High-performance Gel Filtration Chromatography Research Articles

Separation of polyethylene glycols and their amino-substituted derivatives by high-performance gel filtration chromatography at low ionic strength with refractive index detection

The chromatographic analysis of polyethylene glycols (PEG) and their amino-substituted derivatives, with an average molecular mass of 2000 and 3350 is described. The PEG derivatives were perfectly separated by TSK-GEL G4000PW XL column, which was widely used in high-performance size-exclusion chromatography (SEC), at low ionic strength with refractive index detection. It was shown that ion-exchange interactions were mainly involved in the retention mechanism of these compounds on TSK-GEL G4000PW XL column, since retention volume decreased when salt concentration were added to the mobile phase and varied with the pH of the eluent. Additionally, size exclusion for PEG chains plays a role. Organic modifiers also had effect on chromatographic behaviors of PEG compounds.

Combined data from LDL composition and size measurement are compatible with a discoid particle shape

The size of LDL is usually reported as particle diameter, with the implicit assumption that it is a spherical particle. On the other hand, data obtained by cryoelectron microscopy and crystallographic analysis suggest that LDL shape may be discoid. We have investigated LDL particle geometry by combining data on LDL lipid composition with size measurement. The mean LDL diameter of 160 samples was measured by high-performance gel-filtration chromatography (HPGC), and particle volume was calculated from its lipid composition. Assuming a spherical shape, diameters calculated from volume correlated poorly with values obtained by HPGC (R(2) = 0.36). Assuming a discoid shape, particle height was calculated from volume and HPGC diameter. Diameter (20.9 +/- 0.5 nm) and height (12.1 +/- 0.8 nm) were not significantly related to each other (r = 0.14, P = 0.09) and accounted for 23% and 77%, respectively, of the variation in particle volume. In multivariate regression models, LDL core lipids were the main determinants of height (R(2) = 0.83), whereas free cholesterol in the shell, which contributes only 5-9% to LDL mass, was the main determinant of diameter (R(2) = 0.54). We conclude that combined data from composition and size measurements are compatible with a discoid particle shape and propose a structural model for LDL in which free cholesterol plays a major role in determining particle shape and diameter.

Associations of LDL size with in vitro oxidizability and plasma levels of in vivo oxidized LDL in Type 2 diabetic patients.

Oxidative modification of low-density lipoprotein (LDL) is believed to be a key step in the genesis of atherosclerotic lesions. The presence of small, dense LDL is associated with accelerated atherosclerosis and is common in diabetic patients. The aim of this study was to investigate the relationship of in vitro LDL oxidizability and circulating in vivo oxidized LDL with LDL particle size in Type 2 diabetic patients and healthy control subjects. The study group consisted of 58 elderly well controlled Type 2 diabetic patients and 58 control subjects with normal glucose metabolism. LDL particle size was measured by high-performance gel-filtration chromatography. In vitro oxidizability of LDL was measured by monitoring conjugated diene formation and plasma levels of circulating oxidized LDL were determined by ELISA. In vitro susceptibility of LDL to oxidation was not related to plasma levels of in vivo oxidized LDL, nor to LDL particle size. In the diabetic patients, but not in the control group, an inverse relation between LDL size and in vivo oxidized LDL was observed (r=-0.35, P=0.007). This relation was strengthened after controlling for LDL-cholesterol concentration (r=-0.52, P<0.001). In agreement with the view that small, dense LDL accelerates atherosclerosis, an inverse relationship was observed between LDL size and circulating in vivo oxidized LDL in Type 2 diabetic patients. Our results also suggest that in vitro susceptibility to oxidation is not a suitable surrogate measure for in vivo LDL oxidation.

LDL Particle Size by Gradient-Gel Electrophoresis Cannot Be Estimated by LDL-Cholesterol/Apolipoprotein B Ratios

LDL particles have been shown to be heterogeneous in size, density, and composition. Heterogeneity of LDL particles with respect to size has been demonstrated by analytic ultracentrifugation (1), density-gradient ultracentrifugation, gradient-gel electrophoresis (GGE), high-performance gel-filtration chromatography, dynamic light scattering, and electron microscopy. Among these methods, GGE is the most reliable and widely used, but it has the drawback of being labor-intensive. Recently, the ratio of the LDL-cholesterol (LDL-chol) concentration to the LDL-apolipoprotein B (LDL-apo B) concentration has been used as an alternative index of hyperapobetalipoproteinemia and small dense LDL (2)(3)(4)(5). We therefore studied the relationship between LDL particle size as measured by GGE and as estimated by the LDL-chol/LDL-apo B ratio. LDL particle size was quantified by the above two methods in samples from healthy controls (n = 49) and from hyperlipemic subjects (n = 81). The …

On-line coupling of high performance gel filtration chromatography with imaged capillary isoelectric focusing using a membrane interface

On-line coupling of high performance gel filtration chromatography with imaged capillary isoelectric focusing using a membrane interface

On-line coupling of high performance gel filtration chromatography with imaged capillary isoelectric focusing using a membrane interface.

A high performance liquid chromatography system, a sample preparation device, and an imaged capillary IEF (CIEF) instrument are integrated and multiplexed on-line. The system is equivalent to two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), by transferring the principle of 2-D separation to the capillary format. High performance liquid chromatography (HPLC) provides protein separation based on size using a gel filtration chromatography (GFC) column. Each eluted protein is sampled and directed to a novel microdialysis hollow fiber membrane device, where simultaneous desalting and carrier ampholyte mixing occurs. The sample is then driven to the separation column in an on-line fashion, where CIEF takes place. The fluidic technology used by our 2-D system leads to natural automation. The coupling of the two techniques is simple. This is attributed to high speed and efficiency of the sample preparation device that acts as an interface between the two systems, as well as the speed and simplicity of our whole column absorption imaged CIEF instrument. To demonstrate the feasibility of this approach, the separation of a mixture of two model proteins is studied. Sample preparation and CIEF were complete in just 4-5 min, for each of the eluted proteins. Total analysis time is about 24 min. Three-dimensional data representations are constructed. Challenges and methods to further improve our instrument are discussed, and the design of an improved horseshoe-shaped sample preparation sample loop membrane interface is presented and characterized.

Synthesis and application of peptide dendrimers as protein mimetics.

The multiple antigenic peptide (MAP) is a novel approach to preparing peptide immunogens. The MAP consists of an inner core matrix built up of a large layer of Lys residues and a surface of peptide chains attached to the core matrix. Because of its dendrimeric structure, MAP can be very useful as a template for assembling potential peptide surfaces. Two methods for preparing MAP systems are described in this unit. The direct approach for preparing MAP systems is presented in the first two protocols, including the procedure for b-butyloxycarbonyl (Boc) chemistry and the procedure for 9 -fluorenylmethyloxycarbonyl (Fmoc) chemistry. An indirect approach for preparing MAP systems, in which peptide and core matrix are synthesized separately and conjugated by several ligation methods, is then described. The cMAP approach is also executed using either the direct or indirect approach, but requires an additional cyclization step to constrain the peptides after synthesis. The synthesis of cMAP is described, and the preparation of cyclic peptides is illustrated. A support protocol describes the ninhydrin assay to assess the completeness of the coupling reaction. In most cases, MAP systems can be used directly after simple dialysis or desalting. Some immunological studies, however, require purified MAPs. Additional support protocols describe MAP system purification by dialysis and high-performance gel-filtration chromatography.

Pig splenic nerve: peptides derived from chromogranins by proteolytic processing during axonal transport

We have investigated the proteolytic processing of chromogranin A, chromogranin B and NESP55 (a novel chromogranin-like protein) during axonal transport using pig splenic nerve as a model. We have also studied the presence of chromogranin-derived peptides in the perfusate during electrical stimulation of this nerve. High-performance gel filtration chromatography followed by radioimmunoassay (RIA) revealed that chromogranins are proteolytically processed to varying degrees during axonal transport. For chromogranin A and NESP55, the precursor is still present in the proximal part of the nerve, whereas in the distal part and nerve terminals, intermediate-sized peptides and the free peptides GE-25 and GAIPIRRH dominate, respectively. For chromogranin B, the precursor has already been processed to an intermediate-sized peptide in the proximal part of the nerve, which is also present in the distal parts together with the free peptide PE-11. For chromogranin B and NESP55, only the free peptides PE-11 and GAIPIRRH, or in the case of chromogranin A, the free peptide GE-25 plus an intermediate-sized one, are released from the terminals into the splenic perfusate. These results demonstrate that chromogranins are processed to smaller peptides during axonal transport.

Measurement of LDL particle size in whole plasma and serum by high performance gel-filtration chromatography using a fluorescent lipid probe

We have recently described a technique for measuring LDL size by high performance gel-filtration chromatography (HPGC) with UV detection (Scheffer et al., Clin Chem 1997;43:1904-12). A drawback of this method is the necessity of LDL isolation before chromatography. We now describe a modification of this method based on selective detection of lipoproteins by postcolumn labeling with parinaric acid, a fluorescent lipid probe. Measuring the size of isolated LDL by HPGC in 56 subjects, we obtained diameters of 25.72 +/- 0.60 nm with UV detection and of 25.74 +/- 0.58 nm with fluorescence detection. The modified method is suitable for LDL size measurement in whole plasma or serum. LDL sizes measured in whole plasma correlated strongly with the respective values in isolated LDL (r = 0.976) but were on average 0.18 nm larger (P < 0.001). CVs for within- and between-series imprecision were <0.25%. The present method requires only 5 microL plasma or serum without sample preparation and is suitable for the unattended analysis of large series of samples.

Purification and characterization of NADPH-dependent carbonyl reductase, involved in stereoselective reduction of ethyl 4-chloro-3-oxobutanoate, from Candida magnoliae.

A NADPH-dependent carbonyl reductase was purified to homogeneity from Candida magnoliae AKU4643 through four steps, including Blue Sepharose affinity chromatography. The enzyme catalyzed the stereoselective reduction of ethyl 4-chloro-3-oxobutanoate to the corresponding (S)-alcohol with a 100% enantiomeric excess, which is a useful chiral building block for the chemical synthesis of pharmaceuticals. The relative molecular mass of the enzyme was estimated to be 76,000 on high performance gel filtration chromatography and 32,000 on SDS polyacrylamide gel electrophoresis. The enzyme reduced alpha-, beta-keto esters and conjugated diketones in addition to ethyl 4-chloro-3-oxobutanoate. The enzyme activity was inhibited by quercetin and HgCl2, but not by EDTA. The N-terminal amino acid sequence of the enzyme showed no apparent similarity with those of other oxidoreductases.

Detection of glycosaminoglycans as a copper(II) complex in high-performance liquid chromatography

Glycosaminoglycans including heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate and hyaluronan were analyzed by high-performance gel filtration chromatography. Detection was achieved at 240 nm based on the formation of copper(II) complex in copper sulfate solution at low pH. Detection of the copper(II)-heparin complex is sensitive, permitting the analysis of as little as 10 −7 g. This method was also successfully applied to the analysis of the chemically derivatized glycosaminoglycans (de-N-acetylated and/or oversulfated chondroitin sulfate) that cannot be depolymerized by enzymes such as heparin and chondroitin lyases.

Measurement of low-density lipoprotein particle size by high-performance gel-filtration chromatography

We describe a new technique for measuring LDL size by high-performance gel-filtration chromatography (HPGC). LDL was subjected to chromatography, and the column effluent was monitored at 280 nm. The retention time of the LDL peak was used to calculate the LDL diameter. We compared the HPGC method with gradient gel electrophoresis (GGE) on 2-10% nondenaturing polyacrylamide gels. In a group of 60 non-insulin-dependent diabetes mellitus patients, LDL size as measured by HPGC and GGE was highly correlated (r = 0.88, P <0.001). Good reproducibility, high precision, and the possibility of analyzing large series of samples are the main advantages of the automated HPGC method. Within-run and between-run CV for LDL size measured by HPGC were <0.1% and 0.2%, respectively. There was a significant inverse association between LDL size measured by HPGC and the logarithm of plasma triglycerides (r = -0.84, P <0.001), and a significant positive association with the LDL free cholesterol/protein ratio (r = 0.89, P <0.001).

Functional domains of Escherichia coli single-stranded DNA binding protein as assessed by analyses of the deletion mutants.

A series of C- and N-terminal deletion mutants of Escherichia coli single-stranded DNA binding protein (SSB) was constructed, purified, and characterized in terms of ability to self-multimerize and to bind to DNA. High-performance gel filtration chromatography revealed that the amino acids 89-105 play a key role in the maintenance of homotetramer for native SSB of 177 amino acids. Interestingly, all of the N-terminal deletion mutants studied here were eluted as octamers, indicating that the N-terminal 11 residues are involved in the prevention of the formation of octamers. The binding of SSB and its deletion mutant proteins to single-stranded d(T)n was examined by gel mobility shift assay and circular dichroism spectroscopy. C-terminal deletion mutant proteins, SSB1-135 and SSB1-115, maintained high affinity and may be wrapped by single-stranded DNA (ssDNA) in the same way as in the case of native SSB. In contrast, deletion of the C-terminal region (residues 89-115) or N-terminal region (residues 1-11) caused a dramatic decrease in the binding affinity. Furthermore, two different stoichiometries of SSB in the complexes with d(T)64, but not with d(T)32, were observed for native SSB, SSB1-135, SSB1-115, and SSB37-177, suggesting that the (SSB)65 and (SSB)35 binding modes, as previously demonstrated [Lohman, T. M., & Overman, L. B. (1985) J. Biol. Chem. 260, 3594-3603; Bujalowski, W., & Lohman, T. M. (1986) Biochemistry 25, 7799-7802], occurred at lower and higher SSB concentrations, respectively. A functional map for SSB molecule was presented and discussed.

A Method for the High Performance Gel Filtration Chromatography Characterization of Inorganic Colloids

Colloids are potentially important adsorbents in natural environments and their characterization is a necessary step in understanding their chemistry and geochemistry. A method for determining the relative molecular weight distribution of alumina, silica, and hydroxyaluminosilicate colloids by high performance gel filtration chromatography utilizing a G3000 PWXL column and a refractive index detector is reported. Analytical parameters that affect the molecular weight distribution of the inorganic colloids are discussed. The optimum analytical conditions for performing this analysis were found to be an ionic strength of 0.1 M(as NaNO 3), a pH of 5, a flow rate of 0.8 mL/min, and a colloid concentration of 400 mg/L.

Role of ectoparasitoid venom in the regulation of haemolymph ecdysteroid titres in a host noctuid moth

The effects on haemolymph ecdysteroid titre of a host noctuid moth in response to parasitism or injection of extracts of venom from the external parasitoid wasp, Eulophus pennicornis, were ascertained for the penultimate (fifth) instar larva of the tomato moth, Lacanobia oleracea. Unparasitized larvae showed a marked peak of ecdysteroid titre (85 ng/ml ecdysone equiv.) at approximately 48 h into the fifth stadium. This major ecdysteroid peak was eliminated, or its timing was significantly postponed (by at least 48‐60 h), when the larvae were parasitized by E. pennicornis. Parasitized larvae failed to moult and eventually died. Saline-injected larvae showed a normal, but slightly delayed (ca. 12 h) major peak of ecdysteroid titre at 64‐66 h (ca. 140 ng/ml) that followed a smaller peak (56 ng/ml) ca. 12‐14 h earlier. As with parasitism, these ecdysteroid peaks were effectively eliminated, or significantly delayed, when larvae were injected with 0.7 gland equivalents of E. pennicornis venom. Venom-injected larvae failed to moult to the sixth instar, whereas saline-injected larvae moulted and developed normally. High-performance gel filtration chromatography was used for the initial purification of active venom components. Bioassays of the size-fractionated venom gland proteins by injection into fifth instar larvae showed that the suppression of moulting effect was confined to just two fractions (out of 12) with apparent Mr between 43,000 and 158,000. Arch. Insect Biochem. Physiol. 35:169‐178, 1997. PUBLISHED 1997 WILEY-LISS, INC.

Purification and Cloning of the GTP Cyclohydrolase I Feedback Regulatory Protein, GFRP

The activity of GTP cyclohydrolase I, the initial enzyme of the de novo pathway for biosynthesis of tetrahydrobiopterin, the cofactor required for aromatic amino acid hydroxylations and nitric oxide synthesis, is sensitive to end-product feedback inhibition by tetrahydrobiopterin. This inhibition by tetrahydrobiopterin is mediated by the GTP cyclohydrolase I feedback regulatory protein GFRP, previously named p35 (Harada, T., Kagamiyama, H., and Hatakeyama, K. (1993) Science 260, 1507-1510), and -phenylalanine specifically reverses the tetrahydrobiopterin-dependent inhibition. As a first step in the investigation of the physiological role of this unique mechanism of regulation, a convenient procedure has been developed to co-purify to homogeneity both GTP cyclohydrolase I and GFRP from rat liver. GTP cyclohydrolase I and GFRP exist in a complex which can be bound to a GTP-affinity column from which GTP cyclohydrolase I and GFRP are separately and selectively eluted. GFRP is dissociated from the GTP agarose-bound complex with 0.2 NaCl, a concentration of salt which also effectively blocks the tetrahydrobiopterin-dependent inhibitory activity of GFRP. GTP cyclohydrolase I is then eluted from the GTP-agarose column with GTP. Both GFRP and GTP cyclohydrolase I were then purified separately to near homogeneity by sequential high performance anion exchange and gel filtration chromatography. GFRP was found to have a native molecular mass of 20 kDa and consist of a homodimer of 9.5-kDa subunits. Based on peptide sequences obtained from purified GFRP, oligonucleotides were synthesized and used to clone a cDNA from a rat liver cDNA library by polymerase chain reaction-based methods. The cDNA contained an open reading frame that encoded a novel protein of 84 amino acids (calculated molecular mass 9665 daltons). This protein when expressed in Escherichia coli as a thioredoxin fusion protein had tetrahydrobiopterin-dependent GTP cyclohydrolase I inhibitory activity. Northern blot analysis indicated the presence of an 0.8-kilobase GFRP mRNA in most rat tissues, the amounts generally correlating with levels of GTP cyclohydrolase I and tetrahydrobiopterin. Thus, mRNA levels were relatively high in liver and kidney and somewhat lower in testis, heart, brain, and lung. These results suggest that GFRP is widely expressed and may play a role in regulating not only phenylalanine metabolism in the liver, but also the production of biogenic amine neurotransmitters as well as nitric oxide synthesis.

The source of the bile: The legacy of Mathias Mokel

HepatologyVolume 24, Issue 1 p. 274-276 Hepatology ElsewhereFree Access The source of the bile: The legacy of Mathias Mokel D M Heuman M.D., D M Heuman M.D. Medical College of Virginia and McGuire Department of Veterans Affairs Medical CenterSearch for more papers by this author D M Heuman M.D., D M Heuman M.D. Medical College of Virginia and McGuire Department of Veterans Affairs Medical CenterSearch for more papers by this author First published: July 1996 https://doi.org/10.1002/hep.510240145Citations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References 1 Somjen GJ, Gilat T. A non-micellar mode of cholesterol transport in human bile. FEBS Lett 1983; 156: 265– 268. 2 Somjen GJ, Gilat T. Contribution of vesicular and micellar carriers to cholesterol transport in human bile. J Lipid Res 1985; 26: 699– 704. 3 Hardison WG, Apter JT. Micellar theory of biliary cholesterol excretion. Am J Physiol 1972; 222: 61– 67. 4 Cohen DE. Studies of biliary lipid secretion and aggregation in model and native biles (Ph.D. dissertation). Cambridge, MA: Harvard University, 1986. 5 Cohen DE, Angelico M, Carey MC. Quasielastic light scattering evidence for vesicular secretion of biliary lipids. Am J Physiol 1989; 257: G1– G8. 6 Somjen GJ, Marikovsky Y, Lelkes P, Gilat T. Cholesterol-phospholipid vesicles in human bile: an ultrastructural study. Biochim Biophys Acta 1986; 879: 14– 21. 7 Ulloa N, Garrido J, Nervi F. Ultracentrifugal isolation of vesicular carriers of biliary cholesterol in native human and rat bile. Hepatology 1987; 70: 235– 244. 8 Amigo LC, Covarrubias C, Nervi F. Rapid isolation of vesicular and micellar carriers of biliary lipids by ultracentrifugation. J Lipid Res 1990; 31: 341– 347. 9 Cohen DE, Carey MC. Rapid (1 hour) high performance gel filtration chromatography resolves coexisting simple micelles, mixed micelles, and vesicles in bile. J Lipid Res 1990; 31: 2103– 2112. 10 Crawford JM, Mockel G-M, Crawford AR, Hagen SJ, Hatch VC, Barnes S, Godleski JJ, Carey MC. Imaging biliary lipid secretion in the rat. ultra-structural evidence for vesiculation of the hepatocyte canalicular membrane. J Lipid Res 1995; 36: 2147– 2163. 11 Möckel G-M, Gorti S, Tandon RK, Tanaka T, Carey MC. Microscope laser light-scattering spectroscopy of vesicles within canaliculi of rat hepatocyte couplets. Am J Physiol 1995; 269: G73– G84. Citing Literature Volume24, Issue1July 1996Pages 274-276 ReferencesRelatedInformation

Dye-affinity labelling of bovine heart mitochondrial malate dehydrogenase and study of the NADH-binding site

The ability of the reactive dichlorotriazine dye Vilmafix Blue A-R (VBAR) to act as an affinity label for bovine heart L-malate dehydrogenase (MDH) was studied. VBAR binds specifically and irreversibly to MDH (k3 0.16 min-1; KD 14.4 microM). The inactivation of the NADH-dependent enzyme by VBAR is competitively inhibited by NAD+, NADH and ADP. Quantitatively inhibited MDH contained approx. 1 mol of dye per mol of active site. The inhibition is irreversible and activity cannot be recovered either on incubation with 10 mM NAD+, 10 mM NADH or 10 mM ADP, or by extensive dialysis or gel-filtration chromatography. Data obtained from high-performance gel-filtration chromatography and analysed by Scatchard plot suggested the presence of two coenzyme-binding sites per MDH dimer. Tryptic digestion of VBAR-labelled MDH followed by reverse-phase HPLC analysis revealed one VBAR-labelled peptide. It appears that each subunit features the same peptide bearing the modifying residue involved in MDH labelling. The pKa of the modifying residue is 8.05. Both total acid hydrolysis of VBAR-labelled MDH followed by HPLC and TLC analysis, and molecular-modelling studies suggest that the modifying residue is Lys-81 and/or Lys-217.

β-Carotene and protein complex found in microsomal membranes of bovine corpora lutea

The role of β-carotene in the fertility of ruminant animals has long been acknowledged (Friesecke, 1978). Initially this was thought to be due to its action as a vitamin A precursor but recently β-carotene itself has been considered responsible for improving reproductive performance (Hurley &amp; Doane, 1989). The mechanism by which β-carotcne acts is unclear, but as its concentration in the corpus luteum was typically found to be 70μg per gram of tissue, its biological action is probably exerted in this area.β-Carotene has been studied in the bovine corpus luteum using Raman spectroscopy, high performance liquid chromatography (HPLC) and gel filtration chromatography. The structure of β-carotene produces a characteristic Raman spectrum and by utilising an imaging technique, photographs of isolated luteal cells were obtained indicating the regions of β-carotene within them. Differential centrifugation was used to obtain pure subcellular fractions of luteal cells.

β-Carotene and protein complex found in microsomal membranes of bovine corpora lutea

The role of β-carotene in the fertility of ruminant animals has long been acknowledged (Friesecke, 1978). Initially this was thought to be due to its action as a vitamin A precursor but recently β-carotene itself has been considered responsible for improving reproductive performance (Hurley &amp; Doane, 1989). The mechanism by which β-carotcne acts is unclear, but as its concentration in the corpus luteum was typically found to be 70μg per gram of tissue, its biological action is probably exerted in this area.β-Carotene has been studied in the bovine corpus luteum using Raman spectroscopy, high performance liquid chromatography (HPLC) and gel filtration chromatography. The structure of β-carotene produces a characteristic Raman spectrum and by utilising an imaging technique, photographs of isolated luteal cells were obtained indicating the regions of β-carotene within them. Differential centrifugation was used to obtain pure subcellular fractions of luteal cells.