J. Biol. Chem., Vol. 256, Issue 2, 547-550, Jan, 1981
Localization of the two protease binding sites in human alpha 2- macroglobulin
F Pochon, V Favaudon, M Tourbez-Perrin and J Bieth
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.
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