Denaturation of Ricin D in Frozen Aqueous Solutions

Abstract

Ricin D was denatured in frozen acidic solutions accompanied by the insolubilization. Kinetic studies indicated that the insolubilization of ricin D occurred during the incubation of the frozen solution at – 51°C ~ – 5°C, but not in the freezing process at – 80°C or the thawing process. Such an insolubilization of ricin D in the frozen system was accelerated by lowering the pH below pH 4.0, and the optimum temperature was found at – 29°C. The rate of the insolubilization of ricin D decreased on the addition of NaCl or on elevation of the temperature from – 29°C to – 5°C, or on increasing the protein concentration above 0.15%. The freeze-denaturation of ricin D was prevented by saccharides, and lactose, with a higher affinity for ricin D than galactose, suppressed the insolubilization of ricin D more effectively than galactose. A slight suppressive effect of lactose was found for insolubilization of a derivative of ricin D in which the saccharide-binding ability of the low affinity saccharide-binding site was destroyed by N-bromosuccinimide-oxidation. It is suggested that the denaturation of ricin D in the frozen acidic solution is principally due to the concentration of protons and solutes into liquid regions surrounded by ice crystals. Spectroscopic data also suggest that the protonation of the ionizable group(s) with pK_a = 4~5 may be closely associated with the denaturation of ricin D in the frozen state below pH 4.0.