Fractionation Column Chromatography Research Articles

Bacterial Mutagenicity of Fractions from Chloroform Extracts of Ceylon Cinnamon

Further to a previous report (Food Chem. Toxicol. 22:109–112, 1984) describing the presence of mutagens in a chloroform extract of Ceylon cinnamon (Cinnamomum zeylanicum Nees), we report here our findings on the mutagenicity of silica gel column chromatographic fractions from this extract. Mutagenicity was evaluated by the rec assay using Bacillus subtillis strains H17 (rec+) and M45 (rec−). Fractions collected after elution with chloroform, chloroform-ethanol (98:2) and (95:5) exhibited mutagenicity, while those obtained by ellution with chloroform-ethanol (85:15), (75:25) and ethanol showed no such activity.

A New Common Polysaccharide Antigen of Strains of Pseudomonas aeruginosa Detected with a Monoclonal Antibody

A monoclonal antibody, E87, that binds to various serotype strains of Pseudomonas aeruginosa was produced by a hybridoma cell line prepared by fusion of mouse plasmacytoma P3-X63-Ag8-U1 with spleen cells of mice immunized with P. aeruginosa strain IFO3080 (serotype M). E87 bound to approximately 80% of the tested strains of various serotypes of P. aeruginosa. The antigen recognized with E87 was found in the lipopolysaccharide and was eluted in void volume fractions of Sephadex G50 column chromatography after acetic acid treatment. This antigen was eluted in the fraction containing substances of lower molecular weight than the O side chain by Sephacryl S-300 column chromatography, and this fraction was found to be the peak fraction of hexose. This antigen was not separated from the O side chain by Sephacryl S-300 column chromatography of untreated lipopolysaccharide. Colorimetric analyses and thin-layer chromatography showed that this antigen consisted mainly of rhamnose and ribose (molar ratio, 10:1).

Lasioderma chemistry sex pheromone of cigarette beetle (Lasioderma serricorne F.)

A chemical study of the sex pheromone of the cigarette beetle was carried out. Seven components were isolated from active fractions of column chromatography of the female extract, and their structures were elucidated by spectroscopic evidence and confirmed by synthesis to be (4S,6S,7S)-4,6-di-methyl-7-hydroxynonan-3-one (serricornin) (I), 2,6-diethyl-3,5-dimethyl-3,4-dihydro-2H-pyran (anhydroserricornin) (II), 4,6-dimethylnonan-3,7-dione (III), 4,6-dimethylnonan-3,7-diol (IV), 4,6-dimethyl-7-hydroxy-4-nonen-3-one (V), (2S,3R)-2,3-dihydro-3,5-dimethyl-2-ethyl-6-(l-methyl-2-oxobutyl)-4H-pyran-4-one (serricorone) (VI) and (2S,3R)-2,3-dihydro-3,5-dimethyl-2-ethyl-6-(1-methyl-2-hydroxybutyl)-4H-pyran-4-one (serricorole) (VII).These structural features suggested that the occurrence of these components might be related to the polyketide biosynthesis. The behavioral bioassay and BAG experiments revealed the biological role of each component in the copulatory behavior of this insect.

CAMP-independent casein kinase mediates phosphorylation of many T3-dependent phosphoproteins in rat liver cytosol.

Administration of T3 (20 micrograms/100 g BW) for 3 days increases phosphorylation of several proteins in rat liver cytosol in vitro. To help elucidate the mechanism of T3-induced phosphorylation, we studied which protein kinase(s) mediate phosphorylation of endogenous cytosolic proteins. Five different protein kinases were obtained by DEAE+ cellulose column chromatographic fractionation of liver cytosol. When their ability to phosphorylate heat-inactivated cytosol was investigated, casein kinase, a cAMP independent protein kinase, showed the strongest effect. Casein kinase, purified by phosphocellulose chromatography, phosphorylated more than 10 cytosolic proteins. Several T3-dependent (and cAMP independent) phosphoproteins were included among these. One protein with Mr 39 X 10(3), of which phosphorylation is stimulated by T3 within five hours after injection, was the most active substrate for casein kinase. The results suggest that casein kinase is the enzyme responsible for phosphorylation of many rat liver cytosolic proteins and that several phosphoproteins, apparently under T3-regulation, might be phosphorylated by this enzyme.

7-glycidoxycoumarin (GOC): A fluorophotometric epoxide substrate for the assay of glutathione s-transferase activity

7-Glycidoxycoumarin (GOC), a new fluorophotometric epoxide substrate for glutathione S-transferase (GSH TFase), was conjugated regiospecifically with GSH at pH 6.5 in rat liver cytosol to yield S-(2-hydroxy-3-(7′-coumaroxy)-1-propyl)glutathione which was isolated by HPLC and identified with an authentic specimen by 13CNMR spectroscopy. The conjugation product formed in the incubation media consisting of GOC, GSH, and 9000 g supernatant fractions from various tissues of the rat, was directly determined by photometry of fluorescence emission at 388 nm at an excitation wavelength of 328 nm after removal of the unreacted substrate and its enzymic hydrolysis product, 7-(1′,2′-dihydroxy-3′-propoxy)coumarin, by simple extraction with isobutyl alcohol in the presence of a saturating amount of sodium chloride. Stability of GOC at pH 6.5 markedly retarded its autoconjugation with GSH and made the fluorophotometric method sensitive enough to assay small GSH TFase activities in gel column chromatographic fractions as well as in various tissues of the animals. Apparent K m and V max for GOC in rat liver cytosol were 55 μM and 7.41 nmole/mg protein/min, respectively. GSH conjugation of GOC was catalyzed by at least two isozymes, E and AA, of hepatic GSH TFases.

Isolation and characterization of cytochrome [formula omitted] from photosynthetic bacterium [formula omitted] R-26

Cytochrome c 1 of photosynthetic bacterium R . sphaeroides R-26 has been purified from isolated cytochrome b - c 1 complex to a single polypeptide, using a procedure involving Triton X-100 and urea solubilization, calcium phosphate column chromatography and ammonium sulfate fractionation. The purified protein contains 30 nmoles heme per mg protein and has an apparent molecular weight of 30,000, as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis. Bacterial cytochrome c 1 is soluble in aqueous solution in the absence of detergent and has spectral characteristics similar to mammalian cytochrome c 1 . The amino acid compositions of these two proteins, however, are not comparable.

The chemistry of toxic principles from Maytenus nemerosa

Maytenus nemerosa has been fractionated systematically by following the toxicity of extracts, partitioned solutions and column chromatographic fractions against cultured KB cells. Several compounds were isolated in this fashion, including 3-oxo-20(29)-lupen-30-al, β-amyrin, 29-hydroxyfriedelan-3-one, 30-hydroxy-20(29)-lupen-3-one, 30-hydroxyfriedelan-3-one and lup-20(29)-ene-3β,30-diol, as well as tingenone, 20-hydroxytingenone and galactitol. 3-Oxo-20(29)-lupen-30-al was shown to be cytotoxic for the first time; the structural basis of this cytotoxicity was investigated, in part, by bioassay of four products obtained by chemical transformation of single, isolated principles.

Uridine phosphorylase from Acholeplasma laidlawii: purification and kinetic properties.

Uridine phosphorylase was purified 1,370-fold from sonicated extracts of Acholeplasma laidlawii by ammonium sulfate precipitation, DEAE-Sephadex column chromatography, hydroxylapatite chromatography, and Sephadex G-200 fractionation. The molecular weight of the enzyme as determined by gel filtration was approximately 65,000. [U-14C]ribose-1-phosphate (Rib-1-P), prepared enzymatically from [U-14C]inosine, was utilized in initial velocity studies of uridine synthesis, which indicated a sequential reaction with a KmUra of 110 microM and a KmRib-1-P of 17 microM. The kinetics of uridine cleavage were assessed at a saturating cosubstrate concentration, resulting in a KmUrd of 170 microM and a KmPi of 120 microM. These results indicate that an intracellular flux from uracil to uridine is kinetically feasible. However, such flux would be metabolically unproductive, since the low affinity of uridine kinase (KmUrd = 3.2 mM) precludes the operation of uridine phosphorylase and uridine kinase in tandem to convert uracil to UMP. We conclude that uridine phosphorylase performs only a catabolic function in A. laidlawii.

Cytochromes, rubredoxin, and sulfur metabolism of the non-thiosulfate-utilizing green sulfur bacterium Pelodictyon luteolum

Two soluble c-type cytochromes (c-553 and c-555) and the nonheme iron-containing protein rubredoxin of the non-thiosulfate-utilizing green sulfur bacterium Pelodictyon luteolum were highly purified by ion exchange column chromatography, gel filtration and ammonium sulfate fractionation. Both cytochrome are small and basic hemoproteins, while rubredoxin is an acidic small nonheme iron protein. Cytochrome c-553 has a molecular weight of 13,000 determined by Sephacryl S-200 chromatography and of 10,700 by electrophoresis on SDS acrylamide gel, an isoelectric point at pH 10.2, a redox-potential of +220 mV. It shows maxima at 413 nm in the oxidized form, and the characteristic three maxima in the reduced state (α-band at 553 nm, β-band at 523 nm, γ-band at 417 nm). The best purity index (A280/A417) obtained was 0.18. Cytochrome c-555 (best purity index obtained: A280/A418=0.17) has an isoelectric point at pH 10.5, a molecular weight of 9,500 (by electrophoresis on SDS acrylamide gel) and a redox-potential of +160mV. The reduced form of this cytochrome shows the typical bands of c-type cytochromes at 555 (551) nm (α-band), 523 nm (β-band) and 418 nm (γ-band), while the oxidized form has the γ-band at 413 nm.

Cytochromes of the green sulfur bacterium Chlorobium vibrioforme f. thiosulfatophilum. Purification, characterization and sulfur metabolism

Three cytochromes of the thiosulfate-utilizing green sulfur bacterium Chlorobium vibrioforme f. thiosulfatophilum were highly purified by ion exchange column chromatography and ammonium sulfate fractionation. All three cytochromes are located in the soluble fraction. Cytochrome c-551 (highest purity index obtained: A280/A416=0.39) shows maxima at 551 nm (α-band), 521 nm (β-band), and 416 nm (γ-band) for the reduced form. This cytochrome is an acidic protein with a molecular weight of 32,000, a redox potential of 150 mV, and an isoelectric point at pH 6.0. Cytochrome c-553 (highest purity index obtained: A280/A417=0.8) is also an acidic protein with maxima at 553,5 nm, 523,5 nm and 417 nm for the reduced form, a molecular weight of 63,000, a redox potential of 90 mV, an isoelectric point at pH 6.3, and it contains FAD as flavin component. It is autoxidizable and participates in sulfide oxidation, but cannot catalyze the reverse reaction. The cytochrome c-555 (highest purity index obtained: A280/A418=0.16) is a small basic protein with maxima at 555 nm, 523 nm and 418 nm (reduced form), a molecular weight of 12,500, an isoelectric point between pH 10 and 10.5, and a redox potential of 155 mV. The ratio of the cytochrome contents to each other is constant and does not change when the organism has only thiosulfate or sulfide as the main electron donor in the medium.

38] Fructose-bisphosphate aldolase from human erythrocytes

Publisher Summary This chapter describes an assay for fructose-bisphosphate aldolase from human erythrocytes that is based on the oxidation of NADH by dihydroxyacetone phosphate in a coupled system containing triosephosphate isomerase and α-glycerophosphate dehydrogenase. The purification process involves washing of erythrocytes and hemolysis, batch process, column chromatography, and reverse ammonium sulfate fractionation. Isoelectric focusing reveals the presence of one form of aldolase in the erythrocyte with an isoelectric point of 8.9. Sedimentation equilibrium ultracentrifugation and gel filtration chromatography reveals a native molecular weight of 158,000. The enzyme, like other mammalian aldolases, appears to be a tetramer with identical or near identical subunits. The amino acid composition is very similar to that of the human heart and rabbit muscle enzymes. The human erythrocyte aldolase appears to be a type A isozyme or muscle-type aldolase.

Cytochromes of the non-thiosulfate-utilizing green sulfur bacterium Chlorobium limicola

Flavocytochrome c-553 of the non-thiosulfateutilizing green sulfur bacterium Chlorobium limicola strain 6330 was partially purified by ion exchange column chromatography and ammonium sulfate fractionation (highest purity index obtained: A 280/A 417 red=0.96). It is autoxidizable and located in the soluble fraction. This hemoprotein contains a flavin component and one heme per molecule. The dithionite reduced spectrum reveals the typical maxima of a c-type cytochrome: α=553,5 nm; β=523 nm; γ=417 nm, while the oxidized form shows a γ-band at 410 nm and two shoulders at 440 nm and 480 nm indicating the flavin component. The flavocytochrome is a basic protein with an isoelectric point at pH 9.0 (± 0.5), a redox potential of 65 mV, a molecular weight of 56,000. It participates in sulfide oxidation and shows neither adenylylsulfate reductase nor sulfite reductase activity. C. limicola further contains a soluble cytochrome c-555 (highest purity index obtained: A 280/A 412 ox=0.13; isoelectric point between pH 9.5 and 10) and the non-heme iron-containing proteins rubredoxin and ferredoxin, but lacks cytochrome c-551. Besides these soluble electron transfer proteins a membrane-bound c-type cytochrome (α=554,5 nm) can be detected spectrophotometrically.

Column chromatography fractionation of complex waste water sample extracts for measurement of ppt levels of 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin

AbstractThe novel application of azulene as a visual monitor of column chromatography performance during fractionation of complex waste water extracts for measurement of 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) at part‐per‐trillion concentrations is described. TCDD elutes directly behind azulene, the blue visual aid, in the 6% ethyl ether/hexane fraction during Florisil column chromatography.

In Vitro Microbial Degradation of Bituminous Hydrocarbons and In Situ Colonization of Bitumen Surfaces Within the Athabasca Oil Sands Deposit

Bituminous hydrocarbons extracted from the Athabasca oil sands of north-eastern Alberta were adsorbed onto filter supports and placed at sites in the Athabasca River and its tributaries where these rivers come in contact with the oil sands formation. Colonization of the hydrocarbon surfaces at summer and winter ambient temperatures was examined by scanning and transmission electron microscopy as well as by epifluorescence microscopy of acridine orange-stained cross sections. Ruthenium red and alkaline bismuth stains visualized an association of bacteria with the hydrocarbon surface which was mediated by bacterial polysaccharides. Bacteria apparently lacking a glycocalyx were also found closely associated with the surface of the hydrophobic substrate and in channels within the substrate. A solvent precipitation and column chromatographic fractionation of the bitumen was followed by cross-tests for growth on the fractions by various isolated sediment microorganisms, as determined by epifluorescence count. All fractions except the asphaltenes supported the growth of at least two of the isolates, although fractionation of degraded bitumen revealed that the saturate, aromatic, and first polar fractions were preferentially degraded.

Application of Azulene as Visual Aid to Monitor Column Chromatographic Fractionation of Samples for Pesticide and Polychlorinated Biphenyl Determination

Abstract Environmental samples are cleaned up by Florisil column chromatography, using azulene as a visual aid to monitor column performance. Azulene migrates as a blue band in the 6% ethyl ether-hexane fraction, and the gas chromatograph electron capture detector (GC-ECD) is nonresponsive at the use concentration. This method complements the standardization of Florisil columns by weight adjustment based on adsorption of lauric acid. Spiking of sample with azulene provides a convenient real-time monitor for column channeling, mistakes in polarity of mobile phases, particular sample composition, column overloading, and dynamic changes in the adsorptivity of the stationary phase between lauric acid calibration and use. Using this visual aid, the analyst is alerted to unexpected column behaviors. Normally, polychlorinated biphenyls (PCBs) elute in the 6% fraction, in front of the blue indicator. Experiences with azulene and industrial effluent samples demonstrated that PCBs can elute in the 100% hexane fraction. GC-ECD analysis of the hexane eluate revealed a PCB characteristic chromatogram, not observed with the original extract. Thus, monitoring column performance avoided reporting a false negative result, i.e., no PCBs in the 6% ethyl ether-hexane fraction.

Purification and immunochemical analysis of cytochrome c from Neurospora crassa

Procedures were refined and improved for the purification of cytochrome c from Neurospora crassa, for development of specific antisera to the protein, and for immunochemical identification of cytochrome c after in vivo synthesis and radiolabeling with 3H and 35Fe. The cytochrome c was obtained in high yields via column chromatography and salt fractionation, and the product was pure as judged by spectrophotometric and electrophoretic criteria. Effective, specific antisera to cytochrome c could be obtained only after the monomeric protein antigen was crosslinked with glutaraldehyde; immunoprecipitation of cytochrome c synthesized in vivo was achieved efficiently with a double-antibody procedure.

Purification and characterization of mitochondrial cytochrome P-450 associated with cholesterol side chain cleavage from bovine corpus luteum.

The cytochrome P-450scc associated with cholesterol side chain cleavage was purified to homogeneity from bovine corpora lutea mitochondria by fractional column chromatography on various ion exchange columns and on a heptyl-Sepharose hydrophobic column. Purification was 75-fold, recovery was 10%, the specific concentration of P-450 was about 15 nmol/mg of protein, and the specific content of heme was 16 nmol/mg of protein. The purified protein migrated as a single band on native polyacrylamide gel electrophoresis and as a single major band with an insignificant minor band (less than 1%) on SDS-gel electrophoresis. The molecular weight of the major band was 48,000. The optical absorption spectra of the preparation exhibited Soret maxima at 416, 416, and 450 nm for the oxidized form, reduced form, and CO-reduced complex, respectively. Apparently homogeneous enzymes were purified with a molecular weight 2, 4, 6, and 8 times the molecular weight of the single unit. The enzymatic activities of the reconstituted systems under optimal conditions were 16 nmol of cholesterol cleaved/mol of P-450. The 2-unit protein was about 5% as active either because it is an incomplete portion of the natural enzyme or because the molecular environmental conditions were not optimal. No differences in biophysical characteristics between cytochrome P-450sec from corpus luteum and adrenal sources could be identified.

The Wormicidal Substances Of Fresh Water Fishes On Clonorchis Sinensis: III. Seasonal variation on the wormicidal substances of mucous substances of Carassius carassius

The epidermal mucous substances of C. carassius which were extracted by ether were separated into various spots on the silica gel thin layer chromatography and separated into various fractions in the silica gel column chromatography, using petroleum ether (30 %) and chloroform(70 %) as a solvent. The wormicidal effects of the spots and the fractions on the cercaria, the liberating metacercaria and the adult of C. sinensis, were observed by month giving attention to ambient water temperature. 1. Among the four spots of the thin 1ayer chromatography of the ether extracts from C. carassius, the uppermost first spots (Rf. 0.924~0965) had a strong wormicidal effect on the cercaria in May, June, July, August and September, and the cercaria which came in contact with the materials was killed in 29~49 min., but in other months (Rf. 0.612~0.752) it was killed only after 50~115 min. 2. In column chromatography of ether extracts from C. carassius, the first fractions which had a wormicidal effect killed the metacercariae within 38~163 min. in May, June, July, August, and September, but it took about 120~458 min. to kill them in other months, meanwhile the wormicidal time of the fractions on the adults was 445~835 min. in May, June, July, August and September, but was 878~2,130 min. in other months. 3. The Rf. value of the uppermost first spot of the thin layer chromatography of ether extrart from the mucous substance of C. carassius which was reared in the aquarium controlled at 23 degrees C was 0.79, and its first fraction of column chromatography killed the metacercaria and the adult of C. sinensis within 315, and 1,850 min. respectively. As summarized above, the wormicidal materials of the epidermal mucous substances of C. carassius had no relation to ambient water temperature, but the secretion period of this wormicidal materials was the same as the liberating period of the cercariae from Parafossarulus manchouricus.

A centrifugal analyser technique for measuring isoenzymes of acid phosphatase in column-chromatography fractions

A column-chromatographic technique for separation of acid phosphatase (ACP) isoenzymes has been combined with a rapid and sensitive automated method for detecting ACP using a Centrifichem 300. The substrate used is p-nitrophenyl phosphate (PNPP), in a citrate buffer of pH 6.5. At this pH the ACP activity is not significantly less than optimal, while the extinction coefficient of PNPP is high enough to detect changes in OD using a direct computer analysis of the Centrifichem OD readings. Three main peaks of isoenzyme activity are found; the second peak, containing isoenzyme 2, is the major fraction found in prostatic tissue and serum from patients with prostatic cancer. Serum from patients with normal values for total ACP was used to determine a normal range for isoenzyme 2. The value of this test is the sensitivity with which small amounts of isoenzyme 2 can be detected, increasing the possibility of detecting small elevations in prostatic ACP long before high total ACP indicates that prostatic cancer has metastasized.

Purification and properties of pig brain guanine deaminase

Guanine deaminase (guanine aminohydrolase, EC 3.5.4.3) from pig brain was purified to homogeneity by column chromatography and ammonium sulphate fractionation. Homogeneity was established by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulphate (SDS). The molecular weight of 110 000 was determined by gel filtration and sucrose density gradient centrifugation. SDS polyacrylamide gel electrophoresis indicated subunits of a molecular weight of 50 000. The amino acid composition, the isoelectric point and the number of -SH groups were determined. 5,5′-Dithiobis-(2-nitrobenzoic acid) reacts with about seven -SH groups in the native enzyme, but upon denaturation with SDS, 10 -SH groups react with this former reagent. Using electrolytic reduction, 44 half-cystines were determined in accordance with the number of cysteic acid residues determined by amino acid analysis after performic acid oxidation. The K m values determined for substrates of the enzyme were 1.1 · 10 −5 M for guanine in 0.1 M Tris · HCl buffer (pH 8.0) and 3.3 · 10 −4 M for 8-azaguanine in 0.1 M phosphate buffer, pH 6.4. The P K a values determined for ionizable groups of the active site of the enzyme were near pH 6.2 and pH 8.2. The chemical and kinetic evidence suggests that cysteine and histidine may be essential for the catalysis.