RABBIT CERULOPLASMIN - PURIFICATION AND PARTIAL CHARACTERIZATION

Rabbit ceruloplasmin (Cp) was purified after solid ammonium sulfate pr ecipitation to 60% final saturation by a two-step column chromatograph y procedure, utilizing DEAE-Sephadex A-50 and changing the NaCl concen tration in the buffer to 0.16 M to achieve the isolation of the protei n. The purified Cp was used to prepare antibodies in guinea pigs that were used afterwards to determine Cp concentration in normal rabbits a nd in rabbits with an experimentally induced chronic anemia. The molec ular,weight of rabbit Cp determined by SDS-PAGE was 125,000 and a high molecular weight Cp of 200,000 comprising 8% of the total purified pr otein was also found. An optical density ratio (610 nm/280 nm) of 0.04 75 and a molar extinction coefficient of 7625 were obtained. Copper de terminations yielded a value of 0.24% that corresponds to 5 copper ato ms per molecule. The staining of Cp following disc-electrophoresis in polyacrylamide gels also showed a two band pattern.