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 simplified procedure for purification of cytochrome P-450 by preparative ampholine gel for isoelectric focusing

The purification process for cytochrome P450 is very complicated, involving five or more column chromatography steps for the final preparation. This paper describes a reduction in the number of the steps; it can be easily purified from pig testis microsomes with improved the yield. As the first step, DEAE-Toyopearl column chromatography is performed only once and then, as the second step, the partially purified cytochrome P450 is completely purified by a preparative Ampholine PAG-plate Gel for Isoelectric Focusing. The combination reduced the purification to a two-step procedure.     

Preparative isoelectric focusing in ampholine electrofocusing columns versus immobiline polyacrylamide gel for the purification of biologically active leukoregulin

Preparative vertical and rotating horizontal (Rotofor) ampholine column and immobiline flat bed polyacrylamide gel isoelectric focusing were evaluated for the isolation of the biologically active acidic form of leukoregulin, a 50,000-Da glycoprotein lymphokine with tumor growth inhibitory activity. Leukoregulin secreted by normal human lymphocytes was concentrated by 10,000 nominal molecular weight size exclusion ultrafiltration and by DEAE anion exchange chromatography using step elution with 0.02 M Tris-HCl: 0.1 M NaCl, pH 7.4. Preparative isoelectric focusing was carried out in a 110-ml vertical column containing 1% ampholines in a pH 4-6 gradient at 15 W constant power for 16-18 h, in a Rotofor 55-ml horizontal column containing 2% ampholines in a pH 4-6 gradient at 12 W constant power for 4-6 h, or in an immobiline pH 4.5-6.5 gradient within a 5% polyacrylamide 120 X 110 X 5-mm flat bed gel at 3 W constant power for 16-18 h. Recovery of biologically active leukoregulin from the vertical and horizontal ampholine columns was similar. The pH 4.9-5.2 fractions from the Rotofor ampholine column contained 4-7% and the fractions from the immobiline gel contained 4% of the leukoregulin activity applied to the electrofocusing column or gel, respectively. Analytical immobiline isoelectric focusing of the leukoregulin in the pH 4.9-5.2 fractions from the Rotofor column demonstrated that a single silver staining band with a pI of 5.1 can be obtained by this rapid method of preparative isoelectric focusing.     

Ultrathin-layer isoelectric focusing in polyacrylamide gels on cellophane.

A method of ultrathin-layer isoelectric focusing in 0.12-, 0.24-, or 0.36-mm polyacrylamide gel layers polymerized on a sheet of cellophane as support is deseribed. The gel adheres firmly to the cellophane during all operation steps, is protected from fracture, and can be handied very conveniently. Resolution is markedly improved in ultrathin gels in comparison with the conventional 1- to 2-mm-thick gels. Staining and destaining are completed in a substantially shorter time than so far achieved. The ultrathin gels can be easily dried on the cellophane, a perfectly transparent record being obtained for future reference and for densitometric evaluation. Results are presented for a number of commercial proteins and legume seed proteins. The advantages of ultrathin-layer isoelectric focusing are discussed.     

Microheterogeneity of mammalian haptoglobins in isoelectric focusing.

1. Human haptoglobin type 1-1, porcine haptoglobin, and equine haptoglobin were isolated and purified. 2. These haptoglobins were similar in polyacrylamide gel electrophoresis and in subunit structure but showed microheterogeneity in isoelectric focusing. 3. Isoelectric points of human haptoglobin as determined with photopolymerized gels were found to be 4.03-4.24, of porcine haptoglobin 4.0-4.30, and of horse haptoglobin 3.80-4.15, respectively. 4. Results obtained with chemically polymerized gels were 0.08-0.3 pH units higher. 5. Examined haptoglobins differed also in the ability of complex formation with hemoglobin, in sialic acid content and in antigenic specificity.     

Methods of isoelectric focusing

The method of isoelectric focusing has been avoided by many workers because of expense, technical difficulty, and problems of interpretation. Inexpensive, easy, and interpretable results are possible using equipment and reagents commonly available. Methods which allow these results are presented and explained.     

Isoelectric focusing in layers of granulated gels. II. Preparative isoelectric focusing.

A method for preparative isoelectric focusing of 0.1-10 g amounts of proteins is described. For anticonvective stabilization of the pH gradient, layers of granulated gels (E.G. Sephadex or Bio-Gel) of variable length, width and thickness were used either on glass plates or in troughs. Load capacity, defined as the amount of protein per ml gel suspension, was determined to be 5-10 mg per ml for total protein, irrespective of the pH range of the carrier ampholytes. For single proteins load capacities of 0.25-1 mg per ml were found for pH 3-10 carrier ampholytes, and 2-4 mg per ml for narrow pH range ampholytes. Experiments on a quartz plate followed by densitometric evaluation in situ at 280 nm have demonstrated that it is possible to proceed from analytical thin-layer isoelectric focusing to preparative separations without loss of resolution, just by changing the dimension of the gel layer and increasing the protein load. Improved resolution which facilitates isolation of isoelectrically homegenious components could be achieved on a 40 cm long separation distance. The geometry of a layer is favourable to heat dissipation and this permits the use of high voltage gradients. Recovery of the focused proteins is high an elution simple. The efficiency of the method is illustrated by examples showing separations of single proteins and protein mixtures.     

Electrofocusing of heparin: fractionation of heparin into 21 components distinguishable from other acidic mucopolysaccharides.

Twenty-one fractions have been demonstrated in each of 15 different commercially available heparins subjected to electrofocusing. These fractions show a molecular-weight range from 3000 to 37,500 with a constant interval between molecular weights. Degradation of each fraction by purified enzymes of Flavobacterium heparinum yielded identical end products, suggesting chemical identity. Only fractions with a molecular weight of 7000 and up had significant anticoagulant activities. The phenomenon of electrofocusing of mucopolysaccharides is dependent upon pH, molecular weight, and ampholyte availability. Chemical composition of the mucopolysaccharide is also an essential factor since N- and O-desulfation of heparin markedly changed the focalization pattern. The pattern produced when heparin is subjected to electrofocusing is not duplicated by any other naturally occurring acidic mucopolysaccharide tested. Heparitin sulfate D shows some similarities to heparin and it is probable that heparitin sulfate D is a normal contaminant of heparin preparations (this assumption is supported by molecular-weight and anticoagulant activity determinations). The technique is specific and reproducible and unequivocally distinguishes heparin from other acid mucopolysaccharides.     

Preparative isoelectric focusing in agarose

The use of agarose gels as supporting media for flat-bed preparative isoelectric focusing was applied to the fractionation of serum proteins in the pH range 3.5–6, and red cell hemolysates in the pH range 3–8. The agarose gels are easy to prepare, give linear pH gradients, and do not appear to produce molecular sieving effects. Up to 1 g serum proteins can be loaded on the gels, with recoveries between 68 and 82%. Nucleoside phosphorylase from red cell lysates was recovered with 76% yield, indicating that no appreciable denaturation of this enzyme had occurred. Preparative isoelectric focusing in agarose gels provides a useful alternative to existing techniques of preparative isoelectric focusing in sucrose gradients or granulated gels.     

Lymphocytosis produced by heparin and other sulphated polysaccharides in mice and rats

Lymphocytosis has been produced in mice and rats using heparin and other sulphated polysaccharides. Two hours after heparin (50 mg/kg ip) the concentration of lymphocytes in mouse blood increased threefold; it fell to control levels after 9 hr. The height of the lymphocytosis was related to the dose of heparin injected. After intravenous heparin in rats there was a comparable lymphocytosis maximal 1 hr after injection. In mice other negatively charged sulphated polysaccharides also caused lymphocytoses, which were greater and occurred later with increase in molecular weight of the substance injected. Results in rats were similar. No lymphocytosis followed the injection of negatively charged phosphated dextrans, positively charged DEAE dextran, or neutral dextran. There was no correlation between the effect of these substances on lymphocytes and their effect on coagulation of blood, hepatic phagocytosis, or the immune response to sheep red blood cells.     

Flat bed gel isoelectric focusing of neuraminidases.

Technical details and preliminary results are described for a new flat bed gel isoelectric focusing method to study charge heterogeneity in neuraminidases. The method combines simplicity and high resolving power with the capacity to analyse multiple samples in a single experiment. Isoelectric points are obtained which agree favourably with those gained by other methods.     

Heterogeneity of staphylococcal enterotoxin A on isoelectric focusing and disc electrophoresis.

Heterogeneity of purified staphylococcal enterotoxin A, obtained from a culture supernatant of Staphylococcus aureus, strain 13N-2909, was demonstrated by isoelectric focusing. The toxin was composed of three immunologically identical fractions with isoelectric points of 6.5, 7.0 and approximately 8.0. Heterogeneity of the toxin was also shown by disc electrophoresis. At pH 8.0 and 9.4 two major bands and a faint minor band were observed, while at pH 4.3 only one band was observed. The faster-moving band for the anode in disc electrophoresis at pH 9.4 was found to correspond with the pI 6.5 component from isoelectric focusing, while the slower-moving band corresponded with the pI 7.0 component. From the results of the electrophoretic migration tests of the toxin, the components corresponding to the two major bands found in disc electrophoresis at pH 9.4 were considered to be charge isomers.     

Binding of polyanions to carrier ampholytes in isoelectric focusing.

The binding of carrier ampholytes to polyanions is markedly pH-dependent: it is very strong at pH 3, rather weak at pH 5 and abolished at pH 7. Binding is affected by the type of negative charge, its density and spatial orientation on the polyanion. On the basis of the type of negative charge, the binding strength decreases in the following order: polyphosphate greater than polysulphate greater than polycarboxylate. Given the same type of negative charge, the binding is dependent on charge density and its space orientation: thus polyglutamic acid forms stronger complexes than polygalacturonic acid. The minimum length of the polyanion eliciting a measurable binding appears to be of the order of about six negative charges, as demonstrated with hexametaphosphate.