Direct kinetics of bait region cleavage of α‐2‐macroglobulin by a rapid quenching method

Abstract

The rate of bait region cleavage of human c~-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. ~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 HCI. 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 k~ = 40 s -l and k 2 = 2 s ~.c~-2-Macroglobulin; Bait region cleavage; Rapid quenching method

The kinetics of conformational changes of α2‐macroglobulin determined by time resolved X‐ray solution scattering

“…This proteinase-dependent rate-limiting step may or may not be the cleavage of the bait region. Recently we measured the rate of bait region cleavage using SDS-PAGE [25] and pH-indicating dye (in preparation), which showed the existence of cleavage that was faster than the rate shown here. These results suggest that the cleavage of a second bait-region or an unknown proteinase-dependent reaction following bait region cleavage is the rate-limiting step for the conformational change rather than the first bait region cleavage.…”