Self-interaction of dynein fromTetrahymena cilia

Summary The molecular mass (M_r) and enzymic activity of the larger dynein species fromTetrahymena thermophila has been studied in the high (600mm) to low (40mm) ionic strength range. The apparent M_r is found to vary with both ionic strength (by sedimentation velocity and quasi elastic light scattering analysis) and with protein concentration at low ionic strength (by sedimentation equilibrium analysis). These data indicate a strong self-interaction, resulting in dimer formation under low salt conditions. There is no evidence for the formation of species of higher than dimeric mass. A molecular mass for the dynein monomer of 1.64×10⁶ daltons has been determined, a value rather lower than previous published estimates.The ATPase activity of dynein increases with increasing ionic strength. The possible relationship between this effect and the self-association phenomenon is discussed.     

Innermolekular vernetzte Makromoleküle (Mikrogel): Synthese,Eigenschaften und Nachweis in Gemischen

Emulsionpolymerisation of styrene with divinylbenzene gives spherical particles of nearly uniform diameter, with molecular weights M between 5.10⁵ and 3.10⁷, with various degrees of crosslinking density and with many endgroups. Light scattering, viscosimetry, gelpermeation, diffusion and rheological measurements show, that intramolecular crosslirrking has significant and partly unknown effects: particles of a definite swelling degree have second virial coefficients A₂, which are nearly independent of M. This is contrary to the theory for unsolvated spheres. The FOX -FLORY -constant ? increases with intramolecular crosslinking to the value calculated for spheres, which is larger than that for coils of linear molecules. Gel permeation measures the diameter of the particles in a similar way as hydrodynamics but different from light scattering. At low concentrations the solutions of such gels behave viscosimetrically like dispersions of spheres. At higher concentrations the gel particles deswell and show a concentration dependence of viscosity between that of hard spheres and linear molecules. The viscosity of the solution and its shear dependence are smaller than that of linear molecules, the shear stress τ_w in the turning point of the curves log η vs. log τ being also much smaller. The osmotic pressure at high concentrations is a swelling pressure and depends on the degree of crosslinking. Intramolecular crosslinks reduce the melt viscosity, if the molecular weight of the chain segments between branching points is M_e > 15000. At very high crosslinking densities the melt viscosity is increased. Modulus and density of glassy gel polymers are nearly normal, but the stress at breaking is decreased with increasing intramolecular crosslinking. Particles have higher swelling degrees and intrinsic viscosities as well as lower FOX -FLORY -constants, if they are polymerised in the presence of toluene. Gels cannot be separated quantitatively from linear molecules by sedimentation or by evaluating an abnormal angular dependence of scattered light. But one may calculate the amount of gel and its degree of crosslinking, if one knows the behaviour of linear molecules and measures M_w [η] and a third independent quantity, e.g. melt viscosity, non-NEWTON ian behaviour or the concentration dependence of the viscosity of solutions.     

Etude par diffusion de la lumière du système ternaire solvant/polydiméthylsiloxane/poly(méthacrylate de méthyle)

The ternary system solvent (1)/polydimethylsiloxane (2)/poly(methyl methacrylate) (3) was investigated by light scattering. First the measurements were realized on mixtures containing low-molecular-weight polydimethylsiloxane: M₂ ? M₃. In a second step, high and comparable molecular weights of both polymers were chosen for the measurements. The thermodynamic interaction parameter χ₂₃ was determined from light scattering data (spinodal) and demixtion curves as a function of concentration and molecular weight of the polymers. Also the variation of the angular repartition of the scattered light was studied and the results were compared with calculated values.     

Characterization of different oligomeric species of the Yersinia enterocolitica outer membrane protein YadA

The oligomeric structure of the plasmid-encoded outer membrane protein YadA of Yersinia enterocolitica was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and sucrose gradient sedimentation, respectively. The apparent molecular weight (M _r) of the oligomeric 200-kDa YadA species detected by SDS-PAGE varied from 152,000 to 240,000 depending on the respective acrylamide concentration. The atypical electrophoretic behavior of the 200-kDa YadA species results from an exceptionally high relative free mobility as revealed by the Ferguson plot. In contrast, the apparent M _r of 53,000 of the YadA monomer was independent of the acrylamide concentration. An additional oligomeric 116-kDa YadA species was detected by SDS-PAGE when membrane preparations of Y. enterocolitica were solubilized in SDS at 37 °C. The gel-purified 116-k Da YadA species was completely converted to the 200-kDa species by heating at 100 °C and to the monomeric form (M _r 53,000) by heating in the presence of 10 M urea without reducing agents, respectively. This suggests that the 116-kDa YadA species represents the native oligomeric form of YadA, whereas the 200-kDa species is only generated from native YadA during denaturation in SDS. The significance of the 116-kDa YadA species is also supported by the rather slow sedimentation at about 6 S of detergent-solubilized YadA in sucrose gradients, which probably contains only two or three monomers.     

Herstellung und Molmassencharakterisierung von poly[(dimethyliminio)ethylen(dimethyliminio)methylen-1,4-phenylenmethylen-dichlorid]

The ionene poly[(dimethyliminio) ethylene(dimethyliminio) methylene-1,4-phenylenemethylene dichloride] was synthesized and characterized. Number-, mass- and z-average molar masses M _n, M _w and M _z, and molar mass distribution were determined using equilibrium ultracentrifugation. With detection by means of Schlieren, interference, and UV optics. M _w was additionally determined by static light scattering. All measurements yield within the experimental error the same value of M _w viz. M _w = 12,2 · 10³ g/mol. This value agrees well with the value from an analysis of kinetical data, viz. M _w = 11,4 · 10³ g/mol. Molar mass distribution w(M) was determined from the concentration profile extrapolated to concentration γ₀ = 0; it is of a Wesslau-type and relatively broad. The non-uniformity U = M _w/M _n ? 1 ≈ 3. According to the theory of polycondensation a Schulz-Flory type of distribution should be obtained. A Schulz-Flory distribution is not achieved because the monomers p-xylylene dichloride and N,N,N′,N′-tetramethylethylenediamine exhibit higher reactivity in the initiation reaction than the homologs during chain propagation which results in a broader molar mass distribution than a Schulz-Flory distribution.     

Characterization of DEAE-dextran by means of light scattering and combined size-exclusion chromatography/low-angle laser light scattering/viscometry

Ten DEAE (2-(diethylamino)ethyl) dextran samples were investigated by means of static and dynamic light scattering, viscometry and size-exclusion chromatography (SEC) in combination with on-line small-angle laser light scattering (LALLS) and viscometry (VISC). In dilute solution the behavior of DEAE-dextran was compared with that of unsubstituted dextran and the molecular weight M dependences of the radius of gyration R_g, hydrodynamic radius R_h, intrinsic viscosity [η], second virial coefficient A₂ and z-average diffusion coefficient D _z were determined. The relationships for DEAE-dextran dissolved in a 0,8 molar sodium nitrate solution were nearly the same as for dextran dissolved in water with 0,05 wt.-% sodium azide and gave the same exponents. The molecular weight dependence of the intrinsic viscosity cannot be described by a Kuhn-Mark-Houwink relationship with a constant exponent. The slope in the plot of log [η] versus log M decreases with increasing molecular weight which indicates the occurrence of branching. By means of SEC/LALLS/VISC measurements the molecular weight distributions were determined. The distributions were calculated (1) directly from the light scattering signal, (2) from a calibration line obtained by light scattering data of a DEAE-dextran sample with a broad distribution and (3) from the intrinsic viscosity distribution obtained by the on-line viscosity/refractive index detector in combination with the [η]-M relationship. In order to get the correct molecular-weight dependence of the intrinsic viscosity it is necessary to determine the molecular weight distribution directly by LALLS (technique 1) and to combine this with the appropriate intrinsic viscosity data from the viscometer. Only the third technique, which is an extension of technique 1, gave satisfactory results over the whole molecular weight region observed.     

Streulichtmessungen an polymerlösungen zur bestimmung der verteilungsbreite des molekulargewichtes

By increasing the experimental accuracy of the classical light scattering technique it became possible to determine the width U = M_w/M_n ? 1 of the molecular weight distribution of polymers. For this purpose a very stable laser and a lock-in-amplifier were used allowing an accuracy of 0,2–0,3% in the scattering intensity. The numerical evaluation of the polydispersity is done by a Gauß-Newton procedure analysing the small curvature of the reciprocal scattering function. In an appropriate molecular weight range U is obtained with an error between 0,01 and 0,1 depending on the width of the molecular weight distribution of the sample tested.     

How is the z-Average Molar Mass Related to the Sedimentation Data?

Nowadays, students, professors, and researchers in the field of polymer education and research know how different average molar masses of polymer chains are defined in textbooks, but not everyone knows that these different averages actually come from different experimental methods, not purely from different mathematical definitions and calculations. Some of the detailed knowledge have been lost in literature and even textbooks during the polymer development process. The z-average molar mass (M_z) is one of the typical examples; namely, why it is called the z-average and how it is related to the concentration profile inside an ultracentrifuge cell. Currently, it is rather difficult, if not impossible, to find how to derive the final equation used in the calculation of M_z. It is the purpose of this paper to show a simple and nontedious derivation of how to obtain M_z from a sedimentation experiment, very different from the original old paper published by Lansing and Kraemer in 1935. It is also experimentally shown how to calculate M_z by using the ultracentrifuge data from a mixture of three polystyrene standards.     

Analysis of Heparan Sulfate from the Engelbreth-Holm-Swarm (EHS) Tumor

The size of the heparan sulfate chains from the Engelbreth-Holm-Swarm (EHS) tumor heparan sulfate proteoglycan (PG) was measured by several techniques in order to resolve uncertainty about their size and the chains were chemically characterized for comparison with other basement membrane heparan sulfate PGs. Heparan sulfate size was determined by gel filtration (M_r = 5.5 – 6.0 X 10⁴), by equilibrium sedimentation centrif-ugation (M_w = 6.8 × 10⁴), and by end group analysis (M_n = 7.1 × 10⁴). A higher molecular weight (HMW) (M_w = 2.13 × 10⁵) calculated from scattering measurements may reflect chain-chain interactions. Forty percent of newly synthesized chains eluted on gel filtration as a lower molecular weight (LMW) shoulder and in vivo turned over faster than the larger species.

A large heparan sulfate PG was present after 4 hours of in vivo ³⁵SO₄ labeling in both a low density form and a high density, slightly smaller form with large heparan sulfate chains (M_r ～ 8.0 × 10⁴). Heparan sulfate PG of intermediate size (K_av = 0.3–0.65, Sepharose CL-4B) and of smaller size (K_av = 0.75, CL-4B) were found predominantly as high density species. These PGs contained chains (M_r = 3.5 × 10⁴ and M_r = 1.2 × 10⁴, respectively) which were partially sensitive to chondroitinase ABC (CABC) and may include a hybrid heparan sulfate/chondroitin sulfate PG. Heparan sulfate chains, possibly intracellular degradation products, were also found.

Heparan sulfate chains were normal in N-sulfation (58% of hexosamine residues) and in iduronate content (～ 30%). N-sulfation started within two disaccharides of the linkage region. The EHS heparan sulfate was unusually low in O-sulfation (10% of the total sulfation) and no 6–0 sulfated, N-acetylated glucosamine residues adjacent to N-sulfated block regions were found.     

The conformational coil-globule transition of polystyrene in cyclohexane solution

The phase diagram of a monodisperse high molecular weight polystyrene (M _w = 3.3 · 10⁶, M _w/M _n < 1.06) solution in cyclohexane was thoroughly studied by means of light scattering, cloud point, coexisting phases volume ratio, coexisting phases composition and scanning calorimetry methods, and binodal and spinodal curves were obtained. Coordinates of the UCST (T = 306.2 K, ?₂ = 1.88 · 10^?2) determined by different methods were similar. It was found that the first-order phase transition curve obtained by calorimetric scanning at 10^?6 K/s accurately fits the spinodal of the system within the concentration range of semidilute and concentrated solutions and has a horizontal part in dilute solutions where the temperature of phase transition was found to be 303.86 K, independently of concentration. At the same temperature a deflection point was found on the concentrated branch of the spinodal. The results were taken as calorimetric evidence of a coil-globule phase transition in the polystyrene solution. It was shown that the collapse of the polystyrene coils takes place in the metastable system between the binodal and the spinodal in the same way as in the homogeneous solution above the binodal.     

Behaviour of ring-shaped polymers in dilute solution. Quantitative calculation and experimental verification for molecular dimensions and second virial coefficient

Relationships between the ratios of the mean-square radii of gyration g_s and the second virial coefficients g_A2 of ring-shaped and linear polymers with the degree of polymerization P of the polymer and its Peterlin exponent ε in dilute solution were derived theoretically and numerical values calculated. The second virial coefficients of ring-shaped and of linear polystyrene at 25°C in toluene were determined using light scattering. By verifying the theoretical calculation based on the experimental determination of this polymer-solvent system with light scattering and other methods reported in the literature, the agreement between theoretical calculation and experimental results is shown. The correlation equations for linear (l) and ring-shaped (r) polystyrene in toluene at 25°C A_{2, 1} = 8,06 · 10^?3 · M^0,224 and A_2,r = 6,68 · 10^?3 · M^0,218 were obtained using light scattering.     

Studies of lyotropic liquid crystalline mesophases of some novel regioselective substituted cellulose derivatives, 1. Synthesis and characterization

Novel regioselective substituted cellulose derivatives, suitable for investigations of their liquid crystalline phase behaviour, were synthesized. The derivatives, tricarbanilates with varying substituents at the phenolic residue, were characterized according to their molecular weight and molecular weight distribution by means of size exclusion chromatography coupled with low angle laser light scattering (SEC/LALLS). A simple new method is presented, which allows the calculation of the constants K and a of the Staudinger-Mark-Houwink (SMH) equation, the viscosity-average molecular weight M _η as well as the intrinsic viscosity [η] from the light scattering calibration curve of an SEC experiment. The calculated values of the intrinsic viscosity are in good agreement with those derived from measurements with a low-shear viscometer. A plot [η] vs. weight-average molecular weight M _w shows, that a substitution at the C-2 and C-3 position of the glucosic unit with 3-chlorocarbanilate leads to a well pronounced enlargement of the polymer dimensions which is mirrored by larger values of [η] at the same M _w as compared with other cellulose derivatives.     

Small angle neutron scattering (SANS) of cyclic and linear polystyrene in toluene

Samples of ring and open chain polystyrene in dilute perdeuterated toluene solution were measured by small angle neutron scattering up to a value of the scattering vector Q = 2 nm^?1. The molar masses 12000 ≤ M/(g/mol) ≤ 22000, the mean square radii of gyration 〈R²〉 and the second virial coefficients A₂ of the samples were determined. The results are compared to theories which describe the dependencies 〈R²〉 = f(M) and A₂ = f(M) for both cyclic and linear chain molecules. A qualitative agreement between theory and experiment is obtained.     

A general method for the determination of “monomolecular property versus molar mass relationships” from measurements on polymolecular polymers

“Monomolecular property (p) versus molar mass (M) relationships” of the type p = K_p · M^ap as would result from an experimental determination of p and M of a polymer-homologous series of strictly monomolecular polymers can also be obtained from a polymer-homologous series of polymolecular samples or fractions, provided their correct corresponding averages p and M are determined (Principle of Corresponding Averages). In order to obtain the monomolecular intrinsic viscosity [η] vs. M relationship (Mark-Houwink relationship), the corresponding averages weight-average intrinsic viscosity ([η]_w ≡ [η]) and viscosity-average molar mass (M_[η]) of a series of polymer-homologous, polymolecular polymer samples or fractions have to be determined experimentally. Accordingly, the corresponding monomolecular relationships sedimentation coefficient s vs. M, diffusion coefficient D vs. M, and mean-square radius of gyration 〈r²〉 vs. M are obtained by the determination of a series of couples of the corresponding averages s vs. M , D vs. M , and 〈r² 〉 vs. M , respectively. The experimental determination of the corresponding averages presented is possible by different methods, including the determination of the molar mass distribution.     

Characterization of cellulose in solvent mixtures with N‐methylmorpholine‐N‐oxide by static light scattering

Cellulose was dissolved in a solvent mixture containing N‐methylmorpholine‐N‐oxide (NMMNO), diethylenetriamine (DETA) and water. The weight‐average molar masses M_w, radii of gyration R_G, and second virial coefficients A₂ of 9 different cellulose samples were determined by means of static light scattering (SLS) at room temperature. The influence of two different dissolution procedures was studied on four samples. The M_w results were compared with results obtained by gel permeation chromatography (GPC) with multi angle laser light scattering (MALLS) detection. The original samples and samples recovered from the solvent mixture by precipitation were tricarbanilated and dissolved in tetrahydrofuran for the GPC analysis. The SLS results led to the conclusion that cellulose was molecularly dispersed in the solvent mixture NMMNO/H₂O/DETA. The M_w dependence of the radii of gyration showed a common power law behavior with an exponent which is identical to the theoretical value for flexible, linear chains in good solvents. The observed differences between SLS and GPC results, especially for the high‐molecular samples, can be explained by a molecular degradation of cellulose during the dissolution in NMMNO/H₂O/DETA. The different dissolution procedures and the use of potassium pyrophosphate as a stabilizer showed no significant effect on the determined results.     

Investigations of the crystallization of polyethylene by means of simultaneous small-angle and wide-angle X-ray scattering

The isothermal crystallization of linear polyethylene fractions was studied by means of simultaneous measurements of wide-angle X-ray scattering (WAXS) and small-angle X-ray scattering (SAXS) employing synchrotron radiation. From WAXS the overall degree of crystallinity x_c was determined. From SAXS, the scattering power Q was evaluated and compared with several different models for the crystallizing system. When the scattering arises solely from the supermolecular structures, such as spherulites, Q is proportional to x_s·x_cL·(1 ? x_cL), where x_s is the fraction of material transformed into such structures and x_cL is the degree of crystallinity within these structures. By comparing x_c with Q it was possible to separate the primary and secondary crystallization and to show that secondary crystallization takes place within the lamellar stacks where the crystals become thicker and the amorphous layers become thinner. The process of recrystallization during annealing of crystalline samples was studied in the same way. Other models were treated in a similar manner.     

Solution properties and flexibility of poly(2-biphenylyl methacrylate)

Dilute solution properties of poly(2-biphenylyl methacrylate), (poly[1-(2-biphenylyloxycarbonyl)-1-methylethylene], POB) were studied by viscosity, sedimentation, light scattering and osmotic pressure in different solvents. The solvent dependence of intrinsic viscosity, [η], Huggins constant, K_H, Mark-Houwink exponent, a, and expansion factor, α_η, is very weak, although the chemical nature of the solvents is quite different (CHCl₃, tetrahydrofuran, benzene, 1,4-dioxane). For poly(4-biphenylyl methacrylate) (poly[1-(4-biphenylyloxycarbonyl)-1-methylethylene], PPB) and other aromatic polymethacrylates the changes of [η], K_H, a and a_η are very high. The unperturbed dimensions calculated from viscometric and light scattering data by different methods, are in excellent agreement with one another and good agreement with those obtained from sedimentation data. POB has a higher flexibility factor (σ = 2,8) than PPB (σ = 2,7). In our opinion this is due to the fact that the 2-biphenylyl group is closer to the main chain than the 4-biphenylyl group. The very high value of σ explains the weak solvent dependence of [η], K_H, a and α_η in the case of poly(2-biphenylyl methacrylate).     

Serodiagnosis of Chagas' disease using monoclonal antibody against Trypanosoma cruzi-specific Mr 25000 antigen

A mouse monoclonal antibody TCF87, prepared previously, was reactive with Trypanosoma cruzi-specific M_r 25000 antigen regardless of strain. The M_r 25000 antigen was recognized by all sera from chagasic patients living in different areas of South America, when examined by Western immunoblotting analysis. Although many antigens of T. cruzi epimastigotes were also recognized by sera from patients with leishmaniasis, the M_r 25000 antigen of T. cruzi did not react with leishmaniasic sera. These results indicate that M_r 25000 antigen recognized by TCF87 is valuable as a diagnostic antigen for Chagas' disease. When a competition enzyme-linked immunosorbent assay using TCF87 was carried out, all sera from Chagas' disease patients showed positive inhibition. By contrast, all patients with leishmaniasis or other parasitic diseases were scored as seronegative. The present study suggests that competition enzyme-linked immunosorbent assay using monoclonal antibody against the M_r 25000 antigen of T. cruzi will be useful for serodiagnosis of Chagas' disease in areas where leishmaniasis is co-endemic.     

A new procedure for the determination of molar mass distribution from sedimentation equilibrium runs in an analytical ultracentrifuge (AUC), 1. Measurements with interference optics

A new procedure for the determination of the number-, weight- and z-average molar masses M?_n, M?_w and M?_z and a model molar mass distribution W(M) has been developed from measurements of the sedimentation equilibrium. The method is based on the determination of the experimentally measured reduced concentration profile by extrapolation to zero concentration and direct calculation of the molar mass distribution W(M) by nonlinear regression of the integral equation according to the Simplex procedure. Results obtained in different solvents for the well characterized polystyrene NBS 706 with broad distribution are reasonable. Generally, it can be concluded that the suggested procedure is a suitable possibility for the determination of molar mass average values and the molar mass distribution of polymers if the latter is a log-normal, Schulz-Flory or Poisson distribution.