International Journal of Biological Macromolecules 26 (1999) 167–171 Micropurification of - and -crystallins from rabbit aqueous humor Maria Grazia Leone a , Luciano Saso a , C. Yan Cheng b , Bruno Silvestrini a, * a Department of Pharmacology of Natural Substances and General Physiology, Uniersity of Rome ‘La Sapienza’, P.le Aldo Moro 5, 00185 Rome, Italy b The Population Council, Rockefeller Uniersity, 1230 York Aenue, New York, NY 10021, USA Received 5 January 1999; received in revised form 20 March 1999; accepted 23 March 1999 Abstract 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 g each of - and -crystallins were obtained from 20 ml of rabbit aqueous humor by C 8 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 V 8 . © 1999 Elsevier Science B.V. All rights reserved. Keywords: Aqueous humor; Cataract; Crystallin; Micropurification; Open-angle glaucoma 1. Introduction It has been known for a long time that cataract may be accompanied by weight decrease of the lens [1,2] partly due to leakage of crystallins (C) [3,4], as confi- rmed by the increase of their concentration in the aqueous humor (AH) [5–11]. The role played by soluble proteins in obstructing the aqueous outflow is not clearly understood [12 – 14] and C, which are known to form high molecular weight aggregates in the lens [15], could play a pathological role in certain types of open-angle glaucoma [16]. In this connection, it is known that the protective action of -C, an exceptionally stable protein [17], against denaturation and aggregation of -C and -C [18–20] may change upon aging [21]. However, study of the stability and the tendency to aggregate of -C and -C in the AH have not been performed yet, mainly due to the lack of a suitable purification procedure from this fluid, which contains very low levels of C [5,9] mixed with almost all serum proteins [22]. Here we report a two-step method for the purifica- tion of microgram quantities of - and -C from rabbit AH. Abbreiations: ACN, acetonitrile; AH, aqueous humor; CAPS, 3-cyclohexylamino-l-propanesulfonic acid; C, crystallin; HPEC, high- performance electrophoresis chromatography; HPLC, high-perfor- mance liquid chromatography; kDa, kilodalton; M r , molecular weight; PAGE, polyacrylamide analytical gel electrophoresis; PVDF, polyvinylidene fluoride; Rt, retention time; SDS, sodium dodecyl sulfate; TFA, trifluoroacetic acid. * Corresponding author. Tel.: +39-6-49912948; fax: +39-6- 49912480. E-mail address: silverstrini@uniromal.it (B. Silvestrini) 0141-8130/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 4 1 - 8 1 3 0 ( 9 9 ) 0 0 0 7 8 - 1