Purification of lactic acid dehydrogenase from crude bovine heart extract by pH-peak focusing counter-current chromatography

pH-peak focusing counter-current chromatography (CCC) was applied to the purification of lactic acid dehydrogenase (LDH) from a crude bovine heart extract using a cross-axis coil planet centrifuge (CPC). The experiment was performed with two sets of polymer phase systems composed of 16% (w/w) polyethylene glycol (PEG) 1000-12.5% (w/w) potassium phosphate buffer and 15% (w/w) PEG 1540-15% (w/w) ammonium sulfate each at various pH values. The best result was achieved from the PEG 1540-ammonium sulfate polymer phase system by adding a retainer (10 mM acetic acid) to the upper stationary phase and an eluter (100 mM sodium hydroxide) to the lower mobile phase. At a flow-rate of 0.5 ml/min, LDH was eluted as a sharp peak which was well resolved from other proteins. Collected fractions were analyzed by the LDH enzymatic activity and by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis to detect contaminated proteins. LDH was purified directly from crude bovine heart extract in a concentrated state.     

高速逆流双水相色谱法纯化卵白蛋白

生物大分子的液_固色谱纯化过程中固相载体会产生产物吸附、变性等不良影响。高速逆流色谱无需固相载体 ,且具有高分便率和高回收率的优点 ,其中有机相 水相体系在分离天然产物中应用广泛 ,而应用双水相体系分离生物大分子尚处于研究阶段。双水相高速逆流色谱体系的建立与仪器设备及操作工艺条件密切相关 ,因此利用多分离柱高速逆流色谱仪 ,研究了PEG1000-无机盐双水相体系对标准蛋白质混合物以及卵白蛋白的分离。pH值和PEG浓度对不同种类蛋白质的分配系数影响不同 ,实验发现在pH9.2的150% (W/W)PEG1000 170% (W/W)磷酸钾盐体系中 ,细胞色素C、溶菌酶和肌红蛋白的分配系数差异较大 ,且分布合理 ,因而采用该体系在 0 8mL min流速 ,85 0r min转速的条件下 ,成功分离了细胞色素C、溶菌酶和肌红蛋白的混合物。实验也发现在pH9 2的 16 0 % (W/W)PEG10 0 0 17 0 % (W/W)磷酸钾盐体系中 ,鸡蛋清样品中的主要蛋白质成分:卵转铁蛋白、卵白蛋白和溶菌酶的分配系数差异最大 ,因而采用该体系在 1 8mL min流速、85 0r mi转速的条件下,200min内从鸡蛋清样品中成功分离卵白蛋白,其电泳纯度为100%,收率为95%.     

Aqueous-aqueous two-phase systems composed of low molecular weight of polyethylene glycols and dextrans for counter-current chromatographic purification of proteins

New aqueous-aqueous two-phase systems composed of relatively low molecular weight polymers such as polyethylene glycol (PEG) (Mr: 1000-4000) and dextran (Mr: 10,000 and 40,000) were evaluated for purification of proteins by counter-current chromatography (CCC). The compositions of aqueous two-phase systems were optimized by measuring parameters such as viscosity and volume ratio between the two phases. CCC purification of a glucosyltransferase (GTF) from Streptococcus mutans (SM) cell-lysate was successfully demonstrated with a 7.5% PEG 3350-10% dextran T40 system containing 10mM potassium phosphate buffer at pH 9.0. After CCC purification, both PEG and dextran contained in the CCC fractions were easily removed by ultrafiltration in a short period of time. The fractionated column contents containing GTF were analyzed by enzymatic activity as well as sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The recovery of the enzyme from CCC fraction was over 95% as estimated by enzymatic activities.     

Purification of glucosyltransferase from cell-lysate of Streptococcus mutans by counter-current chromatography using aqueous polymer two-phase system

Counter-current chromatography (CCC) using a cross-axis coil planet centrifuge (X-axis CPC) was applied to the purification of glucosyltransferase (GTF) from a cell-lysate of cariogenic bacteria. The purification was performed using an aqueous polymer two-phase system composed of 4.4% (w/w) polyethylene glycol (PEG) 8000-6% (w/w) dextran T500 containing 10mM phosphate buffer at pH 9.2 by eluting the upper phase (UP) at 1.0ml/min. The bacterial GTF in the cell-lysate of Streptococcus mutans was selectively retained in the dextran-rich lower stationary phase. The column contents were diluted and subjected to hydroxyapatite (HA) chromatography to remove the polymers from the GTF. Fractions eluted with 500mM potassium phosphate buffer were analyzed by GTF enzymatic activity as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The GTF purity in the final product was increased about 87 times as that in the cell-lysate with a good recovery rate of about 79% through this purification process.     

Pepsin extraction from bovine stomach using aqueous two-phase systems: molecular mechanism and influence of homogenate mass and phase volume ratio

Pepsin partitioning, a gastric acid protease, in aqueous two-phase systems of polyethyleneglycol/potassium phosphate, sodium citrate and ammonium sulphate was assayed using polyethylenglycol of different molecular mass. Pepsin was found to be partitioned towards the polymer-rich phase in all the systems, which suggests an important protein-polymer interaction due to the highly hydrophobic character of the protein surface exposed to the solvent. The pepsin partitioning behavior was explained according to Timasheff's preferential interaction theory. The process was driven entropically with participation of structured water around the polyethyleneglycol ethylenic chains. The best pepsin recovery was observed in the systems polyethyleneglycol molecular mass 600. These systems were chosen in order to assay the bovine stomach homogenate partition and to compare different working conditions such as the top-bottom phase volume ratio and homogenate proportions in the total system. The best purification factors were obtained with PEG600/potassium phosphate with low top-bottom volume ratio using 15% of bovine stomach homogenate in the system total mass.     

Rapid fractionation of bovine transferrin using immobilized gangliosides.

Bovine transferrin (BTF) was fractionated from bovine whey using ganglioside affinity chromatography. After loading the immobilized matrix with a 2% whey solution, the matrix was washed with sodium acetate buffer at pH 4 containing 1 M NaCl before elution of BTF with sodium phosphate buffers at pH 7. Concanavalin-A affinity and ion exchange chromatography were used for further purification. The ganglioside column showed a 74.2% BTF recovery from whey and BTF was enriched to 61% purity with ion exchange chromatography. Bovine transferrin was identified by SDS-PAGE and western analysis. The Concanavalin-A affinity and ion exchange chromatography steps enriched BTF in the samples and removed other whey proteins from ganglioside purified fractions. These results indicate that immobilized ganglioside can be used to fractionate BTF from bovine whey. Our novel ganglioside affinity chromatography is rapid and efficient for the fractionation of BTF from whey.     

Purification and in situ immobilization of lipase from of a mutant of Trichosporon laibacchii using aqueous two-phase systems

The crude intracellular lipase (cell homogenate) from Trichosporon laibacchii was subjected to partial purification by aqueous two-phase system (ATPS) and then in situ immobilization by directly adding diatomites as carrier to the top PEG-rich phase of ATPS. A partition study of lipase in the ATPS formed by polyethylene glycol–potassium phosphate has been performed. The influence of system parameters such as molecular weight of PEG, system phase composition and system pH on the partitioning behaviour of lipase was evaluated. The ATPS consisting of PEG 4000 (12%) and potassium phosphate (K₂HPO₄, 13%) resulted in partition of lipase to the PEG-rich phase with partition coefficient 7.61, activity recovery 80.4%, and purification factor of 5.84 at pH of 7.0 and 2.0% NaCl. Moreover, the in situ immobilization of lipase in PEG phase resulted in a highest immobilized lipase activity of 1114.6 U g^?1. The above results show that this novel lipase immobilization procedure which couples ATPS extract and enzyme immobilization is cost-effective as well as time-saving. It could be potentially useful technique for the purification and immobilization of lipase.     

Synthesis of a disulfide affinity adsorbent for purification of estrogen receptor

Synthesis of an estrogen affinity adsorbent containing a disulfide linkage between the steroid and stationary matrix permitted facile purification of high affinity estrogen binding proteins. Following affinity chromatography of either antibody directed against estrone 17-carboxymethyloxime — bovine serum albumin or immature calf uterine cytoplasmic estrogen receptor proteins, the specifically bound protein was recovered by incubating the adsorbent with 2-mercaptoethanol. Crude antibody and uterine cytosol was prepared for affinity chromatography in buffer containing 10^?3 to 10^?2M cystamine (S-S) to block SH-containing proteins, in order to protect the adsorbent against protein-mediated S-S ag SH exchange. Cystamine was found to markedly stabilize crude cytosol receptor protein by 200–300% compared with preparations obtained under ordinary conditions. Disulfide affinity adsorbents are versatile in that they can be used either under conventional conditions of specific protein recovery, or with 2-mercaptoethanol which removes the ligand and bound protein from the stationary matrix quantitatively.     

Affinity separation by protein conjugated IgG in aqueous two-phase systems using horseradish peroxidase as a ligand carrier

A novel affinity separation method in an aqueous two-phase system (ATPS) is suggested, using protein conjugated IgG as a ligand. For verification of the proposed approach, horseradish peroxidase (HRP) and human IgG was used as a ligand carrier and affinity ligand, respectively. The partition of the affinity ligand, human IgG, was controlled by the conjugation of HRP. Two ATPSs, one consisting of potassium phosphate (15%, w/w) and polyethylene glycol (PEG, M.W. 1450, 10%, w/w) and the other of dextran T500 (5%, w/w) and PEG (M.W. 8000, 5%, w/w), were used. The conjugated human IgG-HRP favored a PEG-rich top phase, whereas human IgG, rabbit anti-human IgG and goat anti-mouse IgG preferred a salt or dextran-rich bottom phase. Using the conjugated human IgG-HRP, rabbit anti-human IgG was successfully separated into a PEG-rich top phase from the mixture with goat anti-mouse IgG. The appropriate molar ratio between human IgG-HRP and rabbit anti-human IgG was around 3:1 and 1:1 for the salt and dextran-based ATPS, respectively. The dextran-based ATPS showed a better recovery yield and purity than the salt-based ATPS for the range of test conditions employed in this experiment. The yield and purity of the recovered rabbit anti-human IgG were 90.8 and 87.7%, respectively, in the dextran-based ATPS, while those in the salt-based ATPS were 78.2 and 73.2%.     

Modified Two-Phase System for Partition of Bacillus macerans Spores

An aqueous two-phase system made with polyethylene glycol (PEG) 1000 and potassium phosphate gives much higher recovery of Bacillus macerans spores in the upper phase (PEG rich) than does a similar system utilizing PEG 4000. The upper phase completely rejects vegetative cells, which collect at the interface. The system may be useful in purifying spores of other species. Scanning electron micrographs of B. macerans spores cleaned in this system show no obvious attached sporangial fragments. The micrographs show that the ridged coat may form polygonal structures at the poles, as previously observed in Bacillus polymyxa spores.     

Two-step process for initial capture of plasmid DNA and partial removal of RNA using aqueous two-phase systems

In this paper, a two-step process for initial capture of plasmid DNA (pDNA) and partial removal of RNA using polyethylene glycol (PEG) and di-potassium hydrogen phosphate aqueous two-phase systems (ATPS) has been investigated. A Kühni-type ATPS extraction column was prepared with 50 ml (12% (w/w) PEG 1450, 12% (w/w) phosphate) of stationary phase and loaded with crude mobile phase (26% (w/w) PEG 1450, 4% (w/w) phosphate and 70% (w/w) lysate) at a flow rate of 6 ml min⁻¹ at an impeller speed of 200 rpm. The experiment was terminated after 100 min, and after complete resettling of the phases, 45 ml of stationary phase was harvested. During a subsequent second extraction step contained 18% (w/w) PEG 300 and 14% (w/w) phosphate, a proportion of RNA, which was also concentrated during the column process, was removed. It was demonstrated that the recovery of pDNA in the second bottom phase was 89.4%, which was similar to the initial recovery after column extraction (92.1%).     

Comparative studies on the molecular weight of glutathione S-transferases from mammalian livers and an insect.

1. A comparative study was conducted on the molecular weights of glutathione S-transferases in the housefly and liver of the mouse and rat using Sephadex G-100 gel chromatography. 2. The values varied depending upon the buffers used in gel filtration. Molecular weights of 44,600, 53,600 and 43,000 daltons respectively were obtained with 0.01 M potassium phosphate buffer, pH 6.7; 0.05 M Tris-HCl buffer, pH 8.0; and 0.05 M Tris-HCl buffer containing 0.1 M KCl, pH 8.0, respectively. 3. There was no difference in the molecular weights of the enzymes obtained from the insect and from the mammalian livers. Purified enzymes eluted in the same fractions as those from the crude extracts, suggesting little modification in the molecular size of the enzymes during purification. 4. The presence of a large volume of stabilizer(s) in the enzyme solutions applied to the column delayed the elution of the activity peaks and resulted in erroneous values. Therefore, different literature values of molecular weights for glutathione S-transferases may be the result of different buffers and stabilizers used in gel filtration and probably do not represent a real difference in molecular size.     

Isolation of alpha-1-antitrypsin from human plasma by partitioning in aqueous biphasic systems of polyethyleneglycol-phosphate

The partitioning of alpha-1-antitrypsin was assayed in biphasic aqueous systems containing potassium phosphate and two polyethyleneglycols of molecular mass 600 and 1000, respectively. In order to isolate the alpha-1-antitrypsin from serum plasma, the partitioning behaviour of human serum albumin, its principal contaminant, was also studied. Several aqueous two-phase systems with different partitioning properties were obtained by varying the PEG1000/PEG600 mass proportion. In systems with PEG1000/PEG600 mass ratio of 8, the optimal difference between the partition coefficients of both proteins was found. Under such conditions, a satisfactory purification was carried out by a three-step extraction procedure. By applying this method the alpha-1-antitrypsin specific activity increased severalfold (nearly 10 times) with a yield of 43%.     

Extractive fermentation for enhanced gellan-hydrolysing enzyme production by Bacillus thuringiensis H14

A new extractive fermentation process using PEG and potassium phosphate aqueous two-phase system (ATPS) was developed for enhanced production of gellan-hydrolysing enzyme by Bacillus thuringiensis H14. Five different Bacillus sp. were tested for their ability to synthesize gellan-hydrolysing enzyme. Bacillus thuringiensis H14 was found to be the best organism for gellan-hydrolysing enzyme production. The enzyme showed maximum activity at pH 7.5 and 40 °C. The partition studies of gellan-hydrolysing enzyme in the system using PEG X (X = 9000, 6000, 4000) and potassium phosphate–water and PEG–sodium citrate–water system indicated at PEG (4000)– potassium phosphate–water is the best system for partitioning of gellan-hydrolysing enzyme into the PEG phase (K = 4.99). Gellan-hydrolysing enzyme production by Bacillus thuringiensis H14 was studied in ATPSs composed of PEG X (X = 9000, 6000, 4000) and potassium phosphate. The top phase is continuous and rich in PEG while the bottom phase is dispersed and is rich in phosphate, microbial cells being mainly retained in the bottom phase. The gellan-hydrolysing enzyme produced during fermentation partitioned into the upper PEG phase and total gellan-hydrolysing enzyme produced was 2.12, 2.29 and 2.40 times higher than that of homogeneous fermentation when the fermentations were carried out using PEG 9000–potassium phosphate–water, PEG 6000–potassium phosphate–water, PEG 4000–potassium phosphate–water systems respectively.     

The in vitro metabolism of Eulan WA New by liver homogenates from freshwater fish

The rate of formation of pentachloro-2-aminodiphenylether (5-PAD) from pentachloro-2-(chloromethylsulphonamido) diphenylether (6-PCSD) by goldfish liver homogenates was studied in a variety of buffers. The highest rate was observed when the homogenate was prepared and assayed in phosphate buffer containing dithiothreitol. The 5-PAD forming activity of liver homogenate preparations from a variety of freshwater fish (goldfish, carp, pike, rainbow trout, perch and eel) showed contrasting species differences. These results could be correlated with information available on the toxicity of the mothproofing agent Eulan WA New (which contains PCSDs) to these fish.     

Simplified two-stage method to B-phycoerythrin recovery from Porphyridium cruentum

A simplified two-stage method for B-phycoerythrin (BPE) recovery from Porphyridium cruentum was developed. The proposed method involved cell disruption by sonication and primary recovery by aqueous two-phase partition. The evaluation of two different methods of cell disruption and the effect of increasing concentration of cell homogenate from P. cruentum culture upon aqueous two-phase systems (ATPS) performance was carried out to avoid the use of precipitation stages. Cell disruption by sonication proved to be superior over manual maceration since a five time increase in the concentration of B-phycoerythrin release was achieved. An increase in the concentration of crude extract from disrupted P. cruentum cells loaded to the ATPS (from 10 to 40%, w/w) proved to be suitable to increase the product purity and benefited the processing of highly concentrated disrupted extract. Kinetics studies of phase separation performed suggested the use of batch settlers with height/diameter (H/D) ratio less than one to reduce the necessary time for the phases to separate. The proposed ATPS stage comprising of 29% (w/w) polyethylene glycol (PEG) 1000g/mol, 9% (w/w) potassium phosphate, tie-line length (TLL) of 45% (w/w), volume ratio (V(R)) of 4.5, pH 7.0 and 40% (w/w) crude extract loaded in a batch settler with H/D ratio of 0.5 proved to be efficient for the recovery of 90% of B-phycoerythrin at the top PEG-rich phase. The purity of B-phycoerythrin increased up to 4.0 times after the two-stage method. The results reported here demonstrate the potential implementation of a strategy to B-phycoerythrin recovery with a purity of 3.2 (estimated by the absorbance relation of 545-280nm) from P. cruentum.     

Synthesis and application of a poly(ethylene glycol)-antibody affinity ligand for cell separations in aqueous polymer two-phase systems

The possibility of producing biospecific affinity ligands for separating cells in two polymer aqueous phase systems on the basis of cell surface antigens was investigated. Rabbit anti-human erythrocyte IgG was reacted with cyanuric chloride-activated monomethyl poly(ethylene glycol) (PEG) fractions (molecular weights approximately 200, 1900, and 5000) at various molar ratios of PEG to protein lysine groups. The partition coefficient of the protein in a Dextran/PEG two-phase system increased with increasing degree of modification and increasing PEG molecular weight. There was a concomitant loss in ability to agglutinate human erythrocytes. The ability of the modified IgG to bind to a DEAE-cellulose column was almost eliminated by reaction with the PEG 5000, and was decreased to a lesser extent by PEG 1900. This PEG 1900-modified IgG substantially increased the partition of fresh or fixed human erythrocytes into the PEG-rich phase of a suitable phase system, while having no effect on rabbit cell partition. The partition increase could be inhibited by unmodified anti-human red cell IgG but not by nonspecific unmodified human IgG, demonstrating that the ligand effects were specific for the cell type against which the antibody was raised. A mixture of rabbit and human erythrocytes, which ordinarily have very similar partitions in the phase systems used, could be separated on a countercurrent distribution apparatus using the modified IgG. These results demonstrate the feasibility of producing immunologically specific affinity partition ligands for cell separation.     

Partitioning of beta-galactosidase fusion proteins in PEG/potassium phosphate aqueous two-phase systems.

Four different beta-galactosidase fusion proteins have been partitioned in poly(ethylene glycol) (PEG) 4000/potassium phosphate aqueous two-phase systems. The partition coefficients (K) of staphylococcal protein A-beta-galactosidase (SpA beta gal) (K = 3.5) and staphylococcal protein A-streptococcal protein G-beta-galactosidase (AG beta gal) (K = 2.8) were compared with the partition coefficients of their constituent molecules, beta-galactosidase, SpA, and protein AG. It was found that by fusing beta-galactosidase to the smaller proteins SpA and protein AG, their partition coefficients were increased four to five times. Experimental data were fitted into, and found to agree with, the Albertsson partition model of interacting molecules. The compatibility with PEG and potassium phosphate of beta-galactosidase, SpA, and two different versions of the SpA beta gal protein, displayed as precipitation curves, showed a relationship to the protein partition coefficients in PEG/potassium phosphate systems. High solubility in one phase component was accompanied by preferential partitioning to the phase rich in the same component in the PEG/potassium phosphate system. Also, a changed linker region in SpA beta gal resulted in a more soluble protein. This, together with the improved K values of the target proteins by fusion, shows that it is possible to use beta-galactosidase as an affinity handle.     

Technical improvement in the purification of enzymes from red algae using an aqueous two-phase partitioning system

The effective elimination of phycobiliproteins from crude enzyme preparation of the red alga Caloglossa continua (Okamura) King et Puttock (Ceramiales, Florideophyceae) was investigated in an aqueous two‐phase partitioning system (ATPS) by changing the concentrations of polyethylene glycol (PEG) and ammonium sulfate (AS). The phycobiliproteins shifted from the AS‐rich lower phase to the PEG‐rich upper phase in high PEG and AS concentrations. The best ATPS condition for the elimination of phycobiliproteins from the lower phase was obtained by the combination of 20% (weight/volume; w/v) PEG and 16% (w/v) AS. However, the recovery of aldolase and mannitol‐1‐phos‐phatase activities was significantly reduced. For purification of the enzymes, a combination of 15% (w/v) PEG and 16% (w/v) AS was the best ATPS condition, because a high specific activity and recovery of the enzymes were obtained. Under these conditions, 98% of the phycobiliproteins were removed from the lower phase. Therefore, the ATPS proved to be a very useful method as a first step in the purification of enzymes from red algae.     

Subcellular localization of non-specific carboxylesterases, acylcarnitine hydrolase, monoacylglycerol lipase and palmitoyl-CoA hydrolase in rat liver

The subcellular distribution and sidedness on the membranes of four chemically and genetically distinct esterases (esterases ES-3, ES-4, ES-8, ES-15) in rat liver was investigated using selective substrates. (1) Rat liver homogenate was divided into nine subcellular fractions by differential centrifugation techniques. The cell fractions were assayed for the enzymatic hydrolysis of acetanilide (ES-3), propanidid, palmitoyl-CoA and monopalmitoylglycerol (ES-4), methyl butyrate and octanoylglycerol (ES-8), and decanoylcarnitine (ES-15). With all substrates, the highest specific activities were found in the rough and smooth endoplasmic reticulum fractions. This localization of the esterases was confirmed by labelling the cell fractions with the specific, covalently binding inhibitor bis(4-nitro[14C]phenyl) phosphate. The enzymatic hydrolysis of the palmitoyl esters in differing cell fractions did not completely parallel that of propanidid. This confirms the well-known existence of palmitoyl-CoA hydrolases other than esterase ES-4. (2) Density gradient fractionations with crude mitochondria indicated that a low amount of at least one of these carboxylesterases was an integral part of these organelles too. (3) Proteinase treatment reduced the non-specific esterase activities as well as lipase activities versus dioctanoylglycerol, acylcarnitines and palmitoyl-CoA only in detergent-disrupted microsomal vesicles. This might indicate a lumenal orientation of these enzymes. However, of the charged substrates palmitoylcarnitine and palmitoyl-CoA only the latter one showed the typical latency to be expected for a hydrolysis in the lumen of the endoplasmic reticulum.