502 



A. TSUGITA AND H. I'RAENKEL-CONRAT 



specific exchange was usually possible. Ccitain changes, involving resi- 

 dues near the .V-acetyl end or the C-teiiuinus, could be ascertained by 

 application of similar methods to the intact protein. 



AVe shall now proceed to consitler the results of these studies (see 

 Table II). The first important observation is that many mutants (about 



Fig. 6. Chromatugnipliic separation of peptides in soluble fraction of tryptic 

 digest of TMV protein. Buffer syslei7i: 



1. 40 ml collidine + 40 ml pyridine + 1.5 ml acetic acid (pH 8.2) (180 ml). 



2. Gradient (by Antegrade) (Technicon Chromatography Co., New York) Cham- 

 bers 1 and 2: 40 ml collidine + 40 ml pyridine + 8.0 ml acetic acid (pH 7.3); 

 3: O.l N acetic acid; 4- 0.5 N acetic acid; 5: 0.5 N acetic acid; 6: IN acetic acid; 

 7: 2N acetic acid; 8 and 9: glacial acetic acid (each chamber contains 120 ml). 



Column, 0.9 X 150 cm, Dovvex 1x2 (200-400 mesh) ; flow rate 30 ml/hour by 

 Mini pump (Milton Roy Co.); fraction, 3.3 ml; 80 mg protein. 



The numbers identify the main component of each peak with the secjuential 

 peptide number on the polypeptide chain; 1 is not present because it is the 

 N-terminal I-peptide removed by centrifugation prior to chromatograph3\ 



50% of those analyzed) did not differ from connnon TMV in their amino 

 acid composition. Thus, the original report of Wittmann (1959a) con- 

 cerning a mutant w'hich showed the same composition as common TMV 

 has been confirmed. 



Another class of mutants was observed which showed the replace- 

 ment of 1-3 amino acids. In no case was the total number of i-esidues 

 found to be changed. Certain amino acids were observed to be frequently 

 substituted, and frequently by a particular one; others have not as yet 

 been found to be replaced. Although our studies covered mutants ob- 

 tained with three different types of reactions (deamination, alkylation, 

 and bromination), the extent and the nature of the observed changes 

 were similar. Thus, 1 deamination, 4 bromination, and 3 methylation 

 mutants showed the replacement of 1 proline by a leucine, which in- 



