Purification and characterization of undegraded human ceruloplasmin

✍ Scribed by Michel Noyer; Francis E. Dwulet; Yu Lee Hao; Frank W. Putnam

• Publisher: Elsevier Science
• Year: 1980
• Tongue: English
• Weight: 760 KB
• Volume: 102
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(80)90181-5

✦ Synopsis

Human ceruloplasmin, which is usually cleaved by limited proteolysis into three major fragments during preparation (M, = 18,650,5O,OOO, and 70,OOO) was isolated in good yield as an undegraded single-chain protein (M, = 135,000). The cryosupernatant from fresh frozen plasma (100 liters) was fractionated with polyethylene glycol (PEG 4OOO) at +s"C yielding a ceruloplasmin-enriched fraction in the 20% PEG supernatant. Three steps of chromatography on DEAE-Sephacel, hydroxyapatite, and Sephadex G-200 produced a homogeneous protein with maximal enzymatic activity and the A&A ZB,, ratio of 0.046 corresponding to 98-100% purity. Two forms of ceruloplasmin having this absorbance ratio were obtained; Form I was predominant and was studied further. The procedure separated both forms from apoceruloplasmin and degraded ceruloplasmin. The single-chain ceruloplasmin (Form I) had an NH,-terminal sequence of Lys-Glu-Lys-His-Tyr-Tyr-Be-, the same as for the 70,000 fragment, and is suitable for structural study by sequence analysis and physicochemical methods.