Two-stage method for purification of ceruloplasmin based on its interaction with neomycin |
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Authors: | A V Sokolov V A Kostevich D N Romanico E T Zakharova V B Vasilyev |
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Institution: | Research Institute of Experimental Medicine, Russian Academy of Medical Sciences, ul. Pavlova 12, 197376 St. Petersburg, Russia. biochemsokolov@gmail.com |
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Abstract: | 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 A
610/A
280 ∼ 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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