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PubMedDate not supplied

Carboxymethyl cellulose as a new heterobifunctional ligand carrier for affinity precipitation of proteins.

Affinity precipitation is a technique that imparts selectivity to the widely used primary purification step of precipitation of proteins from crude extracts. Hetero-bifunctional affinity precipitation involves use of reversibly soluble/insoluble polymers that can be used as backbones to conjugate affinity ligands for specific separations. A variety of such polymers have been reported in literature. In this work we report development of carboxymethyl cellulose (CM cellulose) as a cheap, readily available and versatile reversibly soluble polymer system. Available CM cellulose as sodium salt could be quantitatively precipitated from its aqueous solution in presence of about 50 mM calcium and 7.2% w/v polyethylene glycol-4000, and could be resolubilised in the working buffer in absence of calcium, polyethylene glycol or both. Effectiveness of the CM cellulose-calcium-polyethylene glycol system was demonstrated by purifying lactate dehydrogenase from porcine muscle extract using covalently conjugated Cibacron blue dye-ligand. By careful choice of conditions that suppressed non-specific interactions, the system was shown to be an effective affinity precipitation polymer system inspite of the polyelectrolytic nature of CM cellulose. Up to 23 fold purification of the enzyme from crude extarct was obtained in one single precipitation sequence.

Animals↗

PubMed2000

Examination of bioaffinity immobilization by precipitation of mannan and mannan-containing enzymes with legume lectins.

The interaction of four lectins from crops of the legume family with Saccharomyces cerevisiae alpha-mannan, and also with two glycoenzymes containing mainly alpha-mannan moieties, has been studied. The interaction was characterized by a quantitative precipitation assay. The results of precipitation differ with respect to both quality (the point of maximum precipitation) and of the quantity (the amount of aggregated lectin and saccharide). The lectin concanavalin A [Con A, from jack bean (Canavalia ensiformis)] was observed to form more extensive precipitates with Saccharomyces cerevisiae mannan and glycoenzymes than did lectins from Lens culinaris (lentil) and Pisum sativum (garden pea), while in the case of Vicia faba (broad or fava bean) no interaction was found with either the examined mannans or with glycosylated enzymes. The complete precipitation of invertase and glucoamylase with Con A (enzymes and also Con A; up to 100%) was achieved at a Con A glycoenzyme molar ratio of 20.2 and 2.3 respectively, whereby about 85% of precipitated and also of initial activities of glycoenzymes were determined in the aggregates. More valuable results were achieved by the technique of enzyme immobilization called 'multiple bioaffinity layering' which is based on the stepwise biospecific adsorption of the glycosylated enzymes and Con A on a matrix precoupled with Con A. A 3-fold repetition of the layering procedure afforded up to a 10-fold increase in catalytic activity of the immobilized invertase, in contrast with a 2.1-fold increase in catalytic activity of the immobilized glucoamylase.

Affinity Labels↗

PubMed1976

A study of the physicochemical interactions between biliary lipids and chlorpromazine hydrochloride. Bile-salt precipitation as a mechanism of phenothiazine-induced bile secretory failure.

Since chlorpromazine hydrochloride [2-chloro-10-(3-dimethylaminopropyl)-phenothiazine hydrochloride] is commonly implicated in causing bile-secretory failure in man and is secreted into bile, we have studied the physicochemical interactions of the drug with the major components of bile in vitro. Chlorpromazine hydrochloride molecules are amphiphilic by virtue of possessing a polar tertiary amine group linked by a short paraffin chain to a tricyclic hydrophobic part. At pH values below the apparent pK (pK'a 7.4) the molecules are water-soluble cationic detergents. We show that bile salts in concentrations above their critical micellar concentrations are precipitated from solution by chlorpromazine hydrochloride as insoluble 1:1 salt complexes. In the case of mixed bile-salt/phosphatidylcholine micellar solutions, however, the degree of precipitation is inhibited by the phospholipid in proportion to its mole fraction. With increases in the concentration of chlorpromazine hydrochloride or bile salt, micellar solubilization of the precipitated complexes results. Sonicated dispersions of the negatively charged phospholipid phosphatidylserine were also precipitated, but dispersions of the zwitterionic phospholipid phosphatidylcholine were not. Chlorpromazine hydrochloride efficiently solubilized these membrane phospholipids as mixed micellar solutions when the drug:phospholipid molar ratio reached 4:1. Polarizing-microscopy and X-ray-diffraction studies revealed that the precipitated complexes were amorphous and potentiometric studies confirmed the presence of a salt bond. Some dissociation of the complex occurred in the case of the most polar bile salt (Ks 0.365). As canalicular bile-salt secretion determines much of bile-water flow, we propose that complexing and precipitation of bile salts by chlorpromazine hydrochloride and its metabolites may be physicochemically related to the reversible bile-secretory failure produced by this drug.

Animals↗

PubMed1998

Prevention of precipitation of phenytoin in an infusion fluid by hydroxypropyl beta-cyclodextrin.

Injection of phenytoin is often diluted with infusion fluids before administration, which may lead to precipitation of the drug due to changes in pH and/or vehicle. It is not possible to add cyclodextrins to the original injections to prevent precipitation of the drug, because the quantities required would be impractical (>100% w/v). However, from a knowledge of the solubility of phenytoin in cyclodextrin solutions, it is possible to add sufficient amounts of a soluble cyclodextrin to infusion fluids to maintain the solubility of phenytoin after the original injection is diluted to clinical concentrations in the fluid. From solubility measurements, theoretical amounts of hydroxypropyl beta-cyclodextrin (HPCD), sufficient to prevent precipitation of phenytoin, were added to 0.9% w/v sodium chloride solution, adjusted to pH 7.0. Phenytoin injection was diluted to clinical concentrations in the cyclodextrin/NaCl solutions. The mixtures, together with analogous mixtures containing phenytoin, but no HPCD, were stored at 25 degrees C for 3 days. In the presence of the cyclodextrin no precipitates of phenytoin were observed, but in its absence an immediate massive precipitation of phenytoin occurred. This is a flexible method for using soluble cyclodextrins to prevent precipitates of drugs in infusion fluids, provided the cyclodextrin used is not toxic.

2-Hydroxypropyl-beta-cyclodextrin↗

PubMed2001

Bacterially mediated precipitation in marine stromatolites.

Stromatolites are laminated, lithified (CaCO3) sedimentary deposits formed by precipitation and/or sediment accretion by cyanobacterial-bacterial mat communities. Stromatolites have been associated with these communities as far back as the Precambrian era some 2+ billion years ago. The means by which microbial communities mediate the precipitation processes have remained unclear, and are the subject of considerable debate and speculation. Two alternative explanations for microbially mediated precipitation include: (i) cyanobacterial photosynthesis increases pH in a system supersaturated in respect of CaCO3, resulting in CaCO3 precipitation and then laminated lithification, and (ii) decomposition of cyanobacterial extracellular organic matter (e.g. sheaths, mucilage and organic acids) by microheterotrophs leads to release of organic-bound Ca2+ ions and CaCO3 precipitation. We evaluated these explanations by examining metabolically active, lithifying stromatolitic mat communities from Highborne Cay, Bahamas, using microautoradiography. Microautoradiographic detection of 14CO2 fixation and 3H organic matter (D-glucose and an amino acid mixture) utilization by photosynthetically active cyanobacteria and microheterotrophs, combined with community-level uptake experiments, indicate that bacteria, rather than cyanobacteria are the dominant sites of CaCO3 deposition. In the oligotrophic waters in which stromatolites exist, microheterotrophs are reliant on the photosynthetic community as a main source of organic matter. Therefore, autotrophic production indirectly controls microbially mediated precipitation and stromatolite formation in these shallow marine environments.

Bacteria↗

PubMed1971

Spontaneous precipitation of brushite in urine: evidence that brushite is the nidus of renal stones originating as calcium phosphate.

Further evidence that brushite plays a regulatory role in renal stone formation was provided by the identification of brushite as the first precipitate that appears in supersaturated urine by spontaneous precipitation. Calcium chloride was added to induce supersaturation in urine specimens from twelve subjects with and twelve subjects without nephrolithiasis. The first precipitate in all specimens with pH below 6.9 was identified as brushite by x-ray diffraction and shown to have a calcium-phosphorus ratio of approximately 1.0. The activity product of [Ca(2+)] x [HPO(4) (2-)] necessary to produce a precipitate ranged from 2.2 to 3.5 times the solubility product of brushite, but the range and mean were the same for both groups of subjects. The activity product of [Ca(2+)] x [HPO(4) (2-)] in the supernatant (after spontaneous precipitation) was not significantly different from that obtained after incubation of the same urine specimen with synthetic brushite. These results provide conclusive evidence that brushite constitutes the solid phase formed by spontaneous precipitation from acidic urine supersaturated with respect to calcium and phosphorus; they suggest that the nidus for calcium-containing renal stones is brushite as well.

Calcium↗

PubMed1991

Condensation and precipitation of chromatin by multivalent cations.

The condensation and the precipitation of rat liver chromatin upon addition of spermine4+, spermidine3+, hexamminecobalt(III)3+ and Mg2+ cations have been studied using solubility, fluorescence, circular dichroism, melting curves, electric dichroism and spermidine binding measurements, made on both soluble and precipitated complexes. The soluble complexes obtained with tetra- and trivalent cations were depleted from all histones and enriched in other proteins, particularly high mobility group proteins 1 and 2, which brings about an important enhancement of tryptophan fluorescence without modification of its two lifetimes 5.1 and 1.2 ns. In the precipitates the non-histone proteins are eliminated. Under precipitation by Mg2+ ions, the distribution of proteins remains practically unchanged. The electric dichroism and the melting curves indicate that the soluble complexes between polyamines and chromatin undergo important condensation and, at high ratios of cation over phosphate, are constituted by heterogeneous assemblies of non-histone proteins and DNA. On the contrary, the insoluble complexes seem to retain the main features of original chromatin. Precipitation by Mg2+ ions reveal much less drastic changes than those produced by polyamines. Precipitation by spermidine occurs when one cation is bound per eight nucleotides, which in addition to the histone positive charges brings about a complete neutralization of chromatin phosphates.

Animals↗

PubMed2001

Prediction of metal precipitates in tannery sludge leachate based on thermodynamic calculations.

Thermodynamic calculations were performed in order to predict the formation of metal precipitates during the tannery sludge leaching. Gibbs free energy deltaG=RTln(Qc/Kc) of precipitation reaction was used to examine the formation of precipitates during the leaching process. The values of activity equilibrium constant (Ka) of various precipitation reactions were adopted from the literature. The Ka values were corrected to obtain the corresponding values of concentration product (Qc) and concentration equilibrium constant (Kc) using the activity coefficients (gamma) of the ions. The activity coefficients (gamma) of the ions was calculated using Davies equation Lngammai=-1.172Zi2((I0.5/(1+I0.5))-0.3I) (aqueous solution, 25 degrees C, 1 atm, I>0.3 mol x l(-1)). The values of ionic strength (I) at different sludge solids concentration and leaching pH were obtained by measuring the concentration of all ionic species in the leachate. The thermodynamic calculations indicated that the possible metal precipitates formed during the leaching process were (am)Cr(OH)3, (am)CrPO4, (am)Fe(OH)3, alpha-FeOOH(goethite), FePO4.2H2O, (am)AIPO4-2H2O, CaSO4.2H2O(gypsum). The solubility of these precipitates was found to decrease with the increase in the ionic strength (or sludge solids concentration). The results of computations were supported by the experimental observations.

Chemical Precipitation↗

PubMed2004

Preparative separation of proteins using centrifugal precipitation chromatography based on solubility in ammonium sulfate solution.

A preparative modification of the centrifugal precipitation chromatography (CPC) is described. The sample-loading capacity is improved in the present system by the use of convoluted tubing containing dialysis tubing instead of a dialysis membrane placed between a pair of disks equipped with mirror-imaged spiral grooves as in the original design. The system uses, basically, the same principle of as the original CPC, in that a concentration gradient of precipitant is generated under a centrifugal force field. The protein sample injected into the CPC column is exposed to an increasing concentration of the precipitant where it precipitates at various portions of the column according to its solubility. The gradient is then gradually lowered so that the sample undergoes dissolution and precipitation many times within the column; the proteins finally elute from the column according to their solubilities. A basic study was performed using this machine to separate human albumin and 3-globulin using ammonium sulfate (AS) as precipitant. Preliminary results indicate that this method can separate 500 mg of protein.

Ammonium Sulfate↗

PubMed2006

Ultrastructural study of iron oxide precipitates: implications for the search for biosignatures in the Meridiani hematite concretions, Mars.

Two terrestrial environments that have been proposed as analogs for the iron oxide precipitation in the Meridiani Planum region of Mars include the Rio Tinto precipitates and southern Utah marble concretions. Samples of two typical Utah iron oxide concretions and iron oxide precipitates in contact with biofilms from Rio Tinto have been studied to determine whether evidence could be found for biomediation in the precipitation process and to identify likely locations for fossil microorganisms. Scanning electron microscopy, energy dispersive X-ray, and gas chromatography-mass spectrometry (GC-MS) were used to search for biosignatures in the Utah marbles. The precipitation of iron oxides resembles known biosignatures, though organic compounds could not be confirmed with GC-MS analysis. In contrast, textural variations induced by biological activity are abundant in the modern Rio Tinto samples. Although no compelling evidence of direct or indirect biomediation was found in the Utah marbles, the ultrastructure of the iron oxide cement in the concretion suggests an inward growth during concretion precipitation from an initially spherical redox front. No indication for growth from a physical nucleus was found.

Chemical Precipitation↗

PubMed1978

Ristocetin precipitation test: a new simple test for detection of fibrin monomer and fibrin degradation products.

The ristocetin precipitation test was designed as a simplified test to detect fibrin monomers and fibrinogen/fibrin degradation products (FPD/fdp). The ristocetin precipitation test is positive in plasma samples containing either fibrin monomer (greater than 5--10 microgram/ml) or early fdp (greater than 50--100 microgram/ml). The ristocetin precipitation test is negative in plasma with fibrinogen concentrations to 1,000 mg/dl or fibrinogen degradation products FDP) and late fdp to 400 microgram/ml. The ristocetin precipitation test is positive in plasmas collected from rabbits after the infusion of thrombin (2.7 u/kg) or thrombin and streptokinase (10,000 u/kg); the test is negative in plasmas from animals treated with streptokinase or saline solution alone. The ristocetin precipitation test is negative in normal human plasmas and plasmas from patients who have primary firbinogenolysis, but positive in plasmas from patients with disseminated intravascular coagulation. These results suggest that the restocetin precipitation test can be a useful test for the detection of plasma fibrin monomers and early fdp.

Animals↗

PubMed2000

Calcium concentration and artificial precipitates in human pancreatic juice.

We studied the role of the increase in the calcium concentration in pure pancreatic juice of alcoholic noncalcified chronic pancreatitis. Pure pancreatic juice was obtained endoscopically. The pancreatic juice from patients with chronic pancreatitis was adjusted to pH 7.5; then the calcium concentration was adjusted to 0.4, 2.9, 5.4, or 10.4 mmol/L. Artificial precipitates were produced by incubation of the samples at 37 degrees C for 6 hours. Proteins in the artificial precipitates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the protein patterns were compared with the patterns of natural protein plugs from patients with chronic pancreatitis. The amount of the precipitate increased as the added calcium increased. The protein patterns of SDS-PAGE of the artificial precipitates were similar to those of protein plugs. Albumin, a-amylase, lipase, trypsinogen, and chymotrypsinogen were identified by immunoblotting both in the precipitate and in the protein plug. The increased calcium concentrations in pancreatic juice induced the formation of precipitates whose protein composition was similar to that of protein plugs. An increased calcium concentration in human pancreatic juice may play an important role in the pathogenesis of protein plugs.

Adult↗

PubMed1999

Protein crystallization with a combination of hard and soft precipitants.

Much effort and progress have been made in understanding the nucleation and crystallization of globular proteins, and many techniques have been developed to crystallize proteins in the past decades. The advantages of the use of combined precipitants in protein crystallization have been much appreciated. Unfortunately, there is still no theory or empirical guide on how to combine so many precipitants and how to use combined precipitants, although many proteins have been crystallized successfully using combined precipitants. This report gives a proposal about how to use conventional precipitants to obtain protein crystals, based on a novel idea of hard and soft precipitant combinations.

Animals↗

PubMed2005

The role of cognitive-motor function in precipitation and inhibition of epileptic seizures.

PURPOSE: To examine the effects of cognitive-motor function on EEG discharges and the neuropsychological mechanisms of seizure induction in patients sensitive to cognitive-motor tasks. METHODS: Four hundred eighty patients with epilepsies were subjected to cognitive tasking, termed "neuropsychological EEG activation (NPA)." It consisted of reading, speaking, writing, written calculation, mental calculation, and spatial construction. Furthermore, patients showing a provocative NPA effect were subjected to a detailed NPA protocol to identify the possible precipitating factors, which consisted of simple hand movements, action programming requiring hand movement, and thinking activity not requiring hand movement. RESULTS: NPA had an inhibitory effect on EEG discharges in 133 (63.9%) of 208 patients with discharges in the awake EEG. Conversely, NAP had a provocative effect in 38 (7.9%) of 480 patients. In 32 of the 38 patients, the precipitating factor was action programming. Among them, five showed a precipitating factor restricted to linguistic activity, and the remaining 27 were affected by various action-programming factors including both linguistic and praxic activities. In four of 38 patients, the precipitating factor was thinking, predominantly linguistic tasks in one patient and spatial tasks in three patients. No patient had a precipitating factor identified as motor activity. CONCLUSIONS: These results suggest that cognitive-motor function has an inhibitory effect on EEG discharges in the majority of epilepsy patients and a provocative effect in some patients, and that seizures of the patients showing a provocative NPA effect are precipitated by action programming or thinking activity.

Adolescent↗

PubMed1984

Determination of IgE-containing immune complexes in human sera. Evaluation of polyethylene glycol precipitation of monomeric and complex IgE and of the detectability of IgE in the complexes.

We studied the polyethylene (PEG) precipitability of monomeric human IgE, and of human IgE artificially complexed with rabbit anti-human IgE. At conditions where precipitation of monomeric IgE did not occur, from 0.2 to 20% of the complexed IgE was precipitated. The PEG precipitability of the complexes was inversely related to the IgE/anti-IgE ratio used for preparation of the complexes. From 1.5 to 19.2% of the IgE in the redissolved precipitates could be detected by use of a two-site IgE immunoradiometric assay, the percentage being highest for complexes formed at equivalence. We conclude that exact quantitation of circulating IgE immune complexes (IC) probably is impossible by any PEG precipitation assay. However, the optimized assay was found to be useful for identification of IgE IC in sera with total IgE concentrations below 5,000 U/ml. IgE IC were found in 5/20 sera from patients with Felty's syndrome, in 5/39 sera from patients with extrinsic allergy and high levels of specific IgE, and in 1/17 sera from immunized wasp allergics. No IgE IC were found in 20 normal human sera.

Antigen-Antibody Complex↗

PubMed1986

Ileal resection: effect of cimetidine and taurine on intrajejunal bile acid precipitation and lipid solubilisation.

We have investigated whether pH-dependent bile acid precipitation limits lipid solubilisation after ileal resection, and whether treatment with cimetidine, or taurine improves solubilisation. Nine ileal resection patients were treated with placebo, cimetidine and taurine in random order for two weeks each. Upper jejunal content was aspirated and pooled according to pH for three hours after a standard Lundh test meal. On placebo, 50% of the bile acids were precipitated at pH less than 5, compared with only 26% at pH greater than 6, whilst aqueous-phase lipid concentration tended to be lower at pH less than 5 than at pH greater than 6 (5.1 vs 8.2 mmol/l). On cimetidine mean pH rose, particularly during the third hour (6.6 vs 5.8, p less than 0.05), associated with a reduction in bile acid precipitation (13.9 vs 33.1%, p less than 0.05), and an increase in aqueous-phase lipid concentration (10.4 vs 6.6 mmol/l, p less than 0.05). On taurine, the proportion of taurine conjugated bile acids increased (67 vs 22%, p less than 0.01), but there was no significant change in bile acid precipitation or lipid solubilisation. Lower jejunal samples were aspirated similarly from five of these patients on no treatment, and all were at pH greater than 6; apparent 'precipitation' was reduced (16.4 vs 28.1%), but lipid solubilisation did not improve. These findings suggest that pH dependent bile acid precipitation can limit lipid solubilisation within the jejunum after ileal resection, and that these effects can be reduced by cimetidine but not by taurine. Cimetidine may have a role in ileal resection patients with severe steatorrhoea unresponsive to dietary fat restriction.

Adult↗

PubMed1993

Defluoridation of drinking water by co-precipitation with apatite.

The two-step brushite saturation, apatite precipitation method which has previously been shown to be effective for the partial defluoridation of water containing 10 ppm F has now been tested at lower concentrations. The method also reduced F concentrations in the range 1-5 ppm, but was less efficient at lower F levels. Although co-precipitation as fluorapatite is the intended mechanism here, experiments indicated that F adsorption on the seed material added to the defluoridation effect. When the contribution of co-precipitation was isolated it was found that this mechanism could, on its own, account for a reduction of 2.2-2.6 ppm F in water initially containing 3-5 ppm F. A computer program which determines equilibrium F concentrations when supersaturated solutions are induced to precipitate fluorapatite was used to estimate the maximum possible F removal from water by co-precipitation. Using saturation with brushite as a starting point for precipitation, F reductions similar to those found experimentally were predicted. Computer simulations also suggest that any desired reduction in F concentration may be achieved by adding sufficient Ca and phosphate and/or by raising the initial pH.

Adsorption↗

PubMedDate not supplied

Fatal microvascular pulmonary emboli from precipitation of a total nutrient admixture solution.

BACKGROUND: Paroxysmal respiratory failure and death occurred in two young adult females with pelvic infections. Autopsy revealed an amorphous material containing calcium obstructing the pulmonary microvasculature of each patient. Both patients received an identical total nutrient admixture (TNA) solution before their deaths. METHODS: Infusion of TNA into an animal model was undertaken in an effort to reproduce the clinical effect. Laboratory investigation was also performed to isolate a precipitate and identify the factors contributing to precipitation. RESULTS: A nonvisible precipitate containing calcium, phosphorus, and organic material was isolated from the TNA solution. Infusion of the formulation into healthy pigs resulted in sudden death within 4 hours. Alteration of the amino acid component, mix sequence, agitation technique, and mixing container influenced precipitate formation. CONCLUSION: Pulmonary embolization of a precipitate containing calcium phosphate resulted in the death of two patients. The pH of the amino acid component, transient elevation of calcium and phosphorus concentrations during mixing, and the lack of agitation during automated preparation of the formulation were identified as the etiologic factors producing the fatal precipitate.

Adult↗