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Query: UNIPROT:P01189 (beta-endorphin)
 21,003 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cation-exchange high-performance liquid chromatography (HPLC) was used to increase the sensitivity and specificity of the radioimmunoassay of plasma beta-endorphin. Proteins were precipitated from a 0.5 to 2.5 ml sample of plasma with 60% acetonitrile at pH 4.7. The supernatant was subjected to cation-exchange HPLC. Gradient elution with volatile buffers was used to separate beta-endorphin from beta-lipotropin. The beta-endorphin fraction (1.8 ml) was concentrated by lyophilization and subjected to radioimmunoassay. In healthy pregnant women at labour plasma concentration of beta-endorphin varied from 105 to 403 pg/ml. In healthy non-pregnant women plasma concentration of beta-endorphin was low, exceeding the detection limit (4 pg/ml) of the assay in only one of the 7 subjects studied.
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PMID:Rapid extraction and separation of plasma beta-endorphin by cation-exchange high-performance liquid chromatography. 609 2

Sensitivity in the 10-100 pg range for enkephalins, beta-endorphin, tyrosine (T), 12 tyrosylglycine (T-G) and tyrosylglycylglycine (T-G-G) was attained by using a high-performance liquid chromatographic (HPLC) method with electrochemical detection which is at least 100 times more sensitive than HPLC with UV detection. The chromatographic conditions on a reversed-phase C18 silica column were 50 mM sodium phosphate buffer (pH 2.1) (A) in acetonitrile-methanol (1:1) (B), isocratic mixture, flow-rate 0.6-1 ml/min, UV detection at 205 nm, electrochemical oxidation potential + 1.25 V. The separation of T, T-G and T-G-G was obtained by using 10% B while the separation of the pentapeptide, enkephalins required 40% B. Separation of enkephalins from beta-endorphin was attained at a shorter retention times did not exceed 15 min. This method can be used to determine tissue levels and pharmacodynamics of enkephalins and beta-endorphin. A highly specific measurement of the different enzymes involved in the metabolism of enkephalin has been achieved.
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PMID:Analysis of enkephalins, beta-endorphins and small peptides in their sequences by highly sensitive high-performance liquid chromatography with electrochemical detection: implications in opioid peptide metabolism. 631 25

This study was designed to assess practically the suitability of different C18 reversed-phase radially compressed polythene cartridges (Radial-Pak, Waters Assoc.) in two types of radial-compression systems, for the separation and analysis of various neuropeptides at both high (less than 5 micrograms) and low (greater than 100 pg) levels in biological extracts and to compare them with well established techniques using stainless-steel columns. A solvent system fully compatible with both radially compressed and steel columns is described. The completely volatile mobile phase (acetonitrile gradient containing trifluoroacetic acid) allows ultraviolet detection below 215 nm, gives good resolution and is readily compatible with the further radioimmunoassay and bioassay of collected fractions. The efficiency of radially compressed 5 and 10 microns "capped" and "non-capped" C18 silica supports and slurry-packed steel columns has been assessed by: (1) separation and recovery of a complex standard mixture of neuropeptides; (2) separation and subsequent identification of degradation products formed during the incubation of neurotensin with rat cortical synaptosomes; (3) analysis of alpha-melanotropin and corticotropin-(18-39) in tissue culture media containing varying amounts of foetal calf serum; and (4) characterization of corticotropin-like immunoreactivity in human cerebrospinal fluid. The Z-module fitted with the capped 10-microns irregular C18 silica cartridge gave better resolution than with the mu Bondapak steel column but the selective retention was similar. The back-pressures in the Z-module are much reduced (approximately 13 bar at 1 ml/min); therefore, flow-rates may be increased and analysis times greatly reduced. In order to obtain good resolution with the RCM-100 module which uses a non-capped stationary phase, a salt must be added (e.g. 15 mM sodium chloride) to the mobile phase to reduce polar interactions between the peptide and the free silanol groups on the stationary phase. This makes the solvent non-volatile and therefore less useful.
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PMID:High-performance liquid chromatography of neuropeptides using radially compressed polythene cartridges. 632 86

alpha-Melanotropin (alpha-MSH) is a linear tridecapeptide (Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2), that is primarily known for its ability to stimulate melanosome dispersion within integumental melanocytes (F. J. H. Tilders, D. F. Swaab and T. B. van Wimersma Greidanus (Editors), Frontiers of Hormone Research, Vol. 4, Karger, Basel, 1977; J. Ramachandran, S. W. Farmer, S. Liles and C. H. Li, Biochim. Biophys. Acta, 428 (1976) 347). In our efforts to understand the relationships of structure and conformation to the biological activities of alpha-MSH, we have prepared a series of diastereoisomeric analogues based on the highly potent analogue Ac-[Nle4, D-Phe7]-alpha-MSH4-11-NH2 (T. K. Sawyer, V. J. Hruby, B. C. Wilkes, M. T. Draelos, M. E. Hadley and J. Bergsneider, J. Med. Chem., 25 (1982) 1022). These analogues differed only in the amino acid substituted in the seven position, which was thought to be a critical residue for the biological activity of alpha-MSH. The chromatographic behavior of these analogues was examined on a C18 Vydac (16-micron) reversed-phase column with five different mobile phases. The selectivity (alpha) for the analogues was compared in 0.10% trifluoroacetic acid (TFA), 0.10% heptafluorobutyric acid (HFBA) and 0.25 M triethylammonium phosphate (TEAP) using either acetonitrile or methanol as the organic modifier. With only one exception all analogues substituted with a D-amino acid in the seven position were eluted prior to their L-amino acid counterparts.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Reversed-phase high-performance liquid chromatography studies of alpha-MSH fragments. 652 85

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

The overall objective of this research was to develop a sensitive, specific, and stability-indicating HPLC assay for the determination of the [Nle4-DPhe7]alpha-melanocyte-stimulating hormone analog known as Melanotan-1 (MT-1) in biological matrices, i.e., cell culture transport media and human plasma. Separation was accomplished isocratically within 8.0 min using a C8 reversed-phase column. The mobile phase consisted of 0.1 M phosphate buffer-acetonitrile (80:20, v/v) with 18 microliters/l triethylamine at pH 2.50. The flow-rate was 1 ml/min with detection at 214 nm. Standard curves (n = 5) were linear over the concentration range 100-1000 ng/ml. The precision, accuracy, intra- and inter-day variations were good with C.V.s typically within 8.7% for concentrations greater than 100 ng/ml. This method was applied to a study of the transport of MT-1 in the Caco-2 cell monolayer model.
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PMID:High-performance liquid chromatographic assay for Melanotan-1 ([Nle4-DPhe7]alpha-melanocyte-stimulating hormone) in biological matrices. 854 13

Products formed during cyanogen bromide (CNBr) digestion of alpha-endorphin, beta-endorphin, and horse heart myoglobin are examined using reversed-phase high-performance liquid chromatography and electrospray mass spectrometry. It is demonstrated that unstable intermediate reaction products may be formed, as well as oxidized products when the CNBr reaction is performed in 0.1% TFA in water/acetonitrile (6:4 v/v) and that, under other conditions commonly employed for the CNBr cleavage reaction, unstable intermediate products are also generated. The formation of the expected cleavage products is found to be improved by adjusting the hydrolysis conditions. The structure of the intermediate formed from alpha-endorphin is examined using electrospray mass spectrometry in combination with low-energy collision-induced dissociation and tandem mass spectrometry and is shown to have a cyclic hydrated homoserine iminolactone part. The results obtained in this study explain the formation of partially cleaved proteins in the case of Met-Thr-containing sequences, which likely have a cyclic hydrated homoserine iminolactone part instead of the putative homoserine residue.
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PMID:Characterization of unstable intermediates and oxidized products formed during cyanogen bromide cleavage of peptides and proteins by electrospray mass spectrometry. 884 40

Recently, an endogenous digitalis-like factor (EDLF) was shown to be stimulated in corticotropin (ACTH) hypertension in the rat. We have shown that mammalian plasma contains a vasoconstrictor Na,K-ATPase inhibitor, which cross-reacts with an antibody to amphibian EDLF, marinobufagenin. In the present experiment, the effect of 8 days of intramuscular ACTH treatment (0.5 mg/kg/day) of male Fisher 344 x NB rats on blood pressure, plasma ouabain-like and marinobufagenin-like immunoreactivity, and on the activity of Na,K-ATPase in aortic sarcolemma were studied. The ACTH treatment for 8 days resulted in increased systolic blood pressure (151 +/- 12.4 v 121 +/- 4.0 mm Hg, P < .01), inhibition of Na,K-ATPase in aortic sarcolemma (2.99 +/- 0.35 v 5.43 +/- 0.17 micromol ADP/mg(prot)/h), and increases in plasma concentration of marinobufagenin-like (0.44 +/- 0.06 v 0.21 +/- 0.05 nmol/L), but not ouabain-like (0.09 +/- 0.01 v 0.10 +/- 0.04 nmol/L) immunoreactivity. In dissociation enhanced lanthanide fluoroimmunoassay (DELFIA), serial dilutions of plasma from ACTH-treated rats extracted with 25% and 80% acetonitrile, respectively, demonstrated parallelism to the calibration curves of ouabain and marinobufagenin. These findings suggest that an endogenous bufodienolide Na,K-ATPase inhibitor, rather than an endogenous ouabain-like compound, is increased after 8 days of treatment of rats with ACTH.
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PMID:Plasma marinobufagenin-like and ouabain-like immunoreactivity in adrenocorticotropin-treated rats. 968 40

The isocratic and gradient elution behaviour of beta-endorphin and glucagon, two polypeptides known to exist in amphipathic alpha-helical conformations in lipophilic environments, have been examined under reversed-phase high-performance liquid chromatographic (RP-HPLC) conditions with low pH, aquo-acetonitrile mobile phases. The effects of changes in the volume fraction, psi, of the organic solvent modifier and temperature, T, on the magnitudes of the S and log k(o) values of these two polypeptides, obtained from the plots of logarithmic capacity factor (log k') vs. psi using isocratic elution conditions have been determined. These data have then been compared to the corresponding S and log k(o) values, obtained from the plots of logarithmic median capacity factor (log k) versus the median volume fraction of the organic solvent modifier (psi) derived from the linear gradient elution data, using the same n-butyl silica sorbent and related aquo-acetonitrile mobile phase conditions. As apparent from these studies, substantial differences occur in the temperature-dependent trends and magnitudes of the corresponding S and S values, or the log k(o) and log k(o) values, when these parameters are derived from experimental data acquired by these two different elution methods. Moreover, when gradient elution data for beta-endorphin and glucagon are utilised, the extrapolated values of the intercept and slope of the plots of log k vs. 1/T (corresponding to an apparent change in the median enthalpy of association, deltaH(o)assoc, or an apparent change in the median entropy of association, deltaS(o)assoc) substantially deviated from the values obtained for the thermodynamic parameters, deltaH(o)assoc and deltaS(o)assoc, derived from the log k' vs. 1/T plots using the corresponding isocratic data. These findings thus have important implications for biophysical and thermodynamic investigations when gradient elution data are employed to assess the molecular basis of the interaction of polypeptides with non-polar ligates.
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PMID:Comparison between the isocratic and gradient retention behaviour of polypeptides in reversed-phase liquid chromatographic environments. 1048 Feb 29

In this paper, we describe a general procedure to evaluate the thermodynamics of the interaction between polypeptides and hydrophobic ligands in the presence of aquo-organic solvent mixtures. These studies address experimental requirements for the determination of the linear free energy relationships, derivation of partition coefficients or other extrathermodynamic parameters such as contact areas, or assessment of the conformational changes that may occur when polypeptides or proteins interact with immobilized nonpolar ligands. Not unexpectedly from thermodynamic arguments, the trends and magnitudes of free energy parameters, such as the enthalpy of association, as previously derived in many studies from gradient elution reversed-phase high-performance liquid chromatographic (RP-HPLC) measurements are often different from the data for the same parameters derived from equilibrium binding or microcalorimetric determinations. To reconcile these divergencies and to more closely examine the thermodynamic basis of the interaction of polypeptides with nonpolar ligands, the dependency of the logarithmic capacity factor, ln k', on temperature, T, for several polypeptides (bombesin, beta-endorphin, glucagon) have been investigated using a n-butylsilica and acetonitrile-water or methanol-water mixtures of defined solvent compositions. With low-pH, acetonitrile-water mixtures, the van't Hoff plots, i.e., the plots of ln k' versus 1/T, were nonlinear over the range of T = 278-358 K, although within a narrow temperature range, e.g., from T = 278-308 K, the experimental data for these polypeptides could be approximated by a linear relationship. This nonclassical van't Hoff behavior was associated with interactive processes that involved temperature-dependent enthalpic, entropic, and heat capacity changes. In contrast, with low-pH, methanol-water mixtures, the van't Hoff plots showed dependencies that were essentially linear over the range of T = 278-358 K. The slopes of the van't Hoff plots with acetonitrile-water and methanol-water mixtures at a defined T value and solvent composition were significantly larger than those found for the corresponding experiments carried out under gradient elution RP-HPLC conditions. From these plots of ln k' versus 1/T, the changes in the apparent enthalpy of association (delta H++assoc) and the apparent entropy of association (delta S++assoc) for the interaction of these polypeptides with the solvated n-butyl ligands at different T and solvent compositions have been determined. For these polypeptides, both delta H++assoc and delta S++assoc exhibited linear dependencies on the volume fraction, phi, of the organic solvent over a narrow range of T, but the slopes of these plots were dependent on the T range examined. The dependencies of the slope term, S, and the intercept term, ln ko, derived from the plots of ln k' versus phi as a function of T, have also been investigated. A new relationship linking the S values with delta H++assoc and delta S++assoc as a function of T and phi has been derived and validated. In addition, the relationship between S, delta H++assoc, delta S++assoc, the apparent change in heat capacity, delta C++assoc, and the accessible surface area, delta Atot, of these polypeptides has been examined, thus providing a linkage of these thermodynamic and extrathermodynamic parameters to the partition coefficient, P, and the molecular properties of these polypeptides. The results confirm that entropy-enthalpy compensation effects participate in the interaction of polypeptides with hydrophobic ligands. This investigation has confirmed that the use of solvent-water mixtures of defined composition, rather than the more convenient practice of using gradient elution methods, is essential if thermodynamically consistent values of the binding affinities and partition coefficients are to be quantitatively derived. (ABSTRACT TRUNCATED)
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PMID:Investigations into the thermodynamics of polypeptide interaction with nonpolar ligands. 1056 77


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