Microheterogeneity of human kininogen by isoelectric focusing and crossed immunoelectrophoresis.

LMW kininogen was isolated from whole human plasma by gel filtration on Sephadex G-200 (Kav 0.34) followed by DEAE-chromatography according to earlier established methods. Further purification was performed with specific Sepharose-antibody columns to remove protein contaminants, avoiding procedures which may denature kininogen. The microheterogeneity was investigated by isoelectric focusing in column in the pH-gradients 3.5-10, 4-6 and 3.5-5. Kininogen components were determined by single radial immunodiffusion against monospecific anti-human kininogen serum, in comparison with focusing of whole plasma. 40% of isolated as well as whole plasma kininogen focused at pI 4.5; the respective focusing ranges were pI 4.4-4.7 (60--80%) and pI 4.3-4.6 (92%). The results were verified by crossed immunoelectrophoresis. The pI 4.5 component is apparently the main native form of human kininogen as shown by focusing of whole human blood bank plasma. Earlier described difficulty of separating kininogen and alpha2HS-glycoprotein was verified by crossed immunoelectrophoresis which showed approximately seven kininogen components after focusing in polyacrylamide gel electrophoresis at pI 4.5-5.0 and four alpha 2HS components at pI 4.2-4.6.     

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.     

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.     

Microheterogeneity of agonist and antagonist glucocorticoid receptor complexes detected by isoelectric focusing and modifications induced by receptor activation

Rat-liver glucocorticoid receptor was incubated with either [3H]triamcinolone acetonide or [3H]RU 486, a well known antiglucocorticoid. Once formed, the steroid-receptor complexes were analyzed by isoelectric focusing in agarose gel slabs. A careful slicing of the receptor tracks revealed the presence of three distinct radioactive peaks focused at the following pI values: 5.3 +/- 0.2 (n = 17) and 4.4 +/- 0.1 (n = 17). All these peaks correspond with receptor isoforms as suggested by control experiments. The receptor state was analyzed after focusing by a chromatographic assay on DNA-cellulose, DEAE-trisacryl and hydroxyapatite minicolumns. The peak of pI 4.4 apparently corresponded to the non-transformed receptor and was greatly stabilized in the presence of RU 486, whereas the peaks of pI 4.8 and 5.3 were probably made of transformed receptor and meroreceptor. These results were confirmed by autoradiographic studies after isoelectric focusing of receptor molecules covalently labelled with [3H]dexamethasone mesylate. Thus, the rat-liver glucocorticoid receptor appeared to be a rather acidic protein which became less acidic after transformation by heat, displaying a pI shift which was strongly reduced in case of steroid-receptor complexes formed with the antiglucocorticoid RU 486.     

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.     

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.     

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.     

An isoelectric focusing study of plasma membrane actin.

Plasma membranes were purified from secondary chick embryo fibroblasts labeled with [³⁵S]methionine for 1 or 18 h. The total cell homogenate, postnuclear supernatant and plasma membrane fractions were analyzed by two-dimensional electrophoresis (isoelectric focusing followed by SDS-slab gel electrophoresis). The α, β, and γ isoelectric variants of actin were present in similar proportion in membranes, supernatant, and cell homogenate as determined by incorporation of ³⁵S into each species of actin. These results indicate that the plasma membrane actin of chick fibroblasts is heterogeneous and that no isoelectric variant of actin is unique to the plasma membrane.     

The isoelectric focusing problem analytic solution

The focusing problem under isoelectric focusing has been solved analytically precisely. The solutions determine the law of the fraction moving and narrowing in the instant electric field gradient. This is especially actually because of developing the calculation methods in electrophoretical experiment.     

Current trends in capillary isoelectric focusing of proteins

Isoelectric focusing (IEF) in thin capillaries is reviewed here. After an introduction on the genesis and chemistry of the carrier ampholyte buffers, different approaches to IEF are discussed and evaluated. The classical approach consists on IEF under conditions of suppressed electroosmotic (EOF) flow, usually obtained by covalently bonding hydrophilic polymers to the inner capillary wall. The other approach consists of IEF in dynamically (and partially) coated capillaries, so as to allow a reduced EOF flow to coexist with the IEF process, so that focusing and transport of the train of stacked bands occurs simultaneously. The various experimental parameters: focusing, elution and detection steps, pI measurements, as well as typical drawbacks, such as isoelectric precipitation are evaluated. The review ends with some examples of analytical separations, at the moment mostlyl limited to focusing of native hemoglobins (normal and point mutants). These separations are compared with those obtained by slab-gel IEF and in immobilized pH gradients.     

Transient state isoelectric focusing: theory

Mathematical theory is developed to describe the transient state of isoelectric focusing (pH-gradient electrophoresis) in a linear pH gradient under highly idealized conditions. This theory makes it possible to predict the concentration profile (distribution) for the protein or other amphoteric species of interest as a function of time, when the sample is applied in a zone of infinitesimal thickness at one end of the column, or in a uniform distribution throughout the column. Further, the position of the centroid, and the second moment around the mean, σ², (square of the standard deviation of peak width) are described as a function of time, irrespective of the initial distribution of the protein in the column. Three arbitrary stages of the “focusing” experiment are considered: (1) Focusing, wherein the sample is applied to a preformed pH gradient; (2) Defocusing, which occurs when the electrical field is abolished after an arbitrary time (usually after the concentration profile has begun to approach its steady state) and diffusion is allowed to occur. (3) Refocusing, which occurs after the electrical field is reapplied. Although stages 1 and 3 are conceptually identical aside from the difference in initial conditions, they may differ in several important respects in practice, both with regard to technical problems of measurement, and with regard to the closeness of conditions to the stated assumptions.This theory should make it possible to predict the time necessary to achieve any desired degree of focusing, i.e., approach to the steady-state distribution. Further, this theory and the techniques of analytical scanning isoelectric focusing provide the basis for measurement of the apparent diffusion coefficient (D), the derivative of velocity with respect to position, and if the field strength is known, the slope of the mobility-pH curve at the isoelectric point, {$\text{dM}\text{d(}\text{pH}\text{)}$}.     

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.     

Microheterogeneity of molecular forms of arginase in mammalian tissues

Two isoforms of arginase, A1 and A2, were found in rat liver, submaxillary gland and kidney as well as beef kidney. In beef liver, however, A2 was the only detectable form. Two additional forms, A3 and A4, found only in rat kidney were probably artifactitious. A1 and A2 exhibited chromatographic and immunological microheterogeneity. While A1 in rat liver and submaxillary gland was excluded by DEAE-cellulose (pH 8.3) and retained on CM-cellulose (pH 7.5), that (A'1) in beef and rat kidneys was excluded by both ion-exchangers. A2 in all tissues was retained on DEAE-cellulose, but not on CM-cellulose. Both A1 and A2 in rat liver and beef kidney, A1 from rat submaxillary gland and A2 from beef liver were precipitated by antibodies to rat and beef liver arginases. None of the forms in rat kidney (A1, A2, A3 and A4) showed any cross-reactivity to either antibody. Rat submaxillary gland A2 was precipitated by anti-rat liver arginase, but activated by anti-beef liver arginase. While the major molecular forms were A1 in rat liver and submaxillary gland and A2 in beef liver and rat kidney, the two forms occurred in equal proportions in beef kidney. It appears that different isoforms might function as components of the urea cycle in the liver of different mammals and of the arginine catabolic pathway in different extrahepatic tissues.     

Subsetting of acetylcholine receptor-reactive antibodies by preparative isoelectric focusing.

The antibodies produced against most foreign antigens are composed of a family of immunoglobulins, a family composed of members that are of a number that often reflects the size/complexity of the molecule that stimulates their production. In other words, such responses involve the activation of a "polyclonal" B lymphocyte population. The antibody products of the B cells, although all capable of binding the original antigen, bind at various immunogenic sites (epitopes) on that antigen. Such differences in antigen-binding fine specificity is determined by amino acid residues in the antibody variable region domains found associated with the antigen combining site and tend to have a complimentary biochemistry with the molecule for which they are intended to interact. Furthermore, in addition to amino acid differences that dictate the isotypes and allotypes of antibody molecules, differences in the amino acids that compose the variable regions can produce differences in net charge of particular antibody molecules; thus, families of polyclonal antibodies, all reactive with the same antigen but with different fine specificities, can be separated and, as shown below, purified based on their isoelectric points by preparative isoelectric focusing (pIEF).     

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.