Concentration and fractionation by isoelectric trapping in a micropreparative-scale multicompartmental electrolyzer having orthogonal pH gradients. Part 2

A micropreparative-scale multicompartmental electrolyzer called ConFrac has been developed and tested for isoelectric trapping separations. ConFrac can be operated in pass-by-pass mode or recirculating mode, using either asymmetrical feeding (feed enters only the anodic or the cathodic flow-through compartment) or symmetrical feeding (feed enters both the anodic and the cathodic flow-through compartment). Symmetrical feeding results in higher processing rates and is the preferred operation mode. Residence time in the flow-through compartments is set as a compromise between processing rate and temperature rise in the effluent. Ampholytic components have been isolated from 5 to 50 mL volumes of micromolar feed solutions and hundredfold concentrated into 100-μL collection compartments. Samples containing ampholytic analytes in highly conducting salt solutions were readily desalted and fractionated in ConFrac in one operation. pH transients formerly observed in other isoelectric trapping devices were observed in ConFrac as well. The pH transients were caused by the unequal ion transmission rates of the anodic- and cathodic-side buffering membranes.     

Rapid isoelectric trapping in a micropreparative-scale multicompartment electrolyzer

A multicompartmental electrolyzer called membrane-separated wells for isoelectric focusing and trapping (MSWIFT) has been developed and tested for micropreparative-scale isoelectric trapping separations. In the MSWIFT, the length of the heat conduction path from the center of the compartments to the wall is less than 1 mm. The compartments are made from 99.8% nonporous alumina that has a high heat conductivity and a high specific heat capacity, leading to adequate Joule heat dissipation even at power loads as high as 5 W. The length of any compartment parallel to the electric field (the intermembrane distance) can be selected to be multiples of 1.5 mm, leading to compartment volumes that are multiples of about 60 muL. A maximum of 20 (1.5 mm long) separation compartments can be readily assembled in the current version of MSWIFT. The MSWIFT has been used to desalt samples, isolate small ampholytic components (amino acids, peptides, and dyes), prefractionate complex protein mixtures and enrich minor components; these separations were achieved in 20-60 min.     

Preparative-scale isoelectric trapping by recursive electrophoresis in a compartmentalized system having orthogonal primary and secondary pH gradients. Part 1--construction and standard operation

The performance of the current preparative-scale isoelectric trapping systems is limited by the serial arrangement of the separation compartments. A new system has been developed that achieves trapping by recursive electrophoresis in a compartmentalized system (T-RECS). T-RECS features (i) parallel-connected elementary separation heads with independent electrode compartments, feed compartments, and harvest compartments, (ii) orthogonal primary pH gradients and secondary pH gradients, (iii) directionally controlled convective analyte transport along the primary (resolving) pH gradients, and (iv) electrophoretic analyte transport along the secondary (harvesting) pH gradients. The operation of T-RECS has been quantitatively characterized via separation of small molecule ampholytes.     

Preparative-scale isoelectric trapping by recursive electrophoresis in a compartmentalized system having orthogonal primary and secondary pH gradients. Part 2--operation in cascade mode

A parallel multicompartmental electrolyzer recently developed for preparative-scale isoelectric trapping separations, trapping by recursive electrophoresis in a compartmentalized system, was set up to operate as a cascade of binary separations to produce at least one pure target ampholyte (or more, with additional separation heads) without other ampholytes ever entering (even transiently) the harvest stream. This mode of operation avoids the need for exhaustive electrophoresis and the accompanying long separation times brought about by the exponentially decreasing concentrations over the course of batch separations. Continuous operation can be achieved in the cascade mode by continuously feeding the sample into the first separation head configured with three flow-through compartments and continuously harvesting one (or more) target components in additional separation heads configured with two flow-through compartments, attached to the respective branching points.     

Analysis of recombinant proteins by isoelectric focusing in immobilized pH gradients.

Isoelectric focusing in immobilized pH gradients (IEF-IPG) was used to analyze three different recombinant proteins. Recombinant leech hirudin (65 amino acids, three disulfide bonds) expressed in Saccharomyces cerevisiae as a secreted protein and purified by anion-exchange and reversed-phase chromatography proved to be homogeneous with regard to its isoelectric point (pI). In addition, the theoretical pI, calculated on the basis of the primary structure, corresponded precisely to the measured pI of 4.30. IEF-IPG was further employed to follow the stability of recombinant hirudin at pH 9, indicating that deamidation occurred under these conditions. A variant of recombinant human alpha 1-antitrypsin (AAT) (389 amino acids, one cysteine residue) expressed in Escherichia coli and purified by anion-exchange, metal chelate and hydrophobic-interaction chromatography appeared to be homogeneous by polyacrylamide gel electrophoresis under reducing and denaturing conditions as well as by various high performance liquid chromatography methods. However, some heterogeneity was detected by IEF-IPG between pH 5-6. The measured pI values of 5.43-5.58 were slightly lower than the calculated pI based on the primary structure (5.72). This indicated deamidations of Asn or Gln residues. A recombinant Schistosoma mansoni parasite antigen, p28 (210 amino acids, one cysteine residue) obtained after intracellular expression in Saccharomyces cerevisiae and affinity purification on glutathione agarose was analyzed by IEF-IPG in a pH 7.3-8.3 gradient. It appeared to be heterogeneous with regard to its pI, with the major component having a pI of 7.81 compared to the calculated value of 7.17.(ABSTRACT TRUNCATED AT 250 WORDS)     

Apolipoprotein E phenotyping by isoelectric focusing in immobilized pH gradients and silver staining.

A simple and high resolution procedure of apoprotein E (apo E) phenotyping by isoelectric focusing with immobilized pH gradients and silver staining is described. This method needs delipidated very low density lipoproteins (isolated from 1 mL of serum) but obviates immunoblotting as well as neuraminidase treatment in routine applications because the sialylated forms are clearly separated. Immunoblotting (with polyclonal and monoclonal anti-apo E antiserum), cysteamine and neuraminidase treatment, and pI markers allowed the localization of three main alleles, xi 2, xi 3, xi 4 and the detection of variants or rare alleles (6/450 determinations). The serum amyloid A (SAA) apolipoproteins (SAA1,SAA2) could be characterized unequivocally (especially with E3 and E4). Silver staining proved more sensitive than Coomassie Brilliant Blue and needs only 5 micrograms of protein in the sample. The results of 403 normo-or hyperlipidemic patients are shown. In the group of 191 normolipidemic patients (cholesterol less than 6.40 mmol/L triglycerides less than 2 mmol/L), the relative frequency of the xi 3 allele (0.83) is higher than in other reports on Caucasians (about 0.77) whereas the xi 4 allele is lower. As previously described, we find a high frequency of the 4/3 phenotype in hypercholesterolemia and 3/2 in hypertriglyceridemia. The high frequency of the E2/E2 phenotype, usually associated with hyperlipidemia, and variants in complex hypertriglyceridemia makes the apo E phenotyping necessary in many cases of dyslipidemias.     

Column isoelectric focusing in natural pH gradients generated by biological buffers

Using 2 or 3 simple Good zwitterionic buffers at a 16 or 18 mmol/L final column concentration of the mixture, natural pH gradients of 4 to 8 and 3 to 9.5, respectively, were generated in a liquid LKB column. The pH gradients, stabilized by an anticonvective sucrose gradient, were linear, reproducible and stable in the electric field up to 5h. The pH gradients were used for isoelectric focusing of a number of impure proteins such as human hemoglobin, bovine serum albumin and chicken egg white lysozyme. The protein components could be well separated in the gradient, were easily recovered and appeared to be quite pure when analyzed by sodium dodecyl sulfate-gel electrophoresis. Furthermore, the pH gradient 4-8 was effectively used to isolate one of the acidic isozyme (pI 5.6) components of mouse liver alcohol dehydrogenase (EC 1.1.1.1) in an enzymatically active state, suggesting that the procedure does not denature proteins. The low cost, the ease with which the pH gradients are formed, their linearity, stability for a sufficient period to allow proteins to reach equilibrium and their subsequent recovery from buffer eluates should make the procedure interesting for electrofocusing of proteins.     

Nonamphoteric isoelectric focusing: II. Stability of borate-glycerol pH gradients in recycling isoelectric focusing

By complexing polyols with borate in recycling isoelectric focusing and by varying the ratio of polyol to borate over the useful pH range of 4.0-6.0, it is possible to control pH. Twelve solutions of 0.1 M boric acid and varying glycerol concentration were used to vary pH in a twelve-compartment commercial recycling isoelectric focusing (RIEF) system. Various concentrations of boric acid were tested as anolyte, and various Tris(hydroxymethylamino)methane-borate buffer systems were tested as catholyte. Electroosmosis, hydrogen ion flow, and fluid balancing were characterized in two glycerol gradients; one was maintained at 0.06 pH/fraction and the other at 0.12 pH/fraction. In the latter case, ovalbumin (pI4.70) migrated to the pH 4.61 and 4.72 compartments. It is concluded that the borate-glycerol system can be adequately stabilized in RIEF for isoelectric purification of certain proteins.     

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.     

Shifts of isoelectric points between cellular and secreted antibodies as revealed by isoelectric focusing and immobilized pH gradients

Charge microheterogeneity of monoclonal antibodies, as revealed by isoelectric focusing in carrier ampholytes, has been known for a long time. Here we demonstrate, in the case of monoclonals against the gp-41 of the HIV-1 virus, that this heterogeneity is already present within the cell sap of hybridoma cells during antibody synthesis. When the monoclonals are secreted extracellularly, the same isoelectric point (pI) spectrum is maintained, but there is marked redistribution of the relative isoform abundance towards the lower pI components. This suggests in vivo processing of such forms, possibly via glycosylation or deamidation. The secreted antibodies are also analyzed by immobilized pH gradients (IPG), where they demonstrate an even more extensive heterogeneity, due to the marked increment in resolving power. Single bands are purified by preparative IPGs in a multicompartment electrolyzer and are shown to be stable with time. Thus, artefactual heterogeneity produced by the focusing technique is completely excluded and cellular processing is clearly established.     

Electrophoretically silent hemoglobin mutants as revealed by isoelectric focusing in immobilized pH gradients

The applications of isoelectric focusing in immobilized pH gradients to the analysis of (i) human hemoglobin mutants, (ii) animal hemoglobin mutants (from cattle, sheep, dog and mouse), and (iii) tryptic digests of alpha and beta chains, are discussed and evaluated. Immobilized pH gradients appear to be an excellent tool for screening of genetic polymorphism and for detecting "silent mutants", i.e. those substitutions involving amino acids with nonionizable side chains. At present, not even capillary zone electrophoresis, claimed to have a resolving power equivalent to 1 million theoretical plates, has shown a resolution capability comparable to that of immobilized pH gradients, at least in the field of protein separation.     

pH transients during salt removal in isoelectric trapping separations: a curse revisited

The pH transients that occur during isoelectric trapping separations as a result of the removal of nonampholytic ionic components have been re-examined. Salts containing strong electrolyte anions and cations, both with equal and dissimilar mobilities, have been studied using anodic and cathodic buffering membranes whose pH values were both equidistant and nonequidistant from pH 7. The direction and magnitude of the pH transient (acidic or basic) was found to depend on both the mobilities of the anion and cation (mu(anion)/mu(cation)) and the pH difference between pH 7 and the pH of the buffering membranes (|pH(memb) (anodic) - 7|/|7 - pH(memb) (cathodic)|). When |pH(memb) (anodic) - 7|/|7 - pH(memb) (cathodic)| = 1, mu(anion)/mu(cation)<1 leads to an acidic pH transient, mu(anion)/mu(cation) = 1 eliminates the pH transient and mu(anion)/mu(cation)>1 leads to a basic pH transient. When mu(anion)/mu(cation) = 1, |pH(memb) (anodic) - 7|/|7 - pH(memb) (cathodic)|<1 leads to a basic pH transient, |pH(memb) (anodic) - 7|/|7 - pH(memb) (cathodic)| = 1 eliminates the pH transient and |pH(memb) (anodic) - 7|/|7 - pH(memb) (cathodic)|>1 leads to an acidic pH transient. By selecting appropriate anodic and cathodic buffering membranes to adjust the |pH(memb) (anodic) - 7|/|7 - pH(memb) (cathodic)| value, pH transients caused by dissimilar anion and cation mobilities can be avoided.     

Microheterogeneity of human serum transferrin: a biological phenomenon studied by isoelectric focusing in immobilized pH gradients

The heterogeneity of human transferrin results from (i) differences in iron content, (ii) genetic polymorphism and (iii) differences in the carbohydrate moiety. This article primarily deals with the last phenomenon, the microheterogeneity of human transferrin. Owing to the comparatively simple carbohydrate structure of human transferrin and the high resolving power of isoelectric focusing in immobilized pH gradients, microheterogeneous forms of transferrin can be separated. Differences between samples can be quantitated by crossed immunoelectrophoresis. Examples of the differences between the microheterogeneity patterns of transferrin in several biological fluids and the changes that can be observed in diseases such as rheumatoid arthritis, idiopathic hemochromatosis and Kahler's disease are presented. Special attention has been focused on changes occurring during pregnancy.     

Subtypes of the human complement component C3 revealed by isoelectric focusing in immobilized pH gradients

The phenotyping of the third component (C3) of human complement has been performed by isoelectric focusing in immobilized pH gradients followed by immunoblotting on nitrocellulose filter membrane. This powerful technique reveals variations of C3* and C3*F alleles not detected by agarose electrophoresis. The limits of the resolving power of isoelectric focusing in immobilized pH gradients for C3 analysis are shown to depend upon the high molecular weight of this protein. The notion of “suptypes” is discussed. Finally, the importance of subtyping for medical applications and for determination at the molecular level of interacting protein mechanisms is underlined.     

Microheterogeneity of human plasma lecithin: cholesterol acyltransferase examined by isoelectric focusing in immobilized pH gradients

The microheterogeneity of highly purified human plasma lecithin:cholesterol acyltransferase (LCAT) has been examined by electrophoresis in immobilized pH gradients in Immobiline-polyacrylamide gels of the pH ranges 4-7 and 4.2-4.9. Seven isoforms were obtained with LCAT isolated from pools of normal plasma. Using this technique the apparent pI values at 15 degrees C for the isoforms in the pH 4.2-4.9 gradient were 4.37, 4.42, 4.48, 4.53, 4.60, 4.67 and 4.74. (SD = +/- 0.03 for all). The most intensely stained band in the isoform pattern corresponded to the isoform with a pI value of 4.48.     

Preparative-scale fractionation by isoelectric trapping under nondenaturing conditions: separation of egg white protein isoforms on a modified Gradiflow unit

pH-biased isoelectric trapping was used to separate proteins from egg white at the preparative level (80 mg), into discrete protein fractions based on isoelectric point. The problems of isoelectric precipitation that are common for the separation of complex protein mixtures under isoelectric conditions were mitigated by using single-component isoelectric buffers within the sample separation compartments. This combined with the mild process conditions of the Gradiflow unit that was modified for binary isoelectric trapping separations, ensured that biological activity was maintained. This was verified by measurement of the trypsin protease inhibitory activity of the extract and separated fractions. Furthermore, the high resolving power of this system under preparative conditions was demonstrated by separation of three protein isoforms using isoelectric membranes with differences of 0.025 pH units from each other.     

Determination of the operational pH value of a buffering membrane by an isoelectric trapping separation of a carrier ampholyte mixture

The operational pH value of a buffering membrane used in an isoelectric trapping separation is determined by installing the membrane as the separation membrane into a multicompartmental electrolyzer operated in the two-separation compartment configuration. A 3相似文献   

Charge heterogeneity of recombinant pro-urokinase and urinary urokinase, as revealed by isoelectric focusing in immobilized pH gradients

When analysing homogeneous preparations of recombinant pro-urokinase and urinary urokinase by isoelectric focusing (IEF) in immobilized pH gradients, an extreme charge heterogeneity was detected (at least ten major and ten minor bands in the pH range 7–10). This extensive polydispersity was not caused by different degrees of glycosylation, or by IEF artefacts, such as binding to carrier ampholytes or carbamylation by urea. A great part of this heterogeneity could be traced back to the existence of a multitude of protein molecules containing Cys residues at different oxidation levels (-SH, -S-S-, even cysteic acid). Owing to the very large number of Cys residues in pro-urokinase (24 out of a total of 411 amino acids) and to the relatively high pI of its native forms (pI 9.5–9.8; the native form is believed to contain all Cys residues as -S-S- bridges), the presence of SH or cysteic acid residues would increase the negative surface charge, as even SH groups would be extensively ionized. In pro-urokinase, part of the heterogeneity was also due to spontaneous degradation to urokinase and possibly also to cleavage into lower-molecular-mass fragments. When all these causes of heterogeneity were removed, the pI spectrum was reduced to only four, about equally intense, bands. The cause of this residual heterogeneity is unknown.