Use of isoelectric focusing in studying the composition of allergens from mites

The method of the isoelectric separation of allergens isolated from Dermatophagoides ticks with the use of isoelectrofocusing has been improved. 10-12 protein fractions with isoelectric points from 3.50 to 6.55 have been isolated. Allergens obtained from D. farinae and D. pteronyssinus grown on two different nutrient media have been found to contain common protein components. In D. pteronyssinus allergen the presence of a greater number of protein fractions has been noted in the region of pH 5.25-6.00 than in D. farinae allergen.     

Device for miniscale isoelectric focusing of proteins

A simple device is developed for mini-scale electrofocusing of proteins. The main apparatus consists of only two glass tubes joined by a small tubing. No special cooling system, stopcocks, stands, etc., are needed. Even the need for a peristaltic pump for fractionation is eliminated. The apparatus does not require very high voltages and the amount of Ampholines is drastically reduced. The model can be used for analytical as well as semi-quantitative purposes.     

Parvalbumins for calibrating acidic isoelectric focusing gels

Partially purified fish and amphibian parvalbumins are compared to several proteins commonly used in commercial standard mixtures for calibrating isoelectric focusing gels. Parvalbumins are proffered as useful standards for acidic ranges on the basis of conformity to a set of five criteria.     

A modified column for sperm isoelectric focusing

A modified U-shaped column is described for efficient isoelectric focusing of spermatozoa, other cells, and protein. The washed spermatozoa of the rabbit showed a PI of 4.4. After sonication, heads and tails focused at same pH, indicating similar and equal charge.     

High-molecular-weight carrier ampholytes for isoelectric focusing of peptides

A method is described for the synthesis of high-molecular-weight carrier ampholytes for preparative isoelectric focusing of peptides. A giant polyethylene imine ($\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{40 000–60 000}$) is mixed with a linear gradient of acrylic acid in a flow-through system and let to react at 80°C for 70 h. Giant carrier ampholytes ($\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{range}\text{50 000–90 000}$) are thus obtained. These compounds interact very strongly among themselves, probably not by hydrogen bonds or hydrophobic interactions but ionic bonds. In fact, the aggregates are split by high salt (NaCl) or by zwitterionic compounds (Gly, taurine) or at acidic or alkaline pHs. They appear to interact only weakly and reversibly with proteins and no interactions are apparent with model dipeptides (His-Ser, His-Met, His-Phe and His-Lys).     

Mathematical model for transient isoelectric focusing of simple ampholytes

A mathematical model describing transient processes in isoelectric focusing (IEF) of L biprotic ampholytes is presented. The model is a generalization of our previous research on steady slate in IEF and consists of L nonlinear partial differential equations coupled with 2L+2 algebraic equations. Constraints imposed by the mode of operation, viz., constant current. voltage or power, are described. Due to the nonlinearity of the equations, analysis of the model requires computer simulation. Model equations suitable for computer implementation are derived.     

Stable storage conditions of Immobiline chemicals for isoelectric focusing

Most of the problems connected with the use of the Immobiline chemicals (a set of six, non-amphoteric, acrylamido buffers having pK values in the pH 3.5-9.5 interval) can be attributed to the alkaline species (with pK values 6.2, 7.0, 8.5 and 9.3). These compounds, to varying degrees are subjected to two degradation pathways: (a) hydrolysis of the amido bond, producing free acrylic acid and a diamine, the latter unable to be incorporated into the polyacrylamide matrix; (b) spontaneous auto-polymerization, producing a number of oligomers up to n-mers, able to aggregate and precipitate large proteins. Storage of their water solutions as frozen aliquots, a method widely employed, only partially alleviates the problem. Addition of trace-amounts of inhibitors, as lately adopted by the manufacturer, could only reduce the problem of auto-polymerization, but not block the hydrolysis of the amido bond. A new solution has been found, which abolishes both phenomena: storage in n-propanol. As demonstrated by gas chromatography, HPLC analyses and two-dimensional separations of complex samples, storage in organic solvent completely abolishes both hydrolysis and auto-polymerization and allows production of highly reproducible focusing patterns.     

Polymorphism of the haptoglobin peptides by isoelectric focusing electrophoresis and isoelectric point determinations

Summary In this investigation, the authors developed two new procedures: a micromethod for haptoglobin purification and the isoelectric focusing electrophoresis on slab polyacrylamide gel for peptide subtyping. These technics are adapted to the study of large sample series for population genetic surveys. The improvements obtained enabled us to disclose in an easy and highly reproducible way the Hp and ₂ peptide chains. Electrophoretic separation of the ₂ FS, SS, and FF chains were greatly improved. Their frequencies estimated in a sample already investigated by the conventional PAGE presented higher values than previously described. New Hp and ₂ mutants were also detected. For the first time, isoelectric points of the Hp peptides were determined; the values obtained are discussed with regard to their known amino acid structure.     

Iodine stain for detection of peptides after isoelectric focusing

Iodine stain is used for the detection of peptides after isoelectric focusing has been developed. Ultrathin gels (240–360 μm) are cast and, after focusing, dried at 110°C on a filter paper sheet. The paper-pasted gel is then exposed to iodine vapors for a few seconds to a few minutes, depending on the peptide load. White peptide zones are visivle on a brown, uniform background. The reaction is fully reversible and can be used also for small-scale preparative purification of peptides. Better than 80% recoveries of peptide from the gel can be obtained by elution in 80% acetic acid.     

Studies on the structure of sphingomyelinase. Amino acid composition and heterogeneity on isoelectric focusing.

Sphingomyelinase, purified to apparent homogeneity from human placenta, is an acidic protein, as judged from its amino acid composition and by isoelectric focusing of the carboxymethylated protein. The amino acid composition is characterized by an approximately equal content of hydrophobic and polar amino acid residues. The reduced-alkylated polypeptides were separated into two groups. Most of the polypeptides were heterogeneous with pI values of 4.4-5.0, but an additional more minor component was observed at pI 5.4. Liquid isoelectric focusing resolved the purified enzyme into a single major component (pI 4.7-4.8), a minor component (pI 5.0-5.4) and a plateau region of activity (pI 6-7). On thin-layer isoelectric focusing, the protein profile obtained from each of these regions was the same. In addition, the substrate specificity, Km values and effect of inhibitory substances were identical. We conclude that sphingomyelinase is an acidic, microheterogeneous protein that likely exists as a holopolymer of a single major polypeptide chain. the heterogeneity of the intact protein on isoelectric focusing appears to reflect this microheterogeneity, which is influenced by a tendency to associate with itself and with detergents such as Triton X-100.     

Use of isoelectric focusing to define major histocompatibility complex class I polymorphism in goats

We have used biochemical methods to extend and improve serological class I typing using a panel of 77 Swiss goats of the Saanen breed, comprising dam-offspring combinations from six half-sib sire families and several unrelated animals. Of these animals class I molecules were precipitated from cell lysates with the mAb B1.1G6 and HC10. Immunoprecipitates were analysed by SDS-PAGE and 1D-IEF. There was a good agreement between class I serological types and IEF banding patterns. We have identified three new class I specificities and subdivided the Bel 7 specificity. IEF has enabled us to make planned immunizations to produce antisera to the new specificities. New evidence for the expression of a second class I locus product in the Be7 haplotype has been found.     

Gel isoelectric focusing of wheat alcohol dehydrogenase

Summary Alcohol dehydrogenase (ADH) of different hexaploid wheat subspecies and varieties was investigated by isoelectric focusing in polyacrylamide gels. With this technique six ADH isoenzymes can be separated, while by the standard electrophoretic technique only three are visible. The ADH pattern revealed by isoelectric focusing is in full accordance with the hypothesis that the active ADH isozymes in hexaploid wheat are dimers composed of six possible combinations of subunits coded by triplicate structural genes.     

Identification of abnormal hemoglobins by isoelectric focusing

Using IEF on slabs of acrylamide gel was adapted for screening of abnormal Hemoglobins which are at the same level by electrophoresis on cellulose acetate strips. This method is fast, inexpensive and allowed the simultaneous analysis of 70 samples of whole blood. The characterization technique of IEF allowed us to distinguish some rare variants like Hb O Arab, HbD and T gamma in B 0-thalassemia.     

Use of the desirability function method for assessing water quality by periphyton composition

Water quality generalized index according to periphyton community obtained by the method of desirability function has been used to estimate the Moscow River pollution. To reveal the indicator species by this method one-year observation period is required. The indicator species list presented here can be recommended for the calculation of generalized index according to the periphyton data for the Moscow River.     

A thin-gel isoelectric focusing method for quantitation of protein S-thiolation

A thin-gel isoelectric focusing method has been developed for analysis of protein S-thiolation (formation of mixed disulfides with low molecular weight thiols). The method is rapid and it can be used with 3 to 5 micrograms of a pure protein, or 15 to 20 micrograms of tissue extract protein. It is possible to detect a modification of the protein sulfhydryl by either charged or uncharged thiols, and to determine the quantity of different S-thiolated protein species in a modified sample. The method was used to quantitate the amount of S-thiolation of phosphorylase b in a reaction with oxidized glutathione that produced four S-thiolated forms of the enzyme. The method was also used to detect S-thiolation of two proteins in a cardiac tissue extract treated with diamide. One of the protein bands was shown to be S-thiolated with both cysteine and glutathione, while the other band was S-thiolated only with glutathione.     

Some optical properties of carrier ampholytes for isoelectric focusing.

Some optical properties of carrier ampholytes are herewith described. Newly synthesized Ampholines do not possess asymmetric carbon atoms. pH 3–5 and pH 8–10 ranges, synthesized before 1970, rotate the plane of polarized light since they were “reinforced” with glutamic and aspartic acid, in the acidic side, and lysine in the basic region.The various pH ranges possess characteristic chromophores, whose absorbance is strongly pH dependent. These chromophores, when excited at appropriate wavelengths, exhibit a fluorescence emission spectrum, typically reproducing the pH dependence of the corresponding uv spectra.The optical properties here described can be useful in studying Ampholinesmacromolecules interactions. Due to the widespread use of optical scanning in situ of focusing systems, care has to be taken not to mistake Ampholine peaks at 285, 310, 315, 340, and 365 nm for the substance under study.