Direct kinetics of bait region cleavage of alpha-2-macroglobulin by a rapid quenching method

The rate of bait region cleavage of human alpha-2-macroglobulin by chymotrypsin was determined by a rapid quenching method under conditions where the bimolecular encounter between the two reactants was not rate-limiting. alpha 2M was first mixed with a 30 molar excess of chymotrypsin in a sequential stopped-flow apparatus and after programmed time intervals the activity of chymotrypsin was quenched with 1 N HCl. The fraction of uncleaved subunits was quantitated by SDS-PAGE under reducing conditions. The result indicated that the bait region cleavage proceeded following a two-exponential decay curve with respective rate constants of k1 = 40 s-1 and k2 = 2 s-1.