Modification of a single disulfide bond in trypsinogen and the activation of the carboxymethyl derivative.

Sodium borohydride reduction of native trypsinogen produced a specifk cleavage of only 1 of the 6 disulfide bonds. The partially reduced protein was converted to the 14C-carboxymethyl derivative, and tryptic hydrolysis produced two unique peptides in 72 % yield which contained the radioactive label. The composition of the two purified peptides from a completely carboxymethylated sample agreed with the amino acid sequence around Disulfide 179 to 203. (Residue numbers refer to the amino acid sequence of trypsinogen.) Activation of carboxymethyl trypsinogen with trypsin and with enterokmase formed almost fully active molecules toward the active site titrant p-nitrophenyl-p’-guanidinobenzoate. However, the rate of activation of the modified zymogen was much lower than with trypsinogen. Furthermore, the relative activity of the modified enzyme toward synthetic ester substrates was no greater than one-fifth of the normal rate. Disulfide 179 to 203 is the disulfide loop containing the active site serine of trypsin, and the altered enzyme activity suggests that a small change occurred in the orientation of amino acid residues which are part of the active site.