Quasi-elastic light scattering by diffusional fluctuations in RNase solutions

Using measurements of quasi-elastic light scattering spectra, we have investigated diffusional fluctuations of RNase. The diffusion coefficient for individual protein molecules, together with the corresponding calculated effective molecular radius R_eff, were determined. Between room temperature and the point of irreversible denaturation at 63.5°C, R_eff increased from 20-250 A. This is comparable to the plateau in R_eff of 300 A reached after about 200 min following chemical denaturation in 10 M urea. The measurements indicated the presence of a large size component even in the freshly prepared and chromatographically purified solutions. From the diffusion constants deduced for this large component we obtained effective sizes from 1000-5000 A. Concentration and temperature dependent measurements exclude the possibility that these large particles are impurities and indicate that they are the result of aggregations of RNase molecules.     

Quasi-elastic light scattering studies on pyruvate oxidase.

Quasi-elastic or dynamic light scattering has been used to examine the translational diffusion properties of the enzyme pyruvate oxidase (pyruvate: ferricytochrome beta 1 oxidoreductase, EC 1.2.2.2.). Controlled proteolysis of the enzyme converts the native form of the enzyme to a protease-activated form which has a specific activity about 20-fold greater than the native oxidase. Light scattering studies indicate no significant change in the size or shape of pyruvate oxidase as a result of this proteolytic activation. In both cases the enzyme may be characterized as a hydrated sphere with a Stokes radius of about 53A. The sedimentation velocity-diffusion technique was used to obtain the molecular weight of this tetrameric enzyme, about 252 000 with a value of f/f0 of 1.25.     

Quasi-elastic laser light scattering from solutions and gels of hemoglobin S.

Quasi-elastic light scattering has been used to examine solutions and gels of deoxyhemoglobin S. The autocorrelation function is found to decay with a characteristic exponential relaxation which can be ascribed to the diffusion of monomer (64,000 molecular weight) hemoglobin S molecules. In the absence of polymers, the relaxation time is in good agreement with previous measurements of the diffusion coefficient for solutions of normal human hemoglobin. In the presence of the polymer phase, a large (greater than 200-fold) increase in the scattered intensity is observed but no contribution to the decay of the autocorrelation function from the motion of the aligned polymer phase can be detected. Heterodyning between the time-independent scattering amplitude from the polymers and the time-dependent scattering of the diffusing monomers results in a twofold increase in the relaxation time arising from monomer diffusion.     

Quasi-elastic light scattering studies of membrane motion in single red blood cells.

Studies of red blood cells (RBCs) and RBC ghosts, using a quasi-elastic light scattering (QELS) microscope spectrometer, have identified the membrane as the primary source of the light scattering signal. This is the first report in which motion of the cell membrane has been demonstrated to be the primary source of the QELS signal from a cell. Cytoplasmic changes induced in the RBC by varying the osmotic strength of the medium were also detected using this technique. Comparison of the data from white blood cells (WBCs) with the RBC data demonstrated significant differences between different types of cells.     

Quasi-elastic light scattering from fibrinogen and fibrin intermediate structures

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 M_w) 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 $\text{M}{}_{\mathrm{w}}\text{L}{}_{\mathrm{w}}$ of 3860 and 5460 g mol^?1 nm^?1 were found, respectively. No angular dependence of the apparent diffusion coefficient D_app = Γ/q² was found for the monomers and the fibrin clot; all other samples from (ii) showed an angular dependence if M_w ? 1.76 × 10⁶, were Γ is the first cumulant of the time correlation function and q = (4π/λ) sin?/2. A plot of D_app/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.     

Quasi-elastic light scattering. Application to biological problems]

The origin of the light scattered from macromolecular solutions is considered and a discussion is made around the use of laser and light beating spectroscopy to analyze the scattered field spectrum. Several experiments are reviewed where quasi elastic light scattering has been applied.     

Quasi-elastic light scattering by calf thymus DNA and lamdaDNA.

The quasi-elastic light-scattering (homodyne) time-correlation functions of calf thymus and λDNA are shown to contain contributions from at least two relaxation processes. A method of asymptotic analysis is described and used to obtain an estimate of the longest relaxation time as well as the “average” relaxation time and the mean-squared dispersion in this average. Most theories of scattering from macromolecules in the limit of inifinite dilution predict that the longest relaxation time is due to translational self-diffusion. The data obtained, however, indicate that the longest time is not simply related to the translational self-diffusion coefficient of unaggregated macromolecules. It is also shown that the longest relaxation time of λDNA decreases in the later stages of the denaturation transition region. Some possible mechanisms for the origin of this long time are discussed, including a model of restricted motion of a molecule by its neighbors.     

Quasi-elastic light scattering from migrating chemotactic bands of Escherichia coli.

We report the observation of migrating chemotactic bands of Escherichia coli in a buffer solution. The temporal development of the bacterial density profile is observed by the scattered light intensity as the band migrates through a stationary laser beam. We have made a preliminary analysis of the observed band profile with help of the Keller-Segel theory. The model accounts for only some aspects of the observed time evolution of the density profile. The microscopic motility characteristics of the E. coli in the band are simultaneously studied by photon correlation. The measured correlation functions are analyzed to obtain the spatial dependence of the half-width within the band. A simple analytical model is proposed to account for the contribution of the twiddle motion to the correlation function. By analyzing the correlation function as a superposition of straight-line and twiddle motions, we obtain a satisfactory agreement between the theory and the measured angular dependence of the line shape. As a consequence we are able to extract a parameter beta, which measures the average fraction of twiddling bacteria in the center of the band at a given time.     

Quasi-elastic light scattering of Artemia ribosomes sedimented in a CsCl density gradient

It is impossible to measure the diffusion coefficient of macromolecules directly and accurately by quasi—elastic light scattering, when aggregates cannot be eliminated from the solutions to be investigated. Nevertheless, a simple method can be applied to overcome this problem in many cases. Aggregates are separated from the monomeric macromolecules by rate-zonal sedimentation in a CsCl density gradient in a transparent centrifugation tube; the monomers are then located by laser light scattering intensity measurements; photon correlation spectroscopy of the scattered light finally yields their diffusion coefficient. The viscosity of aqueous CsCl solutions at different temperatures and concentrations allows a good separation by centrifugation and a low uncertainty in the reduction of the measured diffusion coefficient to standard conditions.The application of the method to eukaryotic large ribosomal subunits is described as an example.     

Quasi-elastic light scattering study of salt induced conformational transitions of chromatin subunit

The translational diffusion coefficient D_T of monodisperse solutions of 146 base pairs (bp) core particles was studied by the quasi-elastic light scattering technique. When the salinity was raised a change of D_T from 1.9 × 10^?7 cm² s^?1 to 3.2 × 10^?7 cm² s^?1 was detected at about 2 mM NaCl, followed by a smooth decrease of D_T beyond 0.6 M NaCl. The measurements of particle concentration and scattering vector effects on the D_T showed that the influence of interactions between particles can be disregarded. The interaction between particles and counterions is also discussed and does not appear to be the origin of the actual changes in D_T. These transitions of D_T are hence related to changes of shape and size of the particles. It is shown that the single transition at low salinity corresponds to a conformational change while the variation of D_T at high salinity can be interpreted by a destabilization of the edifice. In different regions of salinities, the observed values of D_T can lead to reasonable hydrodynamic models.     

Surface rheological properties of hyaluronic acid solutions

Using an oscillating ring surface rheometer, surface shear rheological studies of hyaluronic acid solutions at physiological pH have demonstrated the elastico-viscous nature of the surface films. The properties of these surface films change with time and are shown to be related to bulk concentration, ionic strength and pH. This ageing behaviour can be explained on the basis of molecular conformational changes and molecular segmental kinetics. The results are discussed in relation to the postulated function of hyaluronic acid in synovial fluid.     

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.