14] TOBACCO MOSAIC VIRUS 257 



found to be somewhat erratic. Digestion of the acetic-acid prepared protein 

 with 1% trypsin (Worthington, cryst.) at 37°, i.e., near its point of heat 

 d^naturation, for about 8 hours has given the most reproducible resuUs. 

 The ninhydrin analyses indicate the splitting of a maximum of about 12 

 bonds (leucine equivalents). The C-terminal groups found in hydrazinolysis 

 experiments in the acid-soluble fraction of the digest were, besides the 

 original C-terminal threonine, in average about 9 arginine, 1-3 lysine, and 

 0-25 residues of leucine and serine. This indicates a high degree of specificity 

 for the enzyme used, but falls slightly short of quantitative digestion [ex- 

 pected: 11 arginine, 2 lysine groups^'']; but the high correction factors, par- 

 ticularly for arginine,^^ lessen the strictly quantitative significance of these 

 values. The A^-terminal groups found'^''-^^ are about one each of aspartic 

 and glutamic acid, serine, threonine, phenylalanine, and tyrosine, possibly 

 two of glycine, and less than one of alanine, valine and leucine, in general 

 quantitative accord with the sum of C-terminal residues. 



Chymotrypsin digestion liberated about 14 leucine equivalents. C-terminal 

 residues were (besides the original threonine) phenylalanine (6 residues), 

 leucine and isoleucine (3), tyrosine (3), tryptophan (about 1) [total of these 

 amino acids: 8, 13+9, 4 and 2^"], and traces (0-2) of serine, glycine, valine 

 and proline. The A^-terminal residues found in such digests were aspartic 

 and glutamic acid (about 4 and 2), glycine and serine (2), and about one 

 each of threonine, valine, alanine, arginine, leucine and phenylalanine. 



(2) For separation of the peptides of TM V digests Dowex 50-X-2 columns, 

 as advocated by Hirs, Moore and Stein, ^^ have been used with some suc- 

 cess. ^^ Several sharp peaks, however, could be resolved into two or three 

 components by subsequent paper chromatography. At present Dr D. Gish 

 is subjecting the digests to countercurrent distribution and appears to get 

 good resolution of the mixture, although here also seemingly homogeneous 

 peaks may occasionally contain more than one component. A two-dimensional 

 paper electrophoresis — paper chromatography technique is being appHed to 

 the problem by Knight and Woody. ^^ A reproducible pattern of spots indi- 

 cating about 12 peptides is revealed by the ninhydrin and/or the chlorine- 

 iodide test.^^ This technique has been used to survey a number of virus 

 strains in comparative fashion. ^^ Indistinguishable peptide patterns were 

 obtained for strains of very similar amino acid composition (masked strain 

 vs. common TMV), while differences were observed for strains showing dif- 

 ferent composition. 



Most of the peptides obtained from similar digests by the various tech- 

 niques of isolation discussed above have been cross-identified. 



(3) The amino acid composition of most peptides has been determined 

 by the Levy fluorodinitrobenzene (FDNB) method^^-^'' after desalting the 

 preparations according to Hirs et al.,^^ or more recently by the method of 

 the Haugaards.^^ The TV-terminal amino acids were determined by standard 

 procedures.^'' The C-terminal amino acid was generally estabhshed by the 



Rps 



