298 P. Alexander, S. F. Cousens and K. A. Stagey 



No alteration could be seen in the chromatogram of the 

 mesyloxy-treated proteins; this is to be expected since the 

 esters formed with the carboxyl groups would be broken down 

 again under the conditions used to hydrolyse the protein 

 into its constituent amino acids. 



The epoxide- and nitrogen mustard-treated proteins gave 

 rise to additional spots on the chromatogram which are pro- 

 bably due to iV-alkylated lysine or histidine (Fig. 1). The 

 new spots from proteins treated with bifunctional reagent are 

 close to the origin and this would be consistent with the cross- 

 linking of two amino acids to give a large molecule which 

 diffuses slowly. 



The reaction with protein SH groups was not studied in 

 serum albumin since this contains less than one SH group per 

 molecule and ovalbumin was used instead. In the native pro- 

 tein no significant reaction could be obtained (Table II) but 



Table II 



Reaction of alkylating agents with the — SH group 

 3% egg albumin before and after denaturation 



Alkylating asent* ^/« ^^ ^'"^''^* ''''''^'^ 



^ ^ ® Native protein Denatured protein-f 



(C1CH2CH2)2NC6H4 •(CH2)3 COOH 14 88 



Chlorambucil (1-7% solution) 



CH3-S02-0-C2H6 (2-6% solution) 55 



CHg— CH • CH3 (5 % solution) 100 



* Reaction time 4 hours at 37° C. t Benatured by giianidine. 



\o/ 



this was to be expected since these groups had earlier been 

 found inaccessible to thiol reagents. After denaturation with 

 guanidine, the — SH groups reacted extremely readily with 

 the three reagents tested. With mustard gas, Bacq (1946) 

 showed that the rate of combination increased rapidly as the 

 pH was raised from 6 to 9 and this is in agreement with the 

 theoretical prediction that the — S~ form is the reactive 

 species. 



