The reaction of human alpha 2-macroglobulin with alpha-chymotrypsin. A stopped-flow kinetic investigation.
The kinetics of the conformational changes of human alpha 2-macroglobulin (alpha 2M) induced by reaction with pure alpha-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 alpha 2M 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 alpha 2M (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 degrees C), phase I (k1 approximately 19.6 min-1) and phase II (k2 approximately 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 (k3 approximately 0.77 min-1) and Chy-FITC (k3 approximately 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 alpha 2M is able to form reversible complexes with the above-related chymotrypsin-alpha 2M adducts. Only the alpha 2M site A core seems to be involved in this parallel process. In addition the conformational state of the chymotrypsin-alpha 2M complexes is shown to depend on the pH, with a pKa of 6.4.
D002918:Chymotrypsin / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013861:Thiocyanates; D014364:Tryptophan; D000511:alpha-Macroglobulins / Q000378:metabolism*
D005452:Fluoresceins; D005456:Fluorescent Dyes; D013861:Thiocyanates; D000511:alpha-Macroglobulins; D014364:Tryptophan; D002918:Chymotrypsin; D016650:Fluorescein-5-isothiocyanate