94 R. MARKHAM 



its iiifectivity fairly rapidly at 37°C., but apparently without any gross 

 change in its chemistry. It is quite evident that our ideas about virus structure 

 and infectivity are oversimplified. 



The next purification step was precipitation with ammonium sulfate 

 (200 gm./hter), followed by successive precipitations with this salt. The 

 virus is deposited on the addition of salt as an amorphous precipitate, which 

 is more soluble in the cold than at room temperature. This fortunate property 

 enables one to centrifuge a solution of the amorphous material in a refrigerated 

 centrifuge and remove some of the impurities. On further standing, the 

 crystalline form of the virus separates out as beautiful rhombic dodecahedral 

 crystals (Fig. 9). These are very slow to dissolve in cold water, and may be 

 washed if necessary. They can be dissolved very readily in 50 % (v. /v.) 

 ethanol, but will dissolve in water after a time. (The solubility is large. It 

 is only the rate of solution that is small.) 



Stanley (1940), when he came to investigate the virus, elected to omit 

 the heating step, which caused so much loss of infectivity, and used Datura 

 as host. He also used high-speed centrifugation rather than saltmg out for 

 the isolation, although the last step has no obvious advantage, because it is 

 usual to finish the preparation by crystalhzing the virus. 



The yields are somewhat variable but are of the order of 50 to 100 mg. /liter 

 of sap. 



B. Chemical Properties 



The virus contains about 15 % of ribonucleic acid (P = 1.58 %), which 

 has been analyzed by Markham and J. D. Smith (1951b) and de Fremery and 

 Knight (1955). The relative proportions of adenine, guanine, cytosine, and 

 uracil are 25, 28, 21, and 25 on a molar basis. Analyses of the nucleic acid 

 had to be made on the whole virus, because the former is very tightly bound, 

 presumably because of the large numbers of electrovalent links between 

 the protein and the nucleic acid (although, as in turnip yellow mosaic virus, 

 the basic amino acids present are insufficient to neutralize all the phosphate 

 groups). 



The protein of the virus has been analyzed by ion exchange chromato- 

 graphy by de Fremery and Knight, who actually analyzed three isolates 

 which had slightly different symptomatology but had almost identical 

 compositions. The results obtained, in grams of residues per 100 grams of 

 virus, were: alanine, 4.8; arginine, 5.8; aspartic acid, 9.3; cysteine, 0.6; 

 glutamic acid, 5.0; glycine, 4.1; histidine, 1.2; isoleucine, 2.8; leucine, 9.0; 

 lysine, 3.1; methionine, 0.9; phenylalanine, 3.8; proline, 2.7; serine, 5.5; 

 threonine, 8.1; tryptophan, 0.6; tyrosine, 3.3; vahne, 7.2; and amide, 1.16. 

 Estimated colorimetrically, arginine was 6.4. The glycine figure contains an 



