HUMAN ALPHA(2)-MACROGLOBULIN STRUCTURE - LOCATION OF CYS-949 RESIDUES WITHIN A HALF-MOLECULE MEASURED BY FLUORESCENCE ENERGY-TRANSFER

To localize the pair of thiol-ester-derived cysteine-949 residues within a half-molecule of alpha2M and estimate the internal diameter of this alpha2-macroglobulin trap, we have measured the separation between these cysteines. We unexpectedly found that the two cysteines of intermediate form alpha2M had different reactivity, which permitted selective modification of one cysteine with dansyl, and the other with fluorescein fluorophores. From fluorescence energy transfer measurements, we calculated a separation of 41 +/- 10 angstrom between these fluorophores. This indicates that the Cys-949 residues are probably located at the perimeter of the trap with an internal diameter at least as large as this separation.