378 B. MAGASANIK 



h. Salt Fractionation 



The original isolation of tobacco mosaic virus was achieved by salt 

 fractionation.^" The procedure recently described for the preparation of 

 turnip yellow mosaic virus by fractional precipitation with ammonium 

 sulfate is given in detail. ^^ 



(1) Preparation of Turnip Yellow Mosaic Virus.^^ The virus has been prepared 

 from a number of cruciferous plants, namely radish, Bronica carrinata, turnip, Chinese 

 cabbage, and from a specimen of broccoli found infected naturally. For general use 

 either turnip or Chinese cabbage is suitable and, generally speaking, old or "hard" 

 plants give better yields than rapidly growing "soft" plants. Yields up to 1 g. of virus 

 per liter of sap have been obtained from stunted turnip plants grown in the open, but 

 the usual jdelds from glasshouse plants vary from 200 to 500 mg. per liter. 



Plants infected with the virus are minced, either fresh or after freezing overnight, 

 and the sap is expressed. If field turnips are used, it is necessary to use a hydraulic 

 press to remove the last half of the sap. The pH of the sap is usually about 5.7. The 

 sap is clarified by adding slowly, with stirring, 300 cc. of 90% ethanol to each liter. 

 This quantity of ethanol is fairly critical and should be measured carefully. 



The copious precipitate which forms is centrifuged off at once at 3500 r.p.m. for 

 15 minutes. To the supernatant liquid, a volume of saturated ammonium sulfate equal 

 to half the volume of purified sap is added, and the solution is allowed to stand, 

 preferably overnight. After a few hours, large numbers of small octahedral crystals 

 are to be found in the liquid, and these increase in size with time. Many small, highly 

 refractive, birefringent crystals are also to be seen, especially in the sap from old 

 turnip plants, but these are inorganic and are removed later. 



The crystalline precipitate is centrifuged off (30 minutes at 3500 r.p.m.), but the 

 supernatant liquid frequently deposits a second crop of crystals on standing for a 

 day or two, and so may be kept. In addition, it is very difficult to remove all the small 

 crystals by centrifuging as they sediment rather slowly because they are not very 

 dense. 



The precipitate which contains the virus and much insoluble material is resus- 

 pended in a small volume of water (one-tenth to one-quarter of the original sap 

 volume) or buffer solution, and is centrifuged again to remove insoluble material. 

 Precipitation of the virus followed by resolution of the crystals and centrifuging to 

 remove debris is repeated several times. In favorable circumstances the virus prepara- 

 tion may be fairly clean by this stage. 



Several methods may be used for further purification. The virus is not digested by 

 trypsin and treatment with commercial pancreatic extract (10 mg./cc), and incuba- 

 tion for a few hours with a few drops of chloroform as preservative will remove some 

 of the contaminants. The enzyme is removed by several recrystallizations of the 

 virus. 



A treatment which is of use in removing brown pigment, if present, is crystalliza- 

 tion of the virus from dilute alcohol solutions. A solution containing some 5 mg./cc. 

 of virus and a trace of salt is cooled to 0° C. and 0.25-0.30 vol. of absolute alcohol is 

 added gradually with stirring. On adding drop by drop a solution containing 20 cc. 

 of absolute alcohol, 10 cc. of glacial acetic acid, and water to make 100 cc, the virus 

 solution becomes turbid, and this turbidity disappears on allowing the solution to 



2« W. M. Stanley, Science 81, 644 (1935). 



21 R. Markham and K. M. Smith, Parasitology 39, 330 (1949). 



