Screening of crystallin-crystallin interactions using microequilibrium dialysis.

PURPOSE

It has been hypothesized that short-range, protein-protein interactions of crystallin are necessary for the maintenance of lens transparency. Because of their probable weak nature, it has been difficult to both detect and quantitate the nature of these interactions. To determine if interactions exist between alpha-crystallin and gamma-crystallin under true equilibrium conditions, we have used microequilibrium dialysis.

METHODS

Total alpha-crystallin and gamma-crystallin were prepared from soluble proteins of fetal bovine lenses by HPLC and gel filtration chromatography. The proteins were added to one side of a microequilibrium dialysis cell, comprised of two chambers separated by a membrane with 100 kDa molecular weight cut-off. After reaching equilibrium, the amount of free gamma-crystallin and the amount of gamma-crystallin bound to alpha-crystallin was determined by HPLC and reverse phase analysis of both chambers. Selected gamma-crystallin that bound to alpha-crystallin was further purified by ion exchange chromatography, and then incubated with alpha-crystallin, to verify the specificity of their binding.

RESULTS

Analysis of both microequilibrium dialysis chambers incubated at different times at 37 degrees C indicated that equilibrium was reached at 4 days. When total alpha-crystallin and gamma-crystallin were incubated for this time period, significant binding was observed between alpha-crystallin and the IIIA, II, and IVA species of gamma-crystallin. These interactions were confirmed by microequilibrium dialysis determinations containing alpha-crystallin and purified gamma-crystallin species.

CONCLUSIONS

These results show that microequilibrium dialysis can be used to demonstrate significant noncovalent interactions of alpha-crystallin and gamma-crystallin under true equilibrium conditions.