The effects of Depo-Provera on serum protein levels in Nigerian women

The concentration of 13 serum proteins were determined in 50 women who had received the 3-monthly intramuscular injection of Depo-Provera for contraception over a mean period of 18 months and 40 women of comparable ages who served as controls. Sera from fasting subjects were used to determine the levels of each specific protein by quantitative immunodiffusion technique. Treatment with Depo-Provera produced increased serum levels of albumin, alpha 1-acid glycoprotein, alpha 2-macroglobulin, haptoglobin IgG; reduced levels of alpha 1-antitrypsin, transferrin, C3c, C4 but no change in serum IgA, IgM, C-reactive protein and ceruloplasmin. The significant alterations were observed in serum proteins that are notably synthesized by the liver, an observation consistent with the influences which gonadal hormones exert on the metabolic activities of this organ.     

Detection of serum proteins by native polyacrylamide gel electrophoresis using Blue Sepharose CL-6B-containing stacking gels

Analysis of serum proteins by native polyacrylamide gel electrophoresis is difficult because albumin is abundant in serum and interferes with the resolution of other proteins, especially alpha-antitrypsin which has mobility that is very similar to that of albumin. We present here a method in which serum proteins are separated by polyacrylamide gel electrophoresis using stacking gels containing Blue Sepharose CL-6B, which has a high affinity for albumin, lipoproteins, kinases, and pyridine-nucleotide-dependent oxidoreductases. During electrophoresis, proteins that bind to Blue Sepharose CL-6B stay in the stacking gel and do not migrate into the separating gel. As a consequence, certain proteins, including alpha(1)-antitrypsin, can be detected as clear bands. This method overcomes the requirement for fractionation of serum samples prior to electrophoresis to remove albumin and allows the simultaneous analysis of many samples.     

A stoichiometric analysis of bovine serum albumin-gossypol interactions: a fluorescence quenching study.

The interaction of gossypol with bovine serum albumin, human serum albumin and n-bromosuccinimide-modified bovine serum albumin has been followed by fluorescence quenching measurements. The presence of a high affinity site (association constant K = 2.2 x 10(6) M-1) for gossypol on bovine serum albumin and human serum albumin is indicated. The stoichiometry of binding for the high affinity site was evaluated using Job's method of continuous variation, thereby suggesting the formation of 1:1 complex. Modification of the tryptophan residues on bovine serum albumin does not affect the binding of gossypol to either high or low affinity site of albumin.     

Binding of radioiodinated human beta-endorphin to serum proteins from rats and humans, determined by several methods

Binding of immunoreactive radioiodinated human beta-endorphin (125I-beta-EP) to rat serum was demonstrated by gel filtration of 125I-beta-EP in pooled rat serum on Sephadex G-200. Two radioactive peaks associated with proteins eluted from the column. The first peak eluted at the void volume containing lipoproteins, alpha 2- and beta 2-macroglobulins, and the second peak at the fraction of albumin. Binding of 125I-beta-EP to albumin was directly proved by gel filtration of 125I-beta-EP in buffer containing 4% human serum albumin on Sephadex G-200. Equilibrium dialysis was not applicable to investigating the interaction of 125I-beta-EP with serum proteins, because of the intense nonspecific adsorption to the semipermeable membrane and the degradation of the peptide during dialysis. Therefore, in order to quantitatively evaluate the binding of 125I-beta-EP in sera from rats and humans, we utilized four other methods (ultrafiltration, charcoal adsorption, polyethylene glycol precipitation and equilibrium gel filtration). These methods corresponded well with each other and indicated 35-44% binding of 125I-beta-EP in rat serum. Binding of 125I-beta-EP in normal human serum was 36%, determined by ultrafiltration. Serum protein binding of 125I-beta-EP was concentration independent over the concentration range studied (1-1000 nM).     

Properties and immunochemical reactivities of carboxy-modified human serum albumin.

Seven carboxy modified and four amino modified derivatives of human serum albumin have been prepared and studied by optical rotatory dispersion measurements, gel filtration, immunoelectrophoresis, immunodiffusion and by use of an ammonium sulphate technique. It is found that carboxy groups are of major importance for the maintenance of the structure and function of human serum albumin, and that carboxy modification has a much more profound effect than amino modification has to the same relative extent.     

On the possible presence of a beta 2-microglobulin-like protein in extracts of livers from normal chickens and chickens with erythroblastosis-III. Immunological relationship to serum albumin of a small molecular weight human liver protein

Human liver contains a small molecular weight protein (SMWP) previously shown to be biochemically homologous with a chicken liver protein in terms of its amino acid residues. This human protein, which reacts immunologically as a human serum protein, has been tested for its reactions against a battery of antisera that react specifically with many of the human serum proteins. Material prepared from four human livers gave strong reactions in double gel immunodiffusion with an antiserum against human albumin. One of the liver preparations reacted weakly with antiserum against human ferritin; this ferritin is assumed to be a contaminant. Because of the biochemical homology of human liver SMWP with chicken liver SMWP the latter would be expected to react immunologically as serum albumin.     

Gas-phase hydrolysis of proteins and peptides

A simplified gas-phase hydrolysis procedure for proteins and peptides is described. The apparatus consists of a glass vacuum desiccator, a ceramic plate, and a Teflon ring. The method was shown to give reproducible compositions for hydrolysis of human serum albumin and microanalysis of α-melanocyte stimulating hormone including the quantitation of as little as one residue of tryptophan. It minimizes sample handling and allows for the simultaneous hydrolysis of a large number of samples.     

Immunosedimentation: A combination of ultracentrifugation using acrylamide gel followed by immunodiffusion in agarose gel

A technique is described which combines ultracentrifugation in acrylamide-containing density gradients, with immunodiffusion in agarose gel. The use of this procedure to determine sedimentation parameters of antigens in a protein mixture is discussed, and the performance of the technique is illustrated with the immunosedimentation analysis of human serum proteins.     

Development of sheep embryos in vitro in a medium supplemented with different batches of serum albumin

Variability in different lots of commercial serum albumin affects mammalian embryo development in culture. The composition of commercial preparations of ovine, bovine and defatted bovine serum albumin and a fraction of ovine serum containing proteins with a mean molecular weight of 65 kDa (fraction 3) was examined by polyacrylamide gel electrophoresis. All preparations were heavily contaminated with serum proteins other than albumin. Day-6 sheep morulae were cultured for 48 h in a basal bicarbonate-buffered salt solution supplemented with the commercial preparations of ovine, bovine or defatted bovine serum albumin. These three albumin preparations differed in their abilities to support the development of morulae into expanded blastocysts, but these differences disappeared when the basal medium was also supplemented with a component of ovine serum containing substances with molecular weights of less than 10 kDa. In the latter case, the three commercial albumin preparations and fraction 3 of ovine serum all supported full development in about 40-60% of morulae.     

Tryptophan content of serum albumin

Tryptophan content of serum albumin was determined spectrophotometrically. The method used for the determination of tryptophan gave consistent results. Results show that the tryptophan contents of bovine and human serum albumin are significantly different from chicken serum albumin. Bovine and human serum albumins, however, are not significantly different from each other. A large difference in tryptophan content was found between two samples of chicken serum albumin. This suggests that the tryptophan content of serum albumin may not be constant for any given species. For these reasons, tryptophan content should not be used to estimate the molecular weight of serum albumin.     

Protein analysis with tetra-substituted sulfonated cobalt phthalocyanine by the technique of Rayleigh light scattering

This is the first report of cobalt-tetrasulfonatophthalocyanine (CoTSPc) as a probe of Rayleigh light scattering (RLS) to determine proteins at nanogram levels. A highly sensitive method has been developed for the determination of proteins by the light scattering technique on a common spectrofluorimeter, based on the fact that the weak RLS of CoTSPc can be greatly enhanced in the presence of proteins. Under optimum conditions, the linear ranges of the calibration curves were 0.10-34.3 microg x mL(-1) for both human serum albumin and bovine serum albumin, with detection limits of 15.5 and 13.9 ng x mL(-1), respectively. Moreover, there is almost no interference of any amino acids and metal ions. The method has been applied to the direct determination of total proteins in human serum samples, and the results were satisfactory with clinical data provided.     

On-chip microextraction for proteomic sample preparation of in-gel digests

Despite the high sensitivity and relatively high tolerance for contaminants of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) there is often a need to purify and concentrate the sample solution, especially after in-gel digestion of proteins separated by two-dimensional gel electrophoresis (2-DE). A silicon microextraction chip (SMEC) for sample clean-up and trace enrichment of peptides was manufactured and investigated. The microchip structure was used to trap reversed-phase chromatography media (POROS R2 beads) that facilitates sample purification/enrichment of contaminated and dilute samples prior to the MALDI-TOF MS analysis. The validity of the SMEC sample preparation technique was successfully investigated by performing analysis on a 10 nM peptide mixture containing 2 m urea in 0.1 m phosphate-buffered saline with MALDI-TOF MS. It is demonstrated that the microchip sample clean-up and enrichment of peptides can facilitate identification of proteins from 2-DE separations. The microchip structure was also used to trap beads immobilized with trypsin, thereby effectively becoming a microreactor for enzymatic digestion of proteins. This microreactor was used to generate a peptide map from a 100 nM bovine serum albumin sample.     

Two-dimensional electrophoresis of plasma proteins without denaturing agents.

A technique of two-dimensional polyacrylamide gel electrophoresis for the separation of plasma proteins is described. Human plasma proteins were separated by isoelectric focusing followed by electrophoresis in a 4 to 21% linear gradient gel slab. No denaturing agent was used throughout the procedure, so that the analysis of native proteins is possible. Two-dimensional patterns obtained from normal human plasma samples were recorded as "staining density maps," which are similar to contour line maps, and more than 230 protein spots were counted reproducibly on each "staining density map." This technique permits the simultaneous estimation of pI's and approximate molecular weights of native proteins on the slab gel. Applications of this technique to an IgA myeloma plasma sample and a porcine serum sample are described.     

The binding of L-tryptophan to serum albumins in the presence of non-esterified fatty acids.

Bovine, human and rat serum albumins were defatted and palmitic acid, oleic acid and lauric acid added in various molar ratios. The binding of L-tryptophan to these albumins was measured at 20 degrees C in a 0.138 M salt solution at pH 7.4, by using an ultrafiltration technique, and analysed in terms of n, the number of available tryptophan-binding sites per albumin molecule, with apparent association constant, k. 2. n and k were 0.90 and 2.3x10(-4)M(minus-1) respectively for defatted bovine serum albumin and 0.87 and 9.7x10(-3)M(-minus-1) for human albumin. Addition of palmitic acid did not decrease n until the molar ratio, fatty acid/bovine albumin, approached and exceeded 2. The decrease in k was small and progressive. In contrast, lauric caused a marked decrease in n and k at ratios as low as 0.5. A similar distinction between the effects on n of palmitic acid and oleic acid and those of lauric acid was seen for human albumin. k for human albumin was not significantly affected by fatty acids under the conditions studied. 3. It is concluded that primary long-chain fatty acid sites interact only weakly with the tryptophan site on albumin and that inhibition of tryptophan binding occurs when secondary long-chain sites are occupied. Primary medium-chain fatty acid sites are distinct from primary long-chain sites but may be grouped with secondary long-chain sites. 4. The relationship between free and bound tryptophan in samples of rat plasma (Stoner et al., 1975) is discussed in terms of a similar but limited study of rat albumin.     

Comparability of some serum protein determinations by radial immunodiffusion, laser nephelometric and turbidimetric assays employing Q-antisera SEVAC

Quantitation of IgG, IgA and IgM immunoglobulins and C3, C4 components in human serum samples by the radial immunodiffusion technique and by the nephelometric and turbidimetric assays was compared using the linear regression analysis method. Comparisons of the two methods run in polyethylene glycol showed close agreement between methods and a relatively high degree of correlation between the parameters studied. Compared to the radial immunodiffusion technique, nephelometry and turbidimetry gave good correlation between parameters, but the agreement between tests was worse, especially in the case of C3 component determinations in fresh samples of patients' sera. All tests were carried out using the Q-antisera and Control human serum preparations SEVAC.     

Identification of N-acetylglucosamine-binding immunoglobulins in chicken egg yolk and serum distinct from the major mannose-binding immunoglobulins.

An N-acetyl-D-glucosamine (GlcNAc)-binding protein of 170 kDa has been isolated from hen serum and egg yolk. Another GlcNAc-binding protein of higher molecular mass was present only in the serum. The 170 kDa protein co-electrophoresed and co-chromatographed in gel filtration with a chicken IgG, and behaved identical to chicken IgG in double immunodiffusion with goat anti-chicken gamma chain antiserum. The sugar-binding hierarchy for the serum and yolk binding proteins, determined with bovine serum albumin neoglycoproteins, was GlcNAc greater than N-acetyl-D-galactosamine greater than glucose = galactose = L-fucose greater than mannose. This hierarchy was unlike any previously reported GlcNAc-binding proteins. The larger serum binding protein component was shown to be an IgM by double immunodiffusion with goat anti-chicken mu chain antiserum. The serum and yolk GlcNAc-binding proteins comprise a unique set of sugar-binding immunoglobulins distinct from the previously reported hen serum and yolk mannose-binding proteins (Wang et al., 1986).     

A robust, streamlined, and reproducible method for proteomic analysis of serum by delipidation, albumin and IgG depletion, and two-dimensional gel electrophoresis

Serum is a readily available source for diagnostic assays, but the identification of disease-specific serum biomarkers has been impeded by the dominance of human serum albumin and immunoglobulins (Igs) in the serum proteome. There is a need to reduce the technical variation in serum processing and analysis to allow for a reproducible analysis of large cohorts. To this end, we have developed a rapid and reproducible procedure for sample preparation and high-resolution two-dimensional gel electrophoresis to analyze human serum. Serum is centrifuged at high speed to remove lipids and aggregated proteins, incubated with protein G resin to remove IgG, precipitated with NaCl/ethanol to deplete albumin, and slowly resolubilized in a sodium dodecyl sulfate (SDS)/N-(2-hydroxyethyl)piperazine-2'-(2-ethanesulfonic acid) (HEPES) buffer. The delipidated and IgG/albumin depleted serum proteins are focused on pH 4-7 linear large immobilized pH gradient strips, and then resolved by Bis-Tris SDS-polyacrylamide gel electrophoresis. The robustness and reproducibility of the optimized procedure was determined for three individual serum samples on three consecutive days. An image analysis of the nine silver-stained gels demonstrated that the intensity and localization of protein spots are highly reproducible. Our IgG and albumin depletion procedure will aid in screening the patient sera for normal biological variation and disease-specific biomarkers.     

Isolation and characterization of a folate receptor from human placenta

While folate binding proteins have been described in serum and a variety of tissues, the function of these proteins is unknown. A particulate folate binding protein from human placenta has been isolated and characterized following solubilization with Triton X-100. The protein was purified 61,000-fold using affinity chromatography on pteroylglutamic acid-Sepharose as the major purification step. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified protein gave a single band with a Mr = 38,500. Stoichiometry of binding indicated that 1 mol of folate was bound per mol of protein. The protein was a glycoprotein that contained 12% carbohydrate. Antiserum was raised in a rabbit, and on immunodiffusion, gave a single precipitin line with the purified placental folate binding protein. Immunoprecipitation studies using this antiserum indicated that the purified placental folate binding protein shared antigenic determinants with both the large Mr and small Mr folate binding proteins from human milk. Immunofluorescent studies with this antiserum and human erythrocytes revealed the presence of an immunologically similar protein on the plasma membrane of these cells suggesting that this protein may function as a folate receptor.     

Characterization of protein-bound gold in rat urine following aurothiomalate administration and of rat and human albumin-gold-thiomalate

The metabolites of gold in the urine of rats given the antiarthritic drug aurothiomalate were investigated by gel permeation chromatography, electrophoresis, and chemical studies. Following a single dose of aurtothiomalate, the excreted gold was protein-bound in the high-molecular-weight (greater than or equal to 150,000 dalton) and serum albumin fractions. Electrophoresis confirmed the presence of albumin, but showed that the other proteins present differ from those in normal or in vitro aurothiomalate-incubated rat sera. The pattern of the proteins establishes that the proteinuria was of the glomerular type. The alterations in the gold distribution produced by incubation of the urine with the low-molecular-weight thiol penicillamine and with exogenously added aurothiomalate indicated the existence of a labile equilibrium of gold among protein binding sites in the urine. Incubation of rat and human sera and commercially prepared serum albumins with aurothiomalate increased the electrophoretic mobility of the albumin. The significance of this change in electrophoretic mobility with respect to two models of gold binding by serum albumin is discussed.     

Partially Folded Glycated State of Human Serum Albumin Tends to Aggregate

The interaction of reducing carbohydrates with proteins leads to a cascade of reactions that are known as glycation or Maillard reactions that results in the formation of advanced glycation end products. We studied the impact of incubation with various sugars for 4 weeks on the behaviour of human serum albumin incubation using CD, fluorescence, UV?CVis spectrophotometry and polyacrylamide gel electrophoresis. Three weeks of incubation of human serum albumin with sugars resulted in the formation of an intermediate state with negative CD peaks at 222 and 208 nm characteristic of ??-helix. The form also retained tertiary contacts but with altered tryptophan environment and high ANS binding indicative of molten globule state. Further incubation of human serum albumin for 4 weeks resulted in the formation of an intermediate form with negative CD peak at 217 nm, characteristic of ??-sheet, decreased ANS fluorescence and increased thioflavin T fluorescence characteristic of an aggregated state. Prolonged exposure of human serum albumin to reducing sugars thus exerts greater deleterious effects on its structure and formation of aggregates.