Comparative circular dichroism studies of an anti-fluorescein monoclonal antibody (Mab 4-4-20) and its derivatives.
This study presents circular dichroism (CD) spectra of a high-affinity monoclonal anti-fluorescein antibody (Mab 4-4-20), its Fab fragments, and corresponding single-chain antibody (SCA). In the region 200-250 nm, the differences in the CD spectra between these proteins reflect the uneven distribution of chromophores (tryptophan and tyrosine) rather than a major conformational change. On the basis of near-UV CD spectra, binding of the hapten fluorescein to these protein antibodies elicits an increased asymmetry in the microenvironment of the chromophoric residues in contact with the hapten and also perturbs the interface between VL and VH domains. The hapten-binding site provides a chiral microenvironment for fluorescein that elicits a pronounced induced fluorescein CD spectrum in both the visible and UV regions. In contrast to the parent molecules, SCA is thermolabile. Our results demonstrate that (1) UV CD spectra are useful for assessing the chromophoric microenvironment in the binding portion of antibodies and (2) the extrinsic fluorescein hapten CD spectra provide information about the interaction of hapten with the binding pocket.
Tetin SYu
Department of Microbiology, University of Illinois, Urbana-Champaign 61801.
Mantulin
W W
WW
Denzin
L K
LK
Weidner
K M
KM
Voss
E W
EW
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D001666:Binding Sites, Antibody; D002942:Circular Dichroism; D019793:Fluorescein; D005452:Fluoresceins; D006241:Haptens / Q000378:metabolism; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry; D051379:Mice; D008958:Models, Molecular; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D000911:Antibodies, Monoclonal; D005452:Fluoresceins; D006241:Haptens; D007140:Immunoglobulin Fab Fragments; D014443:Tyrosine; D014364:Tryptophan; D019793:Fluorescein