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.
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism*; D003619:Dansyl Compounds; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D006801:Humans; D046911:Macromolecular Substances; D008970:Molecular Weight; D010179:Pancreas / Q000201:enzymology; D011485:Protein Binding; D011487:Protein Conformation; D013861:Thiocyanates; D000511:alpha-Macroglobulins / Q000378:metabolism*
D003619:Dansyl Compounds; D005452:Fluoresceins; D046911:Macromolecular Substances; D013861:Thiocyanates; D000511:alpha-Macroglobulins; D002918:Chymotrypsin; D016650:Fluorescein-5-isothiocyanate