Criteria Of Purity Research Articles

Purification and characterization of glucosamine-6-phosphate deaminase from dog kidney cortex

Glucosamine-6-phosphate deaminase (EC 5.3.1.10) from dog kidney cortex was purified to homogeneity, as judged by several criteria of purity. The purification procedure was based on two biospecific affinity chromatography steps, one of them using N-ϵ-amino- n-hexanoyl- d-glucosamine-6-phosphate agarose, an immobilized analog of the allosteric ligand, and the other by binding the enzyme to phosphocellulose followed by substrate elution, which behaved as an active-site affinity chromatography. The enzyme is an hexameric protein of about 180 kDa composed of subunits of 30.4 kDa; its isoelectric point was 5.7. The sedimentation coefficient was 8.3S, and its frictional ratio was 1.28, indicating that dog deaminase is a globular protein. The enzyme displays positive homotropic cooperativity toward d-glucosamine-6-phosphate (Hill coefficient = 2.1, pH 8.8). Cooperativity was completely abolished by saturating concentrations of GlcNAc6P; this allosteric modulator activated the reaction with a typical K-effect. Under hyperbolic kinetics, a K m value of 0.25 ± 0.02 m m for d-glucosamine-6-phosphate was obtained. Assuming six catalytic sites per molecule, k cat is 42 s −1. Substrate-velocity data were fitted to the Monod's allosteric model for the exclusive-binding case for both substrate and activator, with two interacting substrate sites. The K dis for N-acetyl- d-glucosamine-6-phosphate was estimated at 14 μ m.

Malate dehydrogenase from Chlorobium vibrioforme, Chlorobium tepidum, and Heliobacterium gestii: purification, characterization, and investigation of dinucleotide binding by dehydrogenases by use of empirical methods of protein sequence analysis

Malate dehydrogenase (MDH; EC 1.1.1.37) from strain NCIB 8327 of the green sulfur bacterium Chlorobium vibrioforme was purified to homogeneity by triazine dye affinity chromatography followed by gel filtration. Purification of MDH gave an approximately 1,000-fold increase in specific activity and recoveries of typically 15 to 20%. The criteria of purity were single bands on sodium dodecyl sulfate (SDS) and nondenaturing polyacrylamide electrophoresis (PAGE) and the detection of a single N terminus in an Edman degradation analysis. MDH activity was detected in purified preparations by activity staining of gels in the direction of malate oxidation. PAGE and gel filtration (Sephadex G-100) analyses showed the native enzyme to be a dimer composed of identical subunits both at room temperature and at 4 degrees C. The molecular weight of the native enzyme as estimated by gel filtration was 77,000 and by gradient PAGE was 74,000. The subunit molecular weight as estimated by SDS-gradient PAGE was 37,500. N-terminal sequences of MDHs from C. vibrioforme, Chlorobium tepidum, and Heliobacterium gestii are presented. There are obvious key sequence similarities in MDHs from the phototrophic green bacteria. The sequences presented probably possess a stretch of amino acids involved in dinucleotide binding which is similar to that of Chloroflexus aurantiacus MDH and other classes of dehydrogenase enzymes but unique among MDHs.

Effects of film growth mode on magnetic exchange coupling in trilayer structures (abstract)

In situ temperature-dependent spin-polarized cascade electron spectroscopy is used to study exchange coupling processes between a magnetic substrate and a magnetic overlayer separated by a nonferromagnetic spacer layer. Relative Auger intensities of the overlayer/substrate configurations as a function of overlayer coverage together with a model by Ossicini are used to evaluate the degree to which these trilayer structures grow by a continuous layer growth mode. Measurement of the spin polarization and Auger spectroscopy are used during film growth as criteria of chemical film purity. Evidence will be given to show that the oscillatory magnetic coupling behavior as a function of Cr thickness of a NiFe/Cr/NiFe trilayer changes significantly depending on the deposition temperature, which may be associated with microstructural changes. Competing coupling mechanisms are thought to account for this behavior. Data will also be presented to suggest how trace quantities of residual gas sorbates, presumably acting as surfactants, can dramatically alter the magnetization behavior of ultrathin films. Significant increases in magnetization of a 5-Å Fe overlayer film in a Fe/Ta/Fe trilayer structure were observed and are attributed to changes in wetting characteristics at the Fe/Ta interface which cause morphological changes in the film structure. Conversion of weakly interacting magnetic islands to a long-range ferromagnetic continuous film medium exhibiting remanence and a steady-state Brillouin functional M(T ) behavior is thought consistent with these observations. The possible significance of these kinds of observations to some conflicting data in the literature will be mentioned.

Chlorophyll b in solution: fluorescence lifetimes, absorption and emission spectra as criteria of purity

The fluorescence lifetime τ F of chlorophyll b (chl b) was measured in various solvents. The excitation wavelength was varied and the fluorescence was measured at various wavelengths in the spectral range 650–710 nm. For freshly prepared pure chl b solutions, τ F, does not depend on the excitation and emission wavelengths. However, for contaminated chl b solutions, a significant dependence of τ F on the emission wavelength is observed. In this case there are also clear changes in the molar absorption coefficients of the main absorption bands which correlate with the formation of degradation products of chl b.

Pureté, rigidité, et morphismes entiers

Bousfield and Kan have shown that a ring morphism with domain Z {\mathbf {Z}} is rigid; we say that a ring morphism is rigid if it admits a factorization by an epimorphism, followed by a pure morphism. A ring A A is said to be rigid if every morphism with domain A A is a rigid one. Our principal results are: the rigid domains are the Prüferian rings A A , with Dim ⁡ ( A ) ≤ 1 \operatorname {Dim} (A) \leq 1 , and the Noetherian rigid rings are the Z.P.I. rings. The quasi-compact open sets of an affine rigid scheme, having as underlying ring a domain or a Noetherian ring, are affine and schematically dense if they contain the assassin of the ring. Every injective integral ring morphism with rigid domain is a pure morphism. We give two criteria of purity for integral injective morphisms. As a consequence of these results we obtain the following properties: if A A is a normal ring, containing the field of rationals, or is a regular ring, containing a field, every injective integral morphism with domain A A is a pure one. For a reduced ring, we define the category of reduced modules and show that any injective integral morphism is pure with respect to the category of the reduced modules.

Systematic preparative methods for petroporphyrin purification

The isolation of porphyrins for geochemical research requires multistage concentrations from ≤0.1% to >95% purity. In meaningful studies of porphyrin concentration the criteria of enrichment, recovery, purity, and time, need to be considered. The present study was therefore undertaken with the aim of optimizing these parameters, in the systematic concentration of 31 ppm of mixed vanadyl porphyrins (VOP) from 2 kg of powdered New Albany shale. The following preparative scale operations were carried out: extraction, deasphaltation, medium-performance liquid chromatography (MPLC), precipitation, and solid phase bonded sulfonic acid (RSO 3 H) extraction, which are frequently used to precede high-pressure liquid chromatography (HPLC). The overall scheme gave a 9000-fold enrichment; 60% overall recovery, 23% pure VOP, and required approximately 80 h (2 weeks) of time

Purification and partial characterization of a 65-kDa platelet aggregation-associated protein antigen from the surface of Streptococcus sanguis.

Cells of Streptococcus sanguis express a collagen-like immunodeterminant (class II antigen) on their cell walls that induces aggregation of platelets in plasma. These platelet aggregation-associated proteins (PAAPs) are recovered in cell-free preparations obtained from cells of S. sanguis after 5 min of sonic or limited trypsin treatment. Pretreatment of platelet-rich plasma with these soluble preparations selectively inhibits platelet aggregation in response to S. sanguis cells. A PAAP antigen was isolated and purified from minimal tryptic digests of S. sanguis cells using (i) immunoaffinity chromatography or (ii) gel filtration and ion-exchange chromatography. A monospecific rabbit antiserum was prepared against PAAP (from procedure ii) and used to verify identity with PAAP fragments in different preparations. Criteria of purity included single precipitins in immunoelectrophoresis and crossed immunoelectrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western immunoblot, and COOH (Lys)- and NH2 (Pro)-terminal analyses. The 65-kDa (p65) antigen isolated by immunoaffinity chromatography had 50-fold greater specific inhibitory activity in S. sanguis-induced PRP aggregation than the original tryptic digest and about 1.4 times that recovered by sequential column chromatography. Amino acids of the p65 PAAP fragment constituted 89.5% of the total dry weight, with glycine, lysine, and glutamic acid predominant. Lesser amounts of proline were also noted. Monosaccharides, including glucose and galactose, comprised 4.0% of the total. A platelet interactive determinant of p65 was localized to a 23-kDa tryptic fragment after further trypsin treatment. Amino acids of this 23-kDa fragment constituted 99.8% of the total dry weight. In their native state on the cell wall of platelet-interactive strains of S. sanguis, platelet aggregation-associated proteins are probably assembled on fibrils as polyvalent agonists.

The meaning of pain to young hispanic, African-American, and europeanamerican children

This chapter presents the procedure for assay and purification of thiaminase I. Thiaminase I catalyze the decomposition of thiamine by a base-exchange reaction involving a nucleophilic displacement on the methylene group of the pyrimidine moiety. The enzyme activity is determined by a modification of the method of Douthit and Airth, which employs aniline as the base in the exchange reaction. The formation of the product, N-(4-amino-2-methylpyrimidin-5-ylmethyl)-aniline, is measured spectrophotometrically by an increase in optical density at 248mμ. In addition to Bacillus thiaminolyticus, another bacterium, Clostridium thiaminolyticum, produces thiaminase I. This enzyme has also been found in the viscera of freshwater fish and shellfish, as well as in bracken ferns. The spectrum of bacterial thiaminase I is that of a simple protein with an absorption maximum at 277mμ and a minimum at 252mμ. The enzyme purified from Bacillus thiaminolyticus is homogeneous by three criteria of purity—namely, ultracentrifugation, polyacrylamide gel electrophoresis, and immunodiffusion in agar gel.

Student Teachers’ Concepts of Purity and of States of Matter∗

Abstract The concepts of purity and of states of matter are fundamental ideas in science, especially chemistry. Well defined states of matter are exhibited by chemically pure materials and so for accurate classification, learners must use an accepted framework for purity. This work attempts to provide a description of student teachers' frameworks of purity and the related concepts of the states of matter. Data were collected by asking subjects to sort everyday materials firstly by state of matter, and secondly by perceived purity. Subjects also gave their criteria for the sorting. The tasks were followed by group discussion which was recorded on videotape. The results show that most of the subjects used ‘natural’ as their criterion of purity. Difficulties in classifying states of matter relate to the macroscopic properties of the materials. Few subjects used a particulate theory in either of their classifications. Implications for teaching are drawn from the results. ∗This is a revised and extended versio...

Investigation of the Quaternary Structure and of the Active Site of Methane Monooxygenase from Methane-Oxidizing Bacteria Methylococcus capsulatus (strain M) by Physico-Chemical Methods

Methane monooxygenase (MMO, E.C. 1.14.13.25) was purified from membranes of the methane-oxidizing bacterium Methylococcus capsulatus (strain M) and separated into two components: monooxygenase and NADH-oxidoreductase. The individual components did not oxidize methane. Oxidation of the substrate was observed in the presence of two components. MMO specific activity was 3600 nmol CH4/min/mg of the protein. The degree of purification was 170. The criterion of purity was sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which showed that the monooxygenase and NADH-reductase consist of subunits with a molecular weight of 35 ± 3 kDa and 45 ± 3 kDa, respectively. From gel chromatography on Sephadex G-200, a molecular weight of 180 kDa was found for the native NADH-reductase, suggesting a tetrameric structure for this enzyme. The quaternary structure of the NADH-reductase and monooxygenase were investigated by electron microscopy. NADH-reductase is constructed from four identical subunits with molecular w...

Use of Macrosorb kieselguhr composite and CM-Sepharose Fast Flow for the large-scale purification of l-asparaginase from Erwinia chrysanthemi

An incompressible kieselguhr-agarose composite, derivatized with carboxymethyl groups, Macrosorb KAX-CM, is compared with a conventional agarose gel, CM-Sepharose Fast Flow, for the large-scale purification of l- asparaginase from Erwinia chrysanthemi. The Macrosorb KAX-CM has better pressure/flow characteristics than CM-Sepharose Fast Flow, but both matrices are identical on criteria of product purity. CM-Sepharose Fast Flow has a greater capacity than Macrosorb KAX-CM and enzyme is eluted in a smaller volume.

Honey Protein as Internal Standard for Stable Carbon Isotope Ratio Detection of Adulteration of Honey

Using the difference in stable carbon isotope ratio between a honey and its protein fraction permits objective evaluation of possible adulteration of honey with small amounts (7-20%) as well as larger amounts of corn or cane sugar. The present uncertainty in interpretation of results from pure honey with delta 13C values outside the generally accepted limits for pure honey of -27.5% to -23.5% is eliminated; likewise TLC testing to resolve questionable samples with delta 13C values between -23.5 and -21.5% is not needed. Fifty certified samples of pure honey were used to establish criteria for purity, and 38 other samples with delta 13C values in the "questionable" or "adulterated" range for the AOAC official method were tested. A difference of 1.0% or more between honey and protein fractions is proposed to indicate adulteration.

Synthesis of N-isopropyl-p-123I-amphetamine and biodistribution in rats

The described labelling and purification preparation of N-isopropyl-p-131I-amphetamine /131I-IMP/ represents a fast and efficient method to obtain a compound that fulfils all criteria of purity for its in-vivo application. After isotope exchange and quality control131I-IMP could be obtained with radiochemical yields in the range between 68% and 78% with a radiochemical purity of 98–99%. As demonstrated in animal experiments the cerebral affinity of IMP offers a possibility for the diagnosis of brain diseases in clinical studies when the product is labelled with123I.

Water soluble DACHPt(II) complexes: Problems of purification; Stability of complexes with nitrogen-containing ligands

Two previously reported water soluble 1,2-diaminocyclohexaneplatinum(II) antitumor complexes with nitrogen-containing dicarboxylato ligands (N-substituted iminodiacetato(1,2-diaminocyclohexane)platinum(II) and aminomalonato(1,2-diaminocyclohexane)platinum(II)) were discovered to have significant residual impurities in the chemical formulations. Upon further purification each complex was found to be significantly less active in biological systems than previously reported. Each complex is stable in aqueous solution. This experience suggests that commonly accepted criteria for chemical identification and purity are inadequate for this type of complex. We hypothesize that tridentate bonding between the nitrogen-containing dicarboxylato group and platinum renders these complexes chemically stable and biologically inert.

A study of the purification of methyl methacrylate suggests that the “thermal” polymerisation of this monomer is initiated by adventitious peroxides

Various procedures for purifying methyl methacrylate (MMA) have been examined, and the rate of thermal (“uncatalysed”) polymerisation at 40°C has been used as a criterion of monomer purity. Even when the most rigorous purification procedure was used, the rate at 40° is comparable with that of the thermal polymerisation of styrene. The decay in rate with time, as measured from the conversion curves, is not consisted with the decay in monomer concentration with conversion, and is interpreted in terms of decay of the concentration of an adventitious initiator. On this basis, the rate constant for initiator decomposition could be calculated. In separate experiments, peroxide/hydroperoxide was shown to be present in the purified monomer and the concentration was estimated by iodimetry. Rates of polymerisation could then be calculated from the initiator rate constant, its concentration, and literature values of the rate constants for MMA propagation and termination. These rates of polymerisation proved to be reasonably consistent with the observed rates, and it was concluded that the “thermal” polymerisation of MMA is peroxide/hydroperoxide initiated. Detailed examination of the results indicated the possibility of two distinct initiating species; there is evidence that they are peroxides with widely different half-lives. A critical examination of these results and other published results on the thermal polymerisation of MMA shows that there is no acceptable evidence for a very low “thermal” rate. Rates smaller than those obtained in the present work have not been obtained unless inhibitors are added to the monomer.

1] Preparation and purification of staphylococcal alpha toxin

Publisher Summary This chapter describes the preparation and purification of staphylococcal alpha toxin. Staphylococcal alpha toxin is an extracellular protein produced by most strains of pathogenic Staphylococcus aureus . It is selectively hemolytic with a marked preference for rabbit red blood cells. It induces dermonecrosis, spastic muscle paralysis, and it is lethal for laboratory animals. The alpha toxin genome is cloned and sequenced and the complete amino acid sequence deduced therefrom. Earlier purification procedures suffered from incomplete separations of alpha toxin from delta toxin. The method of purification of alpha toxin described in the chapter is based on selective adsorption and elution from glass-pore beads. The glass-pore bead method has the advantage of being both simple and rapid and giving high yields of alpha toxin that are free of other toxin products and identical in properties to the major form of the toxin obtained by the preparative gel electrophoresis procedure. Two criteria of purity of the alpha toxin preparation are routinely assessed: (1) the specific hemolytic activity and (2) the physical homogeneity as revealed by acrylamide gel electrophoresis.

Purification and some properties of component B of the 4-chlorophenylacetate 3,4-dioxygenase from Pseudomonas species strain CBS 3.

4-Chlorophenylacetate 3,4-dioxygenase system from Pseudomonas sp. CBS 3 consists of two protein components, a red-brown iron-sulfur protein (component A) which functions as dioxygenase and an orange-colored reductase (component B). Component B was purified by a five-step procedure. Criterion of purity was sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which also showed that the protein consists of one polypeptide species with a molecular weight of 35,000. With gel chromatography on Sephadex G-100 also, a molecular weight of 35,000 was found for the native enzyme, suggesting a monomeric structure for the reductase enzyme. The isoelectric point was determined as pH 4.8. The visible absorption spectrum of the homogeneous protein exhibits maxima at 336, 394, and 458 nm. One mol of FMN, 2.1 mol of iron, and 1.7 mol of acid-labile sulfide were found in one mol of component B. The EPR-spectrum of the reduced form of the enzyme (with NADH) showed two types of signals. The signal at g values of 2.03, 1.94, and 1.90 was assigned to an iron-sulfur cluster of the [2Fe-2S]-type. The properties of the other signal type at g = 2.004 are characteristic of flavosemiquinone radical which does not show coupling to an other paramagnetic center. The apparent Km values for 2,6-dichlorophenolindophenol, cytochrome c, and NADH were calculated from Lineweaver-Burk plots as 6.3, 2.3, and 32 microM, respectively. Inhibitors of the reductase are metal-chelating reagents and sulfhydryl-inhibiting compounds. Component B reduces several redox compounds: 2,6-dichlorophenolindophenol, potassium hexacyanoferrat III, cytochrome c, methylene blue, and nitro blue tetrazolium.

Isolation, characterization, and localization of antigen gamma, a serine proteinase of the "kallikrein-family" in the rat submandibular gland.

A trypsin-like serine proteinase, antigen gamma, immunologically partially identical to glandular kallikrein when run against anti-rat glandular kallikrein antiserum in immunoelectrophoresis, was purified from the rat submandibular gland. The enzyme was purified by a two-step chromatography procedure, ionexchange chromatography followed by gel filtration. The criteria for purity were one band in SDS-polyacrylamide gel electrophoresis and in immunoelectrophoresis, respectively. Antigen gamma had a molecular mass of 25,000 Da and consisted of two polypeptide chains with molecular masses of 14,000 and 11,000 Da. The preparation contained several isoenzymes with pI ranging from 4.1 to 4.5. The enzyme showed high specific enzyme activity against the substrate D-valyl-L-leucyl-L-arginine-4-nitroanilide (S-2266), some trypsin-like and kininogenase activity, but no angiotensin converting enzyme, kininase, or tonin activity. Amidolytic activity was increased and stabilized by the presence of detergent in the assay buffer. The pH-optimum of antigen gamma amidolytic activity was about 10. Antigen gamma was inhibited by SBTI and PMSF, whereas aprotinin had to be added in a more than 100 times higher concentration than for glandular kallikrein. The binding pattern of antigen gamma to plasma proteins was different from that of tonin and glandular kallikrein. Antiserum against antigen gamma was raised in rabbits and characterized against rat submandibular gland homogenate. Immunohistochemistry showed antigen gamma in the secretory granules of the submandibular gland granular tubular cells but only adhering to the luminal cell wall in the striated and main excretory ducts. Antigen gamma was not detected in the sublingual or parotid gland or in the kidney. Antigen gamma was demonstrated by immunoelectrophoresis in rat submandibular gland saliva. The concentration was higher in sympathetically than in parasympathetically induced secretion.

Assay, purification and characterizaton of a recombinant malaria circumsporozoite fusion protein by high-performance liquid chromatography

The immunodominant repeat region of the malaria circumsporozoite protein from Plasmodium falciparum was purified from a recombinant Escherichia coli to study as a potential subunit vaccine. The recombinant protein, R32Leu-Arg, is composed of 32 tetrapeptide repeat sequences from the circumsporozoite protein (R32) linked to the dipeptide, Leu-Arg. R32Leu-Arg was purified by a series of precipitation steps including temperature, ammonium sulfate, and acid pH treatments; followed by reversed-phase high-performance liquid chromatography (RP-HPLC). An automated RP-HPLC assay was developed to measure the R32Leu-Arg concentration during both fermentation and purification. This assay was used in a variety of applications including measurement of production levels of the antigen during fermentation, evaluation of the protein purification process, quantitation of protein recovery, and as one criterion of protein purity. With minimal changes, the assay conditions were easily adapted to the semi-preparative level to produce 200 mg of purified product. The purified product was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; amino acid composition; and analytical size-exclusion and RP-HPLC.

10] Isolation and characterization of insoluble and soluble elastins

This chapter discusses isolation and characterization of insoluble and soluble elastins. Elastin demonstrates remarkable functionality, correlating with macroscopic and microscopic structure and with molecular composition. Its ubiquitousness necessitates our understanding of these relationships, because tissue-specific extraction methods are necessary for its purification. Lack of agreement on criteria of purity stems from its complex supramolecular organization and intricate relationships with other macromolecules primarily of connective tissues. Purity is assessed by absence of collagen and collagen-like features (methionine, hydroxylysine, high hydroxyproline), absence of carbohydrate, presence of high content of non polar amino acids (glycine, alanine, proline, valine), and low content of polar amino acids (aspartate, glutamate, lysine, histidine, arginine).this chapter also provides assessment of results of various major methods. Immunologic methods such as radio immunoprecipitation and rocket immunoelectrophoresis may prove to be at least as sensitive in determining the amount of material present. However, because of the insoluble nature of mature elastin, many of these methods are not useful unless elastin is present in a degraded soluble form. Cross-link analysis may also be helpful for quantitation if hydrolyzed samples can be used.