Isoelectric precipitation of soybean protein using carbon dioxide as a volatile acid

A novel process is presented for the isoelectric precipitation of soy protein, using carbon dioxide as a volatile acid. By contacting a soy meal extract with pressurized carbon dioxide, the solution pH was decreased to the isoelectric region of the soy proteins. Complete precipitation of the precipitable soy proteins could be achieved for protein concentrations up to 40 g/l at pressures less than 50 bar. Isoelectric precipitation with a volatile acid enabled accurate control of the solution pH by pressure and eliminated the local pH overshoot, usual in conventional precipitation techniques. The advantage of the improved precipitation control was reflected by the morphology of the precipitate particles. Protein aggregates formed by CO₂ were perfectly spherical whereas protein precipitated by sulfuric acid had an irregular morphology. The influence of process variables to control particle size is discussed.     

Precipitation of porcine insulin with carbon dioxide

Recent works have pointed to the use of volatile electrolytes such as carbon dioxide (CO₂) dissolved in aqueous solutions as a promising alternative to the precipitating agents conventionally used for protein recovery in the food and pharmaceutical industries. In this work we investigated experimental and theoretical aspects of the precipitation of porcine insulin, a biomolecule of pharmaceutical interest, using CO₂ as an acid‐precipitating agent. The solubility of porcine insulin in NaHCO₃ solutions in pressurized CO₂ was determined as a function of temperature and pressure, with a minimum being observed close to the protein isoelectric point. A thermodynamic model was developed and successfully utilized to correlate the experimental data. Insulin was considered a polyelectrolyte in the model and its self‐association reactions were also taken into account. The biological activity of insulin was maintained after precipitation with CO₂, although some activity can be lost if foam is formed in the depressurization step. Biotechnol. Bioeng. 2009;103: 909–919. © 2009 Wiley Periodicals, Inc.     

Effect of divalent ions on protein precipitation with polyethylene glycol: mechanism of action and applications.

Polyethylene glycol (PEG) is extensively employed for protein purification by fractional precipitation. Efficiency of precipitation is highest when the solution pH is near the isoelectric point of the target protein. At pH values far from the isoelectric point of the target protein, proteins develop a net positive or negative charge and are not more resistant to precipitation. We have found that divalent cations (Ba2+, Sr2+, and Ca2+) or divalent anions (SO4(2-)) significantly change the pattern of PEG precipitation when the ion is chosen so as to counteract the expected net charge on the target protein. At moderate (5-50 mM) concentrations of Ba2+, negatively charged proteins can be precipitated from solution at pH values as high as 10 with efficiency unchanged from precipitation at pH values near their isoelectric point values. The mechanism of PEG precipitation of protein at these high pH values appears to be unchanged from the mechanism operative at the protein isoelectric point. Precipitation is rapid and the capacity for protein precipitation is high. There is no detectable coprecipitation of small molecules (AMP, ATP, and NADH) or soluble proteins (carbonic anhydrase) induced when large quantities of protein are precipitated by this method. The purification of bovine carbonic anhydrase from erythrocyte lysate is more efficient at pH 10 in the presence of Ba2+ than is conventional PEG precipitation carried out at the isoelectric point of carbonic anhydrase. Application of these observations should broaden the utility of protein purification by fractional precipitation with PEG.     

蝶蛹金小蜂毒液蛋白不同提取分离方法的比较

为了建立蝶蛹金小蜂Pteromalus puparum毒液抑制寄主血细胞免疫活性组分合适的分离纯化方法,就等电点沉淀法、乙醇沉淀法、75%硫酸铵沉淀法、75%硫酸铵沉淀法+40℃加热处理法,以及75%硫酸铵沉淀法分别与3种不同滤膜的分子大小截留法的组合等7种方法对毒液蛋白分离效果及活性的影响进行了比较。结果表明：等电点沉淀法获得的组分抑制寄主菜粉蝶Pieris rapae离体血细胞延展和包囊的活性最强,乙醇沉淀法次之,75%硫酸铵沉淀法最弱。从蛋白组分的SDS-PAGE图谱来看,等电点沉淀法获得毒液组分相对最纯,仅有3条主要谱带,分子量大小在45～116.2 kDa范围内;乙醇沉淀法次之,有5条主要谱带,分子量大小在24～116.2 kDa范围内;硫酸铵沉淀法的谱带组成与毒液蛋白粗提液相似。3种分子大小截留法获得的毒液组分的活性分析表明,强活性组分分子量大小可能都大于100 kDa。综合认为,7种方法中以等电点沉淀法提取分离蝶蛹金小蜂毒液蛋白相对为最适。     

Ethanol Causes Protein Precipitation—New Safety Issues for Catheter Locking Techniques

Objective

The ethanol lock technique has shown great potential to eradicate organisms in biofilms and to treat or prevent central venous catheter related infections. Following instillation of ethanol lock solution, however, the inherent density gradient between blood and ethanol causes gravity induced seepage of ethanol out of the catheter and blood influx into the catheter. Plasma proteins so are exposed to highly concentrated ethanol, which is a classic agent for protein precipitation. We aimed to investigate the precipitating effect of ethanol locks on plasma proteins as a possible cause for reported catheter occlusions.

Methods

Plasma samples were exposed in-vitro to ethanol (concentrations ranging from 7 to 70 v/v%) and heparin lock solutions. In catheter studies designed to mimic different in-vivo situations, the catheter tip was placed in a plasma reservoir and the material contained within the catheter was analyzed after ethanol lock instillation. The samples underwent standardized investigation for protein precipitation.

Results

Protein precipitation was observed in plasma samples containing ethanol solutions above a concentration of 28%, as well as in material retrieved from vertically positioned femoral catheters and jugular (subclavian) catheters simulating recumbent or head down tilt body positions. Precipitates could not be re-dissolved by dilution with plasma, urokinase or alteplase. Plasma samples containing heparin lock solutions showed no signs of precipitation.

Conclusions

Our in-vitro results demonstrate that ethanol locks may be associated with plasma protein precipitation in central venous catheters. This phenomenon could be related to occlusion of vascular access devices locked with ethanol, as has been reported. Concerns should be raised regarding possible complications upon injection or spontaneous gravity induced leakage of such irreversibly precipitated protein particles into the systemic circulation. We suggest limiting the maximum advisable concentration of ethanol to 28 v/v% in catheter lock solutions.     

Supercritical fluid precipitation of recombinant human immunoglobulin from aqueous solutions

Supercritical carbon dioxide was used as an antisolvent for producing recombinant human immunoglobulin G (rIgG) particulate powders. Liquid carbon dioxide (CO(2)) was premixed with ethanol to create a single-phase, modified supercritical fluid (SCF). The modified SCF was then vigorously mixed with a pharmaceutically acceptable, aqueous formulation of rIgG, and the mixture was immediately atomized into a pressurized vessel where rapid expansion of the modified SCF extracted the aqueous phase, resulting in precipitation of the protein powder. The process was reproducible, and resulting powder products were characterized by their aqueous solubilities, and the spectroscopic profile, molecular integrity, and antigen binding activity of the individual soluble fractions. Molecular integrity was assessed via size-exclusion high-performance liquid chromatography (SEC), whereas antigen binding activity was determined using an enzyme-linked immunosorbent assay (ELISA). Attempts to characterize particle size and morphology were confounded due to the extremely deliquescent nature of the powders, causing them to absorb moisture rapidly and become gummy. Operational conditions were optimized to a point which yielded powders that were completely soluble, and had ultraviolet (UV) spectroscopic and SEC profiles indistinguishable from those of the reference standard starting solution from which the powders were derived. Antigen binding activities of the powders, however, were 相似文献   

Rapid and effective focusing in a carrier ampholyte solution isoelectric focusing system: a proteome prefractionation tool

Preparative (solution) isoelectric focusing (sIEF) is a proven technique for proteome prefractionation, but carries limitations which include the risk of protein loss from isoelectric precipitation, poor focusing, and excessively long separation times. This report describes a simple yet effective method to achieve rapid focusing (as fast as 1 h) and maximize protein recovery using a carrier ampholyte sIEF system. Cathodic drift was not present over the time course of the experiment using our eight-chamber device, and we demonstrate the effectiveness of this device for focusing proteome mixtures. We also discuss an MS-compatible acidic wash protocol, which is shown to enhance the recovery of proteins following sIEF, thus, improving detection by LC-MS/MS. These approaches overcome significant shortcomings of the technique, enabling effective prefractionation prior to MS analysis.     

On the mechanism of the cold ethanol precipitation method of plasma protein fractionation

Given the negligible difference in the value of the dielectric constant of water at 20°C and that of ethanol solutions at low temperatures, the often advanced expanation for the precipitation of plasma proteins by the cold ethanol process, as being due to a reduction of the dielectric constant and the resulting increase in interprotein charge interactions, is not tenable. It is shown by a surface-thermodynamic approach that, upon dehydration by ethanol, isoelectric serum albumin molecules as well as isoelectric serum gamma globulin molecules will attract each other to a sufficient degree by van der Waals forces to become insoluble in the ethanol-water mixtures used.     

A comparison of the dodecyl sulfate-induced precipitation of the myelin basic protein with other water-soluble proteins

The interactions of sodium dodecyl sulfate with a number of proteins were examined at a variety of pH values ranging from 4.8 to 11.6 The dodecyl sulfate-induced precipitation of some of these proteins was observed within a relatively limited range of total dodecyl sulfate concentration. Most of the basic proteins precipitated at low pH but as the isoelectric point of the protein was approached the amount of protein that precipitated decreased. Bovine myelin basic protein was unique in that it precipitated at all pH values examined both above and below its isoelectric point. Thus, the dodecyl sulfate-induced precipitation of myelin basic protein appears to be different from the dodecyl sulfate-induced precipitation of most proteins. A comparison of protein precipitation at equivalent dodecyl sulfate: protein molar or weight ratios revealed very little difference in the precipitation behavior of the proteins studied. When the bovine myelin basic protein was cleaved at its single tryptophan residue, the N-terminal fragment (1–115) formed insoluble dodecyl sulfate complexes at pH values ranging from 4.8 to 9.2. The C-terminal fragment (116–169) precipitated almost completely at pH 4.8 but to a lesser extent at pH 7.4 and 9.2 Equimolar mixtures of the N- and C-terminal fragments precipitated in the presence of dodecyl sulfate at pH 7.4 and 9.2 to an extent greater than the C-terminal fragment alone but comparable to the N-terminal fragment alone or the whole basic protein. These results suggest: (a) that the mechanism by which dodecyl sulfate induces the precipitation of myelin basic protein may be unique compared to other proteins and (b) that the intact myelin basic protein is not necessary for its precipitation by dodecyl sulfate.     

Membrane proteins of Escherichia coli K-12: two-dimensional polyacrylamide gel electrophoresis of inner and outer membranes.

Protein compositions of the inner and outer membranes of Escherichia coli K-12 have been analyzed by two-dimensional gel electrophoresis in which proteins are separated according to apparent isoelectric point (first dimension) and to apparent molecular weight (second dimension). Membrane proteins except for a pair of major outer membrane proteins (proteins Ia and Ib) were found to be solubilized effectively by lysis buffer containing urea, Triton X-100, ampholines and 2-mercaptoethanol. The latter two proteins could be solubilized after precipitation of membrane fraction with trichloroacetic acid; they formed a pair of spots at an acidic region on the electropherogram. Another major protein of the outer membrane, protein II, was also identified. Most of the inner and outer membrane proteins were shown to be focused at a pH range between 4 and 6.5. Specific protein patterns characteristic for both the inner and outer membranes could thous be visualized by the present system. At least 120 and 50 protein species were detected for the inner and outer membranes, respectively.     

Enzymatic synthesis of L‐lactic acid from carbon dioxide and ethanol with an inherent cofactor regeneration cycle

Efficient conversion of carbon dioxide is of great interests to today's endeavors in controlling greenhouse gas emission. A multienzyme catalytic system that uses carbon dioxide and ethanol to produce L ‐lactate was demonstrated in this work, thereby providing a novel reaction route to convert bio‐based ethanol to an important building block for synthesis biodegradable polymers. The synthetic route has a unique internal cofactor regeneration cycle, eliminating the need of additional chemical or energy for cofactor regeneration. Lactate was successfully synthesized with 41% of ethanol converted in a batch reaction, while a turnover number of 2.2 day⁻¹ was reached for cofactor regeneration in a reaction with continuous feeding of ethanol. A kinetic model developed based on reaction kinetic parameters determined separately for each reaction step predicted well the reaction rates and yields of the multienzyme reaction system. Biotechnol. Bioeng. 2011;108: 465–469. © 2010 Wiley Periodicals, Inc.     

Fabrication of nickel and chromium nanoparticles using the protein cage of apoferritin

The iron storage protein, apoferritin, has a cavity in which iron is oxidized and stored as a hydrated oxide core. The size of the core is about 7 nm in diameter and is regulated by the cavity size. The cavity can be utilized as a nanoreactor to grow inorganic crystals. We incubated apoferritin in nickel or chromium salt solutions to fabricate hydroxide nanoparticles in the cavity. By using a solution containing dissolved carbon dioxide and by precisely controlling the pH, we succeeded in fabricating nickel and chromium cores. During the hydroxylation process of nickel ions a large portion of the apoferritin precipitated through bulk precipitation of nickel hydroxide. Bulk precipitation was suppressed by adding ammonium ions. However, even in the presence of ammonium ions the core did not form using a degassed solution. We concluded that carbonate ions were indispensable for core formation and that the ammonium ions prevented precipitation in the bulk solution. The optimized condition for nickel core formation was 0.3 mg/mL horse spleen apoferritin and 5 mM ammonium nickel sulfate in water containing dissolved carbon dioxide. The pH was maintained at 8.65 using two buffer solutions: 150 mM HEPES (pH 7.5) and 195 mM CAPSO (pH 9.5) with 20 mM ammonium at 23 degrees C. The pH had not changed after 48 h. After 24 h of incubation, all apoferritins remained in the supernatant and all of them had cores. Recombinant L-ferritin showed less precipitation even above a pH of 8.65. A chromium core was formed under the following conditions: 0.1 mg/mL apoferritin, 1 mM ammonium chromium sulfate, 100 mM HEPES (pH 7.5) with a solution containing dissolved carbon dioxide. About 80% of the supernatant apoferritin (0.07 mg/mL) formed a core. In nickel and chromium core formation, carbonate ions would play an important role in accelerating the hydroxylation in the apoferritin cavity compared to the bulk solution outside.     

聚丙烯酸分离纯化苦瓜种仁碱性蛋白的方法及影响因素

以苦瓜籽为材料,研究了聚丙烯酸分离纯化苦瓜种仁碱性蛋白的方法及影响因素。等电点沉淀试验表明,柠檬酸、盐酸分别调节苦瓜种仁粗提液pH至6.0、4.0时,各有14.62%和32.49%的苦瓜种仁蛋白被沉淀。醋酸的等电点沉淀作用呈现阶段性特点,pH6.0和4.0时分别有26.17%和38.72%的苦瓜种仁蛋白被沉淀。醋酸、盐酸和柠檬酸处理的1mL苦瓜种仁粗提液(pH4.0),1%PAA选择性沉淀碱性蛋白(等电点pI为8.65~9.30)的最佳用量分别为100μL、120μL和100μL。醋酸调节苦瓜种仁粗提液pH分别至5.0、4.0和3.0,等电点沉淀后的上清液用PAA沉淀碱性蛋白,当PAA(1%)用量为160μL/mL提取液时,pH5.0和3.0样液分别有33.77%和43.56%蛋白质被沉淀;当PAA用量为120μL/mL提取液时,pH4.0样液中30.83%蛋白质被沉淀。PAA-蛋白质复合物溶解于碱性溶液(pH>9.0),当溶液NaCl浓度为3.0%时,溶液蛋白质浓度最高。PAA选择性沉淀的苦瓜种仁碱性蛋白经SephadexG-75柱层析分离,分别在175min和300min出现主峰Ⅰ和Ⅱ。SDS-PAGE和IEF分析表明主峰Ⅰ的分子量约为30kD,pI值约为9.5,主峰Ⅱ的分子量约为10kD,pI值约为9.3。     

Selective precipitation of proteins from guanidine hydrochloride-containing solutions with ethanol

The solubility of guanidine hydrochloride in ethanol and conversely the low solubility of proteins have been used as the basis for a procedure for recovering proteins from guanidine-containing solutions by selective precipitation. Yields of greater than 94% were observed with as little as 28 ng protein and of 98% for larger quantities of protein up to 20 mg/ml. The precipitations were independent of molecular weight for proteins in the range of 6-100 kDa and could be run in as little as 5 min at room temperature. Unlike the conventional desalting methods for removing guanidine, ethanol precipitation is rapid, efficient, and can be applied simultaneously to a large number of samples. The approach should have a wide range of applications.     

The synthesis of amino acids by Methanobacterium omelianskii

1. Methanobacterium omelianskii was grown on (14)CO(2) and unlabelled ethanol, or on [1-(14)C]- or [2-(14)C]-ethanol and unlabelled carbon dioxide. The cell protein was hydrolysed and certain of the amino acids were isolated and degraded. 2. Carbon from both carbon dioxide and ethanol is used for biosynthesis of amino acids, and in most cases ethanol is incorporated as a C(2) unit. Ethanol carbon atoms and carbon dioxide carbon atoms apparently enter the same range of compounds. Ethanol and carbon dioxide are equally important as sources of cell carbon. 3. The origins of carbon atoms of aspartate, alanine, glycine, serine and threonine are consistent with the synthesis of these amino acids, by pathways known to exist in aerobic organisms, from pyruvate arising by a C(2)+C(1) condensation. The proportion of total radioactivity found in C-1 of lysine, proline, methionine and valine is consistent with synthesis of these amino acids by pathways similar to those found in Escherichia coli. Isoleucine is probably formed by carboxylation of a C(5) precursor formed entirely from ethanol. Glutamate is formed by an unknown pathway.     

Effect of temperature on the separation of soybean 11 S and 7 S protein fractions during bipolar membrane electroacidification

The purpose of this study was to evaluate the effect of temperature (10 and 27 degrees C) on the efficiency of bipolar membrane electroacidification (BMEA) to fractionate soybean proteins. BMEA is a technology derived from electrodialysis, based on the isoelectric precipitation of proteins. It appears that temperature has a significant effect on the selective precipitation of the soybean protein fractions, mainly 11 S and 7 S, during BMEA. At 27 degrees C, the precipitation profile of the four protein fractions is situated in a pH range from 6.6 to 4.4, with no possibility of separating any of theses fractions. However, at 10 degrees C, the 11 S globulin precipitates at a higher pH than at 27 degrees C, pH 6.7 vs 5.9, allowing the fractionation of 11 S from the other fractions. Using electroacidification it is possible to obtain a precipitate solution enriched in the 11 S fraction (71.8% of 11 S and 10.8% of 7 S) and a supernatant solution enriched in the 7 S fraction (46.6% of 7 S and 4.6% of 11S).     

Effects of flooding under hydrostatic pressure on the respiratory metabolism of germinated wheat seeds

Germinated wheat seeds ( Triticum aestivum L. cv. Barqai) that had been subjected to short hydrostatic pressure treatments (0.3–1.2 MPa) changed their normal metabolism into one which is characterized by a high ethanol production, a low O₂ consumption and a low CO₂ evolution. Alcoholic fermentation could account for ca half of the CO₂ evolved from the pressurized seeds. The level of acetaldehyde was low, though significantly higher in the pressurized seeds than in the controls. Subjection of wheat seeds to osmotic stress under aerobic conditions lowered their O₂ uptake and CO₂ evolution but did not induce ethanol production. Exposure of pressurized seeds to NaCl stress did not alter their ethanol production beyond that which had been induced by pressure. Ethanol production by pressurized seeds increased following either the addition of sucrose or by excision of the embryos from the endosperms. More electrolytes leaked into the embedding solution from pressurized seeds than from control seeds. Exogenous ethanol was toxic to wheat seeds at concentrations as low as 343 m M . The effects of hydrostatic pressure and of the consequently induced ethanol production on the mortality of flooded seeds is discussed.     

Dye-promoted precipitation of serum proteins. Mechanism and application.

Immobilized dyes have been used primarily for purification of nucleotide dependent enzymes and proteins from plasma and other sources. Due to their low costs, high protein binding capacity and resistance to degradation dyes bear the potential as ligand for affinity separation of proteins on a large scale. In this paper dyes have been used for precipitation of proteins. Using albumin, prealbumin, alpha 1-acid glycoprotein and immunoglobulin G as model proteins we could demonstrate that dye-promoted precipitation depends on several factors which include the structure of the dye, the pH of the solution, the dye/protein molar ratio and the intrinsic properties of the proteins. It revealed that most of the dyes tested were endowed with the precipitating potential. The efficacy of precipitation was found to increase with the complexity of the dye structure. However, the amount of a dye required for total precipitation was found to be different for a given protein. Electrostatic as well as hydrophobic forces are involved in the mechanism of precipitation. It was demonstrated that by optimizing the conditions, mixtures of proteins can be resolved by dye-promoted precipitation. The high sensitivity of the reaction offers the possibility of using this method for rapid concentration of very diluted protein solutions.     

超声波-微波协同盐提莲子蛋白

探索利用超声波微波协同盐提湘莲蛋白质最佳提取条件。最佳工艺条件为超声波功率50w,微波功率600W,料液比1：12（g／mE）、38℃、0．15mol／LNaCl盐溶液提取15min,最佳提取率可达88．9％。以CO2为沉淀剂,乙醇为助溶剂,研究加压CO2和乙醇对蛋白质的协同沉淀作用。在常温、5MPa加压CO2酸沉8％（质量分数）的湘莲蛋白质乙醇液（体积分数20％乙醇）0．5h时,莲子蛋白沉淀率可达81．2％。结果表明,用文中方法提取湘莲蛋白,湘莲蛋白收率为72．2％,纯度达93％.     

A quantitative description of the growth of Saccharomyces cerevisiae CBS 426 on a mixed substrate of glucose and ethanol

Saccharomyces cerevisiae CBS 426 was grown aerobically in continuous culture with a mixture of glucose and ethanol as the carbon source. The flows of biomass, glucose, ethanol, oxygen, and carbon dioxide were measured. A model for growth with two substrates was derived. Application of this model to the above-mentioned system yielded values for Y_ATP and P/O. The joint confidence regions for these parameters were calculated. The relevance to industrial production of bakers' yeast is discussed.