Disaggregation of Tobacco Mosaic Virus by Bovine Serum Albumin Kapila Wadu-Mesthrige, Biswajit Pati, W. Martin McClain,* and Gang-Yu Liu* Department of Chemistry, Wayne State University, Detroit, Michigan 48202 Received October 10, 1995. In Final Form: May 13, 1996 X We have used atomic force microscopy to study tobacco mosaic virus deposited on mica by the evaporation of films of dilute solutions of TMV. Solutions of TMV in distilled deionized water deposit TMV aggregates similar to those seen by electron microscopy on other substrates. However, solutions containing both TMV and bovine serum albumin (BSA) deposit unaggregated, randomly oriented TMV rods. High-resolution AFM images taken under 2-butanol reveal that numerous BSA molecules are attached to each TMV rod. We think that the attachment of BSA particles to TMV rods changes the intervirus interactions and results in dispersed TMV both in solution and on mica. Introduction A growing body of evidence indicates that some colloid solutions are far from homogeneous, with the suspended particles showing an intrinsic tendency to distribute themselves into sizable clusters and voids of irregular shape, even at rather high dilution. 1-3 Earliest experi- mental evidence came from dynamic light scattering, which often shows a distribution of diffusion coefficients in very monodisperse colloid systems. 1 Perhaps more convincingly, Ito et al. report very recently 2 that under the scanning confocal optical microscope, they can directly visualize slow-moving voids and irregular clusters of monodisperse microspheres. 2 Theorists have begun to discuss these findings in terms of an apparent long range attraction, far longer than van der Waals forces, but still shorter than the Coulomb repulsion between the identi- cally charged particles. 3,4 The origin of this force is controversial, but the simplest physical picture is that colloid particles and their counterions sometimes find it energetically or entropically advantageous to cluster in order to share their counterion atmospheres. This many- body clustering is not predicted by the pairwise or few- body potentials that have been the basis of most theoretical work on polyionic solutions. We present here some new information about the clustering of one particular colloid, the tobacco mosaic virus (TMV). We have used atomic force microscopy (AFM) to study TMV solutions dried on mica. The clustering of TMV on graphite films and other electron microscopy substrates is a familiar phenomenon, but one cannot conclude from this evidence that the clusters exist in solution. There is a possibility that the edge of the drying solution droplet drags the viruses together, concentrating them and causing them to aggregate only on the substrate. But we report now conditions under which a drying film deposits randomly oriented rods of TMV. The condition is the presence of a large excess of bovine serum albumin (BSA). Because of the random deposition, there is no reason to suspect that in these solutions the colloid particles are intrinsically aggregated. Therefore, this study suggests that, in the future, comparative studies of TMV solutions with BSA-TMV solutions might resolve the question of intrinsic TMV aggregation in dilute solution, via the sharing of ionic atmospheres, or indeed by any other mechanism. We now review briefly the basic facts about TMV. The length and diameter of TMV rods are 300 nm and 18 nm, respectively, as characterized by X-ray diffraction, 5-7 and its molecular weight is 40 × 10 6 Da. 8 It has 2130 identical proteins in its coat, which wraps helically around its single strand RNA genome of 6390 nucleotides. From electro- phoretic measurements, TMV has an isoelectric point of 3.3. 9-11 Therefore, the TMV surface has net negative charge under neutral pH in aqueous solutions. There are discrepancies in previous studies about the surface charge density, which ranges from 200 to 1000 elementary charges per virus. 12 At high concentrations, TMV forms a gel of remarkable stability. 4,13,14 At moderate concentrations, it forms unstable solutions that separate into two phases. The lower phase is a nematic liquid crystal, and the upper phase has a TMV concentration of 2.45 mg/mL. This concentration is called the “critical overlap concentration”. The supernatant has usually been assumed to be a homogeneous solution of independent, orientationally random rods. When we refer to “dilute solution”, we mean a dilution of the critical overlap concentration. Experimental Section TMV was extracted from infected tobacco leaves following a standard procedure. 15 The virus concentration was determined using UV-visible absorption spectroscopy taking ǫ ) 3.06 mL mg -1 cm -1 at 260 nm. 16 Virus stock solutions were made by repeatedly resuspending a virus pellet in particle-free MilliQ purified water. BSA, fraction V, which is essentially fatty acid and salt free, was purchased from Sigma Chemicals and used without further purification. For the AFM study, about 10 μL of aqueous solution containing TMV was placed on a freshly X Abstract published in Advance ACS Abstracts, July 1, 1996. (1) (a) Patkowski, A.; Gulari, Er; Chu, B. J. Chem. Phys. 1980, 73, 4178. (b) Lin, S. C.; Lee, W. I.; Schurr, J. M. Biopolymers 1978, 17, 1041. (c) Drifford, M.; Belloni, I.; Dablietz, J. P.; Chattopadhay, A. K. J. Colloid Interface Sci. 1985, 105, 587. (d) Sedlak, M.; Amis, E. J. J. Chem. Phys. 1992, 96, 817. (2) Ito, K.; Yoshida, H.; Norio Ise, Science 1994, 263, 66. (3) Schmitz, K. S. Acc. Chem. Res. 1996, 29, 7. (4) Odijk, T. Macromolecules 1994, 27 (18), 4998. (5) Stubbs, G. J. Acta Cryst. 1974, A30, 639. (6) Namba, K.; Patlanayek, R.; Stubbs, C. J. Mol. Biol. 1989, 208, 307. (7) Zenhansean, F.; Adrian, M.; Emch, R.; Taborelli, M.; Jobie, M.; Descouts, P. Ultramicroscopy 1992, 42-44, 1168. (8) Stryer, L. Biochemistry, 3rd ed.; W. H. Freeman and Company: New York, 1988; Chapter 34. (9) Unwin, P. N. T.; Klug, A. J. Mol. Biol. 1974, 87, 641. (10) Jeng, T.-W.; Crowther, R. A.; Scrubbs, G.; Chin, W. J. Mol. Biol. 1989, 205, 251. (11) Ginoza, W.; Atkinson, D. E. Virology 1955, 1, 253. (12) Deggelmann, M.; et al. J. Phys. Chem. 1994, 98, 364. (13) Brenner, S. L.; McQuame, D. A. Biophys. J. 1973, 13, 301. (14) Milleman, B. M.; Irving, T. C.; Nickel, B. G.; Loosley-Millman, M. E. Biophys. J. 1984, 45, 551. (15) Rochon, D. In Vivo and In Vitro Encapsidation of Host RNA by TMV Coat Protein Ph.D. Thesis, Wayne State University 1985. (16) Boedtke, H.; Simmons, N. S. J. Am. Chem. Soc. 1957, 80, 2550. 3511 Langmuir 1996, 12, 3511-3515 S0743-7463(95)00850-X CCC: $12.00 © 1996 American Chemical Society + +