Separation of collagen cyanogen bromide-derived peptides by reversed-phase high-performance liquid chromatography.

A new technique for separation of the CNBr cleavage products of collagen is described. It involves the use of a 30 nm pore reversed-phase high-performance liquid chromatography column eluted with a linear gradient of acetonitrile/water containing 0.01 M-heptafluorobutyric acid. The separation of type I, type II and type III CNBr peptides is described. Resolution is particularly good for the low-molecular-weight peptides. The method is fast, quantitative and sensitive, and the complete volatility of the eluent facilitates the recovery of the separated peptides.     

Poliovirus sampling by using sodium dodecyl sulfate/EDTA-pretreated chromatography paper strips

To achieve the goal of poliovirus eradication, surveillance of endemic areas is a crucial step in the poliovirus eradication program. Currently, six countries still have endemic poliovirus. We have tested a novel method which uses SDS/EDTA-treated chromatography paper strips to collect and transport poliovirus-containing stool samples. The SDS/EDTA-treated paper strips were soaked with different dilutions of poliovirus-containing feces and stored at different temperatures. After storing the SDS/EDTA paper strips for 5 months at 37 degrees C, poliovirus RNA could be successfully amplified using RT-PCR. Infectivity of wild-type poliovirus type 1, 2, and 3 was lost upon contact with the SDS/EDTA-treated strips. This easy, inexpensive, and biosafe chromatography paper strip method for the collection and transportation of poliovirus samples can be of use in poliovirus surveillance and polio vaccination programs.     

Determination of boronophenylalanine in biological samples using precolumn o-phthalaldehyde derivatization and reversed-phase high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method for the detection of boronophenylalanine is described. Determination was obtained by precolumn reaction of o-phthalaldehyde with a mixture of standard amino acids containing boronophenylalanine and separating the corresponding o-phthalaldehyde derivatives, using a Kromasil C-18, 250 x 4.6 mm, 5-microm particle size column, a step gradient with two buffers, a flow rate of 1.2 ml/min, a column temperature of 23 degrees C, and fluorimetric detection (excitation and emission wavelengths of 330 and 430 nm, respectively). The use of such a method for assaying boronophenylalanine in biological samples was tested in neutralized perchloric acid blood and cerebral tissue extracts of rats treated with intracarotid administration of 300 mg/kg of body weight boronophenylalanine. Results of these experiments showed that the present HPLC method represents a valid alternative to currently available analytical techniques for assaying boronophenylalanine based on boron determination in terms of reproducibility, recovery, or sensitivity. Therefore, it is suggested that the present method may routinely be used in all preclinical and clinical studies in which quantification of circulating and tissue concentrations of boronophenylalanine is critical for the application of boron neutron capture therapy.     

Method for rapid removal of sodium dodecyl sulfate from polyacrylamide gels

A method is described for the isolation of hepatic microsomes by polyethylene glycol 6000 fractionation of the postmitochondrial fraction of liver homogenate. The procedure is simple and rapid requiring two centrifugation steps at 8000g for 10 min. The preparation has peptide patterns and levels of drug metabolic and other enzymatic activity similar to those of the microsomal fraction isolated by high-speed centrifugation and is referred to as polyethylene glycol 6000 microsomes. It is clarified with detergents and can serve as the starting material for the purification of microsomal proteins.     

Segmented condensation of peptides on a solid phase using subtilisin complexed with sodium dodecyl sulfate

The subtilisin-sodium dodecyl sulfate complex was shown to catalyze the coupling of peptide segments on a solid phase in organic medium. By a two-stage enzymic condensation of peptide fragments on aminosilochrom (A) containing Met-Ala-Gly as a spacer, Dnp(or Boc)-Ala-Ala-Leu-Ala-Ala-Glu(OMe)-Met-Ala-Gly-A and Z-Ala-Ala-Glu(OMe)-Ala-Ala-Leu-Met-Ala-Gly-A were obtained. It was shown that the condensation products can be split off from the support using the Met residue cleavage by BrCN.     

Quantification of sodium dodecyl sulfate in microliter biochemical samples by gas chromatography

A novel gas chromatography (GC) method has been developed to accurately quantitate sodium dodecyl sulfate (SDS) in aqueous biochemical samples. This method is based on the quantitative conversion of SDS to 1-dodecanol in the GC injection port at elevated temperature, and the thermal degradation product 1-dodecanol was analyzed to determine SDS concentration. It was found that the addition of guanidinium chloride (GnHCl) to SDS samples (via direct dilution with GnHCl/MeOH solution) is necessary to ensure accurate quantitation. The presence of GnHCl enables quantitative conversion of SDS to 1-dodecanol, improves sensitivity, and virtually eliminates interference from proteins and other chemicals commonly present in biochemical samples. The method features direct analysis of diluted SDS samples, is free from interference, and is capable of quantifying less than 1 ng SDS in biochemical samples. It is also suitable for samples with limited volume, with as little as 1 microl sample being sufficient for quantitation.     

Separation and quantitation of leukotrienes by reversed-phase high-performance liquid chromatography

A rapid and sensitive reversed-phase HPLC procedure is reported which allows the simultaneous separation and quantitation of LTC₄, 11t-LTC₄, LTD₄, LTB₄, 12epi,6t,8c-LTB₄, 6t-LTB₄ + 12epi,6t-LTB₄, two trihydroxy-eicosatetraenoic acids tentatively identified as 20-OH-LTB₄ and 20-OH,12epi,6t,8c-LTB₄ and several not yet identified 15-series leukotrienes produced by the cytosol of porcine polymorphonuclear leukocytes.     

Optimum separation condition of peptides in reversed-phase liquid chromatography

An efficient optimization method was suggested to separate biologically active peptides by RP-HPLC. In this work, the binary mobile phase of water and acetonitrile was used with the buffer of trifluoroacetic acid (TFA). The elution profiles were calculated by the plate theory based on the linear and quadratic equations of retention factor, lnk=A+BF, lnk=A+BF+CF(2), and F was the vol.% of acetonitrile. We modified the plate theory to calculate elution profile in both isocratic and gradient mode. From the final calculated results, the first mobile phase composition was water in 0.1% TFA/acetonitrile in 0.1% TFA, 81/19vol.%, then after 7-8 min, the second composition of mobile phase was linearly changed to 79/21vol.%, and finally after 8 min, it was kept at the isocratic mode. In the experimental conditions, the agreement between the experimental data and the calculated values was relatively good.     

Separation of proteins by ion-exchange chromatography using gradient elution

Chromatographic separation of proteins by the gradient elution method using DEAE Toyopearl 650^® was carried through. The concentration gradient was effected by changing the ionic strength of NaCl in the carrier buffer solution. Bovine serum albumin and hemoglobin were used as model proteins for separation. The experimental chromatogram was compared with theoretical results of Yamamoto et al. [1, 2]. Adsorption equilibria of the proteins onto the carrier were measured and expressed by a function of the ionic strength. The retention volume and peak width of the resulting chromatogram can be calculated from the equilibrium data using the Yamamoto theory. The calculated results agreed well with the experimental data.The method presented in this paper will be useful to predict the viability of ion-exchange chromatography in protein separation.List of Symbols c kg m^–3 concentration in the liquid phase - c _s kg m^–3 concentration in the solid phase - D _s m² s^–1 intraparticle diffusivity - d _p m particle diameter - E _z m² s^–1 longitudinal diffusivity of the protein - E _{z I} m² s^–1 longitudinal diffusivity of ionic strength - H /(1 – ) - I kmol m^–3 ionic strength - I _O kmol m^–3 initial ionic strength - I _p kmol m^–3 ionic strength at the peak - I _s kmol m^–3 ionic strength in the solid phase - I/V mol (dm³)^–2 slope of the ionic gradient elution - m distribution coefficient - m distribution coefficient at I - m _I distribution coefficient for ionic strength - Q cm³s^–1 flow rate - R m particle radius - R _s degree of separation - r m radial position inside particles - t s time - u m s^–1 linear velocity - V cm³ eluted volume of liquid - V _p cm³ eluted volume of liquid at the peak - V _T cm³ volume of the packed bed - W cm³ peak width - Z m bed height - z m vertical position in the bed - z _p m peak position from the inlet of the bed - (t) delta input at time - void fraction - ₁ s first moment - ₂ s² second central moment - s superficial space time     

Separation of the major adrenal steroids by reversed-phase high-performance liquid chromatography

The major adrenal steroids were separated by multistep gradient elution with a reversed-phase high-performance liquid chromatography system, employing water and 1-propanol as solvents. With this solvent system, a wide range of 21 5-ene-3 beta-ol and 4-ene-3-one steroids can be resolved in a single chromatogram, which was not possible with previously published gradient solvent systems. In particular, intermediate steroids of the biosynthetic pathway, 17 alpha-hydroxyprogesterone, 17 alpha-hydroxypregnenolone, and dehydroepiandrosterone, were separated with baseline or sufficient resolution to allow accurate quantitation. Using the 1-propanol-water gradient, the separations of 5-ene and 4-ene steroids were compared on different octadecylsilyl packings. Optimum resolution was obtained with a fully covered, spherical particle. The 1-propanol-water gradient was compared to a previously published methanol-water gradient in the analysis of steroidogenesis by adrenocortical cell cultures. HPLC analysis of the steroid production was quantitatively the same with both gradient solvent systems. However, qualitatively, the methanol-water gradient system did not resolve the above-mentioned intermediate steroids.     

Separation of peptides by reverse-phase high-performance liquid chromatography using propyl- and cyanopropylsilyl supports

The separation of peptides and proteins by reverse-phase high-performance liquid chromatography with cyanopropylsilyl and large-pore propylsilyl supports, together with aqueous trifluoroacetic acid/acetonitrile gradients, was studied. Operating parameters (trifluoroacetic acid concentration, flow rate, and gradient slope) were evaluated using different enzymatic digests of horse cytochrome c and bovine serum albumin. Peptides ranging in size from five amino acids to 68 kDa could be separated on the propylsilyl column in a single chromatographic run. The cyanopropylsilyl column is suitable as a supplement to the use of the large-pore column for medium size (5-20 amino acids) peptides. The chromatographic supports and conditions presented here offer a simple, sensitive, and rapid separation system for a wide size range of peptides and proteins. They extend the versatility of separation methodology for these molecules.     

Rapid assay for gamma-carboxyglutamic acid in urine and bone by precolumn derivatization and reversed-phase liquid chromatography

A reversed-phase high-performance liquid chromatographic assay for the analysis of gamma-carboxyglutamic acid (Gla) in urine and bone protein hydrolyzates is described. The method employs precolumn derivatization with o-phthalaldehyde and mercaptoethanol. Gla was quantified by reference to an internal standard (beta-carboxyaspartic acid). The "within-run" coefficient of variation of the assay for Gla in urine was between 2.1 and 3.4%, and that for bone protein hydrolyzates was 3.2%. The "between-run" coefficient of variation ranged from 4.1 to 5.5%. There was good agreement between the measurement of urinary Gla by high-performance liquid chromatography and amino acid analyzer. Free Gla could not be detected in serum.     

Desalting and concentration of proteins in dilute solution using reversed-phase high-performance liquid chromatography

A rapid method for desalting and concentrating dilute protein solutions using short reversed-phase columns (3-4 cm) has been described. The recovery of proteins is usually 90-100%. The method is simple and rapid and allows the desalting and concentration of protein samples simultaneously. A wide variety of proteins in the range up to 80 kDa can be desalted in microgram to milligram amounts, and volumes up to 1 liter can be concentrated to a few milliliters by a single injection.     

Simultaneous determination of arginine and seven metabolites in plasma by reversed-phase liquid chromatography with a time-controlled ortho-phthaldialdehyde precolumn derivatization

In an attempt to simultaneously detect molecules generated through the metabolism of l-arginine, a high-performance liquid chromatography method with on-line time-controlled preinjection reaction of ortho-phthaldialdehyde derivatization was developed. Plasma concentrations of citrulline, N(G)-hydroxy-l-arginine, N(G)-monomethyl-l-arginine, asymmetric N (G), N (G)-dimethyl-l-arginine, symmetric N (G), N (G')-dimethyl-l-arginine, ornithine, and agmatine were analyzed within 35min, using only 20microl of sample, pretreated by a simple cold ethanol cleanup procedure. Plasma samples of 35 healthy human volunteers were analyzed and results were comparable to other published data. All detection parameters of the method demonstrate that it is a reliable and efficient means for the comprehensive determination of arginine and its metabolites, making this approach suitable for routine clinical applications.     

Assessment study on the high-performance liquid chromatography-type hydroxyapatite chromatography in the presence of sodium dodecyl sulfate.

The HPLC-type hydroxyapatite chromatography in the presence of sodium dodecyl sulfate (SDS) was assessed with special attention to the behavior of the surfactant. A significant amount of SDS was found to be adsorbed to the hydroxyapatite packed in the column from the starting buffer, 50 mM sodium phosphate buffer, pH 7.0, only when the buffer contained SDS in a concentration at or above its critical micelle concentration. When the phosphate buffer concentration was increased while the SDS concentration was kept at 1 mg/ml, the adsorbed surfactant was desorbed in advance of the release of proteins. Polypeptides derived from proteins could be successfully separated only when the column had been thoroughly equilibrated with the above-mentioned starting buffer solution. When a protein polypeptide complexed with SDS, which had been similarly equilibrated, was applied to the column, an amount of SDS corresponding to 75-90% (w/w) of the surfactant originally bound to the polypeptide was released upon its binding to the hydroxyapatite. On the other hand, porin, an Escherichia coli outer membrane protein, retaining its trimeric native structure in the presence of SDS, released a significantly smaller amount of SDS. When the membrane protein was denatured to give a single polypeptide, it behaved in a manner similar to that of the other protein polypeptides. The mechanism of binding of the protein polypeptides was discussed on the basis of these results. The native and denatured entities of porin could be efficiently separated as the result of the difference in their mode of interaction with the hydroxyapatite.     

Separation of tryptic phosphopeptides of ribosomal origin by reversed-phase high-performance liquid chromatography

Phosphorylation sites for cyclic AMP-dependent kinase in ribosomal proteins and their synthetic analogues were converted to tryptic phosphopeptides and analysed by reversed-phase high-performance liquid chromatography (RP-HPLC) using gradients of acetonitrile in water and 0.1% trifluoroacetic acid. Tryptic variants differing by only NH₂-terminal basic amino acid redidues or phosphoryl groups were not always well resolved under these conditions. The different phospho forms could be resolved by RP-HPLC and thin-layer cellulose mapping was found to be the most effective strategy for the absolute purification of trytic phosphopeptides from crude tryptic digests.     

Differential refractometric determination of binding of sodium dodecyl sulfate to protein using high-performance gel chromatography

When sodium dodecyl sulfate (SDS) is added to a high-performance gel chromatographic column equilibrated with a buffer solution containing SDS at a level above the critical micelle concentration, the surplus SDS migrates as micelles giving a sharp peak. The presence of an unfolded protein in the sample solution gives a polypeptide peak in advance of the SDS micelle peak. As the result of SDS binding to the polypeptide, the SDS micelle peak is attenuated in comparison to that in the absence of protein. Thus the amount of SDS bound to the polypeptide can be determined accurately and simply from the decrease in the area of the SDS micelle peak. This approach is particularly useful for precise determination of bound SDS, which is pertinent to understanding the state of the protein polypeptide-SDS complex under the conditions of SDS-polyacrylamide gel electrophoresis.