224 C. B. ANFINSEN [13 



The techniques of hydrolysis just described by Drs Hirs, Moore and Stein 

 may be difficult to apply in the case of polypeptide chains longer than that 

 of ribonuclease since the number of overlapping peptide fragments to be 

 considered might become forbiddingly large. We have recently described a 

 method by which the action of trypsin may be restricted to those peptide 

 bonds in which arginine constitutes the /^-terminal side.^ This procedure 

 leads to the formation of peptide fragments, one more in number than the 

 number of arginine residues in the chain. The reactions involved are sum- 

 marized in the following equation : 



Trypsin 



HBr 



Cbzo Cbzo Cbzo 



I I I 



(A^-terminal) Lys Arg Lys .... (C-terminal) - 



(residue) (residue) 



Cbzo Cbzo Cbzo 



I I I 



iV-term Lys Arg+NHoR Lys (C-term.) > 



HCOOH 

 benzyl bromide +C02+unsubstituted peptide fragments (as HBr salts) 

 (Cbzo - CgHs . CH2 . OC— O— ) 



As indicated, carbobenzoxylation of the e-amino groups of lysine prevents 

 the hydrolysis of lysyl bonds by trypsin. Following digestion, the carbo- 

 benzoxy groups are removed to yield a mixture of peptides, which may be 

 separated by one of a variety of methods, for subsequent degradative ex- 

 periments. 



It has been the common experience of many investigators that the trypsin 

 digestion of native proteins proceeds very slowly due, presumably, to the 

 steric hindrances introduced by the presence of disulfide bridges. Hitherto, 

 the rupture of these bridges has been generally accomplished by performic 

 acid oxidation which, unfortunately, causes extensive destruction of trypto- 

 phan residues. The full usefulness of the carbobenzoxylation method de- 

 pends, therefore, on the use of techniques for the cleavage of S-S bridges 

 under conditions which are not likely to damage portions of the sequence 

 containing tryptophan. As an approach to this problem, the disulfide bridges 

 of ribonuclease (which contains no tryptophan) and of lysozyme (containing 

 6-7 residues of tryptophan) have been reductively cleaved with thioglycollic 

 acid in 8m urea and the resulting sulfhydryl groups have been converted 

 to their »S-carboxymethyl derivatives by treatment with iodoacetic acid,^ 

 as shown in the equations opposite. 



Fraenkel-Conrat et al. have previously described a reductive cleavage of 

 lysozyme where SH groups were masked by treatment with iodoacetamide.^ 

 The use of iodoacetic acid, however, appears to have a distinct advantage 

 since the final carboxymethylated derivative is quite soluble over a wide 

 range of pH values in contrast to the amide form. 



