Chain conformation and bioactivity of water‐soluble polysaccharide extracted from Rhizoma Panacis Japonici

A water‐soluble α‐(1→4)‐D ‐glucan heteropolysaccharide with 37% degree of branch extracted by base from Rhizoma Panacis Japonici, coded as RPS3, was fractionated into six fractions by the method of nonsolvent addition. Their weight‐average molecular mass (M_w), polydispersity index (M_w/M_n), and radius of gyration (〈s²〉_z^1/2) were determined with laser light scattering (LLS) and size exclusion chromatography combined with LLS. The structure of the fraction was determined by methylation analyses and ¹³C NMR. The dependences of intrinsic viscosity ([η]) and 〈s²〉_z^1/2 on M_w were established as [η] = 0.71 M_w^{0.27 ± 0.01} (cm³/g) and 〈s²〉_z^1/2 = 1.53 M_w^{0.27 ± 0.02} (nm) in the M_w range from 5.62 × 10⁴ to 3.05 × 10⁶ (g/mol) for RPS3 in 0.15M NaCl aqueous solution at 25°C. On the basis of the current theory of the polymer solution, the fractal dimension (d_f), unperturbed chain dimension (A), and characteristic ratio (C_∞) were calculated to be 3.0, 1.48 Å, and 15.1, respectively. The results revealed that the RPS3 chains existed as spherical conformation in the aqueous solution. Transmission electron microscope further provided the evidence of the sphere shape of the RPS3 and its fractionated molecules in water. In vitro cytotoxicity assay indicated that the fractions could inhibit the tumor cells and showed no harm to normal cells at low dose. The bioactivity was relative with molecular mass of the samples. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 383–390, 2010. This article was originally published online as an acceptedpreprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office atbiopolymers@wiley.com     

Characterization of rodlike RNA fragments.

Native calf thymus DNA was sheared by sonication in a viscous solvent to the molecular-weight range from 3 × 10⁴ to 3 × 10⁵ daltons, and fractionated by gel chromatography. Number and weight average molecular weights (M?_n and M?_w) were determined for individual fractions by electron microscopy; the ratio M?_w/M?_n for the peak fraction is approximately 1.1. Sedimentation coefficients (s⁰_20,w) of these fractionated samples show an approximately linear dependence on the logarithm of the molecular weight M?_w. This behavior is that expected for rodlike molecules, and is in quantitative agreement with the theory of Yamakawa and Fujii [(1973) Macromolecules 6 , 407–415] for the sedimentation coefficient of a wormlike chain with a persistence length of 625 Å, a diameter of 25 Å, and a mass per unit length of 195 daltons/Å. It appears that the wormlike coil model, without excluded volume, can represent the sedimentation behavior of DNA over the entire conformational range from rigid rod to flexible coil, using the above parameters. Equilibrium melting curves were determined for various fractions in aqueous 2.4 M tetraethylammonium bromide. A substantial broadening of the transition and decrease of the melting temperature were observed with decreasing molecular weight. Empirical expressions have been obtained relating both the transition temperature and breadth in this solvent to molecular weight.     

Effect of Calcium Ions on Hydrodynamic Properties of Pentameric and Decameric C-Reactive Protein in Solution

The molecular mass and sedimentation coefficient of native C-reactive protein in solution were determined by analytical ultracentrifugation in the presence and absence of calcium ions. Pentameric C-reactive protein was shown to be the major macroscopic form of this protein in solution. The removal of calcium ions from solution caused decompaction of the protein accompanied by changes in its hydrodynamic parameters. The sedimentation coefficient s ⁰ _20,w of pentameric C-reactive protein in solution containing 2 mM Ca²⁺ (6.6S) exceeded that for C-reactive protein in solution containing 2 mM EDTA (6.4S). Analysis of average molecular masses M _w and M _z obtained from sedimentation data demonstrated that the solution of highly purified protein was not homogeneous. As shown by intermolecular crosslinking, the solution also contained the 241-kDa decamer of C-reactive protein (9.5S) as a separate macroscopic form, whose share hardly reached 10% in the presence of 2 mM Ca²⁺ and increased after removal of calcium ions. The decamers were shown to result from intermolecular association of the pentamers.     

Crystallization and Characterization of Polyamine Oxidase from Penicillium chrysogenum

Polyamine oxidase was purified and crystallized with an overall yield of 35% from mycelial extract of Penicillium chrysogenum by a procedure involving ammonium sulfate fractionation, and DEAE-cellulose and Sephadex G-200 column chromatographies. The crystalline enzyme was homogeneous, as judged by disc gel electrophoresis and ultracentrifugation. The sedimentation coefficient (s_{20, w}⁰) of the enzyme was determined to be 6.9S, and diffusion coefficient (D_{20, w}) to be 4.2 × 10^?7 cm² sec^?1. The enzyme showed a molecular weight of about 160,000 by gel filtration method and ultracentrifugal analysis, and it was composed of two identical subunits. The enzyme was a flavoprotein with absorption maxima at 275, 375 and 450 nm. The prosthetic group was identified to be FAD. The enzyme oxidized spermine, and slightly oxidized spermidine. Diamines and monoamines were not oxidized.     

Stomatal responses to humidity and temperature in darkness

Stomatal responses to leaf temperature (T_l) and to the mole fractions of water vapour in the ambient air (w_a) and the leaf intercellular air spaces (w_i) were determined in darkness to remove the potential effects of changes in photosynthesis and intercellular CO₂ concentration. Both the steady‐state and kinetic responses of stomatal conductance (g_s) to w_a in darkness were found to be indistinguishable from those in the light. g_s showed a steep response to the difference (Δw) between w_a and w_i when w_a was varied. The response was much less steep when w_i was varied. Although stomatal apertures responded steeply to T_l when Δw was held constant at 17 mmol mol^?1, the response was much less steep when Δw was held constant at about zero. Similar results were obtained in the light for Δw = 15 mmol mol^?1 and Δw ≈ 0 mmol mol^?1. These results are discussed in the context of mechanisms for the stomatal response to humidity.     

The boundary layer and the apparent responses of stomatal conductance to wind speed and to the mole fractions of CO2 and water vapour in the air

The experiments and simulations reported in this paper show that, for stomata sensitive to both CO₂ and water vapour concentrations, responses of stomatal conductance (g^w_s) to boundary layer thickness have two components, one resulting from changes in intercellular CO₂ concentration (χ^c_i) and another from changes in leaf surface water vapour saturation deficit (D^w_s). The experiments and simulations also show that the boundary layer conductance (g^w_b) can significantly alter the apparent response of g^w_s to ambient air CO₂ mole fraction (χ^c_a) and water vapour mole fraction (χ^w_a). Because of the feedback loop involved the responses of g^w_s for χ^c_a and χ^w_a each include responses to both χ^c_i and D^w_s. The boundary layer alters the state of the variables sensed by the guard cells—i.e. χ^c_i and D^w_s—and so it is a source of feedback. Thus, when scaling up from responses of stomata to the response of g^w_s for a whole leaf, the effect of the boundary layer must be considered. The results indicate that, for given responses of g^w_s to χ^c_i and D^w_s, the apparent responses of g^w_s to D^w_a and χ^c_a depend on the size of the leaf and wind speed, showing that this effect of the boundary layer should be considered when comparing data measured under different conditions, or with different methods.     

Studies on Sugar-isomerizing Enzyme Purification,Crystallization and Some Properties of Glucose Isomerase from Streptomyces sp.

Glucose isomerase was purified by means of acetone fractionation, DEAE-cellulose column chromatography, DEAE-Sephadex column chromatography and crystallization. The purified enzyme appeared to be homogeneous on ultracentrifugation and electrophoresis. The sedimentation coefficient, s_20,w, the diffusion coefficient, D_20,w, and partial specific volume of the enzyme were 8.0S, 4 × 10^?7cm²/sec and 0.69 ml/g, respectively. The molecular weight of the enzyme was estimated to be 157,000 from the sedimentation and diffusion measurements. The crystalline glucose isomerase contained cobalt and magnesium ions. The properties of the enzyme were also studied.     

Structure and physico-chemical properties of bacteriophage G: III. A homogeneous DNA of molecular weight 5 × 108

The physico-chemical properties of the DNA released from bacteriophage G (active on Bacillus megatherium) are described. Phage G, an unusually large bacteriophage, has a nucleic acid content of 4 to 6 × 10⁸ daltons.Sedimentation velocity analysis at low angular speed and examination by electron microscopy, indicate that a single DNA molecule, sedimenting with s_{20, w}⁰ = 125 ± 1.5 S and at least 200 ± 20 μm long, is released upon thermal or osmotic shock. Melting temperature data and chromatographic analysis indicate a mean base composition of 70% A + T. CsCl and Cs₂SO₄ buoyant density data, circular dichroism spectra and sensitivity to specific nucleases indicate that phage G DNA is similar to the DNAs from T-even phages and is more glucosylated than phage T6 DNA. Direct glucose determination indicates a 185% molar ratio of glucose to cytosine. Linear density extrapolated from literature data and contour length measurement yield a lower limit for the molecular weight of phage G DNA of 4.9 × 10⁸. Comparison of this value with the s_20,w⁰ measured with the analytical ultracentrifuge seems to confirm the validity of the empirical relationship proposed by Freifelder (1970), between s_{20, w}⁰ and molecular weight, over a larger range than that previously known. A possible systematic error in defect in length determination, however, prevents a discrimination between this and other empirical formulae proposed by various authors, which predict a molecular weight that is 20 to 25% higher.     

Physicochemical parameters of partially hydrolyzed S. salivarius levan fractions

The fractions obtained from the partially hydrolyzed branched Streptococcus salivarius levan were examined in solution. Sedimentation coefficients, S⁰, intrinsic viscosities, [η], weight-average molecular weights, M _w, and radii of gyration were obtained from sedimentation velocity, viscosity, and light-scattering measurements. Double logarithmic plots of [η] vs M _w and S⁰ vs M _w each yielded two linear segments intersecting at M _w ≈ 10⁵. Hydrodynamic data suggest that fractions of M _w > 10⁵ behave as compact spheres, whereas for M _w < 10⁵, the particles are best characterized as linear random coils. Calculations based on theories of random coils and spheres support the above observations.     

Physicochemical studies on xylinan (acetan). III. Hydrodynamic characterization by analytical ultracentrifugation and dynamic light scattering

A laboratory-made sample of the polysaccharide xylinan (acetan) has been further characterized with respect to (i) purity, (ii) molar mass and polydispersity, and (iii) gross conformation by a combination of hydrodynamic measurements (sedimentation velocity and equilibrium analytical ultracentrifugation, viscometry, and dynamic light scattering) in aqueous NaCl (I = 0.10 mol·L⁻¹). Sedimentation velocity diagrams recorded using Schlieren optics revealed highly pure material sedimenting as a single boundary [s^o_20.w = 9.5 ± 0.7) S; k_s = (273 ± 112) mL/g]. The hypersharp nature of these boundaries is symptomatic of a polydisperse and highly nonideal (in the thermodynamic sense) system. Low speed sedimentation equilibrium in the analytical ultracentrifuge using Rayleigh interference optics and two different types of extrapolation procedure (involving point and whole-cell molar masses) gave a weight average molar mass M_w of (2.5 ± 0.5) × 10⁻⁶ g·mol⁻¹ and also a second virial coefficient, B = (2.8 ± 0.7) × 10⁻⁴ mL·mol·g⁻², both values in good agreement with those from light scattering-based procedures (Part II of this series). A dynamic Zimm plot from dynamic light scattering measurements gave a z-average translational diffusion coefficient D^o_20.w = (3.02 ± 0.05) × 10⁻⁸ cm²·s⁻¹ and the concentration-dependence parameter k_D = (370 ± 15) mL/g. Combination of s^o_20.w with D^o_20.w via the Svedberg equation gave another estimate for M_w of ≅ 2.4 × 10⁶ g/mol, again in good agreement. Both the Wales-van Holde ratio (k_s/[η]) ≅ 0.4 (with [η] = (760 ± 77) mL/g) and the ρ-parameter (ratio of the radius of gyration from static light scattering to the hydrodynamic radius from dynamic light scattering) as ρ > 2.0 all indicate an extended conformation for the macromolecules in solution. These findings, plus Rinde-type simulations of the sedimentation equilibrium data are all consistent with the interpretation in terms of a unimodal wormlike coil model performed earlier. © 1996 John Wiley & Sons, Inc.     

Structure of native proteoglycan aggregates from chick limb-bud chondrocytes

Laser light-scattering has been used to investigate the size of native proteoglycan aggregates (PGA-aA1) from day-8 chick limb-bud chondrocyte cultures isolated under associative extraction and purification conditions in 0.4M guanidinium chloride (GdnHCl) solution. Dynamic light-scattering measurements yielded a hydrodynamic radius, R_s, of 244 ± 10 nm for PGA-aA1 in 0.4M GdnHCl, and a weight-average molecular weight (M _w) of 150 ± 50 × 10⁶ was obtained from a Zimm plot. Disaggregation in 4.0M GdnHCl aqueous solution yielded proteoglycan subunits (PGS) with R_s = 39 ± 2 nm, M _w = 1.6 ± 0.3 × 10⁶, which reassembled in 0.4M GdnHCl to form “reconstituted native” aggregates (PGA-raA1) with R_s = 121 ± 6 nm, M _w = 17 ± 3 × 10⁶. A second specimen of PGA-aA1 had R_s = 192 ± 10 nm, M _w = 100 ± 10 × 10⁶. The latter value was estimated from an empirical relationship between M _w and R_s. After dissociation, this specimen reassembled to form PGA-raA1 with R_s = 85 ± 5 nm, M _w = 12 ± 1 × 10⁶. These data are compared with those for a specimen of reconstituted aggregate (PGA-A1) that had been extracted under dissociative conditions and then reaggregated by dialysis to 0.4M GdnHCl aqueous solution, for which R_s = 138 ± 9 nm, M _w = 45 ± 8 × 10⁶. From these values, we have calculated the weight-average number of subunits per aggregate N_w: 111 for PGA-aA1 and 12 for raA1 (70 and 7 for the second PGA-aA1 and PGA-raA1 specimen, respectively) as compared to 32 for PGA-A1. The numbers of subunits per aggregate were also determined from electron micrographs of spread specimens. The latter results show the same trends as those obtained by light scattering, but lead in each case to lower numbers of subunits per aggregate. These data demonstrate conclusively that PGA samples exhibit a higher degree of aggregation in solution than visualized in typical electron microscopy (EM) preparations, probably due to disaggregation during EM specimen preparation. Since N_w determined both by light scattering (LS) and by EM are larger for native versus reconstituted aggregate samples, our data point to a more compact aggregation of subunits along the hyaluronic acid (HA) chains in the former.     

Purification and characterization of beef liver dihydrofolate reductase.

Beef liver dihydrofolate reductase has been purified to homogeneity by using a methotrexate affinity column followed by gel filtration to remove several higher molecular weight proteins. Tightly bound dihydrofolate is removed by hydroxylapatite chromatography. The overall purification is 13,000-fold; the specific activity is 26 units·mg^?1, approximately 25 times higher than previously reported. The enzyme has been shown to be homogeneous by the following criteria: (i) discontinuous gel electrophoresis, (ii) sodium dodecyl sulfate-gel electrophoresis, (iii) velocity sedimentation, (iv) equilibrium sedimentation, and (v) methotrexate titration. The amino acid composition has been determined. Notable features include a single cysteine, three tryptophan and three histidine residues. The N-terminal amino acid is leucine. The molecular weight determined by equilibrium sedimentation is 22,500. The s_20,w⁰ is 2.08 × 10^?13 S and D_20,w⁰ = 10.93 cm²·s^?1. A frictional coefficient of 1.04 indicates that the enzyme is essentially spherical. An isoelectrical point of 6.80 was measured.     

Purification, characterization and reassembly of the bacteriophage T4D tail sheath protein P18.

P18, the sole component of T4 tail sheath, has been isolated in a monomeric active form from extended sheaths of intact tails which were dissociated at low ionic strength. The molecular weight of P18 is determined to be 65,000 from sedimentation equilibrium and 73,000 from sodium dodecyl sulphate/gel electrophoresis. Combining the diffusion constant (D_20,w = 5·5× 10^?7cm²s^?1)and the sedimentation constant (s⁰_20,w = 4·2 S) a value of 67,000 is obtained. The circular dichroism spectra reveal a striking similarity of the structure of P18 in the monomeric state and in the extended sheath conformation.The purified P18 is found to reassemble into extended sheaths if the core-baseplate complex is present, forming normal length tails. Structures similar to polysheath are formed in the absence of core-baseplates.     

Polymorphisme et caractère glycoprotéique des lectines de Ricinus communis purifiées par chromatographie d'affinité

Two lectin fractions (S^o_{20 w} = 6,8 and 4,9 S) were purified from Ricinus communis seeds. The purification was carried out in four steps : ammonium sulfate fractionation, affinity chromatography on Sepharose 4 B, gel filtration on Sephadex G 150 and chromatography on CM cellulose. The purified lectins were glycoproteins whose chemical composition was determined. Amino terminal analysis of the two fractions revealed glycine and serine. Polyacrylamide gel electrophoresis of the higher molecular weight fraction allowed the separation of several components with different affinity for PAS staining.     

Levans. II. Light-scattering and sedimentation data of Streptococcus salivarius Levan in water.

The molecular weights and radii of gyration of Streptococcus salivarius levan fractions were obtained from light-scattering measurements in water. Sedimentation coefficients and partial specific volumes of the fractions were also obtained. Double logarithmic plots of [η] versus M?_w and S⁰ versus M?_w yielded slopes having values of 0.17 and 0.62, respectively. The data and various calculated parameters show that levan from Streptococcus salivarius is highly branched and behaves hydrodynamically as a compact particle of spherical symmetry.     

Studies on polyribonucleotides. Synthesis of a polyinosinic: 6-thioinosinic acid copolymer

The enzymatic synthesis of a high molecular weight (s_20,w ～10) copolymer of inosinic and 6-thioinosinic acids [poly(I:s⁶I)] has been achieved. Poly (I:s⁶I) forms a double stranded complex with poly C which has a Tm significantly lower than a poly I · ply C complex of equivalent molecular weight.     

Chromatographic Separation, and Characteristics of Nucleic Acids from HeLa Cells

The application of the phenol-duponol method to extraction of nucleic acids from HeLa cells is described. Chromatography of the phenol extract on an esterified bovine serum albumin column with a salt gradient of sodium chloride gives separation of soluble RNA, DNA, and two different high molecular RNA fractions. Ultracentrifugation of the DNA eluted from the column gives a sedimentation coefficient (s₂₀^o,w) of 38, which agrees with ultracentrifugation data on the phenol extract. The eluted RNA appears polydisperse at low ionic strength, but at high ionic strength and after alcohol precipitation two fractions with the sedimentation coefficients of 16 and 25 to 29, respectively, were obtained.     

Photon correlation spectroscopy, total intensity light scattering with laser radiation, and hydrodynamic studies of a well fractionated DNA sample.

A Malvern laser light-scattering instrument has been modified for use at scattering angles down to 5° and both total intensity and quasi-elastic scattering experiments. A sample of sheared, length-fractionated calf-thymus DNA was characterized by sedimentation, viscosity and electron microscopy. Quasi-elastic scattering and absolute intensity determinations were performed with the laser instrument and intensity determinations only with a Fica conventional light-scattering photometer. The total intensity experiments gave M?_w = (3.75 ± 0.15) × 10⁶ and 〈R²〉^1/2_z = (206.9 ± 10.3) nm which yielded a value for the persistence length, allowing for polydispersity, of 66 ± 6nm. The quasi-elastic experiments at scattering angles below 20° gave D⁰_{20, w} = (2.23 ± 0.06) × 10^?8 cm²/sec which combined with S⁰_{20, w} = 15.6 in the Svedberg equation gave M?_w = (3.73 ± 0.18) × 10⁶. In addition, from the higher angle data we extracted a value of the longest intramolecular relaxation time, τ1 of 17.5 msec. This is not in particularly good agreement with τ1 predicted by the Zimm–Rouse theory using our other experimental parameters. The disagreement may be due to the restricted applicability of the Zimm–Rouse spring-bead model as a quantitative representation of DNA molecules. Alternatively, it may be due to present difficulties in the unambiguous interpretation of molecular motions from the experimental autocorrelation functions.     

Structure and symmetry of B. stearothermophilus pyruvate dehydrogenase multienzyme complex and implications for eucaryote evolution.

The pyruvate dehydrogenase multienzyme complex was purified from B. stearothermophilus. The enzyme was found to be of high molecular weight (s_20,w⁰ = 75S) and to contain four different types of polypeptide chain, with subunit molecular weights estimated as 57,000, 54,000, 42,000 and 36,000, respectively. The subunit of molecular weight 57,000 was shown to derive from the lipoate acetyltransferase component (EC 2.3.1.12), whereas the subunit of molecular weight 54,000 was identified as lipoamide dehydrogenase (EC 1.6.4.3). The other two polypeptide chains are likely to be the subunits of pyruvate decarboxylase (EC 1.2.4.1). The purified lipoate acetyltransferase component was also of high molecular weight (s_20,w⁰ = 35S), and both it and the intact enzyme complex were readily visualized in negatively-stained preparations in the electron microscope. The lipoate acetyltransferase component, in particular, clearly showed the 5 fold, 3 fold and 2 fold rotation axes of a regular pentagonal dodecahedron with a diameter of 23 nm. The symmetry of the enzyme complex is apparently icosahedral. In all these properties the enzyme from B. stearothermophilus (Gram-positive) strikingly resembles the pyruvate dehydrogenase complex from the mitochondria of eucaryotic cells, and stands in marked contrast to the enzyme from E. coli (Gram-negative). A growing body of evidence indicates that the quaternary structures of enzymes from Gram-positive bacteria and the mitochondria of eucaryotes share distinctive common features that set them apart from the corresponding enzymes from Gram-negative bacteria. Adopting the serial endosymbiosis theory for the evolution of the mitochondrion, it follows that the forerunner of mitochondria may have been a Gram-positive rather than a Gram-negative bacterium.     

Preparation and solution properties of pullulan fractions as standard samples for water-soluble polymers

A series of pullulan fractions with molecular weights in the range 5 × 10³ to 8 × 10⁵ were prepared. The weight-average molecular weight (M_w) of all the samples was determined by sedimentation equilibrium. The hydrodynamic properties of pullulan in aqueous solution were investigated by viscometry and ultracentrifugation. The experimental results indicate that pullulan molecules in water are fairly stable and behave as expanded random coils when M_w is above 2 × 10⁴. The molecular weight distributions of the fractions were measured by gel filtration. The ratio M_w/M_n was close to 1·1, except for a sample with the highest M_w.It is concluded that the pullulan fractions prepared by the present work are well characterized and have a narrow molecular weight distribution. They may be useful as standard samples for studies of water-soluble polymers.