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 s uch as cataract, possibly contributing to aqueous outflow pathway obstructi on, leading to glaucoma. Whether the stability and the tendency of aqueous crystalline to aggregate are different in patients with certain forms of op en-angle glaucoma has not so far been established, mainly due to the lack o f a suitable purification procedure from this fluid in which crystallins ar e present at very low concentration together with dozens of other proteins. About 4 mu g each of beta- and gamma-crystallins were obtained from 20 ml of rabbit aqueous humor by C-8 reversed-phase high-performance liquid chrom atography (HPLC) and high-performance electrophoresis chromatography (HPEC) . The identity of the proteins was confirmed by amino acid analysis followi ng sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretic blotting onto polyvinylidene fluoride membranes, with or w ithout previous digestion with Staphylococcus aureus protease V-s. (C) 1999 Elsevier Science B.V. All rights reserved.