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The Wendler version of the Torgov-Ananchenko scheme of total steroid synthesis was shown to be applicable to the preparation of 6-oxaestra-1,3,5(10),8,14-pentaenes. Conditions for cyclodehydration of secosteroids thus obtained were found, which ensured isolation of the target compounds in a high yield without using chromatographic purification methods. [ABSTRACT FROM AUTHOR]

Significant amounts of vitamin B 12 (about 116.3∼⃒556.3 µg/100 g) were determined in a Japanese salted and fermented salmon kidney “Mefun.” A corrinoid compound was purified to homogeneity from Mefun and partially characterized. TLC and HPLC patterns of the purified corrinoid compound were identical to those of authentic vitamin B 12 , but not to inactive corrinoids. The vitamin B 12 found in Mefun was not derived from concomitant vitamin B 12 ‐synthesizing bacteria, but had accumulated in the salmon kidney. Gel filtration experiments demonstrated that most of the vitamin B 12 found in Mefun was recovered in the free vitamin B 12 fractions. These results indicate that Mefun would be an excellent vitamin B 12 (free form) source for elderly persons with food‐bound vitamin B 12 malabsorption. [ABSTRACT FROM AUTHOR]

The cytochrome P450 (Cyt-P450) proteins from the fat body and midgut of the cotton bollworm, Helicoverpa armigera, were respectively partially purified by a set of purification procedures including differential centrifugation, solubilization of CHAPS, protein precipitation by PEG precipitation and DE-32 column chromatography. The Cyt-P450 was detected by methods of CO difference spectrum and SDS-PAGE. Fraction of detergent solubilized microsomes from the fat body of H. armigera was purified more than 17-fold. Three protein bands were detected by SDS-PAGE with molecular masses of 70 600, 63 300 and 571 200Da. It is possible that the proteins with molecular mass of 63 300 and 571 200Da were the isozymes of Cyt-P450. [ABSTRACT FROM AUTHOR]

Abstract: Tagetitoxin is a non-host specific toxin produced by Pseudomonas syringae pv. tagetis in host species of the Asteraceae family and in liquid culture by certain strains of the bacterium. A purification protocol involving anion exchange and partition chromatography was developed that yielded tagetitoxin purified to homogeneity. Based on dilution end-point (nanograms injected in 50μL that caused just detectable apical chlorosis in sunflower seedlings), the protocol resulted in a 2200-fold purification of tagetitoxin. The dilution end-point of purified tagititoxin was 10ng/50μL (295nM) and the I50 for inhibition of Escherichia coli RNA polymerase was 0.1μg/mL (147nM). Electrospray ionization mass spectrometry in 50% methanol:H2O indicated that the molecular weight of tagetitoxin is 678. Preliminary characterization of tagetitoxin structure was performed using 1D and 2D NMR spectroscopy. The results indicate that the previously proposed structure of tagetitoxin [Mitchell RE, Coddington JM, Young H. A revised structure for tagetitoxin. Tetrahedron Lett 1989; 30:501–504. [10]] is incorrect. [Copyright &y& Elsevier]

Ion‐Exchange centrifugal partition chromatography using benzalkonium chloride as a strong exchanger (SIXCPC) was successfully used to purify rosmarinic acid from a crude extract that was produced by callus culture. The purification process was carried out on a gram scale using the ternary biphasic system CHCl 3 : n ‐BuOH∶water 4.5:1:4.5 v/v/v in the ascending mode (mobile aqueous phase and stationary organic phase). Two particular points are discussed: the influence of benzalkonium chloride on ternary solvent system stability and the advantage of injecting the analytes as sodium salts rather than molecular acids. [ABSTRACT FROM AUTHOR]

In our search for bioactive compounds from the whole plantof Picria felterraeLour., three new phenylethanoid glycosides, picfeosides A-C (1-3), along with five known phenylethanoid glycosides, namely wiedemannioside (4), acteoside (5), acteoside isomer (6), cis-acteoside isomer (7), and cis-acteoside (8), were isolated using several chromatographic purification steps, including semipreparation HPLC on RP-18. The structures of the new compounds were elucidated on the basis of extensive spectroscopic analysis.(Managing editor: Wei WANG) [ABSTRACT FROM AUTHOR]

A facile, preparative low-pressure chromatographic process was developed for purification of paclitaxel from crude extract of Taxus spp. The process consisted of normal-phase and a reversed-phase chromatography without utilization of any liquid-liquid partition step. The normal phase chromatography was designed lo separate paclitaxel from a majority of unwanted compounds to obtain an initial purification. Silica gel and Al2O3, were compared as packing materials for this step. The result demonstrated that basic Al2O3 was better than silica gel to give a higher throughput. A further advantage of the Al2O3 was its ability of removing l0-deacetyl-7-epipaclitaxel. a compound difficult to be separated from the paclitaxel in subsequent purification. The chromatographic fraction of A2O3 was purified by a subsequent reversed-phase column chromatography, employing a novel uniform porous microsphere, named PST, as the packing material. Combination of the two steps of column chromatography and one step of crystallization was able to purify paclitaxel, from 0.46% to more than 98% with the total recovery- of 71%. This integrated chromatographic procedure was reproducible, efficient, and simple to use. [ABSTRACT FROM AUTHOR]

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

By selecting the different combination schemes, a simple, fast and highly efficient method for separation and purification of nerve growth factor (NGF) from venom of Chinese cobra is reported in this paper. This purification process consists of a two-step chromatographic separation on DEAE-Sepharose F.F. anion-exchange medium followed by a Sephadex G-50 gel filtration. On reducing and non-reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), the nerve growth factor obtained with this process proved to be homogeneous and its molecular weight was separately estimated to be approximately 14.5 and 29.0 kD, which was consistent with that reported in literature; and on high performance size-exclusion chromatography and reversed-phase chromatography, its purity was about 99%. The yield of this purification method was 0.51% and the nerve growth factor obtained had the activity of eliciting neurite outgrowth from chick embryonic dorsal root ganglia. The optimum concentration of nerve growth factor was 5–100 ng/ml and the minimal concentration eliciting neurite outgrowth from chick embryonic dorsal root ganglia was 5.0 ng/ml. [Copyright &y& Elsevier]

Acetyl esterase (acetic-ester acetylhydrolase, EC 3.1.1.6) from citrus peel, whose natural role is not well known, catalyses, in vitro, the hydrolysis of acetyl groups from a wide range of substrates. This enzyme was extracted from Mediterranean orange peel, largely available in Italy, and purified 190-fold by a single chromatographic step on Sepabeads FP-HG. SDS polyacrylamide gel electrophoresis of the purified enzyme showed a major protein band, corresponding to a molecular mass of 45 kDa. Both free and immobilised enzyme were used in biotransformations. The enzyme removed the acetyl group in the 3 position of β-lactamic antibiotics, such as cephalosporin C and the intermediate 7-aminocephalosporanic acid with ≥98% conversion and 91-93% product yield. [ABSTRACT FROM AUTHOR]

A suitable combination of preparative scale separation methods results in effective clean-up of the ecdysteroids of Silene italica ssp. nemoralis (Waldst. and Kit.) Nyman. The isolation of minor ecdysteroids from the partially purified extract is based on the use of both droplet counter-current chromatography and low-pressure reversed-phase liquid chromatography. The purification is completed by preparative thin-layer chromatography and preparative high-performance liquid chromatography to obtain the minor ecdysteroids, such as 2-deoxy-20-hydroxyecdysone, shidasterone, 2-deoxy-polypodine B, makisterone C, and 9α,20-dihydroxyecdysone. [ABSTRACT FROM AUTHOR]

In the last five years, the acceptance and implementation of packed-column supercritical fluid chromatography-mass spectrometry (pSFC-MS) to drug discovery applications has gained momentum. This article describes the pros and cons of pSFC-MS and attempts to demonstrate its broad applicability to such fields as high-throughput analysis, purity assessment structure characterization and purification. Finally, an outlook for the future of this technique is presented. [ABSTRACT FROM AUTHOR]

A new counter-current chromatography (CCC) method with an upright coil planet centrifuge, which holds four identical multilayer coil columns in the symmetrical positions around the centrifuge axis, was applied to the isolation and purification of celastrol from the roots of Celastrus orbiculatus Thunb. The crude celastrol was obtained by elution with light petroleum from ethanol extracts using 15 cm × 5 cm i.d. silica gel flash chromatography. Preparative CCC with a two-phase system composed of light petroleum (bp 60–90 °C)–ethyl acetate–tetrachloromethane–methanol–water (1:1:8:6:1, v/v) was successfully performed, yielding 798 mg celastrol at 99.5% purity from 1020 mg of the crude sample in one step separation. [Copyright &y& Elsevier]

Epigallocatechin (EGC) was prepared by the degallation of Epigallocatechin-O-3-gallate (EGCG). EGCG was completely converted to EGC and gallic acid by adding 4 mg tannase/100 mg EGCG at the concentration of 2 mg/mL under pH 6.0, at 35°C for 30 min. EGC was then separated from the reaction mixture by HSCCC, using a two-phase solvent system composed of hexane–ethyl acetate–water (1/9/10, v/v/v). Finally, 1.3 g of EGC at 97% purity was prepared from about 2.3 g of EGCG at 85% purity, indicating that EGC was almost completely recovered by HSCCC. [ABSTRACT FROM AUTHOR]

A modified method for preparative purification of trans-resveratrol (trans-3,4 ',5-tri-hydroxystilbene) from vine stems is presented. It includes two new procedures added to the known scheme of resveratrol separation from ligneous organs of vine by ethanol--water extraction: (1) treatment of ethanolic solution of vine stem stilbenoid extract with diethyl ether for precipitation of a dark brown matter from the extract; (2) preparative column chromatography on polyamide carrier for separation of resveratrol from the crude ethanolic extract. As a result, 93.8% resveratrol concentrate with 0.7% of its dimer viniferin impurity was obtained. Resveratrol and viniferin contents in the stems of various frost-hardy hybride cultivars of Vitis vinifera grown open air in Estonia ('Hasaine Sladki', 'Jubilei Novgoroda', Vitis vinifera cv., 'ES 12-7-98', 'Marechal Joffre', 'Zilga') were determined for the plant material collected in July and in October. Lignified stems collected in October turned out to be an excellent source for producing vine stem stilbenoid (resveratrol and viniferin) concentrates for use as healthy ingredients in functional food for preventing heart diseases and atherosclerosis. [ABSTRACT FROM AUTHOR]

We described a novel purification method for a recombinant glucocorticoid receptor (GR) in detail. The purification procedure consists of sequential chromatographies using common ion-exchange columns (Mono Q and Mono S). This procedure is based upon a new finding that the activated GR binds both to a Mono Q column and to a Mono S column at the same pH. The entire chromatographies took about 3 h and GR represented 97% of the purified protein sample. This purification protocol will be applicable to the purification of native GR, point-mutated recombinant GR and other nuclear receptors. [Copyright &y& Elsevier]

Optimized procedures for the affinity purification of soybean agglutinin (SBA) from soybean flour, and its further immobilization, were developed. Lectin purification on galactosyl-Sepharose yielded 44.5±3.5 mg of pure SBA/50 g of flour. To prepare SBA adsorbents, the lectin was immobilized onto 1-cyano-4-(dimethylamino)pyridinium tetrafluoroborate (CDAP) activated Sepharose with high yields (77%). Feasibility of the use of this improved SBA adsorbent for affinity purification of Streptococcus pneumoniae capsular polysaccharides from strain 14 (CPS-14) at laboratory scale was demonstrated. Using SBA-Sepharose adsorbent (7.0 mg lectin per ml), amounts of 6.3 mg of pure CPS-14 per cycle were produced, the adsorbent being reused up to four times without loss of capacity. [Copyright &y& Elsevier]

Recombinant DNA-derived proteins and, in particular, human pituitary hormones, are increasingly used for research, diagnostic and therapeutic purposes. This trend has demanded new synthetic approaches and improved purification techniques. The type and sequence of the purification steps have to be selected in accordance with the cloning and protein expression strategy, the host organism and cellular localization of the protein of interest, with a view to producing the desired product at a required purity, biological activity and acceptable cost. This review article describes and analyzes the main synthetic and purification strategies that have been used for the production of recombinant human growth hormone, prolactin, thyrotropin, luteinizing hormone and follicle-stimulating hormone, giving special consideration to the few published downstream processes utilized by the biotechnology industry. Practically all types of prokaryotic and eukaryotic organisms utilized for this purpose are also reviewed. [Copyright &y& Elsevier]

Protein C deficiency (inherited and acquired) has a relatively high incidence rate in the general population worldwide. For many years, protein C deficient patients have been treated with fresh frozen plasma, prothrombin complex concentrates, heparin or oral anticoagulants, which all have clinical drawbacks. We report the production process of a highly purified human protein C concentrate from 1500 l of cryo-poor plasma by a four-step chromatographic procedure. After DEAE-Sephadex adsorption, protein C was separated from clotting factors II, VII and IX by DEAE-Sepharose FF and further purified, using a new strategy, by an on-line chromatographic system combining DMAE-Fractogel and heparin-Sepharose CL-6B. In addition, the product was treated against viral risks by solvent-detergent and nanofiltration on 15-nm membranes. The protein C concentrate was essentially free of other vitamin K-dependent proteins. Proteolytic activity was undetectable. Neither activated protein C, prekallikrein activator, nor activated vitamin K-dependent clotting factors were found resulting in good stability of the protein C activity. In vitro and in vivo animal tests did not reveal any sign of potential thrombogenicity. The final freeze-dried product had a mean protein C concentration of 58 IU/ml and a mean specific activity of 215 IU/mg protein, corresponding to over 12 000-fold purification from plasma. Therefore, this concentrate appears to be of potential benefit for the treatment of protein C deficiency. [Copyright &y& Elsevier]

The aim of this work was to test a recycling method for imidazole used in immobilized metal affinity chromatography (IMAC) as eluent for recombinant histidine-tag (His-tag) protein. After evaluating two supports, the method was optimized with a mixture of bovine serum albumin, sodium chloride and imidazole. Recycling was performed with an eluate fraction from IMAC of His-tag enhanced green fluorescent protein produced in our laboratory and pure imidazole was recovered in water and was analyzed after being freeze-dried. The imidazole was then reused as eluent in IMAC without any modification in its structure or behavior. This procedure can be used for large-scale chromatography. [Copyright &y& Elsevier]

Affinity chromatography is potentially the most selective method for protein purification. The technique has the purification power to eliminate steps, increase yields and thereby improve process economics. However, it suffers from problems regarding ligand stability and cost. Some of the most recent advances in this area have explored the power of rational and combinatorial approaches for designing highly selective and stable synthetic affinity ligands. Rational molecular design techniques, which are based on the ability to combine knowledge of protein structures with defined chemical synthesis and advanced computational tools, have made rational ligand design feasible and faster. Combinatorial approaches based on peptide and nucleic acid libraries have permitted the rapid synthesis of new synthetic affinity ligands of potential use in affinity chromatography. The versatility of these approaches suggests that, in the near future, they will become the dominant methods for designing and selection of novel affinity ligands with scale-up potential. [Copyright &y& Elsevier]

Experiments were carried out to evaluate the fractionation of proteins and peptides according to mass. Model mixtures were separated by either reversed-phase or ion-exchange chromatography with mass spectrometry-compatible mobile phase additives. Fraction collection was triggered by the mass/charge ratio of each one of the components of the mixture. Chromatography was additionally monitored with a UV–Vis detector in order to compare the new technique with generally accepted in separations. The results indicated that adequate purification is achieved by this new technique. Fraction collection triggered by changes in the mass/charge ratio reduces sample handling and analysis time. This study demonstrates the utility of mass-directed fractionation of peptides and proteins when mass spectrometry-compatible mobile phase additives are used. [Copyright &y& Elsevier]

An alkaline phosphatase was purified from Pinctada fucata, a kind of pearl oyster, by chromatography on DEAE-32 cellulose, Sephadex G-150 and DEAE A-25. The specific activity of the enzyme was 2040 U mg−1. The kinetics characteristics of the enzyme have been studied. The product HPO42− and the product-analog WO43− competitively inhibited the enzyme activity. Positive monovalent cations had no effect on the enzyme activity, while positive bivalent cations had different effects on the enzyme: Mg2+, Ca2+, Co2+ and Mn2+ activated the enzyme while Zn2+, Cu2+ and Cd2+ inhibited the enzyme. [Copyright &y& Elsevier]

A facile multigram scale preparation of (1R,3S,4S)-2-azabicyclo[2.2.1]heptane-3-carboxylic acid via stereoselective synthesis of the corresponding α-amino ester hydrochloride is detailed. Hitherto applied protocols for the synthesis of this cyclic proline analogue involving a tedious chromatographic purification step could thus be considerably improved upon. The specific rotation of the α-amino acid reported in the literature has been revised. [Copyright &y& Elsevier]

pH-Zone-refining counter-current chromatography was successfully applied to the separation of the main components of Food Color Red No. 106 (R-106, acid red, Color Index No. 45100). A 300-mg quantity of sample was separated using the following two-phase solvent system: n-butanol–water, 40 mM sulfuric acid in organic stationary phase and 30 mM ammonia in aqueous mobile phase. The obtained fractions were analyzed by high-performance liquid chromatography and fast atom bombardment mass spectrometry. The separation yielded 261.9 mg of main component of acid red with purity of 99.9%. [Copyright &y& Elsevier]

Peroxidase from turnip roots was purified using metal affinity chromatography up to a specific activity of 337 units/mg protein with 3.02 RZ and 63.5% recovery. After purification, the enzyme showed 2–3 bands on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the purified enzyme was found to be 37–39 kD with matrix assisted laser desorption ionization mass spectrometer (MALDI-MS). The enzyme showed maximum activity in phosphate buffer, pH 6.0, and lowest activity in borate buffer at the same pH. The Km of the enzyme was found to be 7.07×10-4 mM. Turnip peroxidase also contains an iron moiety which is found to be about 0.28%. The enzyme showed 50% inhibition of its specific activity with ethylene diamine tetraacetic acid (EDTA). [ABSTRACT FROM AUTHOR]

Preparative scale purification of PEG-protein conjugates by centrifugal precipitation chromatography is discussed. By utilizing the transport of ammonium sulfate across a membrane and the centrifugal force, an ammonium sulfate gradient is formed and used for differential precipitation of PEG-protein conjugates. Native proteins can be separated from PEG-protein conjugates by using this technique. [ABSTRACT FROM AUTHOR]

Centrifugal partition chromatography linked with evaporative light scattering detection has been investigated to achieve the large scale-up isolation of oraposide which is a phenylpropanoid glycoside having a radical-scavenging activity. A two-phase solvent system has been studied to improve the yield of pure product and to decrease the cost of purification. A comparison with preparative HPLC has been made. [ABSTRACT FROM AUTHOR]

Abstract. A new purification procedure is introduced, which uses the advantages of column chromatography and vacuum filtration. Potassium polyacrylate is used as stationary phase. This method is based on the idea that the size of the existing cavities in the polymer increases during a swelling process in distilled water. The cavities am big enough to entrap the nanoparticles, but allow for a free movement of nanotubes and bundles. [ABSTRACT FROM AUTHOR]

In this research, a process for developing normal-phase liquid chromatography solvent systems has been proposed. In contrast to the development of conditions via thin-layer chromatography (TLC), this process is based on the architecture of two hierarchically connected neural network-based components. Using a large database of reaction procedures allows those two components to perform an essential role in the machine-learning-based prediction of chromatographic purification conditions, i.e., solvents and the ratio between solvents. In our paper, we build two datasets and test various molecular vectorization approaches, such as extended-connectivity fingerprints, learned embedding, and auto-encoders along with different types of deep neural networks to demonstrate a novel method for modeling chromatographic solvent systems employing two neural networks in sequence. Afterward, we present our findings and provide insights on the most effective methods for solving prediction tasks. Our approach results in a system of two neural networks with long short-term memory (LSTM)-based auto-encoders, where the first predicts solvent labels (by reaching the classification accuracy of 0.950 ± 0.001) and in the case of two solvents, the second one predicts the ratio between two solvents (R2 metric equal to 0.982 ± 0.001). Our approach can be used as a guidance instrument in laboratories to accelerate scouting for suitable chromatography conditions. [ABSTRACT FROM AUTHOR]

Column chromatography on aminopropyl-derivatized silica and precipitation of a complex with perchloric acid have been evaluated as methods for the purification of di-tert-butylcyclohexano-18-crown-6 (DtBuCH18C6), a compound frequently employed for the selective extraction of strontium from acidic nitrate media. Both methods are shown to provide a simple and effective means of eliminating inactive sample components (i.e., impurities or stereoisomers incapable of extracting strontium) from the crown ether and enriching the material in 4(z),4′(z) cis-syn-cis DtBuCH18C6, a stereoisomer capable of highly efficient strontium extraction. [ABSTRACT FROM AUTHOR]

The F1 complex of the ATP synthase of Streptomyces lividans was isolated and purified. The procedure involved the solubilization of F1 from membranes with buffer of low ionic strength in the presence of EDTA, ion-exchange chromatography and gel filtration. The purified F1 complex from S. lividans (SLF1) consists of five subunits α, β, γ, δ and ε with molecular masses of 58000, 50000, 36000, 28000 and 13000, respectively and exhibits immunological cross-reactivity with the F1 portion purified from Escherichia coli (ECF1). The enzymatic properties of SLF1 were determined by the use of microtiter-plate-based assay and compared with data obtained for ECF1. ATPase activity of SLF1 (specific activity: 20–30 U/mg) was only observed in the presence of high concentrations of Ca2+ (to mM). Stimulation of the ATPase activity by Mg2+ was not detectable; quite to the contrary, Mg2+ inhibited the Ca2+-stimulated activity of SLF1. SLF1 was re-bound to F1-stripped membranes of S. lividans, but not to F1-stripped membrane vesicles of E. coli. In contrast, ECF1 could be cross-reconstituted with F1-stripped membranes of S. lividans; however, a structural but not a functional reconstitution of the hybrid F1Fo complex was observed. [ABSTRACT FROM AUTHOR]

Reports on the purification and characterization of brain pyridoxal kinase from sheep brain. Use of ammonium sulphate fractionation, diethylaminoethanol-cellulose chromatography, affinity chromatography and Sephadex G-100 gel filtration; Molecular mass of the native enzyme.

A protein, which facilitates assembly ora nucleosome-like structure in vitro, was previously partially purified from mouse FM3A cells [Ishimi, Y. et al. (1983) J. Biochem, (Tokyo) 94, 735-744]. The protein has been purified to approximately 80% from FM3A cells by using histone-Sepharose column chromatography. It sedimented at 4.6 S and had a molecular mass of 53kDa. A preincubation of core histones with the 53-kDa peptide before DNA addition was necessary for the nucleosome assembly. The 53-kDa peptide bound to core histones and formed a 12-S complex. This complex contained stoichiometrical amounts of the 53-kDa peptide and core histones, and the core histones in this complex were composed of equal amounts of H2A, H2B, H3 and H4 histones. The nucleosomes were assembled by adding pBR322 DNA to the 12-S complex. When mononucleosome DNA and core histones were mixed in the presence of the 53-kDa peptide, formation of a 10.5-S complex was observed. The complex contained DNA and core histones in equal amounts, while no 53-kDa peptide was detected in the complex. From above results it is suggested that the 53-kDa peptide facilitates nucleosome assembly by mediating formation of histone octamer and transferring it to DNA. Rat antibody against the 53-kDa peptide did not bind to nucleoplasmin from Xenopus eggs. The relationship between the 53-kDa peptide and nucleoplasmin is discussed. [ABSTRACT FROM AUTHOR]

The results reported in the present paper and N-terminal sequence homologies established by other authors strongly support the assumption that the gastric protease previously called bovine pepsin I or bovine pepsin B belongs to the aspartate protease group and corresponds to gastricsin (or pepsin C) (EC 3.4.23.3) from other species. Bovine gastricsin was prepared from commercial extracts of adult bovine veils by a procedure involving DEAE-cellulose chromatography, gel filtration on Sephacryl S-200 and a further DEAE-cellulose chromatography. The preparation thus obtained was shown to be free of chymosin and bovine pepsin A by immunodiffusion, selective inactivation in urea and isoelectric focusing. Us molecular weight was estimated by gel filtration to be 32800. Bovine gastricsin displayed microheterogeneity on isoelectric focusing with pi values of the components ranging from 3.5 to 4.0. Chromatography of bovine gastricsin on hydroxyapatite separated three fractions, none of them being homogeneous by isoelectric focusing. Concanavalin-A—Sepharose 4B bound bovine gastricsin to some extent, but without any significant fractionation. Proteolytic activity could be detected directly on the isoelectric focusing gel for all the components of gastricsin and its fractions from hydroxyapatite and concanavalin-A—Sepharose 4B. Bovine gastricsin and its fractions from hydroxyapatite have similar amino acid compositions, different from those of bovine chymosin and pepsin A but obviously related to those of human, simian and porcine gastricsins. Bovine gastricsin which is inactivated by reaction with diazoacetyl-DL-norleucine methyl ester and with 1,2-epoxy-(p-nitrophenoxy)propane in a 1:1 and 1:2 stoichiometry, respectively, is able to hydrolyse a synthetic hexapeptide, Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe, used as reference substrate for aspartate proteases, and exhibits a low activity towards N-acetyl-L-phenylalanyl-L-diiodotyrosine. Its specific clotting activity with χ-casein as substrate is only half of that of chymosin and pepsin A. [ABSTRACT FROM AUTHOR]

Chromatography on DEAE-cellulose and gel filtration on Sephadex revealed that pyrazon dioxygenase from pyrazon-degrading bacteria consists of three different enzyme components. No component alone oxidizes the phenyl moiety of pyrazon, only when the three components are combined can oxidation be detected. Following electron paramagnetic resonance and ultraviolet measurements the protein nature of the three components was determined: component A1 (molecular weight about 180000, red-brown in colour) is an iron-sulphur protein. The existence of approximately two moles of iron and two moles of inorganic sulphur per mole of protein was demonstrated. This enzyme component was purified to homogeneity in disc elcctrophoresis. Component A2 is a yellow protein of a molecular weight of about 67000. FAD was shown to be the prosthetic group of this protein. Component B (molecular weight about 12000, brown in colour) is a protein of the ferredoxin type, which was purified to homogeneity, as demonstrated by disc electrophoresis. A hypothetical scheme for the cooperation of the three components is proposed: component A2 accepts as cosubstrate NADH and functions as a ferredoxin reductase. The ferredoxin, component B, has the function of an electron carrier. The conversion of the substrates is effected by component A1, the terminal dioxygenase. [ABSTRACT FROM AUTHOR]

Poly(ADP-ribose) polymerase with a high specific activity was obtained from Ehrlich ascites tumor cells by extraction of nuclei with 175 mM potassium phosphate, followed by chromatography on DNA-agarose. Electrophoretic analysis indicated that the preparation contained two proteins, one of which was shown to catalyze the synthesis of poly( ADP-ribose). As expected from results obtained by other workers, the synthesis was inhibited by nicotinamide and thymidine. and stimulated by DNA. Addition of histones gave inhibition of the synthesis, unless DNA was present in the reaction mixture. [ABSTRACT FROM AUTHOR]

Abstract: A novel and synthetically facile production of 2-substituted pyrrolidines from commercially available nitriles is reported herein. This methodology is operationally simple, and only requires the use of an extraction and a single chromatographic purification to furnish the title compounds in high purity and very good yields. [Copyright &y& Elsevier]

32P-labelled N-formyl methionyl transfer RNA from Escherichia coli strain CA265 was purified by chromatography on a column DEAE Sephadex followed by chromatography on benzoylated DEAE cellulose. Nucleotides from ribonuclease T1 and pancreatic ribonuclease digests of tRNAF were separated and their sequences determined. tRNAF consists of 77 nucleotides of which pCG is 5′-terminal and CAACCOH is 3′-terminal. [ABSTRACT FROM AUTHOR]

Silver-111 (111Ag, t 1/2 = 7.47 d) is a β − emitter suitable for targeted cancer therapy due to favourable decay properties. The production of no-carrier added 111Ag via Isotope Separation On-Line (ISOL) technique is being investigated at the Legnaro National Laboratories of the Italian Institute of Nuclear Physics (ISOLPHARM project). Stable Cadmium-111 (111Cd) is co-produced as isobaric contaminant, hence a chemical separation process must be developed to selectively harvest 111Ag. In this study, a chromatographic procedure employing the commercially available CL resin was investigated by using stable Ag+ and Cd2+. Results indicate that CL resin allows to efficiently separate Ag+ from Cd2+ and recover the former with high yields. • Silver-111 (t 1/2 = 7.47 days) is a β − emitting radionuclide suitable for cancer therapy. • During 111Ag production via ISOL method, 111Cd is co-produced. • Chromatographic method using commercially available CL resin was developed to separate silver from cadmium. [ABSTRACT FROM AUTHOR]

Abstract: Histone octamers are the basic building blocks of chromatin and are platforms for diverse genetic mechanisms. We report a simple method for preparing recombinant histone octamers by overexpressing all four histones from a single polycistronic vector followed by standard chromatography under native conditions. This approach reduces the time needed for the octamer preparation to a single day and should be applicable to making a variety of unmodified and modified histone octamers. [Copyright &y& Elsevier]

Abstract: Rapid and efficient one-pot synthesis of thiopyrano[2,3-b]quinolines is described from the reaction of 3-formyl-quinoline-2-thiones with acrylonitrile using economical organic base Et3N at room temperature. The reaction proceeded smoothly via domino Michael addition/cyclization reactions and did not require dry solvent, inert atmosphere and column chromatography purifications. [Copyright &y& Elsevier]

Abstract: A facile and high-yielding route for the synthesis of solasodine from diosgenin is devised. Ring opening of steroidal spiroketal under mild conditions with trifluoroacetyl trifluoromethanesulfonate (TFAT) provides an applicable protocol to prepare key intermediates 4 or 3-Ac-solasodine, which can potentially serve as a platform for the selective functionalization of C(3)–OH and N–H of solasodine. The simple operations without purification by column chromatography make this method suitable to scale up. [Copyright &y& Elsevier]

• Robust and fast HPLC-UV method for quantification of anidulafungin and micafungin. • SPE improved the signal-to-noise ratios of chromatograms. • Higher sensitivity allows for quantification of lower concentrations in body fluids. • Appropriate approach for pharmacokinetic studies with critically ill patients. The echinocandins anidulafungin (ANID) and micafungin (MICA) are recommended for treatment of invasive Candida infections. As target-site concentrations of antimicrobial agents are crucial for eradication of pathogens, we established and validated high-performance liquid chromatography-UV detection (HPLC-UV) assays for quantification of ANID and MICA in human plasma, ascites fluid, pleural effusion, and in cerebrospinal fluid (CSF). Sample pre-purification was performed by protein precipitation with acetonitrile followed by solid phase extraction. For both assays, intra- and interday precision, and accuracy fulfilled the requirements for bioanalytical methods issued by the European Medicine Agency (EMA). The lower limit of quantification was 0.01 mg/L for both drugs. At 25 °C, ANID and MICA concentrations declined by up to 70% within 24 h. Concentrations remained stable over 24 h at 4 °C and over four weeks at −80 °C. In conclusion, the developed methods are fit for the assessment of target-site pharmacokinetics of ANID and MICA in clinical studies. [ABSTRACT FROM AUTHOR]

Abstract: A facile, mild and rapid solid phase synthetic route free of column chromatographic purification to the synthesis of soluble monodisperse long-chain oligo(1,4-phenyleneethynylene)s is presented. [Copyright &y& Elsevier]