasma) for LVP. Pig plasma contains negligible amounts of AVP and OXY; LVP is much higher (0.28 +/- 0.19 ng/ml). In pig tissue, LVP predominates (6.95 ng/g wet weight) compared to AVP (1.45) and OXY (1.50). Spectral analysis is necessary to identify the bioactive peptide peaks among interfering substances and to increase the sensitivity four-fold. The method described here is useful for the simultaneous determination of LVP, AVP and OXY in the nanogram range and can be extended to picogram levels by employing PAD spectral analysis techniques.
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PMID:Isocratic high-performance liquid chromatography-photodiode-array detection method for determination of lysine- and arginine-vasopressins and oxytocin in biological samples. 205 Jul 61

Although the posterior pituitary is known to contain the PRL releasing activity or factor (PRF), its chemical identification has been a matter of dispute. In the present study, we purified PRF in porcine posterior pituitary extracts to chemically determine the primary structure. PRF activity was assessed during purification by the release of immunoreactive PRL from superfused rat pituitary cells. Two hundred seventy porcine posterior pituitaries were boiled, homogenized, and extracted with 2 M acetic acid. The acid extract was precipitated with 67% acetone, and the supernatant was absorbed onto a C18 column. The column was eluted step-wise with 10, 20, 30, 40, 50, and 60% acetonitrile (CH3CN) in 0.1% trifluoroacetic acid (TFA). The greatest PRF activity was recovered in the 30% CH3CN/0.1% TFA fraction and was further purified by ion-exchange chromatography on SP-Sephadex, followed by gel-filtration on Sephadex G-50. The Sephadex G-50 fractions with major PRF activity were finally purified by two cycles of reverse phase HPLC, yielding a single peak of PRF. Amino acid, as well as sequence analyses, indicated that the highly purified PRF was oxytocin. Authentic oxytocin showed the same chromatographic behavior and biological activity as those of the isolated peptide. In another experiment, desalted crude extracts of rat and porcine posterior pituitary tissues were directly chromatographed by reverse phase HPLC, and each fraction was assayed for PRF activity. Only two areas showed PRF activity; the largest activity coeluted with oxytocin and the smaller one co-eluted with vasopressin. The fractions which coeluted with oxytocin also showed oxytocin immunoreactivity, as examined by RIA. The results clearly indicated that the major PRF in these posterior pituitary extracts was oxytocin.
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PMID:Oxytocin is the major prolactin releasing factor in the posterior pituitary. 229 52

Reversed-phase, high-performance liquid chromatography (HPLC) was carried out to characterize rat hypothalamic corticotropin-releasing factor (CRF) using synthetic ovine CRF as a marker. Samples were injected onto a stainless steel column (4 X 250 mm) packed with Hitachi gel 3053. The column was eluted using a gradient elution of increasing acetonitrile concentration, in a mixture of NaCl-HCl at a flow rate of 1.0 ml/min, monitoring the column effluent at 220 nm with an UV detector. Fractions eluted every 1-2 min were collected and lyophilized for subsequent CRF bioassay and radioimmunoassay. When various neuropeptide mixtures including synthetic ovine CRF were injected onto the column, synthetic ovine CRF was separated from the other neuropeptides with a gradient of 0-60% acetonitrile in 0.1 M NaCl-0.01 N HCl or 0-08% acetonitrile in 0.05 M NaCl-0.01 N HCl. The median eminence extracts showed two main peaks of CRF bioactivity on HPLC. One (small CRF) coeluted with arginine vasopressin and oxytocin markers, and the other (big CRF) appeared near the position of synthetic CRF and was divided into two peaks. One coeluted with synthetic ovine CRF and the second eluted after synthetic CRF, showing high CRF activity. Three or four peaks of CRF immunoreactivity appeared on HPLC and the main peak appeared after synthetic ovine CRF marker. Our results suggest that rat CRF is different from ovine CRF, and the total lipophilicity of amino acid residues of rat CRF may be higher than that of ovine CRF.
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PMID:Characterization of rat hypothalamic corticotropin-releasing factor by reversed-phase, high-performance liquid chromatography with synthetic ovine corticotropin-releasing factor as a marker. 660 51

High performance liquid chromatography (HPLC) was used for the separation of many neuropeptides. Chromatography was carried out using a Hitachi Model 638 high performance liquid chromatograph. Peptides and samples from tissue dissolved in an aqueous buffer were injected into a stainless-steel column (4 X 250mm) packed with Hitachi #3053 (octadecylsilane). The aqueous buffer consisted of NaH2PO4 and H3PO4. After a loading phase (0% organic solvent) of 1 min, the peptides were sequentially eluted at room temperature using a gradient of organic solvent (acetonitrile or methanol, 0-60%). The eluted polypeptides were detected by UV absorbance at 220nm, and then they were collected for subsequent bio and radioimmunoassay using a fraction collector. The gradient of methanol or acetonitrile in 0.02M NaH2PO4, 0.1% H2PO4 was useful for separating small molecular peptides. The gradient of acetonitrile in 0.05-0.1M NaH2PO4, 0.1% H2PO4 was useful for separating many neuropeptides including ACTH related peptides. Retention times of chromatographed polypeptides showed good reproducibility. Good reproducibility was also found in peak areas of these peptides. A linear relationship was observed between the doses of peptides and their peak areas. The extracts of rat pituitary neurointermediate lobe showed several peaks of UV absorbance on PHLC; some of them coincided with AVP, oxytocin, alph-MSH, CLIP and beta-endorphin but others were unidentified. AVP immunoreactivity showed one peak which coincided with the AVP peak of UV absorbance, but ACTH immunoreactivity showed 5-6 peaks. Thus, many polypeptides were well separated using HPLC by changing the eluting condition. The simplicity, speed, good reproducibility and good quality of the separations render this technique suitable for purification and quantitative analysis of neuropeptides, and the combination of HPLC, radioimmunoassay and bioassay gives very fine analysis of neuropeptides.
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PMID:[The separation of neuropeptides by high performance liquid chromatography and its application to the analysis of peptides in the rat pituitary neurointermediate lobe (author's transl)]. 680 25

Analysis of peptides by reverse-phase high-pressure liquid chromatography would be simplified if retention times could be predicted by summing the contribution to retention of each of the peptide's amino acid side chains. This paper describes the derivation of values ("retention coefficients") that represent the contribution to retention of each of the common amino acids and end groups. Peptide retention times were determined on a Bio-Rad "ODS" column at room temperature with a linear gradient from 0.1 M NaclO(4), pH 7.4 or 2.1, at 0 min to 60% acetonitrile/0.1 M NaclO(4) at 80 min. The NaclO(4), a chaotropic agent, was added to improve peak shape and to minimize conformational effects. Retention coefficients for the amino acids were computed by using a Hewlett-Packard 9815A calculator programmed to change the retention coefficients for all amino acids sequentially to obtain a maximum correlation between actual and predicted retention times. Correlations of 0.999 at pH 7.4 and 0.997 at pH 2.1 were obtained for 25 peptides including glucagon, oxytocin, [Met]enkephalin, neurotensin, and somatostatin. This high degree of correlation suggests that, for peptides containing up to 20 residues, retention is primarily due to partition processes that involve all the residues. Although steric or conformational factors do have some effect on retention, the data suggest that under the above chromatographic conditions the retention of peptides containing up to 20 residues can be predicted solely on the basis of their amino acid composition. This possibility was tested by using data taken from the literature.
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PMID:Prediction of peptide retention times in high-pressure liquid chromatography on the basis of amino acid composition. 692 13

Methodology is described for characterization of the kinetics and equilibria of thiol/disulfide interchange reactions of the disulfide bonds in the neurohypophyseal peptide hormones arginine vasopressin and oxytocin and the related peptides pressinoic acid and tocinoic acid. Thiol/disulfide interchange reaction mixtures are analyzed by reversed-phase high-performance liquid chromatography. The effect of mobile-phase composition and pH on the HPLC capacity factors for the native disulfide and reduced dithiol forms of each peptide was examined. In each case, the capacity factor decreases as the acetonitrile content of the mobile phase increases. For each disulfide/dithiol peptide pair, the capacity factor is larger for the dithiol form of the peptide, indicating that the hydrophobic side chains of the linear peptide are more accessible for interaction with the hydrophobic stationary phase. To illustrate application of the methodology, rate and equilibrium constants are reported for the thiol/disulfide interchange reactions of cysteine with arginine vasopressin at pH 7.0. Cysteine reacts with arginine vasopressin to form two mixed disulfides, which in turn react with another molecule of cysteine to give the dithiol form of arginine vasopressin and cystine. Rate and equilibrium constants were determined for each step by analysis of reaction mixtures by HPLC. The results are compared to rate and equilibrium constants for reaction of cysteine with oxidized glutathione.
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PMID:Characterization of the thiol/disulfide chemistry of neurohypophyseal peptide hormones by high-performance liquid chromatography. 825 69

The retention behaviour and selectivity of selected basic, neutral and acidic peptides have been studied by capillary electroendoosmotic chromatography (CEC) with Hypersil C8, C18, Hypersil mixed-mode, and Spherisorb C18/SCX columns, 250 (335) mm x 100 microns, packed with 3 microns particles, and eluted with mobile phases composed of acetonitrile-triethylamine-phosphoric acid (TEAP) at pH 3.0 using a Hewlett-Packard Model HP3DCE capillary electrophoresis system. The selected peptides were desmopressin (D), two analogues (A and B) of desmopressin, oxytocin (O) and carbetocin (C). The peptides eluted either before or after the electroendoosmotic flow (EOF) marker, depending on the concentration of acetonitrile used and the buffer ionic strength. The retention and selectivity of these peptides under CEC conditions were compared to their behaviour in free zone capillary electrophoresis (CZE), where the separation mode was based on the electrophoretic migration of the analytes due to their charge and Stokes radius properties. In addition, their retention behaviour in RP-HPLC was also examined. As a result, it can be concluded that the elution process of this group of synthetic peptides in CEC with a TEAP buffer at pH 3.0 is mediated by a combination of both electrophoretic migration processes and retention mechanisms involving hydrophobic as well as silanophilic interactions. This CEC method when operated with these 3 microns reversed-phase and mixed-mode sorbents with peptides is thus a hybrid of two well-known analytical methods, namely CZE and RP-HPLC. However, the retention behaviour and selectivity of the selected peptides differs significantly in the CEC mode compared to the RP-HPLC or CZE modes. Therefore this CEC method with these peptides represents an orthogonal analytical separation procedure that is complimentary to both of these alternative techniques.
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PMID:Separation of selected peptides by capillary electroendoosmotic chromatography using 3 microns reversed-phase bonded silica and mixed-mode phases. 1048 34

The successful coupling of capillary electrochromatography (CEC) to an ion trap mass spectrometer via a nanoelectrospray interface (nESI) is described. Using a conductively coated tip butted to the end of a CEC column, it was possible to obtain a stable spray without any sheath liquid being employed. Selected small peptides were separated with CEC columns (100 microm i.d./25 cm long) packed with 3 microm Hypersil C8 or C18 bonded silica particles with an eluent composed of ammonium acetate/acetonitrile. Peptide mixtures of desmopressin, peptide A, oxytocin, carbetocin and [Met(5)]-enkephalin were detected in the mid-attomole range, which is the lowest amount analyzed using CEC combined with MS detection. It was also observed that sensitivity can be compromised at higher separation voltages. We demonstrate that CEC/nESI-MS, at the current stage of development, represents one of the most sensitive systems for peptide analysis.
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PMID:Capillary electrochromatography/nanoelectrospray mass spectrometry for attomole characterization of peptides. 1093 36

Oxytocin and arginine-vasopressin (AVP) are secreted into the blood in low concentrations. To analyze these peptides, we investigated two common extraction procedures, acetone-ether precipitation and C(18)-SepPak columns. Recovery from both procedures approached 70-80% of the spiked amount, though the SepPak columns were more efficient. C(18)-SepPak columns were used to sequentially separate oxytocin from AVP by eluting oxytocin first with 98% acetone followed by elution of AVP with 80% acetonitrile. Surface-enhanced laser desorption-ionization time-of-flight mass spectrometry (SELDI-TOF MS) was used to analyze oxytocin and AVP extracted with C(18)-SepPak columns from an autistic patient's plasma sample. We conclude that C(18)-SepPaks provide more consistent and efficient peptide extraction from serum or plasma that augments both quantitative and qualitative analysis by radioimmunoassay and SELDI-TOF MS.
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PMID:Extraction of oxytocin and arginine-vasopressin from serum and plasma for radioimmunoassay and surface-enhanced laser desorption-ionization time-of-flight mass spectrometry. 1286 46


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