THE BIOCHEMISTRY OF PLANT VIRUSES 39 



C. Clarification 



The most important single stage in virus purification is the clarification of 

 the sap. Even centrifuging is not adequate for the removal of the pigments, 

 proteins, and other large particles which it contains; therefore it is necessary 

 to subject the sap to some chemical or physical treatment to eliminate most 

 of these substances. Furthermore, it is necessary to do this at this early stage 

 in the purification, because plant proteins become less easy to get rid of as 

 the isolation proceeds. 



There are only two methods which are of general use in clarification. One 

 is particularly satisfactory, although it has not been employed until recent 

 years: that is the addition of 300 ml, of 90 % ethanol to each liter of strained 

 sap with \'igorous stirring. This procedure causes the immediate formation 

 of a coagulum which may be centrifuged off at low speed, leaving a golden, 

 slightly cloudy fluid which contains the virus. A skin of chlorophyll-containing 

 fatty material may form on the top of the fluid when it is centrifuged, but 

 this may be removed by pouring the supernatant fluid through muslin. 

 There are two contraindications to the use of ethanol for clarification. There 

 may be viruses which cannot tolerate this level of ethanol, which is as low 

 as wiU permit successful clarification, but so far none are known to be so 

 very sensitive. The other is that the virus may be precipitated by weak 

 ethanol at the pH of the sap (for most plants this is somewhat on the acid 

 side of neutrality). This possibility has to be considered because, at a slightly 

 higher ethanol concentration, the turnip yellow mosaic virus is precipitated 

 at between pH 4 and 5, and this was indeed part of the origmal purification 

 procedure for this virus. 



The other useful method for clarifying sap depends upon the fact that 

 when the latter is heated to 55°C, a coagulum forms almost instantly and 

 may then be removed by centrifuging. Many viruses will tolerate 55°C. for 

 a short time, and so this is a rapid and convenient j)rocedure. Some viruses 

 are intolerant of these temperatures, and some saps, such as those from 

 cruciferous plants, do not clarify readily by heat. It is also significant that 

 plant pathologists have found that the "thermal inactivation point" i,e,, the 

 temperature which a virus in expressed sap will just not tolerate for 5 

 minutes (a concept which is not altogether scientific), of many viruses which 

 occur in low concentration is of the order of 55°C, It is now known that in 

 at least one of these instances the virus is not so unstable when purified, and 

 it is probable that the "inactivation" is actually due to the entrainment 

 of the virus in the coagulum. This possibiHty of entrainment is ever present 

 and probably accounts for much of the difficulty in purifying many viruses 

 which are present in low concentrations. 



Another popular clarification procedure, and one which would be better 

 abandoned, consists of the addition of a basic substance, usually K2HPO4, 



