Purification of C1 inhibitor. A new approach for the isolation of this biologically important plasma protease inhibitor.
C1 inhibitor (C1-INH) acts to inhibit active enzymes of both the classical complement and Hageman factor-dependent pathways. Previously reported C1-INH purification procedures were multistep and most have been associated with significant loss in specific functional activity. We have developed a simple chromatographic procedure which yields a pure C1-INH protein from normal human plasma with a specific activity equal to or greater than the starting sample. Briefly, protease inhibitor-treated, pooled human citrated plasma was fractionated with polyethylene glycol (PEG 4000); the supernatant fraction that remained soluble at 16% was obtained. The inhibitor was precipitated with 45% PEG. The resulting precipitate was solubilized and chromatographed on DEAE Sephacel using a linear salt gradient. The eluted fractions containing the C1-INH and other contaminants were pooled and dialyzed against the starting buffer of the next chromatographic step. A unique separation procedure using zinc ion chelate-coupled agarose was employed as the second chromatographic step. The eluted C1-INH, after zinc ion chromatography, displayed a significant enhancement in purity and maintained a specific functional activity twice that of plasma. The final procedure utilized immunoadsorption chromatography using an anti-contaminant column. Under reducing conditions on sodium dodecyl sulfate polyacrylamide gel electrophoresis, the purified C1-INH migrated as a single band with an apparent molecular weight of 90,000-105,000, but under non-reducing conditions, a doublet with apparent molecular weights of 94,000-100,000 and 85,000-93,000 was seen. C1-INH antigenic concentrations were measured and shown to be correlated in serum, citrate plasma, and EDTA plasma from 16 normal subjects.
D002614:Chelating Agents; D002846:Chromatography, Affinity / Q000379:methods*; D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D003174:Complement C1 Inactivator Proteins / Q000097:blood / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007122:Immunoelectrophoresis; D007163:Immunosorbent Techniques; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000097:blood; D015032:Zinc
D002614:Chelating Agents; D003174:Complement C1 Inactivator Proteins; D011480:Protease Inhibitors; D015032:Zinc