Abstract
The interaction of ricin D with urea was studied spectroscopically. The fluorescence intensity of ricin D increased with increasing urea concentration up to 5 M, but it decreased greatly in 7 M urea accompanied by a red shift of the spectrum. The UV -difference spectra of ricin D induced by urea at concentrations below 6 M versus that in the absence of urea had maxima at 280 and 288 nm, and a trough around 300 nm, while those induced by urea at concentrations greater than 7 M had troughs at 284 and 292 nm. It is suggested that spectroscopic changes of ricin D induced by low concentrations of urea are ascribed to the perturbation by urea of the surface-localized chromophores, but those induced by high concentrations of urea are due to the exposure of chromophores on the surface of the protein molecule by conformational change. Together with the CD data, the critical concentration of urea to induce the conformational change of ricin D was estimated to be 6 M. The saccharide-binding ability of ricin D was retained in urea at concentrations up to 4 M, but it decreased greatly in 6 M urea. From the time-dependent changes in the UV-difference spectra of ricin D and its complex with lactose induced by 7 M urea, it is suggested that urea first interacts with ricin D in such a manner as to perturb the surface-localized chromophores and then induces a conformational change that is prevented by binding with lactose.
