Determination of size and charge distributions by combination of quasi-elastic light scattering and band transport

A convenient method for the determination of size and charge distributions of polydisperse samples is described. It includes three steps: (a) band transport to disperse the components of the sample in a scattering cell; (b) total intensity scattering to determine the concentration and mobility at each location; and (c) quasi-elastic light scattering (QLS) to characterize the hydrodynamic sizes. Combining the results of (b) and (c) yields size and charge distributions. Since complete resolution of the various components of the sample cannot occur in step (a), a method is described to estimate the average form factor, from the local mean size and the local degree of polydispersity obtained by QLS. The potential of this method is demonstrated by the results of its application to two radiocolloids which have broad size distribution (50- to 600-nm diameters).     

Effect of ionic strength on the diffusion coefficient of chondroitin sulfate and heparin measured by quasielastic light scattering

The normal modes for a mixture of charged macromolecules and electrolyte solution are calculated. We derive a generalized Debye relaxation time and the apparent diffusion coefficient of the macroion, which is shown to increase from its Stokes value, obtained in excess of added salt, with decreasing ionic strength. We test our result with experimental data for macromolecules with different charge densities: heparin and chondroitin sulfate. Besides, we show for this latter molecule that while the diffusion coefficient is increased, the scattered intensity is decreased but not by the same factor. Our results are compared with other theories developed in quasielastic light scattering.     

Conformational change of core particles studied by quasielastic light scattering

The diffusion translational coefficient D_T of core particles in monodisperse solutions has been measured by the quasielastic light scattering method in a large scale of salinities over the range 6.10⁻⁴ to 2M Na⁺ or K⁺. The observed values of D_T are independent of particle concentration in the range 0.1–2 mg/ml and do not vary with the scattering vector q corresponding to scattering angles between 40°–120°. When the salinity is progressively raised an increase of D_T from 1.9.10⁻⁷ cm²s⁻¹ to 3.2.10⁻⁷ cm²s⁻¹ was observed at about 2.10⁻³ M NaCl followed by a decrease of D_T beyond 0.6 M NaCl.The various possible causes of the changes of D_T such as interactions between particles or between particles and salt ions are discussed. We show that the single low ionic strength change is due to a conformational transition of the core particles, while the second variation of D_T accompanies the disorganization of the core particles.     

The osmotic response of large unilamellar vesicles studied by quasielastic light scattering

Large unilamellar vesicles of two phosphatidylcholines, one saturated (DMPC) and the other unsaturated (DOPC), prepared by the reverse-phase evaporation method were studied using the quasielastic light scattering technique. The accurate sizing obtained by this technique showed an osmotic response for the two kinds of vesicles when the salinity of the external medium was diluted. The elastic moduli of lipid vesicles bilayers in the liquid phase were then estimated according to the elasticity theory of spherical shells taking into account salt leakage data known from the literature.     

A hydrodynamic study of collagen fibrillogenesis by electric birefringence and quasielastic light scattering

Neutral soluble collagen was extracted from lathyritic rat skin under proteolysis-inhibited conditions. Purified solutions were characterized by electric birefringence and heterodyne beat quasi-elastic light-scattering techniques under conditions where the monomeric form was stable (at 4 degrees C in 0.032 M phosphate buffer at pH 7.04). Solutions were then heated and the birefringence and light scattering followed during the fibrillogenesis reaction. The monomer presents a translational diffusion coefficient of 0.85 X 10(-7) cm2/s and a rotary diffusion coefficient of 1150 +/- 50 s-1; these values are consistent with a rodlike molecular model of 220 +/- 10 nm length and 4 +/- 1 nm diameter, substantially different from electron microscopic values of 290 and 1.5 nm, respectively. We propose that at pH 7.04 and relatively high ionic strength, the collagen monomer unit must exhibit substantial deviation from a completely rigid and extended rodlike structure. During the entire lag phase in a thermally induced fibrillogenesis reaction, the relaxation times for both translational and rotational motion remain virtually unchanged. The monomer polarity is also unchanged, as shown by reverse pulse birefringence data. No intermediate size soluble aggregates, such as dimers or trimers, have been detected between monomer and very large aggregates or fibrils during the process, although early multistep assembly products (dimers, trimers) could have been seen if present. These data suggest a model for fibrillogenesis emphasizing a monomer-related nucleation event, such as internal stiffening or conformational transition, followed by a rapid continuous growth up to large fibrils.     

The effect of histone H1 on the compaction of oligonucleosomes. A quasielastic light scattering study

The structural properties of H1-depleted oligonucleosomes are investigated by the use of quasielastic laser light scattering, thermal denaturation and circular dichroism and compared to those of H1-containing oligomers. To obtain information on the role of histone H1 in compaction of nucleosomes, translational diffusion coefficients (D) are determined for mono-to octanucleosomes over a range of ionic strength. The linear dependences of D on the number of nucleosomes show that the conformation of stripped oligomers is very extended and does not change drastically with increasing the ionic strength while the rigidness of the chain decreases due to the folding of linker DNA. The results prove that the salt-induced condensation is much smaller for H1-depleted than for H1-containing oligomers and that histone H1 is necessary for the formation of a supercoiled structure of oligonucleosomes, already present at low ionic strength.     

Molecular weight distribution of carrageenans by size exclusion chromatography and low angle laser light scattering

A procedure to determine the absolute weight average molecular weight and molecular weight distribution of carrageenans by high pressure aqueous size exclusion chromatography coupled with low angle laser light scattering is described. Experimental parameters are successively discussed, particular attention being focused on the absence of shear degradation during elution. The distribution curves were highly reproducible in time and weight average molecular weights integrated along the chromatogram were in good agreement with static light scattering results. A large difference in the molecular weight range between native (food-grade) and acidic degraded carrageenan samples was observed. Weight average molecular weights were found to be in good correlation with viscosity values, for degraded as well as undegraded products. It is also shown that the method described can help people using carrageenans in pharmacological studies by providing information on the real molecular weight distribution of the products they are employing.     

A study of the diffusion of compact particles in polymer solutions using quasi-elastic light scattering

This paper describes an investigation, using quasi-elastic light scattering, of the diffusion of polystyrene spheres through solutions of dextran. The diffusion coefficient, D, of the spheres is shown to vary inversely with the volume fraction, φ, of dextran according to $\text{D =}\text{D}{}_0\text{(1 + νφ + κφ}{}^2\text{)}$. Changes in the molecular weight of dextran are shown to reflect changes in the macromolecular shape parameter, ν, and the interaction parameter, κ. This result differs from previous studies which suggested an $\text{exp}\text{(?}\text{B}\text{φ}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{)}$ dependence and no molecular weight dependence [8].     

Characterization of the size distribution of unilamellar vesicles by gel filtration, quasi-elastic light scattering and electron microscopy

The size and size distribution of unilamellar phospholipid vesicles present in unsonicated phosphatidic acid and mixed phosphatidic acid/phosphatidylcholine dispersions were determined by gel filtration, quasi-elastic light scattering and freeze-fracture electron microscopy. The vesiculation in these dispersions was induced by a transient increase in pH as described previously (Hauser, H. and Gains, N. (1982) Proc. Natl. Acad. Sci. USA 79, 1683–1687). The resulting phospholipid dispersions are heterogeneous consisting of small unilamellar vesicles (average radius r < 50 nm) and large unilamellar vesicles (average r ranging from about 50 to 500 nm). The smallest vesicles with r = 11 ± 2 nm are observed with dispersions of pure phosphatidic acid, the population of these vesicles amounting to about 80% of the total lipid. With increasing phosphatidylcholine content the radius of the small unilamellar vesicles increases and at the same time the population of small unilamellar vesicles decreases. The average radius of small unilamellar vesicles present in phosphatidic acid/phosphatidylcholine dispersions (mole ratio, 1:1) is 17.5 ± 2 nm, the population of these vesicles amounting to about 70% of the total lipid. By a combination of gel filtration, quasi-elastic light scattering and freeze-fracture electron microscopy it was possible to characterize the large unilamellar vesicles. This population is heterogeneous with its mean radius also increasing with increasing phosphatidylcholine content. After separating the large unilamellar vesicles from small unilamellar vesicles on Sepharose 4B it can be shown by quasi-elastic light scattering that in pure phosphatidic acid dispersions 80–90% of the large unilamellar vesicle population consist of vesicles with a mean radius of 170 nm. In mixed phosphatidic acid/phosphatidylcholine dispersions this radius increases to about 265 nm as the phosphatidylcholine content is raised to 90 mol%.     

Hydration of ds-DNA and ss-DNA by neutron quasielastic scattering

Quasielastic neutron scattering measurements were performed in hydrated samples of ds-DNA and ss-DNA. The samples were hydrated in a high relative humidity atmosphere, and their final water content was 0.559 g H(2)O/g ds-DNA and 0.434 g H(2)O/g ss-DNA. The measurements were performed at 8 and 5.2 A for the ds-DNA sample, and at 5.2 A for the ss-DNA sample. The temperature was in both cases 298 K. Analysis of the obtained data indicates that in the ds-DNA sample we can distinguish two types of protons-those belonging to water molecules strongly attached to the ds-DNA surface and another fraction belonging to water that diffuses isotropically in a sphere of radius 2.8 A, with a local diffusion coefficient of 2.2 x 10(-5) cm(2) s(-1). For ss-DNA, on the other hand, no indication was found of motionally restricted or confined water. Further, the fraction of protons strongly attached to the ds-DNA surface corresponds to 0.16 g H(2)O/g ds-DNA, which equals the amount of water that is released by ds-DNA upon thermal denaturation, as studied by one of us (G.M.) by differential scanning calorimetry. This value also equals the difference between the critical hydration values of ds-DNA and ss-DNA, also determined by DSC. These results represent, thus, a completely independent measurement of water characteristics and behavior in ds- and ss-DNA at critical hydration values, and therefore substantiate the previous suggestions/conclusions of the results obtained by calorimetry.     

Dependence of the internal relaxation time for DNA on salt type as inferred by quasielastic light scattering

Quasielastic light scattering methods were used to study calf thymus DNA in solutions of LiCl, NaCl, NH₄Ac, and NH₄Cl. Plots of the reciprocal relaxation time (1/τ) vs sin²(θ/2), where θ is the scattering angle, exhibit two linear regions, in accordance with theories for semiflexible polymers based on the t → 0 approximation. In these theories the slope of the linear region at low angles is associated with the translational diffusion coefficient (D_t), whereas the slope of the linear region at high angles is associated with the segmental diffusion coefficient (D_s = kT/?_s). The midpoint of the “transition” between these two linear regions is associated with the mean displacement between segments (b). Data presented here indicate that the Rouse-Zimm parameters b and ?_s are significantly different for DNA in 0.4M NH₄Cl relative to the other salts at comparable ionic strengths. It is suggested that this difference reflects local solvent structure and that both b and D_s are sensitive to the local water structure.     

Molar mass distribution of hydroxypropyl cellulose by size exclusion chromatography with dual light scattering and refractometric detection

Physico-chemical properties of cellulose derivatives are of considerable interest in many technical applications, for example, in the food and drug industry. Efficient and careful characterisation of these properties is thus highly desirable. In this study, two different size exclusion chromatography (SEC) systems, connected on-line either to a low-angle laser light scattering detector (LALLS) or to a multi-angle laser light scattering detector (MALLS), are employed for size characterisation of three batches of hydroxypropyl cellulose (HPC) from different manufacturers. All three samples turned out to have a weight average molar mass around 100,000 g/mol, but considerable differences concerning conformational properties were found. Two of the samples contained compact components, presumably aggregates of HPC, which were clearly detected by both SEC-systems. The third sample, obtained from another manufacturer, did not show any indication of aggregation. Both SEC-MALLS and SEC-LALLS are proven to be efficient techniques for characterisation of complex polysaccharides like HPC containing mixtures of solvated polymer chains, as well as micelle-like aggregates.     

Absolute molecular weight and molecular weight distribution of guar by size exclusion chromatography and low-angle laser light scattering

A procedure to determine the absolute weight-average molecular weight (M_w) and molecular weight distribution (MWD) of guar by aqueous size exclusion chromatography coupled with low angle laser scattering is described. It is shown that for a rigorously purified sample of guar solution the values for M_w and MWD are 2·2×10⁶ and 1·9 respectively. The effect of sample preparation and purification on these molecular parameters are discussed. Limitations and challenges in the aqueous size exclusion chromatography of complex water soluble polymers such as guar are also explored.     

Structure and mobility of actin filaments as measured by quasielastic light scattering, viscometry, and electron microscopy

Actin filaments of different lengths were prepared by polymerizing actin in the presence of various concentrations of gelsolin, a protein which accelerates actin polymerization by stabilizing nuclei from which filaments grow and which binds to their fast growing ends. The lengths of the actin filaments following polymerization were measured by electron microscopy and showed that the number-average filament length agreed with the predicted length if each gelsolin molecule acted as a seed for the growth of an actin filament. The distribution of lengths was independent of the actin:gelsolin ratio and was similar to that of actin filaments polymerized in the absence of gelsolin (Lw/Ln = 1.8). The mobility of these filaments in solution was studied by quasielastic light scattering and by viscometry. The translational diffusion constant determined by quasielastic light scattering was in agreement with the infinite dilution values calculated from the dimensions and the distribution of lengths determined by electron microscopy for relatively short filament lengths. Under conditions where overlap of the rotational domains of the filaments would be expected to occur, the measured diffusion rates deviated from their predicted dilute solution values and the solution viscosity increased abruptly. The dependence of the diffusion constant and the solution viscosity on the length of the actin filaments can be explained in terms of a theory that describes the restraints on diffusion of independent rigid rods in semi-dilute solution. The results suggest that the rheology of actin filaments can be accounted for by steric restraints. The length of cytoplasmic actin filaments in some cell types is such that these steric constraints are significant and could produce large changes in physical properties with small changes in filament length.     

Effect of tryptophan oligopeptides on the size distribution of POPC liposomes: a dynamic light scattering and turbidimetric study

A chemical regulation of POPC liposome size distribution was investigated, based on the affinity of indole-containing compounds for phosphocholine membranes. In particular, tryptophan oligopeptides have shown interesting properties of size regulation, both when liposomes were formed in their presence and when the peptides were added to a preformed liposome suspension. Combining dynamic light scattering (DLS) and turbidimetric data, it was possible to show how such peptides had an influence on the size distribution of spontaneously formed liposomes prepared by the thin film hydration, reverse-phase evaporation and ethanol (or methanol) injection methods. In the presence of Trp-Trp or Trp-Trp-Trp, a disappearance of large vesicle aggregates was observed, as suggested also by light microscopy analysis. On the contrary, no effect was detected using extruded vesicles. Turbidimetric titration allowed the determination of the relative efficacy of the size regulators, Trp-Trp-Trp being about 20 times more powerful than the dimer, while the monomer had no effect. In addition, other indole-containing compounds and the antimicrobial peptide indolicidin were tested, showing similar behaviours. Discussing the results according to the current knowledge about the preference of Trp residues for interfacial regions in lecithin bilayers, this study confirms the relevant role of tryptophan in the biomembrane binding properties of many peptides and introduces a new behavior in the field of liposomes-peptides interactions.     

Determination of the elastic constants of collagen by Brillouin light scattering

The high-frequency elastic properties of rat-tail tendon collagen have been investigated by means of Brillouin (inelastic) light scattering. Longitudinally and transversely polarised elastic waves of frequency about 10¹⁰ Hz have been observed propagating at various angles to the fibre axis of stretched, partially dried tendon. Assuming that the elastic properties of tendon are transversely isotropic, these measurements enable the five elastic constants for such a system to be determined. In particular the ratio of the Young's modulus for strain parallel to the axis to that for strain perpendicular to the axis ($\text{E}{}_{\mathrm{\parallel }}\text{E}{}_{\mathrm{\perp }}$) is found to be 1.43 and the ratio of the shear modulus to E_∥ is 0.28. In wet collagen only the longitudinal branch has been observed and in this case the ratio $\text{E}{}_{\mathrm{\parallel }}\text{E}{}_{\mathrm{\perp }}$ increases to 1.82. The absolute value for E_∥ in dry collagen is 11.9 GN m^?2 reducing to 5.1 GN m^?2 in wet collagen. An interpretation of these results in terms of the expected vibrations of the collagen molecular assembly is given. Possible applications to the determination of the mechanical properties of collagen composite materials such as bone are discussed as well as some measurements on silk and α- and β-keratins, which are fibrous proteins of different molecular conformation to collagen.     

Determination of liposome size distribution by flow cytometry

BACKGROUND: An essential parameter that describes the quality of liposome suspensions is the mean size, respectively the size distribution. Currently several analytical methods including laser light scattering techniques (LLST) are being employed. METHODS: Here we present an alternative technique using flow cytometry (FCM) to characterize uni- and polydisperse suspensions. As model liposomes preparations containing dipalmitoylphosphatidylcholine (DPPC) were used. A constant number of particles (1,500/s) in the fluid stream and a representative number of 10,000 particles of each sample was measured. Fluorescence-labeled latex beads were measured identically, and their side scatter signals were calibrated and correlated to the results obtained with liposome vesicles. RESULTS: Evaluation of the measurement and validation of the FCM results in comparison to LLST confirm the reliability of results obtained with our method. Latex beads in the range of 100-1000 nm were used for calibration to classify liposomes. Although measurement characteristics and calculation in both methods are basically different, very good agreement of the results was achieved. CONCLUSIONS: Demonstration of stability, reproducibility, and reliability of results make the employment of this method acceptable for an adequate routine analysis technique.