Conformational studies on the tryptic digestion fragments of human immunoglobulin G

Abstract

Conformational studies on Fab(t) and Fc(t) (normal and myeloma) have been carried out by circular dichroism, difference spectra, sedimentation, and viscosity measurements. Both Fab(t) and Fc(t) were largely unfolded in acid (pH 2.1) and alkali (pH 11.7), and the unfolding was greater in alkali than in acid for Fab(t). However, for Fc(t) the circular dichroism spectral change was slightly higher in acid (pH 2.1) than in alkali (pH 11.7). It was also noted that the disorganizations were not complete under these conditions. Similar types of disorganizations were observed in heat denatured Fab(t) and Fc(t). Difference spectral studies demonstrated the presence of a significant number of buried tyrosine and tryptophan residues in both fragments, and a large proportion of the residues became exposed on acid denaturation. The results led us to conclude that both Fab(t) and Fc(t) may be characterized by structures of different stabilities. There seem to be small stable structured regions containing some of the β structures while the other portions are more susceptible to denaturation. Also, there are regions in these proteins which are rich in hydrophobic interactions. Sodium dodecyl sulfate refolded both Fab(t) and Fc(t) into partial helical structures, although the accessibility and rotational freedom of the side chain aromatic chromophores were increased. In 6 m GuHCl containing 0.1 m 2-mercaptoethanol, complete disorganization of Fab(t) was demonstrated despite the fact that its circular dichroism spectra around 217 nm differed from the spectra of random coil polyamino acids. In spite of certain differences, the behavior of Fab(t) and Fc(t) towards different denaturing agents was similar pointing out homology in their basic structural features.