Abstract
The variable regions of five anti-fluorescein monoclonal antibodies were sequenced and corresponding binding sites were studied by means of: steady state fluorescence, circular dichroism (CD) and computer-aided modeling. Steady state fluorescence quenching studies determined that four of the antibodies (FL43.1, FL43.4, FL43.6 and FL55.3) had Kd values (dissociation constants) in the nanomolar range and that the fifth antibody (FL43.11) had a significantly lower Kd in the 10-7M-1 range. Fluorescence quenching studies also suggested that two of the antibodies (FL43.11 and FL55.3) had an exposed tryptophan in the binding site that is not quenched upon fluorescein binding. CD studies of antibody-fluorescein complexes performed in the 400 nm-600 nm range indicated that fluorescein is in contact with tryptophans and/or tyrosines in the binding pocket of all antibodies studied. CD data also supported that fluorescien binds in a significantly different environment with FL55.3 than with the other four antibodies. The variable domains of the antibodies were sequenced by using PCR; and the light chains of four of the antibodies (FL43.1, FL43.4, FL43.6 and FL55.3) were determined to be identical. The sequences of the heavy chains indicated that there were differences in the heavy chain variable domains. These results indicated that there were some constant tyrosines and tryptophans in all of the antibodies, as well as differences, within the variable domains of the antibodies. XABgen computer algorithm was used to model the binding sites. The combination of data acquired in this study supports that H:47W and L:50Y are direct contacts in the binding site with fluorescein in all of the antibodies. There is an extra exposed tryptophan in FL55.3 that accounts for the low tryptophan quenching upon ligand binding. Site directed mutagenesis experiments and x-ray crystallography experiments could determine the specific roles of the tryptophans and tyrosines in the binding pocket.
Harris, Jonathan S (1996). Computer-aided modeling of the binding sites of anti-fluorescein antibodies. Master's thesis, Texas A&M University. Available electronically from
https : / /hdl .handle .net /1969 .1 /ETD -TAMU -1996 -THESIS -H3662.