Purification of a 43,000 dalton aggregate generated from alpha-crystallin.
A procedure is presented for the purification of an aggregate of covalently linked polypeptides of alpha-crystallin. Using either iron catalyzed oxidation or UV irradiation, 6-12% of calf lens alpha-crystallin can be converted to a 43,000 Da aggregate containing non-reducible cross-linked polypeptides as indicated by SDS-PAGE under reducing conditions. The 43,000 Da aggregate generated by Fe2+ oxidation can be isolated to approximately 85% homogeneity with respect to its relative molecular weight on SDS-PAGE. The procedure can be performed in two steps; 1) gel filtration of the oxidized alpha-crystallin under deaggregating and reducing conditions, and 2) preparative SDS-PAGE of the 43,000 Da aggregate-enriched peak obtained by filtration. The 43,000 Da aggregate band can be recovered from the polyacrylamide gels using a new preparative electroelution technique. 1.0 +/- 0.3 mg of purified 43,000 Da aggregate can be obtained from 100 mg of Fe2+ oxidized alpha-crystallin. The described methodology will facilitate further characterization of this 43,000 Da alpha-crystallin aggregate.
McDermott
M J
MJ
Department of Ophthalmology, College of Physicians and Surgeons, Columbia University, New York, NY 10032.
Chiesa
R
R
Spector
A
A
D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D003459:Crystallins / Q000302:isolation & purification* / Q000528:radiation effects; D004591:Electrophoresis, Polyacrylamide Gel; D005290:Ferric Compounds / Q000378:metabolism; D005296:Ferrous Compounds / Q000378:metabolism; D007525:Isoelectric Focusing; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010455:Peptides / Q000378:metabolism; D013997:Time Factors
D003459:Crystallins; D005290:Ferric Compounds; D005296:Ferrous Compounds; D010455:Peptides