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

The effects of pH and ligand binding on the stability of abrin II, a hetero dimeric ribosome-inactivating protein, and its subunits have been studied u sing high-sensitivity differential scanning calorimetry. At pH 7.2, the cal orimetric scan consists of two transitions, which correspond to the B-subun it [transition temperature (T-m) 319.2 K] and the A-subunit (T-m 324.6 K) o f abrin II, as also confirmed by studies on the isolated A-subunit. The cal orimetric enthalpy of the isolated A-subunit of abrin II is similar to that of the higher-temperature transition. However, its T-m, is 2.4 K lower tha n that of the higher-temperature peak of intact abrin II. This indicates th at there is some interaction between the two subunits. Abrin II displays in creased 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 p ronounced at pH 7.2 than at pH 4.5. This suggests that ligand binding stabi lizes the native conformation of abrin II. Analysis of the B-subunit transi tion temperature as a function of lactose concentration suggests that two l actose molecules bind to one molecule of abrin II at pH 7.2. The presence o f two binding sites for lactose on the abrin II molecule is also indicated by isothermal titration calorimetry. Plotting Delta H-m (the molar transiti on enthalpy at T-m) against T-m yielded values for Delta C-p (change in exc ess 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 calculat e the thermal stability of abrin II and to surmise the mechanism of its tra nsmembrane translocation.