Micropurification of beta- and gamma-crystallins from rabbit aqueous humor

Soluble crystallins are normally present in the aqueous humor, originating from the lens, and their concentration may increase in certain conditions such as cataract, possibly contributing to aqueous outflow pathway obstruction, leading to glaucoma. Whether the stability and the tendency of aqueous crystallins to aggregate are different in patients with certain forms of open-angle glaucoma has not so far been established, mainly due to the lack of a suitable purification procedure from this fluid in which crystallins are present at very low concentration together with dozens of other proteins. About 4 microg each of beta- and gamma-crystallins were obtained from 20 ml of rabbit aqueous humor by C8 reversed-phase high-performance liquid chromatography (HPLC) and high-performance electrophoresis chromatography (HPEC). The identity of the proteins was confirmed by amino acid analysis following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretic blotting onto polyvinylidene fluoride membranes, with or without previous digestion with Staphylococcus aureus protease V8.