Preliminary Studies on the Aggregation Process of Alpha-Crystallin

Abstract

The mechanism by which α-crystallin subunits form the native 800 kD aggregate is currently unknown. Experiments were performed to investigate the mechanism for this process. Gel-filtration Fast Performance Liquid Chromatography (FPLC) and Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE), with and without cross-linking with glutaraldehyde, indicate that α-crystallin undergoes a concentration-dependent aggregation process. The denaturation of α-crystallin, and its subsequent renaturation and reaggregation, lead to the formation of several different species. At very low concentrations (< 0.5 μm), only monomeric and/or dimeric species exist. With a ten-fold increase in α-crystallin concentration from 0.05 μmto 0.5 μm, the amount of the monomeric/dimeric species increases to a plateau coincident with the appearance of a tetrameric species at 0.5 μm. With an additional ten-fold increase in concentration from 0.5 μmto 5 μm, the amount of the tetrameric species increases and levels off to its own plateau coincident with the appearance of the native 800 kD α-crystallin aggregate at 5 μm. The amount of the native species is extremely small at this concentration, but increases sharply and linearly with increasing concentration, while the concentrations of monomeric/dimeric and tetrameric species remain constant. The concentration at which the relative amount of the native species begins to increase sharply is within the range of the critical micelle concentration previously characterized.