Higher glycation of beta L- and beta S-crystallins in the anterior lens cortex and maximum glycation of gamma-crystallins in the bovine lens nucleus, demonstrated by frozen sectioning, isoelectric focusing and lectin staining

The aim of the current study was to demonstrate glycation of beta L-, beta S- and gamma-crystallins in the young bovine lens. To establish which of the crystallins are glycated and where they are located in the lens, we carried out microsectioning of the lens, followed by isoelectric focusing (IEF). Four bovine lenses of 1.183 +/- 0.070 years were frozen-sectioned into equator and 11 layers. Water-soluble crystallins were separated by IEF and stained: (1) with Coomassie brilliant blue for proteins; (2) with the lectin concanavalin A, followed by horseradish peroxidase and diaminobenzidine, for glycated proteins. Experiments were performed with crystallins and proteins in native form, in the absence of denaturants. The crystallins were separated by IEF into alpha-crystallins of high molecular weight (HM), alpha L-, beta H-, beta L-, beta S- and gamma-crystallins. In the lectin staining experiments, only HM, beta L-, beta S- and gamma-crystallins were positive, whereas the alpha L- and beta H-crystallins were negative. Contrary to the glycated gamma-crystallins in the lens nucleus, the beta S- and beta L-crystallins were predominantly glycated in the anterior cortex and to a somewhat lower extent also in the posterior cortical regions. The degree of glycation (total densitometric readings of lectin-stained bands/Coomassie-blue-stained bands) is as follows: total gamma-crystallins 2.44, beta S-crystallins 0.77 and beta L-crystallins 0.28. Though glycation in the bovine lens is very low, lectin staining is sufficiently sensitive to detect the various glycated crystallins. The degree of glycation of gamma-crystallins was 3 times higher than that of beta S-crystallins and 9 times higher than that of beta L-crystallins.     

SS-interchanged and oxidized isomers of bovine serum albumin separated by isoelectric focusing

Column isoelectric focusing separates commercial bovine serum albumin in 5 fractions with isoionic points in the vicinity of that of mercaptalbumin (pI 5.24). About 20% of the bovine albumin have isoionic points higher than mercaptalbumin and are split into two fractions, both recognized as SS-interchanges isomers: (1) pI 5.39 is the "aged" albumin described by Nikkel and Foster (1971, Biochemistry 10, 4479); (2) pI 5.45 represents a further degree of SS-interchange, catalyzed by small amounts of cysteine in the solution ('cysteine-aged' albumin). In 6 M urea the "cysteine-aged" albumin is electrofocused to the same pH value as mercaptalbumin. In 6 M urea 40% of commercial albumin focuses in 3 fractions with isoionic points lower than mercaptalbumin. This percentage will increase during incubation at oxidizing conditions ("oxidized" albumin). Electrofocused in water the oxidized fractions have isoionic points at pI 5.28, 5.18 and 5.12, respectively. The shifts in isoionic point of the "oxidized" albumins are caused by irreversible changes in the primary structure. Although the free SH group of albumin is oxidized during the oxidation reaction, the observed changes in isoionic points are caused by modifications of some other amino acid residues. Both "cysteine-ageing" and "oxidation" are inhibited by alkylation of the SH group. "Cysteine-ageing" is furthermore inhibited when the bovine albumin is "oxidized".     

A comparative study of vertebrate eye lens crystallins using isoelectric focusing and densitometry

1. The crystallin proteins of numerous species belonging to different classes of vertebrates have been studied. 2. Species-specific crystallin patterns are revealed which unequivocally characterize the different species. 3. A marked variability in the number and percentage of alpha-, beta- and gamma-crystallins were found in the various species. 4. The gamma-crystallin family, with a meagre number of common bands, has proved to be most representative of the species. The beta-crystallins, with their greater number of common bands, have been best preserved throughout vertebrate evolution. 5. From the similarity coefficient matrix a dendrogram is drawn up, a visual phylogenetic summary of the interrelationships between the vertebrates considered. 6. In the Discussion, other aspects are considered, such as lens morphology, functionality, animal age, post-synthetic modifications and genetic factors.     

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.     

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.     

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.     

Study of haptoglobin-hemoglobin complexes by titration curves, capillary electrophoresis and capillary isoelectric focusing

A novel method is described for monitoring complex formation between macromolecules, based on combined isoelectric focusing-electrophoresis in capillaries. The example studied is the binding of serum haptoglobin (Hp) to hemoglobin (Hb). A known amount of Hb is focused in a capillary in a pH 6-8 range (pI of Hb = 7.0) and thus kept temporarily "immobilized" in the electrophoretic chamber. Subsequently, increasing amounts of ligand (Hp) are loaded cathodically and allowed to sweep past the focused Hb zone. As the complex formed has a pI value well-outside the bounds of such a pH gradient (the 1:1 molar Hb-Hp complex has a pI of 5.5, the 1 to 1/2 molar Hp-Hb complex has a pI of 5.0) it escapes immobilization and moves past the detector window, where it is monitored and quantified. Since the detector is set at 416 nm, where only Hb absorbs, and since the molar extinction coefficient of Hb is well known, it is quite easy to calculate the molar amount of Hb bound to the complex. As an additional check, the amount of unreacted Hb can now be mobilized by disrupting the pH gradient and allowing this residual free Hb to also reach the detector and be quantified. The method is easy, fast, simple and fully automated and thus could represent a valid alternative to existing methods in clinical chemistry for quantifying the amount of Hp in human sera in pathological conditions, such as hemolytic anemias and transfusion reactions.     

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.     

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.     

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.     

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.     

Subtyping of group-specific component and protease inhibitor by rapid isoelectric focusing on PhastSystem

A rapid method on PhastSystem was used to investigate the distribution of group-specific component (GC) and protease inhibitor (PI) subtypes and their gene frequencies from 190 unrelated healthy donors of the Han population in Beijing. Laboratory-made gels (pH 4.5-5.4 and pH 4.2-4.9) were used for analysis of GC and PI, respectively. Sample loading was 1.5 microliters. The separation and visualization time was 0.5 h in each. Gene frequencies were as follows: GC*1F = 0.4891, GC*1S = 0.2432, GC*2 = 0.2678; rare GC variants were discovered in seven cases. The results for PI were: PI*M1 = 0.7542, PI*M2 = 0.1808, PM*M3 = 0.0650. Good agreement between the observed and expected values in both GC and PI subtyping (for GC, sigma chi 2 = 1.4043, 0.7 < P < 0.8; for PI, sigma chi 2 = 1.1233, 0.7 < P < 0.8) was obtained.     

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).     

Quantitative analysis of ovalbumin by thin-layer isoelectric focusing (author's transl)

A new method for quantitation of ovalbumin by thin layer isoelectric focusing in polyacrylamide gel is described. This technique separates ovalbumin from avian-magnum homogenates or egg white and allows the quantitative determination of each ovalbumin fraction. Considering the very small amount (7 mul) of sample needed for an analysis, the sensitivity and the reproducibility of the proposed method are good. The application of this technique to quail egg white ovalbumin analysis leads to the characterization of four fractions A3, A2, A1 and A0 containing, respectively, 0, 1, 2 and 3 atoms of phosphorus per mole and with isoelectric points that range from 4.95 (A3) to 4.65 (A0).