Nonrandom Conformation of a Mouse Igg2a Monoclonal-Antibody at Low Ph

Publication Type:

Journal Article


European Journal of Biochemistry, Volume 201, Number 1, pp. 223-232 (1991)




circular-dichroism; tryptophanyl residues; proteolytic cleavage; secondary structure; rabbit igg; proteins; fluorescence; acid; exposure; phosphorescence


The pH dependence of the conformation of a mouse IgG2a,kappa monoclonal antibody (MN12) was investigated by several physical techniques, including fluorescence spectroscopy, near-ultraviolet and far-ultraviolet CD, and electric-field-induced transient birefringence measurements. The intensity of the intrinsic tryptophan fluorescence remained constant in the pH range from 3.5 to 10.0. A conformational alteration in the MN12 molecule was observed in the pH 3.5 and 2.5, as reflected by a substantial enhancement of the fluorescence quantum yield. This effect was more pronounced at high ionic strengths. The fluorescence emission was unaltered, indicating that the acid-induced conformational state is different from a completely unfolded state. This was confirmed by CD and fluorescence polarisation measurements. Iodide and acrylamide fluorescence quenching studies indicated a gradually increasing accessibility of MN12 tryptophan residues with decreasing pH. At low pH precipitation was observed in the presence of iodide. One rotational relaxation time (0.16-0.18-mu-s) was observed for MN12 by electric-field-induced transient birefringence measurements at low ionic strength. After exposure of MN12 to low pH for 1 h, the relaxation time was increased to 0.23-mu-s; a further increase to 0.30-mu-s was observed after 24 h. The combined results suggest an acid-induced expansion and enhanced flexibility of MN12, which eventually leads to irreversible aggregation.