Criteria Of Purity Research Articles

The anthocyanins of apples and pears: The occurrence of acyl derivatives

AbstractThe major pigment in red peel of some apple cultivars was confirmed as cyanidin 3‐galactoside and minor pigments identified were cyanidin 3‐glucoside, 3‐arabinoside and 3‐xyloside as well as acylated derivatives of all four glycosides. The minor fractions which were separated by paper chromatography varied in composition according to the methods of extraction and purification. The efficiency of their extraction was increased by the use of sulphur dioxide in the extracting medium (acetone). Possible loose association of anthocyanins and proanthocyanidins was indicated, and the instability of the latter during gel filtration with an acid solvent was demonstrated. Minor pigments were negligible in the pears examined. Some further criteria of anthocyanin purity are suggested.

Bulk separation of neurones and glia: A comparison of techniques

Three different techniques of preparation of neurones and neuropil (glia) from rat cortex, those of Rose, Norton and Poduslo, and Sellinger and Azcurra, were compared according to the proposed criteria of purity, integrity and yield. Morphologically the cells prepared by the Norton and Poduslo and Sellinger and Azcurra methods appeared better and the fractions were purer than Rose method I, though not Rose method II. Based on carbonic anhydrase ratios, the glial contamination of neurones was 11% in Rose cells, 24.8% in Norton and Poduslo cells and 8.3% in Sellinger and Azcurra cells. Lactate dehydrogenase activity was 25% in Sellinger and Azcurra cell suspension and 37% in Norton and Poduslo cell suspension, of that in the Rose suspension. Oxygen consumption and carbon dioxide production in Sellinger and Azcurra suspension was only 15% and in Norton and Poduslo cell suspension 50%, of that in the Rose technique. The metabolic damage observed in the Sellinger and Azcurra preparation could be partially averted by alteration of the disaggregation medium. The relative utility of the techniques is discussed.

Analyse spectrophotometrique des chlorophylles et des pheophytines a et b en milieu hydroacetonique: Détermination des extinctions molaires

Molar absorptivities of purified chlorophylls a and b were determined in aqueous acetone media. For each chlorophyll, magnesium was titrated with EDTA to a voltammetric end-point, and the porphyrin ring was titrated with copper(II) in anhydrous acetic acid, the end-points being simultaneously determined spectrophotometrically and potentiometrically. The identical results of these two titrations provide a criterion of purity for the studied chlorophylls. Moreover, these results permit the rigorous determination of the molar absorptivity of one of these pigments at selected wavelengths.

Isolement, purification et propriétés de deux zymogènes gastriques bovins. Propriétés des protéases correspondantes

Summary This communication describes the isolation and purification of two zymogens from bovine abomasum, showing characteristic properties of pepsinogens. u —Stability above pH 7. —No proper proteolytic activity at pH 5.25. —Activation at low pH, probably corresponding to the separation of a basic part of the molecule. —Stability of the corresponding enzymes below pH 6. Elution diagrams, starch and agar gel electrophoresis and N-terminal residue analysis are given as criteria of purity. The proteolytic activity against hemoglobin as a function of pH and the inactivation at alcaline pH of the two corresponding pepsins are compared with those of rennin and swine pepsin.

Isolation and partial characterization of lysozyme from baboon milk

Lysozyme (mucopeptide N-acetylmuramylhydrolase, EC 3.2.1.17) was isolated in 50% yield from baboon milk. Four criteria of purity were met. Amino acid analysis indicated a molecular weight of 15 000, with three histidine and no methionine residues; otherwise the composition was broadly similar to that of human milk lysozyme.

Criteria of purity for guanidine hydrochloride

In addition to ultraviolet absorption spectrum, melting point determination has been established as a simple and reliable method for evaluating the purity of guanidine hydrochloride. A reasonably pure sample appears to have a melting point at 187 – 187.5°C. A number of guanidine hydrochloride (GuHCl) samples, including some commercial products for which the claim of ultrapurity is made, have been compared on the basis of their ultraviolet absorption spectra, m.p., and titration curves. Some samples that had acceptable ultraviolet absorption spectra were found to be seriously contaminated by impurities.

The thermal analysis of “argentic oxynitrate” and silver oxides

The precise stoichiometry of “argentic oxynitrate” has been elucidated by analytical and thermal analytical techniques. A decomposition path of the oxynitrate has been proposed and values of the enthalpy changes obtained. TG has been used as the criterion of purity of silver(II) oxide and new values for the heat of formation of silver(I) and silver(II) oxide are reported.

Differential scanning calorimetric assessment of high purity

The value and limitations of differential scanning calorimetry in the assessment of high-purity substances has been examined. In favorable cases, good agreement has been secured for polycyclic hydrocarbons between DSC purity values and GC assay values. For some halogenated benzoic acids, used as microanalytical reference standards, good agreement has been obtained between DSC purity values and acid—base titration results. DSC studies on cholesterol and urea, which have limited thermal stability, are presented. With the available instrument and technique, the practical upper limit of absolute DSC purity values may be 99.95 mole%, although higher numerical values can be obtained. Because the DSC technique is “blind” to equilibrium solid solution formation, DSC values should not be used as a sole criterion of purity; this recommendation is of special importance for compounds purified by fractional solidification processes.

Purification of lysozymes by retention on Bio Gel CM 30

An improved method for the purification of lysozymes, in high yield, is described. The lysozyme from human milk, mare milk or hen egg-white, retained by Bio Gel CM 30, was chromatographed on a second column of Bio Gel CM 30 — Asbestos. After desalting, a chromatography on Amberlite CG 50 gave purified lysozymes. Criteria of purity included amino acid analysis, a constant specific activity, chromatography on Sephadex G-100 and analytical gel electrophoresis. A human milk lysozyme B was isolated in some of the experiments. Mare milk lysozyme was purified for the first time. Une méthode améliorée pour la purification des lysozymes en peu d'étapes, avec un rendement élevé, est décrite. Les lysozymes du lait humain, lait de jument ou blanc d'œuf de poule, absorbés par Bio Gel CM 30, ont été chromatographiés sur une deuxième colonne de Bio Gel CM 30 — Amiante. Après dessalage, une chromatographie sur Amberlite CG 50 donne des lysozymes purifiés. La pureté a été vérifiée par la composition en acides aminés, la constance de l'activité spécifique, la chromatographie sur Sephadex G-100 et l'électrophorèse analytique sur gel d'acrylamide. Dans plusieurs cas, un lysozyme humain B a été isolé. Le lysozyme du lait de jument a été purifié pour la première fois Wir beschreiben hier eine verbesserte, besonders ertragreiche Methode zur Reinigung von Lysozymen in wenigen Etappen. Lysozyme von Frauenmilch, Stutenmilch und Hühnereiweiss, von Bio Gel CM 30 zurückgehalten, wurden durch eine zweite Säule mit Bio Gel CM 30 — Asbest chromatographiert. Nach Entsalzung erhält man durch Chromatographie an Amberlit CG 50 gereinigte Lysozyme. Die Aminosäurenzusammensetzungen, die konstante spezifische Aktivität, die Chromatographie durch Sephadex G-100 und die analytische Akrylamid-Electrophorese bestätigten den Reinheitsgrad. In einigen Versuchen wurde ein menschliches Lysozym B isoliert. Das Stutenmilchlysozym wurde zum ersten Mal gereinigt.

Užiti kyselin, louhů a enzymů k extrakci nukleovýcn kyselin z pylu

Various methods for extracting nucleic acids from pollen were tested to find a suitable procedure for obtaining a pure preparation of nucleic acids uncontaminated by polysaccharides and polyphosphates without the use of ion exchangers. Extraction was carried out with perchloric acid, potassium hydroxide, ribonuclease and deoxyribonuclease, sodium tetraborate, and combinations of these. In all fractions, residues of precipitates and residues of extracted pollen matter, the quantity of RNA, DNA, proteins and concomitants,i.e. Polysaccharides and polyphosphates, was determined. The purity of preparations was checked by means of UV-spectra. The criterion of nucleic acid purity was agreement between the nucleic acid amounts calculated on the basis of measurement of absorption in UV-region, orcinol reaction and content of phosphorus. It was found that in our material only a few methods would be applicable and with great limitation, because many polysaccharides and polyphosphates appeared in nucleic acid fractions.

Ultraviolet absorption spectrum of RNA polymerase

Received 8 July 1970 1. Introduction One of the criteria of purity of a preparation of RNA polymerase (EC 2.7.7.6) is its 280/260 rq~ absorbance ratio. Enzyme preparations with a ratio below 1.65 are assumed to contain some contami- nating nucleic acids or their derivatives [l] . How- ever, no nucleic acid was detected in a purified en- zyme preparation of absorbance ratio 1.25 [2], either by the Dische method (limit of detection 1%) or the orcinol procedure (limit of detection 0.6%). From the data of Warburg and Christian [3] ,

The isolation of polysomes from rat liver. Contamination of polysomes by ribonucleoprotein of nuclear origin

Polysomes isolated by methods involving Triton X-100 were contaminated by cosedimenting rapidly labeled material to a considerably greater extent than if the detergent sodium deoxycholate was used. A method for obtaining uncontaminated polysomes by using Triton X-100 was developed and criteria for purity were established. The contaminating material sedimented on the average somewhat slower than the polysomes. The sedimentation rate was not influenced by EDTA. Nuclear ribonucleoprotein particles containing RNA of DNA-like base composition have a sedimentation rate and a buoyant density similar to those of the contaminating material. They were not attacked by EDTA nor by nonionic detergents, whereas sodium deoxycholate reduced the particles to slowly sedimenting material by removal of protein from their RNA moiety. Nonionic detergents were able to protect against the action of sodium deoxycholate. Rapidly labeled material leaked out from the nuclei during the homogenization procedure. It cosedimented with the polysomes in sucrose gradients without being actually bound to them. It is suggested that the radioactive material contaminating polysome preparations is of nuclear origin. Part of it may have been present in the cytoplasm in vivo.

Studies on Poly Adenosine Diphosphate-Ribose: VIII. A METHOD FOR PURIFICATION OF POLY ADENOSINE DIPHOSPHATE-RIBOSE

Abstract A method was developed for isolation and purification of radioactive poly ADP-ribose formed by a nuclear enzyme from rat liver. The procedure involved: (a) Pronase digestion and phenol extraction, (b) pancreatic RNase and pancreatic DNase digestion, (c) a first gel filtration through a Sephadex G-50 column, (d) micrococcal nuclease and spleen phosphodiesterase digestion, and (e) a second gel filtration. The over-all recovery of acid-insoluble radioactivity in the procedure was more than 50% and one-third of the acid-insoluble radioactivity was eluted just after the void volume from the Sephadex G-50 column. Increase of the specific radioactivity (counts per min per OD260) and decrease of the ratio of absorption at 280 mµ to that at 260 mµ were used as criteria of purity of poly ADP-ribose. At the final step of purification, the specific radioactivity of poly ADP-ribose was 80-fold that of the crude extract after Pronase digestion and phenol extraction. The specific activity of purified poly ADP-ribose and that of α-32P-ATP used as a precursor were 1.26 x 105 cpm per OD260 and 0.99 x 105 cpm per OD260, respectively. The A280:A260 ratio of absorption of purified poly ADP-ribose at the final step was 0.26. Purified poly ADP-ribose was completely hydrolyzed by snake venom phosphodiesterase and did not yield any nucleotide other than 5'-AMP and PR-AMP on Dowex 1 column chromatography. Experiments with 14C-ATP and NMN with a 32P-labeled whole nuclear preparation showed that poly ADP-ribose after the second gel filtration was devoid of 32P radioactivity. This indicates that poly ADP-ribose at this step was already free from contaminating RNA and DNA.

Molar Absorptivity Values for Aflatoxins and Justification for Their Use as Criteria of Purity of Analytical Standards

Abstract Measurements of molar absorptivities in methanol were carried out by two laboratories on samples of anatoxins Bi and Gi prepared and purified independently in four laboratories and on samples of aflatoxins B2 and G2 prepared and purified independently in three laboratories. Molar absorptivities of pure aflatoxins Bi, B2, G1, and G2 in benzene-acetonitrile (98 + 2) were determined at two laboratories. With the exception of aflatoxin G2, no significant difference between aflatoxin samples could be demonstrated. Molar absorptivity values and absorbance ratios for each aflatoxin, based on these data, are given. Statistical 95% confidence limits were established for a single determination of molar absorptivity. A statistical study was made of the components of variance which contribute to the accuracy of the determination of molar absorptivity. A comparison of the fluorescence intensities of spots of various aflatoxin preparations, developed on silica gel-coated thin layer plates, with the amount of aflatoxin in each spotting solution as determined by absorbance measurements, demonstrated the validity of using absorbance and fluorescence comparison measurements for aflatoxin quantitation.

Gas—liquid chromatography of Caramiphen hydrochloride and its analogues

Abstract : Parpanit (Caramiphen hydrochloride, 2'-diethylaminoethyl I-phenylcyclopentanecarboxylate hydrochloride) and many of its analogues have been prepared and studied as possible antidotes for anticholinesterase poisoning. The feasibility of using gas-liquid chromatography both as a criterion of purity for these compounds and as a means for their quantitative analysis has been briefly examined. Hydrochloride salts of this type decompose to the corresponding free bases during GLC and consequently it is possible to determine quantitatively either the free base or hydrochloride by GLC analysis. (Author)

Ribonucleoside diphosphate reductase. Purification of the two subunits, proteins B1 and B2.

Ribonucleoside diphosphate reductase from Escherichia coli B consists of two non‐identical subunits, called proteins B1 and B2. After purification from extracts of a partially derepressed thymine‐requiring mutant, both subunits were obtained in essentially pure form.Criteria of purity included the demonstration of constant specific enzyme activity in peaks from final chromatographic or electrophoretic steps and the behaviour of the proteins during ultracentrifugation and analytical gel electrophoresis.Determinations of molecular weights by sedimentation equilibrium centrifugation gave values between 160000 and 200000 for protein B1 and 78000 for protein B2. Evidence was obtained for some dissociation of protein B1 during centrifugation.Separately, each subunit was completely inactive; together, under the proper conditions, they catalyzed the reduction of the 5′‐diphosphates of cytidine, uridine, adenosine and guanosine to the corresponding deoxyribonucleotides.With the exception of deoxyadenosine 5′‐triphosphate, the regulatory effects of nucleoside triphosphates were identical to those observed earlier with less pure preparations of enzyme. The general inhibitory effect of deoxyadenosine 5′‐triphosphate at concentrations higher than 1 μM was obtained as before. In addition, lower concentrations of this nucleotide had a novel stimulatory effect on the reduction of cytidine diphosphate.

2-Keto-4-hydroxyglutarate Aldolase of Bovine Liver: Purification, criteria of purity, and general properties

Abstract 2-Keto-4-hydroxyglutarate aldolase, which catalyzes the reversible cleavage of 2-keto-4-hydroxyglutarate to yield pyruvate and glyoxylate, has been purified 1300-fold from bovine liver extracts. The purified enzyme appears to be homogeneous when examined by electrophoresis on starch gel or cellulose polyacetate (Sepraphore III) or by chromatography on columns of Sephadex. A molecular weight of 120,000 is obtained with a calibrated column of Sephadex G-200. Two protein bands, both of which are enzymatically active, are observed in polyacrylamide gel electrophoresis. Under certain conditions, two protein peaks are also seen in the ultracentrifuge; the individual components are estimated to have molecular weights of 113,000 and 208,000. The enzyme has a pH optimum of 8.8, is not stimulated by divalent metal ions, and is not inhibited by a number of metal chelating agents. No absolute requirement for added thiol compounds can be shown, but enzymatic activity is strongly inhibited by p-hydroxymercuribenzoate, N-ethylmaleimide, or iodoacetate.

Defective bacteriophage PBSH in Bacillus subtilis. I. Induction, purification, and physical properties of the bacteriophage and its deoxyribonucleic acid.

PBSH, a defective phage of Bacillus subtilis strain 168, is described. Conditions are given for optimal induction of the prophage with mitomycin C. After a latent period of 90 min, cells were lysed and phage-like particles were released with a burst size of approximately 100 to 400 phage per bacterium. Since no known host supports phage replication after infection, burst size was determined by electron microscope count. Purification procedures and criteria for purity are described. The molecular weight of deoxyribonucleic acid (DNA) extracted from PBSH was estimated by length measurement and sedimentation. No circular DNA molecules were found by either technique. PBSH DNA molecules are linear, double-stranded, and of homogeneous molecular weight, about 12 x 10(6) daltons. There is no evidence for single-strand breaks. The majority of PBSH DNA molecules show a sedimentation behavior dependent on ionic strength. It is inferred that most of the DNA molecules are less hydrodynamically rigid than native DNA having a similar average base composition and molecular weight. Possible reasons for the sedimentation behavior are discussed.

The isolation and properties of a fungicidal compound present in seedlings of Vicia faba

The isolation of a fungicidal compound in 0.001% yield from healthy broad bean seedlings by extraction with benzene is described. After column chromatography, purification was achieved by low temperature recrystallisation from hexane, using ultra-violet light absorption, inhibition of spore germination ofAlternaria brassicicola and thin layer chromatography as criteria of purity. The compound has been given the name wyerone. Its in vitro activity against phytopathogens and dermatophytes and its protectant and eradicant activity against certain fungal pathogens of plants are reported.

Application of Craig Countercurrent Extraction Techniques to the Purification of Dyes

The theory of countercurrent extraction as a criterion of purity is described. Application of the theory fully established the purity of two disperse dyes that had been separated by the countercurrent technique. The use of the method to show that a mixture of materials is such is discussed. Its limitation with regard to ionic compounds is also discussed.