Air-reoxidation of reduced, denatured chymotrypsinogen A covalently attached to a solid matrix

Bovine chymotrypsinogen A was covalently attached to porous succinylglass beads via the zymogen's amino groups. The bound zymogen was completely reduced in 8 M urea, then allowed to reoxidize, and activated to chymotrypsin. Comparison of the k_o (catalytic coefficient) of this preparation with that of a similar preparation which had never been exposed to reductant showed a 53% recovery of esterolytic activity towards N-benzoyl-L-tyrosine ethyl ester. Values of K_m and K_o for non-reduced, matrix-bound zymogen, following its activation to chymotrypsin, were 12.8 × 10⁻⁵ M and 11.0 sec⁻¹, respectively. Corresponding values for reduced, air-reoxidized preparations were 6.8 × 10⁻⁵ M and 3.1 sec⁻¹.