The reaction of human α 2 ‐macroglobulin with α‐chymotrypsin

The kinetics of the conformational changes of human α 2 ‐macroglobulin (α 2 M) induced by reaction with pure α‐chymotrypsin, have been analyzed using three fluorescent probes, namely protein tryptophan groups and the dye 6‐(4‐toluidino)‐2‐naphthalenesulfonate, to monitor alterations of the α 2 M structure, and a covalent conjugate of chymotrypsin and fluorescein isothiocyanate (Chy‐FITC). The main reaction sequence exhibits a triphasic time course with any of the labels used. Each phase is first‐order. The fixation of a single molecule of chymotrypsin to one protease‐binding site of α 2 M (site A) initiates the whole process and determines the access to the second site (site B). Of the three exponential phases of the reaction (20°C), phase I ( k 1 ∼ 19.6 min −1 ) and phase II ( k 2 ∼ 5.3 min −1 ) belong to site A. Phase III is related to site B transformation. It contains two steps with different responses from tryptophan ( k 3 ∼ 0.77 min −1 ) and Chy‐FITC ( k 3 ∼ 0.19 min −1 ) fluorescence measurements. The point to be stressed is that site A and site B, while presumably identical in the native form, are not equivalent with regard to their fluorescence and kinetic properties. However, the activation energy ( E = 30.1 ± 2.7 kJ mol −1 ) is the same for the three phases of the reaction. When present in sufficient excess, free chymotrypsin or native α 2 M is able to form reversible complexes with the above‐related chymotrypsin‐α 2 M adducts. Only the α 2 M site A core seems to be involved in this parallel process. In addition the conformational state of the chymotrypsin‐α 2 M complexes is shown to depend on the pH, with a p K a of 6.4.