Thermal stability and mode of oligomerization of the tetrameric peanut agglutinin: A differential scanning calorimetry study

Peanut agglutinin is a homotetrameric legume lectin. The crystal structure of peanut agglutinin shows that the four subunits associate in an unusual m anner, giving rise to open quaternary structure [Banarjee, R., et al.(1994) Proc. Natl, Acad. Sci. U.S.A. 91, 227-231]. The thermal unfolding of peanu t agglutinin has been characterized by differential scanning calorimetry an d gel filtration to elucidate its thermal stability and its mode of oligome rization. The unfolding process is reversible and could be described by a t hree-state model with two transitions occurring at around 331 and 336 K. Fo r the tetramer, the ratio of Delta H-c/Delta H-v for the first transition i s close to 4 and for the second transition is close to 0.25, suggesting tha t 4 and 0.25 cooperative unit(s) of the tetramer are involved in the first and second transitions, respectively. The agreement between the model-indep endent Delta H-v(S) determined from the values of the temperatures of the p eak maximum (T-p1) with the protein concentration with the values of Delta H-v obtained from the fit of the data to the transition confirms that the f irst peak is associated with the dissociation of peanut agglutinin tetramer s (A(4)) to "folded" monomers (4A), whereas the second peak describes the u nfolding (4U) of these monomers. The overall process for the thermal unfold ing of peanut agglutinin could therefore be summarized as A(4) double left right arrow 4A double left right arrow 4U. Gel filtration studies confirm t his process, as peanut agglutinin elutes as a tetramer up to 50 degrees C, and at and above 56 degrees C (T-m of first transition), it elutes at a pos ition commensurate with that of the folded monomer of peanut agglutinin. Th e unfolding behavior of peanut agglutinin in the presence of saturating amo unts of carbohydrate ligands is similar to that observed for the unligated form. The temperature of maximal stability of the peanut agglutinin tetrame r at pH 7.4 is calculated to be around 33 degrees C with a maximal free ene rgy of stabilization of 8.70 kcal/mol. The results demonstrate that unfoldi ng of peanut agglutinin goes through two distinct phases with folded monome r being the intermediate.