Denaturation of concanavalin A by urea at acid pH

The denaturation of dimeric concanavalin A induced by urea at pH 3 has been studied using optical activity and sedimentation velocity. Under the conditions employed Mn+2 and Ca+2 are dissociated from the protein, but the basic structural elements are little changed from those prevailing in the functional lectin at pH 5.5 [H.E. Auer and T. Schilz, preceding paper in this issue]. The protein passes through three stages as the urea concentration is varied from 0 to 10 M. Below 4 M urea the only effect observed is the loss of optical activity of the aromatic amino acid residues. At 4 M, a conformational change occurs producing extensive aggregation, which persists to 7 M. At 8-10 M urea a disordered monomeric protein molecule prevails. The protein could be reactivated provided that dilution to native conditions was very rapid and the protein concentration remained very low. Kinetics of denaturation were monitored by optical activity at 218, 225 and 283 nm. Transients with one, two or three components were observed, which were resolved by nonlinear regression according to sequential first-order decay laws. First order character was confirmed by independence of the kinetic parameters from protein concentration over a two- to four-fold range. Enthalpies and entropies of activation for the various steps were also determined. The transients at the three wavelengths monitor changes in beta structure, beta turns and aromatic groups, respectively. The urea dependence of the rate constants is unique in most cases. It is concluded that different structural elements of the concanavalin A molecule unfold independently from one another.