Physicochemical characterization of the First World Health Organization International Standard for low molecular weight heparin derivatives.

High-field (300 MHz) 1H NMR spectral analysis and particle size distribution analysis employing the quasielastic light scattering (QELS) technique were performed on samples of the 1st International Standard for low molecular weight (LMW) heparin derivatives recently selected by the World Health Organization (WHO). We propose that the results of these analyses, which showed that the material is highly homogeneous in particle size and retains spectral features characteristic of its porcine mucosal origin, form an appropriate basis for physicochemical comparison between the "Standard" and other LMW heparin preparations.

The formation of multilamellar vesicles from saturated phosphatidylcholines and phosphatidylethanolamines: morphology and quasi-elastic light scattering measurements.

The aggregation properties of diacyl phosphatidylcholines (PC) and phosphatidylethanolamines (PE), with linear symmetrical saturated chains, were characterized at temperatures below and above the lipid solid to fluid transition. PEs in the solid state form bundles of closely apposed flat bilayer stacks which at the solid to fluid transition temperature fold into closed multilamellar vesicles. On the other hand, PCs in the solid state form extended multilayer sheets. At the solid to fluid transition, multilamellar vesicles appear to "bud off" from the surface. Quasi-elastic light scattering (QELS) measurements indicated that for the PEs, bundle size is independent of acyl chain length n, but that the sizes of vesicles which form at the solid to fluid transition are positively correlated with n. The results of temperature jump experiments showed that once the transition temperature was reached, vesicle formation was largely complete within 30 s.

Chemical composition, particle size range, and biological activity of some low molecular weight heparin derivatives.

Several low molecular weight (LMW) heparin sodium derivatives from different sources, as well as some related regular heparin sodium preparations, were examined for chemical composition by high field (300 MHz) 1H NMR spectroscopy, for particle size range by quasi-elastic light scattering (QELS) methods, and for anti-coagulation potency and anti-factor Xa activity by the standard U.S. Pharmacopeial assays described for regular heparin. The NMR spectra provided insight into possible modes of depolymerization used to generate the LMW heparins, as well as into the presence of dermatan sulfate or other chemical contaminants. The QELS analysis permitted the heparin preparations to be characterized and compared by virtue of their distinctive particle size distributions.

Examination of a possible role for dermatan sulfate in the aggregation of commercial heparin samples.

A wide range of commercial heparin preparations were examined by the technique of quasi-elastic light scattering (QELS) for size distribution and evidence for aggregation in relation to determined dermatan sulfate (DS) content. No correlation could be found between DS content and state of aggregation. The possible interaction of DS with heparin was further studied by QELS in a control experiment in which known quantities of DS were added to a heparin sodium preparation known to be low in DS. The effect of increasing DS content was to slightly decrease the measured most probable size of the samples and also to decrease the size spread. The biological activity (as measured by the official test) of the heparin samples, including those treated with additional DS, was found to fall within the accepted limits, independent of the aggregation state of the samples. Overall, there is no direct, observable effect that links DS to the observed aggregation of commercial heparin samples, although DS itself is known to self-associate.

Use of light scattering to estimate the fraction of spherical particles in a mixture.

Two polarization configurations for the light scattered from a mixture of small particles are used to estimate the fraction of spherical particles it contains. The mixture is composed of 0.3-microm alumina polishing powder and 0.106-microm polystyrene spheres. The alumina is an ensemble of randomly oriented irregularly shaped particles. The fraction of spheres in the scattering volume is varied and compared with that deduced from the optical information.

Aggregation of commercial heparin samples in storage.

The size distribution of heparin aggregates in commercial heparin preparations was examined with the technique of quasi-elastic light scattering. The size distributions were initially examined to determine if any relationship existed between the physical state of the heparin preparation, its age, and its biological activity. It was found that commercial heparin samples change their aggregation state in storage. The amount of aggregation appears to be related to the amount of time in storage and to the storage history. Storage of the samples under conditions of refrigeration and handling represents the storage history that most noticeably increases the aggregation state of the heparin preparations. These aggregates, once formed, appear to be stable. The biological activity of the heparin samples (as measured by the official test) was found to still fall within the accepted limits, independent of the aggregation state of the samples. It is not known what effect, if any, a change in the physical state of the commercial preparation should have on its biological activity.

Interaction of hepatic asialoglycoprotein receptor with dimyristoyl phosphatidylcholine vesicles.

The hepatocyte membrane asialoglycoprotein receptor (ASGP-R) was extracted from rabbit liver, purified, and then incubated with preformed vesicles of dimyristoyl phosphatidylcholine. The association of protein with lipid was dependent on vesicle size and the best results were achieved with small vesicles of about 20 nm diameter. The ligand binding capacity of ASGP-R-vesicle complexes was also measured and found to be approximately sevenfold greater than free receptor in aqueous buffer and twofold greater than receptor solubilized in Triton X-100. Most likely, the reconstitution procedure used in these experiments does not result in transmembrane insertion of the receptor. ASGP-R probably resides on the surface of the vesicle, held there primarily by weak hydrophobic forces.

A low angle quasi-elastic light scattering investigation of Chlamydomonas reinhardtii.

This paper outlines the results of the first homodyning low angle scattering experiment known to have been reported for motile particles. Low angle scattering QELS studies were carried out on a wild strain of Chlamydomonas reinhardtii at scattering angles of 3.3 degrees, 2.6 degrees and 1.7 degrees. The resultant experimental autocorrelation functions and scaling curves were compared with a theoretical model that had been successful previously at scattering angles greater than 15 degrees.

A quasi-elastic light scattering and cinematographical comparison of three strains of motile Chlamydomonas reinhardtii: a wild type strain, a colchicine resistant mutant and a backward swimming mutant.

Quasi-elastic light scattering and cinematographical techniques were used to investigate the motility of three strains of Chlamydomonas reinhardtii: a wild cell type (WI), a colchicine resistant mutant (CO), and a backward swimming mutant (BA). Mathematical models were developed which reproduced the experimental autocorrelation functions for each of the three types of cells. These models were based on the physical characteristics of the cells' motion and the values of the parameters in the models were compared with those values that could be obtained from cinematography. Mean progressive speeds for these cells were found to be 84 microns s-1 (WI), 70 microns s-1 (CO) and 30 microns s-1 (BA).

Lateral forces in the filament lattice of vertebrate striated muscle in the rigor state.

The repulsive pressure between filaments in the lattice of skinned rabbit and frog striated muscle in rigor has been measured as a function of interfilament spacing, using the osmotic pressure generated by solutions of large, uncharged polymeric molecules (dextran and polyvinylpyrrolidone). The pressure/spacing measurements have been compared with theoretically derived curves for electrostatic pressure. In both muscles, the major part of the experimental curves (100-2,000 torr) lies in the same region as the electrostatic pressure curves, providing that a thick filament charge diameter of approximately 30 nm in rabbit and approximately 26 nm in frog is assumed. In chemically skinned or glycerol-extracted rabbit muscle the fit is good; in chemically skinned frog sartorius and semitendinosus muscle the fit is poor, particularly at lower pressures where a greater spacing is observed than expected on theoretical grounds. The charge diameter is much larger than the generally accepted value for thick filament backbone diameter. This may be because electron microscope results have underestimated the amount of filament shrinkage during sample preparation, or because most of the filament charge is located at some distance from the backbone surface, e.g., on HMM-S2. Decreasing the ionic strength of the external solution, changing the pH, and varying the sarcomere length all give pressure/spacing changes similar to those expected from electrostatic pressure calculations. We conclude that over most of the external pressure range studied, repulsive pressure in the lattice is predominantly electrostatic.

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.

Effects of hyperosmotic solutions on the filament lattice of intact frog skeletal muscle.

The effect of increasing the osmotic strength of the extracellular solution on the fifament lattice of living frog sartorius and semitendinosus muscle has been studied using low-angle x-ray diffraction to measure the lattice spacing. As the extracellular osmotic strength is increased, the filament lattice shrinks like an osmometer until a minimal spacing between the thick filaments is reached. This minimal spacing varies from 20 to 31 nm, depending on the sarcomere length. Further increase in the osmotic strength produces little further shrinkage. The osmotic shrinkage curve indicates, for both muscles, an osmotically-inactive volume of approximately 30% of the volume in normal Ringer's solution. Shrinkage appears to be independent of temperature and the type of particle used to increase the osmotic strength (glucose, sucrose, small ions). The rate at which osmotic equilibruim is reached depends on muscle size, being slower for greater muscle diameters. Equilibrium spacings are approached exponentially with time constants ranging from 20 to 60 min. Independent of osmotic equilibrium, the lattice tends to shrink slowly by approximately 3% over the first few hours after dissection, probably because of a leakage of K+ ions from inside the muscle cells. This can be partly prevented by using an extracellular solution which contains a higher concentration of K+ ions or which is hypoosmotic. The volume of the muscle filament lattice (1.155d10(2) . S) is constant over a very wide range of sarcomere lengths, and is equal to approximately 3.6 x 10(6) nm3 for a range of amphibian muscle types.