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.     

Histone-induced conformational changes in DNA as probed by quasi-elastic light scattering.

Quasi-elastic light scattering as measured by intensity fluctuation (self-beat) spectroscopy in the time domain can be profitably used to follow both the translational diffusion D and the dominant internal flexing mode τ_int of DNA and its complexes with various histones in aqueous salt solutions. Without histones, DNA is found to have D = 1.6 × 10^?8 cm²/sec and τ_int ? 5 × 10^?4 sec in 0.8 M NaCl, 2 M urea at 20°C. Total histone as well as fraction F2A induce supercoiling (D = 2.6 × 10^?8 cm²/sec, τ_int ? 2.8 × 10^?4 sec) whereas fraction F1 induces uncoiling (D = 1.0 × 10^?8 cm²/sec, τ_int ? 9.4 × 10^?4 sec). Upon increasing the salt concentration to 1.5 M the DNA–histone complex dissociates (D = 1.8 × 10^?8 cm²/sec). Upon decreasing the salt concentration to far below 0.8 M, the DNA–histone complex eventually precipitates as a chromatin gel.     

Inelastic light scattering studies of solutions of the chloroplast coupling factor (CF1) at high and low ionic strength

The translational diffusion coefficient of CF₁ at low and high protein concentration as well as at different ionic strength (0.05 – 1.65 M) wsa determined by means of quasi-elastic light scattering experiments. The diffusion coefficient changes from D_20,w^o = 3.12 × 10^?7 cm² · sec^?1 at 0.05 M, pH 7.8, 20°C, to D_20,w^o = 3.52 × 10^?7 cm² · sec^?1 at 1.6 M, pH 7.8, 20°C. At high enzyme concentration (20 mg/ml) and under crystallization conditions (Paradies, BBRC 91: 685, 1979) CF₁ behaves as a solution of “true” hard spheres, whereas at low salt concentration the ionic atmosphere has a larger spatial extent, resulting in a higher effective hydrodynamic radius (R_H = 65 Å).     

Dynamic light scattering as a probe of superhelical DNA-intercalating agent interaction

Polarized dynamic light-scattering measurements on superhelical pBR322-plasmid DNA solutions in 0.2M NaCl, 2 mM NaPi, pH 7.0, 2 mM EDTA result in a translational diffusion coefficient D = (3.77 ± 0.10) × 10^?8 cm²/s for the native molecule. Modeling the DNA, in the simplest approximation, as a 10 × 440-nm effective hydrodynamic rigid rod yields a good fit to the apparent diffusion coefficient angular-dependence data up to 70°; the model fails at higher angles, probably due to the effects of flexibility or branching of the rod. Diffusion coefficient titration experiments with a platinum complex intercalating agent (PtTS) result in a titratable superhelix density of σ = ?0.079 ± 0.008 under our experimental conditions, corresponding to about 34 superhelical turns in the native DNA. The DNA contour length predicted by our two independent results, the rod dimensions and the number of superhelical turns, is in excellent agreement with the contour length calculated from the number of base pairs, supporting the hydrodynamic approximation of an effective rodlike structure for this small DNA molecule in solution.     

Dynamic light scattering from thin rigid rods: Anisotropy of translational diffusion of tobacco mosaic virus

An Exact theoretical expression for the apparent diffusion coefficient D_app(K) of a thin rigid rod with arbitrary anisotropy of its translational diffusion diffusion coefficient is derived from the first cumulant of its dynamic structure factor. D_app(K) is predicted to reach a limiting plateau value at extermely large values of KL, where K is the scattering vector and L the rod length. Howerver, that limiting plateau value is approached only very slowly along a quasi-plateau with a very gradual slope. Dynamic light-scattering studies have been performed on tobacco mosaic virus from K² = (0.4–20) × 10¹⁰ cm^?2 using 632-8-nm laser radiation. The present data yield D₀ = (4.19 ± 0.10) × 10^?8 cm²/s (corrected to 20,w conditions) and, with literature data to establish L = 2980 Å and the rotational diffusion coefficient D_R = 318s^?1, yield also Δ ≡ D_∥ ? D_⊥ = (1.79 ± 0.38) × 10^?8 cm²/s. The experimental data closely follow the curve of D_app(K) vs K² calcuated for these parameters. The present value of D₀ substantially exceeds all previous dynamic light-scattering values, but is in good aggreement with previous sedimentation data, which were confirmed for the presemt sample. The anisotropy ratio Δ/D₀ = 0.43 ± 0.09 is in accord with theoretical predictions based on the modified Kirkwood algorithm, despite the fact the D₀ lies significantly below its corresponding theoretical value. The present data largely predlude the possibility that both D₀ and Δ/D₀ could simultaneously match their theoretical predictions. We present a detailed comparison of the experimental data with the calculations of Tirado and Garcia de la Torre based on the modified Kirkwood algorithm and with the Broersma formulas.     

Static and dynamic light scattering from dilute insulin solutions

Static and dynamic light-scattering measurements are reported on zinc-insulin at room temperature (21 ± l°C) and pH = 6.88 in 0.1M NaCl aqueous solution. The experiments were performed at very low concentration, in the range 0.12 × 10^?4 to 0.90 × 10^?4 g cm^?3. Within experimental error, we find no evidence for a critical micellar concentration in this system. The aggregation phenomenon starts immediately after preparation of the solutions, and takes several days to come to stable equilibrium. The concentration dependence of the diffusion coefficients, D _z, = D_o (1 — k_DC), is negative, and k_D was observed to decrease as a function of time, while the aggregate size was found to increase. The equivalent concentration coefficient, ?2BM _W, obtained from static light scattering, showed a similar behavior, and, within experimental error, was found to be numerically equal to k_D. From the relation found between the diffusion coefficient at infinite dilution and the molecular weight of the aggregates, log D₀ = ?0.240 log M _w ? 5.077, we deduce that the insulin aggregates are compact structures with a characteristic radius of 0.71 Å/(dalton)^1/3, surrounded by a hydration layer of a thickness of 8.0 Å. The equilibrium aggregation number is approximately 10.     

Transient electric birefringence of T-even bacteriophages. II. T4B in the presence of tryptophan and T4D.

Measurements of electric birefringence, sedimentation velocity, and biological adsorption rate are used to study the properties of bacteriophage T4B in the presence of excess tryptophan. The adsorption rate determined in borate buffer pH 9 (at 25°C) increases from 0.003 × 10^?8 ml min^?1 (0.025 M) to 0.130 × 10^?8 ml min^?1 (0.150 M). The Kerr coefficient, rotational diffusion coefficient, and the sedimentation coefficient of the phage are also dependent on buffer concentration and reach plateau values above 0.12 M given by K_sp = ?(275 ± 18) × 10^?9 OD^?1 cm² statvolt^?2, D_25,w = 133 ± 4 sec^?1, and s_20,w = 818 ± 11 S. From a comparison of electric birefringence measurements of T4B and T4D it is concluded that T4D and T4B (in the presence of excess tryptophan) exhibit a similar hydrodynamic behavior. The change in physical parameters is solely due to a shift in fiber configuration. At high buffer concentrations the fibers make an angle of approximately 3π/4 with the sheath and the permanent dipole moment is about 200,000 D. This dipole moment is roughly ten times as large as that of a phage particle with nonextended fibers. This difference may be due to a change in hydrodynamic center upon fiber extension or to the presence of positive charges on the fiber tips, or both. At intermediate buffer concentrations the phage population behaves as if it were monodisperse. Probably not all six fibers are extended under such conditions.     

Measurement of diffusion coefficient and electrophoretic mobility with a quasielastic light-scattering–band-electrophoresis apparatus

We have constructed an apparatus for the simultaneous measurement of electrophoretic mobility, μ, and diffusion coefficient, D, of macromolecules and cells. It combines band electrophoresis in a vertical, sucrose-gradient stabilized column, with quasielastic laser light-scattering determination of the diffusion coefficient of the species within the band. The entire electrophoresis cell is scanned through the laser beam of the quasielastic laser light-scattering apparatus by a vertical translation stage. Total intensity light-scattering measurement at each point in the cell gives the macromolecular concentration at that point. Solvent viscosity and electrical potential are measured at each point in the cell. Application of this apparatus to resealed red blood cell ghosts and to bovine hemoglobin indicates that measurements of field, viscosity, and migration distance are reliable, and that electroosmosis is insignificant. Application to T4D bacteriophage gives μ_20,w = (?1.05 ± 0.05) × 10^?4 cm²/V sec and D_20,w = (3.35 ± 0.10) × 10^?8 cm²/sec for fiberless particles, and μ_20,w = ?(0.59 ± 0.03) × 10^?4 cm²/V sec and D_20,w = (2.86 ± 0.09) × 10^?8 cm²/sec for whole phage with 6 fibers. Approximate analysis of these results with the Henry electrophoresis theory for spheres in dicates that each fiber contributes about 193 positive charges to the phage particle, compared with 327 from amino-acid analysis. The advantages and disadvantages of this apparatus, relative to conventional electrophoresis and to electrophoretic light scattering, are discussed.     

Dynamic light scattering of the bacteriophage T4B: determination of translational diffusion coefficients and centres of rotation

Using dynamic light scattering, the translational diffusion coefficient (D_T) and the distance between the hydrodynamic centre and the centre of the head (r₀) of the bacteriophage T4B have been determined. For a particle with retracted tail fibres we found D_T20.w =2.88 (2.88 ± 0.02) × 10^?8cm²s^?1 and r₀ = 52 ± 1 nm. For a phage with fully extended tail fibres D_T20w = (.210 ± 0.02) × 10^?8cm²s^?1 and r₀ = 112 ± nm. These data were obtained by interpreting the correlation function using a theory which takes into account the influence of the lollipop shape of the phage. In the literature this influence has not been taken into account, which has led to erroneous values of diffusion coefficients for T4B and other phages. The sedimentation coefficient of T4B phage is 1040 ± 5 S (fibres retracted) or 829 ± 4 S (fibres extended). With the above mentioned diffusion coefficients, these values correspond to a molecular weight of 236 × 10⁶ ± 3 × 10⁶. Finally, the theory used in this study is applied to other bacterial viruses, to correct reported values of the translational diffusion coefficients and of the corresponding molecular weights of these viruses.     

Li+ counterion self-diffusion in ordered DNA

The anisotropic self-diffusion coefficient of ⁷Li⁺ (I = 3/2) counterions has been studied in hydrated, macroscopically oriented Li-(B)DNA fibers at relatively high water contents, corresponding to approximate DNA-DNA helix axis distances of 22–35 Å, using the pulsed field gradient hmr spin-echo method. Self-diffusion coefficients parallel (D_∥) and perpendicular (D_?) to the DNA helix axis increase with increasing salt content and with increasing DNA-DNA helix axis distance. The observed anisotropy D_∥/D_? decreases from 1.6 to 1.2 with the DNA-DNA separation increasing from 22 to 35 Å in the salt-free sample. This result can be understood by the obstruction effect caused by the DNA molecules themselves. The values of the Li⁺ self-diffusion coefficients in the most water-rich system with no added salt (corresponding to an approximate distance of 35 Å between the DNA helix axes) were D_∥ ～ 1.15 × 10^?10 m² s^?1 and D_? ～ 0.98 × 10^?10 m² s^?1, compared to 9.14 × 10^?10 m² s^?1 for the diffusion of Li⁺ in an aqueous solution of LiCl (～ 2.1M). The possible occurrence of restriction effects in the DNA fibers have also been studied by determining the self-diffusion coefficient at different effective diffusion times. The self-diffusion coefficient of Li⁺ in the sample with the largest DNA-DNA helix axis distance seems to be independent of the effective diffusion time, which indicates that the lithium ions are not trapped within impermeable barriers. The possibility of diffusion through permeable barriers has also been investigated, and is discussed. © 1994 John Wiley & Sons, Inc.     

Translational and rotational diffusion of tobacco mosaic virus from polarized and depolarized light scattering

The translational and rotational dynamics of tobacco mosaic virus in sodium phosphate buffer (pH =7.5) solutions has been investigated by polarized and depolarized light scattering Rayleigh linewidth studies. For concentrations ranging from 1.75 × 10^?4 g ml^?1 to 0.25 × 10^?4 g ml^?1 the translational diffusion coefficient (D_T) has been found to be slightly concentration dependent and extrapolation to zero concentration gives D₀^20°C = 0.34 ± 0.01 × 10^?7 cm²S^?1. A full analysis of the polarized spectra obtained at high and low scattering angles and the depolarized spectra at near zero scattering angles has enabled these techniques to be compared and the rotational diffusion constant D_R to be determined. At a solution concentration of 1.75 × 10^?4 g ml^?1 a mean value is found to be D_R^20°C = 350 ± 30s^?1. These values of D_T and D_R are in approximate agreement with calculations based on models of the tobacco mosaic virus molecule as a cylindrical rod.     

Hydrodynamic properties of mucins secreted by primary cultures of Guinea-pig tracheal epithelial cells: determination of diffusion coefficients by analytical ultracentrifugation and kinetic analysis of mucus gel hydration and dissolution

We have used two different approaches to determine hydrodynamic parameters for mucins secreted by guinea-pig tracheal epithelial cells in primary culture. Cells were cultured under conditions that promote mucous cell differentiation. Secreted mucins were isolated as the excluded fraction from a Sepharose CL-4B gel filtration column run under strongly dissociating conditions. Biochemical analysis confirmed the identity of the high molecular weight material as mucins. Analytical ultracentrifugation was used to study the physical properties of the purified mucins. The weight average molecular mass (M _w ) for three different preparations ranged from 3.3×10⁶ to 4.7×10⁶ g/mol (corresponding to an average structure of 1 – 2 subunits), and the sedimentation coefficient from 25.5 to 35 S. Diffusion coefficients ranging from 4.5×10^–8 to 6.4×10^–8 cm²/s were calculated using the Svedberg equation. A polydispersity index (M _z /M _w ) of ∼1.4 was obtained. Diffusivity values were also determined by image analysis of mucin granule exocytosis captured by videomicroscopy. The time course of hydration and dissolution of mucin was measured and a relationship is presented which models both phases, each with first order kinetics, in terms of a maximum radius and rate constants for hydration and dissolution. A median diffusivity value of 8.05×10^–8 cm²/s (inter-quartile range = 1.11×10^–7 to 6.08×10^–8 cm²/sec) was determined for the hydration phase. For the dissolution phase, a median diffusivity value of 6.98×10^–9 cm²/s (inter-quartile range = 1.47×10^–8 to 3.25×10^–9 cm²/sec) was determined. These values were compared with the macromolecular diffusion coefficients (D _20,w ) obtained by analytical ultracentrifugation. When differences in temperature and viscosity were taken into account, the resulting D _37,g was within the range of diffusivity values for dissolution. Our findings show that the physicochemical properties of mucins secreted by cultured guinea-pig tracheal epithelial cells are similar to those of mucins of the single or double subunit type purified from respiratory mucus or sputum. These data also suggest that measurement of the diffusivity of dissolution may be a useful means to estimate the diffusion coefficient of mucins in mucus gel at the time of exocytosis from a secretory cell. Received: 10 March 1998 / Accepted: 27 March 1998     

Electron microscopy of human interphase nuclei

Quantitative electron microscopy was used to analyze surface-spread, critical-point-dried human interphase nuclei and chromatin. The following information is presented: (1) Unstimulated interphase nuclei of lymphocytes from peripheral blood have a mean dry mass of 50.30×10^?12 g. The mean dry mass of stimulated nuclei of lymphocytes was determined to be 59.34×10^?12 g, a significant statistical difference from the unstimulated ones. (2) Mean diameter of chromatin fibers and mean fiber mass per micron were 199ű15% coefficient of variation (C.V.) and 5.95×10^?16g×29% C.V., respectively. (3) A line of regression of fiber mass on fiber diameter for 83 fibers indicated that a 200-Å fiber has a mass of 5.86×10^?16g/μ, or almost the same as the mean fiber mass of 5.95× 10^?16g/μ. (4) With the value 7×10^?12g for the DNA content of an unstimulated lymphocyte nucleus, a total length of 215 cm is calculated for the DNA double helix. When this length is compared to the mean length of chromatin fiber per nucleus (7.59 cm), a ratio of 28.3 to 1 results, which is called the DNA-packing ratio. (5) This DNA-packing ratio of 28.3 is reasonably close to the packing ratio of 26.9 suggested from model calculations for the second DNA supercoil in a 200-Å chromatin fiber.     

Hydrodynamic properties of mushroom tyrosinase

The hydrodynamic properties of mushroom tyrosinase were determined at pH 6.5 using a Sephadex G-200 column. From the comparison of its gel-filtration behaviour with those of standard proteins, the following parameters were calculated: MW (122 500 ± 1%), Stokes' radius (42.75 × 10^?8 cm²/sec), diffusion coefficient (5.048 × 10^?7 cm²/sec) and frictional ratio (1.26). These values suggest a globular conformation of this enzyme.     

Isolation,characterization, and magnesium-induced self-association kinetics of discrete aggregates of RecA protein from Escherichia coli

Dynamic and static intensity light scattering techniques were employed to identify conditions allowing preparation of homogeneous solutions of distinct oligomeric states of RecA protein. These hydrodynamically distinguishable oligomer populations of RecA protein were obtained in homogeneous pure quantities sufficient for physical studies. Results indicate two fairly narrow distributions of RecA oligomers comprised on average of 42 ± 3 and 18 ± 1 RecA monomers. These structures, denoted RecA₄₂ and RecA₁₈, respectively, could be obtained reproducibly in milligram quantities and were stable for at least one week. This enabled reliable characterizations of their hydrodynamic properties by dynamic and total intensity light scattering. These measurements revealed RecA₄₂ had an average translational diffusion coefficient, D₂₀(L) = 8 ± 2 × 10⁻⁸ cm²/s, molecular weight, M_r = 1.6 ± 0.1 × 10⁶, and radius of gyration, R_G = 465 ± 29 Å. The smaller aggregate, RecA₁₈, had D₂₀(S) = 20.5 ± 2.5 × 10⁻⁸ cm²/s. M_r = 7.0 ± 0.4 × 10⁵, and R_G = 300 ± 20 Å. Heating RecA₁₈ at 37°C overnight resulted in conversion to a species with hydrodynamic properties indistinguishable from RecA₄₂, called RecA_18/42. Conversion of RecA₄₂ to RecA₁₈ occurred almost instantaneously by 50% dilution at 38°C or very slowly with incubation at 4°C for at least 39 days. Self-association reactions of the three starting oligomeric states (RecA₁₈, RecA₄₂, and RecA_18/42) induced by MgCl₂ were monitored at several temperatures by dynamic light scattering. Results of these experiments provided evaluations of kinetic activation parameters of the self-association reactions. The activation parameters found for each starting oligomeric state of the protein were significantly different, revealing the variable influence of MgCl₂ on the activation barriers to RecA self-association. Highly aggregated equilibrium solutions that ultimately form in solutions of each starting oligomeric species, incubated in MgCl₂ at 38°C for four days, were characterized by total intensity light scattering. Interpretations of these data in terms of characteristic behavior of random polymers suggests the surface morphologies of these highly associated equilibrium states formed from RecA₄₂ and RecA_18/42 are similar but contrast with that of RecA₁₈. Calculated values of the translational diffusion coefficient D₀ were obtained for oligomeric structures modeled as helical arrays of connected monomer spheres. Best agreement with experimentally determined diffusion coefficients required that constituent monomer spheres of RecA₄₂ have radii 33–40% larger than the monomer spheres of RecA₁₈. Results suggest the hydrodynamically distinct oligomeric forms of RecA may reside in conformational states with different surface exposure of hydrophobic residues, which results in substantial differences in local solvation/hydration. © 1996 John Wiley & Sons, Inc.     

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.     

Translational diffusion coefficient of α-chymotrypsinogen A by laser-light-scattering spectroscopy

The translational diffusion coefficient of a pure sample of α-chymotrypsinogen A is measured by laser light scattering to give a value of D_20,w⁰ = (8.40 ± 0.15) × 10^?7 cm²/sec.     

Dynamic light scattering from collagen solutions. I. Translational diffusion coefficient and aggregation effects

Solutions of native collagen extracted from rat tail tendons in neutral salt solution have been studied by dynamic light scattering. The spectra obtained are consistent with the presence in solution of both single rod-shaped collagen molecules and aggregates of molecules. No contribution to the spectrum has been detected at any scattering angle from rotational diffusion of single molecules, although a measurable broadening effect is expected at high angles. The translational diffusion coefficient D of single molecules, calculated from the broader spectral component, shows an anomalous dependence on collagen concentration with a maximum value of D_20,w = 8.6 ± 0.2 × 10^?12 m²/sec near the concentration 0.04% by weight. Above 0.05% D falls linearly with increasing concentration and takes the value D _20,w = 8.1 ± 0.2 × 10^?12 m²/sec at 0.064% collagen.