Screening of crystallin-crystallin interactions using microequilibrium dialysis

Abstract

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.

Figure 1.

Elution profile of lens soluble proteins, using a TSK G3000SW gel filtration column. Peaks designated by horizontal bars were collected for microequilibrium dialysis studies or for further purification. Flow rate of TSK Buffer was 1.0 ml/min.

Figure 2.

Elution profiles of γ-crystallin species in “empty” microequilibrium dialysis chambers. Representative C₁₈, reverse phase profiles of total γ-crystallin in the “empty” chamber of the microequilibrium dialysis apparatus after injection of γ-crystallin into the “full” chamber. Each peak is designated by a separate number. Peak 1 separated into two peaks after four days incubation. A: Profile of γ-crystallin in empty chamber after incubation for 1 day. B: Profile of γ-crystallin in empty chamber after incubation for 4 days.

Figure 3.

Area of each γ-crystallin peak from the empty and full chambers, after injection of total γ-crystallin into the full chamber. Each point is the mean of three determinations; the error bars represent the standard deviation. Units were obtained after integrating the peaks from Figure 2. A: One day incubation. B: Four day incubation.

Figure 4.

Elution profile of γ-crystallin using a S300 cation exchange column. Approximately 1 mg of γ-crystallin in 300 μl of Buffer A was resolved using a gradient of 100% Buffer A for 10 min, followed by a 0-30% linear gradient of increasing concentration of Buffer B for 15 min. Bold letters and numbers designate fractions collected and used for microequilibrium dialysis studies.