Isolation of nonlabile human ceruloplasmin by chromatographic removal of a plasma metalloproteinase.
Ceruloplasmin (EC 1.16.3.1) is a copper-containing alpha 2-glycoprotein and a member of the acute phase reactant family. Fragmentation of ceruloplasmin during purification and storage has hampered studies of its structure, but it has been shown to be a 132-kDa monomer. Combining two published chromatographic steps with additional gel filtration and fast protein liquid chromatography (FPLC) steps, we now report a procedure that yields a highly purified and nonlabile protein. Human plasma was subjected to QAE-Sephadex A-50 chromatography, precipitated with ammonium sulfate, and chromatographed on a hydroxyapatite column. The resulting protein was > 95% pure but highly unstable as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; at 37 degrees C the 132-kDa protein disappeared completely within 48 h. Further purification of ceruloplasmin by Sephadex G-50 chromatography and Mono Q FPLC yielded a protein that was essentially pure by multiple criteria and completely stable even after incubation at 37 degrees C for 4 weeks. When purified ceruloplasmin was reconstituted with fractions eluted from the Sephadex G-50 column, a single fraction induced proteolytic degradation. The degradation of ceruloplasmin by this fraction was inhibited by EDTA and 1,10-phenanthroline, indicating that a plasma metalloproteinase is responsible for degradation of ceruloplasmin.
Ehrenwald
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Department of Cell Biology, Cleveland Clinic Research Institute, Ohio 44195.
Fox
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PL
D000645:Ammonium Sulfate; D002570:Ceruloplasmin / Q000302:isolation & purification*; D011232:Chemical Precipitation; D002845:Chromatography; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004355:Drug Stability; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008666:Metalloendopeptidases / Q000097:blood*
D017886:Durapatite; D002570:Ceruloplasmin; D008666:Metalloendopeptidases; D000645:Ammonium Sulfate