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1.
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 [so20.w = 9.5 ± 0.7) S; ks = (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 Mw 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 Do20.w = (3.02 ± 0.05) × 10−8 cm2·s−1 and the concentration-dependence parameter kD = (370 ± 15) mL/g. Combination of so20.w with Do20.w via the Svedberg equation gave another estimate for Mw of ≅ 2.4 × 106 g/mol, again in good agreement. Both the Wales-van Holde ratio (ks/[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.  相似文献   

2.
Bacteriophage φ6 has been studied by small-angle X-ray scattering, intensity-fluctuation spectroscopy, analytical ultracentrifugation, and spectroscopy. The sedimentation coefficient (s200, w) is 375 S, the diffusion coefficient (D200, w) is 2.66 · 10?8 cm2/s. Using the Svedberg equation and an estimate of the partial specific volume, the Mr is 1.49 ± 0.32 · 108.A simple model which describes φ6, is a central sphere consisting of RNA and protein of radius 330 Å and an outer shell of low electron density 40 Å thick. The RNA may form five concentric shells in the region r = 140?290 A?  相似文献   

3.
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 μ20,w = (?1.05 ± 0.05) × 10?4 cm2/V sec and D20,w = (3.35 ± 0.10) × 10?8 cm2/sec for fiberless particles, and μ20,w = ?(0.59 ± 0.03) × 10?4 cm2/V sec and D20,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.  相似文献   

4.
This paper reports the first detailed study of the physicochemical properties of a fatty acid synthetase multienzyme complex from a mammalian liver. Fatty acid synthetase from pig liver was purified by a procedure including the following main steps: (i) preparation of a clarified supernatant solution (50,000 g), (ii) ammonium sulfate fractionation, (iii) DEAE-cellulose chromatography to separate 11 S catalase from the 13 S fatty acid synthetase, (iv) a preparative sucrose density gradient step to remove a 7 S impurity, and (v) a calcium phosphate gel step to remove an unusual yellow 16 S heme protein to yield a colorless preparation. The purified fatty acid synthetase was colorless and showed a single symmetrical peak in sucrose density gradient and conventional sedimentation velocity experiments. Fatty acid synthetase was very stable at 4 °C in the presence of 1 mm dithiothreitol and 25% sucrose. Extrapolation to zero protein concentration yielded values of So20,w = 13.3 S and Do20,w = 2.60 × 10?7cm2/s for the sedimentation and diffusion coefficients of the enzyme. Frictional coefficient values of 1.55 and 1.56 × 10?7 cm, respectively, were calculated from the values for the sedimentation and diffusion coefficients. Based on these frictional coefficient values, the Stokes radius of the enzyme was calculated to be 82.4 Å. Sedimentation and diffusion coefficient data yielded a molecular weight value of Mw (sD) = 478,000 and sedimentation equilibrium data yielded a value of Mw = 476,000. Preliminary intrinsic viscosity measurements at 20 °C gave a value of 7.3 ml/g, indicating that the enzyme is somewhat asymmetric. This is supported by the value of 1.58 calculated for the frictional ratio and by the fact that the values for the sedimentation and diffusion coefficients are both slightly lower than expected for a globular protein of molecular weight 478,000. The enzyme possesses about 90 SH groups per molecule, assuming a molecular weight of 478,000. The ultraviolet absorption spectrum of the enzyme shows a maximum at 280 nm and an unusual shoulder at 290 nm. The fluorescence spectrum of the enzyme is dominated by tryptophan fluorescence and, over the excitation range of 260–300 nm, there is a single emission maximum at 344 nm.  相似文献   

5.
Using dynamic light scattering, the translational diffusion coefficient (DT) 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 DT20.w =2.88 (2.88 ± 0.02) × 10?8cm2s?1 and r0 = 52 ± 1 nm. For a phage with fully extended tail fibres DT20w = (.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.  相似文献   

6.
Quasi-elastic light scattering measurements have been carried out for (i) human and bovine fibrinogens under identical conditions, (ii) for a large number of fractionated and unfractionated fibrin intermediates at various pH (10.0 to 10.5), (iii) for three intermediates polymerized at pH 7.4 and stabilized at pH 10.5 and (iv) for a gelled clot. Human and bovine fibrinogen proved to have noticeably different diffusion coefficients (same Mw) indicating a longer rod on the average for the human than for the bovine fibrinogen. This finding is in agreement with measurements of the integrated light scattering from these fibrinogens where a mass per unit length MwLw of 3860 and 5460 g mol?1 nm?1 were found, respectively. No angular dependence of the apparent diffusion coefficient Dapp = Γ/q2 was found for the monomers and the fibrin clot; all other samples from (ii) showed an angular dependence if Mw ? 1.76 × 106, were Γ is the first cumulant of the time correlation function and q = (4π/λ) sin?/2. A plot of Dapp/D versus q2〈S2〉 gave curves which, for the low molecular weight samples, correspond to ellipsoids. For the longer fibrils a dynamic behaviour in between that of a long rigid rod and random coil was found and indicates a certain flexibility of the fibrils. The diffusion coefficients from (ii) decay with increasing molecular weight and can be described by Perrin's theory for ellipsoids when M2 < 2 × 106 while a beter agreement with Kirkwood's theory of long rods is obtained for the longer fibrils. The hydrodynamic behaviour of (iii) indicates short and unbranched rods of considerable thickness.  相似文献   

7.
The molecular weight of the adenovirus type 2 hexon was calculated from sedimentation equilibrium, light scattering and sedimentation and diffusion experiments. The extinction coefficient, E1 cm1%, was determined to be 14.3 at 279 nm, from quantitative nitrogen and carbon analyses combined with the N,C content calculated from the amino acid composition. Other parameters determined were: the partial specific volume, \?gn = 0.738 cm3 g?1; the refractive index increment, (?n?c)T,P = 0.193 cm3 g?1 at 435.8 nm; the sedimentation coefficient, s20,w0 = 13.0 S; and the diffusion constant, D20, w0 = 3.32 × 10?7 cm2 s?1. All molecular weights were between 355,000 and 363,000. Crystal density measurements were made on native and glutaraldehyde cross-linked crystals and the molecular weights calculated from these data were compared with the precise molecular weight determined by physico-chemical methods.Only one polypeptide of molecular weight 120,000 was found in reduced, or reduced and alkylated, hexon. Four or six organomercurial molecules were bound per 120,000 molecular weight of native hexon upon titration with 2-chloromercuri-4-nitrophenol and 2-chloromercuri-4,6-dinitrophenol, respectively. With 5,5′-dithiobis (2-nitrobenzoic acid) only one SH-group per 120,000 could be titrated in native hexon, but after denaturation in 1% sodium dodeeyl sulphate five more SH-groups reacted per 120,000 molecular weight. Thus there are three identical polypeptides of molecular weight 120,000 per hexon of total molecular weight 360,000.  相似文献   

8.
The molecular weight and polypeptide chain stoichiometry of the native pyruvate dehydrogenase multienzyme complex from Escherichia coli were determined by independent techniques. The translational diffusion coefficient (Do20,w) of the complex was measured by laser light intensity fluctuation spectroscopy and found to be 0.90 (±0.02) × 10?11m2/s. When this was combined in the Svedberg equation with the measured sedimentation coefficient (so20,w = 60.2 (±0.4) S) and partial specific volume (v? = 0.735 (±0.01) ml/g), the molecular weight of the intact native complex was calculated to be 6.1 (±0.3) × 106. The polypeptide chain stoichiometry (pyruvate decarboxylase: lipoate acetyltransferase: lipoamide dehydrogenase) of the same sample of pyruvate dehydrogenase complex was measured by the radioamidination technique of Bates et al. (1975) and found to be 1.56:1.0:0.78.From this stoichiometry and the published polypeptide chain molecular weights estimated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis, a minimum chemical molecular weight of 283,000 was calculated. This structure must therefore be repeated approximately 22 times to make up the native complex, a number which is in good agreement with the expected repeat of 24 times if the lipoate acetyltransferase core component has octahedral symmetry. It is consistent with what appears in the electron microscope to be trimer-clustering of the lipoate acetyltransferase chains at the corners of a cube. It rules out any structure based on 16 lipoate acetyltransferase chains comprising the enzyme core.The preparation of pyruvate dehydrogenase complex was polydisperse: in addition to the major component, two minor components with sedimentation coefficients (so20,w) of 90.3 (±0.9) S and 19.8 (±0.3) S were observed. Together they comprised about 17% of the total protein in the enzyme sample. Both were in slowly reversible equilibrium with the major 60.2 S component but appeared to be enzymically active in the whole complex reaction. The faster-sedimenting species is probably a dimer of the complex, whereas the slower-sedimenting species has the properties of an incomplete aggregate of the component enzymes of the complex based on a trimer of the lipoate acetyltransferase chain.  相似文献   

9.
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 〈R21/2z = (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 D020, w = (2.23 ± 0.06) × 10?8 cm2/sec which combined with S020, 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.  相似文献   

10.
《FEBS letters》1986,200(1):226-230
Magnesium binding to cation-depleted blue bacteriorhodopsin (b-bR) was studied spectrophotometrically as well as by following stopped-flow kinetics. There exist three kinetically different steps in the binding process, yielding purple bacteriorhodopsin (p-bR). Since only the firtst step is dependent on the concentration of the reactants, the reaction scheme
can be proposed as the simplest model, with MgbR being the first intermediate and ΣI denoting a set of successive intermediates. According to this model k1, k−1 and k2 are calculated to be 2.8 × 104 M−1 · s−1, 5.0 × 10 s−1 and 1 × 10−2 s−1, respectively.  相似文献   

11.
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, Rs, 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 Rs = 39 ± 2 nm, M w = 1.6 ± 0.3 × 106, which reassembled in 0.4M GdnHCl to form “reconstituted native” aggregates (PGA-raA1) with Rs = 121 ± 6 nm, M w = 17 ± 3 × 106. A second specimen of PGA-aA1 had Rs = 192 ± 10 nm, M w = 100 ± 10 × 106. The latter value was estimated from an empirical relationship between M w and Rs. After dissociation, this specimen reassembled to form PGA-raA1 with Rs = 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 Rs = 138 ± 9 nm, M w = 45 ± 8 × 106. From these values, we have calculated the weight-average number of subunits per aggregate Nw: 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 Nw 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.  相似文献   

12.
Static and dynamic light scattering measurements were made of solutions of pGem1a plasmids (3730 base pairs) in the relaxed circular (nicked) and supercoiled forms. The static structure factor and the spectrum of decay modes in the autocorrelation function were examined in order to determine the salient differences between the behaviors of nicked DNA and supercoiled DNA. The concentrations studied are within the dilute regime, which is to say that the structure and dynamics of an isolated DNA molecule were probed. Static light scattering measurements yielded estimates for the molecular weight M, second virial coefficient A2, and radius of gyration RG. For the nicked DNA, M = (2.8 ± 0.4) × 106g/mol, A2 = (0.9 ± 0.2) × 10−3 mol cm3/g2, and RG = 90 ± 3 nm were obtained. For the supercoiled DNA, M = (2.5 ± 0.4) × 106 g/mol, A2 = (1.2 ± 0.2) × 10−3 mol cm3/g2, and RG = 82 ± 2.5 nm were obtained. The static structure factors for the nicked and supercoiled DNA were found to superpose when they were scaled by the radius of gyration. The intrinsic stiffness of DNA was evident in the static light scattering data. Homodyne intensity autocorrelation functions were collected for both DNAs at several angles, or scattering vectors. At the smallest scattering vectors the probe size was comparable to the longest intramolecular distance, while at the largest scattering vectors the probe size was smaller than the persistence length of the DNA. Values of the self-diffusion coefficients D were obtained from the low-angle data. For the nicked DNA, D = (2.9 ± 0.3) × 10−8 cm2/s, and for the supercoiled DNA, D = (4.11 ± 0.21) × 10−8 cm2/s. The contribution to the correlation function from the internal dynamics of the DNA was seen to result in a strictly bimodal decay function. The rates of the faster mode Γint, reached plateau values at low angles. For the nicked DNA, Γint = 2500 ± 500 s−1, and for the supercoiled DNA, Γint = 5000 ± 500 s−1. These rates correspond to the slowest internal relaxation modes of the DNAs. The dependence of the relaxation rates on scattering vector was monitored with the aid of cumulants analysis and compared with theoretical predictions for the semiflexible ring molecule. The internal mode rates and the dependence of the cumulants moments reflected the difference between the nicked DNA and the supercoiled DNA dynamical behavior. The supercoiled DNA behavior seen here indicates that conformational dynamics might play a larger role in DNA behavior than is suggested by the notion of a branched interwound structure. © 1996 John Wiley & Sons, Inc.  相似文献   

13.
Activated white cells use oxidants generated by the heme enzyme myeloperoxidase to kill invading pathogens. This enzyme utilizes H2O2 and Cl, Br, or SCN to generate the oxidants HOCl, HOBr, and HOSCN, respectively. Whereas controlled production of these species is vital in maintaining good health, their uncontrolled or inappropriate formation (as occurs at sites of inflammation) can cause host tissue damage that has been associated with multiple inflammatory pathologies including cardiovascular diseases and cancer. Previous studies have reported that sulfur-containing species are major targets for HOCl but as the reactions are fast the only physiologically relevant kinetic data available have been extrapolated from data measured at high pH (>10). In this study these values have been determined at pH 7.4 using a newly developed competition kinetic approach that employs a fluorescently tagged methionine derivative as the competitive substrate (k(HOCl + Fmoc-Met), 1.5×108 M−1 s−1). This assay was validated using the known k(HOCl + NADH) value and has allowed revised k values for the reactions of HOCl with Cys, N-acetylcysteine, and glutathione to be determined as 3.6×108, 2.9×107, and 1.24×108 M−1 s−1, respectively. Similar experiments with methionine derivatives yielded k values of 3.4×107 M−1 s−1 for Met and 1.7×108 M−1 s−1 for N-acetylmethionine. The k values determined here for the reaction of HOCl with thiols are up to 10-fold higher than those previously determined and further emphasize the critical importance of reactions of HOCl with thiol targets in biological systems.  相似文献   

14.
J Greve  J Blok 《Biopolymers》1973,12(11):2607-2622
Measurements of the electric birefringence of suspensions of T4B in the absence of tryptophan and of fiberless T4 particles show that both kinds of particles are hydrodynamically equivalent. Their rotational diffusion coefficients corrected to 25°C and water viscosity (D25,w) are 280 ± 9 sec?1 and 295 ± 10 sec?1, respectively. These corrected rotational diffusion coefficients are almost independent of buffer concentration and temperature. The sedimentation coefficient (s20,w) of T4 B is equal to 1023 ± 12 S, a value which is likewise independent of buffer concentration. By analysis of the field strength dependence of the steady-state birefringence and by reversing pulse experiments it could be shown that the orientation in an electric field is largely due to a permanent dipole moment. This dipole moment is somewhat dependent on buffer concentration and amounts to about 24,000 debye for T4B and 95,000 debye for fiberless T4. An approximate calculation shows that the difference in dipole moment may be ascribed to positive charges on the fiber tip (at least ten per fiber), to negative charges along the fiber or (and) positive charges on the fiberless particle at those places where the fibers are attached in normal particles.  相似文献   

15.
The lateral diffusion of the excimer-forming probe pyrene decanoic acid has been determined in erythrocyte membranes and in vesicles of the lipid extracts. The random walk of the probe molecules is characterized by their jump frequency, vj, within the lipid matrix. At T = 35°C a value of vj = 1.6 · 103 s?1 is found in erythrocyte membranes. A somewhat slower mobility is determined in vesicles prepared from lipid extracts of the erythrocyte membrane. Depending on structure and charge of the lipids we obtain jump frequencies between 0.8 · 108 s?1 and 1.5 · 108 s?1 at T = 35°C. The results are compared with jump frequencies yielded in model membranes.The mobility of molecules perpendicular to the membrane surface (transversal diffusion) is investigated. Erythrocyte ghosts doped with pyrene phosphatidylcholine were mixed with undoped ghosts in order to study the exchange kinetics of the probe molecule. A fast transfer between the outer layers of the ghost cells (τ12 = 1.6 min at T = 37°C) is found. The exchange process between the inner and the outer layer of one erythrocyte ghost (flip-flop process) following this fast transfer occurs with a half-life time value of t12 = 100 min at T = 37°C.The application of excimer-forming probes presumes a fluid state of the membrane. Therefore we investigated the phase transition behaviour using the excimer technique. Beside a thermotropic phase transition at T = 23°C and T = 33°C we observed an additional fluidity change at T = 38°C in erythrocyte ghosts. This transition is attached to a separation of the boundary lipid layer from the intrinsic proteins. No lipid phase transition is observed in liposomes from isolated extracts of the erythrocyte membrane with our methods.  相似文献   

16.
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 (D20,w = 5·5× 10?7cm2s?1)and the sedimentation constant (s020,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.  相似文献   

17.
The kinetics and mechanism of a linear trihydroxamic acid siderophore (deferriferrioxamine B, H4DFB+) ligand exchange with Al(H2O)63+ to form mono(deferriferrioxamine B)aluminum(III) (Al(H2O)4H3DFB)3+ have been investigated at 25 °C over the [H+] range 0.001−1.0 M and I = 2.0 M (HClO4/NaClO4) by 27Al NMR. Kinetic results are consistent with Al(H2O)4(H3DFB)3+ formation and dissociation proceeding through a parallel path mechanistic scheme involving Al(H2O)63+(k2/k−1) and Al(H2O)5(OH)2+(k2/k−2) where k1 = 0.13 M−1 s−1, k−1 = 8.7 × 10−3 M−1 s−1, k2 = 2.7 × 103 M−1 s−1, and k−2 = 9.6 × 10−4 s−1. Relative complex formation rates at Al(H2O)63+ and Al(H2O)5OH2+, and comparison with kinetic data for a series of synthetic hydroxamic acids, suggest that an interchange mechanism is operative. These results are also discussed in relation to kinetic data for the corresponding iron(III)-deferriferrioxamine B system.  相似文献   

18.
Quasi-elastic light scattering has been used to characterize the oligomeric properties of solutions of glycerol-cycled bovine microtubule protein, and the properties of the 30 S oligomeric species and 6 S tubulin heterodimer prepared by gel filtration on Sepharose 6B. It is shown that in dimer preparations, as little as 0.04% by number of 30 S rings would account for the difference between an observed mean diffusion coefficient D20, W = 3.1 × 10?7 cm2 s?1 and the value of D20, W = 5.1 × 10?7 cm2 s?1 calculated for tubulin dimer of Mrel 100,000. The 30 S ring has an observed diffusion coefficient of D20, W = 0.49 × 10?7 cm2 s?1. These values are not changed significantly by the presence of 4 m-glycerol, indicating the persistence of 6 S and 30 S forms for dimer and ring, respectively.Mixtures of ring and dimer components of this preparation behave as a non-interacting two-component system, indicating the absence of substantial re-equilibration between the species at 5 °C and pH 6.5.The effect of salt on ring and microtubule protein samples indicates partial dissociation, consistent with the formation of additional intermediate oligomeric forms.In quasi-elastic light scattering measurements adapted to kinetic studies, changes in the oligomeric composition of microtubule protein are detected in the early stages of the reversible assembly process at pH 6.5. A 25% decrease in scattered light intensity, without significant change in mean diffusion coefficient, indicates the lability of the ring oligomeric structures, which undergo partial transformation to alternative oligomeric species under these assembly conditions.  相似文献   

19.
The micellar properties of gangliosides in water solutions were investigated by quasielastic light scattering measurements. GM1 and GD1a gangliosides were isolated from calf brain, purified to more than 99% and dissolved in 0.025 M Tris—HCI buffer (pH 6.8) at 37°C. The average intensity of scattered light and the intensity correlation function were measured by an apparatus including a 5145 Å argon laser and a real-time digital correlator. The scattered intensity data allowed the derivation of an upper limit to the critical micelle concentration (c0) and the evaluation of the molecular weight (M) of the micelle. The intensity correlation function gave the diffusion coefficient D, and hence the hydrodynamic radius RH, and also contained information on the polydispersity of the sample. We find co < 1 × 10?6 M for both GM1 and GD1a, M = 532 000 ± 50 000 and RH = 63.9 ± 2 A? for GM1, and M = 417 000 ± 40 000 and RH = 59.5 ± 2 A? for GD1a. The mixture 3:1 of the two gangliosides gave intermediate values for all examined parameters. The presence of cations, within the physiological concentration range. and, in particular of Ca2+, did not influence significantly the values of co and the main features of the micelle.  相似文献   

20.
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 s20, w0 = 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 s20,w0 measured with the analytical ultracentrifuge seems to confirm the validity of the empirical relationship proposed by Freifelder (1970), between s20, w0 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.  相似文献   

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