Buoyant density sedimentation of macromolecules in sodium iothalamate density gradients.

Nucleic acids, bacteriophages, phage capsids, and a DNA-capsid complex have been centrifuged to an equilibrium buoyant density in sodium iothalamate density gradients. Nucleic acids have comparatively high hydrations and are less dense than proteins in these gradients. Sodium iothalamate gradients can be used to separate DNA from RNA, single-chain DNA from double-chain DNA and to separate bacteriophage T7 and λ deletion mutants from the respective wild-type phage.The DNA packaged in bacteriophage T7 appears to be less hydrated than free DNA in sodium iothalamate gradients. There is evidence that the hydration of DNA packaged in phage T7 is restricted by the volume of the phage head. The total volume of phage T7 was estimated to be 1.32 × 10⁻¹⁶ ml. The volume available to package phage T7 DNA was estimated to be 2.2 times the volume of the B form of T7 DNA.     

Active enzyme sedimentation, sedimentation velocity, and sedimentation equilibrium studies of succinyl-CoA synthetases of porcine heart and Escherichia coli

Succinyl-CoA synthetases from Escherichia coli and porcine heart muscle have been viewed as prototypes of two classes of the enzyme. The bacterial enzyme has been reported to be an alpha 2 beta 2 tetramer, with many suggestions in the literature for cooperative interactions between active sites that may contribute to its catalytic efficacy. In contrast, gel filtration experiments of others have indicated that the heart enzyme is a simple alpha beta dimer, with no evidence of dimerization or interaction between like sites. All previous estimates of molecular size of these enzymes have been carried out at concentrations that are much higher than those that are used during activity measurements. The present study was carried out to confirm the differences in the quaternary structures of these two species of succinyl-CoA synthetase and to extend our knowledge of these structures to very low concentrations to enable correlation of their subunit structures with their catalytic properties. Conventional sedimentation velocity centrifugation with both enzymes indicates behavior typical of noninteracting globular proteins with no evidence of size heterogeneity. The sedimentation coefficients at infinite dilution (s20,w) have been determined to be 7.04 S and 4.55 S for the E. coli and porcine heart enzymes, respectively. Sedimentation velocity measurements have been extended to very low enzyme concentrations (typical of those used in activity measurements) by active enzyme centrifugation experiments, in which we have determined the rate of sedimentation of a zone of active enzyme through a chromogenic substrate solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Seston sedimentation

SUMMARY. Three experiments are described in which the rates of loss from suspension of Lycopodium spores introduced at the water surface in a large butyl rubber enclosure ('Lund Tube') were monitored. Rates of arrival at the mud surface and in sediment traps placed in the water column were also measured. Results obtained by each method are compared with the predetermined intrinsic sinking behaviour of the individual particles. The experiments, which were carried out under differing conditions of water-column stability (isothermal mixing, thermal microstratification, and autumnal destratification), demonstrate the importance of turbulent mixing in determining the effective settling rate of seston. A simple predictive model is developed to relate sinkinglosses to the vertical extent of broadly defined water layers. Recoveries in sediment traps can give an accurate synopsis of net sedimentation when they are operated in non-turbulent water layers, but trap performance in turbulent flow is significantly modified by hydrodynamic perturbation generated by the traps themselves.     

The evaluation of standard sedimentation coefficient in band sedimentation of macromolecules

Standardization in the calculation of the sedimentation coefficient of macromolecules by means of band techniques is discussed. When a sample of macromolecules suspended in a solvent is layered on to a denser bulk solution, the macromolecules do not sediment in this solution alone, but sediment in a mixture of bulk solution and sample solvent. This is caused by the diffusion between sample solvent and bulk solution. Experimental evidence of this process is shown during band sedimentation of ribosomes when the variation of the density and the viscosity along the cell is measured. The calculation shows that in the experimental conditions frequently used, standardization made in the usual way leads to a sedimentation coefficient which is largely overestimated; while standardization yields the correct coefficient if the diffusion effect of the sample solvent in to the bulk solution is taken into account, together with possible deuteration effects. A method to calculate the standard coefficient with the aid of a computer is proposed.     

Convection-free boundary sedimentation

A simple technique is described that prevents the occurrence of convective flows in boundary sedimentation experiments. Superimposed density gradients employed as in band centrifugation are evaluated quantitatively. The usefulness of the method is demonstrated by comparison of stabilized and nonstabilized experiments run under identical conditions, with considerable variations in temperature introduced on purpose. The technique is shown to be of special value for the investigation of polydispersity problems.     

Flexibility of tropocollagen from sedimentation and viscosity

Sedimentation constant and intrinsic viscosity were measured on purified tropocollagens extracted from earthworm-cuticle and lathyritic ratskin. A cartesian diver viscometer was used to make viscosity measurements at small shear stress and to avoid the effects of surface forces. By comparing the experimental data with the hydrodynamic theories of wormlike-coil of Ullman a value of 1300 Å has been assigned for the persistence length of these tropocollagens. Other factors which may affect the estimate are discussed.     

Continuous monitoring of erythrocyte sedimentation process: a new possible mechanism of erythrocyte sedimentation

An automated system is constructed to record the complete course of erythrocyte sedimentation process. In this system a light source and a paired photodetector are employed to monitor the change of light transmittance at the junction of plasma and the sedimenting red blood cell column, thus providing a continuous record of erythrocyte sedimentation as a function of time. Differentiation of this sedimentation--time curve yields a velocity--time curve of erythrocyte sedimentation. Frequently recorded "spikes" on top of the velocity--time curve imply the episodes of very rapid fall of erythrocytes in the sedimentation tube that cannot be explained by the currently accepted theory of erythrocyte sedimentation based mainly on Stokes' law, and a new mechanism of rouleau coalescing and fracturing is proposed to account for them.     

Analyses of sedimentation equilibrium data

A numerical procedure is presented which can quite adequately compute the molecular weight averages as a function of solute concentration from sedimentation equilibrium data for homogeneous systems and for monomer-dimer associating systems with a possible extension to heterogeneous systems where monotonic variation in the weight average molecular weight is observed such as in weakly associating or dissociating systems. The procedure utilizes the method of orthogonal polynomials for curve fitting which allows for a rapid determination of best fit with minimal round off error. The procedure is particularly applicable in cases where the concentration of solute at the meniscus can be considered to be neither appreciable and reasonably well determined as in low speed sedimentation equilibrium experiments, nor essentially zero as in high speed sedimentation equilibrium experiments where the calculations become somewhat more simplified. The use of moderate speed sedimentation equilibrium has the advantage of providing a more broad concentration distribution in the centrifuge cell which yields more extensive information concerning dissociating systems yet still provides results at low solute concentrations where most solutes can be considered to be behaving ideally.     

Improved activity sedimentation method

The activity sedimentation method of Cohen is improved by use of a sucrose gradient to stabilize the sedimenting band. Approximate means of analyzing the data are outlined consistent with the limitations of the method. The molecular weight of a newly discovered glutamic dehydrogenase from Mycoplasma laidlawii is also estimated to be 250,000 ± 10%.