Two-Stage Method for Purification of Ceruloplasmin Based on Its Interaction with Neomycin

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A. V. Sokolov^*, V. A. Kostevich, D. N. Romanico, E. T. Zakharova, and V. B. Vasilyev

Research Institute of Experimental Medicine, Russian Academy of Medical Sciences, ul. Pavlova 12, 197376 St. Petersburg, Russia; fax: (812) 234-9489; E-mail: biochemsokolov@gmail.com

^* To whom correspondence should be addressed.

Received October 24, 2011; Revision received February 26, 2012

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A two-stage chromatography that yields highly purified ceruloplasmin (CP) from human plasma and from rat and rabbit serum is described. The isolation procedure is based on the interaction of CP with neomycin, and it provides a high yield of CP. Constants of inhibition by gentamycin, kanamycin, and neomycin of oxidase activity of CP in its reaction with p-phenylenediamine were assayed. The lowest K_i for neomycin (11 µM) corresponded to the highest specific adsorption of CP on neomycin-agarose (10 mg CP/ml of resin). Isolation of CP from 1.4 liters of human plasma using ion-exchange chromatography on UNO-Sphere Q and affinity chromatography on neomycin-agarose yields 348 mg of CP with 412-fold purification degree. Human CP preparation obtained with A₆₁₀/A₂₈₀ ~ 0.052 contained neither immunoreactive prothrombin nor active thrombin. Upon storage at 37°C under sterile conditions, the preparation remained stable for two months. Efficient preparation of highly purified CP from rat and rabbit sera treated according to a similar protocol suggests the suitability of our method for isolation of CP from plasma and serum of other animals. The yield of CP in three separate purifications was no less than 78%.

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KEY WORDS: ceruloplasmin, neomycin, affinity chromatography

DOI: 10.1134/S0006297912060107

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