Acid-induced dissociation of alpha A- and alpha B-crystallin homopolymers.
Homopolymers were constructed from the alpha A and alpha B 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 alpha A and around 140 kDa for the alpha B dissociation products. The pKa for this dissociation was 3.8 +/- 0.2 for alpha A and 4.1 +/- 0.1 for alpha B homopolymers. Further decreases in pH, to 2.5, resulted in the presence of only denatured alpha B polypeptides, whereas the alpha A dissociation products remained intact. Fractionation of the acid dissociation products from the alpha A 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 alpha A 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 alpha A 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.
Stevens
A
A
National Vision Research Institute of Australia, Carlton, Victoria.
Augusteyn
R C
RC
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002417:Cattle; D002942:Circular Dichroism; D003459:Crystallins / Q000737:chemistry* / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000737:chemistry
D000596:Amino Acids; D003459:Crystallins; D013438:Sulfhydryl Compounds