ACID-INDUCED DISSOCIATION OF ALPHA-A-CRYSTALLIN AND ALPHA-B-CRYSTALLIN HOMOPOLYMERS

Homopolymers were constructed from the alphaA and alphaB polypeptides isolated from the lens protein alpha-crystallin. As the pH is lowered from 7.0 to 3.4, these homopolymers dissociate to smaller species with molecular masses ranging from 80 to 250 kDa for the alphaA and around 140 kDa for the alphaB dissociation products. The pK(a) for this dissociation was 3.8 +/- 0.2 for alphaA and 4.1 +/- 0.1 for alphaB homopolymers. Further decreases in pH, to 2.5, resulted in the presence of only denatured alphaB polypeptides, whereas the alphaA dissociation products remained intact. Fractionation of the acid dissociation products from the alphaA homopolymer at pH 2.5 yielded stable species with molecular masses of 220 +/- 30, 160 +/- 20, and 90 +/- 10 kDa. The majority of the population at acid pH consisted of the 160 kDa species. Conformational analysis of these species revealed that most of the secondary structure of the original alphaA homopolymer was retained but that the tertiary structure was perturbed. Fluorescence quenching and energy transfer measurements suggested that the molecule had undergone acid expansion, with the greatest perturbation observed in the smallest particles. The results from this work suggest that alphaA homopolymers are heterogeneous populations of aggregates of a ''monomeric'' molecule with a molecular mass of 160 kDa. This ''monomeric'' molecule may be formed from the association of two tetrameric units.