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



protein.) When the reaction mixture containing the degraded virus was sub- 

 sequently titrated back to the original pH (usually pH 7-0), more alkali 

 was required than that equivalent to the acid used to reach the pH at which 

 the virus was degraded. The difference was similar to the alkali consump- 

 tion observed in the earlier experiments, 2* and corresponded to about 1-5 

 /xmole per 18 mg virus. Subsequent titrations between pH 7-0 and 3-5 re- 

 quired equivalent amounts of acid and alkah, and followed the same curve 

 as the first back titration (Fig. 2). An alternative procedure is to perform 



Fig. 2. Alkali consumption during degradation of TMV by 1 % sodium dodecyl sulfate 

 below pH 4, as indicated by back titration to original pH. 



the customary degradation with SDS at about pH 7-5 and 40° in a pH-stat 

 and note the amount of alkali required to maintain a constant pH. Such 

 experiments have given an average alkali consumption of 1-9 /xmole/ 18 mg 

 virus (Fig. 3). 



A working hypothesis which could account for these observations is as 

 follows: A certain number of carboxyl groups of the virus (probably 2 per 

 subunit) are hydrogen bonded in the undissociated form to some other, 



OH-X— 



y I 



possibly tyrosine, groups ( — C = 0- — H ). This bond is so stable as to 

 prevent the carboxyl groups from dissociating until disaggregation of the 

 macro-molecule re-establishes their normal pK and titrability. Carboxyl- 

 tyrosine bonds have been proposed by Edelhoch for pepsin, ^^ and are in 

 that case beheved to be stable up to' about pH 6, at which point pepsin 

 becomes denatured. In the case of TMV one would have to assume an 

 extension of the pH stability range of such groups to almost pH 9. Inter- 



