Circular Dichroism Study of the Antibody Combining Site

The circular dichroism (CD) of the Fv fragment derived from myeloma protein 315 possessing anti-dinitrophenyl (Dnp) activity was studied. The CD spectrum in the far-ultraviolet region exhibits a negative band at 217 nm which is characteristic also of the Fab’ and the 7S monomer, and indicates β structure. In addition a new positive band, characteristic only of Fv(the variable fragment), appears at 233 nm. The 217- and 233-nm bands are not affected by hapten binding. On the other hand, all the CD bands in the region 250–310 are enhanced upon binding of the hapten, N∈-Dnp-lysine. The difference spectra in this region of bound and free protein are very similar for Fv, Fab, and the intact protein. Besides this newly observed Cotton effect in the protein absorption range, hapten binding produced large extrinsic Cotton effects in the 310- to 500-nm range, similar to those described earlier by Glaser, M., and Singer, S. J. [(1971), Proc. Nat. Acad. Sci. U. S. 68, 2477], This extrinsic Cotton effect was found to be identical in shape, sign, and magnitude in the 7S monomer and the Fab′ and Fv fragments. It is concluded that the combining site in Fv has the same native features as in the intact protein.