Physico‐Chemical Properties of Bovine Chymotrypsinogen B

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Abstract
A comparative structural study of chymotrypsinogens A and B at acidic pH has shown in both cases an isomerisation I′⇌II′ occurs due to the unmasking of an abnormal carboxylate ion. Chymotrypsin Bπ does not bind the proflavine which is a competitive inhibitor for trypsin and chymotrypsin Aα. The acidic transition observed for the zymogens also exists for the corresponding enzymes (I⇌II). The unmasking of the abnormal carboxylate ions involves a dissociation of the chymotrypsin Aα or trypsin‐proflavine complexes. The thermal transitions of chymotrypsinogen B have been studied and compared with those of chymotrypsinogen A, trypsinogen and chymotrypsin Bπ. The kinetics of denaturation by urea of chymotrypsinogen B, neochymotrypsinogen B, chymotrypsins Bπ and Bα were studied at different pH. The transformations of chymotrypsinogen B into neochymotrypsinogen and of chymotrypsin Bπ into chymotrypsin Bα are accompanied by a decreased stability in urea. The activation of chymotrypsinogen B to chymotrypsin Bπ increases the stability of the molecule. However, the rates of denaturation of states II′ and II of chymotrypsinogen and chymotrypsin Bπ are much more similar than the rates of denaturation of states I′ and I. The implications of this result are discussed. Ca2+ binds specifically to chymotrypsinogens A and B and to trypsinogen. (pKCa2+= 3.0, 3.1, and 3.2 respectively), probably on identical sites. The protection afforded by this binding against urea denaturation is of the same magnitude for trypsinogen and chymotrypsinogen A; it is much lower for chymotrypsinogen B. This observation together with the experiments concerning proflavine binding shows that chymotrypsinogen A shares with trypsinogen certain functional similarities that it does not share with chymotrypsinogen B. The reactivity of phenol side‐chains has been studied by titration and specific modification with acetylimidazole. The localisation of the normal and abnormal tyrosines in the sequences of chymotrypsinogens A and B is discussed. The reactivity of indole side‐chains was examined with 2‐hydroxy‐5‐nitrobenzyl bromide. The four indoles of trypsinogen and the eight indoles of chymotrypsinogens A and B are masked in the native forms (I′).