INFECTIVITY OF TOBACCO MOSAIC VIRUS 435 



B. Preparation of Protein 



The method most generally employed for the isolation of native TMV 

 protein makes use of alkali for the degradation of the virus. Careful treatment 

 near 0°C. with various buffers of pH 10-10.5 has given extensive splitting 

 of the virus with little denaturation of the protein (Schramm et al., 1955). 

 Alkanolamine buffers appear advantageous for this purpose (Newmark and 

 Myers, 1957). Ammonium sulphate can again be used to separate the protein 

 from the nucleic acid. Electrophoresis has also been employed in preparing 

 monodisperse protein fractions (A-protein), with a sedimentation constant 

 of 4;S, and approximate molecular weight of 100,000 (Schramm and Zilhg, 

 1955). 



An alternate degradation method, which in our laboratory has displaced 

 the alkaline degradation, is by means of 67 % acetic acid (Fraenkel-Conrat, 

 1957b). This medium, an excellent solvent for most native and denatured 

 proteins, dissociates the bonds between the two viral components in the cold 

 and causes the precipitation of the free nucleic acid. The supernatant 

 contams only protem, which can be isolated by dialysis. It aggregates as the 

 acetic acid is removed, and can be sedimented in the ultracentrifuge. The 

 protein can then be redissolved in dilute sodium hydroxide to a final pH of 

 7.5 to 8.0. This procedure is remarkably sunple and convenient and yields 

 native protem in excellent yield that corresponds in all properties to the 

 alkali-prepared protem. The nucleic acid fractions, which can be isolated as 

 by-products of these procedures, are not easily freed from contaminating 

 protein, and are, as stated above, of low infectivity. 



C. Structure and Function of Viral Proteins 



As previously stated, the proteins of TMV and TYMV have been found to 

 occur naturally in the form of particles almost indistinguishable in shape from 

 the complete virus. In the case of chemically degraded TMV, the small 

 molecular protein (4 S) has also been shown to retain the ability to reaggre- 

 gate to the general shape of the original virus under appropriate conditions 

 of pH and ionic strength (Schramm and Zilhg, 1955; Franklin, 1955). Thus, 

 the shape of the viruses appears to be primarily a function of protein struc- 

 ture (Klug and Franklin, 1957). TMV protein consists of peptide chains of 

 an approximate molecular weight of 18,000, held together only by hydrogen 

 bonds and other coordinate or ionic linkages; it appears probable that 

 spherical viruses are also constructed from separate submiits (Klug et al., 

 1957). The tendency of many protems to form specific and stable mole- 

 cular aggregates is a well-known phenomenon. However, in only a few 

 instances are proteins known to form particles approaching in size and in 

 clearly defined architecture the virus protein aggregates, such as the TMV 



