Dissociation of Bovine 6S Procarboxypeptidase A by Reversible Condensation with 2,3-Dimethyl Maleic Anhydride: Application to the Partial Characterization of Subunit III (subunit isolation/free carboxypeptidase A zymogen/inactive serine protease/inactivating deletion)

Bovine 6S procarboxypeptidase A can be dissociated into its three subunits by acylation with dimethyl maleic anhydride. The deacylated subunits are obtained in a largely native form due to instability of the bonds to dimethyl maleate at pH values near neutrality. The seven first residues of subunit III are identical to residues 18-24 of bovine chymotrypsinogen B and very similar with the same residues in bovine chymotrypsinogen A and C (subunit II) and also in proelastase A of the African lungfish. Therefore, this subunit is likely to be a chymotrypsinogen or proelastase-A-like zymogen which has lost the ability to be activated on account of a deletion of the N-terminal residues from half-cystine 1 to valine 17. Like other pancreatic zymogens, subunit III appears to possess a weakly functional active site. Bovine 6S procarboxypeptidase A is known to be formed by the noncovalent association of three subunits (1). Subunit I is the immediate precursor of carboxypeptidase A. Upon tryptic activation, subunit II yields a serine endopeptidase (2) resembling porcine chymotrypsin C (3, 4). The apparently inactive subunit III has been assumed to derive from subunit II by autolysis or amino acid replacement in the region of the active site (2). The three subunits are so firmly bound that it has not yet been possible to achieve their dissociation without altering their structure. Dissociation at high pH and room temperature (1) yields a completely denatured subunit I and a still activatable but modified subunit II (5). Acylation of the proteins' NH2 groups by negatively charged succinate groups (6) leads to an activatable subunit I, though the succinates cannot be removed under conditions preserving protein integrity. No information about the nature and structure of subunit III is presently available. Ready dissociation of 6S procarboxypeptidase under very mild conditions has now been obtained by condensation with 2,3-dimethyl maleic anhydride (DMMA), which is known to yield unstable derivatives at pHs near neutrality (7). Subunit III was prepared by this technique in practically pure form and likely is a pancreatic serine zymogen modified by a deletion in the N-terminal region that prevents its activation. MATERIALS AND METHODS 6S Procarboxypeptidase A was purified to homogeneity from pancreas acetone powder (8, 5). Subunit II, either free or as part of the trimer, was activated through incubation with trypsin for 90 min at 00 (enzyme-substrate weight ratio, 1:100) in 50 mM Tris-HCl buffer, pH 8.0, and the resulting chymotryptic activity was evaluated potentiometrically on acetyl-i,-tyrosine ethyl ester (ATEE). Subunit I was activated through incubation for 3 hr at 370 with trypsin (1: 5 w/w or 1: 50 w/w for the trimer or free subunit, respectively) in the same buffer as above made 100 mM in CaCl2. Carboxypeptidase activity was measured spectrophotometrically at 233 nm, using carbobenzoxyglycyl-iphenylalanine (Cbz-Gly-Phe) as substrate. An absorbance (A"m) at 280 nm of 19 was used for the determination of all protein concentrations. The Nterminal sequence of reduced-carboxymethylated subunit III was determined automatically with the aid of a Socosi Sequenator model PS-100 using the Quadrol program.