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.     

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.     

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.     

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.     

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.     

Aggregation of chlorophyll a probed by resonance light scattering spectroscopy.

We report the resonance light scattering (RLS) spectra of chlorophyll a aggregated in a 9:1 solution of formamide and pH 6.8 phosphate buffer. The aggregate formed after 2 h of mixing, referred to as Chl469, shows a strong scattering feature at 469 nm (Soret band) and a much weaker feature at 699 nm (Qy band). A kinetic investigation confirmed that the aggregation process is cooperative, and also detected one intermediate (Chl458) with a strong RLS spectrum but only a weak CD spectrum. We propose that aggregation proceeds via at least three steps: 1) formation of a nucleating species, probably a dimer of chlorophylls; 2) formation of large aggregates with little or no secondary structure (e.g., Chl458); and 3) conformational change to form helical aggregate (Chl469).     

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.     

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 dyanmics 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.     

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.     

Environment-induced changes in DNA conformation as probed by ethidium bromide fluorescence

The interaction of the ethidium cation with calf thymus DNA is investigated in solutions of different ionic strength and temperature by observation of the enhancement of fluorescence of ethidium upon intercalation in the duplex structure. The quantum yield of the fluorescence of the intercalated dye is found to increase either upon lowering the Na+ concentration or upon increasing the temperature. The existence of a correlation between the geometry of the intercalation complex and the features of the secondary structure of DNA is suggested. Binding isotherms under corresponding environmental conditions are also quantitated by fluorescence enhancement and interpreted in terms of the neighbor exclusion model. Large contributions from change in hydration to the thermodynamics of binding are demonstrated by the temperature dependences of the equilibrium constants. The neighbor exclusion range is found to be practically independent of the salt concentration but its value increases from an average of 2.4 around room temperature to 4-5 at 80 degrees C, as inferred from the binding curves in 0.15 and 0.5 M [Na+] or from the DNA hypochromism vs temperature profiles of complexes at 10(-3) M [Na+]. All the data point to a possible sequence-conformation specificity in the intercalation of ethidium which in heterogeneous DNA is mediated by environmental changes.     

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.     

Hydration of NaDNA by neutron quasi-elastic scattering.

Preliminary results of neutron quasi-elastic scattering experiments are reported for hydrated paracrystals of sodium deoxyribonucleic acid (NaDNA). The samples were investigated at two water contents: 3.5 +/- 1.0 and 9.5 +/- 1.5 mol H2O per mole nucleotide. The results of the scattering experiments were almost independent of whether the NaDNA fibers were oriented parallel or perpendicular to the momentum transfer. The data indicate that at the lower hydration the water molecules do not diffuse appreciably on the time scale of the neutron measurements (approximately 3 X 10(-10) s). At the higher hydration the water molecules diffuse isotropically in a sphere of 9 A in diameter with a diffusion coefficient of (5 +/- 2) X 10(-6) cm2 s-1.     

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.     

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.     

Native conformation of human von Willebrand protein. Analysis by electron microscopy and quasi-elastic light scattering

von Willebrand factor (vWF) was analyzed by electron microscopic and quasi-elastic light scattering techniques in order to evaluate the size and shape of this heterogeneous polymeric plasma glycoprotein. Electron micrographs demonstrated that native vWF molecules are flexible, linear polymers, ranging in contour length from 100 to 1300 nm. In their typical configuration, the polymers were coiled upon themselves with maximal diameters ranging from 60 to 200 nm. Individual repeating protomeric subunits were discernible in occasionally noted, uncoiled polymers and measured 100 nm X 1.5-2.0 nm. Quasi-elastic light scattering analysis confirmed that measurements of the size and shape of purified vWF molecules in solution were similar to those obtained with electron microscopic techniques. In addition, the mean Stokes radius and mean radius of gyration assessed by quasielastic light scattering were directly related over a wide range of values, as were the diameter and contour length measured from electron micrographs, suggesting that the overall shape of polymers does not change with increasing size. This study supports the concept that native vWF molecules are flexible, linear polymers. In addition, this study clearly shows that the polymer configuration assessed from electron micrographs is a valid representation of the configuration of the polymer in solution. The data presented also provide the first evidence for a well-defined, repeating protomeric subunit.     

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.     

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.     

Application of constrained regularization analysis to low-angle quasi-elastic light scattering

A constrained regularization procedure has been applied to a low-angle quasi-elastic light scattering system in order to determine particle size distributions. The conditions under which this procedure may be successfully applied to low-angle photon correlation spectroscopy have been characterized. Acquisition of photon count data over a short time period, relative to the long exponential decay constants of correlation functions obtained at low forward angles, resulted in particle size distributions which were stable with regard to peak width and weighted mean particle radius. Irrespective of the number of photon counts obtained, peak resolution and position on the particle size scale were not optimized unless anomalies in the correlation function due to transient increases in the mean photon counting rate were removed from the photon count data prior to autocorrelation. When such measures were taken, reasonable size distributions were obtained for well characterized protein standards and for liposomal suspensions.