Treatment of mobile phase particulate matter in low-angle quasi-elastic light scattering

The method of quasi-elastic laser light scattering (QLS), particularly at low forward scattering angles, has been complicated by the transient presence of Mie or large Rayleigh scattering particles which contaminate the scattering volume. These large contaminating particles have substantial effects on photon correlation spectroscopy because the presence of these larger scatterers tends to decrease the value of the apparent diffusion coefficient of the particle of interest. A method is presented which yields more accurate diffusion constants by autocorrelation of selected photon count periods representative of minimal Mie or large Rayleigh particle contamination. This method was applied to the determination of the apparent diffusion constant for four proteins—ovalbumin, chymotrypsinogen-A, bovine serum albumin, and ribonuclease-A.     

Characterization of acid-denatured DNA by low-angle light scattering

Low-angle light scattering results reported previously demonstrated that measurements on high molecular weight native DNA must be made at angles below 30° in order to obtain correct molecular weights. Earlier light-scattering data obtained on denaturated DNA at angles above 30° showed no change in molecular weight upon denaturation, even though other techniques clearly showed that strand separation occurred. This paper reports low-angle measurements on solutions of calf thymus and T7 DNA denatured under acidic conditions. The results demonstrate that a halving of molecular weight consistent with strand separation is detected by light scattering only when low-angle data are used to obtain correct molecular weights for native material. As expected from theoretical considerations, the scattering from denatured DNA is a linear function of sin²(θ/2), where θ is the angle of observation. This result shows that anticipated experimental artifacts have no significant effect on the low-angle measurements and demonstrates that the curvature in the scattering envelope observed for native DNA below 30° is an inherent property of the native molecule.     

Desmin filaments studied by quasi-elastic light scattering.

We studied polymers of desmin, a muscle-specific type III intermediate filament protein, using quasi-elastic light scattering. Desmin was purified from chicken gizzard. Polymerization was induced either by 2 mM MgCl(2) or 150 mM NaCl. The polymer solutions were in the semidilute regime. We concluded that the persistence length of the filaments is between 0.1 and 1 microm. In all cases, we found a hydrodynamic diameter of desmin filaments of 16-18 nm. The filament dynamics exhibits a characteristic frequency in the sense that correlation functions measured on one sample but at different scattering vectors collapse onto a single master curve when time is normalized by the experimentally determined initial decay rate.     

Refolding and aggregation of bovine carbonic anhydrase B: quasi-elastic light scattering analysis

Bovine carbonic anhydrase B (CAB) is chosen as the model protein to study the phenomenon of protein aggregation, which often occurs during the refolding process. Refolding of CAB from 5 M GuHCl has been observed by quasi-elastic light scattering (QLS), which confirms the formation of a molten globular protein structure as reported previously [Semisotnov, G. V., Rodionova, N. A., Kutyshenko, V. P., Ebert, B., Blanck, J., & Ptitsyn, O. B. (1987) FEBS Lett. 224, 9-13]. QLS analysis reveals the formation of multimeric species prior to precipitation. Activity and cross-linking studies have confirmed the presence of inactive multimeric protein species. The dimer formation has been determined to be the initiating step in the aggregation of CAB during refolding. Activity studies have indicated that the first intermediate observed in the refolding pathway of CAB aggregates to form the inactive dimer. The rate of formation of the dimer has a stoichiometric dependence on the final protein concentration. The dimer formation rate is a function of the final guanidine hydrochloride (GuHCl) concentration to the inverse 6.7 power, which correlates well with the binding of GuHCl to the native protein in 0.60-0.80 M GuHCl. These rate dependencies require the refolding of CAB to be performed at high GuHCl concentrations (1 M GuHCl) and low protein concentrations (less than 1 mg/mL) to avoid the formation of aggregates. Alternatively, refolding can be performed by allowing the first intermediate to form the second intermediate prior to further dilution or dialysis. The aggregation of a hydrophobic first intermediate species is likely to be common to the refolding of other molten globular proteins.     

Motility analysis of circularly swimming bull spermatozoa by quasi-elastic light scattering and cinematography.

The Rayleigh-Gans-Debye approximation is used to predict the electric field autocorrelation functions of light scattered from circularly swimming bull spermatozoa. Using parameters determined from cinematography and modeling the cells as coated ellipsoids of semiaxes a = 0.5 micrometers, b = 2.3 micrometers, and c = 9.0 micrometers, we were able to obtain model spectra that mimic the data exactly. A coat is found to be a necessary attribute of the particle. It is also clear that these model functions at 15 degrees may be represented by the relatively simple function used before by Hallett et al. (1978) to fit data from circularly swimming cells, thus giving some physical meaning to these functional shapes. Because of this agreement the half-widths of experimental functions can now be interpreted in terms of an oscillatory frequency for the movement of the circularly swimming cell. The cinematographic results show a trend to chaotic behavior as the temperature of the sample is increased, with concomitant decrease in overall efficiency. This is manifested by a decrease in oscillatory frequency and translational speed.     

Properties of pancreatic zymogen granules studied by quasi-elastic light scattering

Physico-chemical properties of isolated zymogen granules of the mouse pancreas were studied by means of quasi-elastic light scattering. The average diameter of the granules in 0.3 M sucrose was found to be 1.1 ± 0.1 μm from the correlation time of intensity fluctuation of the scattered light. The average diameter altered depending on the osmolality of the medium in a manner that the alteration was smaller than that expected from the van't Hoff relation. Aggregation of the granules induced by the increase of Ca²⁺ concentration or the decrease of pH in the medium was also detected. The aggregation started at a critical level of 1 mM CaCl₂ or at pH 5.4.     

Measurement of particle sizes by elastic and quasi-elastic light scattering.

A method of measuring an average particle radius in a highly polydisperse dispersion using the wavelength dependence of turbidity is described. For particles which are no larger than 0.3 of the wavelength of light used, a polynomial representation of the scattering cross-section can be used. For larger particles, more extensive numerical calculations are required. The use of the method is illustrated by determining the average particle radius of casein micelles by both elastic and quasi-elastic light scattering techniques. A polydisperse homogeneous sphere model is found to be a reasonably accurate representation of casein micelles. Several modifications of the model which would improve the agreement between the two techniques are mentioned briefly.     

Casein micelle size from elastic and quasi-elastic light scattering measurements.

The average molecular weight, particle radius and size distribution of particles in skim milk from eight cows in mid-lactation have been measured by means of elastic and quasi-elastic light scattering techniques. The properties of sub-micellar casein particles in the milk of each cow were also studied. Particular attention has been given to the effects of particle size heterogeneity in the interpretation of results. The weight average molecular weight of the particles from different cows varied from 2.6-10(8) to 15-10(8) and the corresponding average particle radius varied between 90 and 130 nm. An unusual feature of these particles is their high water content, which was found to vary from 2.4 to 6.4 ml/g with a positive correlation between average particle density and average particle mass. Variations in particle water content can be most readily understood in terms of a gel-like casein micelle.     

Very low-angle light scattering. A characterization method for high-molecular-weight DNA

A very low-angle light-scattering photometer is described with respect to optical features, scattering cell, correction factors, and absolute calibration in the angular range 2°–35°. An improved microfiltration apparatus was used to obtain essentially dust-free aqueous solutions for very low-angle light scattering. The instrument was calibrated with silicotungstic acid, an absolute molecular-weight standard, and the calibration was confirmed with the use of several secondary standards. Very low-angle light-scattering measurements were made to determine the weight-average molecular weight M?_r and z-average radius of gyration R_g,z of a commerical preparation of calf-thymus DNA. Microfiltration of the solutions allowed measurements down to 6°. The value M?_r = 20.0 × 10⁶ obtained by extrapolating 6°–9° data to 0° is more than three times that from 30°–75° data (6.38 × 10⁶) but ～20% smaller than that from 10–35° data (23.7 × 10⁶). The experimental errors in M?_r and R_g,z are estimated to be ±8% and ±14%, respectively. Combined 6°–75° data from two photometers fit well a theoretical scattering curve for a model wormlike coil of the same M?_r as the DNA sample.     

A quasi-elastic light scattering and cinematographic investigation of motile Chlamydomonas reinhardtii.

Quasi-elastic light scattering and cinematographical techniques were used to investigate the motility of Chlamydomonas reinhardtii (wild type). It was found that quantitative information on the trajectory of motion was required for a meaningful interpretation of the autocorrelation functions. Two models for describing the oscillatory motion of the cell were developed; one based on the instantaneous forward-and-backward motion of the cell, and the other based on a sinusoidal perturbation to the average forward motion. Both models gave satisfactory agreement with the shape of the experimentally measured autocorrelation function, thus making it possible to use this measurement to determine mean progressive swimming velocities in a population of greater than 200 cells.     

Rapid estimation of length distributions of microtubule preparations by quasi-elastic light scattering

A new method for determining the length distribution of microtubule preparations, using quasi-elastic light scattering (QELS) is described. The experimental electric field autocorrelation functions are analyzed using a closed-form expression recently described by Hallett, Craig, and Nickel [(1985) Biopolymers 24 , 947–960], which is incorporated into an exponential sampling procedure. The resulting length distributions are compared with those obtained for the same samples with electron microscopy (EM). If standard grid-preparation procedures were used, the EM results yielded shorter length distributions than QELS. When grids were prepared at lower microtubule number densities, less grid washing was required. In these cases, excellent agreement between the EM and QELS techniques were achieved.     

Determining molecular weight distributions of antigen-antibody complexes by quasi-elastic light scattering.

Physiological properties of soluble antigen-antibody (Ag-Ab) complexes depend in part on the size of the complexes. In previous work, the size distribution and structure of model Ag-Ab complexes were determined by electron microscopy. In this study, we used constrained regularization analysis of quasi-elastic light scattering data to estimate molecular weight distributions of model Ag-Ab complexes. A conformational model was necessary to determine appropriate correlations between molecular weight and diffusion coefficient, and to estimate particle structure factors. Porod-Kratky theory proved to be an adequate conformational model for these purposes. The molecular weight distributions determined by constrained regularization compared favorably with distributions obtained either by electron microscopy or by thermodynamic modeling.     

Histone-induced conformational changes in DNA as probed by quasi-elastic light scattering.

Quasi-elastic light scattering as measured by intensity fluctuation (self-beat) spectroscopy in the time domain can be profitably used to follow both the translational diffusion D and the dominant internal flexing mode τ_int of DNA and its complexes with various histones in aqueous salt solutions. Without histones, DNA is found to have D = 1.6 × 10^?8 cm²/sec and τ_int ? 5 × 10^?4 sec in 0.8 M NaCl, 2 M urea at 20°C. Total histone as well as fraction F2A induce supercoiling (D = 2.6 × 10^?8 cm²/sec, τ_int ? 2.8 × 10^?4 sec) whereas fraction F1 induces uncoiling (D = 1.0 × 10^?8 cm²/sec, τ_int ? 9.4 × 10^?4 sec). Upon increasing the salt concentration to 1.5 M the DNA–histone complex dissociates (D = 1.8 × 10^?8 cm²/sec). Upon decreasing the salt concentration to far below 0.8 M, the DNA–histone complex eventually precipitates as a chromatin gel.     

A study of the dynamic properties of actomyosin systems by quasi-elastic light scattering

A laser light source and a digital autocorrelator were employed in the study of the molecular dynamics of acto-heavy meromyosin during the splitting of ATP. Low protein concentrations were used, so that molecular and not gel properties were evident. The addition of Mg²⁺ to acto-heavy meromyosin solutions in the presence of ATP caused a marked widening of the spectrum at high scattering angles. No such change was observed when chemically inactivated heavy meromyosin was used, when actin was cross-linked or when the proteins were in a high ionic strength solution. The data can be interpreted in terms of pronounced change in flexibility of acto-heavy meromyosin induced by active mechanochemical coupling.     

The internal dynamics of gene 32 protein-DNA complexes studied by quasi-elastic light scattering

The hydrodynamic properties of large homodisperse single stranded DNAs complexed with the helix destabilizing protein of phage T4, the product of gene 32 (GP32), have been measured. The results suggest a size of the binding site between 8 and 10 nucleotides/GP32 molecule, in reasonable agreement with earlier work on a complex between GP32 and single stranded 145 base DNA. From static light scattering experiments it is concluded that the persistence length of these complexes is about 30 nm, distinctly smaller than the generally accepted value for double stranded DNA. The quasi-elastic light scattering properties of the DNA-GP32 complexes were determined. The variation of the apparent translation diffusion coefficient Dapp with the scattering vector q was analyzed using the discrete ISMF and Rouse-Zimm models [S.C. Lin et al., Biopolymers 17 (1978) 425]. The model parameters that followed from the fit of Dapp versus q2 and from an extensive global analysis of the actually measured autocorrelation functions agreed with the notion that these DNA-protein complexes are indeed rather flexible. The continuous Soda model [K. Soda, Macromolecules 17 (1984) 2365] could successfully explain the variation of Dapp versus q2, assuming a persistence length of 30 nm and a base-base distance in the complex of 0.44 nm.     

Swimming speed distributions of bull spermatozoa as determined by quasi-elastic light scattering.

88 semen samples from 39 bulls have been investigated by the quasi-elastic light scattering technique. Normal, defective, and dead cells each yielded characteristic autocorrelation functions. The form of these functions indicates that the swimming speed distribution of normal cells is a gamma distribution with two degrees of freedom while that for defective or circular swimmers is a gamma distribution with one degree of freedom. The resulting analysis of the experimental autocorrelation functions yields the fraction of the sample that is normal, the fraction that is defective, and the average speed of each group. The average helical swimming speed of normal cells was found to be 384 micron/s, while the average trajectory speed of the circular swimmers was found to be 103 micron/s. The overall quality of the semen samples as determined by light scattering is compared to quality determination on the same samples by technicians from the artificial insemination industry.     

Molecular weight of T7 and calf thymus DNA by low-angle light scattering

A low-angle light-scattering instrument has been used to measure molecular weights of native calf-thymus and T7 DNA. Molecular weights obtained by extrapolation of angular data to 0° from measurements above 30° are less than molecular weights from extrapolation of data taken at low angles (below 30°). The low-angle molecular weights determined for calf-thymus DNA and for T7 DNA agree well with estimates of weight-average molecular weight obtained with other techniques. The low-angle light-scattering molecular weight for calf-thymus DNA is higher than previously reported values by light scattering at angles above 30°. A concentration dependence in the scattering from DNA solutions is also observed.