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



finding of Gurd and Murray,^^ but this appears to be the first case where 

 lead and to a lesser extent other divalent ions can enter into protein struc- 

 ture by replacing a normally non-dissociable carboxyl hydrogen atom. 



One alternative possibility which could account for the presented observa- 

 tions is that about 5000 secondary phosphate — OH groups ( — R — O — P — 0~), 



/ \ 



ICH] 



rather than carboxyl groups, prevented from dissociation by their involve- 

 ment in structural stabilization, might acquire their normal dissociability 

 upon degradation of the virus. Since each virus particle contains only about 

 10,000 nucleotides, most if not all phosphate-diester linked, this explana- 

 tion appears to be ruled out. Unusual bonds as numerous as the titrations 

 indicate can only be located in the protein. And unusual bonds of some 

 kind or other will probably be required to explain the unusual stability of 

 the intact virus as compared with its major component, the protein. Simple 

 aggregation, as observed with so many proteins, does not generally evoke 

 major changes in character. Yet the virus is stable at temperatures above 

 60°, while the isolated protein becomes denatured below 40°.^^ Differences 

 in resistance towards enzymes and other agents may be expressions of the 

 same phenomenon. The fact that the ease of degradation of the virus differs 

 for different strains, as well as for chemical derivatives of the same strain, 

 also points to the existence of a definite determining and limiting factor in 

 the inter-unit bonding. It has also been noted that the electrophoretic mobili- 

 ties of the virus, as well as of various virus protein aggregates, are often 

 higher, i.e., the protein is less acidic than one would expect from the amino 

 acid composition. An unexplained shift in the opposite direction was ob- 

 served for the enzymatically dethreoninated virus.^ It would appear that 

 carboxyl-masking could be a determining factor in the observed electro- 

 phoretic differences, as well as in immunological and other particle surface 

 properties. 



STUDIES OF THE PRIMARY STRUCTURE OF TMV 



During the past years various studies have been initiated by several investi- 

 gators in the University of California Virus Laboratory to solve structural 

 and peptide sequence problems connected with the TMV protein. The 

 approach is by now almost conventional. The steps involved are, (1) enzy- 

 matic degradation of the protein ; (2) separation of pure peptide fragments ; 

 (3) elucidation of their composition and sequence; and (4) fitting together 

 of the pieces on the basis of overlaps supplied by the two or more sets of 

 peptides obtained through the use of two or more different enzymes. 



(1) Considerable effort has been made to use the SDS-denatured protein, 

 which is a by-product of the nucleic acid preparation, for these studies. 

 However, the susceptibility of this material to enzymatic digestion has been 



