14] TOBACCO MOSAIC VIRUS 251 



below, and gives water-clear solutions except in the isoelectric region (pH 

 3-5-6-5). In that pH range there occurs a remarkable reaction: The solution 

 becomes opalescent and its 'virus-like' appearance is borne out by electron 

 micrographs which show that most of the material is again in the form of 

 rods of great variability in length, but exactly the same diameter as the 

 original virus. The shortest rods often stand upright and appear as circular 

 disks with a central perforation. These have been referred to as doughnuts. 

 Thus, the prime biological activity of this particular protein, namely its 

 tendency to controlled aggregation resulting in a particular macro-structure, 

 has not been destroyed in the course of degradation and separation of the 

 nucleic acid. The sedimentation coefficient of this material in solution (usually 

 determined at about pH 9) resembles that of Schramm's electrophoretically 

 separated A-protein (about 4 S) and suggests a molecular weight of about 

 100,000 corresponding to six-chain aggregates. It appears quite possible but 

 by no means proven that units of such a size are required for the proper 

 functioning of this protein. 



A convenient method for the breakdown of the virus and isolation of the 

 protein has recently been found which has the particular advantage of great 

 simplicity.^' The virus is degraded by means of cold 67% acetic acid, the 

 precipitate which flocculates in a few minutes is centrifuged off (largely 

 nucleic acid), and the protein which alone remains in the water-clear solu- 

 tion is isolated after dialysis. The resulting protein preparation appears indis- 

 tinguishable in all respects from that obtained by alkali spHtting and am- 

 monium sulfate fractionation. This is at present the method of choice in 

 our laboratory.* 



TERTIARY STRUCTURE OF VIRUS PROTEIN 



From the preceding description it is clear that the protein as isolated from 

 dilute alkali or from acetic acid must retain a definite shape which enables 

 it to bridge the gulf between a 100,000 molecular weight 'Pseudoglobulin' 

 and a virus rod particle with a mol. wt. of 50 million. What type of bonds 

 are involved in maintaining its structure? This question has repeatedly been 

 attacked in our laboratory. One amino acid side chain which is very prob- 

 ably implicated in this structure is the cysteine-SH group. This group was 

 found to exist in a form which protects it from many — SH reagents of 

 various types, including oxygen, and thus renders it as stable as the masked 

 — SH groups of many other proteins. ^^^^ On the other hand the — SH 

 group can readily be titrated after unmasking it by the usual denaturing 

 agents (such as guanidine hydrobromide), and represents a convenient means 

 of estimating as to what extent a protein preparation has retained its original 

 native character. It was found to be quantitatively present (one per 18,000 



* It has only recently come to our attention that acetic acid was used by Bawden and 

 Pirie in 1937 for the degradation of TMV, although not as a means of preparing the pure 

 virus protein.18 



