Localization of the two protease binding sites in human alpha 2-macroglobulin

The distance between the two protease binding sites in human plasma alpha 2-macroglobulin has been estimated using singlet-singlet energy transfer experiments. alpha-Chymotrypsin was labeled covalently with donor (dansyl chloride) or acceptor (fluorescein isothiocyanate) groups, and the efficiency of transfer between these dyes was measured within the alpha 2-macroglobulin . (alpha-chymotrypsin)2 complex. The distance between the surface exterior of the protease molecules was calculated to be 4 to 11 A, depending on the assumption made about the equivalence of the binding sites. A catalytically active dimer of alpha-chymotrypsin was prepared using the heterobifunctional reagent N-succinimidyl-3-(2-pyridyldithio)propionate. In contrast with the alpha-chymotrypsin monomer, it binds to alpha 2-macroglobulin with a 1:1 stoichiometry. However, the 1:1 alpha 2-macroglobulin . dimeric alpha-chymotrypsin complex is still able to bind 1 mol of the alpha-chymotrypsin monomer. Energy transfer experiments performed with this ternary complex showed that the distance between the alpha 2-macroglobulin-bound alpha-chymotrypsin molecules is not higher than 4 A, i.e. the two protease binding sites in alpha 2-macroglobulin should be about 44 A apart (center to center) if the anhydrous radius of alpha-chymotrypsin is 20 A.