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



mol. wt.) in the two types of soluble protein described above, ^' but absent, 

 presumably autoxidized, in the detergent type of preparation. 



Further studies of this group have shed new light on its mode of linkage, 

 and probably of masked protein — SH groups generally.^^ For it has be- 

 come evident that the H atom of this masked group can be replaced by 

 CHsHg — or by an iodine atom without apparent disturbance of protein 

 structure. The substitution by iodine is particularly surprising since sulfenyl- 

 iodides are normally quite unstable and decompose within a few seconds 

 in aqueous solvents. In the native virus, in contrast, the sulfenyliodide group 

 is stable, but decomposes rapidly if the virus is denatured by agents such as 

 guanidine hydrobromide. 



It must be concluded from these results that the masked — SH groups 

 (1) occur as such in the protein and (2) probably represent the acceptors 

 rather than donors of hydrogen-bonded structures involving other as yet 



H 



/ 

 unknown groups ( — S- — H X), This linkage must be of unusual firm- 

 ness in the present case since the masked — SH groups of ovalbumin and 

 a few other proteins that were tested did not give stable sulfenyliodide deriva- 

 tives, although their reactivity otherwise resembled that of TMV. 



Another type of tertiary bond, which is at present receiving much atten- 

 tion from protein chemists, is the hydrogen-bonded interaction of the phe- 

 nolic groups of tyrosine residues with the carboxylate ion groups of glutamic 

 or aspartic acid residues. Evidence for the presence of such groups is often 

 based on the spectral shift to longer wavelengths observed for tyrosine so 

 bonded. If this spectral shift is largely reversed in acid solution, and if the 

 pK of this effect is within the range of that of carboxyl groups, then a 

 tyrosine-carboxylate interaction is postulated. ^^'^^ In the case of the native 

 TMV protein the spectral divergence from that of an ad hoc prepared amino 

 acid mixture has already been pointed out^ as well as the correction of this 

 divergence by the use of an acidic solvent. Difference spectra have now 

 been plotted for the protein at pH 7 as compared to pH 2-2, and these 

 were found to resemble closely those obtained with ribonuclease, insulin, 

 etc. (Fig. 1). Thus it appears probable that bonds of the tyrosine-carboxylate 

 type do occur in the isolated virus protein It must be noted, however, that 

 the making and breaking of these bonds appears readily reversible and that 

 they are not stable under some, if any, of the conditions required for the 

 degradation of the virus prior to isolation of the protein. Thus it would 

 appear impossible to attribute to these bonds any major role in maintaining 

 the original folding and shape of the protein. Nor can they play part in the 

 inter-unit bonding, unless originally intermolecular bonds of this type are 

 presumed to have exchanged partners during degradation of the virus to 

 give the possibly weaker intra-molecular bonds observed in the final prepara- 

 tion. A search for spectral differences between the intact virus and that 



