Resolution and quantitation of apolipoproteins A-I and A-II from human high-density lipoprotein by size exclusion high-performance liquid chromatography

Apolipoproteins A-I and A-II, extracted from human high-density lipoprotein (HDL), were resolved and quantified by size exclusion high-performance liquid chromatography on TSK 125 and TSK 250 analytical columns connected in series without the use of chemical denaturants or detergents in the eluent buffer. The columns were pre-equilibrated with a solution containing 0.1 M sodium phosphate, pH 7.2, 0.2 M sodium chloride at a flow-rate of 1 ml/min. Delipidated HDL (1 mg protein per ml) was resolved into two populations of apolipoprotein (apo) A-I: one representing the apo A-I monomer and the other, a self-associated form with a molecular weight of approximately 120,000 daltons. The column eluates were screened for immunoreactivity to apo A peptides, and the identity of each peak was confirmed by sodium dodecyl sulfate--polyacrylamide gel electrophoresis followed by immunoblot analysis. Apo A-I peptides isolated by high-performance liquid chromatography disrupted unilamellar phospholipid vesicles to form smaller phospholipid particles that eluted on gel filtration columns within the size range of HDL. Thus, a rapid method for the isolation and quantitation of non-denatured apolipoproteins from HDL has been developed using size exclusion high-performance liquid chromatography.     

Inhibition of renal permeability towards albumin: a new function of apolipoproteins with possible pathogenetic relevance in focal glomerulosclerosis.

Focal segmental glomerulosclerosis (FSGS) is a degenerative renal disease characterized by the accumulation of extracellular matrix and lipids within the glomerular tuft. It has been proposed that an abnormal renal permeabilization towards proteins induced by a putative plasma factor is, in some way, involved in the pathogenesis of the disease. In this paper, we measured the plasma permeability activity (Palb) in several sera of patients with FSGS and found a mean activity of 0.82+/-0.03 which means a marked increase compared to a mean Palb of 0.16+/-0.03 in normal controls. Coincubation of FSGS and normal serum reduced the permeability activity within the normal range; normal serum added to the incubation medium after the glomeruli had already been exposed to the FSGS serum had no effect, suggesting the presence of inhibitory substances with a direct effect on a circulating substrate. Finally, the antipermeability activity was retained when heated to 60 degrees C but not to 100 degrees C. By serial fractionations of normal serum and reported activity measurements at each step, five natural occurring inhibitors of albumin permeabilization were purified and characterized by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS), as components of apolipoproteins (apo) (apo E2 and E4, apo L, the high Mr apo J and a 28 kDa fragment of apo A-IV). Coincubation of each apolipoprotein with FSGS serum inhibited permeability, but only apo J and apo E2 and E4 were found to be crucial for the process. In conclusion, we have purified from normal serum five inhibitors of permeability induced by FSGS serum, all corresponding to apolipoproteins. An imbalance between permeability factors and apolipoproteins may play a pathogenetic role in FSGS.     

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.     

Analysis of lipoproteins with analytical capillary isotachophoresis

An analytical free flow capillary isotachophoresis procedure, with a discontinuous electrolyte system, for the detailed analysis of lipoproteins in human body fluids has been developed. The technique is based on prestaining whole serum lipoproteins with a lipophilic dye before separation. Human serum lipoproteins are separated into 14 well-characterized subfractions according to their electrophoretic mobility. High density lipoproteins (fraction 1 to 6) are separated into three major subpopulations, the fast migrating high density lipoprotein (HDL) subpopulation, containing mainly apo AI and phosphatidylcholine, the subpopulation with intermediate mobility, consisting of particles rich in apo AII, apo E, and C apolipoproteins, and the slowly migrating HDL subfraction, containing mainly particles rich in apo AI, apo AIV, and lecithin: cholesterol acyltransferase (LCAT) activity. The apo B containing lipoproteins (fraction 7 to 14) can be subdivided into four major functional groups. The first represents chylomicron derived particles and large triglyceride-rich very low density lipoproteins (VLDL). The second group consists of small VLDL and intermediate density lipoprotein (IDL) particles, anf the third and fourth group represent the low density lipoproteins. The isotachophoretic analysis of human serum samples obtained from patients with hyperlipoproteinemias is compatible with the classification according to the Frederickson phenotypes and reflects the respective biochemical abnormalities. Furthermore, several genetic disorders of lipid and lipoprotein metabolism like HDL deficiency syndromes, familial LCAT deficiency, Fish eye disease, hypobetalipoproteinemia and abetalipoproteinemia can be well characterized by analytical capillary iso tachophoresis. In addition to patient analysis we investigated the influence of lipid lowering drugs on the lipoprotein subfraction distribution during therapy with analytical capillary isotachophoresis.     

Two-dimensional electrophoresis of plasma proteins and high density lipoproteins during inflammation

Plasma protein and lipoprotein fractions of five patients were analyzed on day 1, 5, and 15 after severe head injury by combining three types of two-dimensional electrophoresis (2-DE) to obtain information on lipoprotein and apolipoprotein composition. On analysis under nondenaturing conditions in both dimensions on day 5, the samples show modifications of isoelectric point (pI) and molecular weight (Mr) properties of the high density lipoprotein (HDL) fraction in addition to an increase in inflammatory proteins and a return to a normal pattern on day 15. In the second type of 2-DE the samples were analyzed employing isoelectric focusing without denaturant in the first dimension, followed by sodium dodecyl sulfate (SDS) in the second dimension in order to study the protein composition of lipoprotein fractions. On day 5, a decrease of the apolipoproteins apo A-I, apo A-II, and apo C were noted, with simultaneous appearance of an unidentified protein with Mr 12,000 and pI 6.0. In the third type of 2-DE, employing urea and Nonidet P-40 in the first and SDS in the second dimension, the plasma polypeptide composition was studied. The presence of an unidentified polypeptide could be confirmed on day 5, tending to disappear thereafter. This Mr 12,000 component consists of two major spots at pI 5.7 and 6.0 and four minor ones between pI 6.0 and 8.0. These properties suggest that this protein corresponds to serum amyloid A apolipoprotein.     

High-performance liquid chromatography of apolipoproteins in serum high-density lipoproteins

A simple and rapid method for apolipoprotein analysis in serum high-density lipoproteins (HDL) has been developed using high-performance liquid chromatography (HPLC) with sodium phosphate buffer (pH 7.0) containing 0.1% sodium dodecyl sulphate (SDS) as eluent. In contrast to the use of urea solution as an eluent, apolipoproteins can be analysed by applying an incubation mixture of HDL and the eluent buffer. A TSK-GEL column of G3000SW was found to be more profitable than G2000SW or G4000SW for analysis of HDL apolipoproteins. Elution patterns monitored by absorbance at 280 nm using a G3000SW column can give precise quantitative as well as qualitative information about apolipoproteins of molecular weight between 10(4) and 10(5). HPLC patterns of HDL apolipoproteins were compared between individual human subjects with various diseases. Elution profiles for lipid components in an incubation mixture were also examined.     

Feasibility of a recombinant human apolipoprotein E reference material

The aim of this work was to prepare a recombinant apo E material and to determine its suitability as a reference material. We produced human apo E3 using recombinant DNA technology. The cDNA of human apo E3 was cloned in the pARHS bacterial expression vector and used to transfect E. Coli BL21 (DE3) cells. The recombinant protein was then purified in one step by affinity chromatography on a Ni-chelated agarose column under denaturing conditions. The purity of the protein estimated by SDS PAGE was greater than 96%. The physicochemical properties and biological and immunological reactivity of the purified recombinant apo E3 were shown to be close to those of the protein purified from human plasma VLDL. A limited batch of lyophilized apo E material was then prepared. The stability of the lyophilized apo E material examined by temperature accelerated degradation was acceptable. No degradation of the measured apo E was observed after storage of the lyophilized material at +4°?C and –20°?C for 11 months. The reconstituted lyophilized material, in comparison with human fresh serum samples and with apo E purified from human VLDL, showed no major alteration of its immunological reactivity when assayed by immunoturbidimetry or ELISA.     

Purification of biologically active apolipoproteins by chromatofocussing

Chromatofocussing has been used to isolate homogeneous apolipoproteins (apo) from human very-low-density lipoproteins and high-density lipoproteins with protein recovery of 70%. The inclusion of sulfhydryl-reducing agent (dithiothreitol) was required during solubilization of the lipoproteins (following delipidation) to achieve reproducible elution profiles. Removal of polyvalent buffers from apoproteins was rapidly accomplished on small columns of hydroxylapatite. The biological activity of purified apo AI and apo CII was confirmed by assessment of their ability to activate lecithin:cholesterol acyltransferase or lipoprotein lipase, respectively. Functional properties of isolated apo E were assessed by in vitro interaction with the low-density lipoprotein receptor expressed by cultured fibroblasts. Apolipoproteins purified by this rapid procedure exhibit identical physical, chemical and biological properties to those purified by other, more tedious techniques.     

Chromatographic methods for the quantification of free and chelated gadolinium species in MRI contrast agent formulations

Speciation measurements of gadolinium in liposomal MRI contrast agents (CAs) are complicated by the presence of emulsifiers, surfactants, and therapeutic agents in the formulations. The present paper describes two robust, hyphenated chromatography methods for the separation and quantification of gadolinium in nanoemulsion-based CA formulations. Three potential species of gadolinium, free gadolinium ion, gadolinium chelated by diethylenetriamine pentaacetic acid, and gadolinium chelated by 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetic acid, were present in the CA formulations. The species were separated by reversed-phase chromatography (reversed phase high-performance liquid chromatography, RP-HPLC) or by high-pressure size-exclusion chromatography (HPSEC). For RP-HPLC, fluorescence detection and post-column online isotope dilution inductively coupled plasma mass spectrometry (ID-ICP-MS) were used to measure the amount of gadolinium in each species. Online ID-ICP-MS and species-specific isotope dilution (SID)-ICP-MS were used in combination with the HPSEC column. The results indicated that some inter-species conversions and degradation had occurred within the samples and that SID-ICP-MS should be used to provide the most reliable measurements of total and speciated gadolinium. However, fluorescence and online ID-ICP-MS might usefully be applied as qualitative, rapid screening procedures for the presence of free gadolinium ions.     

Sensitive Morphological Characterization of Oriented High-Density Lipoprotein Nanoparticles Using 31P NMR Spectroscopy

The biological function of high-density lipoprotein (HDL) nanoparticles, the so-called good cholesterol that is associated with a low risk of heart disease, depends on their composition, morphology, and size. The morphology of HDL particles composed of apolipoproteins, lipids and cholesterol is routinely visualised by transmission electron microscopy (TEM), but higher-resolution tools are needed to observe more subtle structural differences between particles of different composition. Here, reconstituted HDL formulations are oriented on glass substrates and solid-state ³¹P NMR spectroscopy is shown to be highly sensitive to the surface curvature of the lipid headgroups. The spectra report potentially functionally important differences in the morphology of different HDL preparations that are not detected by TEM. This method provides new morphological insights into HDL comprising a naturally occurring apolipoprotein A-I mutant, which may be linked to its atheroprotective properties, and holds promise as a future research tool in the clinical analysis of plasma HDL.     

High-performance size-exclusion chromatography of porcine colonic mucins. Comparison of Bio-Gel TSK 40XL and Sepharose 4B columns

A high-performance size-exclusion chromatography (HPSEC) method was developed for the separation of porcine colonic mucins using a Bio-Gel TSK 40XL HPSEC column (300 mm x 75 mm). In addition, porcine gastric and bovine submaxillary mucin preparations were used to describe more fully the separation characteristics of the HPSEC column. For comparison, the same preparations were also separated using a Sepharose 4B column (100 cm x 2.6 cm). The colonic and gastric mucins eluted in the void volume (V0) of both columns. Bovine submaxillary mucin was in the elution volume (Ve) of both columns. Analytical HPSEC of fractions (V0 and Ve) of the various preparations obtained by Sepharose 4B chromatography exhibited retention times identical to those for fractions obtained by HPSEC. After separation by both methods, purified mucins were obtained by CsCl2 density gradient ultracentrifugation; analytical HPSEC profiles, protein contents, and monosaccharide compositions of both gastric and colonic mucins from either column were similar. The HPSEC method, however, is ideally suited to separate microgram to milligram quantities of colonic mucin preparations quickly: 2 to 4 h, compared with 24 to 30 h for the Sepharose 4B method.     

Laboratory Production of Human Prolactin from CHO Cells Adapted to Serum-Free Suspension Culture

Human prolactin (hPRL) is a polypeptide with 199 amino acids and a molecular mass of 23?kDa. Previously, a eukaryotic hPRL expression vector was used to transfect Chinese hamster ovary (CHO) cells: this work describes a fast and practical laboratory adaptation of these transfected cells, in ~40?days, to grow in suspension in serum-free medium. High cell densities of up to 4.0?×?10⁶ cell/ml were obtained from spinner flask cultures and a stable and continuous production process was developed for at least 30?days. Two harvesting strategies were set up, 50 or 100?% of the total conditioned medium being collected daily and replaced by fresh culture medium. The volumetric productivity was 5?C7???g hPRL/ml, as determined directly in the collected medium via reversed-phase HPLC (RP-HPLC). A two-step process based on a cationic exchanger followed by size exclusion chromatography was applied to obtain purified hPRL from conditioned medium. Two hPRL isoforms, non-glycosylated and glycosylated, could also be separated by high-performance size-exclusion chromatography (HPSEC) and, when analyzed by RP-HPLC, HPSEC, Western blotting, and bioassay, were found to be comparable to the World Health Organization International Reference Reagent of hPRL. These results are useful for the practical scale-up to the pilot and industrial scale of a bioprocess based on CHO cell culture.     

Molecular size fractionation of soil humic acids using preparative high performance size-exclusion chromatography

High performance size-exclusion chromatography (HPSEC) is useful for the molecular size separation of soil humic acids (HAs), but there is no method available for various HAs with different chemical properties. In this paper the authors propose a new preparative HPSEC method for various soil HAs. Three soil HAs with different chemical properties were fractionated by a Shodex OHpak SB-2004 HQ column with 10mM sodium phosphate buffer (pH 7.0)/acetonitrile (3:1, v/v) as an eluent. The HAs eluted within a reasonable column range time (12-25 min) without peak tailing. Preparative HPSEC chromatograms of these HAs indicated that non-size-exclusion effects were suppressed. The separated fractions were analyzed by HPSEC to determine their apparent molecular weights. These decreased sequentially from fraction 1 to fraction 10, suggesting that the HAs had been separated by their molecular size. The size-separated fractions of the soil HA were mixed to compare them with unfractionated HA. The analytical HPSEC chromatogram of the mixed HA was almost identical to that of the unfractionated HA. It appears that the HAs do not adsorb specifically to the column during preparative HPSEC. Our preparative HPSEC method allows for rapid and reproducible separation of various soil HAs by molecular size.     

Characterization of plasma apolipoproteins by capillary electrophoresis.

The main apolipoproteins of plasma high-density lipoproteins (HDL) and low-density lipoproteins (LDL) were analyzed by capillary electrophoresis. Where possible the results were compared with slab sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Addition of the detergent SDS to the running buffer was essential for separation. Separations were carried out in bare silica and polyacrylamide-coated capillaries. The main apolipoproteins of HDL could be separated in an uncoated capillary filled with borax buffer containing 0.1% SDS. Using the coated capillary, a mixture of HDL and LDL apolipoproteins was resolved in less than 12 min. These preliminary studies indicate that capillary electrophoresis is a promising technique for screening plasma apolipoproteins.     

Humic acid fractionation upon sequential adsorption onto goethite

Mineral-humic complexes are commonly distributed in natural environments and are important in regulating the transport and retention of hydrophobic organic contaminants in soils and sediments. This study investigated the structural and conformational changes of humic acid (HA) and mineral-HA complexes after sequential HA adsorption by goethite, using UV-visible spectroscopy, high performance size exclusion chromatography (HPSEC), Fourier transform infrared (FT-IR) spectroscopy, and solid-state 13C nuclear magnetic resonance (NMR) spectroscopy. The HA remaining in the solution after adsorption showed low polarity index values ((N+O)/C), which indicates that polar functional moieties are likely to adsorb on the goethite surface. In addition, we observed decreased E4/E6 and E2/E3 ratios of unbound HA with increasing number of coatings, implying that aliphatic rich HA fractions with polar functional moieties readily adsorb to the goethite surface. According to IR spectra, carbohydrate carbon would be the important fractions associated with goethite. NMR spectra provided evidence for HA fractionation during adsorption onto the mineral surface; that is, aliphatic fractions were preferentially adsorbed by goethite while aromatic fractions were left in solution. Relatively small molecular weight (MW) HA fractions had a greater affinity for the goethite surface based on analyses of the HPSEC chromatograms, which differs from the results reported in the literature. Finally, our results suggest that the polar aliphatic fractions of HA were mainly adsorbed to goethite via electrostatic attraction and/or ligand exchange reactions.     

Human plasma lecithin:cholesterol acyltransferase. Preparation and use of immobilized p-aminophenylarsenoxide as a catalytic site-directed covalent ligand in enzyme purification.

A method is described for the preparation of p-aminophenylarsenoxide-linked carboxymethyl (CM) Bio-Gel A and its use as a specific, catalytic site-directed affinity chromatography ligand in the final stages of the purification of human plasma lecithin:cholesterol acyltransferase (LCAT) (EC 2.3.1.43). Previous mechanistic studies by us demonstrated that phenylarsenoxide derivatives, which are highly specific for vicinal thiols, could inhibit LCAT via a covalent interaction with the sulphydryl groups of the two catalytic cysteine residues and that this inhibition could be rapidly and completely reversed upon addition of 2,3-dimercaptopropanesulphonic acid. The ligand was covalently linked to CM Bio-Gel A activated through an N-hydroxysuccinyl ester formed by N-hydroxysuccinimide and dicyclohexylcarbodiimide in dry dimethyl sulphoxide; 87% of the added p-aminophenylarsenoxide was coupled to the CM Bio-Gel A in 3 h at 25 degrees C giving 2.3 mg of p-aminophenylarsenoxide per ml of gel. Homogeneous LCAT free of apo A-I, apo E, apo D and albumin was obtained in an 11% yield and purified 15,013-fold overall. A 13-fold purification was obtained by chromatography upon p-aminophenylarsenoxide-CM Bio-Gel A. This method is a useful final step in LCAT purification and will prove valuable in the purification of other proteins and compounds containing vicinal thiols.     

Alternative mobile phases for the reversed-phase high-performance liquid chromatography of peptides and proteins

The use of a high content of acetic acid as mobile phase additive for the reversed-phase high-performance liquid chromatography (RP-HPLC) of several proteins and extracts of biological tissues was evaluated for a divinylbenzene (DVB)-based stationary phase, and the separations obtained with acetic acid gradients in acetonitrile, isopropanol or water were compared with classical polypeptide RP-HPLC on silica C4 with trifluoroacetic acid (TFA)-acetonitrile. The separation patterns for recombinant derived interleukin-1 beta (IL-1 beta) on the C4 column eluted with TFA-acetonitrile and the DVB column eluted with acetic acid-acetonitrile were similar, but only the polymeric column was able to separate the components present in an iodinated IL-1 beta preparation. Neither eluent had any harmful effect on the biological activity of IL-1 beta isolated after RP-HPLC. Several standard proteins could be separated when the polymeric column was eluted with acetic acid gradients in acetonitrile, isopropanol or water and, although the separation efficiency with acetic acid in water was lower than that in combination with classical organic modifiers, insulin, glucagon and human growth hormone (hGH) were eluted as sharp, symmetrical peaks. The recoveries of insulin and hGH were comparable for all three mobile phases (80-90%). The separation patterns obtained from a crude acetic acid extract of a normal and a diabetic, human pancreas analysed using acetic acid gradients with or without organic modifiers were found to be similar and comparable to those obtained on a silica C4 column eluted with an acetonitrile gradient in TFA. The principal differences resulted from the use of different UV wavelengths (215 nm for TFA-acetonitrile, 280 nm for acetic acid). Acetic acid extracts of recombinant derived hGH-producing Escherichia coli were separated on the DVB column eluted with an acetic acid gradient in water. Although the starting material was a highly complex mixture, the hGH isolated after this single-step purification was surprisingly pure (as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis). Consequently several (pure) polypeptides and complex biological samples were separated on a polymeric stationary phase eluted with acetic acid gradients in water without the use of organic modifiers.     

Refolding and simultaneous purification of recombinant human proinsulin from inclusion bodies on protein‐folding liquid‐chromatography columns

Protein‐folding liquid chromatography (PFLC) is an effective and scalable method for protein renaturation with simultaneous purification. However, it has been a challenge to fully refold inclusion bodies in a PFLC column. In this work, refolding with simultaneous purification of recombinant human proinsulin (rhPI) from inclusion bodies from Escherichia coli were investigated using the surface of stationary phases in immobilized metal ion affinity chromatography (IMAC) and high‐performance size‐exclusion chromatography (HPSEC). The results indicated that both the ligand structure on the surface of the stationary phase and the composition of the mobile phase (elution buffer) influenced refolding of rhPI. Under optimized chromatographic conditions, the mass recoveries of IMAC column and HPSEC column were 77.8 and 56.8% with purifies of 97.6 and 93.7%, respectively. These results also indicated that the IMAC column fails to refold rhPI, and the HPSEC column enables efficient refolding of rhPI with a low‐urea gradient‐elution method. The refolded rhPI was characterized by circular dichroism spectroscopy. The molecular weight of the converted human insulin was further confirmed with SDS–18% PAGE, Matrix‐Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry (MALDI‐TOF‐MS) and the biological activity assay by HP‐RPLC. Copyright © 2014 John Wiley & Sons, Ltd.     

One-step purification of a recombinant protein from a whole cell extract by reversed-phase high-performance liquid chromatography

We have developed a one-step facile, flexible and readily scalable purification method for a recombinant protein, TM 1-99 (113 amino acid residues; 12,837 Da) based on reversed-phase high-performance liquid chromatography (RP-HPLC) from an E. coli cell lysate. Following cell lysis, the cell contents were extracted with 0.1% aqueous trifluoroacetic acid (TFA), applied directly under conditions of high sample load to a narrow bore RP-HPLC C(8) column (150 mm x 2.1 mm I.D.) and eluted by a shallow gradient of acetonitrile (0.1%/min). Loads of 23 and 48 mg of lyophilized crude cell extract produced 2.4 and 4.2mg of purified product (>94% pure), respectively, at >94% recovery. Our results show the excellent potential of one-step RP-HPLC for purification of recombinant proteins from cell lysates, where high yields of purified product and greater purity are achieved compared to affinity chromatography. Such an approach was also successful in purifying just trace levels (<0.1% of total contents of crude sample) of TM 1-99 from a cell lysate.