Gel Electrofocusing Research Articles

CHICKEN BLOOD PLASMA PROTEINS: PHYSICOCHEMICAL, NUTRITIONAL AND FUNCTIONAL PROPERTIES

ABSTRACTChicken blood plasma protein was prepared by collecting blood during the slaughter of animals using 0.5% sodium citrate solution as an anticoagulant. The blood cells were separated by centrifugation, and the plasma recovered by freeze drying either before or after dialysis. Disc polyacrylamide gel electrophoresis gave a pattern with nine protein bands, which were reduced to seven bands when the sample and gels were treated with urea. Sodium dodecyl sulfate (SDS) gel electrophoresis furnished nine protein bands with molecular weights ranging from 24,000‐l15,000. Gel electrofocusing revealed three protein bands with isoelectric points of 5.7, 5.3 and 4.8, respectively. Digestibility of the proteins was above 90%, and the protein efficiency ratio (PER) was 2.8 in comparison with 2.5 for casein. Addition of plasma to wheat flour for bread making at 2.5 and 5% levels raised the PER of bread from 0.87 to 1.67 and 2.02, respectively.

Multiple forms of endo-polygalacturonase from Saccharomyces fragilis.

Three forms of endopolygalacturonase from Saccharomyces fragilis (Kluyveromyces fragilis) were separated by a procedure including adsorption on Amberlite IRC-50, CM Sephadex C-50 column chromatography and repeated preparative disc electrophoresis. Each endo-PG was almost homogenoeus as judged by polyacrylamide gel electrofocusing and disc electrophoresis. The three enzyme were designated as enzymes I, II and III. Enzymes I and II were similar but enzyme HI different from I and II in isoelectric point. The three enzymes resembled one another in eznyme action on pectic acid and other properties. All the three enzymes showed macerating activity toward the potato and carrot tissues.

Glutathione thiol esterases of human red blood cells: Fractionation by gel electrophoresis and isoelectric focusing

The number and the substrate specificities of glutathione thiol esterases of human red blood cells have been investigated by gel electrophoresis and isoelectric focusing and staining methods devised for the location of these enzymes on gels. Several glutathione thiol esterase forms, both unspecific (with respect to the S-acyl group of the substrate) and specific were found. Electrophoresis on both polyacrylamide and agarose gels resolved three enzyme components with apparently similar substrate specificity. Isoelectric focusing in liquid column separated two unspecific thiol esterase components with S-lactoylglutathione (p I=8.4) and S-propionylglutathione (p I=8.1) as the best substrates, respectively, and two specific enzymes, S-formylglutathione hydrolase (p I=5.2) and S-succinylglutathione hydrolase (p I=9.0). Isoelectric focusing on polyacrylamide gel resolved nine unspecific glutathione thiol esterase bands (between pH values 7.0 and 8.4). Partially purified glyoxalase II ( S-2-hydroxyacylglutathione hydrolase, EC 3.1.2.6) from erythrocytes or liver still gave three components on electrophoresis and several activity bands on gel electrofocusing. These results indicate that human red cells contain at least four separate glutathione thiol esterases. Glyoxalase II, one of these enzymes, apparently occurs in multiple forms. These were neither influenced by pretreatment of the samples with neuraminidase or thiols nor were interconvertible during the fractionations.

Preparative flatbed electrofocusing in granulated gels with natural pH gradients generated from simple buffers

Techniques are reported for preparative electrofocusing in natural pH gradients formed from simple buffers using flatbeds of granulated gels. The stability and reproducibility of the gradients permit excellent resolution of complex mixtures of proteins. Examples reported in this paper include the preparative separation and recovery of biologically active serum and thyroid proteins.

A three-step method for isolating a few to several hundred milligrams of protein

An operationally simple general protein isolation method was devised from three previously available separation tools, and was tested by application to two demanding fractionation problems and for yield. One test system was the isolation by gel electrofocusing of two model proteins with p I values of 4.6 and 4.8, bovine serum albumin and ovalbumin, with a load of 220 mg each. The other test was the isolation of 10 mg of human growth hormone isohormone B from a mixture of closely migrating other isohormones. The three-step procedure comprises of: (1) separation into zones of homogeneous protein by gel electrofocusing; (2) excision of the zones of homogeneous protein from the gel followed by concentration of the protein to a small volume of solution by means of Steady-State Stacking; (3) purification from polyacrylamide-like contaminants and non-volatile buffers by gel filtration followed by lyophilization. The average overall recovery was 70–80%.

Isolation of human platelet and red blood cell plasma membrane proteins by preparative detergent electrophoresis

High resolution polyacrylamide gel electrophoretic techniques have been applied to the preparative isolation and analysis of plasma membrane proteins and glycoproteins from human platelets and red blood cells. The techniques presented allow relatively simple, direct, rapid and quantitative purification of a broad molecular weight range of membrane proteins, by means of continuous elution preparative gel electrophoresis of proteins solubilized with sodium dodecyl sulfate. Spectrophotometric and fluorophotometric (fluorescamine) profiling, and high resolution gel electrophoretic analysis (SDS-acrylamide gradient slab gels, and gel electrofocusing) of eluted protein species indicate that purified membrane proteins of a broad molecular weight range may be obtained in a one step procedure, and in quantities and concentrations sufficient for further analytical or experimental procedures.

Isozymes of rat urinary kallikrein

Two isozymes of rat urinary kallikrein (A and B) have been purified and studied in detail. The enzymes were separated by chromatography on DEAE-cellulose and distinguished by electrophoresis and electrofocusing in polyacrylamide slab gels. The electrophoretic mobilities were not altered by treatment with neuraminidase but were shifted toward the cathode after complexing with aprotinin. Molecular weights of rat urinary kallikreins A and B were estimated to be 36,500 and 35,500, respectively, by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis. The enzymes did not differ in pH optimum, specific activity or substrate specificity, but differed slightly in heat stability. They were not distinguishable biologically, nor immunologically using an antiserum generated in a sheep against kallikrein B. Both kallikreins were activated by deoxycholate and several nonionic detergents. α-N-tosyl-L-arginine methyl ester (Tos-L-Arg OMe) hydrolysis by both forms was inhibited by aprotinin and stimulated by ovomucoid and lima bean trypsin inhibitors, whereas Tos-L-Arg-OMe hydrolysis was stimulated by soybean trypsin inhibitor at low concentrations and inhibited at high concentrations. Kinetic measurements of α-N benzoyl-L-arginine ethyl ester (Bz-L-Arg-OEt) hydrolysis by the two enzymes showed no significant differences in substrate affinity or in maximal velocity.

Enzymic Mechanisms of Starch Breakdown in Germinating Rice Seeds

The time sequence analysis of the starch digestion pattern of the thin sectioned germinating rice (Oryza sativa L.) seed specimens using the starch film method showed that at the initial stage amylase activity was almost exclusively localized in the epithelium septum between the scutellum and endosperm. Starch breakdown in the endosperm tissues began afterward; amylase activity in the aleurone layers was detectable only after 2 days. Polyacrylamide gel electrofocusing (pH 4 to 6) revealed nearly the same zymogram patterns between endosperm and scutellum extracts, although additional amylase bands appeared in the endosperm extracts at later germination stages (4 to 6 days). These are presumably attributable to the newly synthesized enzyme molecules in the aleurone cells.

The measurement of the Bohr effect of fish hemoglobins by gel electrofocusing

1. Hemolysates from 16 species of Amazon fish and one amphibian were analyzed by gel electrofocusing. The change in isoelectric point upon deoxygenation provided a reliable estimate of the Bohr effect.2. Certain species of fish had single hemoglobin components whose pI increased significantly upon deoxygenation, as in man. Other fish had hemoglobins whose isoelectric points were unaffected by deoxygenation. Six species of fish had at least two hemoglobin components, one of which had a reduced isoelectric point upon deoxygenation indicating a reversed Bohr effect, whereas the other(s) had an increased isoelectric point on deoxygenation, as occurs with the normal alkaline Bohr effect.3. A close correlation was found between the change in isoelectric point with deoxygenation and the Bohr effect determined by oxygen equilibrium measurements.

Physicochemical properties of leucine aminopeptidase from Aspergillus japonica.

Physicochemical properties of the purified leucine amino peptidase from Aspergillus japonica were studied. The molecular weight of this enzyme was estimated to be approx. 31100 by sedimentation equilibrium, and the sedimentation coefficient (S20, W) was determined to be 3.12 S. The extinction coefficient at 278.5 nm, E1%1cm was 7.72 and the isoelectric point was at around pH 8.0 by gel electrofocusing. The helical content was calculated to be 1.7% from the Moffit-Yang plots. The amino acid analyses indicated that the enzyme was composed of 289 amino acid residues and contained one cystine. The amino-terminal amino acid was lysine, and the partial specific volume calculated from amino acid composition was 0.726.

In vitro phosphorylation of bovine adrenal tyrosine hydroxylase by adenosine 3‘:5‘-monophosphate-dependent protein kinase.

We have studied the effects of adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase on the phosphorylative and functional modification of bovine adrenal tyrosine hydroxylase. Incubation of partially purified tyrosine hydroxylase with cAMP-dependent protein kinase in the presence of [gamma32P]ATP and 5 micron cAMP led to a 3- to 5-fold activation of tyrosine hydroxylase and to incorporation of [32P]phosphate into protein. When tyrosine hydroxylase preparations activated by exposure to enzymatic phosphorylating conditions were analyzed by sucrose density gradient centrifugation, polyacrylamide gel electrophoresis, and gel electrofocusing, the radioactivity of 32P was coincident with the activity of tyrosine hydroxylase, suggesting incorporation of 32P from [gamma-32P]ATP into tyrosine hydroxylase. Polyacrylamide gel electrophoresis of the phosphorylated tyrosine hydroxylase preparation in the presence of 0.1% sodium dodecyl sulfate revealed that the 60,000-dalton polypeptide subunit of tyrosine hydroxylase served as the phosphate acceptor.

Purification and characterization of β-fructofuranosidase (invertase) from an oral strain of Streptococcus mitis

β-Fructofuranosidase (EC 3.2.1.26) isolated from the cell extract of Streptococcus mitis ATCC 903 was purified 167-fold. The enzyme appeared homogeneous by gel electrophoresis. In gel electrofocusing, two protein bands, identical with two bands of β-fructofuranosidase activity, were visualized at pI values 4.05 and 4.25. The mol. wt of the enzyme was estimated to be 49,000 and the K m for sucrose was 7.4 × 10 −2M. The enzyme had optimal activity in the pH-range 6.3–7.2. The sulphhydryl reagents 2-mercaptoethanol, p-chloromercuribenzoate, N-ethylmaleimide and iodoacetate and the protein denaturants urea and sodium dodecylsulphate were inhibitory. EDTA and sodium fluoride had no effect on the activity. Inorganic phosphate had a stimulatory effect on enzyme activity. Apparent K m for inorganic phosphate was 2.1 × 10 −2M. The enzyme obeyed Michaelis-Menten kinetics. Variation in pH did not change the shapes of the saturation curves for sucrose. The Hill coefficient was calculated to be 1.0. No stimulatory or inhibitory effect of glycolytic intermediates and nucleotides was found. These results indicate that β-fructofuranosidase is a non-allosteric enzyme.

Purification and properties of three forms of .ALPHA.-glucosidase from germinated green gram (Phaseolus vidissimus Ten.).

Three forms of α-glucosidase have been isolated from 5-day-old green gram (Phaseolus vidissimus Ten.) seedlings, by a procedure including fractionation with ammonium sulfate and polyethylene glycol 6000, DEAE-cellulose column chromatography, SP-Sephadex column chromatography, preparative gel electrofocusing and preparative disc gel electrophoresis. The α-glucosidases isolated were designated as α-glucosidase I, α-glucosidase II–1 and α-glucosidase II–2. They were homogeneous on polyacrylamide disc gel electrophoresis. Their molecular weights were 145,000, 105,000 and 65,000, respectively. The three enzymes hydrolyzed maltose, maltotriose, phenyl α-maltoside, amylose and soluble starch liberating glucose, but did not act on sucrose. Their enzymes hydrolyzed phenyl α-maltoside into glucose and phenyl α-glucoside. They hydrolyzed amylose liberating α-glucose. Maltotriose was the main α-glucosyltransfer product formed from maltose by the three α-glucosidases.

Hindered migration of amino acids toward their isoelectric positions in gel electrofocusing, and facilitation of the migration at high ionic strength

Amino acids with a largep I -p K p difference are known to be poor carrier ampholytes in electrofocusing, exhibiting isoelectric zones with poor conductivity across as many as 4 pH units. Accordingly, radioactive amino acids of this type, e.g., glycine, are found to be distributed over the entire pH gradient formed by Ampholine in electrofocusing gels, while radioactive amino acids like histidine or glutamic acid with small p I - p K p differences form single peaks at or near their p I's. When poor carrier ampholyte amino acids are subjected to gel electrofocusing in 0.1 m KCl, their distribution sharpens into single peaks, at or near the p I, indistinguishable from those of the good carrier ampholyte amino acids. At an intermediate stage of peak coalescence of the original broad distributions of poor carrier ampholyte amino acids, in 0.01 m KCl, acidic and basic peaks of amino acid can be observed, possibly analogous to acidie and basic distributions previously observed with labeled Ampholine. The rate of peak coalescence of anionic amino acids seems higher than that of the cationic species. The mechanism by which high ionic strength facilitates the condensation of poor carrier ampholyte amino acids at their p I remains unknown. Possibly, the current within zones of poor carrier ampholyte amino acids is insufficient, or poor carrier ampholyte amino acids are not sufficiently charged, to allow for electrophoretic migration of the bulk of loaded amino acid to its isoelectric position, unless the current density is increased by electrofocusing at high ionic strength. Alternatively, 0.1 m KCl may interfere with electrovalent interactions between amino acids and isoelectric carrier ampholyte zones, analogous to the action of urea in preventing the interaction between polyanions and carrier ampholytes.

Aging of pyruvate kinase isozymes in rabbit lens

Using acrylamide gel electrofocusing, several bands of pyruvate kinase activity were found, corresponding to “M” bands; the isoelectric points of these bands differed according to the zones of rabbit lens: four bands were seen in the epithelium (where protein synthesis is active), and only two bands in the core of the adult lens (where there is no protein biosynthesis). On the contrary, the four bands exist in the lens core of 12-day-old rabbits. Kinetic results and thermostability experiments confirmed that the bands of the adult lens core correspond to the “M 1” and “M 4” isozymes. It seems that the disappearance of certain molecular forms of pyruvate kinase may be due to their shorter life-span. These modifications may have some physiological or pathological consequences.

Glycopeptides derived from individual membrane glycoproteins from control and Rous sarcoma virus-transformed hamster fibroblasts.

The membrane glycoproteins from control (BHK21/C13) and Rous sarcoma virus-transformed (C13/B4) baby hamster kidney cells grown in medium containing [14C]- or D-[3H]glucosamine have been separated into two distinct classes: a phenol-soluble fraction and an aqueous fraction. The membrane glycoproteins from both BHK21/C13 and C13/B4 partitioned similarly into these two fractions. The phenol and aquesous-soluble glycoproteins differed in their sodium dodecyl sulfate-polyacrylamide gel profiles, polyacrylamide isoelectric focusing profiles, and glycopeptide distribution on Sephadex G-50. A number of aqueous and phenol-soluble glycoproteins from BHK21/C13 and C13/B4 cells were purified to near homogeneity by means of polyacrylamide electrophoresis and gel electrofocusing. These glycoproteins range in molecular weight from 179,000 to 31,000 and have isoelectric points of 7.5 to 3.0. Our results show that the pronase glycopeptides of 20 out of 24 homologous membrane glycoproteins of equivalent molecular weight and isoelectric point from BHK21/C13 and C13/B4 cells are dissimilar as measured by Sephadex G-50 gel filtration.

Properties of D-amino acid oxidase covalently modified upon its oxidation of D-propargylglycine.

Upon oxidation of D-propargylglycine by D-amino acid oxidase, the enzyme is converted by covalent alkylation to catalytic species with different properties from those of native enzyme. At least five distinct modified enzyme species are present in the preparation, as determined by gel electro-focusing. Individual characterization of the components has not yet been attempted. The combined kinetic and spectral properties of the preparation have been studied. The modified enzymes have a marked preference for hydrophobic amino acids: the rates of oxidation decrease in the series D-phenylalanine, D-methionine, D-norleucine, D-norvaline, D-alpha-aminobutyrate, D-alanine. In addition, the observed Kms of the amino acids are increased, especially those of the smaller substrates (D-alanine and D-alpha-aminobutyrate). A primary kinetic isotope effect is observed upon oxidation of amino acids by the modified enzymes, evidence that this catalysis exhibits a different rate-determining step from catalysis by native enzyme. The modified apoenzyme exhibits intense absorbance at 318--320 nm, not present in native enzyme. This chromophore can be partially (75%) removed by treatment of the modified enzyme with hydrazine. However, the activity of native enzyme is not substantially restored by this process, suggesting the existence of superficial alkylations in addition to the modification responsible for the observed changes in kinetic parameters.

A method for density gradient isoelectric focusing in small scale

A method for density gradient isoelectric focusing in small seale is described. It utilizes the same equipment as that ordinarily used for electrofocusing in polyacrylamide gels, thus allowing several samples to be analyzed in the same experiment and the simultaneous electrofocusing in density gradients and gels. Data concerning the stability of the pH gradient and the effect of salt and sample load on the focusing are given. Three different methods for fractionating the gradient are described; in one of these the frozen gradient is sliced with an ordinary gel alicer. This fractionation method is especially advantageous if the gradient contains zones of highly aggregated material, and it also allows the gradients to be stored frozen for later fractionation.

Differential protein synthesis after red light illuminations in germinating fern spores

The effects of red and far red illuminations were studied in spores of the dark germinatingPteridium aquilinum and that of the light dependently germinatingDryopteris filix-mas. Proteins were analysed by electrophoresis according to their molecular weights. The isoesterases were separated by gel electrofocusing. In both species we found two newly synthesized protein bands, probably controlled by the presence of active phytochrome. One of these seems to be identical, the other is different in the two species. Phytochrome control was also detected in the esterase isoenzyme pattern.

Preparative separation of hemoglobins A and S by gel electrofocusing, using selective zone elution by gel transposition between suitable anolytes and catholytes

Preparative electrofocusing on polyacrylamide gels has been limited, until recently, to excision of gel slices, diffusion, and collection of the slice diffusates. An advance was made by the introduction of a method of selective electrophoretic zone recovery by specific changes of anolyte (A. McCormick, L. E. M. Miles, and A. Chrambach, 1976, Anal. Biochem. 75, 314–324). It was shown (a) that selective zone recovery could be achieved by transposition of the gels into either isoelectric ampholytes or charged buffers, (b) that it could be applied to the gram scale, and (c) that zone elution could proceed either continuously or discontinuously. The early study was, however, limited to a trivial model problem, the separation of hemoglobin from bovine serum albumin (BSA). The present study was an attempt to apply a similar selective zone recovery method to a more demanding separation problem, the separation of hemoglobin A from hemoglobin S as well as from other minor components contained in a sickle-trait human hemolysate. The study shows that selective electrophoretic zone elution from a electrofocusing gel 18 mm in diameter is capable of yielding hemoglobin A, separated from hemoglobin S, differing by only 0.2 pH units in isoelectric point. The recovery of hemoglobin A was 70%, with a load of 32 mg of hemoglobin mixture per gel, using discontinuous zone elution into a collection cup.