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PubMed1990

Removal of endotoxin from protein solutions by phase separation using Triton X-114.

Endotoxin contamination of protein solutions was reduced by a phase separation technique using the detergent, Triton X-114. Protein solutions containing endotoxin were treated with Triton X-114 on ice. The solution was then warmed to 37 degrees C, whereupon two phases formed. The Triton X-114 phase, containing the endotoxin, was precipitated by centrifugation. The first cycle of phase separation produced a 1000-fold reduction of endotoxin from contaminated preparations of cytochrome c, catalase and albumin. Complete removal of endotoxin could be achieved by further cycles of phase separation. Each cycle of phase separation resulted in only a 2% loss of protein, and could be completed within 15 min. The small amount of detergent (0.018%) that persisted in protein solution could be removed by gel filtration or absorption. Proteins treated by this procedure retained normal functions. This phase separation technique provides a rapid and gentle method for removing endotoxin from protein solutions.

Catalase↗

PubMed1993

Comprehensive planning of operative strategy for separation of ischiopagus tripus twins with particular reference to quality of life.

A 27-year-old mother was diagnosed by prenatal ultrasonography as having triplets at gestational age 32 weeks. Following cesarean section at 37 weeks, a pair of female babies were noted for the first time to be joined by a common pelvis with three lower limbs. They had separate upper gastrointestinal tracts, which joined in the distal ileum, leading to a common colon, rectum, and a single anus. Each twin had a functioning kidney, with a single ureter leading to a common bladder. A common urethra originating from the bladder neck ran into the urogenital sinus of one baby. Prior to the surgical separation, placement of four tissue expanders and 20 pneumoperitoneums were performed, in order to stretch the parietes for easier approximation of the wound edges. At 13 months of age, separation was performed, requiring 17 hours. The skin and musculature from the conjoined third leg was used as a fillet for abdominal wall closure in each patient. One infant was given the distal half of the colon and an entire anus with a temporary jejunostomy, and the right half of the bladder with the urethra. The other infant was given the proximal half of the colon with a permanent colostomy, and the left half of the bladder with permanent cystostomy using appendiceal pedicle graft (Mitrofanoff's procedure). This is the 10th case of surgical separation in ischiopagus tripus twins reported in the literature, and the seventh successful separation with both patients alive.

Female↗

PubMed1983

Transformations of C21 and C19 steroids in porcine ovarian preparations and the chromatographic separation of the metabolites.

The separation of several C21 steroids, such as 17-hydroxyprogesterone, and androgens, such as testosterone, from the non-polar steroids like 16-androstenes has been achieved on one thin-layer chromatographic plate using a two-dimensional technique. This method has been further developed to include a separation of some oestrogens from C19 steroids. The thin-layer chromatographic development was then utilised to separate the metabolites of porcine ovarian incubations. Homogenised preparations of corpora lutea (5-14 days post ovulation) were incubated separately with [4-14C] testosterone, [4-14C] progesterone and [4-14C] pregnenolone, using NADPH as cofactor. After two-dimensional thin-layer chromatography the metabolites were identified by establishing their radiochemical purity either by repeated thin-layer chromatography or by gas-fraction collection. Pregnenolone was converted to small yields of 4,16-androstadien-3-one (0.13-0.28%) and 5 alpha-androst-16-en-3-one (less than 0.1%) and also to 17-hydroxypregnenolone and 17-hydroxyprogesterone (0.8 and 0.37% respectively). Increased activity of 5-ene-3 beta-hydroxysteroid dehydrogenase/4,5-isomerase was shown by the high yields (73-83%) of progesterone obtained from pregnenolone. This was associated with decreased C-17.20 lyase activity as reflected in the relatively small amounts of 4-androstenedione obtained from both pregnenolone and progesterone. None of the well-known oestrogens or 16-androstenes was formed from progesterone or testosterone, but the latter was converted into 4-androstenedione, in small yield. Both pregnenolone and progesterone gave rise to a metabolite in 1-2% yield (of similar polarity to the 16-androstenes but separated from them by thin-layer chromatography on AgNO3-impregnated plates) which has been tentatively identified as 5 alpha-pregnane-3,20-dione. Incubations of porcine follicular fluid and tissue with labelled pregnenolone or progesterone did not result in the biosynthesis of labelled 16-androstenes.

Androgens↗

PubMed1975

Survival and recovery of physiologic functions after early maternal separation in rats.

Fourteen-day-old rats separated from their mothers have greatly increased survival rates, if provided with a local heat source. Body weight, temperature, cardiac and respiratory rates declined for 3 days in all separated animals, and eye opening was delayed most in those separated at room temperature. In survivors, a second phase followed in which all measures rebounded to levels characteristic of normally mothered infants. Cardiac rate, having fallen 40 percent in the first days after separation, rose to levels higher than normally mothered infants at 21 and 30 days of age. Recovery was not clearly initiated by any singly system studied, although non-survivors showed greater weight and temperature loss and evidence of gross motor deficit during the separation response phase.

Animals↗

PubMed1978

Cardenolide analogues. 3. A fast thin layer chromatographic separation of cardenolide diastereomers.

Cardenolide diasteromeric mixtures may be analytically separated on 2.5 x 6.5 cm x 0.25 mm pre-coated silical gel 60 F-250 (EM) tlc plates (cut from 20 x 20 cm plates). Three successive developments in mixtures of CH2Cl2-EtOAc-MeOH achieve complete separation of diastereomers in less than 15 minutes, and 2--6 micrograms of sample is sufficient. This procedure facilitated the separation of several diastereomeric mixtures, including 20(R) and 20(S)-20,22-dihydrocardenolides. A single development produces separations sufficient for separating starting materials and products of common cardenolide types of reactions.

Cardenolides↗

PubMed1992

Separation and identification of microcystins in cyanobacteria by frit-fast atom bombardment liquid chromatography/mass spectrometry.

In order to separate and identify microcystins, a new analytical method was developed using a frit probe as an interface for fast atom bombardment mass spectral analysis of high performance liquid chromatographic (HPLC) effluents. Two types of HPLC conditions were designed for separation of standard microcystins RR, YR and LR. The HPLC conditions, for example, methanol:0.01% trifluoroacetic acid = 61:39 (containing 0.8% glycerol) as a mobile phase and 0.5 ml/min as a flow rate, provided a base line separation of standard microcystins RR, YR and LR. The HPLC conditions were also effective for separation of the non-toxic geometrical isomers of microcystins RR and LR. The total ion chromatogram of a mixture of standard microcystins showed excellent correlation with the HPLC separation using a u.v. detector. The method was subsequently applied to analysis of microcystins contained in both a culture strain and a field sample, and the procedure from toxin extraction to identification of microcystins was performed within 1 day. The mass chromatogram monitored at m/z 135 that is always observed with abundance in the FAB mass spectra of the purified microcystins, differentiated between microcystins and other types of compounds. This technique allowed the rapid identification of unknown microcystins without standard samples. Additionally, compounds other than microcystins were also found, which would not be seen by u.v. detection at 238 nm.

Chromatography, High Pressure Liquid↗

PubMed1988

Sequence of centromere separation of mitotic chromosomes during human cellular aging.

The sequence of centromere separation of mitotic chromosomes derived from lymphocytes of 17 individuals of various ages was studied. A comparison of the mean CSI (centromeric separation index) values for individual chromosomes in the complement indicated a significant difference between the older and the younger subjects only with regard to the chromosome nos. 6, 11, and 19 in both sexes and the X chromosome exclusively in the female. With age, chromosome no. 6 in both males and females and the X chromosome in females showed premature centromeric separation whereas chromosome nos. 11 and 19 in both males and females displayed delayed centromeric separation. Our study, therefore, indicates that changes in the sequence of centromeric separation of certain chromosomes do occur in lymphocytes during chronological aging in humans.

Adult↗

PubMed1991

Influence of different surfactants on the separation of von Willebrand factor multimers by use of agarose gel electrophoresis and semi dry blotting technology.

The separation of vWF-multimers by use of sodium dodecyl sulfate (SDS) agarose gel electrophoresis and semi dry blotting instead of tank blotting procedure has been studied. We could demonstrate, that multimers in the range of highest molecular weights could not be visualized using this technique. Therefore we have investigated the influence of different types of anionic surfactants on the separation of vWF-multimers and could show that the hydrophilic/lipophilic balance (HLB) is an important value to characterize the capacity of a surfactant to separate vWF-multimers. The best separation quality was achieved with sodium dodecyl benzene sulfonate (SDBS). This surfactant is a superior alternative to SDS in separating vWF-multimers of the highest molecular weights.

Benzenesulfonates↗

PubMed1980

Complete separation by high performance liquid chromatography of metabolites of arachidonic acid from incubation with human and rabbit platelets.

Separation of all major cyclooxygenase and lipoxygenase metabolites of arachidonic acid were obtained by high performance liquid chromatography (HPLC). A C18 reverse-phase column was used in ion suppression mode to separate underivatized metabolites of arachidonic acid isolated from human and rabbit platelets. The metabolites were monitored by measuring radioactivity or ultraviolet light absorption at 192 nm (absorption by double bonds). Comparisons of TLC and HPLC separations demonstrated that the HPLC separation of metabolites of [1-14C]arachidonic acid was quantitative. HPLC also resolved several metabolites that were not detected by scanning of TLC separations.

Animals↗

PubMed1990

Polymer-protein conjugates. II. Affinity precipitation separation of human immunogammaglobulin by a poly(N-isopropylacrylamide)-protein A conjugate.

The conjugate of protein A with poly(N-isopropylacrylamide) was synthesized and utilized in the separation of human immunogammaglobulin. In the separation process, poly(N-isopropylacrylamide)-protein A conjugate binds to the immunoglobulin with high specificity to form the poly(N-isopropylacrylamide)-protein A/immunoglobulin complex. The complex can be conveniently separated by precipitation upon heating above the lower critical solution temperature of the poly(N-isopropylacrylamide)-protein A/immunogammaglobulin complex. The separation capacity of poly(N-isopropylacrylamide)-protein A conjugate for human immunogammaglobulin was studied and it was demonstrated that approximately one out of every four protein A molecules binds to human immunogammaglobulin with a dissociation constant (Ks) of 3 x 10(-6) M. The affinity precipitation separation of human immunogammaglobulin is a rapid process which avoids the need for chromatographic columns. It can also be designed to run in a continuous mode.

Acrylic Resins↗

PubMed1980

Separation of prostaglandins, thromboxane, hydroxy fatty acids and arachidonic acid by high pressure liquid chromatography.

Separation of the major metabolites of arachidonic acid (AA) produced by the cyclo-oxygenase and the lipoxygenases was achieved by using reverse phase high-pressure liquid chromatography. Prostaglandins (PGs), thromboxane B2 (TXB2), and AA were separated on a C-18 radial compression column. An initial isocratic elution resolved the PGs and TXB2 which was followed by a linear gradient in order to elute AA. Variations of the gradient elution shape were required to permit the separation of 12-L-hydroxy-5,8,10-heptadecatrienoic acid, 5-12 and 15-hydroxy-5,8,11,14-eicosatetraenoic acid. The recovery of the labeled AA and its metabolites was investigated. Use of these separation methods and radiolabeled substrates should permit investigators to obtain reproducibly in one chromatographic run adequate separation and quantitation of both PGs and hydroxy fatty acid systems.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗

PubMed1985

Phase separation in Triton X-114 of antigens of transmission blocking immunity in Plasmodium gallinaceum.

The distribution of proteins of mosquito midgut forms of Plasmodium gallinaceum in the detergent-free (aqueous) and detergent-enriched phases was studied using a phase separation technique in Triton X-114. Of the three surface proteins on gametes and newly fertilized zygotes (240, 56, and 54 kDa) immunoprecipitated by transmission blocking monoclonal antibodies, 240 kDa protein was recovered in the aqueous phase, whereas 56 and 54 kDa proteins were found preferentially in the detergent phase. The hydrophobic properties of the 56 and 54 kDa proteins were also shown by their strong tendency to interact with the lipid bilayers and a hydrophobic matrix phenyl-Sepharose. Monoclonal antibody IID3B3 immunoprecipitated all the three proteins from the whole Triton extract but in the phase-separated extracts reacted only with the 240 kDa protein in the aqueous phase and not with the 56 and 54 kDa doublet in the detergent phase. In Western blot analysis also monoclonal antibody IID3B3 reacted only with the 240 kDa protein. The 240 kDa protein in the aqueous phase was retained by monoclonal antibody IID3B3 linked to Sepharose 4B beads and could be eluted either with 0.1 M acetic acid or 50 mM diethylamine. The 56 and 54 kDa doublet in the detergent phase could be bound to and eluted from Sepharose 4B beads-linked monoclonal antibody IID4 or rabbit anti-male P. gallinaceum gamete serum. Two stage-specific glycoproteins of 26 and 28 kDa on the surface of ookinetes of P. gallinaceum were also separated in the detergent phase following Triton X-114 extraction. Phase separation in Triton X-114 offers a simple approach to the separation of a select group of proteins from the bulk of the cellular proteins.

Animals↗

PubMed1992

Selective protein separations using Formed-In-Place anion exchange membranes.

Anion exchange membranes prepared by adsorption of polymers on Formed-In-Place microfiltration substrates were formed and ion-exchange separations of solutions containing two proteins were determined by ion exchange membrane sequential separation procedures, similar to affinity membrane separation procedures. Representative ion exchange separation processes utilizing adsorbed poly(ethylene imine) (PEI) as the ion exchange membrane for the separation of the components of solutions containing two proteins, bovine serum albumin (BSA) and lysozyme and ovalbumin and lysozyme, are described. The stability of the PEI adsorbed layer, binding characteristics of the BSA to the membrane and purification properties of the procedure were determined.

Adsorption↗

PubMed1989

Changes in the molecular topography of the light and heavy chains of type A botulinum neurotoxin following their separation.

Botulinum neurotoxin serotype A, an approx. 150 kDa protein, is composed of two subunits, the light and heavy chains (approximately 50 and approximately 100 kDa, respectively). The neurotoxin's mode of action is believed to depend on coordinated but independent actions of the two subunit chains. The molecular environments of the aromatic amino acid residues of the dichain neurotoxin and the two isolated subunit chains were analyzed using near-ultraviolet circular dichroism (CD) (between 250 and 320 nm) and second-derivative ultraviolet absorption spectroscopy (between 240 and 320 nm) to investigate the conformational variations of the subunit chains in separated and conjugated forms. The mean residue weight ellipticities showed virtually no change (i.e., 1.7%) in the vicinities of Phe (268 nm), and only a small change (11%) around Tyr (279 nm) residues following dissociation of the subunit chains. However, significant changes (23-26%) at 286 nm as well as at 292 nm were noted, suggesting considerable alteration in the conformation of the subunits. Second-derivative ultraviolet absorption spectra indicated the degree of Tyr exposure in the dichain neurotoxin, isolated heavy and light chains at 70.7, 81.5 and 46.4%, respectively. A weighted mean of the degree of exposed Tyr residues in the separated heavy and light chains was 69.6%, virtually same as the 70.7% exposed Tyr residues observed in the intact dichain neurotoxin, indicating no difference in their Tyr exposure upon separation of the two chains. This was corroborated by the CD data which revealed only small changes in the CD signals of Tyr residues, and no alteration in those of the Phe residues following separation of the subunit chains. However, a change in the CD signal at 292 nm suggested that the conformations of Trp-containing segments of the two chains were significantly influenced upon their separation. The heavy and light chains of the neurotoxin therefore appear to exist as two semi-independent domains, in spite of being linked by disulfide and noncovalent bonds, and at least part of their conformations depends on interactions between them.

Botulinum Toxins↗

PubMed1992

Separation of rat pancreatic secretory proteins by cation-exchange fast protein liquid chromatography.

Rat pancreatic secretory proteins were separated by an automated liquid chromatography system utilizing a Mono S cation-exchange column. Optimal resolution was obtained with a multistep salt and pH gradient (0.01-2 M LiCl, pH 5.3-63). A total of fourteen well-separated peaks, as well as several minor peaks, were detected by UV absorption. The main pancreatic enzymes were resolved (two amylases, two chymotrypsinogens, two trypsinogens, proelastase, lipase, prophospholipase A2, procarboxypeptidase A, procarboxypeptidase B, and ribonuclease). In addition, proteins without enzymic activity, such as lithostathine and pancreatitis-associated protein, were identified. Activation of proenzymes did not occur during the separation. At a flow-rate of 0.5 ml/min, ca. 250 micrograms to 5 mg of protein could be applied with equal resolution. The reproducibility of retention volumes and peak areas was high (less than 1% or 5% variation, respectively). When radiolabeled proteins were separated, a comparable pattern of peaks was obtained. The technique described is, therefore, not only useful for analytical and preparative separation of pancreatic proteins but can additionally serve for quantitative determination of the pancreatic isoenzyme pattern.

Animals↗

PubMed1993

Electrophoretic and chromatographic separation methods used to reveal interstrand crosslinking of nucleic acids.

Several electrophoretic and chromatographic techniques, many of which have only been developed recently, provide sensitive methods for the detection and separation of DNA containing interstrand crosslinks such as those produced by many cancer chemotherapeutic drugs and photoactive psoralen derivatives. Most of the methods rely on the fact that the presence of such crosslinks prevent the complete denaturation of the two complimentary DNA strands by heat or alkali. A simple and highly sensitive neutral agarose gel electrophoresis method is particularly applicable to detailed time-course experiments of both total crosslink formation, and the "second-arm" of the crosslink reaction. This method separates denatured single-stranded from double-stranded DNA which has reannealed as a result of an interstrand crosslink. Polyacrylamide gel-based assays using denaturing gels are more suited to the separation of smaller crosslinked DNA fragments and, in particular, small oligonucleotides on high-percentage gels. In addition, they provide methods for the determination of the exact base position and sequence selectivity of crosslink formation. Sephadex chromatography and high-performance liquid chromatography can separate small crosslinked oligonucleotides from non-crosslinked duplexes, and the hydroxyapatite column chromatographic separation of single- and double-stranded cellular DNA can be used to quantitate the level of interstrand crosslinking present in the bulk of the genome. Finally, the analysis of damage by crosslinking agents, and its repair, at the level of specific genes can be achieved by hybridization with specific probes following membrane transfer from neutral agarose gels used to fractionate restricted and fully denatured genomic DNA from drug-treated cells.

Animals↗

PubMed1994

Capillary electrophoresis: separation and quantitation of reverse transcriptase polymerase chain reaction products from polio virus.

In the present study reverse transcriptase (RT) polymerase chain reaction (PCR) products were generated from the RNA of polio virus. The products of the RT-PCR were analyzed by slab-gel electrophoresis (SGE) on 4% agarose gels, and capillary electrophoresis (CE). CE separations were performed in a coated capillary containing a linear polyacrylamide. Samples were injected hydrodynamically or electrokinetically. Detection of the RT-PCR products on CE was by UV absorbance at (254 nm) or by laser-induced fluorescence (LIF). While SGE resulted in adequate separation of 163 and 97 base pair RT-PCR products, separation of the 97, 71 and 53 base pair products was minimal. CE separations showed baseline resolution for all the above PCR products. Finally, it was possible to quantitate the amount of RT-PCR product by developing a standard curve showing a linear relationship between the amount of RNA used in the RT-PCR and the amount of product formed in the RT-PCR. These results suggest the greater resolution and enhanced sensitivity observed, together with the ease of quantitation, make CE a powerful alternative to SGE for the separation and quantitation of PCR products.

Electrophoresis↗

PubMed1994

Application of high-performance membrane chromatography for separation of annexins from the plasma membranes of liver and isolation of monospecific polyclonal antibodies.

The separation of annexins, calcium-binding plasma membrane-associated proteins from rat liver and Morris hepatoma 7777 by high-performance membrane chromatography (HPMC) is described. The annexins with low molecular masses, CBP 33 and CBP 35, and the annexin with a high molecular mass, CBP 65/67, can be separated within 10 min from one another by anion-exchange HPMC under non-denaturing conditions. The separation devices used consist of compact, porous disks (QuickDisk) on the one hand and of bundled membranes made of cellulose fibers (MemSep) on the other. Both have been found to be equally well suited for this separation. The annexins obtained in this way are subsequently bound to epoxy-activated porons disks and used for the separation of monospecific polyclonal antibodies against the annexin CBP 65/67.

Animals↗