Circular dichroism studies on proteolytic cleavage fragments from rabbit IgG

Abstract

The circular dichroism (CD) spectra of proteolytic cleavage fragments derived from rabbit IgG have been studied. In particular, the CD spectra of C γ2 and C γ3 domain-containing fragments have been obtained and compared with their human counterparts. At wavelength below 250 nm, the CD spectra of the Facb, F(ab′) 2, Fc, pFc′ and tFc′ fragments from rabbit IgG were characterized by a negative absorption maximum at 217 nm, indicating the presence of β-pleated sheet structure. The magnitude of this absorption was significantly greater in the spectrum of the F(ab′) 2 fragment. a negative absorption at 255 nm was observed as a shoulder in the CD spectra of intact rabbit IgG and its Fc fragment, but was absent from those of the other fragments, including both the pFc′ and Facb fragments. The structural feature giving rise to the CD transition at 225 nm was shown to be destroyed by plasmin digestion of acid treated IgG, indicating that this transition reflects an interaction between the C γ2 and C γ3 domains of rabbit IgG. A comparison of the CD spectra of human and rabbit Fc fragments revealed conformational similarities, whereas qualitative and quantitative differences in the spectra of the corresponding pFc′ fragments (C γ3 dimer) was observed. The CD spectrum of the rabbit pFc′ fragment was characterized by a negative maximum at 217 nm, whereas the human pFc′ fragments spectrum was characterized by a negative maximum at 225 nm. At wavelengths greater than 250 nm, the CD spectra of both human and rabbit pFc′ fragments were qualitatively similar. Such studies suggest that the human pFc′ fragment, in marked contrast to the rabbit pFc′ fragment, undergoes significant changes in secondary structure upon cleavage from the intact IgG molecule.