ACTIVATION OF BOVINE CHYMOTRYPSINOGEN-A - ISOLATION AND CHARACTERIZATION OF MU-CHYMOTRYPSIN AND OMEGA-CHYMOTRYPSIN

Threonine-neochymotrypsinogen and alanine-neochymotrypsinogen were prepared by limited hydrolysis of bovine chymotrypsinogen A with α-chymotrypsin. The two neochymotrypsinogens were then activated under conditions designed to trap the immediate protein species arising from the cleavage of the Arg15-Ile16 bond. Two trapping procedures were used. In one procedure the neochymotrypsinogen was activated with trypsin under classical rapid activation conditions but in the presence of a competitive inhibitor of chymotryptic action, β-phenylpropionate. In the other procedure, the neochymotrypsinogen was activated with an acid proteinase isolated from Aspergillus oryzae using pH conditions which inhibit autolytic activity. Both were successful in preventing autolytic attack of the initial protein and both yielded identical results. The immediate protein species obtained from activated threonine-neochymotrypsinogen, called M-chymotrypsin, had Thr, lie, and half-cystine as aminoterminal amino acids. The specific esterase activity of μ-chymotrypsin toward N-acetvl-L-tyrosine ethyl ester was two times greater than its stable autolytic product, α1-chymotrypsin. Similarly, the immediate active species of alanineneochymotrypsinogen, called ω-chymotrypsin, had Ala, lie, and half-cystine as amino-terminal amino acids and also displayed an esterase activity which was two times greater than its stable autolytic product, α-chymotrypsin. The first-order rate constants of denaturation in 8 M urea for these two new enzyme species were 0.8 min-1 for μ-chymotrypsin and 1.4 min-1 for ω-chymotrypsin, respectively. These rate constants differ from all previously known species of chymotrypsin. The genesis of μ- and ω-chymotrypsin and their relationship to current schemes for the activation of bovine chymotrypsinogen A are discussed. © 1979, American Chemical Society. All rights reserved.