THE BIOCHEMISTRY OF PLANT VIRUSES 99 



also found, but these are insoluble in water, so that the pellet of crystals 

 obtained on centrifuging may be extracted with water and reprecipitated 

 as crystals. 



The virus can, of course, be prepared by centrifuging the clarified sap, 

 but this method has no obvious advantage over the salt precipitation method, 

 which can, if necessary, be scaled up to the tens of liters by using a Sharpies 

 centrifuge both for clarification and for collecting the crystals. 



The original method for purifying the virus (Markham and K. M. Smith, 

 1946) depended upon an alcohol precipitation step which is only feasible 

 under winter temperature conditions in England, but which might be 

 successful in a cold room in hotter climates. 



B. Further Purification 



Under normal conditions three or four recrystallizations of the virus 

 from ammonium sulfate suffice for the purification. Crystallization follows 

 smoothly after enough salt has been added to make the solution slightly 

 cloudy, and it is virtually impossible to prepare the virus in an amorphous 

 form. The crystals are octahedra (Fig. 12). 



The virus is fairly resistant to enzymatic attack, and pancreatic extract 

 has been used for cleaning up preparations, but this is always accompanied 

 by the absorption of enzymes on to the virus particles. Another method which 

 may be employed for the purification is crystallization out of ethanol 

 solutions. A solution of the virus in water, containing about 5 mg./ml. and 

 having a trace of a salt (such as ammonium sulfate) present, is cooled to 0°C. 

 Absolute ethanol (0.3 vol.), also cooled, is added with stirring. The solution 

 is then acidified by adding a few drops of a solution (ethanol 20 parts, 

 acetic acid 10 parts, water to 100 parts) until the virus solution begins to 

 become cloudy. 



The virus then will crystallize out in the cold in the form of bunches of 

 fine birefringent needles, or occasionally as flat plates, which are exceedingly 

 thin. "When centrifuged down, the crystals dissolve readily in 0.1 M Na2HP04, 

 or on the addition of NH3 to a suspension m water. Contaminants may 

 then be spmi oif. This crystallization takes place at about the isoelectric pH 

 of the \nrus (3.7). 



C. Properties of the Preparation 



The virus dissolves readily in water, and is soluble over a wide pH range. 

 The solutions are opalescent and isotropic and have a characteristic ultra- 

 violet hght absorption. The optical density is about 0.7 for 1 cm. at 0.1 mg./ 

 ml. but varies shghtly (Fig. 13). 



