On the subunit structure of crotoxin: hydrodynamic and shape properties of crotoxin, phospholipase A and crotapotin.

This paper reports physical-chemical properties of the subunit structure of crotoxin, phospholipase A and crotapotin. The native crotoxin has a sedimentation coefficient of 3S and a radius of gyration of R_g = 16.5 Å and a molecular weight of 30,900. Dissociation of the 3S particle results in two proteins of unequal size with sedimentation coefficients of 1.5 S (crotapotin) and 1S (phospholipase A). These dissociated species and the reconstituted complex were investigated by means of hydrodynamic methods including small angle X-ray scattering. The actual frictional ratios were obtained indicating that crotoxin is a sphere with a Stokes' radius of R_o = 22.5 Å and an axial ratio of 1:3, whereas phospholipase A, depending on the degree of association, has a radius of gyration of R_g = 32.4 Å and a high axial ratio of 1:14 for the monomer. Crotapotin has a radius of gyration of R_g = 12.4 Å, indicating an oblate ellipsoid of revolution of an axial ratio of 1:4. Evidently, the crotoxin complex consists of one highly asymmetric molecule (phospholipase A) and an oblate ellipsoid (crotapotin), which reconstitutes to a spherical 3S-particle (crotoxin).     

On the quaternary structure of native rabbit muscle phosphofructokinase.

Small angle X-ray scattering measurements on solutions of native rabbit muscle phosphofructokinase (EC 2.7.1.11; ATP; D-fructose-6-phosphate 1 phosphotransferase) show that the dimer has a radius of gyration of 32.5 Å and a molecular weight of 160,000, and that the biologically active tetramer has a radius of gyration of 51.5 Å and a molecular weight of 320.000. A possible model was calculated from scattering curves of the dimer and tetramer suggesting two hollow cylinders with cell dimensions for the dimer of a height of 78.0 Å and a long half axis of 38.0 Å, and for the tetramer of a height of 155.0 Å and an outer radius of 35.0 Å. The tetramer is formed along the 78.0 Å axis of the dimer by means of an end-to-end aggregation. The overall particle dimensions of the protomer of molecular weight 80,000 is calculated to be 35.0 × 30.0 × 55.0 Å, assuming an elliptical molecule. The distance between the centers of the two dimeric units within the tetramer is 104.5 ± 1.5 Å.     

Speciation of chromium in chromium yeast

High-performance liquid chromatography was used to separate Cr(III) and Cr(VI) in samples with detection by inductively coupled plasma mass spectrometry(ICP-MS). The separation was achieved on a weak anion exchange column. The mobile phase was pH 7.0 ammonium nitrate solution. The redox reaction between Cr(III) and Cr(VI) was avoided during separation and determination. This separation method could be used to separate the samples with large concentration differences between Cr(III) and Cr(VI). The alkaline digestion was used to extract chromium in solid sample, which had no effect on the retention time and the peak area of the Cr(VI). However, the conversion of Cr(VI) from Cr(III) was observed during alkaline digestion, which displayed positive relation with the ratio of Cr(III) and Cr(VI) in samples. Both Cr(III) and Cr(VI) contents of chromium yeasts cultured in media with different chromium additions were determined. The spike recoveries of Cr(VI) for chromium yeasts were in the range of 95–108 %.     

Oxidative phosphorylation and proton translocation in membrane vesicles prepared from Escherichia coli

This paper reports physical-chemical properties of proteins L7 and L12 from E. coli 50 S subunits. Evidence is presented that these two proteins behave in their native state as a dimer of molecular weight 24000. From sedimentation velocity and intrinsic viscosity data the actual frictional ratio of the dimer has been obtained revealing an asymmetric particle which can be described as a rod with cell dimensions of L = 130 Å and a diameter of D = 17.0 Å. From small X-ray scattering the radius of gyration (Rg = 37.0 Å), the thickness factor, and the degree of hydration were determined. This indicates that the extended shape of the dimer is due to the asymmetry of the molecule and not to the hydration.     

Metabolic reduction of chromium,as related to its carcinogenic properties

At variance with Cr(III), Cr(VI) compounds easily cross cell membranes and exert genotoxic effects. No metabolic oxidation of Cr(III) could be detected, whereas Cr(VI) reduction was observed in the presence of body fluids and subcellular fractions of various tissues from several animal species. The differential efficiency of this process may account for the selection of target tissues in Cr(VI) carcinogenesis. For instance, reduction by saliva and gastric juice may explain a lack of carcinogenicity by the oral route; reduction inside erythrocytes may explain a lack of carcinogenicity at a distance from administration sites; reduction by the epithelial-lining fluid of terminal airways and by alveolar macrophages may be consistent with the occurrence of thresholds in lung carcinogenesis. Liver preparations displayed the top efficiency in reducing Cr(VI), whereas skeletal muscle, i.e., a typical target in experimental Cr(VI) carcinogenesis, had no detectable activity. Bronchial tree and peripheral lung parenchyma preparations from almost 100 individuals reduced Cr(VI) to a variable extent. The efficiency of lung parenchyma and of isolated alveolar macrophages was enhanced in cigarette smokers. In rats, Cr(VI) reduction by lung preparations was significantly stimulated by the repeated i.t. instillation of Cr(VI) itself. Among the electron donors (chiefly GSH) and enzymatic mechanisms responsible for the intracellular Cr(VI) reduction, such as cytochrome P-450 reductase, glutathione redactase, and aldehyde oxidase, an important role can be ascribed to cytosolic DT diaphorase activity, usually catalyzing a 2-electron reduction.     

Small-angle X-ray scattering of D-Ribulose-1,5-diphosphate carboxylase from Dasycladus clavaeformis roth (Ag.) in solution

D-Ribulose-1,5-diphosphate carboxylase from $\text{Dasycladus}$ was purified, and the gross dimensions were obtained by means of small-angle X-ray scattering measurements in solution. Dissolved single crystals of this enzyme (called “fraction I protein”) gave the same hydrodynamic parameters as the purified form. The molecular weight was found to be 535,000, and a radius of gyration of R_g = 45.5 Å was determined. The experimental scattering curves revealed a geometrical particle of D-Ribulose-1,5-diphosphate carboxylase with gross dimensions of that of a hollow sphere with outer radius of 56 Å and inner radius of 12 Å. Determinations of the diffusion coefficients lead to the conclusion that the enzyme has a spherical shape of almost uniform density.     

Small-angle x-ray scattering study of lysine transfer RNA ligase from yeast

The scattered X-ray intensities from dilute solutions of lysine transfer RNA ligase, in 0.1 m-phosphate buffer at pH 7.0, have been measured at 21 °. The radius of gyration R (37.5 Å), the molecular weight M (114,000), and the volume V (295,000 ų) were determined.A comparison between the scattering curves obtained from the enzyme and the theoretical scattering curves of different triaxial bodies shows that the shape of the molecule can be represented by an oblate ellipsoid with the semiaxes A = 62.7, B = 50.1 and $\text{C = 23.5}\text{A}\text{?}$.     

EDTA- and puromycin-derived duck- and rabbit globin-messenger ribonucleoprotein complexes isolated by oligo(dT)-cellulose chromatography

Duck- and rabbit globin messenger ribonucleoprotein complexes isolated by oligo(dT) cellulose chromatography reveal an identical protein pattern—two main proteins of molecular weights of 73000 and 49000 daltons and minor components—whether the complexes have been liberated from polyribosomes with the EDTA-or the puromycin-high-salt method. In the globin messenger ribonucleoprotein particles of both species predominantly the protein with a molecular weight of 73000 daltons is attached to poly(A)-containing regions of the messenger RNAs.     

Estimates of heavy metal tolerance and chromium(VI) reducing ability of Pseudomonas aeruginosa CCTCC AB93066: chromium(VI) toxicity and environmental parameters optimization

The potential role of parameters in the reduction of hexavalent chromium [Cr(VI)] by Pseudomonas aeruginosa is not well documented. In this study, laboratory batch studies were conducted to assess the effect of a variety of factors, e.g., carbon sources, salinity, initial Cr(VI) concentrations, co-existing ions and a metabolic inhibitor, on microbial Cr(VI) reduction to Cr(III) by P. aeruginosa AB93066. Strain AB93066 tolerated up to 400 mg/L of Cr(VI) in nutrient broth medium compared to only 150 mg/L of Cr(VI) in nutrient agar. This bacteria exhibited different levels of resistance against Pb(II) (200 mg/L), Cd(II) (100 mg/L), Ni(II) (100 mg/L), Cu(II) (100 mg/L), Co(II) (50 mg/L) and Hg(II) (5 mg/L). Cr(VI) reduction was significantly promoted by the addition of glucose and glycerine but was strongly inhibited by the presence of methanol and phenol. The rate of Cr(VI) reduction increased with increasing concentrations of Cr(VI) and then decreased at higher concentrations. The presence of Ni(II) stimulated Cr(VI) reduction, while Pb(II), Co(II) and Cd(II) had adverse impact on reduction ability of this strain. Cr(VI) reduction was also inhibited by high levels of NaCl, various concentrations of sodium azide and 20 mM of SO₄ ^2?, MoO₄ ^2?, NO₃ ^?, PO₄ ^3?. No significant relationship was observed between Cr(VI) reduction and redox potential of the culture medium. Scanning electron microscopy showed visible morphological changes in the cells due to chromate stress. Fourier transform infrared spectroscopy analysis revealed chromium species was likely to form complexes with certain functional groups such as carboxyl and amino groups on the surface of P. aeruginosa AB93066. Overall, above results are beneficial to the bioremediation of chromate-polluted industrial wastewaters.     

Size and structure of antigen-antibody complexes. Electron microscopy and light scattering studies

Size parameters of model antigen-antibody (Ag-Ab) complexes formed by the interaction of bovine serum albumin (BSA) and pairs of monoclonal anti-BSA antibodies (mAb) were evaluated by quasielastic light scattering, classical light scattering, and electron microscopy (EM). Mean values for the hydrodynamic radius, radius of gyration, and molecular weight were determined by light scattering. Detailed information regarding the molecular weight distribution and the presence of cycles or open chains was obtained with EM. Average molecular weights were calculated from the EM data, and the Porod-Kratky wormlike chain theory was used to model the conformational behavior of the Ag-mAb complexes. Ag-mAb complexes prepared from three different mAb pairs displayed significantly different properties as assessed by each of the techniques employed. Observations and size parameter calculations from EM photomicrographs were consistent with the results from light scattering. The differences observed between the mab pairs would not have been predicted by idealized thermodynamic models. These results suggest that the geometric constraints imposed by the individual epitope environment and/or the relative epitope location are important in determining the average size of complexes and the ratio of linear to cyclic complexes.     

Characterization of the soluble nanoparticles formed through Coulombic interaction of bovine serum albumin with anionic graft copolymers at low pH

A static light scattering (SLS) study of bovine serum albumin (BSA) mixtures with two anionic graft copolymers of poly(sodium acrylate-co-sodium 2-acrylamido-2-methyl-1-propanesulphonate)-graft-poly(N,N-dimethylacrylamide), with a high composition in poly(N,N-dimethylacrylamide) (PDMAM) side chains, revealed the formation of oppositely charged complexes, at pH lower than 4.9, the isoelectric point of BSA. The core-corona nanoparticles formed at pH = 3.00 were characterized. Their molecular weight and radius of gyration were determined by SLS, while their hydrodynamic radius was determined by dynamic light scattering. Small angle neutron scattering measurements were used to determine the radius of the insoluble complexes, comprising the core of the particles. The values obtained indicated that their size and aggregation number of the nanoparticles were smaller when the content of the graft copolymers in neutral PDMAM side chains was higher. Such particles should be interesting drug delivery candidates, if the gastrointestinal tract was to be used.     

Removal of Cr(VI) from aqueous solutions by fruiting bodies of the jelly fungus (Auricularia polytricha)

The aim of this study was to investigate the potential to remove chromium (Cr) from aqueous solutions using the fruiting body of Auricularia polytricha. Batch experiments were conducted under various conditions, and different models were used to characterize the biosorption process. Results showed that, for both fresh and dried fruiting bodies of A. polytricha, removal efficiencies of Cr(VI) and total Cr reached maximum values at pH values of 1 and 2, respectively. The process of Cr(VI) removal by A. polytricha included the sorption process as well as the reduction of Cr(VI) to Cr(III). Spectra of X-ray photoelectron spectroscopy of the biosorbent revealed that most of the Cr loaded on the biomass surface was in the trivalent form. The Freundlich model fitted the isotherm process better than the Langmuir model in the concentration range examined. The pseudo-second-order model well described the adsorption process of Cr onto the biomass. The biosorption capacity of Cr(VI) by fruiting bodies was much higher than that by most of other biosorbents reported. The results suggest that the fruiting bodies of A. polytricha should be a promising biomaterial for Cr removal from water contaminated by the heavy metal.     

Escherichia coli lac repressor is elongated with its operator DNA binding domains located at both ends

From small-angle X-ray scattering experiments on solutions of Escherichia coli lac repressor and repressor tryptic core, we conclude that the domains of repressor that bind to operator DNA lie at the ends of an elongated molecule. The addition of the inducer, isopropyl-β-d-thiogalactoside, to either repressor or core does not produce a measurable structural change, since the radius of gyration of repressor is 40.3 ± 1.9 Å without and 42.2 ± 1.7 Å with isopropyl-β-d-thiogalactoside; the core radius of gyration is 35.4 ± 1.1 Å without ligand and 36.3 ± 1.1 Å with isopropyl-β-d-thiogalactoside. In the context of data from single crystals of repressor and core, the measured radii of gyration are shown to be consistent with a core (or repressor) molecule of dimensional anisotropy 1: (1.5 to 2.0): (3.0 to 4.0). The 5 Å difference in radius of gyration between native and core repressor is interpreted to mean that the amino terminal 59 residues (headpieces) lie at the ends of an elongated repressor molecule. This structure implies that the repressor may have DNA binding sites, consisting of two adjacent headpieces, on each end of the molecule and this binds to the DNA with its long axis perpendicular to the DNA.     

Small-angle x-ray scattering of bovine nasal cartilage proteoglycan in solution

The proteoglycan subunit (PGS) from bovine nasal cartilage was examined in water and in 0.15 N LiCl by small-angle x-ray scattering (SAXS). The molecular weight of 2.5 × 10⁶ and the radius of gyration, R_g = 493 Å, in 0.15 N LiCl, obtained by SAXS, are in good agreement with values reported by others for similar preparations. Values of the radius of gyration of the cross section, mass per unit length, and persistence length of the PGS are also reported. The low value of intrinsic viscosity ([η]) found in 0.15 N LiCl, and a comparison of the experimental distance distribution function to that of the theoretical distance distribution function for sphere, suggest that the PGS in salt solution approaches spherical symmetry. The much higher value of [η] in water suggests a prolate ellipsoid of low axial ratio.