Molecular basis of virology / edited by H. Fraenkel-Conrat.

Date:
[1968]
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Licence: Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)

Credit: Molecular basis of virology / edited by H. Fraenkel-Conrat. Source: Wellcome Collection.

    29/664 (page 11)
    A. Physical Properties 11 which is the well-known Svedberg equation: M = (5) D{\ - Vp) Through such means it is possible to estimate the molecular weight of an unknown virus from the experimental determination of its sedimentation constant, diffusion constant, and partial specific volume. As for methods for the determination of D and K, the reader is again referred to more de¬ tailed descriptions in other articles.Here we only wish to indicate a few basic principles and typical applications to virus problems. Diffusion is the free movement, under the influence of thermal energy, of particles in solution (Brownian movement). If individual particle move¬ ments could be measured over a longer period of time, D could be de¬ termined directly. In the absence of appropriate microscopic techniques, diffusion is usually studied by directing the movement of a large number of solute molecules through the establishment of a concentration gradient. This will result in the net flux of solute molecules from the region of higher to that of lower concentration, leading to a gradually decreasing steepness of the concentration gradient, and eventual equilibration to uniform concentration. Of the methods employed to measure D, the free diffusion method (as contrasted to diffusion through a porous disk) is most often used in the study of viruses. In diff'usion cells of appropriate design (Tiselius electrophoresis cell), a boundary is created between two zones of pure solvent and solution (solvent + solute) respectively. As in the ultracentrifuge, this boundary is visualized with either schlieren, inter- ferometric, or absorption optics. The spreading of the boundary with time is evaluated quantitatively, and must obey certain statistical laws, if the substance under investigation is homogeneous with respect to D. In this sense, the measurement of D with time can be an additional important criterion for viral homogeneity. It must be pointed out that, therefore, the quantitative evaluation of boundary spreading in a sedimentation velocity ultracentrifuge run, can, in principle, be used to determine D. Proper corrections must be made for the effects of radial dilution, sedi¬ mentation (and, consequently, concentration dependence of sedimentation) on the shape of the boundary. These corrections are far from simple. How¬ ever, several investigators have exploited the principal capability of the ultracentrifuge of forming a boundary between solvent and solution by the initial application of a strong centrifugal field,and have made use of so- called synthetic boundary cells.Subsequently, rotor speeds can be reduced to low values in order to minimize any adverse effects of the centrifugal field on the diffusion process. We should like to call special at¬ tention to the paper by Möller.^^ He used the centrifugal field to form the boundaries and subsequent rotor speed deceleration to determine the dif¬ fusion coefficients of TMV, transfer RNA, MS2 bacteriophage, and the faster sedimenting species of E. coli ribosomal RNA. Combination of