The Use of Fast Protein Liquid (Size Exclusion) Chromatography for the Fractionation of Crystallins and the Study of β-Crystallin Aggregation

Abstract

The separation of bovine crystallins on two types of fast protein liquid chromatography® (Pharmacia) size exclusion columns, Superose-6 (fractionation range 5-5.10³ kDa) and Superdex-75 (fract. range 3-70 kDa), and on a combination of these was studied. This type of columns combines the biocompatibility of the conventional soft-gels with the performance characteristics of high performance liquid chromatography. Up to 20 mg protein in 100 μl could be separated within two hours on analytical columns without significant loss of resolution. On preparative columns we could fractionate 75 mg of protein within 6 hours. The effect of column load by variation of both concentration and injection volume is reported. It is shown that size estimation of β-crystallins by size exclusion chromatography, as is often done, gives inconsistent results. The possibility that native β-crystallin is significantly larger than sofar assumed cannot be excluded.