Influence of fatty acid and time of focusing on the isoelectric focusing of human plasma albumin

In the isoelectric focusing of human plasma albumin, two major peaks of pI 4.8 and 5.6 are observed. As fatty acids are removed from the albumin either by defatting before the focusing experiment or gradually during the focusing experiment, the pI 4.8 peak diminishes and the pI 5.6 peak increases. The interpretation of this effect is confirmed by fatty acid analysis before and after focusing. Alkylation of the free sulfhydryl group changes the focusing position of the defatted peak to pI 5.7. Otherwise the isoelectric focusing pattern of human albumin is unaffected by the status of the free sulfhydryl group or by the ampholine concentration.     

Hepatocyte [3H]-palmitate uptake: effect of albumin surface charge modification.

The role of plasma proteins on the cellular uptake of lipophilic substrates has perplexed investigators for many years. We tested the hypothesis that an ionic interaction between the protein-ligand complex and hepatocyte surface may be responsible for supplying more ligand to the cell for uptake. The surface-charged groups on albumin were modified to yield proteins having a range of isoelectric points (ALB, ALBs, ALBm, ALBe had values of 4.8-5.0, 4.5-4.7, 3.0-3.5, 8.4-8.6, respectively). [3H]-Palmitate uptake studies were performed with adult rat hepatocyte suspensions using similar unbound ligand fractions in the presence of the different binding proteins. Mass spectrometry, isoelectric focusing (pI), and heptane:water partitioning were used to determine protein molecular weight, pI, and protein-palmitate equilibrium binding constant, respectively. Hepatocyte [3H]-palmitate clearance in the presence of ALBs and ALBm were significantly lower (p < 0.05) than ALB, whereas [3H]-palmitate clearance in the presence of ALBe was significantly higher (p < 0.05) than ALB. The data were consistent with the notion that ionic interactions between extracellular protein-ligand complexes and the hepatocyte surface facilitate the uptake of long-chain fatty acids.     

Preliminary characterization of a cloned neutral isoelectric form of the human peptidyl prolyl isomerase cyclophilin

We report the cloning of a neutral isoelectric form of the human peptidyl prolyl isomerase, cyclophilin, its expression in Escherichia coli, and its purification and comparison to bovine thymus cyclophilin. The cloned protein exhibited a pI of approximately 7.8 and formed a simple 1:1 complex with cyclosporin A. This cloned form had a pI similar to that observed for the neutral isoform (pI approximately 7.4) of human splenocyte cyclophilin. The bovine thymus proteins exhibited anomalous behavior on CM-cellulose chromatography but were resolved into alkaline (pI approximately 9.3) isoforms and a new neutral (pI approximately 7.8) isoform by isoelectric focusing gel electrophoresis and ultimately into at least four discrete isoforms by capillary electrophoresis. For cyclosporin A binding we observe a Kd of approximately 160 nM for an electrophoretically heterogeneous preparation of the natural bovine protein and approximately 360 nM for the more homogeneous preparation of the cloned human neutral isoform. Stopped-flow measurements of the activation energies for peptidyl-prolyl isomerase activity indicate the recombinant human protein has an activation enthalpy of 3.67 kcal/mol and an activation entropy of -47.3 cal/K-mol for cis----trans isomerization.     

Purification and characterization of human transcortin.

Human transcortin was purified to apparent homogeneity from plasma by a two-step procedure involving affinity and hydroxyapatite chromatography. The affinity gel incorporated denatured bovine serum albumin as the spacer and cortisol hemisuccinate as the ligand. Although isolated transcortin showed a propensity for spontaneous polymerization according to a geometric progression (1, 3, 9) only one band was observed on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Cortisol-binding activity of the isolated protein gave an apparent association constant of 2.5 X 10(8) M-1 at 4 degree C in equilibrium dialysis. Isoelectric focusing of purified native transcortin showed six discrete bands, five between pH 3.75 and 4.15 and another, possibly desialylated, at pH 6.15. Desialylated transcortin also gave six bands on isoelectric focusing, with pI values ranging from 4.90 to 6.30.     

Isolation of a hibernation inducing trigger(s) from the plasma of hibernating woodchucks

Plasma from hibernating woodchucks was desalted utilizing a hollow fiber device having a M. W. cut-off of 5,000. This preparation was fractionated by isoelectric focusing (IEF) in a pH gradient extending from 3.5 to 10.0 resulting in protein components having isoelectric points (pIs) of 4.5, 5.2, 5.5, 6.3, and 7.0. Fraction I (comprised of proteins having pIs of 4.5 and 5.2) induced hibernation within 2 to 6 days in 8 out of 10 summer-active ground squirrels. Fraction II (pI 5.5) and Fraction III (pI 6.3 and 7.0) failed to induce any summer hibernation in 10 animal test groups at identical sample concentrations. Polyacrylamide gel electrophoresis of Fraction I indicated that albumin was a major constituent of this still heterogeneous preparation. Thus, in order to more clearly define the plasma locus of this hibernation inducing trigger(s) (HIT) molecule, whole plasma and/or Fraction I was fractionated by 3 distinct resolving techniques. These included sub-fractionation of Fraction I by isoelectric focusing utilizing a narrower pH gradient extending from 3.5 to 6.0, isotachophoresis of whole plasma and affinity chromatography of Fraction I and whole plasma. A total of 40 summer-active ground squirrels were injected and assayed for HIT activity with fractionated preparations derived by the three previously cited separation techniques. A total of 18 of these summer-active ground squirrels hibernated. However, a much more impressive figure is that 16 out of 21 animals hibernated when injected with resolved hibernating plasma fractions in which albumin was the predominant plasma protein. A total of 8 control animals were injected with vehicle and none of these hibernated.     

Identification of albumin as the serum factor essential for the growth of activated human lymphocytes.

Albumin from human, bovine, or rabbit serum supported the growth of concanavalin A-stimulated human thymus-derived lymphocytes equally well. This activity was completely abolished by pepsin digestion. It was shown for bovine serum albumin that the albumin molecule itself, and neither an impurity nor a factor bound to albumin was essential for the growth of lymphocytes. This conclusion was based on observations that the growth-promoting activity could not be removed from albumin, and that the specific activity of albumin remained unaltered after the following procedures: molecular sieving at pH 7.5 at pH 3.0, and in 8 M urea at pH 6.6; ion exchange chromatography at pH 4.3 and in 8 M urea at pH 7.2; isoelectric focusing; charcoal treatment; acetone precipitation; and reduction with 2-mercaptoethanol in the presence of 8 M urea. Dimeric albumin was found to support growth of lymphocytes as well as monomeric albumin, and mercaptalbumin and non-mercaptalbumin were shown to have equal activity.     

Isoelectric-focusing properties and carbohydrate content of pea (Pisum sativum) legumin

Legumin from pea (Pisum sativum) is a molecule made up of six pairs of subunits, each pair consisting of an `acidic' subunit (mol.wt. about 40000) and a `basic' subunit (mol.wt. about 20000) linked by one or more disulphide bonds. The heterogeneity of legumin has been investigated by isoelectric focusing; undissociated legumin could not be focused satisfactorily, but legumin subunits could be analysed under dissociating conditions. 8m-Urea was not found to be a satisfactory medium for isoelectric focusing of legumin, as the `basic' subunits showed a shift in pI with time of incubation in urea. A new dissociating medium for isoelectric focusing, namely 50% (v/v) formamide, was used for analysis of legumin, which gave pI values of 5.0–5.3 for the `acidic' subunits, and 8.3–8.7 for the `basic' subunits. Both types of subunits were shown to be heterogeneous in charge and molecular weight by two-dimensional analysis employing isoelectric focusing in the first dimension and sodium dodecyl sulphate/polyacrylamide gel electrophoresis in the second. The `basic' and `acidic' subunits of legumin were separated on the preparative scale by ion-exchange chromatography in 50% formamide. Carbohydrate attached to the protein was investigated as a possible cause of the heterogeneity of legumin subunits. However, both a fluorescent-labelling technique and a sensitive radioactive-labelling technique failed to show any carbohydrate bound to legumin subunits, and it was concluded that legumin is not a glycoprotein.     

Artifactual isoform profile modification following treatment of human plasma or serum with protease inhibitor, monitored by 2-dimensional electrophoresis and mass spectrometry

The inclusion of protease inhibitors in serum or plasma samples has been found to significantly impact the isoform profile of selected plasma proteins as seen on 2-dimensional electrophoresis (2-DE) gels. With the addition of a protease inhibitor cocktail, several human plasma protein trains [depleted of albumin and immunoglobulin G (IgG)] exhibited higher isoelectric point (pI) isoforms. This shift was especially apparent for apolipoprotein A1 (apo A1), a relatively high abundance protein. The six protease inhibitor components of the cocktail were individually investigated with albumin and IgG depleted human plasma, and it was shown that the observed effects were caused by 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), a serine protease inhibitor that covalently modifies proteins and/or peptides. Several serine-and/or tyrosine-containing peptides of apo A1 were modified with a concomitant mass increase of 183 Da, which is consistent with the mass increase expected following reaction with AEBSF. These modifications were observed with increasing propensity in the higher pI spots. An increase in both the number and proportion of modified peptides with increasing pI was also observed. A model is proposed for the random or stochastic coupling of AEBSF-derived moieties to serine and/or tyrosine residues throughout apo A1 and potentially other plasma proteins.     

A comparative study of vitamin D binding globulins in milk.

1. The occurrence of 25-hydroxy vitamin D binding protein in human, bovine, monkey and porcine milk was investigated. 2. Sucrose gradient ultracentrifugation revealed the presence of 4.2 S and 5.7 S binding globulins in the whey of human, monkey and porcine milk. 3. Although bovine plasma also contains a 4.2 S globulin only a 5.7 S protein was found in bovine milk. 4. The 4.2 S and 5.7 S globulins in milk could not be resolved by polyacrylamide gel electrophoresis or by isoelectric focusing. 5. Plasma and whey binding proteins of any one species had the same isoelectric point but there were small differences among species (4.5-4.8). 6. Competitive displacement studies showed that the binding proteins in milk have high affinity for 25-hydroxy-cholecalciferol and 24,25-dihydroxycholecalciferol.     

Two-dimensional peptide mapping by polyacrylamide-gel electrophoresis with limited proteolysis in SDS

The peptide mapping method described by Cleveland, et al. (1) was improved to a two-dimensional analysis applicable to minute amounts (less than 0.5 microgram) of proteins. Radioiodinated proteins for analysis were purified by electrophoretic elution of the proteins from polyacrylamide gels into buffer containing 0.1% sodium dodecyl sulfate. The proteins were digested enzymatically in the presence of 0.1% sodium dodecyl sulfate and an excess of nonlabeled bovine serum albumin (0.2 mg/ml) relative to labeled substrate in order to attain reproducibility by maintaining a consistent substrate concentration among different samples. The peptides of these limited proteolytic products were resolved by two-dimensional polyacrylamide gel electrophoresis (isoelectric focusing followed by SDS-gels). The resulting 2D-peptide maps of murine and bovine albumin and a murine lymphocyte membrane protein, Tp100, showed excellent resolution and reproducibility.     

Cardiac fatty acid-binding proteins. Isolation and characterization of the mitochondrial fatty acid-binding protein and its structural relationship with the cytosolic isoforms

In the course of our studies on the structural diversity of the isoforms of cardiac fatty acid-binding proteins (cFABPs), a cardiac-type FABP from the matrix of bovine heart mitochondria was purified to homogeneity and obtained as a single 15-kDa protein with an isoelectric point of 4.9. The primary structures of this protein and of the two isoforms isolated from the cytosol (pI4.9-cFABP and pI 5.1-cFABP) were investigated by means of plasma desorption mass spectrometry and sequencing of peptides. All three proteins are amino-terminally blocked with an acetyl group and shown to be colinear with the sequence deduced from a cDNA clone for bovine heart fatty acid-binding protein (Billich, S., Wissel, T., Kratzin, H., Hahn, U., Hagenhoff, B., Lezius, A. G., and Spener, F. (1988) Eur. J. Biochem. 175, 549-556) except for the residue at position 98. This residue is demonstrated to be the molecular origin of bovine cFABP isoforms since pI 5.1-cFABP contains Asn98 in accordance with the sequence derived from the cDNA, whereas in pI 4.9-cFABP, this position is occupied by Asp98. Moreover, mitochondrial FABP is identical to pI 4.9-cFABP. Molecular masses of pI 4.9-cFABP (14,679 +/- 10 Da) and pI 5.1-cFABP (14,678 +/- 20 Da) determined by plasma desorption mass spectrometry coincide with that calculated from the cDNA (14,673 Da). Hence, residues linked to these proteins by posttranslational modification are not present, and the Asn-Asp exchange is the sole origin of heterogeneity of mitochondrial and cytosolic fatty acid-binding proteins from bovine heart.     

Presence of serum albumin in normal human epidermis: Possible implications for the nutrition and physiology of stratified epithelia

In this paper, using both immunofluorescence and protein biochemistry techniques, we present definitive evidence that plasma proteins such as albumin are present within normal human epidermis. This result confirms several previous reports supporting the idea that relatively large molecules can diffuse through the epidermal basement membrane into epidermis. Our results bring new insights for discussing how hydrophobic ligands or drugs present in the bloodstream and bound to plasmatic carriers can reach epidermal cells of all layers.Abbreviations CHAPS 3-[3-cholamidopropyl dimethylammonio] propane sulfonate - kD kilodaltons - BSA bovine serum albumin - 2ME 2-mercaptoethanol - DTT dithiothreitol - SDS sodium dodecyl sulfate - pI isoelectric point - Mw molecular weight - Tris Tris-(hydroxymethyl) aminomethane - 1D one dimensional - 2D two dimensional - PAGE poly acrylamide gel electrophoresis - MEM Minimal Eagle's Medium     

Burkavidin: a novel secreted biotin-binding protein from the human pathogen Burkholderia pseudomallei

The avidin-biotin technology has many applications, including molecular detection; immobilization; protein purification; construction of supramolecular assemblies and artificial metalloenzymes. Here we present the recombinant expression of novel biotin-binding proteins from bacteria and the purification and characterization of a secreted burkavidin from the human pathogen Burkholderia pseudomallei. Expression of the native burkavidin in Escherichia coli led to periplasmic secretion and formation of a biotin-binding, thermostable, tetrameric protein containing an intra-monomeric disulphide bond. Burkavidin showed one main species as measured by isoelectric focusing, with lower isoelectric point (pI) than streptavidin. To exemplify the potential use of burkavidin in biotechnology, an artificial metalloenzyme was generated using this novel protein-scaffold and shown to exhibit enantioselectivity in a rhodium-catalysed hydrogenation reaction.     

Fast protein chromatofocusing of human very-low-density lipoproteins

Using fast protein chromatofocusing, a high-efficiency column chromatography method with a self-generated pH gradient and focusing effects, soluble human very-low-density lipoprotein (VLDL) apolipoproteins were fractionated between pH 6.3 and 4.0. In the presence of 6 mol/l urea and with a flow rate of 1 ml/min, one run (up to 10 mg of protein) took 30 min. VLDL apolipoproteins were separated in seven peaks. As revealed by SDS-polyacrylamide gel electrophoresis, isoelectric focusing and double-immunodiffusion against mono-specific antisera, fractions corresponded to the following proteins: apolipoprotein C-I, albumin, apolipoproteins A-I, E, C-II plus C-III0, C-III1 and C-III2, respectively. Apolipoproteins were eluted in sharp, well-resolved peaks. The recovery of proteins was 78% of the starting material. With fast protein chromatofocusing, an efficient isolation of single apolipoproteins is possible from small amounts of VLDL apolipoprotein preparations. This technique is superior to the commonly used, time-consuming methods for apolipoprotein isolation.     

Anion exchange fractionation of serum proteins versus albumin elimination

Elimination of albumin, constituting more than 50% of total serum proteins, allows increased protein loads on immobilized pH gradient (IPG) gels and better visualization of low-abundance proteins; however, it may result in the loss of albumin-bound low-abundance proteins. In this study, we report the prefractionation of serum proteins by batch anion exchange chromatography into three fractions: one containing proteins with isoelectric points (pI values) higher than the pI of albumin, a second fraction containing proteins with pI values in the same range as the pI of albumin, and a third fraction containing proteins with pI values lower than the pI of albumin. This procedure uses common instrumentation, is carried out under denaturing conditions, and takes less than 30min. We also report the loss of a clinically established prostate cancer serum biomarker, prostate-specific antigen (PSA), after albumin is eliminated using two commercially available albumin elimination kits: one that uses Cibacron Blue F3GA, which achieves albumin depletion through dye-ligand binding, and one that uses specific albumin antibody. The loss of PSA secondary to albumin elimination exceeded that after batch anion exchange serum sample prefractionation.     

Distribution of proteins inside polyelectrolyte microcapsules depend on pH of medium

Distribution of bovine serum albumin and ferritin inside polyelectrolyte microcapsules was studied by transmission electron and confocal microscopy at the pH range 2-5. It was estimate that protein's distribution depends on isoelectric point (pI) and first polyelectrolyte used for preparation of capsule shell. So peptide is placed in the bulk of capsule if pH values of medium are lower isoelectric point of protein and polycation was used as a first polyelectrolyte layer. If the first polyelectrolyte was polyanion, the protein is located near internal surface of the shell. The protein is situated near internal surface of the shell for both polyelectrolytes when pH is equal to pI.     

Purification of plant complex protein extracts in non-denaturing conditions by in-solution isoelectric focusing

An alternative approach for plant complex protein extracts pre-purification by in-solution isoelectric focusing in non-denaturing conditions is presented. The separation of biologically active proteins, in narrow ranges of isoelectric point (pI) was obtained by a modified OFFGEL electrophoresis. Two different water-soluble protein extracts from Phragmites leaves were fractionated into 24 fractions within a 3–10 pI range at 10 °C in the absence of denaturing/reducing agents. One-dimensional electrophoretic analysis revealed different protein distribution patterns and the effective fractionation of both protein extracts. Peroxidase activity of each fraction confirmed that proteins remained active and pre-purification occurred. Biological triplicates assured the needed reproducibility.     

Superoxide dismutase isoenzymes in bovine and human milk

The presence of superoxide dismutase in bovine and human milk was investigated by ultrafiltration, gel filtration, and isoelectric focusing. Conclusive evidence for the presence of this enzyme in both milks is presented. The molecular weight of the enzyme was estimated by gel filtration on Sephadex G-100 to be 30,000, which is consistent with reported values for the copper, zinc form of superoxide dismutase. In addition, enzyme activity was inhibited by cyanide, thus eliminating the possibility that the enzyme was present in the manganese form. Several isoenzymes were detected by isoelectric focusing in polyacrylamide gel, and the isoenzyme pattern in bovine milk was the same as that found for bovine plasma, suggesting that milk superoxide dismutase originates from plasma. It may be that the presence of copper, zinc superoxide dismutase in milk is important for the maintenance of its oxidative stability.     

Biosynthesis of the common precursor to vasopressin and neurophysin in vitro in transplantable human oat cell carcinoma of the lung with ectopic vasopressin production

Transplantable human oat cell carcinoma cells of the lung with ectopic vasopressin production were incubated with labeled amino acids and immunoreactive neurophysins in cell extracts were analyzed by isoelectric focusing. When the cells were incubated with L-(35S)-cysteine for 20 h, one major peak (isoelectric point; pI=5.3) and several minor peaks (pI=6.1, 5.7, 5.1, 4.9 and 4.7) of labeled proteins were observed. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the relative molecular mass (Mr) of the pI 5.7 protein was estimated to be 20,000 and that of the pI 6.1 species to be 19,000, while the remainder had a Mr of approximately 10,000. The result of the pulse-labeling experiment has clearly shown that the pI 5.7 and 6.1 proteins, which have affinity for concanavalin A, are biosynthetic precursors for the smaller form of neurophysin with a pI 5.3. When subjected to limited proteolysis with trypsin, the pI 5.7 protein generated a Mr 10,000 protein and a smaller peptide. The Mr 10,000 protein thus produced was identified as neurophysin on the basis of its pH-dependent affinity for vasopressin and the migration pattern on isoelectric focusing. The smaller peptide coeluted with synthetic arginine vasopressin and bound to neurophysin suggesting that it possesses a cysteine-tyrosyl sequence at its N-terminus. Similarly, the pI 6.1 protein liberated neurophysin and vasopressin-like peptide after incubation with trypsin. These results suggests that the glycosylated protein with a pI of 5.7 and a Mr of 20,000 is the common precursor to vasopressin and neurophysin in human oat cell carcinoma of the lung with ectopic vasopressin production. The pI 6.1 protein may be an intermediate in the conversion of the precursor to vasopressin and neurophysin.     

pH-dependent binding analysis, a new and rapid method for isoelectric point estimation

Based upon the pH-dependent binding affinity of amphoteric molecules for an ion exchanger, and by taking advantage of batch procedures, a facile method was developed for estimating isoelectric points of these molecules. The new method allows pI measurements to be accomplished within 1 h. Moreover, any possible protein-ampholyte interaction or artifact formation, as may be introduced from the presence of carrier ampholytes when conventional focusing methods are employed, is eliminated by the method. In addition, because of the short processing time, isoelectric points of proteins can be measured at any desired temperature without much risk of protein denaturation. Seven proteins with well-defined isoelectric points were examined by the method. The measured pI values were within a range of 0.2 pH unit or less of the reported values. The precision of pI measurements by the method can be even further improved with the employment of a narrower pH gradient. Since the isoelectric point is an important parameter which governs much of the art of separating proteins, the advent of a simple and rapid method for its measurement would be of use for selecting the proper strategy for protein isolation and purification.