Conformational transitions of gramicidin A in phospholipid model membranes. A high-performance liquid chromatography assessment.

We have investigated the conformation of gramicidin A reconstituted in different phospholipid environments, small unilamellar vesicles, extensive bilayers, and micelles, by exploiting a recently proposed experimental approach based on high-performance liquid chromatography [Ba?ó et al. (1988) J. Chromatogr. 458, 105; Ba?ó et al. (1989) FEBS Lett. 250, 67]. The method allows the separation of conformational species of the peptide, namely, antiparallel double-stranded (APDS) dimers and beta 6.3-helical monomers, and quantitation of their proportions in the lipid environment. Various experimental parameters (e.g., nature of organic solvent, time of incubation in organic solvent, lipid-to-peptide mole ratio, time of sonication, and temperature) commonly involved in sample preparation protocols have been analyzed independently. The results show how the peptide conformation in model membranes is exquisitely dictated by the particular nature of the reconstitution protocol. In addition, we have elucidated the nature of the slow conformational transition of gramicidin toward the channel configuration that takes place upon incubation of the model membranes. This transition has been characterized as a temperature-dependent conversion from APDS dimeric to beta 6.3-helical monomeric forms. Analysis of kinetic data permits an accurate calculation of the rate constant for this process at different temperatures in phospholipid vesicles and micelles. Finally, an explanation is proposed for the laboratory-to-laboratory variation in the observed spectral patterns of inserted gramicidin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of dipalmitoyl phosphatidylcholine in amniotic fluid by high-performance liquid chromatography

A novel method for the determination of dipalmitoyl phosphatidylcholine (DPPC) in amniotic fluid by high-performance liquid chromatography (HPLC) is described. Aliquots of 50 μl of amniotic fluid were hydrolyzed with phospholipase C from Bacillus cereus and the resulting dipalmitoylglycerol analyzed by HPLC. Run-to-run and day-to-day precision for DPPC analysis were 4.2 and 6.1%, respectively, and analysis time was 10 min. Recoveries for DPPC ranged between 92 and 98%. In summarizing, this method provides a high precision and fast turnaround time means for the analysis of DPPC in amniotic fluid.     

A study of the conformational equilibrium of DL-oligophenylalanines in nonpolar solvents in the presence and absence of lipids by high-performance liquid chromatography

The usefulness of high-performance size-exclusion liquid chromatography (HPSEC) for the separation of dimeric and monomeric species of DL-alternating oligophenylalanines is demonstrated for the first time. The experimental data obtained as a function of time fit a simple dimer-monomer equilibrium in a nonpolar solvent such as tetrahydrofuran. A higher extent of monomerization and a decrease in the time required for reaching equilibrium were detected in the presence of either water or phosphatidylcholine. The analysis of the relative proportions of the two separated species under equilibrium conditions has allowed the influence of the oligopeptide chain length on the stability of dimeric species to be determined. The advantages of this methodology, in combination with spectroscopic techniques, in studies on autoassociating peptides are considered.     

Size-exclusion high-performance liquid chromatography in the study of the autoassociating antibiotic gramicidin A in micellar milieu

Gramicidin A (gA) is a polypeptide antibiotic which forms dimeric channels specific for monovalent cations in biological membranes. It is a polymorphic molecule that adopts several different conformations, double-stranded (ds) helical dimers (pore conformation) and single-stranded beta-helical dimers (channel conformation). This study investigated the conformational adaptability of gramicidin A when incorporated into micelles as membrane-mimetic model system. Taking advantage of our reported, versatile, size-exclusion high-performance liquid chromatography (SE-HPLC) strategy that allows the separation of double-stranded dimers and monomers, we have quantitatively characterized the conformational transition undergone by the peptide in the micellar milieu. The importance of both hydrophobic/hydrophilic moieties of the amphipaths in the stabilization of concrete conformational species is demonstrated using detergents with different hydrocarbon chain length and/or polar head. SE-HPLC is a valuable, rapid, accurate technique for the structural characterization of hydrophobic autoassociating peptides that work in lipid environments such as biological membranes.     

Separation of galactolipid molecular species by high-performance liquid chromatography

A technique for the separation, detection, and quantification of molecular species of monogalactosyldiglyceride and digalactosyldiglyceride is described. Use of the technique to analyze the molecular species composition of the galactolipids isolated from Dunaliella salina chloroplasts is presented. The results indicate that the respective compositions of the two lipids are quite different. This suggests that the enzymes involved in galactolipid metabolism are very specific with respect to acyl chain composition and pairing, or that extensive retailoring of constituent acyl chains occurs following formation of digalactosyldiglyceride from monogalactosyldiglyceride.     

Separation of molecular species of sphingomyelin by reversed-phase high-performance liquid chromatography.

A convenient method for the separation of molecular species of sphingomyelin by reversed-phase high-performance liquid chromatography (HPLC) is described. Sphingomyelin species from bovine brain and sheep and pig erythrocytes were resolved into 10-12 separate peaks on a micro -BondaPak C(18) or Nucleosil-5-C(18) reversedphase column with methanol-5 mM potassium phosphate buffer, pH 7.4, 9:1 (v/v) as the solvent. Detection was at 203-205 nm. The sphingomyelin species were primarily resolved due to specific hydrophobic interaction of their fatty acid and sphingoid chains with the alkyl ligand of the stationary phase. The retention time of the sphingomyelin species increased progressively as the number of carbon atoms in the hydrophobic chains increased in the homologous series. The presence of one double bond in the molecule reduced the retention time significantly. Introduction of a second double bond in the fatty acid side chain did not reduce the retention time to the same extent as the first double bond. The presence of a trans double bond in the sphingoid moiety increased the retention time of sphingomyelin more than did a cis double bond in the fatty acid side chain. The differential hydrophobic interaction observed between the ligand of the stationary phase and different alkyl chains of the sphingomyelin species illustrates that reversed-phase HPLC technique can be conveniently used to study the extent of relative hydrophobicity of different types of alkyl chains.-Jungalwala, F. B., V. Hayssen, J. M. Pasquini, and R. H. McCluer. Separation of molecular species of sphingomyelin by reversed-phase high-performance liquid chromatography.     

The metabolism of leukotrienes in blood plasma studied by high-performance liquid chromatography

The metabolism of leukotrienes (B4, C4, D4, and E4) within human plasma was studied and a simple sample preparation is presented. It was demonstrated that leukotriene E4 and leukotriene B4 were stable during incubation at 37 degrees C using the in vitro system. In contrast, leukotriene C4 was metabolized by gamma-glutamyl transpeptidase activities into leukotriene D4 which was further metabolized by dipeptidase activities of plasma into leukotriene E4. The transition state inhibitor of gamma-glutamyl transpeptidase L-serine-borate decreased the metabolism of leukotriene C4 in plasma. Dilution of plasma demonstrated that the dipeptidase was more active compared to the gamma-glutamyl transpeptidase. The metabolizing activities of plasma were functionally characterized by fractionating the plasma proteins.     

Separation of brain monosialoganglioside molecular species by high-performance liquid chromatography

A method for the separation of molecular species of brain monosialogangliosides by high-performance liquid chromatography is described. GM4, GM3, GM2, and GM1 were purified from human brain and their individual molecular species were separated on a C18 reversed-phase column. Peaks were identified by mass spectrometry of the intact ganglioside, by gas-liquid chromatography of the fatty acids, and by high-performance liquid chromatography of the long chain bases. A characteristic elution sequence of molecular species permitted their identification based upon their retention times on the reversed-phase column.     

Determination of omeprazole in rat plasma by high-performance liquid chromatography without solvent extraction

A HPLC method without solvent extraction and using ultraviolet detection at 302 nm for the determination of omeprazole in rat plasma has been validated. Plasma samples after pretreatment with acetonitrile to effect deproteinization were dried under N(2) at 40 degrees C and reconstituted with mobile phase. The standard calibration curve for omeprazole was linear (r(2)=0.9999) over the concentration range of 0.02-3 microgml(-1). The intra- and inter-day assay variability range was 4.8-9.2% and 5.2-10.3% individually. This method has been successfully applied to a pharmacokinetic study of omeprazole in rats.     

A chemical and physical comparison of ferritin subunit species fractionated by high-performance liquid chromatography

Selected chemical and physical properties were measured for different forms of ferritin subunits which had been separated by reverse-phase high-performance liquid chromatography. Ferritin subunits from porcine spleen behaved, on sodium dodecyl sulfatepolyacrylamide gel electrophoresis, as though they were ~ M_r 2000 larger than equine spleen ferritin, whereas no difference in size was observed by gel chromatography in 6 m guanidinium chloride. All subunit species exhibited similar isoelectric focusing properties. In contrast to previous reports, no carbohydrate could be found associated with any of the isolated subunit species. Thus, the aberrant behavior of the porcine ferritin subunits between the two empirical molecular weight estimation methods appears to be the result of factor(s) other than protein intrinsic charge or covalently attached carbohydrate.     

Rapid determination of ranitidine in human plasma by high-performance liquid chromatography without solvent extraction

A simple high-performance liquid chromatographic procedure was developed for the determination of ranitidine in human plasma. The method entailed direct injection of the plasma samples after deproteination using perchloric acid. The chromatographic separation was accomplished with an isocratic elution using mobile phase consisting of 21 mM disodium hydrogen phosphate–triethylamine-acetonitrile (1000:60:150, v/v), pH 3.5. Analyses were run at a flow-rate of 1.3 ml/min using a μbondapak C₁₈ column and ultraviolet detection at a wavelength of 320 nm. The method was specific and sensitive, with a quantification limit of approximately 20 ng/ml and a detection limit of 5 ng/ml at a signal-to-noise ratio of 3:1. The mean absolute recovery was about 96%, while the within- and between-day coefficient of variation and percent error values of the assay method were all less than 8%. The linearity was assessed in the range of 20–1000 ng/ml plasma, with a correlation coefficient of greater than 0.999. This method has been used to analyze several hundred human plasma samples for bioavailability studies.     

The composition of immune sera against Neisseria meningitidis studied by high-performance liquid chromatography

The comparative study of the composition of immune rabbit sera to N. meningitidis, as well as nonimmune sera, has been made by the methods of HPLC and radial immunodiffusion. The quantitative evaluation of the main serum proteins by the two methods has shown the coincidence of the results yielded by these methods. To study the total level of IgM and IgG in the sera under study, a simple and rapid HPLC technique is proposed. The study of the stability of sera during storage (at 4-6 degrees C) has revealed that immune sera show greater stability during storage under such conditions in comparison with sera obtained from nonimmune animals.     

Separation of bilirubin species in serum and bile by high-performance reversed-phase liquid chromatography

A high-performance, reversed-phase liquid chromatographic (HPLC) procedure has been developed for the separation of at least three major bilirubin fractions in bile and four fractions in human serum. This procedure was unlike most others, in that serum was not totally deproteinized prior to injection onto the HPLC column; instead, serum was treated with an excess of sodium sulfate solution to precipitate primarily proteins larger than albumin. Injection of the filtered and diluted supernatant onto a reversed-phase column then resulted in the separation of the bilirubin species in a 24-min gradient elution run. Both the initial aqueous acidic mobile phase and the final isopropyl alcohol-based mobile phase contained 5% methoxyethanol (v/v) to facilitate elution of albumin still present in the treated sample. Bilirubin species eluting from the column were detected by absorbance at 450 nm.Results of a number of chromatographic separations of pathological sera indicated a wide variation in the relative proportions of the four bilirubin fractions observed. A correlation of the sum of the areas of the bilirubin peaks observed by HPLC was found with the total bilirubin value obtained by a standard reference procedure.     

Separation of the major species of interstitial collagen by reverse-phase high-performance liquid chromatography

Results presented here demonstrate a further application of reverse-phase high-performance liquid chromatography to the separation of large proteins. At a pH near 4.5 with a high pyridine concentration, we have completely separated three major types of human collagen (Types I, II, and III) from mixtures. We illustrate the application of this technique to the preparation of Types I and II collagen from lathyritic chick cartilage extracts.     

A sensitive method for quantitative analysis of phospholipid molecular species by high-performance liquid chromatography

A simple and sensitive method was developed for quantitative analysis of phospholipid molecular species. Diacylglycerols were prepared from phospholipids by phospholipase C treatment and converted to the corresponding dinitrobenzoyl derivatives, which could be sensitively detected at 254 nm. The derivatives of 21 molecular species were resolved by high-performance liquid chromatography with an octadecylsilyl reversed-phase column. All the derivatives had the same peak area per mol, and peak areas were proportional to the amounts of the derivatives. Quantification was carried out at the picomole level.     

Determination of 5-fluorouracil in microvolumes of human plasma by solvent extraction and high-performance liquid chromatography

In the present study, a new reversed-phase HPLC method has been developed and validated for the quantitative determination of 5-fluorouracil (5-FU) in human plasma using only 100-μl samples. The sample extraction and clean-up procedure involved a simple liquid–liquid extraction after addition of 5-chlorouracil (5-CU), used as internal standard, with 5 ml ethyl acetate. Chromatographic separations were performed on an Inertsil ODS-3 column (250×4.6 mm ID; 5 μM particle size), eluted with a mobile phase composed of acidified water (pH 2.0). The column effluent was monitored by UV absorption measurement at a wavelength of 266 nm. The calibration curves were constructed over a range of 0.20–50.0 μM and were fitted by weighted (1/x) linear regression analysis using the ratio of peak heights of 5-FU and 5-CU versus concentrations of the nominal standards. Extraction recoveries over the total range averaged 92 and 93% for 5-FU and 5-CU, respectively. The lower limit of quantitation was established at 0.20 μM (26 ng/ml), with within-run and between-run precisions of 4.2 and 7.0%, respectively, and an average accuracy of 109.3%. The within-run and between-run precisions at four tested concentrations analyzed in quintuplicate over a time period of four days were <1.4 and <4.4%, respectively. The accuracy at the tested concentrations ranged from 98.4 to 102.3%. Compared to previously described validated analytical methods for 5-FU, our present assay provides equivalent to superior sensitivity using only microvolumes of sample.     

Evaluation of commercial Ginkgo biloba dietary supplements for the presence of colchicine by high-performance liquid chromatography.

A high-performance liquid chromatographic method with photodiode array detection was developed for the detection of the presence of colchicine in commercial ginkgo products. The method is based on the baseline separation of constituents in ginkgo samples plus reference colchicine. The minimal detectable concentration of colchicine is 1.0 ng on column in the current assay. By analysis of retention time and UV profile of suspect peaks in the sample with those of reference colchicine, none of the nine commercial ginkgo products analyzed contained colchicine.     

Separation and quantitation of molecular species from plant lipids by high-performance liquid chromatography

Monogalactosyl-, digalactosyl-, and sulfoquinovosyl diacylglycerol as well as phosphatidyl glycerol were isolated by conventional TLC and then separately subjected to HPLC for resolution of molecular species. Molecular species emerge in groups from reversed-phase columns during gradient elution. The groups are separated according to the sum of carbon and double bond numbers in fatty acyl pairs in linear relation to elution times. Therefore, it is possible to identify a species group with respect to carbon and double bond numbers by its retention time. The separation is monitored by recording the absorbance at 200 nm which depends on double bond combinations in acyl pairs. Diacylglycerols released from glyco- and phospholipids were separated as rho-anisoyl derivatives according to similar criteria. In this case separation was monitored at 250 nm, at which wavelength the absorbance is directly related to molar proportions. By calculating corrected 200-nm/250-nm absorbance ratios for different molecular species of rho-anisoyl diacylglycerols, relative response factors for different double bond combinations were obtained. The 200-nm absorbances of intact lipid species can be converted to molar proportions by division with these factors.     

Evaluation of high-performance liquid chromatography for the separation and determination of arsenic species by on-line high-performance liquid chromatographic-hydride generation-atomic absorption spectrometry

An on-line high-performance liquid chromatographic-microwave assisted oxidation-hydride generation-atomic absorption spectrometric (HG-AAS) system (using columns of different kinds) has been developed for the determination of arsenite, arsenate, dimethylarsinate (DMA), monomethylarsonate (MMA), arsenobetaine (AsB) and arsenocholine (AsC) in environmental samples. Ion-pair reversed-phase chromatography using tetrabutylammonium phosphate as the ion-pair reagent and anion-exchange chromatography were evaluated and the analytical performances of each are reported. The detection limits were 97–143 and 10–30 μg l⁻¹ for ion-pair reversed-phase and anion-exchange chromatography, respectively. The Hamilton PRP-X 100 anionic column was proposed for the determination of the six species; AsB can be quantitated independently of AsC by taking the difference between readings at pH 6 and pH 10.7. The proposed methods were applied to water samples and sediments and their potential for future application was demonstrated.