Analytical Measurements of Ultracentrifugal Sedimentation

Tiselius, Arne; Pedersen, Kai O.; Svedberg, The

doi:10.1038/140848a0

Cited by 90 publications

(18 citation statements)

References 5 publications

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“…in a separation cell of the type designed by Tiselius, Pedersen, and Svedberg (22), and used in studies on tobacco mosaic virus in this laboratory (23). Mter the centrifuge was stopped the content of the top compartment was removed.…”

Section: Additional Component In Pr8 Virus Preparations Obtained By Dmentioning

confidence: 99%

Biophysical Properties of Preparations of Pr8 Influenza Virus

Lauffer¹,

Stanley²

1944

Journal of Experimental Medicine

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The biophysical properties of several preparations of PR8 influenza virus have been studied. Electron micrographs showed slightly irregular, circular particles with an average diameter of 115 mµ and a standard deviation of the distribution of diameters of 15 per cent. The specific volume was determined with a pycnometer to be 0.79. The sedimentation rate was found to vary inversely with the concentration of virus. The extrapolated value for one preparation was 722 Svedberg units and the value for another preparation was 658 units. Sedimentation studies in sucrose solutions of varying densities showed a non-linear dependence of sedimentation rate upon solvent density, indicating that the density in solution increases with increasing sugar concentration. The virus particles floated in a sugar solution with a density of 1.18. The density in the absence of sucrose was estimated to be about 1.1. The virus was shown not to lose infectivity very rapidly in sucrose solutions. The spreading of the boundary during sedimentation was shown to be represented by a standard deviation of the sedimentation rate equal to 8 per cent of the mean. This corresponds to a distribution of diameters with a standard deviation of 4 per cent of the mean. A more slowly sedimenting component was observed with the ultracentrifuge in the highest concentrations of several of the preparations. The intrinsic viscosities, calculated on the basis of concentrations expressed as grams per cubic centimeter, of several highly purified preparations were determined to be between 11.3 and 16.5. The variation of sedimentation rate with concentration was shown to be due entirely to the variation of solution viscosity with concentration. The viscosity of the virus preparations can be explained as being due in great part to the presence of the slower sedimenting contaminating components which possess a very high intrinsic viscosity. The average particle diameter of the anhydrous PR8 virus particle was calculated from sedimentation and specific volume data to be about 80 mµ. The discrepancy between this value and that obtained from the electron micrographs, the greater size distribution from the electron micrographs, the slight irregularities in some of the particles as observed in the electron micrographs, the behavior of the sedimentation process in sucrose solutions of different densities, and the inactivation of the virus by withdrawal of electrolytes can all be explained in a straightforward manner if it is assumed that the virus particles in solution contain, in addition to the constituents shown by chemical analysis, about 60 per cent by weight of water. It is estimated that such hydrated virus particles are about 100 mµ in diameter.

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Section: Additional Component In Pr8 Virus Preparations Obtained By Dmentioning

confidence: 99%

Biophysical Properties of Preparations of Pr8 Influenza Virus

Lauffer¹,

Stanley²

1944

Journal of Experimental Medicine

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“…in a separation cell similar to that described by Tiselius, Pedersen, and Svedberg (14). The sedimentation of the material was followed optically by the Svensson (15) schlieren method.…”

Section: Methodsmentioning

confidence: 99%

The Sedimentation Rate of the Biological Activities of Influenza a Virus

Lauffer¹,

Miller²

1944

Journal of Experimental Medicine

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The sedimentation rates of the mouse infectivity principles of the PR8 and the F12 strains of influenza A virus were shown by studies in the separation cell to have values which are not significantly different from sedimentation rates of the principal components of purified preparations. It was shown further that the bulk of the chicken red blood cell agglutinating activity and of the chick embryo infectivity of PR8 influenza virus preparations sediments at the same rate as that of the 100 mµ. component. Some activity was shown to be associated with aggregates. These results lend strong support to the assumption that the three biological activities are associated with the particles characterized as spheres with a diameter of about 100 mµ.

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“…After 77 min a t this speed, when the major boundary was approximately halfway between the meniscus of the solution and the partition, the rotor was decelerated and the solution withdrawn from the upper portion of the cell. From the content of toxin in this solution compared with that originally present, the sz0 value of the toxic activity was calculated using the formula given by Tiselius et al [4].…”

Section: Ultracentrifugal Examination Of Eluates From Cy Aluminamentioning

confidence: 99%

A Partial Characterization of the alpha-Toxin of Clostridium oedematiens Type B

1970

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The a-toxin of Clostridium oedematiens type I3 was purified by gel-filtration, adsorption with Cy alumina and ultracentrifugation. The molecular weight of the toxin was found to be 132000. The amino acid composition is given and the electrophoretic behaviour of the toxin is described.It was reported earlier [l] that a high degree of purification of the a-toxin of Cl. oedematiens type B could be achieved by gel-filtration on columns of Sephadex G-100. From the behaviour of the toxin on columns of this type, it was suggested that its molecular weight might lie between 50000 and 100000. The investigation referred to above has now been extended in an attempt to prepare the toxin in a pure state. MATERIALS AND METHODSProduction of the Crude a-Toxin The strain of Cl. oedematiens used (CN755 in the Wellcome Research Laboratories' Culture Collection) was reconstituted from the freeze-dried condition in Robertson's meat broth medium. After 48 h a t 37", the resulting culture (500 ml) was used to inoculate the growth medium (51). Th' is was prepared from a papain digest of horse muscle, containing sodium hydrosulphite and 1O0/, by volume of a sodium sulphate extract of horse muscle [2]. This secondary culture was allowed to grow for 48 h a t 37" and the organisms were subsequently removed by filtration. The culture filtrate contained the crude toxin.Assay of the a-Toxin This was carried out by toxin-antitoxin neutralization tests in mice. The antitoxin, a laboratory substandard which had been calibrated by comparison with the International Oedematiens a standard antitoxin, was diluted so as to contain 8 units per ml. The toxin, crude or at various stages during purification, was titrated by adding to a series of volumes (in the preliminary test differing by 500/,, and in the final tests by 1O0/,) 0.5 ml of the laboratory standard antitoxin. The volume of each of these mixtures was made up to 2 ml with buffered saline, mixed by inversion and allowed to stand for one hour at room 24* temperature. Pairs of mice, each weighing 18-22 g, were then injected subcutaneously with 0.5 ml of each mixture and observed for 72 h. The end point of each titration was that mixture resulting in the death of 50°/, of the mice by the morning of the third day. The test was repeated at least once, usually twice, with volumes of toxin differing by loo/, and the results of the separate tests made with mixtures of the same composition were added together. The limits of error ( P = 0.95) have been estimated to be dz loo/,. Gel-Filtration of the a-ToxinDialysis bags each containing crude toxin (500 ml) were suspended in a jar containing polyethylene glycol (carbowax 20 M). After about 72 h at 4", the volume in each sack had diminished to about 10 ml. The contents of 10 bags were pooled and after removing a dark coloured precipitate by centrifugation, the concentrated toxin was applied to a column (100 x 5.5 em) containing Sephadex G-100 (100 g) packed in phosphate buffer a t pH 7.7, I = 0.1. After the toxin had soaked into the gel. it was washed thr...

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Analytical Measurements of Ultracentrifugal Sedimentation

Cited by 90 publications

References 5 publications

Biophysical Properties of Preparations of Pr8 Influenza Virus

Biophysical Properties of Preparations of Pr8 Influenza Virus

The Sedimentation Rate of the Biological Activities of Influenza a Virus

A Partial Characterization of the alpha-Toxin of Clostridium oedematiens Type B

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