Calorimetric studies on the stability of the ribosome-inactivating protein abrin II: effects of pH and ligand binding

Abstract

The effects of pH and ligand binding on the stability of abrin II, a heterodimeric ribosome-inactivating protein, and its subunits have been studied using high-sensitivity differential scanning calorimetry. At pH7.2, the calorimetric scan consists of two transitions, which correspond to the B-subunit [transition temperature (T_m) 319.2K] and the A-subunit (T_m 324.6K) of abrin II, as also confirmed by studies on the isolated A-subunit. The calorimetric enthalpy of the isolated A-subunit of abrin II is similar to that of the higher-temperature transition. However, its T_m is 2.4K lower than that of the higher-temperature peak of intact abrin II. This indicates that there is some interaction between the two subunits. Abrin II displays increased stability as the pH is decreased to 4.5. Lactose increases the T_m values as well as the enthalpies of both transitions. This effect is more pronounced at pH7.2 than at pH4.5. This suggests that ligand binding stabilizes the native conformation of abrin II. Analysis of the B-subunit transition temperature as a function of lactose concentration suggests that two lactose molecules bind to one molecule of abrin II at pH7.2. The presence of two binding sites for lactose on the abrin II molecule is also indicated by isothermal titration calorimetry. Plotting ΔH_m (the molar transition enthalpy at T_m) against T_m yielded values for ΔC_p (change in excess heat capacity) of 27±2 kJ·mol⁻¹·K⁻¹ for the B-subunit and 20±1 kJ·mol⁻¹·K⁻¹ for the A-subunit. These values have been used to calculate the thermal stability of abrin II and to surmise the mechanism of its transmembrane translocation.