250 H. FRAENKEL-CONRAT AND K. NARITA [14 



an aggregate of much smaller protein and nucleic acid subunits or mole- 

 cules. The minimal molecular weight of the protein subunit, based on amino 

 acid analyses, is about 18,000. One cysteine and two lysine residues occur 

 in all strains of TMV, and one histidine in the H.R. strain, in a unit of about 

 18,000 gms.^'^'' That this approximates the true molecular weight of the 

 smallest unit was strongly suggested by the discovery of Harris and Knight 

 that carboxypeptidase releases only threonine and no other amino acid from 

 the virus and that the rapidly reached maximum amount of threonine cor- 

 responds to one residue per 17,300^-^ or about 2900 residues per virus 

 particle. It has since been established beyond doubt that this threonine 

 residue represents a C-terminal group,^"-^^ and that the virus is actually 

 composed of about 2900 separate peptide chains, held together only by 

 secondary forces. Since 'macro-structure' is the main function of this pro- 

 tein, it is not surprising that those secondary binding affinities are much 

 in evidence, so that the subunits are hard to separate, and even harder to 

 keep separated under conditions favorable for physicochemical characteriza- 

 tion. Thus, only rough approximations of the subunit weight have as yet 

 been made on the basis of sedimentation studies, ^^ but these support the 

 concept that single peptide chains supply the bricks building up the super- 

 molecule. 



DEGRADATION OF VIRUS AND ISOLATION 

 OF PROTEIN 



A variety of methods has been used for the degradation of the intact virus 

 and for the isolation of disaggregated nucleic acid-free protein. Detergents, 

 such as sodium dodecyl sulfate (SDS), represent the most efficient agents 

 for the disruption of all interunit bonds, but the protein obtained from such 

 reaction mixtures shows many undesirable features, some of which will be 

 referred to later. It appears that these agents break not only inter-chain 

 but also intra-chain secondary bonds. Thus the product is not the expected 

 mixture of sticky bricks, i.e., subunits of definite shape, but a mixture of 

 sticky threads, i.e., denatured peptide chains, with a tendency to unlimited 

 random aggregation into a most intractable and insoluble material. 



Gentler methods for the isolation of the protein make use of alkali which 

 was found by Stanley to degrade the virus. ^^ A commonly used procedure 

 was proposed by Schramm et al.^^ in which the virus is solubilized at about 

 pH 10-5 in the cold. Carbonate, glycine, and more recently alkanolamine 

 buffers have been used for this purpose. ^^ Harrington and Schachman have 

 studied the kinetics of this degradation, and have demonstrated the great 

 tendency for reaggregation of split products in the alkaline reaction mix- 

 tures, particularly at higher temperatures.^^ Ammonium sulfate can con- 

 veniently be employed to separate the protein cleanly from the nucleic acid 

 after all types of degradation. The final protein preparation obtained from 

 alkali-degraded virus appears 'native' in various respects, to be discussed 



