Criteria Of Purity Research Articles

Purification and partial characterization of an actin-like protein from cricket early egg plasmodium.

The purification of an actin-like protein from cricket egg yolk plasmodia by different selective extraction procedures, ammonium sulphate precipitation, ion exchange and immunoabsorption chromatography is described. Criteria of purity from analytical ultracentrifugation, SDS-disc electrophoresis, and immunoelectrophoresis are presented. Immunodiffusion analysis was used to control the success of the purification procedures.The molecular weight of the monomeric form is 60000±10%. Polymerization to pearl-chain aggregate structures occurs under different conditions in 0.1 M KCl in the presence of ATP. Vinblastine precipitation leads to similar structures. Possibly related structures and the possible rÔle of this protein in organizing movements in the plasmodial system are discussed.

Cellular compartmentation of brain metabolism and its functional significance

This paper reviews work from our laboratory on the metabolism and interrelations of isolated neuronal and neuropil fractions. The cell preparations are evaluated according to criteria of yield, purity, and integrity. Differences in the levels of activity of six groups of enzymes, of glucose metabolism, amino-acid metabolism, transmitter metabolism, acid hydrolysis, alkaline hydrolysis, and carbonic anhydrase have been followed, and neuronal and glial marker enzymes are proposed. Lysosomes and their enzymes are concentrated in neuronal perikarya. Metabolically, although no major differences in glucose oxidation have been found, there is considerable evidence of compartmentation of amino acids and their metabolism. At short times after injection of 3H-lysine as precursor in vivo, neuronal incorporation is high as compared with neuropil; at longer times the ratio is reversed, and we interpret this as evidence for the presence of a rapidly labeling protein fraction present in the neuromal perikarya but subsequently transported out. Neuronal protein incorporation is suppressed in the visual but not the motor cortex of dark-reared rats and is switched on following exposure to light; there is evidence that the suppressed fraction of neuronal protein synthesis includes the rapidly labeling component. A model for neuronal-glial metabolic interaction and its state-dependence in response to changes in the organism's environment and behavior is sketched out.

Improved isolation procedures for the purple membrane of Halobacterium halobium.

Techniques for purifying teh purple membrane of Halobacterium halobium are given. This purple membrane contains a chromoprotein with a retinal prosthetic group similar to rhodopsin, the chromprotein found in the visual systems of higher invertebrates and vertebrates. The described purple membrane isolation procedures yield a highly purified preparation as determined by transmitting electron microscopy and gel electrophoresis. Critical analysis of the absorption spectra of the purple membrane was also employed to establish criteria of purity for the preparation. The visible absorption spectra of the purified purple membrane preparation in buffer was found to have a maximum at 559 nm which shifted to 567 nm on light exposure. No indication of any spectral perturbation arising from bacterioruberin-containing membrane, the major contaminant in purple membrane preparations, was found. Furthermore, the ratio of protein aromatic amino acid absorbance at 280 nm to chromophore absorbance at 567 nm was found to be 1.5 in light-exposed preparations compared to the previously reported ratio of 2.3.-3 The decrease in the value of this ratio is also indicative of an increase in the purity of the purple membrane preparation.

Formation and excretion of pyrrole-2-carboxylate in man.

A detailed investigation of the purification of pyrrole-2-carboxylate (PCA) from human and rat urine indicates that previously reported mean values overestimate the correct quantity of free PCA by a factor of approximately three for rat urine and approximately five for human urine. Although several criteria of purity were satisfied by a previous method, pyrrole-reactive impurities were still present in the final fractions. These impurities are separated from PCA by chromatography through an amino acid analyzer ion-exchange resin. With the corrected method, normal human values for endogenous urinary PCA in 16 individuals averaged 0.51 mumol/day, with a range of 0.20-1.3 mumol and a SD of 0.31 mumol. The probable source of human PCA is free hydroxy-L-proline, as inferred from the high value for PCA in the urine of a subject with hereditary hydroxyprolinemia, and from the threeto eightfold elevation in PCA excretion by two normal subjects after a large oral load of hydroxyl-L-proline. Subcutaneous administration of [2-(14)C]PCA to a single human subject indicated almost complete conversion of the exogenous compound to derivatives, which are largely excreted in the urine. Data are discussed suggesting that much or all of the PCA in human urine may be formed in urine from a labile precursor, presumably Delta(1)-pyrroline-4-hydroxy-2-carboxylate.

The nuclear reactor and its products

The history of nuclear fission has been a rapid transition from theory to practical application as a weapon and, finally, to beneficial applications under controlled conditions in a fission reactor as a source of power and radioisotopes. The effective use of the enormous capacity of reactors to produce radioisotopes requires an understanding of the reaction cross sections and the influence of the neutron energy spectrum. Practical considerations include the selection of target compounds and isotopic enrichment to meet criteria for safety and product purity. Despite many natural limitations on what they can produce, reactors supply a large number of radionuclides important in nuclear medicine.

A Purity Criterion for Pairs of Linear Transformations

In connection with the study of perturbation methods for differential eigenvalue problems, Aronszajn put forth a theory of systems (X, Y; A, B) consisting of a pair of linear transformations A, B:X → Y (see [1]; cf. also [2]). Here X and Y are complex vector spaces, possibly of infinite dimension. The algebraic aspects of this theory, where no restrictions of topological nature are imposed, where developed in [3] and [5]. We hasten to point out that the category of C2-systems (definition in § 1) in which this algebraic investigation takes place is equivalent to the category of all right modules over the ring of matrices of the form

Enhancement of Optical Rotation of Levodopa by Cyclization

The optical rotation of levodopa is an important purity criterion, since it provides the only means to control the presence of the allegedly toxic and therapeutically inactive D-isomer. The specific rotation, [α]D25, of levodopa is only about −12°, which is insufficient to control optical purity. Barooshian et al. (1) proposed a TLC limit test for d-dopa in levodopa, involving conversion of the enantiomers to diastereomeric dipeptides. Jouin and Saias (2) and Coppi et al. (3) described methods based on stereo- specific enzymatic destruction of levodopa. The BP 1973 (4) provides requirements of [α]36520 -119 to −125° for levodopa drug substance and [α]D20 -38.5 to −41.5° for its dosage forms, the rotation being increased by addition of aluminum sulfate and sodium acetate.

Capsular polysaccharide of Clostridium perfringens Hobbs 10.

A capsular polysaccharide was isolated from a strain of Clostridium perfringens Hobbs 10 type A by cold-water extraction of whole, heavily encapsulated cells. The water-soluble polymer was isolated by alcohol precipitation and purified by treatment with chloroform-butanol, cetytrimethylammonium bromide, and column gel permeation chromatography by using Bio-Gel A-5m agarose. The formation of a single precipitin line, when the isolated polysaccharide was reacted with its homologous antisera by double diffusion in gel, was considered a criterion of immunochemical purity. The purified polymer appeared as a single peak when eluted from diethylaminoethyl-Sephadex with a linear gradient of NaCl. The polysaccharide was composed of glucose, galactose, galactosamine, and iduronic acid in a molar ratio of 4.1:5.1.7:1, respectively. These constituents accounted for 83% of the dry weight. The polysaccharide appeared to have a molecular weight of 40,000 and exhibited aggregation up to 120,000. A trace of peptide material could not be removed during purification.

Isolation and Partial Characterization of Four Host-specific Toxins of Helminthosporium maydis (Race T)

Helminthosporium maydis, race T, produces four host-specific toxins in culture. These have been designated toxins I, II, III, and IV. A method for isolation and purification of the four toxins is presented, and the criteria of purity of preparations of toxins I, II, and III are given. Toxins I and II are chemically similar and yield the same molecular ion when subjected to mass spectrometry, while toxin III appears to be a glycoside of a compound related to toxins I and II. Toxins I, II, and III can be biologically derived from (14)C-mevalonic acid or (14)C-acetate, permitting preparation of (14)C-labeled toxins. Some chemical, spectral, and chromatographic properties of toxins I, II, and III are presented, and these data are discussed relative to the possible structure of the three compounds. In addition, four host-specific toxins have been isolated from corn infected with H. maydis (race T). These toxins are recovered in the same fractions as toxins I, II, III, and IV using the isolation procedure described here. Three of the toxins isolated from infected corn cannot be distinguished from toxins I, II, and III on the basis of infrared spectra or chromatographic mobility.

The structural polypeptides of RNA slow viruses

The structural polypeptides of the RNA slow viruses Visna, Maedi, Progressive Pneumonia Virus, and Zwoegerziekte have been determined by electrophoresis in polyacrylamide gels in a discontinuous SDS system. Identical patterns were obtained of 15 polypeptides of molecular weight ranging from 14,000 to 140,000 daltons. Most of the mass of protein is in three small molecular weight polypeptides; the major polypeptide species has a molecular weight of 25,000. A distinctive glycopeptide of molecular weight 140,000 is present in each virus; three additional glycopeptides were found, one of which is barely detected in radiolabeling experiments. There is one phosphorylated polypeptide. In a comparative study of RNA slow viruses and tumor viruses, 9 of 15 polypeptides of visna virus comigrated with 13 polypeptides of Rous sarcoma virus including the major 25,000 MW polypeptide of each virion. In addition, the isoelectric point of visna was 3.8 in conformity to the isoelectric point reported for Rous sarcoma virus. Distinctive small molecular weight polypeptides and a totally different pattern of glycosylation constitute the main differences of the polypeptides of these two groups of viruses. Extensive studies of the purification procedures for C-type viruses were carried out and criteria for virus purity delineated. The expectations for the relationship of the structural polypeptides to antigenic determinants are discussed.

Residual Fluorescence as an Index of Purity of Reduced Nicotinamide Adenine Dinucleotide

Abstract Determination of fluorescence remaining after reduced nicotinamide adenine dinucleotide (NADH) has reacted with excess acetaldehyde in the presence of alcohol dehydrogenase (EC 1.1.1.1) is useful as a criterion of NADH purity when used in conjunction with other methods for determining purity such as the rate of reaction, the ratio of ultraviolet absorbances at 260 nm and 340 nm, the color, and the chromatographic homogeneity of the preparation. Measurement of residual fluorescence monitors the enzymatically inactive material which absorbs at 340 nm. The specific optical rotations of NADH at several wavelengths are also reported.

Criteria for Mycotoxin Standards

Abstract It is recommended that all samples of mycotoxins to be used as analytical standards be in compliance with certain criteria of purity and that methods be made available for analysts to check the concentration and purity of their standards. General criteria for meeting these goals are described. A procedure for the determination of the concentration and purity of aflatoxin M1 standards, which was used in an IUPAC collaborative study to determine the stability of M1, is recommended for acceptance by the AOAC.

Isolation of brain tubulin by affinity chromatography

An affinity chromatographic procedure for the isolation of tubulin from brain is described. The yield is good and the method rapid and gentle. Criteria of purity are colchicine binding, SDS acrylamide gel electrophoresis, and velocity sedimentation.

The effect of impurities on dynamic surface tension—basis for a valid surface purity criterion

Small impurities in a surfactant can cause major contamination of surfaces of its solutions if they are strongly adsorbed. Foaming (and foam removal) is widely used to purify the surface, but a criterion is needed to evaluate the purity obtained. An idealized calculation shows that dynamic surface tension effects can be used for this purpose. Following rapid expansion of the surface, a pure one returns to the equilibrium surface tension more rapidly than a contaminated one. The delay can be related to the surface enrichment of the impurity, and the extent of the slow equilibrium to the amount of surface contamination. Experiments have indicated that this criterion is quite effective in detecting unsuspected impurities in the surface.

Preparation of 14C-Labeled Aflatoxin B1

Abstract A successful method to obtain 14C-labeled aflatoxin B1 from Aspergillus parasiticus ATCC 15517 was developed. The nitrogen-free resting culture contained mycelia from a 72 hr primary culture, 0.02M glucose, 0.005M (1 mCi/mmole) acetate-1-14C, salts, and trace metals. It was incubated 12 hr at 30°C. Aflatoxin B1-14C was isolated from chloroform extracts of the resting culture by column chromatography on silica gel H eluted under pressure with a continuous gradient of chloroform to chloroform-methanol (98+2). The ultraviolet spectrum of the 14C-labeled aflatoxin was examined. The ratios of the absorbances at 220/265 and 362/265 nm compared to established values and a reference standard were used as criteria of chemical purity. Label integrity was verified by chromatography, hydrogenation to the tetrahydrodeoxoaflatoxin B1, and conversion to the hemiacetal and the epimeric acetates. After purification, 1.37 mg 14C-labeled aflatoxin B1 of specific activity 744 μCi/mmole was obtained from 2 mCi acetate-1-14C of specific activity 1 mCi/mmole.

Surface areas of naturally occurring lipid classes and the quantitative microdetermination of lipids

The surface area (A) of a lipid was directly proportional to the amount of lipid in a surface film (A = k micro moles) measured at constant surface pressure, temperature, and subphase composition. A surface area of 2300 cm(2)/ micro mole was obtained for cholesterol isolated from human adrenal and aorta and for cholesterol from hydrolysates of cholesteryl esters isolated from the same tissues. Unsaturated methyl esters that contained from one to four cis double bonds had the same surface area, 39.4 A(2)/molecule. As a consequence, naturally occurring triglyceride mixtures which had similar saturated-unsaturated fatty acid ratios had the same surface area, 6090 cm(2)/ micro mole. Naturally occurring phospholipid mixtures had the same surface area, 4590 cm(2)/ micro mole, and it appeared that the composition of these mixtures was regulated to control the physical properties of the mixtures. Surface area was much more sensitive than colorimetric procedures for the estimation of cholesterol and triglycerides. The surface area/molecule was a criterion of purity and an expanded surface area/molecule was an indication of autoxidation. Thus, surface area measurements were valuable for both the microdetermination and the characterization of lipid classes.

Phoratoxin, a Toxic Protein from the Mistletoe Phoradendron tomentosum subsp. macrophyllum (Loranthaceae)

In a simplified procedure for isolation of phoratoxin, the dried plant material (stems and leaves) was extracted with 2% aqueous acetic acid. The extract was treated with polyamide to remove colored impurities and subjected to gel filtration on Sephadex G‐25. The high‐molecular‐weight fraction was chromatographed on SE‐Sephadex, using a gradient elution technique, whereby pure phoratoxin was obtained.The material was homogeneous by standard criteria of protein purity. The molecular weight, determined in the ultracentrifuge by the sedimentation equilibrium method, is 5000. Phoratoxin has the following amino acid composition (residues/mole): alanine 2, arginine 3, aspartic acid 4, 1/2‐cystine 6, glycine 6, histidine 1, Isoleucine 3, leucine 1, lysine 4, phenylalanine 1, proline 2, serine 5, threonine 5, tryptophan 1, tyrosine 1, valine 1. The molecular weight, calculated from the amino acid composition, is 4884. The material contains no reducing sugars, amino sugars or sialic acids. The LD50, determined by intraperitoneal injection in mice, is 0.57 ± 0.05 mg/kg bodyweight.

Some biological properties of purified staphylococcal haemolysins

Staphylococcal alpha-, beta-, gamma and delta-haemolysin were extensively purified and several criteria of homogeniety for each were investigated before biological studies were started. The haemolytic spectrum, the lethal effect and the cytotoxicity of each lysin were studied, and several other properties were also compared. The LD 50-dose of beta-lysin was 10–100 μg for mice and rabbits, which means that this lysin, on the basis of weight, is about as potent a lethal toxin as alpha-lysin, while the LD 50-dose for gamma-lysin was about 1 mg and for delta-lysin about 125 mg. For the first time beta- and gamma-lysin were shown to be cytotoxic for mammalian cells (HeLa-cells and human fibroblasts) and gamma-lysin was shown to be lethal for mice and rabbits. Human thrombocytes and their α-granules were rapidly lysed by beta-lysin, while human leucocytes and bacterial protoplasts were resistant. Beta-lysin possesses sphingomyelinase activity. The susceptibility to lysis of different cells by this enzyme also seems to be compatible with the different phospholipid composition of these cell membranes. The importance to use purified toxins fulfilling several criteria of purity for biological studies will be discussed.

STUDIES OF STAPHYLOCOCCAL TOXINS IN RELATION TO TOXIC EPIDERMAL NECROLYSIS (THE SCALDED SKIN SYNDROME)

Summary.— A diffusible product which causes extensive epidermal splitting in new-born mice, has been obtained from phage group II staphylococci in a highly purified form. It is free of staphylococcal α-toxin, δ-toxin and hyaluronidase and has a molecular weight of approximately 33,000. Criteria of purity are presented and the possible role of this factor in experimental T.E.N. is discussed.

3] The preparation of guanidine hydrochloride

This chapter describes the criteria of purity of guanidine hydrochloride (GuHCl) samples and the methods of producing pure samples, based on a laboratory manual. GuHCl has proved to be the most powerful among commonly used protein denaturants and is being used for a variety of purposes. There are two methods for obtaining a GuHCl sample of good quality: the recrystallization of commercial preparation and the conversion of guanidinium carbonate into chloride. Before deciding the method that should be adopted, it must be noted that the purity requirement depends on the type of experiment. In many cases, a sample of the highest quality adhering to solubility, melting point, and absorption spectrum requirements is not needed, and such a sample may be prohibitively expensive, for example, for gel chromatography for the determination of the molecular weight of proteins.