Determination of the isoelectric point of a purified milk clotting enzyme preparation by analytical and preparative isoelectric focusing

Ohne Zusammenfassung

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Determination of the isoelectric point of a purified milk clotting enzyme preparation by analytical and preparative isoelectric focusing

     

Separation of human alloalbumin variants by isoelectric focusing

A technique for the separation of human alloalbumin variants by means of isoelectric focusing in the presence of 8M urea and 60 mM L-serine is described. The potential usefulness of this technique in the detection and classification of genetic heterogeneity at the albumin locus is demonstrated by the differentiation of three human alloalbumin variants of European origin.     

Estimation of isoelectric points of human plasma proteins employing capillary isoelectric focusing and peptide isoelectric point markers

Synthetic UV-detectable peptide pI markers were used to estimate isoelectric point (pI) values of proteins separated by capillary isoelectric focusing (CIEF) followed by cathodic mobilization in the absence of denaturing agents. The pI calculation and quantitative analysis of purified proteins showed the feasibility of these peptides as pI markers and internal standards in CIEF separation of proteins. Estimation of pI values of major proteins in human plasma was performed using the peptide pI markers, and the values were compared with those previously obtained by gel isoelectric focusing (IEF). Sera of immunoglobulin G (IgG) myeloma patients, which showed characteristic peaks of myeloma IgG in their CIEF patterns, were also subjected to the analysis and the pI values of the myeloma proteins have been estimated.     

Recycling isoelectric focusing and isotachophoresis

A new apparatus for large scale preparative isoelectric focusing in free solution is described. Its main characteristic is rapid fluid recirculation with but short residues times in the electric field. For the first time the recycling principle was also applied to preparative isotachophoresis, by incorporating into the system a computer controlled leader counterflow. The rapid recirculation permits application of unusually high electrical power for rapid resolution, as it suppresses fluid flow instabilities arising from electrohydrodynamic effects.     

Conductivity properties of carrier ampholyte pH gradients in isoelectric focusing

The conductivity properties of natural pH gradient created by carrier ampholytes were studied during the process of isoelectric focusing (IEF). IEF was performed in capillaries (10-30 mm long) or in microchips with the same channel length. A 10-30x reduction of the conductivity of the separation medium was observed during the establishment of pH gradient. Results obtained using different IEF voltages indicate that there is a nonlinear relationship between the conductivity of an established pH gradient and the applied electric field. Our theoretical analysis using a simplified model generated values that reasonably agree with the experimental data. In addition, we found that above a certain electric field ( approximately 300 V/cm), resolution does not increase with the applied voltage as predicated; we observed band-broadening and gel breakdown. The approach presented in this work can be used for optimization of the IEF separation and judicious selection of IEF conditions.     

High-efficiency capillary isoelectric focusing of protein complexes from Escherichia coli cytosolic extracts

High-efficiency capillary isoelectric focusing (cIEF) separations of protein complexes obtained from soluble protein fractions are demonstrated. Size-exclusion chromatography was used as a first dimension separation to fractionate putative protein complexes with apparent molecular masses of up to 1,500,000 from an Escherichia coli cytosolic fraction. Non-denaturing cIEF separations using highly hydrophilic polymer-coated capillaries constituted the second dimension. The conditions developed produced reproducible and high-efficiency separations, corresponding to approximately 2 x 10(6) theoretical plates and peak capacities of approximately 10(3) for pH 3-10 cIEF separations in 65 cm long capillaries. Combination of the two non-denaturing separation dimensions permitted isolation and analysis of individual protein complexes from complicated biological samples. Studies indicated that many E. coli complexes were stable on the time scale of the cIEF separations, but were degraded upon more extended periods of storage on ice, necessitating rapid sample processing and fast analysis techniques.     

Capillary isoelectric focusing of erythropoietin glycoforms and its comparison with flat-bed isoelectric focusing and capillary zone electrophoresis

The influence of several operation conditions on separation of recombinant human erythropoietin glycoforms by capillary isoelectric focusing (cIEF) is explored. From this study it is deduced that in order to separate several glycoforms of erythropoietin, urea has to be added to sample, which should not be completely depleted of the excipients used in its formulation. On-line desalting does not provide separation enhancement for samples with high content of salt. Better resolution is obtained using a mixture of a broad and a narrow pH-range carrier ampholytes than with either one used separately. Under the experimental conditions, focusing voltages of 25 kV improve separation compared to lower and higher electric fields. Focusing times shorter than the time necessary for electric current to reach a minimum provide similar separations than longer focusing times at which a minimum value of the current has already been achieved. The optimized method allows the separation and quantitation in 12 min of at least seven bands containing glycoforms of recombinant erythropoietin with apparent isoelectric points in the range 3.78–4.69. Compared to flat-bed isoelectric focusing, cIEF provides better separation of bands of glycoforms in a shorter time, and allows quantitative determination. Capillary zone electrophoresis (CZE) gives rise to resolution of erythropoietin glycoforms similar to that obtained by cIEF. Although CZE requires a longer analysis time, its reproducibility in terms of peak area of glycoforms is better than in cIEF.     

Isoproteins and an isoelectric focusing mutant of human apoprotein serum amyloid A

On isoelectric focusing of human plasma and subsequent immunoblotting, using antii-human serum amyloid A (SAA) antibodies, a genetic variant of SAA was detected in a family of Turkish origin. All affected members of the family were apparent heterozygotes for the mutant protein, which underwent a charge shift of about one charge unit toward the anode. The variant is likely to be a mutant of the most prominent forms of SAA (SAA1 and SAA2, or SAA1 and SAA1 des Arg). The appearance of a genetic variant of two of the six reported SAA-isoforms in human plasma supports the concept of SAA proteins being products of different genes.     

Mass distribution and focusing properties of carrier ampholytes for isoelectric focusing: I. Novel and unexpected results

In an attempt to prepare quasi-isoelectric buffers as BGEs for CE, carrier ampholytes (CAs) (Ampholine, pH 7-9; Servalyt, pH 7-9; Bio-Lyte, pH 8-10 and Pharmalyte, pH 8-10.5) have been subdivided with the Rotofor into 20 fractions, of ca. 0.1 pH unit span, whose composition has been studied by CZE-MS. The results have allowed identifying the number of different molecular mass compounds present in every commercial brand, as well as the number of isoforms (having identical mass, but representing positional isomers) associated with a given M(r) value. Ampholine is composed of 29 species, for a total of 85 different isoforms; Bio-Lyte is made of 43 compounds, for a total of 136 isoforms; Pharmalyte comprises 58 different M(r) chemicals, for a total of 102 isoforms and Servalyt is constituted by 65 species, for a total of 306 compounds (all of these values to be considered as minimum numbers, as detected by the present methodology). Surprisingly, and contrary to theory, a very large proportion (up to 70%) of these species are 'poor carrier ampholytes', in that they are unable to focus and are evenly distributed along the generated pH gradient in the electric field. Paradoxically, the pH gradient is created and sustained by the minority of species (30% for three brands, up to 50% for Pharmalyte) that appear to focus at their pI position into reasonably sharp zones. Even in the narrowest pI fraction, up to 20 different compounds can be detected. It is concluded that very few amines with different useful pK values are utilized for the synthesis and that a new generation of CAs with a more diversified population of amines with proper pK values within the given pH intervals should be sought. Ampholine, the poorest of the commercial brands, appears to be still made with the original synthesis devised by Vesterberg, i.e. by reacting a concoction of oligoamines with alpha,beta-unsaturated acids.     

Thin-layer agarose isoelectric focusing of alkaline phosphatase isoenzymes from human neutrophils

An improved method for thin-layer agarose isoelectric focusing of alkaline phosphatases (AP) from human neutrophils is described. The solubilization of AP isoenzymes was studied with four detergents. The best results were obtained after sonication with Zwittergent 3-12 (1% final concentration), followed by butanol extraction and ultracentrifugation 105,000 x g for 1 h. The cytosol can be stored at 0 degrees C or -80 degrees C, but not at -20 degrees C. Dialysis of the cytosol against a Tris buffer, pH 7.5, was imperative prior to focusing for removal of the detergent. Enzyme visualization is enhanced by incorporation of ZnCl2 (3 mM) into the agarose gel. The focusing patterns consist of two sets of isoenzymes: two main zones with pI 6.4 and 6.8 and minor components with pI 4.2, 4.8 and 5.2.     

Isoforms analysis of recombinant human erythropoietin by polarity-reversed capillary isoelectric focusing

Recombinant human erythropoietin (rhuEPO) has been extensively used as a pharmaceutical product for treating anemia in the clinic. Glycosylation of rhuEPO was crucial for affecting biological activity, immunogenicity, and pharmacokinetics. Because of the heterogeneity of glycan, the structure of rhuEPO was complex with several isoforms. Characterization of isoforms was important for quality control of rhuEPO. Here, an improved cIEF method has been established and validated. A polarity-reversed focusing step was used by reversing both the polarity of the voltage and the catholyte and anolyte vials. A weak base (100 mM ammonium hydroxide solution) was used as a chemical mobilizer to make the acidic bands mobilize stably to the detection window. Compared with CZE method in European Pharmacopoeia, the numbers of isoforms and their peak area percentage were highly consistent. Better reproducibility and higher resolution have been obtained by the improved cIEF method. Moreover, in improved cIEF method, the isoelectric points (pI) of each isoform can be calculated and used for identification. It was also the first time that the cIEF method was fully validated for rhuEPO analysis according to the International Conference on Harmonization (ICH) guidelines.     

Analysis of human apolipoproteins C by isoelectric focusing in immobilized pH gradients

Apolipoproteins C are involved in many ways in the metabolism of plasma lipoproteins. Apolipoproteins C from the delipidated VLDL of 35 controls and 165 normo- and hyperlipoproteinemic patients were analyzed by isoelectric focusing on an immobilized pH gradient, pH 4.0-5.0, with 7 M urea, which raised the apparent pH range to 4.8-5.7. This method is an improvement over conventional isoelectric focusing with carrier ampholytes with regard to both resolution and reproducibility. Due to the high resolution (0.1 pH units per cm) additional apolipoprotein C-III bands: C-III0 A1, C-III0 A2, C-III1 C and C-III2 C (the designations A, anodic, and C, cathodic, refer to direction of migration on IEF in relation to the main band) are described for the first time. The possible artifactual nature of these protein bands could be excluded. Cleavage with neuraminidase and peptidases, immunological detection and/or two-dimensional electrophoresis were used to obtain more information. The additional bands seem, in part, to be hydrolysis products of carboxypeptidase A (C-III1 C, C-III2 C). The appearance of C-III1 C and C-III2C was dependent upon the serum triglyceride concentration. The percent distribution of C apolipoproteins in very low density lipoproteins (VLDL) from control serum agreed with previously published data. Apolipoproteins C can also be focused in immobilized pH gradients from VLDL and serum without delipidation.     

A multivariate approach for the determination of isoelectric point of human carbonic anhydrase isoforms by capillary isoelectric focusing

Human carbonic anhydrase (hCA) IX and XII are isoenzymes which are highly overexpressed in many cancer types. Recently, it has been shown that hCA IX contributes to the acidification of the tumor environment leading to chemoresistance with basic antitumoral drugs. The development of selective hCA inhibitors constitutes a new therapeutic axis. In order to elucidate the specific interactions between hCA and inhibitors, physico-chemical properties of hCA must be evaluated. This work reports the determination of the isoelectric point (pI) of a series of hCA isoforms by capillary isoelectric focusing. First, the method was optimized with synthetic UV-detectable pI markers using a central composite design. The separation was performed in a fused-silica capillary chemically derivatized with hydroxypropylcellulose and using a glycerol-water medium as the anticonvective gel. Three main factors (ampholyte content, focusing time and mobilization pressure) were optimized in order to obtain the best resolution, detection threshold and precision on the pI determination. Then, the model was validated through the analysis of standard proteins mixture having known pI values, before investigating the pI of hCA isoforms.     

Low-molecular-weight human serum proteome using ultrafiltration, isoelectric focusing, and mass spectrometry

Identification of the serum proteome is a daunting analytical task due to the complex nature of the sample which has an extremely large dynamic range of protein components. This report addresses this issue by using centrifugal ultrafiltration to enrich the low-molecular-weight (LMW) serum proteome while decreasing the amount of abundant high-molecular-weight proteins. Reduction of the complex nature of the sample was achieved by fractionation of the LMW serum proteins using solution-phase isoelectric focusing (IEF). Multiple enzyme digestions are performed and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Analysis of the tandem mass spectra resulted in the identification of 262 proteins belonging to LMW serum proteome. Our results demonstrate the effectiveness of this methodology to isolate and identify LMW proteins with improved confidence in the MS data acquired. In addition, our methodology can be combined with other multidimensional chromatography techniques performed on the peptide level to increase the number of identified proteins.     

Estimation of biotinylated lectin by isoelectric focusing

Concanavalin A (Con A) was biotinylated to various degrees using N-biotinyl-omega-aminocaproic-acid-N-hydroxy succinimide ester as the biotinylation reagent, and then analyzed by isoelectric focusing using PhastGel IEF 3-9. The isoelectric points of biotinylated ConAs were found to decrease with increasing concentration of the biotinylation reagent. Analysis by isoelectric focusing followed by dot blotting clearly indicated that the biotinylated ConA with an isoelectric point lower than that of the original ConA by 2.2 +/- 0.6 had the strongest binding activity for ovalbumin.