New Crystalline Forms of Tomato Bushy Stunt Virus

From the first report on the crystallization of tobacco mosaic virus 1 until recently, only 2 additional crystalline forms of purified viruses have been reported, the non-birefringent dodecahedra 2 , 3 of tomato bushy stunt virus (TBS) and the faintly birefringent plates of tobacco necrosis virus. 4 These were obtained by means of crystallization with electrolytes such as ammonium sulfate. A new crystallization technic for large proteins employing hydrophilic colloidal materials such as heparin, starch, etc., has resulted in the crystallization of a hitherto uncrystallized hemocyanin, the usual crystalline form of the anisotropic viruses such as tobacco mosaic virus, and new crystalline forms of TBS 5 and of a tobacco necrosis virus. 6

In continuing studies on the crystallization of TBS by means of heparin and other substances, two previously unobserved crystalline forms of this virus were obtained. One form was obtained using heparin under conditions somewhat different from those reported previously. To a solution of TBS in water at a concentration of 4.0 mg per cc the solid sodium heparinate† was slowly added at room temperature and dissolved with rubbing until an opalescence due to protein precipitation was observed. This appeared at a concentration of about 5% with respect to heparin. The mixture was cooled in an ice-bath until the amorphous precipitate completely redissolved, and was then stored at 4° overnight. Well defined crystals, some of which were 0.2 mm in length, appeared in that time. Warming this mixture to room temperature and rechilling resulted in a new larger crop of smaller crystals of the same shape. The new non-birefringent crystals, shown in Fig. 1, appear to be eight-sided prisms whose ends are tapered by joint triangular planes. After being washed several times with 3 M ammonium sulfate, the crystals were dissolved in water and analyzed for carbohydrate and phosphorus as described previously. 5