Purification of undegraded ceruloplasmin from outdated human plasma.
A method for the rapid isolation of homogeneous undegraded ceruloplasmin from outdated human plasma is reported. The procedure consists of a precipitation step with polyethylene glycol 4000, batchwise adsorption and elution from QAE-Sephadex, and gradient elution from DEAE-Sepharose CL-6B. Ceruloplasmin was purified 1740-fold and the yield from outdated plasma was 67%. The purified ceruloplasmin was found to be homogeneous on anionic polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate-PAGE, isoelectric focusing, and low-speed equilibrium centrifugation. The isoelectric point as determined by isoelectric focusing was 4.4. The purified enzyme was sensitive to storage; when a sample was resubmitted to PAGE after 4 months of storage at 4 degrees C, two bands were obtained and the fast-moving band showed no oxidase activity. The molecular weight estimated by gel electrophoresis and sedimentation equilibrium centrifugation was 130,000.
D000596:Amino Acids / Q000032:analysis; D002570:Ceruloplasmin / Q000302:isolation & purification*; D055598:Chemical Phenomena; D002621:Chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007525:Isoelectric Focusing; D013048:Specimen Handling; D013997:Time Factors; D014461:Ultracentrifugation
D000596:Amino Acids; D002570:Ceruloplasmin