Purification of the Tobacco Etch and Other Viruses of the Potato Y Group. I. S. Damirdagh, Department of Plant Pathology, University of California, Davis 95616; R. J. Shepherd, Department of Plant Pathology, University of California, Davis 95616. Phytopathology 60:132-142. Accepted for publication 25 August 1969. DOI: 10.1094/Phyto-60-132.
A procedure was developed for purification of the tobacco etch and other viruses of the potato Y group without marked aggregation as judged by behavior of the isolated product during sucrose density-gradient centrifugation. Using 0.5 m urea and 0.1% 2-mercaptoethanol in the resuspension buffer during purification was highly beneficial in preventing lateral aggregation. The results suggest that aggregation may be the consequence of hydrophobic interactions.
The purity of virus preparations was assessed by electron microscopy, sucrose density-gradient centrifugation, and polyacrylamide gel electrophoresis. With the latter technique, impure preparations yielded several migrating components in the running gel and heavy staining in the sample gel after electrophoresis. Protein from highly purified virus yielded two components in the running gel without detectable staining in the sample gel after electrophoresis, indicating dimerization due to formation of intermolecular disulfide bonds. Carboxymethylated viral protein migrated as a single component in polyacrylamide gel electrophoresis experiments with either cationic or anionic systems, demonstrating that only a single type of protein is present in the virus.
Also used as a preparative procedure was centrifugation to equilibrium in cesium chloride density gradients—to purify the viruses, to assess the purity of the products isolated, and to estimate buoyant densities. Estimation of buoyant densities by graphical methods gave values of 1.332 g/cc for the tobacco etch virus and 1.336 g/cc for turnip mosaic virus.