Immunochemical studies on lens protein-protein complexes II. On the mechanism of alpha-beta-gamma crystallin complex formation

Abstract

An analysis of the β- and γ-crystallins present in various lens protein complexes of α-β, α-β-γ or only β-γ crystallins was done. This analysis was based on a detailed study of different proteins in the β- and γ-crystallin fractions. Beta and γ-crystallins were isolated from the different complexes by reacting each of them with anti-β crystallin antibodies used as ligands in an immunoadsorbent. The reaction resulted in dissociation of the complexes and binding of the various β-crystallins to the immunoadsorbent, leaving α- and γ-crystallins free in solution. After elution and concentration, the β-crystallins were compared in immunodiffusion tests with the high and low molecular weight β-crystallins. The freed γ-crystallins were first separated from α-crystallin by filtration on Diaflo XM-300 membranes. The filtrates were concentrated and compared with high and low molecular weight γ-crystallins. All of the β-crystallins which complexed with α-crystallin to form the α-β as well as the different α-β-γ crystallin complexes were found to contain β-crystallin molecules I and II, molecules which are typical of high molecular weight β-crystallin fractions. In addition, none of these β-crystallins contained β-crystallin molecule VII which is typically found in low molecular weight β-crystallin fractions. By contrast, all of the β-crystallins which did not complex with α-crystallin but which did form various β-γ crystallin complexes were related to the low molecular weight β-crystallins, as shown by the presence of β-crystallin molecule VII in these complexes and the absence of β-crystallins I and II. β-Crystallin molecules III through VI present in both high and low molecular weight β-crystallin fractions were found in all complexed crystallins. All complexes were also found to contain the same γ-crystallin molecules I and II. The data indicate that with progressing lens age, an initial α-β complex binds free γ-crystallins forming α-β-γ crystallin complexes. The latter complexes are not formed by association of α-β with β-γ complexes.