Subtyping of transferrin by isoelectric focusing in immobilized pH gradients

Isoelectric focusing in immobilized pH gradients ranging from pH 5.05 to 5.60 was used to study the distribution of transferrin (Tf) subtypes and their gene frequencies from 188 unrelated healthy donors of the Han population in Beijing. Six phenotypes (TfC1, TfC2, TfC1C2, TfC1C3, TfC1Dchi and TfC2Dchi) were detected and Tf*C3 has never before been reported in the Han population. Gene frequencies were as follows: Tf*C1 = 0.7420, Tf*C2 = 0.2420, Tf*C3 =0.0027, Tf*Dchi = 0.0133. There is good agreement between the observed and expected values corresponding to the Hardy-Weinberg equilibrium (sum (chi2) = 0.9183, df = 2, 0.5 < P < 0.75). The allele frequencies for Tf*C1, Tf*C2 and Tf*Dchi agreed with those previously reported for the Chinese Han population.     

Theory and practice of isoelectric focusing of interacting systems

The theory of mass transport coupled to reversible protein interactions forms the basis for computer simulation of the isoelectric focusing behavior of several model systems. These include pH-dependent conformational transition, carrier ampholyte-induced interactions and protein-ligand interactions. The computational results compare favorably with experimental observations. In addition, a method is formulated for an isoelectric focusing procedure which enables determination of intrinsic ligand-binding constants for statistical binding of a charged ligand, binding to heterogeneous sites, and cooperative binding.     

pI-dependent isolation of antibody isoforms by semipreparative isoelectric focusing

Polyclonal antibodies were raised against peptides corresponding to residues 1-15, 469-483 and 933-951 of the rabbit skeletal muscle L-type calcium channel alpha 2/delta primary translation product, for use as topological probes. Immunocytochemical comparison of the abilities of the antibodies to bind to the alpha 2 and delta subunits in intact and detergent-permeabilised rat dorsal root ganglion cells enabled the membrane orientation of these regions to be established. The resultant data indicate that the regions containing residues 1-15 and 469-483 of the alpha 2 subunit, and residues 1-17 of the delta subunit, are exposed on the extracellular surface of the membrane, findings consistent with a model that proposes alpha 2 to be entirely extracellular.     

Purification of larval Taenia solium antigens by isoelectric focusing

By preparative isoelectric focusing in a rotating ampholine column, crude cystic membrane (M) or fluid (F) antigens of larval Taenia solium were each separated into 20 fractions. M fractions were less specific and sensitive than F fractions in detecting cysticercosis antibodies in pig serum. Among the F fractions, F15 showed the best potential to serve as a screening antigen. It contained 18 polypeptides, with pI 5.3-8.2 and a specific epitope of 25 kDa which was detected by immunoblotting. Although F15 showed slight cross-reactions with heterologous antisera in double-antibody IgG enzyme-linked immunosorbent assays (ELISA), it yielded the highest absorbance values when tested against homologous antisera. The antigen was used to screen sera samples from 4870 pigs slaughtered in Hong Kong and five other Chinese cities for cysticercosis antibodies by double-antibody ELISA, Falcon Assay Screening Test (FAST)-ELISA and enhanced chemiluminescent immunoassay. The results varied significantly between assays. However, the samples collected from Shenzhen yielded the highest positive rates. Enhanced chemiluminescent immunoassay based on camera-luminometry was found suitable for use under field conditions.     

New set-up for capillary isoelectric focusing in uncoated capillaries

Substituted aminomethylphenol dyes, low-molecular-mass isoelectric point (pI) markers and hemoglobin samples from normal individuals and diabetic patients were used to test a new set-up of capillary isoelectric focusing (cIEF) in uncoated capillaries. In previous cIEF methods, a mixture of sample components and carrier ampholytes was applied in the capillary and analyzed. In the new set-up a fractionated injection protocol is used to apply a 'sandwich' ampholyte-sample-ampholyte plug in the capillary for analysis. This new set-up allows the separation of amphoteric compounds having pI values outside the pH region of the ampholytes applied in the capillary with high precision. The high resolution power of this technique was proven with the analysis of hemoglobin variants.     

Preparative isoelectric focusing in multicompartment electrolyzers: novel, hydrolytically stable and hydrophilic isoelectric membranes

Preparative isoelectric focusing in multicompartment electrolyzers is based on the production of isoelectric membranes of precise isoelectric point, able to buffer at their pI value and to titrate proteins tangent to or crossing the membranes. Up to the present, such membranes have been based on polyacrylamide chemistry; acrylamide, however, is neither stable in acidic nor basic environments. We describe here novel membranes, produced with a unique monomer, N-acryloylaminoethoxyethanol (AAEE). Poly(AAEE) membranes are extremely stable to alkaline hydrolysis (500 times more stable than polyacrylamide) and even more hydrophilic than the latter matrix. This allows production of highly reproducible membranes (these do not change their pI with time, since no acrylic acid is produced by hydrolysis upon storage) which do not adsorb proteins by hydrophobic interaction.     

Recent developments in capillary isoelectric focusing with whole-column imaging detection

Capillary isoelectric focusing (CIEF) is a high resolution technique for protein separation. The on-column single point detector requires a mobilization step which lengthens the analysis time and causes an uneven resolution along the separation column. The real time and whole column imaging detection has been developed for performing CIEF without mobilization. Three types of imaging detection systems have been developed: optical absorption, refractive index gradient, and laser induced fluorescence. This technique provides a fast analysis speed (about 6 min) and a good resolution of 0.03 pH unit level. Using the absorption imaging detector, ampholyte-free IEF in tapered capillary is being demonstrated, which eliminates the interference of the expensive carrier ampholytes for protein detection in UV region. Recent advancements in this imaged CIEF technique as well as its applications are reviewed.     

Mapping of several abnormal hemoglobins by horizontal polyacrylamide gel isoelectric focusing

The relative mobilities of serveral human hemoglobin variants were studied by use of an isoelectric focusing method in a thin-layer horizontal polyacrylamide gel. The technic is described in detail and a preliminary mapping of these variants is presented. It appears that thin-layer gel isoelectric focusing is a method that is rapid, highly reproducible and easily applicable in laboratories concerned with the study of hemoglobinopathies.     

Characterization of serum cholinesterase variants by kinetic analysis and isoelectric focusing

Serum cholinesterase enzyme (E.C. 3.1.1.8) shows a considerable degree of genetically determined heterogeneity. In this communication we describe some kinetic properties of serum cholinesterase variants (Km and Vmax) from individuals predisposed to prolonged apneic periods after administration of the anesthetic, succinylcholine. Isoelectric focusing, in a narrow pH range (pH 4-6) of sera from normal and atypical phenotypes permitted the detection of differences in protein bands near the isoelectric pH of the enzyme.     

Rapid high-performance isoelectric focusing of monoclonal antibodies in uncoated fused-silica capillaries

Rapid (< 5 min) high-performance isoelectric focusing was performed in uncoated fused-silica capillaries to resolve isoforms of monoclonal antibodies and to determine their isoelectric points (pI). The methodology involved the use of a 32 cm (effective length 9 cm) x 50 microns I.D. uncoated capillary. (Hydroxypropyl)methyl cellulose was used as an additive to suppress analyte-wall interaction and to precisely control electroosmotic flow so that focusing and mobilization of focused zones past detector occur simultaneously. Urea was included in the separation medium to solubilize the antibodies that precipitated at their point of focusing. The methods with and without urea used ampholytes pH 5-8 to generate a demonstrate linear gradient between pH 5.4 and pH 7.2, based on the separation of various protein standards. Reproducibility [< 2% (R.S.D.)] of the migration times (corresponding to the detectable isoforms of the antibodies) was obtained by using two sets of reagents and capillaries on three consecutive days. pI values determined from day-to-day with a reference standard were shown to vary by only 0.01 pH unit. The described capillary isoelectric focusing methods provided a rapid, simple and reproducible way of monitoring micro-heterogeneity and pI of the murine monoclonal antibodies investigated.     

Adaptation of capillary isoelectric focusing to microchannels on a glass chip

As a first step toward adaptation of capillary isoelectric focusing (cIEF) to microchannels on a glass chip, we have compared the three most common mobilization methods: chemical, hydrodynamic, and electroosmotic flow (EOF)-driven mobilization. Using a commercial cIEF apparatus with coated or uncoated fused-silica capillaries, both chemical and hydrodynamic mobilization gave superior separation efficiency and reproducibility. However, EOF-driven mobilization, which occurs simultaneously with focusing, proved most suitable for miniaturization because of high speed, EOF compatibility and low instrumentation requirements. When this method was tested in a 200-micron-wide, 10-micron-deep, and 7-cm-long channel etched into planar glass, a mixture of Cy5-labeled peptides could be focused in less than 30 s, with plate heights of 0.4 micron (410 plates/s) upon optimization. For a total analysis time of less than 5 min, we estimate a maximum peak capacity of approximately 30-40. Interestingly, the order of migration was found to be reversed compared to capillary-based focusing.     

Simultaneous multiple analyte detection using fluorescent peptides and capillary isoelectric focusing

Von Frey filaments used for testing mechanical thresholds are mechanically unstable and their use is difficult to standardize. We have therefore constructed a hand-held electronic pressure algometer. The pressure algometer is connected to a computerized data collection system, allowing on-line display of the applied force as well as the application rate. Data stored on the computer can be replayed and further analyzed. Using this apparatus, we have measured the pressure-induced withdrawal thresholds in rats with surgically induced neuropathy. The probe, with a circular tip of 1.0 mm diameter, was applied manually with a pressure increasing by approximately 0.05 N/s. Presurgical thresholds were normally distributed with a mean of 0.415 N, showing no significant difference between paws. During 2 weeks after surgery, the thresholds of the operated side were significantly reduced (range, 0.209-0.318 N), while the thresholds of the non-operated side remained at higher values (range, 0.432-0.491 N). Thresholds of control rats without surgery were in the 0.380-0.520 N range, with no significant difference between paws. In an additional experiment it was shown that interobserver reliability was high, both between withdrawal threshold values obtained and between rates of application used. In conclusion, the electronic algometer allows standardization of testing, detailed documentation of each experiment and provides an objective and accurate method for measuring the reactions of test animals to mechanical stimuli.     

Non-native capillary isoelectric focusing for the analysis of the microheterogeneity of glycoproteins

The purpose of the present study was to compare two measures of Fe absorption, one from single meals and the other from daily diets. Ten ileostomy subjects were given the same low-fibre composite diet for all three meals each day for five consecutive days. After 3 weeks the experiment was repeated with a high-fibre diet. The morning meal constituted one-seventh of the total daily diet intake, the mid-day meal two-sevenths and the evening meal four-sevenths of the total daily diet intake. On days 4 and 5 of each diet period the morning meal was labelled with 55Fe and all three meals were labelled with 59Fe. The activities retained in the subjects 19 d later showed the Fe absorption from the low-fibre diet measured from the morning meals to be almost 80% greater than the average Fe absorption measured from all meals during the same 2 d. With the high-fibre diet the absorption from the morning meals was less than 50% greater than the average for all meals but the difference was not significant. We suggest that all meals of the day should be labelled with radioFe in order to avoid inflating the measures of Fe absorption.     

Development of crosslinked polyamines suitable for synthesizing complex ampholytes for isoelectric focusing

Commercially available ampholytes used in isoelectric focusing applications vary widely from source to source in their resolving power. This study was initiated to develop alternative ampholyte formulations for high resolution preparative and analytical isoelectric focusing. Initial IR spectroscopy studies showed that divalent acid esters would efficiently crosslink available polyamines with complete consumption of ester. Fast atom bombardment mass spectroscopic analysis of resulting crosslinked polyamines showed extensive structural heterogeneity of the resulting polyamine mixture. Conversion of the polyamine mixture to functional zwitterions using alpha,beta-unsaturated carboxylic acids yielded mixtures giving smooth pH gradients in acrylamide gel isoelectric focusing. Further analysis of these mixtures in immobilized pH gradients showed increases in heterogeneity of available carrier species over similar zwitterion mixtures made using only commercially available polyamine monomers. The mixtures were also more heterogeneous than commercially available ampholytes when analyzed by picric acid precipitation in immobilized pH gradients.     

Quantitation of glycated hemoglobins in human adult blood by capillary isoelectric focusing

A precise and reproducible method for assessment of glycated hemoglobin in human adult red blood cells is reported, based on capillary isoelectric focusing (IEF). In order to obtain baseline resolution between adult hemoglobin (Hb A) and its glycated form (Hb A1c), two species which differ by minute delta pI values, < 0.03 pH units, the following procedure was adopted: the focusing mixture consisted of 5% Ampholine, pH 6-8, 0.5% Pharmalyte, pH 3-10, 3% short-chain liquid polyacrylamide and an equimolar mixture of two "separators", 0.33 M beta-alanine and 0.33 M 6-aminocaproic acid. The last two compounds flatten the pH gradient in the pI region of the two Hbs, thus allowing full separation. Additionally, the Hb samples, instead of being pulse-loaded, are uniformly distributed in the background electrolyte. A longer capillary life-time is obtained if all nonbuffering ions are eliminated; thus, as catholyte, 50 mM Lys (pH 9.7) is utilized and as anolyte 50 mM acetic acid (pH 3.5) is adopted. The percentages of Hb A1c, as obtained by capillary IEF, are in good agreement (+/- 6%) with data obtained by one of the standard zone electrophoretic methods in clinical chemistry, i.e., the Helena REP Glyco gel system.     

Assay of trypsin activity by capillary isoelectric focusing with laser-induced fluorescence detection

Capillary isoelectric focusing is a highly effective method for the separation of proteins due to focusing as a function of their pI values in the separation process. This technique is also effective for certain types of peptides that focus well. Fluorescence labeling and subsequent detection by laser-induced fluorescence farther enhance the sensitivity of this technique. This paper demonstrates the utility of this technique in an enzyme assay. A synthetic nona peptide, H-Gly-Cys-His-Glu-Ala-Arg-Ala-Glu-Glu-OH, was labeled with an iodoacetyl derivative of Lissamine rhodamine B at the thiol group of the cysteine residue as a substrate for trypsin. Trypsin catalyzed the cleavage of the Arg-Ala bond of the labeled substrate, which focused at pH 4.8, and liberated a shortened, labeled product, H-Gly-*Cys-His-Glu-Ala-Arg-OH that focused at pH 6.9 (* indicates the label). The product peptide at 3-300 pM was determined with a relative standard deviation of 5.5% (n = 5) by fluorescence detection at 590 nm with excitation by a green line of He-Ne laser. Incubation of trypsin with the substrate for 10 min at 37 degrees C allowed the determination of 50-250 pg of trypsin, with a relative standard deviation of 5.3% (n = 5).