40 E. MARKHAM 



in an amount sufficient to make the final concentration some 1 % (w./v.). 

 This causes a coagulum, which is to some extent dependent upon the presence 

 of divalent cations in the sap and the virus is left in the supernatant fluid. 

 In some cases quite severe losses of infective material ensue and the method 

 has little to recommend it apart from respectable antiquity. 



The most recent innovation in the preparation of clarified sap is the use of 

 butanol in the purification of the tobacco ringspot virus. This procedure, 

 which resembles closely that adopted by Morton (1953) to release soluble 

 enzymes from lipoprotein complexes in mammalian tissues, will be discussed 

 elsewhere, but it is of interest to note here that it is evidently not devoid of 

 risk, because it was found to eliminate the protein associated with the turnip 

 yellow mosaic virus nucleoprotein, which is not a normal plant constituent 

 and which has many of the properties of the virus itself. 



There are, of course, innumerable other ways in which sap clarification 

 might be achieved. Thus, for example, with those viruses which are not 

 precipitated by 0.4 saturation with (NH4)2S04, this salt may be used for 

 the clarification of the sap. 



D. Centrifuge Separation Techniques 



The use of centrifuges for virus purification is usually fairly straight- 

 forward and routine. Provided that the centrifuge is running smoothly and 

 at constant temperature, boundary formation takes place and the bulk of the 

 virus is sedimented in a finite time, which can be calculated accurately for 

 a sectorial cell, and approximately for other types of cell. Sometimes, how- 

 ever, it is necessary to perform separations which are quite difficult and which 

 require special techniques. As an example, both the turnip yellow mosaic 

 virus and the wild cucumber mosaic virus preparations contain two con- 

 stituents, which had to be separated for identification, and the only obvious 

 method for effecting this separation was by means of centrifugation. 



1. Turnip Yellow Mosaic Virus 



In the case of the turnip yellow mosaic virus two components are present 

 having sedimentation coefficients approximately in the ratio of 1 : 2. Conse- 

 quently, if one were to sediment the faster component completely, only 

 about half the slower component would be in the pellet. A second sedimenta- 

 tion would leave one-quarter, and so on. Thus in theory one cannot obtain 

 the faster sedimenting nucleoprotein component completely free from the 

 slower protein component by simple sedimentation. Yet in practice this 

 separation is quite simple. The reason for this is as follows: In an ordinary 

 centrifuge, the boundaries are in a metastable condition. It is not generally 

 appreciated that, because the centrifugal field increases with the distance 



