Site-directed labeling of a monoclonal antibody: targeting to a disulfide bond.
We have designed and synthesized crabescein, the first member of a class of fluorescent labels that add across disulfide bonds. Crabescein is a fluorescein derivative that reports the rotational correlation time of the immunoglobulin G (IgG) segment to which it is covalently bound. Chemical analysis of the IgG labeled with crabescein indicates that the fluorophore is inserted into the third disulfide bond (cysteine-229 of mouse IgG2a) in the hinge region. The rotational correlation time of this labeled macromolecule was measured as a single exponential with a decay constant of 26.8 ns. This is in contrast to the double exponential with decay constants of 14.3 and 0.2 ns for the same IgG when labeled with fluorescein via a conventional labeling reagent in which the probe is bound to the macromolecule by one-point attachments. Thus, crabescein is the prototype of a class of fluorescent and phosphorescent probes that, by virtue of their two-point attachments to proteins, faithfully report on the dynamics of the segment of macromolecule to which they are covalently bound.
D000818:Animals; D000911:Antibodies, Monoclonal; D002460:Cell Line; D004220:Disulfides; D005452:Fluoresceins / Q000138:chemical synthesis*; D005456:Fluorescent Dyes; D006680:HLA Antigens; D006801:Humans; D007074:Immunoglobulin G; D007202:Indicators and Reagents; D051379:Mice; D010446:Peptide Fragments / Q000032:analysis; D014357:Trypsin
D000911:Antibodies, Monoclonal; D004220:Disulfides; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006680:HLA Antigens; D007074:Immunoglobulin G; D007202:Indicators and Reagents; D010446:Peptide Fragments; C049609:crabescein; D014357:Trypsin