Further purification and characterization of serum proteins used to detect cystic fibrosis genotypes by isoelectric focusing

Sera from cystic fibrosis (CF) homozygotes and obligate heterozygotes contain a CF factor (gamma CF factor) not found by isoelectric focusing in thin-layer polyacrylamide gels in most normal control sera. In addition, sera from most obligate heterozygotes lack another protein (bland B, C, or D) that is commonly found in sera from most normal and cystic fibrosis individuals. A standardized, biophysical assay is described that employs isoelectric focusing for the detection of both CF homozygotes and heterozygotes based on the analysis of whole serum for the presence of the gamma CF factor and bands B, C, and D. Results of analyzing sera from selected CF patients by isoelectric focusing indicated that there is a general correlation between the amount of the gamma CF factor and the clinical severity of the disease. Partial purification and characterization of the gamma CF factor and protein bands B, C, and D was accomplished by using DEAE-cellulose chromatography, Sephadex G-200 gel filtration, sequential molecular filtration through a series of Amicon Diaflo ultrafiltration membranes, affinity chromatography, and cellulose acetate electrophoresis. The gamma CF factor is a cationic protein with a pI of 8.46+/-0.05, has gamma electrophoretic mobility, a molecular weight between 3,500 and 10,000, and apparently exists in CF serum in 2 forms (free in solution and complexed to IgG). Bands B, C, and D are cationic proteins with pI values of 7.85 to 8.10, have gamma electrophoretic mobility and a molecular weight of approximately 100,000-150,000.     

Rapid high-performance isoelectric focusing of monoclonal antibodies in uncoated fused-silica capillaries

Rapid (< 5 min) high-performance isoelectric focusing was performed in uncoated fused-silica capillaries to resolve isoforms of monoclonal antibodies and to determine their isoelectric points (pI). The methodology involved the use of a 32 cm (effective length 9 cm) x 50 microns I.D. uncoated capillary. (Hydroxypropyl)methyl cellulose was used as an additive to suppress analyte-wall interaction and to precisely control electroosmotic flow so that focusing and mobilization of focused zones past detector occur simultaneously. Urea was included in the separation medium to solubilize the antibodies that precipitated at their point of focusing. The methods with and without urea used ampholytes pH 5-8 to generate a demonstrate linear gradient between pH 5.4 and pH 7.2, based on the separation of various protein standards. Reproducibility [< 2% (R.S.D.)] of the migration times (corresponding to the detectable isoforms of the antibodies) was obtained by using two sets of reagents and capillaries on three consecutive days. pI values determined from day-to-day with a reference standard were shown to vary by only 0.01 pH unit. The described capillary isoelectric focusing methods provided a rapid, simple and reproducible way of monitoring micro-heterogeneity and pI of the murine monoclonal antibodies investigated.     

Quantitation of glycated hemoglobins in human adult blood by capillary isoelectric focusing

A precise and reproducible method for assessment of glycated hemoglobin in human adult red blood cells is reported, based on capillary isoelectric focusing (IEF). In order to obtain baseline resolution between adult hemoglobin (Hb A) and its glycated form (Hb A1c), two species which differ by minute delta pI values, < 0.03 pH units, the following procedure was adopted: the focusing mixture consisted of 5% Ampholine, pH 6-8, 0.5% Pharmalyte, pH 3-10, 3% short-chain liquid polyacrylamide and an equimolar mixture of two "separators", 0.33 M beta-alanine and 0.33 M 6-aminocaproic acid. The last two compounds flatten the pH gradient in the pI region of the two Hbs, thus allowing full separation. Additionally, the Hb samples, instead of being pulse-loaded, are uniformly distributed in the background electrolyte. A longer capillary life-time is obtained if all nonbuffering ions are eliminated; thus, as catholyte, 50 mM Lys (pH 9.7) is utilized and as anolyte 50 mM acetic acid (pH 3.5) is adopted. The percentages of Hb A1c, as obtained by capillary IEF, are in good agreement (+/- 6%) with data obtained by one of the standard zone electrophoretic methods in clinical chemistry, i.e., the Helena REP Glyco gel system.     

Two-dimensional electrophoretic separation of yeast proteins using a non-linear wide range (pH 3-10) immobilized pH gradient in the first dimension; reproducibility and evidence for isoelectric focusing of alkaline (pI > 7) proteins

The proteome of the yeast Saccharomyces cerevisiae was analysed by two-dimensional (2D) polyacrylamide gel electrophoresis utilizing a non-linear immobilized pH gradient (3-10) in the first-dimensional separation. Cells were labelled by [35S]methionine incorporation in the respiro-fermentative phase during exponential growth on glucose. Gels were run, visualized with phosphoimager technology and all resolved proteins automatically quantified. Proteins were well resolved over the whole pH interval, and evidence for isoelectric focusing on the basic side of the pattern was generated by sequencing of some spots, revealing the 2D positions of Tef1p, Pgk1p, Gpm1p, Tdh1p and Shm2p. Roughly 25% of the spots were resolved at the alkaline side of the pattern (pI > 7). The position reproducibility was high and in the range 1-2 mm in the x- and y-dimension, respectively. No quantitative variation was linked to a certain size or charge class of resolved proteins, and the average quantitative standard deviation was 17 +/- 11%. The obtained immobilized pH gradient based pattern could easily be compared to the old ampholine-based 2D pattern, and the previously reported identifications could thus be transferred. Our yeast pattern currently contains 43 known proteins, all identified by protein sequencing. Utilizing these identified proteins, relevant pI and Mr scales in the pattern were constructed. Normalization of the expression of identified spots by compensating for the number of methionine residues a protein contains allowed stoichiometric comparisons. The most dominant proteins under these growth conditions were Tdh3p, Fba1p, Eno2p and Tef1p/Tef2p, all being expressed at more than 500,000 copies per cell. The differential carbon source response during exponential growth on either glucose, galactose or ethanol was examined for the alkaline proteins identified by micro-sequencing in this study.     

Studies on the glycosidases of semen. Further purification and characterization of two hexosaminidases from bull seminal plasma

Two isozymes of beta-N-acetylglucosaminidase (2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxy glucohydrolase, EC 3.2.1.30) (A and B) from bull seminal plasma were purified to homogeneity by isoelectric focusing having pI values of 5.31 and 6.78. The two proteins were glycoproteins with very similar amino acid composition but isozyme A contained more sialic acid than isozyme B. The molecular weights of isozyme A and B were estimated at 200 000 and 190 000 by gel filtration. Two identical subunits corresponding to molecular weights of 53 000 and 13 400 were obtained from hexosaminidase A and B when subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Similar results were obtained when dissociation of the isozymes was effected with mercaptoethanol, guanidine hydrochloride and urea in presence of sodium dodecyl sulphate and the subunits separated by acrylamide gel electrophoresis. The two isozymes were more stable in frozen conditions than at the refrigerated temperature. Of the divalent ion tested, glucosaminidase and galactosaminidase activities of isozymes A and B were strongly inhibited by Hg2+ and Ag+ thus suggesting the presence of thiol groups in the two proteins. The two isozymes were active on natural substrates; isozyme B being more active than isozyme A.     

A simple method for the determination of isoelectric points of ampholytes with closely spaced pKa values using pressure-mediated capillary electrophoresis

A simple method, based on a modified version of pressure-mediated capillary electrophoresis (PreMCE) has been developed for the determination of the isoelectric points of ampholytes which have closely spaced pKa values. This new pI-determination PreMCE method (i) can be easily executed on most commercial capillary electrophoresis instruments; (ii) it can use small, impure samples, (iii) unlike isoelectric focusing methods in natural pH gradients, it does not require a linear pH gradient, and (iv) it eliminates the pI errors that are due to chromatographic retention on the walls of the separation chamber.     

New set-up for capillary isoelectric focusing in uncoated capillaries

Substituted aminomethylphenol dyes, low-molecular-mass isoelectric point (pI) markers and hemoglobin samples from normal individuals and diabetic patients were used to test a new set-up of capillary isoelectric focusing (cIEF) in uncoated capillaries. In previous cIEF methods, a mixture of sample components and carrier ampholytes was applied in the capillary and analyzed. In the new set-up a fractionated injection protocol is used to apply a 'sandwich' ampholyte-sample-ampholyte plug in the capillary for analysis. This new set-up allows the separation of amphoteric compounds having pI values outside the pH region of the ampholytes applied in the capillary with high precision. The high resolution power of this technique was proven with the analysis of hemoglobin variants.     

Capillary zone electrophoresis of oligonucleotides and peptides in isoelectric buffers: theory and methodology

The use of isoelectric buffers in capillary zone electrophoresis is reviewed. Such buffers allow application of extremely high voltage gradients (up to 1000 V/cm in relatively high bore capillary, e.g. 75 to 100 microm internal diameter), permitting separations of the order of a few minutes and thus favoring high resolution due to minimal, diffusion-driven peak spreading. The fundamental properties of ampholytes are first discussed, such as buffering power (beta) as a function of delta pK, i.e. of the distance between the pI value and neighboring protolytic groups. The highest possible relative beta value (= 2) is obtained for amphoteres possessing a delta pK = 0.6, a condition not met by existing amphoteric species. A novel parameter for ampholyte evaluation is then proposed, namely the beta/lambda ratio, i.e. the ratio between the beta power and conductivity at the pI value. It is additionally shown that the pI is not a constant value, but depends on ampholyte concentration in solution. In addition, at constant concentration, the theoretical pI can change as a function of delta pK. Isoelectric His and, to a lesser extent, Lys have been found to offer unique separations of oligonucleotides in sieving liquid polymers. In the absense of sieving media, isoelectric Asp, in presence of 7 M urea (apparent pH 3.77), permits unique separations of oligonucleotides having the same length but different nucleotide composition. Isoelectric Asp (pI 2.77 at 50 mM concentration) provides a medium of high resolving power for generating peptide maps. In difficult cases, of coincident titration curves, the pH can be moved up to higher values (e.g. pH 3.0 for 30 mM Asp) thus eliciting separation of unresolved peptides at pH 2.77. This was illustrated by running peptide maps of tryptic digests of human beta-globin chains. Also imino diacetic acid (pI 2.33 at 50 mM concentration) allows generation of high resolution peptide maps.     

Electrophoretic separation of recombinant tissue-type plasminogen activator glycoforms: validation issues for capillary isoelectric focusing methods

Attempts were made to validate a capillary isoelectric focusing (cIEF) method for a recombinant glycoprotein as an alternative technique to slab gel isoelectric focusing methods routinely used to monitor such charge heterogeneity. The cIEF method principally separates the charged glycoforms of recombinant tissue-type plasminogen activator (rt-PA) on the basis of their sialic acid content. Nine to ten distinct peaks were consistently resolved, with the profile dependent on the class of ampholyte used. The pI of rt-PA measured with synthetic pI standards was in the range pH 6.5-7.5 with the migration of the standards affected by the presence of the protein. The method showed an acceptable recovery of > 100% and had good sensitivity where 25 ng of protein could be resolved into constituent peaks. Recovery of both major peaks and total protein measured by peak areas was linear over a wide range from 50-1000 micrograms/mL. A detailed study showed that when a capillary had been used for some time, capillary age affected peak migration times and, to a lesser extent, resolution. Peak migration times were stable over a temperature range of 15-30 degrees C, and decreased predictably with increasing voltages (400-600 V/cm) and decreasing N,N,N',N'-tetramethylethylene diamine (TEMED) concentrations (0.4-1.5% v/v). Overall the data indicated that this methodology has the potential to be used in the commercial release of protein pharmaceuticals if variability resulting from capillary age and lot were resolved. Even in its present format the method equals the performance of slab gel IEF whilst offering significant improvements in ease of operation and in time and reagent use.     

Proteinases of rat mast cells. Peritoneal but not intestinal mucosal mast cells express mast cell proteinase 5 and carboxypeptidase A

Six basic proteins of 26 to 38 kDa with isoelectric points (pI) > or = 8.5 were abundant in proteins separated by two-dimensional SDS-PAGE from adult rat peritoneal mast cells (MC). One was identified previously as rat mast cell proteinase (RMCP) 1, a chymase of 26 to 28 kDa, pI > 9.0. Microsequence analyses showed that two polypeptides of about 29 and 30 kDa had NH2 terminal amino acid sequences homologous to mouse MC proteinase 5 (MCP-5), whereas the amino terminals of the 33, 35, and 36 kDa proteins were homologous to MC carboxypeptidase A (MC-CPA). Rabbit Abs produced against synthetic peptides of the identified NH2 terminal sequences were used in immunoblot studies. At least three proteins reacted with Abs to MC-CPA, whereas Abs to MCP-5 detected three adjacent polypeptides, rather than just the two identified by using microsequence analysis. Removal of oligosaccharide side chains using peptide:N-glycosidase F reduced the heterogeneity of each set of three polypeptides (MCP-5 and MC-CPA) to a band of each protein of a lower M(r). The serine proteinase inhibitor [3H]diisopropylfluorophosphate ([3H]DFP) bound to a proteinase of 30 to 35 kDa, which is probably MC tryptase (pI < or = 6.0). Immunoblot analysis of proteins from intestinal mucosal mast cells showed RMCP-2, but not RMCP-1, MCP-5, or MC-CPA. This is the first report of MCP-5 in the rat and of clearly distinguishable glycosylated forms of MC CPA. These proteinases appear to be restricted in their distribution to selected MC populations, but little is known about their functions.     

Effects of renovascular hypertension on myocardial protein patterns: analysis by computer-assisted two-dimensional gel electrophoresis

Hypertensive heart disease caused by renovascular hypertension reflects the response of the heart to an increased afterload and neurohormonal stimulation. We hypothesized that in this condition the composition of the myocardial proteins of rats was altered. To identify yet unknown quantitative and qualitative differences in myocardial proteins in renovascular hypertensive heart disease, we analyzed protein patterns by computer-assisted two-dimensional polyacrylamide large gel electrophoresis. Renovascular hypertension was induced by placing a silver clip on the left renal artery in 9-week-old rat siblings. Sham-operated animals served as controls. Systolic blood pressure (197 +/- 19 mm Hg) and heart/body weight ratios (0.36 +/- 0.04%) were significantly increased in the hypertensive animals. Twenty protein patterns from the left ventricle of five hypertensive and five control rats were compared. The molecular mass and isoelectric point (pI) of proteins spots ranging from 13 to 100 kDa and from 4.5 to 8.5, respectively, were determined using marker proteins. In total, 761 +/- 88 protein spots were resolved in all twenty gels. For the quantitative data analysis a univariate (Mann-Whitney test) as well as a multivariate statistical approach (correspondence analysis) were applied. Only one myocardial protein spot (molecular mass = 41.3 kDa; pI = 6.3) was decreased by more than twofold (p < 0.05) in renovascular hypertension. The vast majority of spots did not indicate a significant alteration of intensity. Left ventricular hypertrophy in early renovascular hypertension induces a form of myocardial hypertrophy that conserves the naturally occurring protein expression pattern.     

The Dictyostelium discoideum proteome--the SWISS-2DPAGE database of the multicellular aggregate (slug)

The cellular slime mold Dictyostelium discoideum is a eukaryotic microorganism which has developmental life stages attractive to the cell and molecular biologist. By displaying the two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) protein map of different developmental stages, the key molecules can be identified and characterised, allowing a detailed understanding of the D. discoideum proteome. Here we describe the preparation of reference gel of the D. discoideum multicellular aggregate, the slug. Proteins were separated by 2-D PAGE with immobilised pH gradients (pH 3.5-10) in the first dimension and sodium dodecyl sulfate (SDS)-PAGE in the second dimension. Micropreparative gels were electroblotted onto polyvinylidene difluoride (PVDF) membranes and 150 spots were visualised by amido black staining. Protein spots were excised and 31 were putatively identified by matching their amino acid composition, estimated isoelectric point (pI) and molecular weight (M(r)) against the SWISS-PROT database with the ExPASy AAcompID tool (http:// expasy.hcuge.ch/ch2d/aacompi.html). A total of 25 proteins were identified by matching against database entries for D. discoideum, and another six by cross-species matching against database entries for Saccharomyces cerevisiae proteins. This map will be available in the SWISS-2DPAGE database.     

SS-interchanged and oxidized isomers of bovine serum albumin separated by isoelectric focusing

Column isoelectric focusing separates commercial bovine serum albumin in 5 fractions with isoionic points in the vicinity of that of mercaptalbumin (pI 5.24). About 20% of the bovine albumin have isoionic points higher than mercaptalbumin and are split into two fractions, both recognized as SS-interchanges isomers: (1) pI 5.39 is the "aged" albumin described by Nikkel and Foster (1971, Biochemistry 10, 4479); (2) pI 5.45 represents a further degree of SS-interchange, catalyzed by small amounts of cysteine in the solution ('cysteine-aged' albumin). In 6 M urea the "cysteine-aged" albumin is electrofocused to the same pH value as mercaptalbumin. In 6 M urea 40% of commercial albumin focuses in 3 fractions with isoionic points lower than mercaptalbumin. This percentage will increase during incubation at oxidizing conditions ("oxidized" albumin). Electrofocused in water the oxidized fractions have isoionic points at pI 5.28, 5.18 and 5.12, respectively. The shifts in isoionic point of the "oxidized" albumins are caused by irreversible changes in the primary structure. Although the free SH group of albumin is oxidized during the oxidation reaction, the observed changes in isoionic points are caused by modifications of some other amino acid residues. Both "cysteine-ageing" and "oxidation" are inhibited by alkylation of the SH group. "Cysteine-ageing" is furthermore inhibited when the bovine albumin is "oxidized".     

Study of haptoglobin-hemoglobin complexes by titration curves, capillary electrophoresis and capillary isoelectric focusing

A novel method is described for monitoring complex formation between macromolecules, based on combined isoelectric focusing-electrophoresis in capillaries. The example studied is the binding of serum haptoglobin (Hp) to hemoglobin (Hb). A known amount of Hb is focused in a capillary in a pH 6-8 range (pI of Hb = 7.0) and thus kept temporarily "immobilized" in the electrophoretic chamber. Subsequently, increasing amounts of ligand (Hp) are loaded cathodically and allowed to sweep past the focused Hb zone. As the complex formed has a pI value well-outside the bounds of such a pH gradient (the 1:1 molar Hb-Hp complex has a pI of 5.5, the 1 to 1/2 molar Hp-Hb complex has a pI of 5.0) it escapes immobilization and moves past the detector window, where it is monitored and quantified. Since the detector is set at 416 nm, where only Hb absorbs, and since the molar extinction coefficient of Hb is well known, it is quite easy to calculate the molar amount of Hb bound to the complex. As an additional check, the amount of unreacted Hb can now be mobilized by disrupting the pH gradient and allowing this residual free Hb to also reach the detector and be quantified. The method is easy, fast, simple and fully automated and thus could represent a valid alternative to existing methods in clinical chemistry for quantifying the amount of Hp in human sera in pathological conditions, such as hemolytic anemias and transfusion reactions.     

Purification of larval Taenia solium antigens by isoelectric focusing

By preparative isoelectric focusing in a rotating ampholine column, crude cystic membrane (M) or fluid (F) antigens of larval Taenia solium were each separated into 20 fractions. M fractions were less specific and sensitive than F fractions in detecting cysticercosis antibodies in pig serum. Among the F fractions, F15 showed the best potential to serve as a screening antigen. It contained 18 polypeptides, with pI 5.3-8.2 and a specific epitope of 25 kDa which was detected by immunoblotting. Although F15 showed slight cross-reactions with heterologous antisera in double-antibody IgG enzyme-linked immunosorbent assays (ELISA), it yielded the highest absorbance values when tested against homologous antisera. The antigen was used to screen sera samples from 4870 pigs slaughtered in Hong Kong and five other Chinese cities for cysticercosis antibodies by double-antibody ELISA, Falcon Assay Screening Test (FAST)-ELISA and enhanced chemiluminescent immunoassay. The results varied significantly between assays. However, the samples collected from Shenzhen yielded the highest positive rates. Enhanced chemiluminescent immunoassay based on camera-luminometry was found suitable for use under field conditions.     

Electrophoretic studies of non-histone proteins from pig lymph nodes

Purified chromatin from pig lymph nodes has been prepared and used for the isolation of chromatin proteins and non-histine proteins. Saccharose nuclei from this tissue have been used for the preparation of nuclear phenol-soluble phosphoproteins. The isolated proteins have been compared by analytical electrophoresis in polyacrylamide gel in sodium dodecyl sulfate. Most chromatin non-histone proteins and nuclear phosphoproteins of pig lymph nodes have a molecular weight about 40 000 daltons and show a significant degree of homology of molecular range from 40 000 to 90 000 daltons. The phosphoproteins have been also analyzed by isoelectric focusing in polyacrylamide gel over the pH range of 3-10. They are distributed throughout the pI range of 5.8-9.0. The chromatin non-histone proteins and phosphoproteins are of acidic nature and contain 0,97 per cent tryptophan.     

Quick and easy molecular weight determination with Macintosh computers and public domain image analysis software

The program "molecular weights" allows a fast and easy estimation of molecular weights (M(r)), isoelectric point (pI) values and band intensities directly from scanned, polyacrylamide gels, two-dimensional protein patterns and DNA gel images. The image coordinates of M(r) and pI reference standards enable the program to calculate M(r) and pI values in a real time manner for any cursor position. The program requires NIH-Image for Macintosh computers and includes automatic band detection coupled with a densitometric evaluation.     

Unique identification of proteins from small genome organisms: theoretical feasibility of high throughput proteome analysis

We evaluate current levels of accuracy for estimation of molecular weight (Mr) and isoelectric point (pI) to proteins on two-dimensional (2-D) gels as well as the distribution and clustering of proteins in the predicted proteome of E. coli. We also examine the ability to find single candidates within the predicted proteome for matching to a protein seen on 2-D gels, based on the current level of accuracy. We discuss the levels of accuracy needed to match predicted proteins to observed proteins based solely on Mr and pI criteria obtained from genomic information, and propose methodology to achieve this level of accuracy. In addition, we will address the future goals of this work since the small genomes of bacteria provide a foundation and stepping stone to similar studies in higher organisms.     

Rabbit red blood cell hexokinase. Purification and properties

Rabbit red blood cell hexokinase (EC 2.7.1.1.) has been purified 300,000-fold by a combination of ion exchange chromatography, affinity chromatography, and preparative polyacrylamide gel electrophoresis. The hexokinase activity has been isolated in 35% yield as a protein that is homogeneous by polyacrylamide and sodium dodecyl sulfate gel electrophoresis. The highest specific activity obtained was 145 units/mg of proteins. The native protein has a molecular weight of 110,000 by gel filtration on Ultrogel AcA 44 and 112,000 by sedimentation velocity on sucrose density gradients. Sodium dodecyl sulfate-polyacrylamide gels gave a molecular weight of 110,000 indicating that hexokinase is a monomer. The enzyme had a pI of 6.20 to 6.30 pH units by isoelectric focusing. The enzyme was specific for Mg . ATP and Mg . ITP as the nucleotide substrates. Several hexokinase with different affinities.     

Binding effects in proton-nucleus elastic scattering

Recombinant human FSH (rhFSH) was obtained by expressing the human FSH alpha- and beta-subunit complementary DNAs in the chinese hamster ovary cell line. Isoforms of rhFSH were resolved into specific isoelectric (pI) fractions by chromatofocusing. rhFSH isoforms ranged from pI 3.0-5.5 with a modal value of pI 4.2. Analysis of the biological activity of specific pI isoforms of rhFSH was undertaken using both the rat granulosa cell aromatase (in vitro) bioassay and a RRA. More acidic isoforms (e.g. pI 3.5) showed significantly lower affinity (P < 0.05) for rat testicular FSH receptors than did the less acidic isoforms (e.g. pI 4.8). Consistent with the receptor binding affinity data, the more acidic fractions resulted in significantly less activation (P < 0.05) of rat granulosa cell aromatase activity, as measured by estrogen production, than did the less acidic isoforms. The observed bioactivities and their correlation with the pI values of the rhFSH isoforms are consistent with observations of differing bioactivities seen in both pituitary and urinary FSH isoforms. These results demonstrate that rhFSH, made in the chinese hamster ovary cell line, is both biologically active and has isoform profiles, and presumably carbohydrate structures, that closely resemble those seen in natural hFSH.