Self-association, conformation and binding equilibria of concanavalin A.

Abstract

The discovery of distinct intact and fragmented forms of Con A, together with the observation that Con A self-associates near neutrality raises questions that may be important when interpreting experiments concerned with the biological actions of the protein. Do intact and fragmented units have the same affinity for carbohydrate? Do intact and fragmented units differ in conformation? Are all dimeric units of a homologous type or do hybrid dimers consisting of one intact and one fragmented unit also exist? Can all dimeric types self-associate to the tetramer form? Do dimer and tetramer species differ in their affinity for carbohydrate? These questions have been made amenable to investigation by the development of a method which separates intact and fragmented species under conditions which do not cause time-dependent or irreversible changes in protein conformation. It is found that intact dimeric units preferentially associate to the tetramer form. Under appropriate conditions of pH and ionic strength, dimer and tetramer species, and therefore fragmented and intact forms, can be separated by chromatography on Bio Gel P-100. Hybrid dimers are not present in appreciable amounts. Both types of homologous dimers (intact and fragmented) have similar affinity for carbohydrate, but dimer and tetramer species show significant differences. The results of near UV circular dichroism studies indicate that fragmented units possess slightly different conformation than intact units. An ionization-linked conformational transition in Con A does not appear to be linked directly with the self-association of the protein between pH 5 and 7. Ligand-induced changes in the conformation of Con A are now being examined in detail. Pflumm et al. (1971) have shown that occupation of the sugar binding site of Con A results in a perturbation of conformation as revealed by near UV circular dichroism measurements. The perturbation is relatively small and does not result in more than 1-2% increase in the rotational relaxation time (Shinitzky et al., 1973). On the other hand, removal of metal ions causes a hydrodynamic change sufficient to increase the frictional coefficient and to decrease the sedimentation coefficient (S20, w) from 3.98 S to 3.78 S. Differences between the native and the apoprotein conformation are now being examined using fluorescence polarization and the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulfonate.