PURIFICATION OF MAMMALIAN RIBONUCLEASE USING IMMOBILIZED HUMAN RIBONUCLEASE INHIBITOR

Affinity chromatography on immobilized ribonuclease (RNase) inhibitor was developed for purification of mammalian RNase. Human placental RNa se inhibitor was conjugated to CNBr-activated Sepharose in the presenc e of dithiothreitol. About 80% of the immobilized RNase inhibitor was capable of binding bovine pancreatic RNase A. The bound RNase A was el uted with 3 M NaCl at pH 5.0. Two 25-kDa and 1.8-kDa RNases, which wer e obtained from human liver using a cellulose phosphate column, were b ound to the immobilized RNase inhibitor and recovered in a pure and ac tive form after treatment of the resin with p-hydroxymercuribenzoate. These enzymes were considered to be nonsecretory-type RNases with diff erent sugar contents. (C) 1996 Academic Press, Inc