Human Serum Protein Fractions Research Articles

Potential of Aqueous Two-Phase Systems constructed on flexible devices: Human serum albumin as proof of concept

Abstract In the present research, the potential use of flexible disposable devices, specifically blood bags, for the fractionation of biological products using Aqueous Two-Phase Systems (ATPS) polymer–salt is studied and demonstrated. Purified human serum albumin (HSA) was used as model protein. Experiments were carried out on ATPS polyethylene glycol (PEG)–potassium phosphate constructed on rigid recipients (conical tubes) and flexible devices (blood bags). The device used for ATPS construction had no significant effect on HSA partition behavior. Protein partition towards the top phase was favored on systems constructed using PEG 1000 g/mol and TLL 45% (w/w), achieving up to 85% recovery. On the other hand a recovery of 92% was achieved at the bottom phase when PEG 3350 g/mol and TLL 25% (w/w) were used. Human serum was used as a complex sample on ATPS experiments. Selective fractionation of human serum proteins on ATPS constructed on flexible devices was achieved. ATPS constructed on blood bags required short equilibrium times (

Observation of interactions of human serum components with transferrin by affinity capillary electrophoresis

Interaction of human transferrin (TF) with human serum components was investigated by affinity capillary electrophoresis. It was found that any peaks of human serum protein fractions did not give migration time change on addition of intact TF to running buffer (50 mM phosphate buffer, pH 7.5), whereas two peaks belonging to α-globulin fraction showed marked acceleration upon addition of desialylated TF. These results provide strong evidence that the sialic acid residue in TF masks its binding ability to serum proteins. The association constants of desialylated TF to these interactive components, estimated based on the double reciprocal plot of migration time change vs. glycoprotein concentration, were at a high level of 10 7 M −1. TF is well known as a ferric ion transfer protein, and hence formation of this protein might be changed by ferric ion. The presence of iron(II) played no essential role in this interaction, though its influence was not negligible.

Application of 2D-HPLC system for plasma protein separation

Summary : The ProteomeLab¥PF 2D protein fractionation system is a rapid, semi-automated, 2 D-HPLC instrument that uses two different methods to separate plasma serum proteins: ion-exchange chro-matography using a wide range of pH in the first dimension and non-porous reverse-phase chromatography inthe second dimension. Because this methodology has only very recently been introduced in proteomic labora-tories, little is known about the characteristics of PF 2D fractionation of human serum proteins. To evaluate thesystem’s application in a clinical laboratory setting, the characteristics of the ion-exchange chromatography-ba-sed separation were analyzed. Following fractionation of human serum proteins on a linear gradient of pH (ran-ging from 8.5 to 4.0), each fraction was collected in a cool module of the instrument. Different fractions obta-ined from the first dimension were then pooled together and loaded on classic 2D gel electrophoresis instru-mentation. The different spots obtained were then checked against the Swiss-Prot Database. A total of 36 hu-man serum proteins were identified in different PF 2D-generated fractions. Some important features of the se-paration system were observed. Different eluted fractions contained different proteins, thus demonstrating thereliability of the fractionation system. The proteins were also fractionated according to the theoretical isoelec-tric point (p

Separation of human serum proteins using the Beckman-Coulter PF2D™ system: analysis of ion exchange-based first dimension chromatography

The ProteomeLab PF2D protein fractionation system is a rapid, semi-automated, 2 D-HPLC instrument that uses two different methods to separate plasma serum proteins: ion-exchange chromatography using a wide pH range in the first dimension, and non-porous reverse-phase chromatography in the second dimension. Because this methodology has only very recently been introduced in proteomic laboratories, little is known about the characteristics of PF2D fractionation of human serum proteins. To evaluate the system's application in a clinical laboratory setting, the characteristics of the ion-exchange chromatography-based separation were analyzed. Following fractionation of human serum proteins on a linear pH gradient (ranging from 8.0 to 4.0), each fraction was collected in a cool module of the instrument. Different fractions obtained from the first dimension were then pooled together and loaded on classic 2D gel electrophoresis instrumentation. The different spots obtained were then checked against the Swiss-Prot Data-base. A total of 36 human serum proteins were identified in different PF2D-generated fractions. Some important features of the separation system were observed. Different eluted fractions contained different proteins, thus demonstrating the reliability of the fractionation system. The proteins were also fractionated according to the theoretical isoelectric point (pI). This was consistent with the evidence that the vast majority of immunoglobulins, characterized by an alkaline pI, were not retained by the column and were eluted in the unbound fraction. This outcome also underlines a practical advantage: fractions eluted from pH 8 to pH 4 contained virtually immunoglobulin-depleted serum proteins. This finding supports an immediate use of the PF2D system in a clinical setting, where abundant proteins should be clearly identified to enable evaluation of other less abundant, but potentially relevant, species.

Adsorption and selectivity characteristics of several human serum proteins with immobilised hard Lewis metal ion–chelate adsorbents

In this investigation, human serum has been used as an example of a crude protein mixture to define the protein binding characteristics and selectivity of several immobilised hard Lewis metal ion affinity chromatographic (IMAC) adsorbents. Specifically, the binding properties of immobilised O-phosphoserine ( im-OPS) and 8-hydroxyquinoline ( im-8-HQ), with immobilised iminodiacetic acid as a control system, have been investigated in combination with the hard Lewis metal ions, Al 3+, Ca 2+, Fe 3+, Yb 3+, and the borderline metal ion, Cu 2+, over the pH range pH 5.5 to pH 8.0 with buffers of 0.5 M ionic strength. The same IMAC adsorbents were also investigated for their protein binding capabilities with buffers of an ionic strength of 0.06 M at pH 5.5 and pH 8.0. The binding behaviour of four “marker” proteins, namely transferrin, α 2-macroglobulin, gammaglobulin and human serum albumin have furthermore been employed to monitor the differences in protein selectivity exhibited by these IMAC systems. The experimental findings confirm that these hard Lewis metal ion IMAC adsorbents function in a “mixed” binding mode with both coordination and electrostatic characteristics evident, depending on the ionic strength and pH of the equilibration or elution buffers. Based on a screening protocol, several members of the im-M n+ –8-HQ and im-M n+ –OPS adsorbent series have been identified with high selectivity for transferrin and α 2-macroglobulin. These hard Lewis metal ion IMAC adsorbents thus provide attractive alternatives for selective fractionation of human serum proteins.

Automated free-solution isotachophoresis: instrumentation and fractionation of human serum proteins.

An automated free-solution isotachophoresis system (FS-ITP) for preparative fractionation of biopolymers is described, operated in a batch mode. The dimension of the separation chamber allows an up to 1200-fold higher sample load compared to separation in capillaries of 180 microm inner diameter as used in analytical capillary isotachophoresis (C-ITP). The preparative capacity of the system is within the milligram range. The method is fully compatible with analytical C-ITP, which is essential for preparative-scale isotachophoresis with regard to optimization of electrolyte systems and the search for suitable spacers. As a model application the fractionation of human serum proteins is reported. The collected fractions were analyzed by C-ITP and agarose gel electrophoresis.

Effect of human serum on alkaline phosphatase induction in cultured human tumor cells.

The continuous cell lines T 24 and HT-29, derived from human bladder and colon carcinomas, produce term-placental and intestinal alkaline phosphatase, respectively. Growth in hyperosmolar medium or exposure to prednisolone or sodium butyrate induces increased enzyme levels, and combinations of inducers elicit synergistic activity increases. The effect of the inducing agents is strikingly diminished when cells are grown in the presence of high concentrations of human serum, and the synergistic increases are essentially abolished. Major human serum protein fractions do not affect alkaline phosphatase induction.

Studies on the interaction between human serum protein fractions and 18O-labeled oxosteroids.

The nature of the interaction between progesterone or testosterone and human albumin as well as the interaction between progesterone and partially purified human transcortin has been studied. Modification of lysine residues of albumin with maleic anhydride resulted in a decreased binding of the steroid as judged from equilibrium dialysis experiments. This suggested that lysine residues in albumin interact with the oxosteroids. In order to check this hypothesis, steroids labeled with 18O in their oxo function (testosterone and progesterone) were synthesized for use as probes of the interactions. However, no loss of label was noted when testosterone or progesterone specifically 18O-labeled in their oxo functions were incubated with albumin. This suggested that no covalent interaction between the steroidal oxo group and albumin took place. This was in contrast to the results obtained with 3,20-18O-labeled progesterone and partially purified transcortin, where a complete loss of 18O label in the protein-bound steroid was found. The nonbound steroid showed an almost complete retention of label. These results indicate a participation of steroid oxo groups in the binding of progesterone to transcortin. Of the possible mechanisms discussed, imine bonds between the steroid and transcortin seem most likely although other types of interactions cannot be ruled out.

CYTOTOXIC PROTEIN MOLECULES GENERATED AS A CONSEQUENCE OF ETHANOL METABOLISM IN VITRO AND IN VIVO

A non-dialysable cytotoxic activity developed in the supernatants of human blood-monocyte-derived macrophages cultured in the presence of ethanol and in the serum of three healthy volunteers who drank 500-700 ml wine over 20-35 min. On 'Sephacryl S-300' gel filtration of the culture supernatants and the serum samples from the subjects who took alcohol, the cytotoxic activity eluted together with albumin molecules. Studies of human serum and of various commercially purchased human serum protein fractions treated with carbon-14-labelled acetaldehyde and nonradioactive acetaldehyde, respectively, provided strong circumstantial evidence that the cytotoxic proteins were albumin molecules that had become complexed with acetaldehyde generated by the metabolism of ethanol. The cytotoxic activity developing in vivo was greatest 6-10 h after the consumption of ethanol started, when blood alcohol levels were normal or only slightly high. The appearance of a circulating long-acting cytotoxic macromolecule after the ingestion of ethanol may be of considerable importance in clarifying the mechanisms underlying ethanol-induced tissue damage.

Salt-promoted adsorption: Recent developments

Efficient fractionation of human serum proteins is accomplished by use of a series of tandem-coupled beds of group-affinity adsorbents. The general fractionation strategy for group fractionation of a complex protein mixture is discussed.

A high-capacity hydrophobic adsorbent for human serum albumin

A simple method, based on salting out hydrophobic interaction chromatography, for the efficient removal of trace amounts of serum albumin from partially purified protein preparations is described. The method is also successfully applied for the purification of albumin from Cohn fraction IV, a by-product obtained from the commercial fractionation of human serum proteins by the ethanol precipitation procedure. About 70% of the adsorbed albumin can be eluted by buffer of low ionic strength and can thus be lyophilized directly, if required. The adsorbent can be used for several cycles of adsorption and desorption without affecting its selectivity or capacity. Its adsorption properties and capacity for serum albumin are compared with those of the commercially available adsorbent Blue Sepharose CL-6B.

Three turbidimetric methods for determining total protein compared.

We used human serum protein fractions to evaluate the sensitivity and bias of three turbidimetric methods for determining concentrations of proteins. Each fraction (Cohn Fractions II, III, IV, and V) was assigned a protein concentration value that was determined by the biuret method, which we calibrated with purified monomer of human serum albumin. All three turbidimetric methods (those involving sulfosalicylic acid/sodium sulfate, trichloroacetic acid, and alkaline benzethonium chloride) gave acceptable results for Fraction V with crystallized human serum albumin as the reference material, but there was bias by each of the three methods for the three globulin fractions. The method involving alkaline benzethonium chloride with measurement at 450 nm had the best sensitivity within the range of linearity and the most consistent bias among the three globulin fractions. These results define the dilemma for valid calibration of these methods for total serum protein in cerebrospinal fluid and urine.

Four methods for determining total protein compared by using purified protein fractions from human serum.

The proportional bias of four methods frequently used for determining low concentrations of protein was evaluated with human serum protein fractions (Cohn Fractions II, III, IV, and V). Each fraction was assigned a protein concentration value as determined by the biuret method, calibrated with purified human serum albumin monomer. All four methods (Coomassie Brilliant Blue dye-binding, the method of Lowry et al., ultraviolet absorption, and immunonephelometry gave acceptable results for Fraction V (albumin). The ultraviolet absorption and the Lowry methods overestimated the three globulin fractions (II, III, and IV), whereas the other two methods underestimated these fractions. The method of Lowry et al. gave the least proportional bias for the globulin fractions.

Fractionation of human serum proteins by immobilized metal affinity chromatography

Human serum was fractionated on zinc and cadmium iminodiacetate and tris-(carboxymethyl)ethylenediamine chelates. Zinc iminodiacetate was found to be useful in the isolation of hemopexin and α 2-macroglobulin. The chromatographic behaviour of serum proteins on zinc iminodiacetate could be modulated by the nature of the eluent; a sulphate-containing solvent increased the binding of serum proteins to immobilized metal ions.

Guidelines for the preparative fractionation of human serum proteins on gradient-eluted columns of concanavalin A-sepharose: elution positions of fourteen well-characterized proteins and evidence for concanavalin A-reactive albumin-IgA and -IgG complexes.

Systematic studies on the fractionation of serum proteins on gradient-eluted columns of concanavalin A-Sepharose have been carried out to determine if the oligosaccharide residues were sufficiently different to permit a reasonable separation and to determine where in the chromatogram these proteins would be eluted. Human whole serum and ammonium sulfate fractions derived therefrom were used in conjunction with 2.1 x 75 cm columns of concanavalin A-Sepharose and a 4 x 400 ml gradient (Varigard) with 0.5 M methyl alpha-D-glucopyranoside as limit buffer. The elution positions and chromatographic limits of 14 well-characterized human serum proteins have been determined by double diffusion of aliquots of the effluent fractions (10X concentrated) in agarose gel against specific antibody and the general chromatographic distribution of the proteins by immunoelectrophoresis. Overall, the results demonstrate that the composition of the oligosaccharide side chain, like differences in molecular size, solubility, and charge density, is a useful parameter in the chromatographic separation of protein from serum. Although it is well-known that albumin is a nonglycoprotein, 1.0% of the protein was tightly bound by concanavalin A-Sepharose. Subsequent experiments showed that albumin binding was due to complex formation with IgA and IgG both of which possess the necessary complement of concanavalin A-reactive residues for strong binding. Sodium dodecylsulfate polyacrylamide gel electrophoresis of 2-mercaptoethanol-reduced albumin-IgA and -IgG complexes produced bands corresponding to the molecular weights of albumin and the heavy and light chains of IgA and IgG whereas unreduced samples were not dissociated. When these complexes were reacted with concanavalin A-Sepharose and treated with 2-mercaptoethanol, free albumin was eluted. The remaining adsorbed glycoprotein(s), IgA and IgG, could be eluted with methyl alpha-D-glucopyranoside. These results strongly suggest that these proteins and albumin are linked via a disulfide bond(s).

Inhibition of human collagenase activity by a small molecular weight serum protein

Fractionation of human serum proteins by gel filtration in Sephadex G-200 revealed two regions of collagenase inhibition which corresponded to α 2-macroglobulin and a smaller serum component which eluted after α 1-antitrypsin. The smaller collagenase inhibitor, having a molecular weight of 40,000 was separated from α 1-antitrypsin by chromatography in Sephadex DEAE A.50. It was found to inhibit human collagenases derived from skin, rheumatoid synovium, gastric mucosa and granulocytes, but not the neutral proteases trypsin and papain. Purified preparations of α 1-antitrypsin inhibiting trypsin and papain had no effect on the collagenase activities. The small collagenase inhibitor may have importance as a regulatory factor in the control of collagenase activity in vivo.

Lack of effect of insulin and blood fractions on glucose-ATP phosphotransferases.

SummaryGlucose-ATP phosphotransferase activities of normal and diabetic rat heart and liver were assayed in the presence of normal and diabetic rat serum and plasma and various human serum protein fractions, and in the presence of insulin. No significant alterations in the activities were observed under a variety of conditions.

The interaction of human serum protein fractions with the starch-iodine complex

When starch-agar gel electrophoretic patterns are treated with iodine solution, decolorization in zones corresponding to the various protein fractions becomes more pronounced with time. This progressive loss of color cannot be ascribed entirely to the amylolytic activity since the starch-iodine reaction is influenced by serum proteins other than amylase. Increasing the starch concentration of iodine solution produces a relatively uniform rise in blue color intensity. However, this chromogenic response is greatly diminished by native human serum albumin or γ-globulin. The ability of these two fractions to dissipate starch-iodine color is lessened if the proteins are pre-iodinated. In the presence of fluoride, an amylase inhibitor, normal and pathologic sera are still capable of decolorizing starch-iodine solution to varying degrees. An interaction of serum proteins with available iodine would appear to account for this phenomenon.

Ultraviolet analysis of human serum proteins in zone electrophoresis

Specific absorption coefficients of electrophoretically separated human serum protein fractions were determined. Fresh normal sera from 5 healthy blood donors were pooled and submitted to starch grain zone electrophoresis using either phosphate buffer at pH8.0 or veronal buffer at pH8.6. Protein fractions were eluted with neutral saline and optical density at 280mμ and nitrogen contents were determined. Specific absorption coefficient, e, at 280mμ was expressed as O. D. of the solution of each protein fraction at a concentration of 1mg protein nitrogen per ml measured in a 1cm cell.Average values of e for serum protein fractions obtained from six runs of electrophoresis with the phosphate buffer and six runs with the veronal buffer were as follows: albumin; 4.2, α-globulin; 6.7, β-globulin; 8.3, and γ-globulin; 9.5.Results obtained by densitometric analysis at 280mμ of agar electrophoretic patterns of human sera were corrected for the above mentioned specific absorption coefficients by multiplying the optical densities of albumin, α-, β-, and γ-globulins by 16, 10, 8, and 7, respectively. Percentage value of each serum protein fraction thus obtained were found to be in good agreement with that obtained by free electrophoresis in 10 cases of normal and 6 cases of pathological sera.

INTERRELATIONS OF HUMAN SERUM PROTEIN FRACTIONS IN HEALTH AND DISEASE.

DURING a recent quantitative investigation of the human serum proteins1, a preliminary report was published of statistical associations between protein fractions of healthy subjects in the course of fluctuations within the normal range2. The results were sufficiently interesting to warrant extension of the material to examine the possible effects of a variety of disease processes on these interrelations. This article reports some results which were obtained from a series of patients with carcinoma, cirrhosis of the liver, myocardial infarction, pneumonia, and rheumatoid arthritis, and compares them with results drawn from normal persons in good health and also in pregnancy.