Features of the milk whey protein partitioning in polyethyleneglycol-sodium citrate aqueous two-phase systems with the goal of isolating human alpha-1 antitrypsin expressed in bovine milk

Partitioning behaviour of the bovine whey proteins (bovine serum albumin, alpha-lactoalbumin and beta-lactoglobulin) and human alpha-1 antitrypsin in aqueous two-phase systems prepared with polyethyleneglycol (molecular masses: 1000, 1450 and 3350)-sodium citrate was analysed at pH 5.2, 6.2 and 8.2. Alpha lactoalbumin concentrated in the polyethyleneglycol rich-phase, while beta-lactoglobulin, bovine serum albumin and alpha-1 antitrypsin showed affinity for the citrate rich-phase. In aqueous two-phase systems of high medium pH and high polyethyleneglycol molecular mass the protein partitioning equilibrium is displaced to the citrate rich-phase. The polyethyleneglycol 1450-pH 5.2 system with a top/bottom phase-volume ratio of 3 showed to have the best capability of recovering the alpha-1 antitrypsin from a mixture prepared with natural milk whey and human alpha-1 antitrypsin. The recovery of this protein in the bottom phase was of 90% and the purity of the obtained product was of 98%. The method appears to be suitable as a starting point to isolate other human proteins expressed in transgenic bovine milk.     

Partition of whey milk proteins in aqueous two-phase systems of polyethylene glycol-phosphate as a starting point to isolate proteins expressed in transgenic milk

Partitioning behaviour of the bovine whey proteins (bovine serum albumin, alpha lactoalbumin and beta lactoglobulin) and alpha-1 antitrypsin in aqueous two-phase systems prepared with polyethyleneglycol (molecular masses: 1000; 1500 and 3350)-potassium phosphate was analysed. Bovine serum albumin and alpha lactoalbumin concentrated in the polyethyleneglycol rich phase with a partition coefficient of 10.0 and 27.0, respectively, while beta lactoglubulin and alpha-1 antitrypsin showed affinity for the phosphate-rich phase with a partition coefficient of 0.07 and 0.01, respectively. An increase of medium pH induced an increase of the partition coefficient of these proteins while the increase in polyethyleneglycol molecular mass induced the opposite behaviour. The system polyethyleneglycol 1500-pH 6.3 showed the best capacity for recovering the alpha-1 antitrypsin with a yield of 80% and a purification factor between 1.5 and 1.8 from an artificial mixture of the milk whey proteins and alpha-1 antitrypsin. The method appears to be suitable as a starting point to isolate proteins expressed in transgenic milk.     

Influence of high concentration monovalent cations on the protein partitioning in polyethyleneglycol 1500-phosphate aqueous two-phase systems

The influence of chloride salts of Na+, Rb+ and Cs+ at concentrations from 0.15 to 1.2M was studied with bovine albumin, trypsin, ovoalbumin and lysozyme partitioning in an aqueous two-phase system formed by polyethyleneglycol 1500 and potassium phosphate at pH 7.4. Monovalent cations favoured the protein transfer to the polyethyleneglycol rich phase in the following order: Rb+ > Na+ > Cs+. Structure making cations as Na+ induced a poor loss of structured water, producing little diminution of the molar partial specific volume of polyethyleneglycol, while Rb+ and Cs+, structure breaking cations, induced a significant decrease in the specific volume of the polyethylene glycol. The increase of available solution free volume in the top phase favours the protein transfer to the polyethyleneglycol rich phase. Na+ and Rb+ induced a slight decrease in the alpha helix content of the proteins, while Cs+ increased the secondary structure for all the proteins. All the cations induced a decrease in the hydrophobic surface of the proteins, this effect was more significant in the presence of Cs+.     

Purification of IgG and albumin from human plasma by aqueous two phase system fractionation

The current shortages in human plasma products at global levels justify the development of new, cost effective plasma fractionation methods. We have developed a fractionation process to obtain immunoglobulin G (IgG) and albumin‐enriched fractions based on polymer‐salt aqueous two phase system (ATPS). A small‐scale (0.02 L) ATPS composed of polyethyleneglycol 3350 (PEG), potassium phosphate and sodium chloride, at pH 6.1, was evaluated and subjected to 50‐fold scale‐up (1 L). Further purification of the fractions was performed using caprylic acid precipitation and ion exchange chromatography. Similar yield and purity were obtained at both small and large scales. IgG precipitated in the PEG rich upper phase at 83% recovery and 2.75‐fold purification factor. An 81% pure albumin fraction was obtained in the salt rich bottom phase with a 91% yield. After polishing, IgG was obtained at a recovery of 70% and a purity of 92%. Corresponding values for albumin were 91% and 90%. This IgG and albumin fractionation technology deserves further evaluation as it may represent a potential alternative to conventional plasma fractionation methods. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1005–1011, 2012     

Comparison between the thermodynamic features of alpha1-antitrypsin and human albumin partitioning in aqueous two-phase systems of polyethyleneglycol-dextran

The partitioning features of human serum albumin and alpha1-antitrypsin in aqueous two-phase systems of dextran and polyethyleneglycol were studied. The effect of factors that affect the electrostatic term of Albertsson equation such as pH, ionic strength, presence of neutral salts as well as those which affect the non-electrostatic term such as polyethyleneglycol mol. wt. and temperature were assayed. At room temperature, the positive entropy and enthalpy changes associated to the partition may be due to a release of part of the structured water in the domain of proteins caused by H-bonds rupture when the proteins are transferred to the upper phase. This behaviour may be explained on the basis of a preferential hydration of the proteins in presence of dextran (bottom phase) and a preferential interaction of polyethyleneglycols with the protein domain (top phase). The electrostatic interactions were similar for both proteins due to the proximity of their isoelectric point and similar dissociation profiles of their prototropic groups.     

Study of the serum albumin-polyethyleneglycol interaction to predict the protein partitioning in aqueous two-phase systems

The theoretical framework based only on the excluded volume forces is not enough to explain the bovine serum albumin partitioning behaviour in aqueous biphasic systems. The goal of this work is to look at the phase separation via the polymer effect on the water structure. Our findings suggest that polyethyleneglycol 600-protein interaction is conducted by van der Waals forces between the hydrophobic surfaces from PEG and protein molecules, which implies the rupture of hydrogen bonds from the structured water in their neighbours. Therefore, the protein will concentrate in the most water-structured phase (polyethyleneglycol) in order to reach the minimal free energy condition. When polyethyleneglycol molecular weight increases, its exclusion from protein surface prevails, thus pushing the bovine serum albumin to the bottom phase.     

Simultaneous purification and polymerization method for bovine serum albumin preparation

Bovine serum albumin (BSA) has various applications in blood group serology and different research purposes. In this study purification of BSA has been compared with human serum albumin (HSA) using modified ethanol precipitation method based on the method of Cohn. The purification process was carried out under controlled conditions, particularly of ethanol concentration, pH, ionic strength and temperature. It was revealed that the produced BSA and HSA have purity more than 95%. It is obvious that HSA can be used, as a drug when the amount of its polymers is less than 5% whereas polymer generation is required in order to enhance the potentiating properties of BSA in agglutination of red cells. We propose here a simple and rapid two-step method for simultaneously purification and polymerization of BSA. By this method simply BSA with desired amount of polymers was obtained by 40% ethanol concentration.     

Features of the acid protease partition in aqueous two-phase systems of polyethylene glycol-phosphate: chymosin and pepsin

The partitioning of chymosin (from Aspergilus niger) and pepsin (from bovine stomach) was carried out in aqueous-two phase systems formed by polyethyleneglycol-potassium phosphate. The effects of polymer concentration, molecular mass and temperature were analysed. The partition was assayed at pH 7.0 in systems of polyethyleneglycol of molecular mass: 1450, 3350, 6000 and 8000. Both proteins showed high affinity for the polyethyleneglycol rich phase. The increase of polyethyleneglycol concentration favoured the protein transfer to the top phase, suggesting an important protein-polymer interaction. Polyethyleneglycol proved to have a stabilizing effect on the chymosin and pepsin, increasing its protein secondary structure. This finding agreed with the enhancement of the milk clotting activity by the polyethyleneglycol. The method appears to be suitable as a first step for the purification of these proteins from their natural sources.     

Myoglobinuria detection by capillary electrophoresis

Capillary electrophoresis was used in this study to separate urinary myoglobin from hemoglobin based on its electrophoretic mobility. Urine was applied directly without any treatment. The separation was accomplished in less than 7 min. Myoglobin extracted from human muscle tissues was separated, in a borate buffer 150 mM, pH 8.7 containing 0.5% polyethyleneglycol at 6 kV, into two peaks (MI and MII) which were also resolved far from hemoglobin. Upon standing at room temperature, MII converted into MI. Horse myoglobin eluted close to MI.The addition of polyethyleneglycol to the buffer enhanced the separation and increased the peak height of myoglobin. Optimum conditions for the separation are discussed. The method is suitable for routine clinical analysis because of its simplicity and speed.     

Studies on the structure and mechanism of action of glycoside hydrolases. I. Purification and study of some factors affecting the activity of Rhizopus arrhizus (1 leads to 3)-beta-D-glucanase

The extracellular (1→3)-β-D-glucanase [(1→3)-β-D-glucan glucanohydrolase, E.C. 3.2.1.6] produced by Rhizopus arrhizzus QM 1032 was purified 165-fold by chromatography. The purified enzyme is basic, has a molecular weight of ≈ 10,000, and is unstable in dilute solution but may be stabilized by addition of human serum albumin. The pH-activity profile for the enzyme in the presence of serum albumin shows a peak at about pH 3.5–3.7 and a shoulder at pH 4.5–4.6, whereas in the absence of serum albumin the optimum pH is at pH 4.5–4.6, indicating the presence of two enzymic species, designated “pH 3. 5 activity” and “pH 4.6 activity”. In the presence of albumin the enzyme activity is resistant to inactivation by a wide range of reagents. Ammonium molybdate is, however, a powerful inhibitor of ldpH 3.5 activity” although a much poorer inhibitor of “pH 4.6 activity”. The enzyme activity is stable during heating at pH 3.5 in the presence of human serum albumin. Thus, 94.5 and 88.5 % of “pH 3.5 activity” and “pH 4.6 activity”, respectively, remained after heat treatment for 30 min at 68°. The enzyme is, however, essentially inactive at this temperature, even in the presence of albumin. To account for this finding, a temperature-dependent conformational change is proposed. The enzyme activity is not stable during heating at pH 4.6 in the presence of serum albumin. K_m values for action on laminaran are 0.54 mg/ml (pH 3.5) and 0.27 mg/ml (pH 4.6). For lichenan the corresponding values are 3.33 and 2.38 mg/ml. The V_max for enzyme action on lichenan is 35–40% higher than for action on laminaran at both pH values. Possible relationships between the two forms of the enzyme are briefly considered.     

Partitioning features of bovine trypsin and alpha-chymotrypsin in polyethyleneglycol-sodium citrate aqueous two-phase systems

The partitioning of bovine trypsin and alpha-chymotrypsin--proteases of similar physico-chemical properties--in different polyethyleneglycol/sodium citrate aqueous two-phase systems was investigated. The effect of different factors such as polyethyleneglycol molecular weight, pH, tie line length, temperature and the presence of an inorganic salt on the protein partition coefficient were analysed. Both a decrease in PEG molecular weight and an increase in pH led to a higher partition coefficient for both enzymes. Aqueous two-phase systems formed by PEG of molecular weight 3350 and citrate pH 5.2 showed the best separation capability which was enhanced in presence of sodium chloride 3%. The transfer of both proteins to the top phase was associated with negative enthalpic and entropic changes.     

Isolation and Properties of an α-Amylase Inhibitor (0.53) from Wheat (Triticum aestivum)

A droplet gel-entrapping method used for enzyme immobilization was improved to simplify the procedure and to increase the enzyme stability. This immobilization technique is suitable for coupled enzyme reactions requiring cofactors. Leucine dehydrogenase (LeuDH) and formate dehydrogenase (FDH) were freeze-dried with bovine serum albumin, dextrin and stabilizers. The freeze-dried enzyme powder was suspended in a methylcellosolve solution containing polyethyleneglycol(#4000)diacrylate, N,N′-methylenebisacrylamide and 2-hydroxyethylacrylate, and the suspension was gelled with initiators. The gel was cut up and the pieces were washed in a buffer to remove the methylcellosolve and the dextrin inside. The maximum conversion ratio for a LeuDH-FDH gel column was determined to be 99.8% by means of the recycling reaction. On longterm operation at 30 °C for leucine production, the initial conversion ratio (7.2%) gradually decreased to 6.6% over the first 10 days. However, the conversion ratio remained almost constant after the 10th day. The effects of flow rate, temperature, pH, and the concentrations of formate, α- ketoisocaproate, ammonium and NAD on the leucine productivity with the gel column were also investigated.     

Purification and immunological characterization of human myocardial MB creatine kinase

Elevated plasma MB creatine kinase (CK) is considered the most sensitive and specific diagnostic indicator of myocardial infarction. However, attempts to purify human MB CK have been unsuccessful. The need for purified human MB CK was further enhanced with the development of a radioimmunoassay for CK isoenzymes which would provide more prompt and specific detection of myocardial infarction. The major protein contaminant of MB CK is albumin which has been difficult to separate due to their similar electrophoretic mobility. Human hearts were obtained within 2 h postmortem and the tissue homogenized in 50 mm Tris-HCl (pH 7.4), 2 mm mercaptoethanol. The CK was recovered from the supernatant (31,000g) by ethanol extraction (50–70%). The resuspended pellet was fractionated on DEAE Sephadex A-50 with a salt gradient (50–500 mm, pH 8.0). The MB fraction contained about 90% albumin. The preparation was bound to an Affigel blue column and contaminating proteins other than albumin were eluted with 50 mm Tris-HCl (pH 8.0), 2 mm mercaptoethanol. MB CK was eluted with 250 mm NaCl, but the albumin remained bound. The MB fraction with a specific activity of 453 IU/mg represented an 80-fold increase in purity and exhibited a single protein band on polyacrylamide gels. Purified MB CK labeled with ¹²⁵I exhibited no binding to human albumin antiserum, but bound to MB CK antiserum, and unlabeled MB CK competitively inhibited binding of ¹²⁵I-MB CK in the radioimmunoassay system exhibiting a sensitivity for detection of plasma MB CK at the nanogram level.     

Increased inhibitory potency of free fatty acid-poor albumin on the released and activity of insulin-degrading enzymes from isolated rat adipocytes and hepatocytes

Isolated rat adipocytes and hepatocytes release protease(s) into the medium which degrade insulin and glucagon. This can be partially inhibited by high concentrations of bovine serum albumin. Free fatty acid-poor albumin prepared by charcoal treatment at pH 3 is a more potent inhibitor than untreated albumin. However, the increase in inhibitory potency depends on the exposure of the albumin to the low pH and not on the removal of the fatty acids. Optimum conditions for this treatment are overnight exposure to pH 3-4 at 37 degrees C. In hepatocytes, but not in adipocytes, the treated albumin also diminishes the release of enzymes into the medium.     

Reduction in Brain Glucose Utilization Rate after Tryptophol (3-Indole Ethanol) Treatment

Abstract: 3-Indole ethanol has been recently identified as the hypnotic agent in trypanosomal sleeping sickness, and because it is formed in vivo after ethanol or disulfiram treatment, it is also associated with the study of alcoholism. When administered intraperitoneally to rats (250 mg/kg) tryptophol induced a sleep-like state that lasted less than an hour (no righting reflex was apparent 2 min after injection, but it returned at 11 min in bovine serum albumin solution, and 47 min in 40% ethanol solution). In ethanol solutions, tryptophol reduced brain cortical glucose utilization by 55% to the basal brain metabolic rate, and this effect lasted less than 1 h. Synergistic effects of tryptophol and ethanol were suggested by the observation that in albumin solution, tryptophol reduced brain glucose utilization by 35%, but a normal rate was not observed until 2 h postinjection.     

Binding of bismuth to serum proteins: implication for targets of Bi(III) in blood plasma

Bismuth complexes have been widely used in clinical treatment as antiulcer drugs. However, different adverse effects have been observed and the diagnosis is generally confirmed by the detection of bismuth in blood or blood plasma. In this study, binding of bismuth to human serum albumin was studied by fluorescence spectroscopy with the binding constant logK(a) to be 11.2. Competitive binding of bismuth to human albumin and transferrin was carried out at pH 7.4 by FPLC and ICP-MS. It was found that over 70% of bismuth binds to transferrin even in the presence of a large excess of albumin (albumin/transferrin=13:1) at pH 7.4, 10 mM bicarbonate. The distribution of bismuth between the two proteins was almost unchanged when Cys(34) of albumin was blocked. However, all bismuth binds to albumin when iron-saturated transferrin was used. Almost all of the bismuth was distributed over the fractions containing transferrin (70%) and albumin (<30%) in serum. The percentage of bismuth associated with transferrin was further increased by 15% with elevated transferrin in serum. Binding of bismuth to transferrin is much stronger than human albumin. Transferrin is probably the major target of bismuth in blood plasma, and it may play a role in the pharmacology of bismuth.     

Isolation of plasma albumin by ethanol extraction is inappropriate for isotope ratio measurements during the acute phase response

Isolation of high-purity albumin from plasma is essential to study albumin kinetics in vivo with tracer techniques. Because of its simplicity ethanol extraction has been repeatedly used for albumin purification. However, it cannot be excluded that this single-step procedure completely prohibits contamination by other proteins, especially those known to be produced at an accelerated rate during the acute phase response. In the present study, we wanted to examine the reliability of ethanol extraction in different clinical conditions and to study the effects of potential impurities on albumin enrichment during stable isotope tracer studies. SDS-PAGE revealed a contaminating protein band at about 25,000 Da in healthy subjects and postoperative patients during the acute phase response, but not in critically ill patients. According to densitometry about 8% of proteins after ethanol extraction were contaminants. To examine potential contaminant effects on tracer enrichment 1-[13C]-leucine was given to healthy subjects and postoperative patients. Blood samples were taken after various amounts of time, and albumin enrichments (tracer/tracee ratios) were determined from isotope ratios obtained by mass spectrometry. Irrespective of the magnitude of tracer enrichment, postoperative tracer/tracee ratios were significantly higher (on average +10%) in samples exclusively analysed by ethanol extraction than in samples which had undergone additional electrophoretic purification. No significant effect of the contaminant was seen in healthy subjects. N-terminal protein sequencing revealed contaminants to mainly consist of apolipoprotein A-1. Its physiology and pathophysiology may sufficiently explain its variable effects of albumin enrichment. Our findings suggest that exclusive ethanol extraction is inappropriate for albumin isolation in tracer studies performed during the acute phase response. Ethanol extraction may also not be advisable in all other situations known to be associated with a rise in apolipoprotein A-1 turnover.     

Immunoprecipitation is inappropriate for the isolation of radiochemically pure albumin from tissues

Rats were given intravenous injections of l-[1-¹⁴C]leucine. Twelve minutes after injection, testes, kidneys, livers, and hepatomas were excised rapidly from one group of animals bearing Morris hepatoma 5123tc. From a second group of rats, the blood was removed 90 min after injection. Tissue extracts and serum were divided into three portions each, and albumin was isolated from each of the three portions by one of three different methods.The three different isolation procedures were the following: (A) pretreatment of the tissue extracts and serum with bovine serum albumin and its specific antiserum and subsequent immunoprecipitation of the rat serum albumin, (B) direct immunoprecipitation followed by dissolving the precipitated rat serum albumin in acid/ethanol, precipitation with ether, and ion-exchange chromatography of the redissolved albumin on CM-cellulose, and (C) a modification of a procedure published previously including fractionation with trichloroacetic acid, ethanol, ether, and ammonium sulfate, chromatography on Sephadex G-100 and DEAE-cellulose, and preparative disc electrophoresis in polyacrylamide gel at pH 10.3 and pH 2.7.With method (A), radiochemically pure albumin can be obtained only from serum. Even though testis and kidney do not synthesize albumin, albumin preparations isolated by this procedure from these organs contain significant amounts of radioactivity. Specific radioactivities measured in albumin prepared by method (A) from the four tissues examined are 5–19 times larger than those in preparations isolated by method (C). Thus, method (A) is inappropriate for the isolation of radiochemically pure albumin from the tissues studied.Procedure (B) is sufficient to obtain radiochemically pure albumin from the serum as well as from the other tissues examined except liver. With liver, this method yields an albumin preparation containing 53% more radioactivity than does albumin isolated with method (C).Method (C) is the only procedure yielding radiochemically pure albumin from all sources, including liver. In liver and hepatoma, the properties of the radioactive impurities are very similar to the properties of albumin itself. Therefore, several purification steps and a careful analysis of the distribution of radioactivity among the albumin fractions after chromatographies and electrophoreses are necessary to separate radioactive impurities from the albumin from homogenates of these two sources.     

Partition and substrate concentration effect in the enzymatic synthesis of cephalexin in aqueous two-phase systems

The kinetically controlled synthesis of cephalexin in aqueous two-phase systems was studied, using immobilized penicillin acylase, 7-amino 3-desacetoxycephalosporanic acid as nucleophile and phenylglycine methyl ester as acyl donor. The organic phases used were 80% (v/v) polyethyleneglycol 400 and 600 and the aqueous phase was 2.5 M (NH₄)₂SO₄. 7-amino 3-desacetoxycephalosporanic acid and cephalexin partition coefficients were determined at pH 7.4 and 7.8, at 14 °C and 20 °C. Highest partition coefficient for cephalexin was obtained for polyethyleneglycol 400–(NH₄)₂SO₄ at pH 7.4 and 20 °C, while the lowest partition coefficient for 7-amino desacetoxycephalosporanic acid was obtained in the same system at pH 7.8 and 14 °C. No significant effect of pH was observed on conversion yield and productivity of cephalexin synthesis; however, higher values were obtained with polyethyleneglycol 400 as organic phase. Higher conversion yields with both biphasic systems were obtained at the lowest temperature, where product hydrolysis was lower; volumetric productivity was higher for the fully aqueous medium (control), being higher at 20 °C. All parameters of synthesis were improved at higher substrates concentrations, obtaining conversion yields of 78.2% and 65.4%, with 60 mM 7-amino desacetoxycephalosporanic acid for the polyethyleneglycol 400–(NH₄)₂SO₄ system and the control, respectively.     

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.