Heterogeneity of human very low density lipoproteins by gel filtration chromatography

Very low density lipoproteins were separated by gel filtration on Sepharose 4B. A decrease in mean particle diameter and flotation rate was seen with increasing elution volumes. The smaller lipoproteins had relatively more protein and phospholipid and less triglyceride than the larger ones. No differences were noted in the relative contents of the various phospholipids or partial glycerides between small and large lipoproteins. Fatty acid patterns of triglycerides and cholesteryl esters were also similar for the various lipoproteins. Relatively more lecithin containing linoleoyl acyl groups was found in smaller lipoproteins of some subjects. More of the protein of smaller lipoproteins was apo-LDL protein. Apo-HDL peptide was lost from the very low density lipoprotein as a consequence of the gel filtration.     

Rapid on-line determination of cholesterol distribution among plasma lipoproteins after high-performance gel filtration chromatography

A high-performance gel chromatography (HPGC) system has been developed which allows the unattended on-line determination of lipoprotein cholesterol distribution (VLDL-C, LDL-C, HDL-C), within 40 min, in microliter quantities of plasma using a single, relatively inexpensive column (Superose 6HR). The FAST cholesterol reagent (Sclavo) and a knitted PFTE Kratos reaction coil (Applied Biosystems) were found to provide optimal sensitivity, linearity, resolution, and dispersion characteristics. Validation is provided by comparison to target values for human quality control reference sera, and by comparing the values obtained by HPGC to the beta-quant method (LRC). The utility of the system is illustrated by comparing profiles from seven different species with normal or elevated plasma cholesterol concentrations. This technique allows rapid analysis of samples, regardless of species, without the use of precipitating agents or the ultracentrifuge. It could also be applied for the direct clinical determination of LDL-cholesterol.     

Association-dissociation studies of bovine and rat liver glutamic dehydrogenase by high-performance liquid chromatography gel filtration

The concentration-dependent association-dissociation tendency of purified bovine liver and rat liver glutamic dehydrogenase (GDH) has been demonstrated by high-performance liquid chromatographic gel filtration. In the concentration range of 100 to 1.0 micrograms bovine GDH/ml molecular species ranged from dimer and unimer to subunimeric forms. The dissociation process of the unimeric hexapeptide, consisting of six polypeptide chains, to the subunimeric tripeptide, consisting of three polypeptide chains, was irreversible without added ionic support, but reversible with added ionic support. In dilute Tris-HCl bovine liver GDH was dispersed to subunimeric sizes. Increasing the ionic strength in 20 mM phosphate as the mobile phase increased dissociation to a subunimeric tripeptide while sustaining as much as 80% of its activity. Activity of a eluting subunimer was verified by the inclusion of reaction substrates (NAD and glutamute) in the mobile phase and quantification of reaction products (NADH) in chromatograms. Gel filtration of GDH in the presence of GTP with NADH rendered a subunimeric tripeptide, largely independent of ionic strength or GDH concentration. Rat liver GDH, differing from bovine liver GDH, was dissociated by gel filtration to an active tripeptide independent of ionic or buffer conditions.     

Purification of bacteriophage DNA by gel filtration chromatography

Two fast and effective methods for high-scale purification of linear phage lambda DNA and circular double-stranded M13 replicative form are presented. A substantial reduction of time is attained by avoiding the long-term CsCl gradient centrifugations and dialysis common to standard procedures. Biologically active DNA preparations, free of chromosomal DNA and RNA, are obtained by including a simple gel filtration chromatography as the last step of purification. Yields are comparable to those from previously described methods.     

Apolipoprotein distribution in human lipoproteins separated by polyacrylamide gradient gel electrophoresis

The heterogeneity of serum lipoproteins (excluding very low density (VLDL) and intermediate density (IDL) lipoproteins) and that of lipoproteins secreted by HepG2 cells has been studied by immunoblot analysis of the apolipoprotein composition of the particles separated by polyacrylamide gradient gel electrophoresis (GGE) under nondenaturing conditions. The reactions of antibodies to apoA-I, apoA-II, apoE, apoB, apoD, and apoA-IV have revealed discrete bands of particles which differ widely in size and apolipoprotein composition. GGE of native serum lipoproteins demonstrated that apoA-II is present in lipoproteins of limited size heterogeneity (apparent molecular mass 345,000 to 305,000) and that apoB is present in low density lipoproteins (LDL) and absent from all smaller or denser lipoproteins. In contrast, serum apoA-I, E, D, and A-IV are present in very heterogeneous particles. Serum apoA-I is present mainly in particles of 305 to 130 kDa where it is associated with apoA-II, and in decreasing order of immunoreactivity in particles of 130-90 kDa, 56 kDa, 815-345 kDa, and finally within the size range of LDL, all regions where there is little detectable apoA-II. Serum apoE is present in three defined fractions, one within the size range of LDL, one containing heterogeneous particles between 640 and 345 kDa, and one defined fraction at 96 kDa. Serum apoD is also present in three defined fractions, one comigrating with LDL, one containing heterogeneous particles between 390 and 150 kDa, and one band on the migration front. Most of serum apoA-IV is contained in a band comigrating with albumin. GGE of centrifugally prepared LDL shows the presence of apoB, apoE, and apoD, but not that of apoA-I. However, the particles containing apoA-I, which, in serum, migrated within the LDL size range and as bands of 815 to 345 kDa, were recovered upon centrifugation in the d greater than 1.21 g/ml fraction. GGE of high density lipoproteins (HDL) indicated that most of apoA-I, A-II, and A-IV were present in lipoproteins of the same apparent molecular mass (390-152 kDa). ApoD tended to be associated with large HDL, and this was also significant for HDL apoE, which is present in lipoproteins ranging from 640 to 275 kDa. GGE of very high density lipoproteins (VHDL) presented some striking features, one of which was the occurrence of apolipoproteins in very discrete bands of different molecular mass. ApoA-II was bimodally distributed at 250-175 kDa and 175-136 kDa, the latter fraction also containing apoA-I.(ABSTRACT TRUNCATED AT 400 WORDS)     

Separation and size determination of human serum lipoproteins by agarose gel filtration

A method is described for the separation of the three major classes of human serum lipoproteins by gel filtration on columns of 4 and 6% agarose gel. After calibration of the columns, the elution volumes of the lipoproteins were used to calculate the molecular sizes and molecular weights of these macromolecules. The technique was employed to demonstrate aggregation of low density lipoprotein following partial delipidation, partial proteolysis, or mild heat denaturation. Agarose gel filtration shows promise as a useful method for the isolation, purification, and characterization of lipoproteins.     

Analysis of IgG glycopeptides by alkaline borate gel filtration chromatography

A protocol for the characterization of IgG glycopeptides is described. Central to this scheme is the novel application of an alkaline borate buffer to gel filtration chromatography. The use of this buffer significantly enhances the resolution of glycopeptides. Furthermore, it results in the separation of a unique size class of glycopeptides derived from IgG secreted by murine hybridomas. Although predominantly neutral, these glycopeptides differ both qualitatively and quantitatively by lectin affinity chromatography from the other glycopeptides which are presumably derived from the Fc portion of IgG.     

The fractional composition of immunoglobulin preparations studied by gel filtration on different carriers and by high-pressure liquid chromatography]

The fractional composition of immunoglobulin preparations produced by different manufacturing enterprises of this country has been studied by gel chromatography in columns packed with different carriers (Sephadex G-200 and ultragel AcA-34) and by high-performance liquid chromatography (HPLC). This study has revealed the nonstandard character of immunoglobulin preparations produced according to the same technological procedure (modified Cohn's method). The fractionation of immunoglobulins on different carriers with the use of different methods has yielded similar results confirmed by the statistical processing of the data. The results obtained in the study of the fractional composition of immunoglobulin preparations evidence that gel filtration with the use of ultragel and HPLC have greater resolving capacity in comparison with the method of gel filtration on traditionally used Sephadex G-200.     

Analysis of self-association of bovine neurophysins by gel chromatography

Analytical gel chromatography has been used to examine self-association of bovine neurophysins I and II under several sets of conditions. The data provide no evidence for associated species larger than the dimer. Association constants and Stokes radii of both monomer and dimer are very similar for both proteins in both 0.1 M KOAc, 0.16 M KCl and 0.1 M KPO4, 0.16 M KCl at pH 5.6 and 25 degrees C. The average values derived for the Stokes radii of the monomer and dimer under these conditions are 14.5 +/- 0.7 and 23.0 +/- 0.4 A, respectively. These results confirm the conclusion of Rholam and Nicolas [(1981) Biochemistry 20, 5837-5843] that the monomer and, to a lesser extent, the dimer are highly assymmetric. The Stokes radius of the monomer calculated by Rholam and Nicolas (op cit.) is approximately 30% larger than the value derived here. This discrepancy is probably the result of end-on penetration of the gel by elongated molecules [Y. Nozaki, N. M. Schechter, J. A. Reynolds, and C. Tanford (1976) Biochemistry 15, 3884-3890]. In contrast to Tellam and Winzor [(1980) Arch. Biochem. Biophys. 201, 20-24], it was found that neurophysin II does not exist solely as the dimer in 0.1 M KPO4, pH 5.6, although substitution of 0.1 M KPO4 for 0.1 M KOAc does increase the association constant by a factor of seven. Addition of 1.4 M LiCl at pH 8.1 also increases the association constant sevenfold, as well as increasing the Stokes radius of the monomer approximately 20%. The effects of ionic strength are consistent with the conclusion of Nicolas et al. [(1978) J. Biol. Chem 253, 2633-2639] that formation of the dimer depends upon hydrophobic bonding.     

Refolding of fusion ferritin by gel filtration chromatography (GFC)

Fusion ferritin (heavy chain ferritin, F_H+light chain ferritin, F_L), an iron-binding protein, was primarily purified from recombinantEscherichia coli by two-step sonications with urea [1]. Unfolded ferritin was refolded by gel filtration chromatography (GFC) with refolding enhancer, where 50 mM Na-phosphate (pH 7.4) buffer containing additives such as Tween 20, PEG, andl-arginine was used. Ferritin is a multimeric protein that contains approximately 20 monomeric units for full activity. Fusion ferritin was expressed in the form of inclussion bodies (Ibs). The IBs were initially solubilized in 4 M urea denaturant. The refolding process was then performed by decreasing the urea concentration on the GFC column to form protein multimers. The combination of the buffer-exchange effect of GFC and the refolding enhancers in refolding buffer resulted in an efficient route for producing properly folded fusion ferritin.     

Rat small-intestinal galactosidases. Separation by ion-exchange chromatography and gel filtration

1. The chromatography of rat small-intestinal beta-galactosidase activities on gel-filtration and ion-exchange columns has been studied. Five different substrates were used to measure beta-galactosidase activity (lactose, phenyl beta-galactoside, o-nitrophenyl beta-galactoside, p-nitrophenyl beta-galactoside and 6-bromo-2-naphthyl beta-galactoside) and the activity was measured at one acid and one more neutral pH value. 2. By gel filtration one acid beta-galactosidase, hydrolysing lactose and the hetero-beta-galactosides at about the same rate, and one more neutral beta-galactosidase, hydrolysing lactose much more rapidly than the hetero-beta-galactosides, were separated. 3. By ion-exchange chromatography the acid enzyme was fractionated into two components. These may be individual enzymes or different forms of the same enzyme.     

Preparation of giant RNA from bovine thyroid gland by agarose gel filtration

Giant RNA's were prepared by a rapid gel-filtration technique. DNAase, pronase and thyroid homogenate were successively placed on the column. Proteins and DNA were degraded during filtration. Void volume contained pure giant RNA's. Giant RNAs' yield was 7 times higher with this method than with phenol method.     

Interpretation of the stokes radius of macromolecules determined by gel filtration chromatography

From a comparison of the gel chromatographic properties of large randomly-coiled polypeptides in 6 M guanidine hydrochloride and of large globular proteins, we found that the distribution coefficient was more closely correlated with the intrinsic viscosity-based Stokes radius than with the translational frictional coefficient-based Stokes radius. This means that the effect of the hydrodynamic flow of dissolved molecules during gel chromatography should be considered. The ratio of transport of solute by bulk flow as compared with that by net diffusion (i.e., Brownian motion) is large under some conditions. On the other hand, we consider that the distribution coefficient obtained in static equilibrium experiments should be determined by the translational frictional coefficient-based Stokes radius, since the solvent does not flow. On this basis, we discuss the meaning of the Stokes radius and the separation mechanism of macromolecules by gel filtration.     

High-performance aqueous gel permeation chromatography of human serum lipoproteins

A new application of high-performance aqueous gel permeation chromatography was developed for the analysis of human serum lipoproteins. A good combination of columns (TSK GEL, type PW and type SW) was found for the separation of serum lipoproteins: very low-density lipoprotein, low-density lipoprotein and high-density lipoproteins. Analyses of serum lipoproteins from individual normal subjects and pathological subjects were performed by this combination of columns. The effects of pH and salt concentration of the eluent on the separation of lipoproteins were also investigated.     

Molecular composition of the fractions of immunoglobulin preparations studied by gel filtration

The method of gel filtration in columns permitted the separation of aggregated fractions into polymers whose content did not exceed 10% and dimers, their content ranging from 3.6% to 22.11%. The preparations were also found to contain fractions of monomers and fragments, Fab-fragments being detected in 10 out of 20 batches under study (4.04-27.36%). The shelf life of all preparations did not exceed 8-12 months. The use of spectrophotometric techniques ensured obtaining the most objective results in the calculation of the percentage of fractions contained in immunoglobulin preparations. The evaluation of the molecular composition of immunoglobulin preparations by the method of gel filtration is conducive to the improvement of their quality.     

A procedure for the purification of beta-lactoglobulin from bovine milk using gel filtration chromatography at low pH

A simple and rapid procedure for the purification of beta-lactoglobulin (β-LG) from bovine milk is described. The procedure exploits the major difference in molecular mass of β-LG and other whey components and the existence of the former in monomeric form at acidic pH. Gel filtration of whey was carried out using a Bio-Gel P10 column at pH 3.0. Residual caseins and other milk proteins were excluded from the gel and β-LG and alpha-lactalbumin (α-LA) emerged as two fully resolved peaks. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) suggested that β-LG was purified to apparent homogeneity, while absorption, fluorescence, and circular dichroism spectroscopy indicated the native-like conformation of the protein. Western blot analysis revealed that the antibodies raised against the purified β-LG in rabbits also readily react with the commercial bovine protein. This procedure requires only 4-5 hr for the purification of about 10 mg of β-LG from a single run while using a small column (2.3 cm x 83 cm) of Bio-Gel P10 and has the potential for scaling up.     

Polyacrylamide gel electrophoresis of visna virus polypeptides isolated by agarose gel chromatography.

The proteins of visna are separated into nine major peaks by agarose gel chromatography in 6 M guanidine hydrochloride (GuHCl). The polypeptides in eack peak were isolated by acid precipitation and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The patterns of SDS-PAGE show that the excluded material from the GuHCl column contains an aggregate of 10 non-glycosylated polypeptides. It is shown that this aggregate represents virus substructures that are not completely solubilized by GuHCl. Two glycoproteins, gp175 and gp115, were isolated from the column eluate. The major glycoprotein gp115 was coeluted with P90, P68, and P61 in GuHCl 4. Each of the four major peaks (GuHCl 5 to 8) contains more than one nonglycosylated polypeptide. However, a small polypeptide, P12, can be isolated in a homogeneous form in the last peak, GuHCl 9. Analysis of the virus proteins (100 microgram) by SDS-PAGE shows that 20 radioactive bands can be recognized. During fractionation of the protein on agarose gel columns followed by analysis with SDS-PAGE, a number of minor polypeptides that were not detected before became clearly recognizable. Thus, the combined use of column chromatography and SDS-PAGE shows that visna virus is composed of 25 proteins.