13] STRUCTURE & ENZYMATIC ACTIVITY OF RIBONUCLEASE 227 



When fully reduced, alkylated ribonuclease was carbobenzoxylated and 

 then subjected to trypsin digestion, results were obtained that completely 

 paralleled those obtained with carbobenzoxylated, oxidized ribonuclease. 

 End-group analysis of trypsin digests of these two forms of the extended 

 ribonuclease chain are shown in Table 3. In both cases, the peptide bond 



Table 3 



N-TERMINAL AMINO ACID ANALYSES OF TRYPTIC DIGESTS 



OF CARBOBENZOXYLATED OXIDIZED RIBONUCLEASE AND OF 



REDUCED AND CARBOXYMETHYLATED RIBONUCLEASE 



The carbobenzoxy groups were subsequently removed with anhydrous hydrogen bromide. 



which joins arginine to a half-cystine residue in the native protein was the 

 most resistant to digestion, although in one case the half-cystine residue was 

 in the form of cysteic acid and in the other was present as ^-carboxymethyl- 

 cysteine. 



Data on lysozyme are presented in Table 4. The reduced, alkylated pro- 

 tein was treated with trypsin in this case without prior carbobenzoxylation. 

 Lysozyme contains eleven arginine and six lysine residues and should there- 

 fore yield eighteen peptides upon trypsin digestion, if all the lysyl and 

 arginyl bonds are intrinsically susceptible to trypsin action (i.e. not adjacent 

 to proline, etc.). Electrophoretic patterns^^ of such a digest are shown in 

 Figs. 1a and 1b. Fig. 1a shows the pattern obtained in 1-5 hours at 35v/cm. 

 (pH 6-5, pyridine, acetate buffer), and Fig. 1b in 2-5 hours under the same 

 conditions. The end-group data in Table 4 show that approximately fifteen 

 peptide bond cleavages are detectable after digestion and that these may, 

 in most instances, be accounted for in the lysyl and arginyl sequences deter- 

 mined to be present in lysozyme by Thompson,^ by Schroeder,^^ and by 

 Acher, et al.^^ It is worthwhile to point out that the time required for com- 

 plete cleavage of the reduced, fully alkylated lysozyme chain was less than 

 five minutes at 37°, employing trypsin at a level of 1 % that of the substrate. 



* A^-dinitrophenyl-iS-carboxymethylcysteine. 



t The values for bis-DNP-lysine, both in lysozyme and in ribonuclease, are sometimes 

 unaccountably low. 



