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.     

Light-scattering spectroscopic study of polysaccharide protein conjugate

By combining gel permeation chromatography (GPC) and light-scattering spectroscopy, including photon correlation and angular distribution of absolute scattered intensity, we were able to characterize immunologically active Haemophilus influenzae type b polysaccharide (HIB Ps) bovine serum albumin (BSA) conjugates in terms of equivalent hydrodynamic radius r_h ～ (6.2 ± 0.6) × 10² Å, apparent radius of gyration r_g ～ (5.4 ± 0.3) × 10² Å, apparent molecular weight M_w ～ (3.5 ± 0.4) × 10⁶ g/mol, and a second virial coefficient A₂ ～ (1.9 ± 0.3) × 10^?4 cm³ mol/g². We could study the effects of each of the processes in the conjugate formation according to the following procedure: BSA (dialysis, modification, fractionation) + HIB Ps → HIB Ps/BSA conjugate (conjugate formation, fractionation). Narrow distributions of HIB Ps BSA conjugate formation can be achieved using fractionated BSA.     

Dilute solution properties of proteoglycan fractions from bovine nasal cartilage

Fresh proteogycans (adult bovine nasal cartilage) isolated from the densest portion of a dissociative density gradient had a weight-average molecular weight of ca. 10⁶ in 4M guanidine hydrochloride (GdnHCI) by light scattering. Fractions of such material obtained by elution with 4M GdnHCI from 2% agarose gel, both normal and cross-linkd, has proteoglycan subunit molecular weights ranging from 0.8 to 2.6 × 10⁶ and root-mean-square radii ranging from 35 to 52 nm in the same solvent. The protein molecular weight per proteoglycan subunit was about 1.2 × 10⁵ and that of keratan sulfate about 1.8 × 10⁵, both independent of total molecular weight. A random-flight “graft copolymer” model having uniform side chains of chondroitin sulfate (40 disaccharides) and keratan sulfate (15 disaccharides) and a random-coil polypeptide back bone was used to estimate the unperturbed radius, whihc was about 19 nm for a mol wt of 1.5 × 10⁶. Experimental light-scattering data for fractions were fitted very well by theoretical curves for the particale scattering factor for both linear and appropriate branched polymers. Examination of coil expansion on the basis of perturbation calculations for branched polymer models suggested that expansion did not account for the experimentally observed radii in terms of unperturbed radii calculated from the model. A possible explanation is that substantial local stiffening of the polypeptide chain due to substitution of side-chain clusters increases the unperturbed radii. The intrinsic viscosity [η] is 4M GdnHCI ranged from 120 to 180 ml/g, and could be interpreted in terms of th eequivalent sphere model; the Flory number has approximately its normal value for flexible linear polymers. The treatment of the sedimentation coefficient by this is less successful, since the Man delkern-Flory parameter β apparently increases with increasing molecular weight; average value are similar to those for flexible polymers, but the variation in β makes this method useful only for rough estimation of molecular weight of proteoglycans. Molecular weights of purified proteoglycans are the same in 0.2M NaCI as in 4M GdnHCI, while crude preparations gave higher molecular weights in 0.2M NaCI, probably because of association due to incomplete removel of “linking” proteins.     

Comparison of nuclear and polysomal RNA fractions from goat brain

Phenol extracted RNA preparations from highly purified nuclei and polysomes of goat brain were fractionated by chromatography on oligo (dT)-cellulose and analyzed by electrophoresis on agarose-acrylamide composite gels. The electrophoretic profile of the polysomal polyadenylated RNA fraction showed a major band with a molecular weight of about 0.62 × 10⁶, which corresponds to the size of the tubulin mRNA. The nuclear polyadenylated RNA fraction also displayed a single major band, with an estimated molecular weight of 0.76 × 10⁶, which appears to be a potential precursor of tubulin mRNA.     

Molecular weight and shape of the phycocyanin hexamer

The hexamer of phycocyanin from Phormidium luridum has been isolated and purified by ammonium sulfate fractionation and gel chromatography. The protein is characterized by the sedimentation constant S°_{20, w} = 10.2S, the diffusion coefficient D_{20, w} = 4.73 × 10^?7 cm²/sec, and intrinsic viscosity [η] = 3.89 ml/g. The molecular weight of the aggregate is 209,000. The shape and dimensions of the hexamer are discussed in terms of a model consisting of subunits arranged with C₆ symmetry. The monomers, assumed to be spherical, are found to have a radius of 22 Å, and the diameter across the hexamer is 132 Å. The latter figure agrees closely with dimensions observed in electron micrographs.     

A newly isolated densonucleosis virus from Pseudoplusia includens (Lepidoptera: Noctuidae)

An icosahedral DNA virus isolated from the soybean looper, Pseudoplusia includens, was characterized. Purified virus had a diameter of 20 ± 1 nm and negatively stained preparations showed a trend to form linear to three-dimensional crystals. The virus had a sedimentation coefficient of 120 ± 3 S and a buoyant density of 1.40 ± 0.01 g/cm³. The DNA content of the virus was 37.8 ± 0.1% and the absorption spectrum showed it to be a typical nucleoprotein. Viral DNA in situ was shown to be single-stranded by staining the virus with acridine orange as well as by reaction to formaldehyde. Evidence of inverted terminal repetition of the DNA was observed by electron microscopy. The terminal repetition comprises ca. 6–7% of the genome. The molecular weight of the ssDNA was 2.0 ± 0.1 × 10⁶ as determined by agarose gel electrophoresis or 2.1 ± 0.1 × 10⁶ as determined by electron microscopy. Four virion proteins with molecular weights of 46.5 ± 0.1, 54.0 ± 0.1, 64.0 ± 0.2, and 87.0 ± 0.1 × 10³ were detected by 9% SDS-polyacrylamide gel electrophoresis. Double-diffusion tests showed the virus to be serologically related but not identical to DNV-1. Ultrathin sections showed that the nucleus of the hemocyte, muscle, hypodermal, and fat body cells contained virus-like particles. The chromatin of an infected nucleus always underwent a margination and the nucleoplasm was often replaced largely by virions.Data indicate that the virus belongs to the Densovirus of the family Parvoviridae.     

Molecular Weight and Intrinsic Viscosity of Konjac Gluco-mannan

For the purpose of making clear the macromolecular chemical properties of konjac gluco-mannan, the light scattering and viscosity measurements were carried out for the aqueous solutions of the partially methylated derivatives, and following results were obtained: 1) The weight-average molecular weight, Mw, and the root mean square of radius of gyration, <S²>^1/2, were 100×l0⁴~120×l0⁴ and 1100~1300 Å, respectively. 2) The 2> versus Mw relationship was represented by the equation of <S²>=4.20×10^?1 Mw^1.08, and this fact suggested a random coil as a molecular form in the solution. 3) The intrinsic viscosity, [η], versus Mw relationship was represented by the equation of [η]=6.37×10^?4 Mw^0.74, which was considered to be useful for the purpose of estimating the molecular weight of konjac gluco-mannan by viscometric method.     

Multiple Genetic Components in Bromegrass Mosaic Virus

Bromegrass mosaic virus has three types of structurally similar virions all of which are required for infectivity. The virions contain either one RNA molecule of molecular weight 1.09×10⁶ or 0.99×10⁶ or two of molecular weights 0.75×10⁶ and 0.28×10⁶. A mixture of the isolated RNAs is infectious and deletion of any but the smallest greatly reduces infectivity. The third largest RNA component contains the coat protein gene.     

Hydrodynamics,size, and shape of bacteriophage T4D tails and baseplates

We have used translational diffusion coefficient measurements and subunit hydrodynamic theory to determine the dimensions and shape of bacterioophage T4D baseplates and tails. The diffusion coefficient of the baseplate, measured by quasielastic laser light scattering (QLS), was determined previously by Wagenknecht and Bloomfield to be D = 8.56 × 10^?8 cm²/s. For the tail, we found D = 5.88 × 10^?8 cm²/s by QLS, and D = 6.02 × 10^?8 cm²/s by combining sedimentation coefficient and molecular weight in the Svedberg equation. These values, which have an uncertainty of ±2.7%, when combined with subunit hydrodynamic theory, enabled us to refine estimates of dimensions obtained by electron microscopy. For the hexagonal baseplate, the vertex-to-vertex distance is about 480 Å, the thickness is 160 Å, and there are six extended short fibers 320-Å long and 40 Å in diameter. When a baseplate of these dimensions is attached to a tail tube-sheath-connector complex 1050-Å long and 240 Å in diameter, the calculated D is 5.93 × 10^?8 cm²/s, within 1% of experiment. This combined use of electron microscopy and hydrodynamics, using the former to ascertain shape, and the latter to obtain solution dimensions, is a powerful approach to the structure of biomolecular complexes.     

Letter: Evidence for non-repetitive subunits in the genome of Rous sarcoma virus

The complexity of Rous sarcoma virus RNA has been determined using molecular hybridization. Relative to poliovirus RNA, the complexity of Rous sarcoma virus is 9·3 × 10⁶ daltons, a value close to its physically-determined molecular weight of about 10⁷. Our interpretation is that the 35 S RNA subunits of the 70 S virus genome are non-repetitive, that is, each possesses a unique nucleotide sequence, although a limited amount of redundancy cannot be excluded.     

Evidence for SV40 Specific RNA containing Virus and Host Specific Sequences

AFTER infection of monkey kidney cells with simian virus 40 (SV40), several species of SV40 specific RNA are synthesized¹. Most SV40 RNA have a molecular weight of about 6×10⁵ and 8×10⁵ as measured by polyacrylamide gel electrophoresis¹. In addition to these classes of RNA, a large heterogeneous SV40 specific RNA species of up to three times the length of the monomeric SV40 DNA molecule has been observed^1–4. Nothing is known about the structure of this large heterogeneous virus specific RNA.     

Formation and characterisitcs of hepatic dexamethasone-receptor complexes of different molecular weight

The dexamethasone-binding receptor protein in rat liver cytosol has a Stokes radius of 61 Å and a sedimentation coefficient of 4.0 S. In contrast, cell nuclei labelled with [³H]dexamethasone in vivo or in vitro (reconstitution experiments with [³H]dexamethasone-labelled cytosol and isolated unlabelled nuclei) contain a high-salt-extractable dexamethasone-receptor complex with a Stokes radius of 30–36 Å and a sedimentation coefficient of 3.2 S. Exposure of liver homogenate or 1000 × g homogenate supernatant to low ionic strenght during preparation of cytosol resulted in conversion of the 61 Å to a 36 Å complex very similar to the intranuclear form of dexamethasone receptor. 61 → 36 Å complex-verting activity was present in both the 100 × g ?10 000 × g sediment of liver homogenate, from which it could be extracted by hypotonic media, and in the liver cell nuclei, from which it could be extracted by hypertonic media. Mild digestion of the 61 Å dexamethasone-receptor complex with trypsin also gave rise to a complex with a Stokes radius of 36 Å. Reconstitution experiments with isolated liver cell nuclei indicated that both the 61 Å and 36 Å dexamethasone-receptor complexes were taken up by the nuclei; reextraction of the nuclei incubated with the 61 Å complex revealed that this form had been converted to the 30–36 Å complex.Further digestion of teh 61 and 36 Å [³H]dexamethasone-receptor complexes with hypotonic extract of the 1000 × g ?10 000 × g sediment of liver homogenate or with trypsin resulted in formation of a third complex with a Stokes radius of 19 Å and a sedimentation coefficient of 2.5 S. The approximate molecular weights of the 61, 36 and 19 Å dexamethasone-receptor complexes were calculated as 102 000, 46 00 and 19 000, respectively, and the frictional ratios of the molecules as 1. 84, 1. 38 amd 1.00, respectively.It is concluded that the nuclear 30–36 Å dexamethasone-receptor complex is formed from the cytosol 61 Å complex by proteolytic digestion and that this latter protein contains at least two sites with a relatively high sensitivity to protelytic cleavage.     

Alfalfa mosaic virus protein polymerization.

The self-association of alfalfa mosaic virus coat protein was studied by sedimentation analysis and electron microscopy under a wide range of conditions. In the depolymerized state the protein exists as a molecular species with a sedimentation constant of roughly 3 S and with a molecular weight of (48.4 ± 1.1) × 10³. This value is, within experimental error, twice the value of the monomer (van Beynum, 1975). The dimer has a very stable configuration, as no evidence was found for a monomer-dimer equilibrium between pH values of 3 and 9 and values of ionic strength up to 1.0. One main type of association product (30 S) was found with a molecular weight of (1.48 ± 0.03) × 10⁶. Therefore this particle accomodates 30 dimers which are arranged according to a point group symmetry of 532. The orientation of the 30 dimers within the icosahedral lattice must be such that lattice dyads coincide with the 2-fold axes of the dimers. Micrographs of the 30 S particles show a diameter of about 123 Å; analysis of linear arrays of these particles shows that at low resolution the particle is a hollow sphere with an average coat thickness of about 40 Å.The influence of pH, ionic strength, protein concentration and the type of buffer on the polymerization was determined to some extent and is discussed. The assembly of dimers into the icosahedral particle is an entropy-driven process (Lauffer, 1975); this is concluded from studying the temperature-dependence of the free energy change. Under favourable conditions (phosphate buffer pH 5.5 and ionic strength 0.5) the average enthalpy and entropy changes for the insertion of one dimer into the lattice are about 6.4 kilocalories per mole and 50 entropy units, respectively, based on the unit mole fraction.     

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.     

DNA conformation in N,N-dimethyl formamide-H2O solutions

Optical density, viscosity, and light scattering measurements for calf thymus DNA in water–N,N dimethyl formamide (DMF) solutions are presented. DMF content varied between 0 and 60% (v/v) and DNA molecular weight varied between 15 × 10⁶ and 0.5 × 10⁶. Complementary measurements of the solubility of adenine, thymine, guanine, and cytosine in H₂O–DMF mixtures are presented. The denaturation temperature of DNA, manifested by about a 35% increase of optical density, is gradually depressed by increasing DMF content. However, a significant increase of OD occurs even before (and even after) the denaturation point, when DMF content is increased isothermally. The intrinsic viscosity also exhibits a large decrease when DMF content is increased both before and after the denaturation point. Light scattering data for high-molecular-weight DNA in the predenaturation range indicate a decrease of the mean-square radius and a constant molecular weight on increasing DMF content. The results, interpreted in terms of the wormlike chain of Kratky and Porod, indicate a large decrease of the persistence length of DNA. For low-molecular-weight DNA, radius and molecular weight increase with DMF content, indicating intermolecular aggregation. The formation of compact structures of native DNA is discussed in terms of an increased solubility of uncharged bases, and a decreased solubility of phosphate and deoxyribose groups, when a less polar environment is provided by the addition of DMF.     

The Effects of Hyaluronic Acid on Macrophage FC Receptor Binding and Phagocytosis Are Independent of the Mode of Depolymerization

In order to determine whether exposure of hyaluronic acid to oxygen radicals caused an alteration in its properties. independent of the change in molecular weight induced. we examined its effect upon macro-phage Fc receptor binding. High molecular weight hyaluronic acid (Healon-Pharmacia) caused a dose dependent inhibition of binding between the concentrations of 0.2–1 mg/ml. At a concentration of 0.3 mg/ml both oxygen radical depolymerized and enzymatically degraded hyaluronic acid caused an inhibition of Fc receptor binding at molecular weights of 1 × 10⁶. 1.5 × 10⁶ and 2 × 10⁶. Oxygen radical degraded hyaluronic acid caused a stimulation of Fc receptor binding at molecular weights of 2 × 10⁵ and 3.5 × 10⁵. and enzyme degraded hyaluronic acid causes stimulation at a molecular weight of 2.5 × 10⁶. Thus this “biological property” of hyaluronic acid is dependent upon molecular weight solely and not upon the mode of depolymerization.     

Light scattering from wormlike chains with excluded volume effects

This paper reports a calculation of the angular dependence of light scattering from wormlike chains with excluded volume effects. The Daniels distribution function, modified for excluded volume effects, is used to compute averages for scattering elements separated by contour lengths which are long with respect to the persistence length of the chain. An expansion in terms of exactly known moments of the distribution for the wormlike coil without excluded volume is used for short contour lengths. The results are applied to scattering from calf thymus (M = 18.1 × 10⁶) and T7 (M = 25.4 × 10⁶) DNA. It is demonstrated that the same values of excluded volume parameter (ε = 0.11) and statistical segment length (1/λ′ = 900 Å) which explain the sedimentation and viscosity behavior of DNA also account satisfactorily for the scattering behavior. Molecular weights and root-mean-square radii estimated by extrapolation from scattering data obtained in the angular region from 10° to 25° will be 5–10% too large for DNA of molecular weight 20 × 10⁶–30 × 10⁶.     

The structure of high molecular weight dextrins obtained from potato starch by treatment with Bacillus macerans enzyme

Bacillus macerans enzyme (BME)-derived high molecular weight dextrins, which are by-products in the course of the industrial production of cylodextrins, were isolated and their chemical structures were characterized.Dextrin I was obtained in a yield of about 24% from BME-hydrolyzate (a mixture of dextrin and cylodextrins, 50% each) of potato starch by fractionation with an ultrafiltrator having a membrane of cut-off molecular weight 2.0 × 10⁴. Dextrin II was obtained in a yield of about 15% from BME-hydrolyzate (a mixture of dextrins and cyclodextrins, 70 : 30) of Dextrin I by the same method.Dextrin I and II consisted of dextrin having molecular weights over 20 × 10⁶ and dextrins having molecular weights 4 × 10³−1 × 10⁵ in the ratio of 80 : 12 and 66: 15, respectively.The results of hydrolysis by β-amylase and methylation analysis indicated that the average, exterior and interior chain lenghts of the dextrins having molecular weights over 20 × 10⁶ and 4 × 10³−1 × 10⁵ from Dextrin I were 16.5, 8.2 and 7.3, and 11.5, 6.9 and 3.6, respectively, than those from Dextrin II were 13.6, 4.7 and 9.9, and 10.4, 5.1 and 4.3, respectively.