Isoelectric focusing of proteins in the native and denatured states. Anomalous behaviour of plasma albumin

1. An analytical technique of isoelectric focusing in thin layers of polyacrylamide gel has been used to determine the isoelectric point, pI, of several proteins in the presence and in the absence of concentrated urea. 2. The presence of urea did not greatly affect pI except for bovine plasma albumin, where an increase of approx. 1pH unit was found. 3. Evidence is presented that this change in the pI of bovine plasma albumin is due to the normalization of certain ionizable groups on unfolding of the protein in urea. 4. Evidence is also presented that prolonged exposure of bovine plasma albumin to urea results in intramolecular disulphide interchange and that, on removal of urea, the new patterns of disulphide bonding stabilize abnormal conformations with pI values intermediate between those of the native and denatured states. 5. The studies demonstrate heterogeneity in bovine plasma albumin based on primary-sequence differences. 6. Isoelectric focusing of proteins in urea appears to be useful in the study of various aspects of protein structure.     

Studies on human alpha-fetoprotein. Isolation and characterization of monomeric and polymeric forms and amino-terminal sequence analysis.

Human alpha-fetoprotein has been isolated from the serum and ascitic fluid of a patient with hepatoma by a combination of immunoadsorbent column chromatography and Sephadex G-150 gel filtration. Human alpha-fetoprotein is a sialylated glycoprotein with an estimated molecular weight of 67 500, composed of a single-chain polypeptide of approximately 580 amino acid residues and 3.6% carbohydrate. It is a negatively charged protein with an acid isoelectric point (pH 4.57). In addition to the monomeric form of alpha-fetoprotein, we have identified human alpha-fetoprotein polymers, including dimeric and trimeric forms, which dissociate to the monomer only upon exposure to disulfide-reducing reagents, implying that their formation is dependent upon intermolecular disulfide bonds. These polymers are found in human alpha-fetoprotein isolated by isoelectric focusing in both the major (pI 4.57) and minor (pI 5.2) alpha-fetoprotein fractions. The first 17 residues of the NH2-terminal amino acid sequence of the hepatoma-derived human alpha-fetoprotein have been identified. Fetal alpha-fetoprotein is indistinguishable from hepatoma alpha-fetoprotein by several criteria, including immunoelectrophoresis, acryalmide gel electrophoresis, and proclivity for dimerization.     

Isolation and characterization of alpha-fetoprotein from the mouse hepatoma BW7756.

Purification of alpha-fetoprotein from mouse hepatoma BW7756 extracts was performed using ammonium sulfate precipitations, gel filtration, ion-exchange chromatography and isoelectric focusing. These procedures produced a 5.6% yield of alpha-fetoprotein with 96% purity. Polyacrylamide slab gel electrophoresis, extended agarose electrophoresis and immunoelectrophoresis demonstrated that mouse hepatoma alpha-fetoprotein migrated at pH 8.6 as a rapid alpha1, or postalbumin globulin. Crossed antibody electrophoresis of the agarose zone containing alpha-fetoprotein failed to demonstrate microheterogeneity. Molecular weight analysis of the mouse hepatoma alpha-fetoprotein on a calibrated Sephadex G-200 column yielded a value of 72 000-73 000 for the native protein. Sodium dodecyl sulfate gel electrophoresis subsequently demonstrated a single polypeptide chain with a molecular weight of 72 000. Amino acid analysis showed the alpha-fetoprotein to be an acidic protein dominated by hydrophobic residues. The total carbohydrate content was 5.5%, and 3 mol of sialic acid were detected per mol of alpha-fetoprotein. Although neutral sugars were the principal class present, galactosamine was the most abundant single sugar detected.     

Effect of pH on antigen binding by clonotypic antibodies with different isoelectric points

Polyclonal rabbit antibodies to thyroxine, human myoglobin, human growth hormone, human thyrotropin, human alpha-fetoprotein, and human thyroglobulin were fractionated into clonotypic antibodies with different isoelectric points by agarose isoelectric focusing or chromatofocusing. The effect of pH on the binding of these antigens by their respective clonotypic antibodies was assessed by radioimmunoassay. The profiles of the pH effect differed both for different antigens and for different pI's of the antibodies used. The pH optima in the radioimmunoassays for protein antigens were found to be expressed as a function of pI and molecular weight of both antigen and antibody molecules.     

Analysis of rat serum apolipoproteins by isoelectric focusing. I. Studies on the middle molecular weight subunits.

Analytical isoelectric focusing (IEF) has been applied to the study of the apolipoprotein components of rat serum high density and very low density lipoproteins. The apolipoproteins were separated on 7.5% polyacrylamide gels containing 6.8% urea, with a pH gradient of 4-6. The middle molecular weight range apolipoproteins were identified on IEF gels by the use of apolipoproteins purified by electrophoresis on gels containing sodium dodecyl sulfate (SDS). The A-1 protein focused as 4 to 5 bands from pH 5.46 to 5.82; the A-IV protein and the arginine-rich protein each focused as 4 to 6 bands from pH 5.31 to 5.46. The low molecular weight proteins focused from pH. 4.43 to 4.83 and are the subject of a separate communication. Comparisons of the IEF method with SDS gel electrophoresis, polyacrylamide gel electrophoresis in urea, and Sephadex chromatography are also reported. Additional studies were also carried out that tend to rule out carbamylation or incomplete unfolding of the proteins in the presence of urea as the causes of the observed heterogeneity.     

Isolation and partial characterization of a alpha 2-beta 1-glycoprotein from horse plasma

A alpha 2-beta 1-glycoprotein was isolated from horse plasma by classical methods. The final product appeared homogeneous by agarose gel and pore limit SDS polyacrylamide gel electrophoresis, immunoelectrophoresis and crossed immunoelectrophoresis. The protein moved in agarose gel electrophoresis just above the beta 1 region and seemed composed of a single polypeptide chain. A highly heterogenic banding pattern, focused between pH 5.1 and 6.5 was revealed by isoelectric focusing. The molecular weights determined by gel filtration on Sephadex G100 and by a pore limit polyacrylamide gel electrophoresis in presence of SDS were 65,000 and 82,300 dalton, respectively. No serological relation was found between the horse alpha 2-beta 1-glycoprotein and human and bovine plasma proteins.     

Microheterogeneity in purified broad bean polyphenol oxidase

Polyphenoloxidase was purified from chloroplasts of broad bean leaves (Vicia faba L.) to apparent homogeneity. The enzyme was composed of two proteins with an apparent mass of 65 and 68 kilodaltons after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isolated enzyme contained covalently attached carbohydrates and bound concanavalin A, Phaseolus vulgaris erythroagglutinin, and Ricinus communis agglutinin lectins. Under native isoelectric focusing, several charged isoforms were present in the pH range of 4 to 6. Many, if not all, of the isoforms separated by isoelectric focusing were glycosylated and bound concanavalin A. All these isoforms shared a 65 kilodalton protein in common, and some of the isoforms were associated with both a 65 and 68 kilodalton protein. Isoforms separated by isoelectric focusing in the presence of 9 molar urea followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a similar pattern of proteins within a slightly higher pH range from 5 to 6.5.     

Multiple forms of sheep serum A-esterase activity associated with the high-density lipoprotein.

Five lipoproteins of sheep serum expressing A-esterase activity, but with differing activities towards four organophosphate substrates, were separated by a combination of gel filtration and ion-exchange chromatography. Each had an Mr of approx. 360,000 and contained a major peptide of Mr 28,000-30,000 that appeared to be present as several isoforms on urea/agarose isoelectric focusing. In every case this peptide split into a number of bands on urea/agarose isoelectric focusing. The bands appear to represent isoforms of the peptide, and four lipoproteins yielded characteristic patterns of bands. This peptide resembles the apolipoprotein A-I of human serum, and available evidence suggests that this is the protein that expresses A-esterase activity. Evidence is presented for the existence of different species of high-density lipoprotein HDL2 particles containing different complements of peptide isoforms and expressing contrasting substrate specificities towards organophosphates.     

A universal method for two-dimensional polyacrylamide gel electrophoresis of membrane proteins using isoelectric focusing on immobilized pH gradients in the first dimension.

Two-dimensional gel electrophoresis with immobilized pH gradients in the first dimension, initially applied for the separation of soluble and total cellular proteins, has been extended to the analysis of membrane proteins. We show that the usual procedures lead to artifacts and irreproducible results due to aggregation and precipitation of proteins and protein-phospholipid complexes during isoelectric focusing (first dimension) and sodium dodecyl sulfate (SDS) gel electrophoresis (second dimension). Optimized solubilization procedures for hydrophobic membrane proteins are presented and the use of dilute samples is shown to be essential to overcome the major problems in isoelectric focusing. Increased volumes of samples dissolved in rehydration buffer are applied by direct rehydration of dry immobilized pH gradient (IPG) gels. Isoelectric focusing in 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) without urea gives good results as does 2% Nonidet-P40 with 8 M urea. Heat denaturation should be avoided. An optimized equilibration procedure for IPG gel strips in SDS sample buffer prior to separation in the second dimension was developed that minimizes loss of proteins and results in high-resolution two-dimensional electropherographic maps with a minimum of streaking. The gel strips are partially dehydrated at 40 degrees C and shortly reswollen in situ on the SDS slab gel in SDS-sample buffer containing agarose.     

Investigation of the heterogeneity of rhesus monkey alpha 1-antitrypsin

Rhesus monkey alpha 1-antitrypsin (n = 144) was examined for heterogeneity by acid starch gel electrophoresis, isoelectric focusing in agarose and agarose gel electrophoresis. In contrast to other studies, no heterogeneity of Rhesus monkey alpha 1-antitrypsin could be documented using specific antisera. Rhesus monkey alpha 1-antitrypsin contained a reactive thiol. The pIs of the major isoforms of Rhesus monkey alpha 1-antitrypsin were 4.63, 4.69, 4.84 and 4.86 at 4 degrees C. No deficiency state of Rhesus monkey alpha 1-antitrypsin was detected. The six protease inhibitors in Rhesus monkey sera cross-reacted with antisera to the six human protease inhibitors.     

Human alpha 1-antitrypsin genetic polymorphism: PI N subtypes

Three new genetic variants (PI types) of alpha 1-antitrypsin are described. They have been compared to previously described phenotypes by several techniques including narrow pH range isoelectric focusing in ultrathin polyacrylamide gels. In this system, the relevant alpha 1-antitrypsin gel bands, identified by crossed immunoelectrophoresis, focused between PI M2, the most cathodal PI M subtype, and PI P BUD, the most anodal PI P subtype. They were therefore considered to be PI N subtypes. Two of them, PI N GRO and PI N YER, could not be separated by isoelectric focusing, but gave a different pattern in agarose gel electrophoresis. None of the new alleles seemed to be associated with disease. The high resolving power of isoelectric focusing is emphasized with respect to the information it may provide concerning amino acid substitutions, while the use of other techniques proved to be of utmost importance in the differentiation of other variants showing similar isoelectric points.     

α1-Acute-phase globulins of rats. Microheterogeneity after isoelectric focusing

1. Improved resolution of mixtures of alpha(1)-globulins was obtained by the use of isoelectric focusing. 2. Because material recovered after isoelectric focusing in polyacrylamide gels behaved in a manner which suggested interaction with components derived from the gel, isoelectric focusing when used for preparative purposes was done in a matrix of Sephadex G-75. 3. By this means material from the individual bands formed by isoelectric focusing in 6m-urea could be isolated. The stability of these substances was examined by further isoelectric focusing. 4. Analysis of material that had been shown to be homogenous by isoelectric focusing in the absence of urea and of that from several individual bands derived from the same sample by isoelectric focusing in 6m-urea showed different proportions of sialic acid but no change in amino acid composition. 5. In the presence of 6m-urea the isoelectric points found were increased by 0.14-0.25 pH unit. After removal of most of the sialic acid with neuraminidase the increase was 0.36-0.72 pH unit. After treatment with 0.025m-H(2)SO(4) at 80 degrees C for 1h, which removed all the sialic acid, the increase was 0.40-0.87 pH unit. 6. Because removal of all the sialic acid did not decrease the number of bands formed by isoelectric focusing the observed heterogeneity could not be caused entirely by the presence of various proportions of sialic acid.     

Carbohydrate composition of rat hepatocyte nuclear membrane as compared to normal, Morris hepatoma 7800, and phenobarbital-induced microsomal membranes

The composition of the nonhistone nuclear proteins of rat liver, brain, thymus, and kidney has been analyzed by isoelectric focusing in polyacrylamide gel. Approximately 20–30 components were separated with a wide range of isoelectric points (pl) in the 3- to 10-pH region.Different extraction procedures applied to liver nuclei removed protein mixtures with similar components present in varying amounts. 8 m Urea 50 mm phosphate, pH 7.6, was the most successful and removed most of the nonhistone protein.The thiol groups of proteins extracted from the nuclei of liver, brain, thymus, and kidney with 8 M urea, 50 mM phosphate were labeled with [¹⁴C]N-ethylmaleimide. Although there was a slight variation in the overall thiol content of these tissue proteins, separation of the mixture by isoelectric focusing and SDS polyacrylamide gel electrophoresis showed complex patterns indicating greater heterogeneity than was apparent from the Coomassie blue dye binding.     

Studies on the characterization of the sodium-potassium transport adenosine triphosphatase. Purification and properties of the enzyme from the electric organ of Electrophorus electricus

An unspecific carboxylesterase was purified 180-fold from acid-precipitated human liver microsomes. The final preparation was homogeneous on disc electrophoresis and polyacrylamide gel electrophoresis in the presence of 6.25 M urea at pH 3.2. A single symmetrical peak was also found on gel filtration and on velocity sedimentation in the analytical ultracentrifuge, whereas slight heterogeneity was observed on isoelectric focusing.The amino acid composition of the purified enzyme is presented. From the results the partial specific volume (0.745 ml × g^?1) and the minimal molecular weight (60,000) could be calculated. Fingerprint maps of tryptic peptides from the carboxymethylated enzyme are shown.The molecular weight as determined by gel filtration, disc electrophoresis, and analytical ultracentrifugation is in the range of 181,000–186,000. For the molecular weight of the subunits a value of 61,500 has been obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis. The equivalent weight of the enzyme has been estimated to be 62,500 from stoichiometry of its reaction with diethyl-p-nitrophenyl-phosphate. Partial cross-linking of the subunits with dimethyl suberimidate and subsequent sodium dodecylsulfate polyacrylamide gel electrophoresis yielded three bands with molecular weights of 60,000, 120,000, and 180,000.From these results it is concluded that human liver esterase is a trimeric protein. It is composed of three subunits of equal size, and there is one active site per subunit.     

Electrophoretic Differences in the Capsid Proteins of Simian Virus 40 Plaque Mutants

The structural proteins of three mutants of simian virus 40 (SV40) which differ in plaque size, temperature sensitivity, oncogenicity, host cell restriction, and immunological properties were studied. The polypeptide components of these SV40 strains could not be distinguished by their polyacrylamide gel electrophoretic patterns. When the dissociated virions of two of the mutants were analyzed by the isoelectric focusing technique in a urea gradient, the capsid protein peaks were found to differ significantly in their isoelectric points. The capsid protein of the small-plaque mutant had an isoelectric point of pH 6.51 as compared with pH 6.28 for the large-plaque strain. Isoelectric focusing of the isolated capsid protein revealed three components, a single major subunit and two minor forms. The coat proteins of two of the mutants, small-plaque and minute-plaque strains, were indistinguishable by this technique. The capsid protein peaks obtained by isoelectric focusing were further analyzed by polyacryalmide gel electrophoresis.     

Multiple forms of human renin. Purification and characterization.

Human renin was purified from a juxtaglomerular cell tumor with a high renin content, 24.2 Goldblatt units/mg of protein. The purification procedure comprised three steps: gel filtration, DEAE-cellulose chromatography, and preparative isoelectric focusing. Five forms of renin amounting to 5.3 mg of enzyme were obtained with isoelectric points of 4.95, 5.10, 5.35, 5.55, and 5.70. They were all glycoproteins. The three major fractions had very similar specific activities, 868, 860, and 809 Goldblatt units/mg of protein. These fractions produced a single band on analytical isoelectric focusing and a single arc on immunoelectrophoresis. On polyacrylamide gel electrophoresis at pH 7.8, each fraction consisted of two renin bands with the same molecular weight, but different net charges. The molecular weight determined by gel filtration and Fergusson plot analysis on polyacrylamide gel was 38,000 to 42,000. The optimum pH determined on N-acetyltetradecapeptide substrate was 6.5, and the Km was 6.8 x 10(-6) M. These parameters were identical with those for standard human kidney renin. Antibodies raised against tumor renin completely inhibited the activity of both tumor and standard renin. Under dissociating conditions (sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel electrophoresis in the presence of 6 M urea), part of the purified enzyme dissociated into two smaller fragments (Mr = 20,000 and 25,000) containing renin activity.     

Two-dimensional isoelectric focusing/sodium dodecyl sulfate gel electrophoresis of protein mixtures containing active or potentially active proteases: Analysis of human exocrine pancreatic proteins

Analysis of human pancreatic juice in two dimensions using isoelectric focusing followed by sodium dodecyl sulfate gel electrophoresis indicated that human pancreatic trypsinogen (IEP_n = 6.4) rapidly autoactivated in the absence of the secretory trypsin inhibitor. The addition of 4 to 6 m urea to the protein sample and 8 m urea to the isoelectric focusing gel inhibited this autoactivation process and allowed the analysis of human exocrine pancreatic proteins. Thirteen discrete proteins were separated by the two-dimensional gel procedure including two forms each for trypsinogen, proelastase, and procarboxypeptidase A, and single forms each for amylase, lipase, procarboxypeptidase B, and chymotrypsinogen. The kinetics of inhibition of human trypsin by 8 m urea in the presence of ethylene glycol bis(β-aminoethyl ether)N,N′-tetraacetic acid indicated that samples containing active proteases could also be analyzed by this procedure.     

Characterization of proteins in the human serum proteome.

This paper presents a multidimensional profile of the human serum proteome, produced by a two-dimensional protein fractionation system based on liquid chromatography followed by characterization with capillary electrophoresis (CE). The first-dimension separation was done by chromatofocusing over a pH range from 8.5 to 4.0, where proteins were separated by their isoelectric points (pI). In this dimension, fractions were collected based on pH. The first-dimension pI fractions were then resolved in the second dimension by high-resolution, reversed-phase chromatography with a gradient of trifluoroacetic acid (TFA) in acetonitrile and TFA in water. A selected protein fraction collected from the second dimension by time was characterized by CE for molecular-weight estimation and for presence of isoforms. Molecular-weight estimation was done by sodium dodecyl sulfate capillary gel electrophoresis, where proteins were separated in the range of 10,000-225,000 Da. Detection of isoforms was done by capillary isoelectric focusing over a pH range of 3-10. A selected second-dimension fraction that contained the putative serum iron-binding protein transferrin was analyzed by these two CE techniques for molecular-weight determination and the presence of isoforms. The combination of two-dimensional protein fractionation and CE characterization represents an advanced tool for proteomics.     

Alpha1-antitrypsin: further genetic heterogeneity revealed by isoelectric focusing.

Alpha1-antitrypsin is a major human serum protein that shows an extensive polymorphism. Genetic heterogeneity has previously been demonstrated by starch gel electrophoresis. By applying analytical isoelectric focusing (pH 3.5--5.0) to this system, we found a common variant, Pi M3, with an isoelectric point between those of Pi M1 and Pi M2. The gene frequency of this variant was .11 in U.S. whites and .054 in blacks. When PiM3 and PiM1 are included in the Pi system, the heterozygosity at the Pi locus is five times greater in whites and 10 times greater in blacks than that detected by earlier electrophoretic techniques.     

Two-dimensional gel electrophoresis of chicken skeletal muscle proteins with agarose gels in the first dimension

An improved method of two-dimensional gel electrophoresis is described. The method is specifically developed for preparing a “protein map” of chicken skeletal muscle, and is found to be applicable to the analysis of most protein constituents including high molecular ones, such as myosin heavy chain, without using any detergents in the first dimension. Omission of detergents from the focusing medium results in two advantages. (i) The first-dimension isoelectric focusing pattern can be recorded by taking a photograph of the gel prior to the second-dimension electrophoresis, so that even a close doublet band in the first dimension, which forms one spot in the second dimension, can be found heterogeneous in component by examining the first-dimension pattern of the same gel. (ii) Since peptides of relatively large molecular weights can be analyzed by first-dimension isoelectric focusing, complex formation between polypeptides with different isoelectric points is demonstrable. For example, troponin T, troponin I, and troponin C are found by two-dimensional gel electrophoresis to form a complex in a 4 m urea solution, and so are troponin I and troponin C in a 5 m urea solution.