Abstract
Quasi-elastic light scattering studies were made of tobacco mosaic virus (TMV) in 10 raM-or 100 m~-Tris-HCl buffer pH 7.2, or in 100 mM-sodium phosphate buffer pH 7.2. The translational diffusion coefficients, extrapolated to zero TMV concentration, were 3.75 x 10 -8 cm2/s in phosphate buffer and 4.50 x 10 -8 cm2/s in Tris buffer; the latter value is in good accord with that calculated for monodispersed TMV. Sedimentation studies showed that one sedimenting component was formed in Tris buffer, but two broad bands were formed in phosphate buffer. Sedimentation coefficients were 192 for monodispersed TMV and 214 for the second component, showing that it was an aggregate. These results indicate that the lower translational diffusion coefficient obtained in phosphate buffer is caused by the presence of TMV aggregates, and that Tris buffer is better than phosphate buffer for the preparation of monodispersed TMV solutions.
Tobacco mosaic virus (TMV) has often been used as a model object for quasi-elastic light scattering studies. In the last few years a number of studies have been published aimed at obtaining a better understanding of the hydrodynamics of a rodlike molecule; translational diffusion coefficients (DT) for TMV were reported in the range of 2.8 × 10 -8 to 3.5 × 10 -8 cm2/s (Dubin et al

Tobacco mosaic virus (TMV) has often been used as a model object for quasi-elastic light scattering studies. In the last few years a number of studies have been published aimed at obtaining a better understanding of the hydrodynamics of a rodlike molecule; translational diffusion coefficients (DT) for TMV were reported in the range of 2.8 × 10 -8 to 3.5 × 10 -8 cm2/s (Dubin et al., 1967; Cumins et al., 1969; Schaefer et al., 1971; Wada et al., 1971 ; King et al., 1973; Schurr & Schmitz, 1973; Thomas & Fletcher, 1978; Loh et al., 1979) . Although most authors have attempted to make their samples as monodispersed as possible to avoid difficulties of data analysis, these values differ significantly from the value of 4-24 x 10 -8 cm2/s calculated from the sedimentation coefficient (194) and the molecular weight of TMV (40 x 106) (Wilcoxon & Schurr, 1983) . Schaefer et al. (1971) presented a theory for the spectrum of light quasi-elastically scattered from rodlike molecules and calculated a value of 4-55 x 10 -8 cmZ/sec for the D~-of the TMV particle (diameter 18 nm and length 300 nm).
In an attempt to resolve the discrepancy between the theoretical and the experimental values, we have done quasi-elastic light scattering and sedimentation studies with TMV in 10 mM-or 100 mM-Tris-HC1 buffer pH 7.2, or in 100 mM-sodium phosphate buffer pH 7.2.
TMV, Japanese strain OM, was purified from systemically infected leaves of Nicotiana tabacum L. var Bright Yellow and purified by polyethylene glycol precipitation and differential centrifugation (Sano & Inoue, 1981) . The TMV samples used were freshly prepared and almost uniform in length as judged by electron microscopy (Sano & Inoue, 1981) . TMV solutions were usually prepared by diluting a small volume of stock TMV solution, kept in optically clean double-distilled water at a concentration of about 10 mg/ml, with filtered solutions of 10 mM-or 100 mM-Tris buffer pH 7.2, or 100 mM-sodium phosphate buffer pH 7.2. For the quasi-elastic light scattering measurements all solutions were optically clarified by injection directly into a cylindrical light scattering cell through a filter (Millipore HAWP, pore size 0.45 ~tm), and the concentration of the optically clarified TMV solutions was determined by spectrophotometry. All other reagents were of the purest grade available.
Quasi-elastic light scattering measurements were made with a Light Scattering Analyser LS-601 (Union Giken Co. Ltd., Japan) at 20 °C, the temperature being kept constant by circulating water. The light source was a He-Ne laser (5 mW, 632.8 nm; NEC Co. Ltd., Japan) and the range of the scattering angles was 25°~< 0 ~< 140 °. The autocorrelator (SAI-43A Correlation and Probability Analyser; Kanomax Co. Ltd., Japan) was connected to the light scattering analyser. The autocorrelation function of the photomultiplier photocurrent was recorded on an oscilloscope (Labo Scope Model 9111; Kanomax) and was analysed by standard computer fitting techniques to obtain the Dx value of TMV. Sedimentation studies were made in a Beckman Spinco Ultracentrifuge Model E by using an An-E rotor at 15000 r.p.m, at 20 °C in single-sector cells. The concentration of TMV was 3 mg/ml and its absorbance at 280 nm was measured in the cell by a photoelectric scanner. Fig. 1 shows the semi-log plot of the autocorrelation function of a TMV solution at a scattering angle of 60 ° in 10 mN-Tris buffer or in 100 mM-phosphate buffer. The observed single-clipped photocount correlation function could be described by a single exponential curve as shown in Fig. l (in both cases the correlation coefficient was 0.999). The Da-values were obtained by the first cumulant of the electric field time correlation function at time t = 0. However, the two autocorrelation functions differed from one another.
The slopes of these straight lines in Fig. 1 indicated -2DTK 2, where K is the magnitude of the wave vector difference between the incident and the scattered light. In order to eliminate the effect of the scattering angle, the autocorrelation functions were measured at a number of scattering angles. The results are shown in Fig. 2 . The apparent translational diffusion coefficient, D2o.w, of TMV obtained from the initial slopes of these curves was 3-75 x 10 -s cm2/s in 100 raM-phosphate buffer and 4.50 x 10 .8 cm2/s in 10 mM-Tris buffer.
These diffusion coefficients were corrected to the values expected at 20 °C in pure water. However, the estimates were made at a single TMV concentration (0.17 mg/ml), and hence these are only apparent diffusion coefficients, i.e. D2o,,~, In order to eliminate the effects of interparticle interaction in the measurements, the concentration dependence of D20,w was measured in 10 mM-or 100 mM-Tris buffer, or in 100 raM-phosphate buffer, at a scattering angle of 25 °. In a region of such low KL values (KL <~ 3), the first cumulant gives the translational diffusion coefficient. The results are shown in Fig. 3 . The D2o,w values showed no detectable concentration dependence at low concentrations of TMV, a result in good agreement with that obtained by King et al. (1973) . The infinite dilution value, o D20,w, obtained by the standard method (King et al., 1973 ; Camerini-Otero et al., 1974; Harding & Johnson, 1985) is the same in 10 m~-Tris buffer and in 100 mM-Tris buffer (D°0,w, 4-50 x 10 -8 cm2/s) and this value is similar to that calculated for the monodispersed TMV particle by Schaefer et al. (1971) and by Wilcoxon & Schurr (1983) . However, the infinite dilution value in phosphate buffer, 3.70 x 10 -8 cm2/s, was lower than that obtained in Tris buffer.
In sedimentation studies only one peak was observed in 10 raM-or 100 mM-Tris buffer (Fig. 4) . Plots of the logarithm of the radial distance against time were linear and the apparent sedimentation coefficient s20,w was 194. However, two broad peaks were observed when TMV was sedimented in 100 raM-phosphate buffer and the apparent sedimentation coefficients obtained were 192 and 214. The faster component was an aggregate of TMV, presumably a dimer. The existence of a TMV dimer at pH values lower than 7 was observed by Allen & Van Holde (1971) , Schurr & Schmitz (1973) and Thomas & Fletcher (1978) . Schurr & Schmitz (1973) suggested that the filtration process successfully removed the small number of end-to-end dimers of TMV that were present in the original stock solution. The filtration process, however, may align the TMV particles to some extent and therefore promote side-by-side aggregation of TMV. Using the usual sedimentation equation for rodlike macromolecules, the sedimentation coefficient is proportional to dZln(2L/d) (where d is the diameter and L the length) so long as TMV particles have the same density (Loh et al., 1979) . If we use the values for the diameter (18 nm) and length (300 nm) of native TMV, the sedimentation coefficient is 232 for an end-toend TMV dimer. For a side-by-side TMV dimer having a diameter of 19.1 nm, i.e. about 1.06 times that of the monomeric TMV the sedimentation coefficient is 214. Therefore the apparent sedimentation coefficient 214 (218 at infinite dilution) is presumably that of a side-by-side dimer of TMV.
These results indicate that the relatively low translational diffusion coefficient observed in phosphate buffer is caused by the presence of the TMV aggregate; Tris buffer therefore appears to be superior to phosphate buffer for the preparation of the monodispersed TMV solution.

{'paper_id': '85312993', 'title': 'Translational Diffusion Coefficient of Tobacco Mosaic Virus Particles', 's2_url': 'https://api.semanticscholar.org/CorpusID:85312993', 'source': 'S2ORC'}