Electrophoretic light scattering as a probe of reaction kinetics

A new method is proposed for determining chemical rate contants in dimerization reactions of globular proteins. Light scattering from a solution of charged macromolecules in and applied electric field gives a series of bands whose widths can be used to deduce the reaction rate contants. This method should be applicable to other types of peactions. First order reactions are also considered.     

Salt effects on internal motions of superhelical and linear pUC8 DNA. Dynamic light scattering studies

The plasmid pUC8 (2717 bp) has been studied in its native superhelical and Eco RI-linearized forms by dynamic light scattering at NaCl concentrations from 1.1 mM to 1 M. The data were analyzed using the biexponential model for the dynamic structure factor described by us in a previous paper (J. Langowski, U. Giesen and C. Lehmann, Biophys. Chem. 25 (1986) 191). As before, we could identify two decay components corresponding to the center-of-mass diffusion and to internal motions of the DNA, where the fast component could be identified as a rotational diffusion contribution in the case for superhelical, but not for linear DNA. We found that the conformation of superhelical pUC8 is not affected by changing the ionic strength, while the amplitude of the internal relaxation increases approx. 2-fold when [NaCl] is raised from 1.1 mM to 1 M. The linearized DNA shows an increase of the diffusion coefficient with ionic strength which is, however, not quite as pronounced as that found by others (Z. Kam, N. Borochov and H. Eisenberg, Biopolymers 20 (1981) 2671), and, together with the unchanged conformation of the superhelical DNA, suggests a persistence length which is not strongly dependent on ionic strength. In contrast to the increasing amplitude of internal relaxation for the superhelical DNA, this amplitude remains constant or decreases slightly for linear DNA on going from 1.1 mM to 1 M salt. Our findings are further discussed with respect to possible models of the interwound form of superhelical DNA.     

Differential scattering of circularly polarized light as a unique probe of polynucleosome superstructures

A theoretical approach to modeling Circular Intensity Differential Scattering (CIDS) of native chromatin as multiple scattering of dipoles is discussed without the Born approximation. The model can explain the experimental data in the literature. It is shown that CIDS contains more structural information than does total light scattering and to a good approximation is independent of the length of the scattering molecules. Finally, CIDS in conjunction with traditional light scattering measurements should aid in discriminating between various alternative models of higher order chromatin structure now being proposed. Generalization of this theoretical study to other complex biomolecular structures, is also briefly discussed.     

Changes in DNA superhelical density monitored by polarized light scattering.

Linear and circular lambda-DNA at different ethidium bromide concentrations have been studied by means of polarized light scattering, namely the S14, S34, S33 and S13 elements of Mueller matrix. While S33 at low angle appears well correlated with the total light scattering evaluated by optical density measurements at 632.8 nm for linear and circular DNA of the same mass, the magnitude and slope of the S14, S34 and S13 signals display significant changes for the circular lambda-DNA depending on the degree of negative superhelical density as induced by the different ethidium bromide concentrations. At the same time, for linear lambda-DNA the signal remains invariant, making explicit for the differential scattering of polarized light the possibility to obtain additional information by its angular dependence. Strikingly also the effect of 0.2% glutaraldehyde versus ethanol fixation on the native lambda-DNA structural properties appears to confirm earlier findings by other well-established probes. Results are discussed in terms of first physical principles and of their potential bearings towards our understanding of the mechanism controlling gene expression.     

Configurational and dynamic properties of different length superhelical DNAs measured by dynamic light scattering

The solution conformation and internal motions of five superhelical DNAs between 2100 and 10200 base-pairs in length have been characterized by dynamic light scattering (DLS). Variations in the diffusion coefficients and rotational relaxation times with molecular weight are both indicative of an anisotropic extended structure of these DNAs; we therefore conclude that under our conditions the interwound superhelical structure prevails. The internal dynamics can be described by a superposition of rotational diffusion and internal relaxation. The latter process is characterized by the internal diffusion of persistence length size segments within the DNA chain and faster bending motions within these segments.     

Dynamic light scattering studies of ribonuclease

Dynamic light scattering has been used to measure the translational diffusion coefficients of bovine pancreatic ribonuclease A as functions of temperature and concentration in the presence of 1 M Guanidine-HCl. Data was collected throughout a temperature range including the folding-unfolding transitions. Evidence of a pretransition "swelling" of the protein was observed. Entropy and enthalpy changes upon unfolding were obtained using a two-state model.     

Intramolecular interference effects in dynamic light scattering: rigid double spirals and superhelical DNAs

A theory is developed for dynamic light scattering (DLS) from rigid double spirals by treating an invisible rigid cylinder with two helical scattering stripes on opposite sides of its cylindrical surface. The exact initial, or first cumulant, diffusion coefficient Dapp (K) is obtained in terms of the translational diffusion coefficients (D parallel and D perpendicular) parallel and perpendicular to the symmetry axis, the rotational diffusion coefficients (DR parallel and DR perpendicular) around the symmetry and transverse axes, the length (L) and radius (b) of the cylindrical surface bearing the stripes, and the pitch (p). Interference effects, namely geometrical antiresonances, between strands, produce deep minima in the static structure factor S (K) and corresponding prominent peaks in Dapp (K). These peaks in Dapp (K) depend sensitively on the rotational dynamics around the symmetry axis, and nearly vanish when DR parallel = 0. Some results for single spirals are also presented. A simpler model in which scattering points are attached at opposite ends of an otherwise invisible thin rigid rod is also treated, and shown to exhibit modest minima in S (K) and corresponding maxima in Dapp (K). Confining this rod to a plane containing K enhances the amplitudes of the oscillations in S (K) and Dapp (K), as expected. Rigid double spirals are employed as crude models for interwound supercoiled DNAs in order to assess the possible occurrence of interference effects. Although native supercoiled DNAs exhibit a cylinder diameter that is much too small to exhibit geometrical antiresonances in the presently accessible range of K2, nearly relaxed supercoiled DNAs are predicted to exhibit their first maximum in Dapp (K) just inside this range. Previously reported data for the effect of Escherichia coli single-strand binding (ssb) protein on the DLS of supercoiled pBR322 DNA cannot be mimicked by a gradual homogeneous reduction of superhelix density with increasing ssb, but instead can be mimicked by inhomogeneous all-or-none binding in which uncomplexed native DNAs and nearly relaxed saturated ssb/DNA complexes coexist in varying proportions. Experimental Dapp (K) and S (K) data for a sample of relaxed pUC8 dimers display, respectively, a broad maximum and a corresponding minimum, in qualitative agreement with rough theoretical predictions.     

Protein-polynucleotide scattering centers as a protein structure probe

When certain basic globular proteins are mixed with nucleic acids near a critical concentration ratio, large, low density scattering centers of about 10⁹ particle weight are created. Scattering from these complexes is altered when thermally inactivated proteins are substituted for enzymes in their native, globular conformation. Scattering data from heat-treated ribonuclease and lysozyme mixed with four different synthetic homopolyribonucleotides are reported. The concentration of nucleic acid necessary to produce maximum scattering from a heat-treated protein sample is shown to be a direct indication of the amount of enzyme that remains biologically active after being heated.     

Differential scattering of circularly polarized light as a unique probe of polynucleosome superstructures. A simulation by multiple scattering of dipoles

A theoretical approach to modeling Circular Intensity Differential Scattering (CIDS) of native chromatin as multiple scattering of dipoles is discussed without the Born approximation. The model can explain the experimental data in the literature. It is shown that CIDS contains more structural information than does total light scattering and to a good approximation is independent of the length of the scattering molecules. Finally, CIDS in conjunction with traditional light scattering measurements should aid in discriminating between various alternative models of higher order chromatin structure now being proposed. Generalization of this theoretical study to other complex biomolecular structures, is also briefly discussed.     

Dynamic light scattering methods for biorhelogy

Several noninvasive light scattering techniques are reviewed that can be utilized in rheological studies of mucus and other soft biological matrices. Included are quasielastic light scattering techniques to determine the compressibility moduli of polymer lattices, resonance methods that can be used to measure the shear modulus of weak gels, assays that probe the swelling and contraction of mucin aggregates, and microscope based light scattering techniques to probe gelation within single cells.     

Protein-polynucleotide scattering centers as a protein structure probe.

When certain basic globular proteins are mixed with nucleic acids near a critical concentration ratio, large, low density scattering centers of about 10(9) particle weight are created. Scattering from these complexes is altered when thermally inactivated proteins are substituted for enzymes in their native, globular conformation. Scattering data from heat-treated ribonuclease and lysozyme mixed with four different synthetic homopolyribonucleotides are reported. The concentration of nucleic acid necessary to produce maximum scattering from a heat-treated protein sample is shown to be a direct indication of the amount of enzyme that remains biologically active after being heated.     

Dynamic light scattering study of muscle F-actin

By use of digital autocorrelation and fast Fourier methods, dynamic light-scattering studies of in vitro reconstituted muscle F-actin were made over a wide range of concentrations, 0.01-2 mg/ml F-actin. Measurements of correlation function [g1(t)]2 showed that a transition from a dilute to a semidilute regime for the Brownian motions of filaments occurred at around 0.3 mg/ml F-actin. Beyond this concentration, profiles of successively measured [g1(t)]2 showed very poor reproducibility. This resulted from the existence of very slow components, which could not be measured with a high statistical accuracy even for a measuring time of 3600 s/run. On the other hand, subtraction of these components automatically by an electronic circuit, [g-1(t)]2, or by computer processing, [g1(t)]2, resulted in a fairly good reproducibility of the profiles. The decay characteristics of [g1(t)]2 (and [g-1(t)]2) were very similar to those of [g1(t)]2 for dilute solutions. A theoretical model will be discussed which could account for the above situation. The time sequence [n(t,T)] of photoelectron counts at a sampling time T of light scattered from semidilute solutions of F-actin was stored on magnetic tapes, and both power spectra S(f) and correlation functions [g-1(t)]2 were computed by taking the ensemble average over many short records with duration 1024T. Since both S(f) and [g-1(t)]2 lacked frequency components lower than 1/(2048T) Hz, their profiles were highly reproducible. An analysis of S(f) confirmed our earlier results which had shown an apparent contradiction to later results by a correlation method. A comparison of S(f) and [g-1(t)]2 based on the same [n(t,T)] clarified the reasons why the bandwidth gamma of S(f) largely differed from the bandwidth gamma of [g1(t)]2 and [g-1(t)]2. The temperature dependence of gamma suggested that F-actin would be flexible and that the flexibility parameter would change with temperature.     

Determination of bismuth in pharmaceutical products using phosphoric acid as molecular probe by resonance light scattering

A novel method for the sensitive determination of bismuth(III) in pharmaceutical products using phosphoric acid as a molecular probe by resonance light scattering (RLS) is discussed. In 0.5 mol/L phosphoric acid (H₃PO₄) medium, bismuth(III) reacted with PO₄^3? to form an ion association compound, which resulted in the significant enhancement of RLS intensity and the appearance of the corresponding RLS spectral characteristics. The maximum scattering peak of the system existed at 364 nm. Under optimal conditions, there was linear relationship between the relative intensity of RLS and concentration of bismuth(III) in the range of 0.06–10.0 µg/mL for the system. A low detection limit for bismuth(III) of 3.22 ng/mL was achieved. The relative standard deviations (RSD) for the determination of 0.40 and 0.80 µg/mL bismuth(III) were 2.1% and 1.1%, respectively, for five determinations. Based on this fact, a simple, rapid, and sensitive method was developed for the determination of bismuth(III) at nanogram level by RLS technique with a common spectrofluorimeter. This analytical system was successfully applied to determine the trace amounts of bismuth(III) in pharmaceutical products, which was in good agreement with the results obtained by atomic absorption spectrometry (AAS). Copyright © 2011 John Wiley & Sons, Ltd.     

Dynamic light scattering of biopolymers and biocolloids.

Widespread applications of dynamic light scattering techniques to the study of macromolecular Brownian motion have yielded not only a valuable store of factual information concerning solution conformations and conformational changes, but have also provided an important window through which to view the dynamics of internal modes of motion. These techniques have coincided with a resurgence of interest in the solution physical chemistry of macromolecules, including hydrodynamic properties, and the profound effect of intermolecular interactions on both the disposition and dynamics of macromolecules in solution.     

Dynamic light scattering from solutions of microtubules.

Calf brain microtubule protein was assembled in vitro to form dilute solutions of microtubules (240 A diameter) having lengths greater than 1 micrometer. The microtubule solutions were examined by dynamic laser light scattering techniques. The angular dependence of the correlation function leads to the conclusion that the correlation function is measuring the translational diffusion constant of the particles. The length dependence of the correlation function, however, shows that the translational diffusion constant is not being measured and that the diffusion constant for the microtubules cannot be straightforwardly determined. These results suggest that a collective property of the rods is being measured by the laser light scattering. Although specific microtubule-microtubule interactions are a possible explanation for the observed results, we present arguments that suggest that the solution can be adequately modeled as a network of entangled polymers.     

Dynamic light scattering studies of irradiated kappa carrageenan

Investigation of the dynamic behavior of irradiated kappa carrageenan (in KCl) as a function of irradiation dose and temperature was done by dynamic light scattering (DLS). The intensity correlation function (ICF) shifted towards shorter relaxation times with increasing radiation dose as a result of radiolysis. The characteristic decay time distribution function, G(gamma), indicates the presence of fast and slow mode peaks respectively at around 0.1-10 ms and 100-1000 ms. A peak broadening of the fast mode peak in G(gamma) appeared with decreasing temperature, indicating that coil-to-helical conformational transition took place. The conformation transition temperature (CTT) decreased with increasing radiation dose. No transition was observed for kappa-carrageenan irradiated at 200 kGy. A new faster relaxation mode appeared at around 0.1-1 ms at temperatures below the CTT. This peak is found in kappa-carrageenan irradiated at doses exclusively between 75 and 175 kGy. The peak height of this mode is largest at 100 kGy which corresponds to the optimum biologic activity of kappa-carrageenan reported previously.     

Dynamic light scattering study of calmodulin-target peptide complexes

Dynamic light scattering (DLS) has been used to assess the influence of eleven different synthetic peptides, comprising the calmodulin (CaM)-binding domains of various CaM-binding proteins, on the structure of apo-CaM (calcium-free) and Ca(2+)-CaM. Peptides that bind CaM in a 1:1 and 2:1 peptide-to-protein ratio were studied, as were solutions of CaM bound simultaneously to two different peptides. DLS was also used to investigate the effect of Ca(2+) on the N- and C-terminal CaM fragments TR1C and TR2C, and to determine whether the two lobes of CaM interact in solution. The results obtained in this study were comparable to similar solution studies performed for some of these peptides using small-angle x-ray scattering. The addition of Ca(2+) to apo-CaM increased the hydrodynamic radius from 2.5 to 3.0 nm. The peptides studied induced a collapse of the elongated Ca(2+)-CaM structure to a more globular form, decreasing its hydrodynamic radius by an average of 25%. None of the peptides had an effect on the conformation of apo-CaM, indicating that either most of the peptides did not interact with apo-CaM, or if bound, they did not cause a large conformational change. The hydrodynamic radii of TR1C and TR2C CaM fragments were not significantly affected by the addition of Ca(2+). The addition of a target peptide and Ca(2+) to the two fragments of CaM, suggest that a globular complex is forming, as has been seen in nuclear magnetic resonance solution studies. This work demonstrates that dynamic light scattering is an inexpensive and efficient technique for assessing large-scale conformational changes that take place in calmodulin and related proteins upon binding of Ca(2+) ions and peptides, and provides a qualitative picture of how this occurs. This work also illustrates that DLS provides a rapid screening method for identifying new CaM targets.     

Dynamic light scattering by kappa- and lambda-carrageenan solutions

The intensity correlation functions of kappa- and lambda-carrageenan in various salt solutions and at different concentrations have been determined with the help of dynamic light scattering. From the first cumulant of these correlation functions the values of the translational diffusion coefficients D have been derived. They increase with macromolecular concentration. The extrapolated values to infinite dilution of the diffusion coefficients increase with increasing salt concentration as expected from the salt concentration dependence of the r.m.s. radii of gyration determined previously by static light scattering. The translational diffusion coefficient of lambda-carrageenan in 0.1 M NaCl is smaller than the corresponding value for the kappa species. This is consistent with the difference in contour length and linear charge density of the two samples used. No satisfactory interpretation for the concentration dependence of the diffusion coefficient seems to be possible at present. Although current theories for the macromolecular and salt concentration dependence of D, taking into account charge effects, seem to be applicable, they do not allow for a consistent interpretation of the data. No specific difference between the solution behaviour of kappa- and lambda-carrageenan has been detected.     

Detection of DNA using cationic polyhedral oligomeric silsesquioxane nanoparticles as the probe by resonance light scattering technique

A novel cationic polyhedral oligomeric silsesquioxane nanoparticle (cationic POSS) was synthesized and successfully used as a new probe for the detection of DNA by resonance light scattering technique (RLS). It was found that the electrostatic interaction of cationic POSS and DNA could obviously enhance the RLS signal, the enhanced RLS intensity at 360 nm was proportional to the concentration of nucleic acids within the range of 0.35-42.82 microg ml-1 for calf thymus DNA, the determination limit (3sigma) was 0.32 ng ml-1. The results showed this method was very sensitive, convenient, rapid and reproducible.