A comparative study of lens gamma crystallins using isoelectric focusing

The isoelectric points of gamma crystallins of the lenses of rats, dogfish, calves, and humans differ, with a range of pH 8.87-6.80. In each species, one gamma crystallin component with an isoelectric point at pH 7.5 is present, but at differing levels. Amino acid compositions of this component were similar among these four species. All pH 7.5 isoelectric point proteins crossreact immunochemically. A common messenger RNA must be present for this gamma crystallin in all of these species.     

The measurement of the intrinsic alkaline Bohr effect of various human haemoglobins by isoelectric focusing

We have used isoelectric focusing to measure the differences between the pI values of various normal and mutant human haemoglobins when completely deoxygenated and when fully liganded with CO. It was assumed that the ΔpI(deox.–ox.) values might correspond quantitatively to the intrinsic alkaline Bohr effect, as most of the anionic cofactors of the haemoglobin molecule are `stripped' off during the electrophoretic process. In haemoglobins known to exhibit a normal Bohr coefficient (ΔlogP₅₀/ΔpH) in solutions, the ΔpI(deox.–ox.) values are lower the higher their respective pI(ox.) values. This indicates that for any particular haemoglobin the ΔpI(deox.–ox.) value accounts for the difference in surface charges at the pH of its pI value. This was confirmed by measuring, by the direct-titration technique, the difference in pH of deoxy and fully liganded haemoglobin A₀ (α₂β₂) solutions in conditions approximating those of the isoelectric focusing, i.e. at 5°C and very low concentration of KCl. The variation of the ΔpH(deox.–ox.) curve as a function of pH (ox.) was similar to the isoelectric-focusing curve relating the variation of ΔpI(deox.–ox.) versus pI(ox.) in various haemoglobins with Bohr factor identical with that of haemoglobin A₀. In haemoglobin A₀ the ΔpI(deox.–ox.) value is 0.17 pH unit, which corresponds to a difference of 1.20 positive charges between the oxy and deoxy states of the tetrameric haemoglobin. This value compares favourably with the values of the intrinsic Bohr effect estimated in back-titration experiments. The ΔpI(deox.–ox.) values of mutant or chemically modified haemoglobins carrying an abnormality at the N- or C-terminus of the α-chains are decreased by 30% compared with the ΔpI value measured in haemoglobin A₀. When the C-terminus of the β-chains is altered, as in Hb Nancy (α₂β^{Tyr-145→Asp}₂), we observed a 70% decrease in the ΔpI value compared with that measured in haemoglobin A₀. These values are in close agreement with the estimated respective roles of the two major Bohr groups, Val-1α and His-146β, at the origin of the intrinsic alkaline Bohr effect [Kilmartin, Fogg, Luzzana & Rossi-Bernardi (1973) J. Biol. Chem. 248, 7039–7043; Perutz, Kilmartin, Nishikura, Fogg, Butler & Rollema (1980) J. Mol. Biol. 138, 649–670]. In other mutant haemoglobins it is demonstrated also that the ΔpI(deox.–ox.) value may be decreased or even suppressed when the substitution affects residues involved in the stability of the tetramer. These results support the interpretation proposed by Perutz, Kilmartin, Nishikura, Fogg, Butler & Rollema [(1980), J. Mol. Biol. 138, 649–670] for the mechanism of the alkaline Bohr effect, and also indicate that the transition between the two quaternary configurations is a prerequisite for the full expression of the alkaline Bohr effect.     

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.     

Comparative study of sturgeon oocyte soluble proteins by isoelectric focusing

1. Soluble caviar (oocyte) proteins of four sturgeon species from the Caspian sea, Acipenser stellatus (Sevrouga), Acipenser guldenstadti (Ossietre), Acipenser nudiventris (Chipe) and Huso huso (Belouga) were studied by isoelectric focusing. 2. Isoelectric focusing patterns of these proteins differ from one species to another and allow the identification of the specific origin of caviar. 3. Moreover, this technique allowed the discrimination of a subspecies, Acipenser guldenstadti persicus from the species. 4. This biochemical characterization of caviar proteins allowing identification of sturgeon species producing caviar could be added to data used in fraud tests.     

A comparative study of Echinococcus granulosus from human and animal hosts in Kenya using isoelectric focusing and isoenzyme analysis

Macpherson C. N. L. and Mcmanus D. P. 1982). A comparative study of Echinococcus granulosus from human and animal hosts in Kenya using isoelectric focusing and isoenzyme analysis. International Journal for Parasitology12: 515–521. The soluble enzyme extracts from protoscoleces obtained from hydatid cysts of human, camel, cattle, sheep and goat origin were compared on the basis of their isoenzyme patterns for GPI and PGM using isoelectric focusing. Consistent GPI and PGM isoenzyme patterns were obtained for larvae of human, camel and sheep material. Cattle material varied occasionally in having an additional cathodic band in some of the GPI patterns. Two distinct isoenzyme patterns were evident in the goat material for both enzymes. The more common goat patterns were similar to those of human, cattle and sheep (Kenya, U.K. and Argentina) material, which were similar to each other. The rare goat patterns were similar to those obtained for camel material. Cyst location in the various intermediate hosts had no effect on the zymograms obtained. Additionally, no alteration in the major banding patterns was observed between the larvae and homologous adults produced by experimental infections. Of 26 naturally infected dogs, 19 produced adult GPI zymograms resembling human/ sheep/goat (common form) experimental infection patterns, three were similar to experimental cattle infections and four had camel/goat (rare form) patterns.     

Segregation of histone fractions from purified rat pancreas nuclei by isoelectric focusing.

The present report describes the purification of nuclei from rat pancreas and the use of isoelectric focusing for the segregation of histone fractions, a heterogeneous mixture with respect to charge. This procedure employs gels of 5% polyacrylamide as support matrices, in 6.25 M urea, and spans a pH range of 7.5-10.8. It was, therefore, found adequate for study of the microheterogeneity of histones. The banding pattern of histone fractions in pancreas development using electrofocusing is presented.     

Immunological and isoelectric focusing study of beta-glucocerebrosidase from normal and Gaucher disease

Comparison of normal and Gaucher disease beta-glucocerebrosidase by agarose isoelectric focusing (IEF) demonstrated additional bands at the pI-6 area seen within the mutated enzyme, while both normal placenta and spleen enzyme preparations manifest only major activity at pI-5. Antiglucocerebrosidase antibodies precipitated both normal and pathological enzymes, however, more antibodies were needed to reach an equivalence with the normal enzymes than with the Gaucher's. Cross reactivity of the IEF isozymes were detected by direct immunodiffusion on the prefocused gel.     

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.     

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.     

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).     

A new BF F variant by polyacrylamide gel isoelectric focusing

Summary Polyacrylamide gel isoelectric focusing followed by electroblotting with enzyme immunoassay was done for the investigation of allotypes of properdin factor B (BF) in serum from 326 Japanese subjects. A new BF F variant tentatively designated BF*Fb1 (b=basic) was detected, the isoelectric point of each band of homozygous BF Fb1 being higher than of BF FF. Family data were in accordance with transmission by mendelian inheritance. The allele frequencies calculated from 326 Japanese subjects were 0.7945, 0.1825, 0.0215, and 0.0015 for BF*S, BF*F, BF*Fb1, and BF*F075, respectively, with that of variant BF*Fb1 being a polymorphic frequency. The distribution of phenotypes fitted the Hardy-Weinberg equilibrium.     

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.     

Two-step purification of cytochrome P-450 from adult pig testis by isoelectric focusing.

Adult testicular cytochrome P-450 was purified by a two-step procedure utilizing preparative isoelectrofocusing. Purification was achieved 1132 times with a yield of 4.82%. 17alpha-hydroxylase activity was shown to be 14.5 nmol of product/min/nmol of P-450. The cytochrome P-450 was determined to have an isoelectric point of 6.45 on analytical isoelectric focusing. The purified cytochrome P-450 was found to be homogeneous and its molecular weight was estimated to be 52000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The carbon monoxide difference spectrum with a peak at 448 nm exhibited the absorption spectrum of a typical cytochrome P-450.     

Selective elution of zones from preparative isoelectric focusing gels by ampholytes or buffers.

Protein zones formed by isoelectric focusing on polyacrylamide gels (IFPA) can be eluted without mechanical disruption of the gels. Specific elution is achieved by replacement of the original anolyte, a strong acid, with an ampholyte of a pI higher than that of the protein which is to be eluted. Alternatively, the anolyte may be a buffer of a pH higher than the pI of the focused protein zone. A rudimentary apparatus and procedures for the application of this method of zone elution are described but are not as yet sufficiently developed to provide a ready-to-use preparative IFPA procedure.     

Characterizing bisexual Artemia populations by isoelectric focusing

Isoelectric focusing (IEF) of protein extracts, -Esterase and Phosphoglucose isomerase, from groups of Artemia adults from different bisexual populations were studied. Both gave clear separation between the Old and New World species. The second was more polymorphic, allowing a discrimination among Mediterranean populations.     

Fractionation of "tricholysine" by isoelectric focusing

A possibility to fractionate fibrinolytically and thrombolytically active complex "tricholysin" into five components, differing in iso-points and in enzymatic activity, is demonstrated by means of isoelectric focusing using ampholine solution within pH range 3.0-10.0. Homogenous fraction IV (isopoint 6.8-7.0) has a low caseinolytic activity and a high specific fibrinolytic and esterase activities. This fraction is characterized with a high ability of plasminogen activation. Serine, threonine, alanine and valine are found to prevail in amino acid composition of the fraction IV, its molecular weight being 39000.     

An isoelectric focusing study of acid phosphohydrolases in rat liver lysosomes.

The differentiation of rat liver lysosomal acid phosphatase, acid ATPase, acid phosphodiesterase, acid ribonuclease, and acid deoxyribonuclease was studied by isoelectric focusing. To prevent autolytic digestion, inhibitors of cathepsins and neuraminidase were used. The proportion of acidic forms of acid phosphatase, acid ATPase and acid phosphodiesterase was increased by the use of extraction medium containing 0.05% Triton X-100. To investigate the identity of acid ATPase and acid phosphodiesterase, the relative activities among the multiple forms of these enzymes, the acid phosphodiesterase/acid ATPase ratio at each activity peak, and the degree of enzyme inhibition by p-chloromercuriphenyl sulfonic acid were estimated. The results suggest that acid ATPase is not identical with acid phosphodiesterase. With extraction medium free of Triton X-100, acid ribonuclease appeared in two forms. However, in addition to these forms, a new form of this enzyme with a more acidic pI (4.22) emerged when extraction medium containing 0.05% Triton X-100 was used. The major peak of acid deoxyribonuclease with pI=8.40-9.39 was obtained regardless of the extracting method.