Analytical and preparative isoelectric focusing of peptides in immobilized pH gradients

A new method for peptide analysis and purification is described, based on isoelectric focusing in immobilized pH gradients. On the analytical scale, the peptide zones can now be revealed by an stain for primary and secondary amino group (e.g. ninydrin, fluorescamine, dansyl chloride) since the buffering species, unlike conventional carrier ampholytes, contain only carboxyl and tertiary amino groups. For preparative purposes, conditions have been described to remove most contaminants (e.g. unreacted monomers, non-cross-linked, short polyacrylamide chains) from the gel matrix before the electrophoretic run. However, ca. 2% of the gel dry mass is still present as extractable material. The focused peptides can be recovered in higly yields (ca. 90%) with a fairly high degree of purity (75%), the contaminants being mostly components eluted from the polyacrylamide gel.     

Agarose isoelectric focusing of plasma low and very low density lipoproteins using the PhastSystem

Human plasma lipoproteins, fractionated by density gradient ultracentrifugation, and very low density lipoproteins, subfractionated by cumulative rate centrifugation, were subjected to agarose isoelectric focusing in small format thin gels prepared in the laboratory for the commercially available PhastSystem (Pharmacia). From preparation of the gels to their staining, the procedure took less than 3 h. The pH gradient was found reproducible and the apparent average pI of individual low density lipoproteins could be measured with a coefficient of variation of less than 5% between and less than 2% within the same run. The method appears especially suitable for the exploration of charge properties of multiple lipoprotein samples, or other large macromolecules as low density lipoproteins and very low density lipoproteins, with considerable economy of time and reagents.     

The carbohydrate content of apolipoprotein E from human very low density lipoproteins.

The carbohydrate content of the E protein of human very low density lipoprotein (VLDL) was evaluated both by colorimetric methods and by gas liquid chromatography of the trifluoroacetylated 0-methyl glycosides. The major unmodified hexose was noted to be galactose with a mole ratio with respect to protein which ranged from 0.81 to 1.54. N-acetyl glucosamine (molar ratios from 0.52 to 1.76) and N-acetyl galactosamine (molar ratios from 0.73 to 1.59) and the respective unacetylated amino sugars were noted for all of the apoproteins evaluated. Sialic acid (molar ratios from 0.79 to 1.69) was a prominent carbohydrate for each of the E protein preparations. When the apoprotein was exposed to neuraminidase with a resultant loss of two-thirds of the sialic acid, the isoelectric focus behavior was found to be unchanged. The E protein isolated from the very low density lipoproteins of Type III patients (dysbetalipoproteinemia) revealed a carbohydrate content similar to the normals or Type IV patients.     

Preparative isoelectric focussing of apolipoproteins C and E from human very low density lipoproteins.

The application of isoelectric focussing on a gel-stabilized layer for the separation of the Tris-urea-soluble apolipoproteins of very low density lipoproteins has been described. This method in one step, allows the separation of most apolipoproteins, which were then analyzed and characterized. Apolipoproteins CII and CI were isolated as single protein bands with apparent pI of 5.0 and 6.5, respectively. Apolipoprotein CII was biologically active and could activate lipoprotein lipase. Apolipoprotein CIII was separated into several protein bands with pI ranging from 4.7 to 5.1 as a function of their number of sialic acid residues. Apolipoprotein E was isolated and characterized into five polymorphic bands with pI of 5.7, 5.8, 5.9, 6.0, and 6.2, respectively.     

High-resolution two-dimensional electrophoresis with isoelectric focusing in immobilized pH gradients

Due to the high reproducibility of pH gradient slope and width, immobilized pH gradients (IPG) have been used as the first dimension of two-dimensional techniques in order to generate maps of constant spot position in the $\text{p}\text{M}{}_{\mathrm{<mtext>r</mtext>}}$. However, when coupling IPG to SDS (sodium dodecyl sulphate) gels two problems were encountered: vertical streaking, due to incomplete zone solubilization in SDS, and horizontal streaking, due to spot fusion along the pH axis caused by the electroendosmosis of the charged Immobiline gels. Two methodical modifications are herewith described to overcome these drawbacks: (a) the SDS equilibrium time of the first-dimension gel has been prolonged to at least 30 min; (b) the SDS electrophoresis gel has been cast together with a starting gel, containing 2.5 mM of each Immobiline species used in the first dimension, which serves as a transition from the charged to the uncharged gel.     

Quantification of apoC-II and apoC-III of human very low density lipoproteins by analytical isoelectric focusing.

ApoC-II and apoC-III of human very low density lipoproteins (VLDL) have been quantified by analytical isoelectric focusing (IEF) between pH 4 and 6 in polyacrylamide gels containing 8 M urea. The isoelectric point of apoC-III0 is pH 4.93; apoC-II, pH 4.78; apoC-III1, pH 4.72, and apoC-III2, pH 4.54. ApoC-I is not found in the pH range between pH 4 and 6. Two minor peptides, apoC-IV and apoC-V, with isoelectric points of pH 4.61 and 4.44, respectively, are apoproteins not previously identified. The sensitivity (5--40 microgram) and reproducibility (+/- 8%) of this method allow quantitative analysis of apoC-II and apoC-III distribution in VLDL.     

Isolation of human monoclonal antibody isoproteins by preparative isoelectric focusing in immobilized pH gradients

A method for preparative isolation of human monoclonal antibody isoproteins is described in the present paper. A human monoclonal antibody directed against the transmembrane protein gp 41 from the human immunodeficiency virus (HIV-1) was used in this study. The antibody belongs to the IgG1 subtype and exhibits antibody dependent cellular cytotoxicity. The resolving power of conventional preparative protein separation techniques such as ion-exchange chromatography, chromatofocusing and lectin affinity chromatography is too poor for a complete separation of isoproteins. The more sophisticated technique of chromatofocusing on FPLC-based material (Mono P, Pharmacia) did not satisfy our expectation. With semipreparative IEF in immobilized pH gradients we were able to prepare the different isoproteins of a human monoclonal antibody in milligram amounts. No significant difference between the single isoproteins with respect to specificity and avidity to the recombinant antigen (rec gp 160) was detected. Therefore, we assume that the separation conditions did not influence the immunochemical nature of the antibody and significant denaturation and/or precipitation of the IgG did not occur. Furthermore the method affords preparative separation with resolution equivalent to analytical runs. Experiments for scale up and further characterization of isoproteins (carbohydrate composition, amino acid analysis, half life times etc.) are in progress.     

Characterization of the oligosaccharide side chain of apolipoprotein C-III from human plasma very low density lipoproteins.

Apolipoprotein C-III1 and apolipoprotein C-III2 each contain one oligosaccharide side chain, bound O-glycosidically to threonine in position 74 of the amino acid sequence. The studies reported in this paper characterize these alkali labile oligosaccharides, thereby demonstrating the complete structure of apolipoprotein C-III. Monosaccharide analysis revealed the following sugar composition: D-galactose/N-acetyl-D-galactosamine/sialic acid 1 : 1 : 1 and 1 : 1 : 2 for apolipoprotein C-III1 and apolipoprotein C-III2, respectively. Treatment of desialylated apolipoproteins with alkaline borohydride released the reduced disaccharide beta-D-galactosyl-(1 leads to 3)-N-acetyl-D-galactosaminitol, which was detected by gas-liquid chromatography. Further studies employing periodate oxidation and Smith degradation indicated that the structure of the trisaccharide from apolipoprotein C-III1 was alpha-N-acetylneuraminyl-(2 leads to 3)-beta-D-galactosyl-(1 leads to 3)-N-acetyl-D-galactosaminitol. The tetrasaccharide structure from apolipoprotein C-III2 is made up of this trisaccharide plus one sialic acid residue linked to C6 of N-acetyl-D-galactosaminitol, as was shown by the assessment of chromogens formed upon alkaline degradation.     

Apolipoprotein E Bethesda. Isolation and partial characterization of a variant of human apolipoprotein E isolated from very low density lipoproteins

A variant of apolipoprotein E, denoted E Bethesda, has been identified in the plasma of a 72-year-old woman with type III hyperlipoproteinemia. An offspring of the proband also has this variant and type III hyperlipoproteinemia. Apolipoprotein E Bethesda was isolated by preparative isoelectrofocusing followed by preparative SDS-polyacrylamide gel electrophoresis from the very low density lipoproteins of the proband's son. The purity and the identity of the preparation were analyzed by analytical SDS-polyacrylamide gel electrophoresis, two-dimensional gel electrophoresis and by immunochemical analysis. Apolipoprotein E Bethesda migrates in the E 1 position and its electrophoretic mobility is not affected by neuraminidase treatment. The protein is shifted to the E3 position after cysteamine treatment. The amino acid composition revealed the presence of two cysteine residues. These data support the concept that the apolipoprotein E Bethesda allele is derived from a mutation of the E2 or E2* allele.     

Preparative isoelectric focusing in immobilized pH gradients. II. A case report

The preparative aspects of isoelectric focusing (IEF) in immobilized pH gradients (IPG) have been investigated as a function of the following parameters: environmental ionic strength (I), gel geometry and shape of pH gradient. As model proteins, hemoglobin (Hb) A and a minor, glycosylated component (HbA1c), with a delta pI = 0.04 pH units, have been selected. The load capacity increases almost linearly, as a function of progressively higher I values, from 0.5 X up to 2 X molarity of buffering Immobiline (pK 7.0) to abruptly reach a plateau at 3 X concentration of buffering ion. The load capacity also increases almost linearly as a function of gel thickness from 1 to 5 mm, without apparently levelling off. When decreasing the pH interval from 1 pH unit (pH 6.8-7.8) to 1/2 pH unit (pH 7.05-7.55) the amount of protein loaded in the HbA zone could be increased by 40%. In 5 mm thick gels, at 2 X pK 7.0 Immobiline concentration, over a 1/2 pH unit span, up to 350 mg HbA (in a 12.5 X 11 cm gel) could be loaded in a single zone, the load limit of the system being around 45 mg protein/ml gel volume.     

Preparative isoelectric focusing of human serum very low-density and low-density lipoproteins.

Ten percent glycerol prevented the usual precipitation of human serum very low-density lipoproteins (VLDL) and low-density lipoproteins (LDL) at their isoelectric points during their preparative isoelectric focusing (IEF), IEF separated VLDL and LDL into two major fractions. The observed optical density peaks are not artifacts caused by binding of Ampholines to VLDL or LDL since no radioactivity accumulated in the fractions containing VLDL or LDL during IEF in the presence of [¹⁴C]Ampholine, and gel filtration completely separated the lipoproteins from [¹⁴C]Ampholine. These results suggest that IEF may separate subspecies of VLDL and LDL under suitable experimental conditions.     

The interaction of human serum albumin in the native and fully reduced states with apolipoprotein E,serum amiloid protein and very low density lipoproteins from human plasma

• 1.1. Complex formation in a solution of apolipoprotein E (apoE) isolated from human plasma very low density lipoproteins (VLDL) and human serum albumin (HSA) in both native and fully reduced states was studied. The existence of a kinetically unstable complex of apoE and native albumin was shown. The complex became more stable with the reduction of the S—S links in the albumin molecules capable of forming aggregates under these conditions.
• 2.2. The interaction between native HSA as opposed to a fully reduced one and isolated VLDL particles was more pronounced, probably, due to the existence of amphipathic alpha-helical regions.
• 3.3. Dissociation of the serum amyloid protein (SAP) oligomeric form in solution and the interaction of the protein with fully reduced HSA owing to the provision with the additional hydrophobic surface was shown. ApoE displaced SAP from the complex with fully reduced albumin.
• 4.4. It is suggested that the ability of the apolipoprotein to interact with albumin is determined by internal stability of the molecular structure of the latter and the complexes detected in vitro may be a new transport form of apolipoproteins in lipid-free form in serum. It is assumed that competitive interactions in the HSA-SAP-apoE system may be involved in the development of secondary amyloidosis.

     

Preparative isoelectric focusing in immobilized pH gradients. I. General principles and methodology

A new method for preparative protein purification is described, based on the use of Immobiline matrices. After electrofocusing, the protein zone of interest is recovered by electrophoretic transfer to a hydroxyapatite gel, from which it is eluted with 0.2 M phosphate buffer, pH 6.8, with yields for the proteins studied in the range 76-98%. For six different proteins, the focusing step gives a common upper limit of approximately 45 mg protein/ml gel as mean concentration in a focused protein zone. It is demonstrated that in practical preparative work, components with a pI difference of 0.007 pH units can be completely resolved, and that on a 5-mm-thick gel of dimensions 240 X 110 mm, samples containing as much as 400 mg of the major protein component can be applied. Focusing of large amounts of a salt-containing sample is demonstrated with the aid of human serum. A theoretical expression is given relating the concentration distribution and maximum protein concentration within a focused zone to the applied voltage, the pH slope used and the zone width. Based on this expression and the finding of an upper concentration limit for a protein we shown how to optimize the parameters in preparative work with immobilized pH gradients in relation to the separation power needed. Finally, it is shown that, in comparison with conventional preparative electrofocusing in polyacrylamide gels, immobilized pH gradients allow a ten-fold increase in load, whilst still giving a resolution comparable to that of analytical isoelectric focusing.