SEPARATION AND LOCALIZATION OF THE 4 CYSTEINE-949 RESIDUES IN HUMAN ALPHA-2-MACROGLOBULIN USING FLUORESCENCE ENERGY-TRANSFER

By use of the intermediate form (I-form) [Gettins, Crews, & Cunningham (1989) Biochemistry 28, 5613-5618], α2-macroglobulin can be specifically labeled with fluorescent probes in a manner that allows the determination of the topology of the four thiol ester derived Cys949 residues within this large tetrameric protease inhibitor. Freshly prepared I-form α2-macroglobulin was reacted with 5-[[2-[(iodoacetyl)-amino]ethyl]amino]naphthalene-1-sulfonate (1,5-I-AEDANS) to produce α2-macroglobulin specifically and stoichiometrically labeled with 1,5-AEDANS (donor) at the two Cys949 SH groups in the first protease interaction site. Upon subsequent reaction of this labeled species with chymotrypsin, the remaining two bait regions and thiol ester linkages were opened, generating two free SH groups on the two Cys949 residues in the second protease interaction site. These SH groups were specifically and stiochiometrically labeled with 5-(iodoacetamido)fluorescein (acceptor). Fluorescence energy transfer from donor to acceptor results in 82% loss of AEDANS fluorescence intensity. By use of an R0(2/3) value of 43.5 Å, calculated from the spectral parameters of this system, an R(2/3) separation between donor and acceptor of 33.9 Å was calculated. From fluorescence anisotropy measurements of both donor and acceptor attached to α2-macroglobulin, upper and lower limits on the separation of 43.4 and 26.1 Å, respectively, were calculated. These separations, small in the context of the α2-macroglobulin tetramer, which has approximate dimensions of 190 × 90 × 90 Å, severely restrict the possible locations of the four Cys949 residues. These residues, and the bait regions which are close to them, must be centrally located at the bottom of the deep cavities that constitute the two protease traps. © 1990, American Chemical Society. All rights reserved.