Application of reversed-phase high-performance liquid chromatography to the separation of apolipoproteins A-IV, A-I and E from rat high-density lipoprotein

Apolipoproteins A-IV, A-I and E from rat high-density lipoprotein (HDL) were successfully purified by reversed-phase high-performance liquid chromatography (RP-HPLC), using a method which we have previously developed for the separation of apolipoproteins A-IV, A-I and E from human lymph chylomicrons [T. Tetaz, E. Kecorius, B. Grego and N. Fidge, J. Chromatogr., 511 (1990) 147]. Since analytical-scale RP-HPLC indicated that the C apolipoproteins from rat HDL coeluted with both apo A-IV and apo A-I, delipidated rat HDL was first subjected to preparative-scale size-exclusion HPLC (HPSEC) on a Serva Si300 column, which effectively separated the C apolipoproteins from all but apolipoprotein E. Fractions from HPSEC which were enriched for apolipoproteins A-IV, A-I or E were directly applied to RP-HPLC on a TSK Phenyl-5PW column. This procedure yielded fractions containing apolipoproteins A-IV, A-I or E which were pure as assessed by N-terminal sequencing and silver staining of sodium dodecyl sulphate-polyacrylamide gels.     

Evaluation of New Microparticulate Packings for Aqueous Steric Exclusion Chromatography

Abstract

Two types of high performance aqueous size exclusion columns have recently been developed, one a rigid spherical silica-based packing containing a new hydrophilic bonded phase (MicroPak TSK Gel Type SW) and the other an organic-based, semi-rigid gel (MicroPak TSK Gel Type PW). Characteristics of MicroPak TSK SW and PW columns were compared to other commercially available aqueous SEC columns packed with similar supports. Chromatographic performance of prepacked columns containing microparticulate support materials were compared for exclusion separations of water-soluble organic polymers, biopolymers, and small water-soluble oligomers. Amino acid probes were used to investigate non-exclusion effects of MicroPak TSK SW and PW columns.     

Characterization of an apolipoprotein C-III mutant by high-performance liquid chromatography and time-of-flight secondary ion mass spectrometry

Apolipoprotein (apo) C-III isoforms from a patient with a mutant apo C-III and from controls were isolated to homogeneity by isoelectric focusing and subjected to proteolytic digestion. The peptides obtained were separated by reversed-phase high-performance liquid chromatography, and their molecular masses were determined by time-of-flight secondary ion mass spectrometry. Molecular masses of peptides derived from apo C-III0, C-III1 and C-III2 were indistinguishable from control preparations, whereas the mutant apo C-III contained a COOH-terminal, carbohydrate-containing peptide with an abnormal retention time in high-performance liquid chromatography and a molecular mass higher by 291 daltons owing to oversialation at position 74 of the amino acid sequence (apo C-III3).     

High-performance size-exclusion chromatography of polypeptides on a TSK G2000SW column in acidic mobile phases

Polypeptides subjected to high-performance liquid chromatography on a TSK G2000SW column in acidic mobile phases of low ionic strength underwent a combination of size exclusion and ion exclusion from the matrix. The distribution coefficient (Kd) of each polypeptide was dependent upon the concentration of ion-pairing agent present and increased as the concentration of ion-pairing agent increased. The fractionation range of the column could thus be manipulated by altering the concentration of acid in the mobile phase. The nature of the ion-pairing acid, in terms of hydrophilic or hydrophobic ion pairing, also had an effect on the chromatography, such that hydrophobic ion-pairing agents lead to increased Kd values, especially in the case of the most basic polypeptides. To account for these results it is proposed that the TSK GSW matrix is cationic at low pH, and that ion pairing suppresses the ion exclusion experienced by cationic polypeptides under these conditions.     

Assay for dopamine beta-hydroxylase in human plasma and rat serum by high-performance liquid chromatography with fluorimetric detection

A highly sensitive assay for dopamine beta-hydroxylase activity in human plasma and rat serum by high-performance liquid chromatography with fluorimetric detection is described. Norepinephrine, formed enzymatically from the substrate dopamine, and epinephrine (internal standard), after clean-up with a cation-exchange cartridge (Toyopak IC-SP M), are converted into the corresponding fluorescent compounds by reaction with 1,2-diphenylethylenediamine. The derivatives are separated by reversed-phase chromatography on TSK gel ODS-80TM. The detection limit for norepinephrine formed enzymatically is 5 pmol per assay tube.     

Assay for tyrosine hydroxylase by high-performance liquid chromatography with fluorescence detection

A sensitive assay method for tyrosine hydroxylase in rat brain and adrenal medulla by high-performance liquid chromatography with fluorescence detection is described. L-DOPA formed enzymatically from the substrate L-tyrosine and alpha-methyldopa (internal standard), after clean-up with small cartridges of an activated alumina and a cation exchanger, Toyopak IC-SP M, are converted into the corresponding fluorescent compounds by reaction with 1,2-diphenylethylenediamine. The derivatives are separated by reversed-phase chromatography on TSK gel ODS-120T. The detection limit for L-DOPA formed enzymatically is 2 pmol per assay tube.     

Analysis of ginseng root and leaf by multiple columns and detections liquid chromatography

Abstract

A multiple columns and detections liquid chromatography system, including size exclusion chromatography (SEC) and reversed phase liquid chromatography (RPLC), for the analysis of macromolecules and micromolecules in ginseng root and leaf was developed. The columns were connected by two switching valves. Macromolecules were separated on a SEC column (TSK gel SuperMultipore PW-H column, 6?mm× 150?mm, 8?μm) by isocratic elution of 50?mM ammonium acetate aqueous solution, 0.3?mL/min of flow rate and detected by evaporative light scattering detection (ELSD). Micromolecules were analyzed on a Poroshell RP column (Agilent Poroshell 120?SB-Aq column, 4.6?mm × 50?mm, 2.7 µm) with gradient elution of water and acetonitrile, 0.6?mL/min of flow rate and detected by ultraviolet detection (UV). As a result, in the macromolecules chromatogram of ginseng root sample showed two main peaks while only one major peak for ginseng leaf. For micromolecules analysis, 17 compounds (3 nucleosides + 14 saponins) and 17 compounds (3 nucleosides + 1 flavonoid + 13 saponins) were found in ginseng root and leaf, respectively. The developed method is helpful for the quality evaluation of ginseng root and leaf.     

绍兴黄酒中ACE活性抑制肽的分离分析

首次将绍兴黄酒中的肽类组分进行大孔吸附树脂柱层析和高效凝胶过滤色谱以及反相色谱的多步提取纯化与抑制血管紧张素转换酶（AcE）活性试验,并首次利用基体辅助激光解吸电离-飞行时间串联质谱分析和液相色谱-电喷雾电离四极杆-飞行时间串联质谱联用分析鉴定出黄酒中4种ACE活性抑制肽的氨基酸序列为：VEDGGV、PST、NT和LY。     

High-Performance Liquid Chromatography of Low-Molecular-Weight Bacterial Metabolites of a Proteinic Nature

The efficiency of the separation of low-molecular-weight bacterial metabolites of a proteinic nature under the conditions of reversed-phase, normal-phase, and exclusion high-performance liquid chromatography (HPLC) on Nucleosil C18, Zorbax ODS, TSK G3000 SW, and Ultrasphere Si columns was studied. The number of components in a low-molecular-weight metabolite fraction (<20 Da) of bacterial strains Bacillus sp. 739 and Bacillus sp. X-b was determined. A procedure is proposed for the determination of polypeptides and low-molecular-weight proteins by normal-phase HPLC on an Ultrasphere Si column using elution with an aqueous ammonia solution (pH 7.4) and UV detection at 220 nm.     

从猪血中分离纯化高纯度的猪血红蛋白

为了从猪血中分离纯化高纯度的猪血红蛋白,建立了通过超滤、DEAE-Sepharose Fast Flow离子交换色谱和Sephadex G-75凝胶 排阻色谱三步法制备高纯度猪血红蛋白的方法,并通过十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、高效凝胶排阻色谱和反相高 效液相色谱方法,对纯化后的猪血红蛋白进行了鉴定。经三步分离纯化后,猪血红蛋白的纯度大于99%,含量为1.328 g/L。     

Separation of peptides by multimode chromatography on a column packed with vinyl alcohol copolymer gel

Several mechanisms of peptide separation in high-performance liquid chromatography were observed to occur on the Asahipak GS-320 packed with vinyl alcohol copolymer. Neutral rather than acidic mobile phases were employed as they were found to result in higher retention of many peptides on the GS-320. For neutral peptides, the retention volume corresponded to the Rekker's hydrophobic fragmental constant, with a correlation coefficient of 0.71. Peptides with acidic residues eluted early, as an effect of ionic exclusion; those with basic residues were retained longer, owing to an ion-exchange effect. The ionic interactions were shown to involve the carboxylic group present on the gel polymer. The net result was found to be excellent separation of hydrophilic as well as hydrophobic peptides, related to differences in their isoelectric points. The combination of these complex mechanisms, together with the size-exclusion effect of the GS-320 gel for separation of proteins and other large molecules and for analysis with a mobile phase high in acetonitrile content, makes possible high-resolution isocratic analysis of peptides, which cannot be achieved on octadecylsilica or ion-exchange columns.     

Simultaneous determination of caffeine and its primary demethylated metabolites in human plasma by high-performance liquid chromatography.

An improved high-performance liquid chromatographic method for the simultaneous determination of caffeine and its three primary metabolites (theophylline, theobromine and paraxanthine) in human plasma is described. The four substances were separated on a reversed-phase column (5 microns TSK gel ODS-80TM, 150 mm x 4.6 mm I.D.) by use of the mobile phase methanol-0.1 M NaH2PO4 (30:70, v/v) with a flow-rate of 0.8 ml/min. Absorbance was monitored at 274 nm. The detection limit was 5 ng/ml for theobromine and caffeine and 10 ng/ml for paraxanthine and theophylline. The linearity and reproducibility were sufficient for drug monitoring of caffeine and its primary methylxanthines.     

Improved high-performance liquid chromatographic procedure for the determination of chlormethiazole levels following solid-phase extraction from plasma

An improved high-performance liquid chromatographic method for the determination of chlormethiazole levels in plasma is described. The drug is extracted from plasma using commercially available reversed-phase extraction columns; recovery values obtained using Sep-Pak C18 and Bond Elut C1, C2, C4, C6, C8, C18 columns are compared. Separation was achieved by reversed-phase chromatography, using a mobile phase consisting of 0.025 M sodium acetate buffer, pH 4.5-acetonitrile (67:33) at a flow-rate of 1.6 ml/min in conjunction with a 15-cm Jones Chromatography Apex ODS column. The analytical column was protected by a Waters Assoc. Guard-Pak module containing a Guard-Pak CN insert. Using ultraviolet detection at 254 nm chlormethiazole levels in the region of 50 ng/ml can be measured with only 500 microliter of plasma.     

Purification of canine prolactin and growth hormone by fast protein liquid chromatography.

Prolactin and growth hormone are two peptide hormone with a very similar structure. A simple method is described for the simultaneous purification of these two peptides from canine pituitary extract by fast protein liquid chromatography. After extraction at pH 5.0 and 9.6 and anion-exchange chromatography on a MonoQ column, prolactin and growth hormone are then separated by gel filtration chromatography on two Superose columns coupled in series. The different fractions of the purification scheme are checked for the presence of the peptide hormones by sodium dodecyl sulphate gel electrophoresis in the Pharmacia PhastSystem. Each hormone is also characterized by its behaviour in a Western Blotting Detection System.     

Anion-exchange chromatography of DNA restriction fragments.

The abilities of several high-performance liquid chromatography (HPLC) anion-exchange packings to separate DNA restriction fragments, ranging in size from 50 to 23,000 base pairs, were studied. The ion exchangers investigated include the porous packings Protein-Pak DEAE-5PW, Nucleogen-DEAE 4000-7, Poros-Q and BakerBond WP-PEI, and the non-porous packings TSK Gel DEAE-NPR, Gen-Pak FAX, and ProPac PA1. The results indicated that the non-porous packings could separate all 18 fragments (less than 600 base pairs) in a pBR322 DNA-HaeIII digest, while of the porous packings, only Nucleogen-DEAE 4000-7 could resolve DNA fragments in this size range. Only Gen-Pak FAX and TSK Gel DEAE-NPR could significantly resolve the very large DNA fragments (125-23,000 base pairs) of a lambda DNA-HindIII digest. The chromatographic parameters governing this separation by Gen-Pak FAX were optimized so that six of eight fragments were resolved. Split-peak phenomena were observed at low flow-rates when employing non-poros packings, but were eliminated by the incorporation of organic modifiers or surfactants, suggesting that, under certain conditions, hydrophobicity may play a significant role in separations on this packing. Gen-Pak FAX also separated 21 of 23 fragments in a 1000-base pair DNA ladder, a performance which, in addition to the quantitative capabilities of HPLC, makes anion-exchange chromatography a powerful method complementary to slab-gel electrophoresis, and perhaps preferable over agarose gel electrophoresis for applications such as the confirmation of plasmid integrity.     

High-performance liquid chromatographic method for the simultaneous purification of cathepsins B, H and L from human liver.

A high-performance liquid chromatographic procedure for the isolation of the three cysteine proteinases, namely cathepsins B, H and L, is described. The method is based on the following four steps. (1) A classical AcA 44 gel permeation separation with a 30-70% ammonium sulphate fraction from the human liver homogenate is used to remove the non-enzymic high-molecular-mass components. (2) Preparative cation-exchange chromatography on a CM-SW TSK column can separate the three proteinases. (3) An anion-exchange step on a semi-preparative DEAE-SW TSK column for the cathepsin H fraction is used to remove a small amount of cathepsins B and L activities. (4) The three separated enzymes are purified on an analytical TSK gel 2000 SW column. The purity of each enzyme is assessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis and electrofocusing on polyacrylamide gels. To check the activities of the purified proteinases, the kinetic constants [Michaelis constant (KM) and catalytic constant (Kcat)] and the ratio Kcat/KM against the fluorigenic substrates Arg-NH-Mec, Z-Arg-Arg-NH-Mec and Z-Phe-Arg-NH-Mec after active-site titration using E-64, were determined. Z-Phe-Phe-CNH2 was also used as a specific inhibitor of cathepsin L. This method requires only 6 g of human liver, and gives a high yield of the three lysosomal cysteine-proteinases: thus, about 150 micrograms of cathepsin B and 50 micrograms each of cathepsins L and H are obtained in a single run.     

Isolation from fetal bovine serum of a peptide similar to the alpha chain of thrombin by reversed-phase high-performance liquid chromatography

During the preparation of erythrotropin from fetal bovine serum, a group of peptides co-eluted with this erythroid cell stimulating factor on semi-preparative reversed-phase high-performance liquid chromatography. They could be subsequently separated by a combination of reversed-phase high-performance liquid chromatography in the presence of heptafluorobutyric acid as ion-pairing reagent and gel permeation high-performance liquid chromatography. One of these peptides has been extensively purified. Partial amino acid sequence analysis indicated that fourteen of the seventeen N-terminal amino acids are identical with the N-terminal sequence of the alpha chain of bovine thrombin. The same isolation procedure could be useful for the identification of other major peptides of fetal bovine serum.     

Determination of phenylethanolamine N-methyltransferase by high-performance liquid chromatography with fluorescence detection

A highly sensitive assay method for phenylethanolamine N-methyltransferase in rat adrenal medulla and brain is described which employs high-performance liquid chromatography with fluorescence detection. Epinephrine formed enzymatically from the substrate norepinephrine and isoproterenol (internal standard), after chromatography on a small cartridge of a cation exchanger, Toyopak SP, are converted into the corresponding fluorescent compounds by reaction with 1,2-diphenylethylenediamine, a selective fluorescence derivatization reagent for catechol compounds. The derivatives are separated by reversed-phase chromatography on TSK gel ODS-120T. The detection limit for epinephrine formed enzymatically is 0.66 pmol per assay tube.     

Anion-exchange fast protein liquid chromatographic characterization and purification of apolipoproteins A-I, A-II, C-I, C-II, C-III0, C-III1, C-III2 and E from human plasma

This paper describes a procedure for the rapid isolation of urea-soluble apolipoproteins (apo) from delipidated human very-low- and high-density lipoproteins using anion-exchange fast protein liquid chromatography. The separation was complete within 30 min and peaks corresponding to apolipoproteins A-I, A-II, C-I, C-II, C-III0, C-III1, C-III2 and E were identified by comparing their chromatographic, electrophoretic and immunological behaviour with that of purified standards of each protein. A second purification step is necessary to obtain pure apolipoproteins. Apo E, which is difficult to purify by conventional chromatography, has been obtained in a good yield. The apo C-II that was obtained produced a symmetrical peak on chromatography but three bands in isoelectric focusing. The method can be upgraded to a preparative scale and offers the possibility of direct purification of apolipoproteins both from high-density lipoproteins and (following preliminary gel chromatography) from very-low-density lipoproteins.     

Efficient purification of human plasma beta 2-microglobulin from the haemodialysate of a patient with chronic renal failure by use of high-performance liquid chromatography

Human plasma beta 2-microglobulin was isolated in a good yield (more than 80%) from the haemodialysate (blood ultrafiltrate) of a patient with chronic renal failure. The isolation procedure consisted of Sephadex G-100 gel filtration and two steps of high-performance liquid chromatography: reversed-phase high-performance liquid chromatography and gel permeation chromatography. The purified beta 2-microglobulin was homogeneous by sodium dodecyl sulphate polyacrylamide gel electrophoresis and two-dimensional electrophoresis.