Affinity Chromatography of Cytosine Deaminase from Escherichia coli with Immobilized Pyrimidine Compounds

Abstract

We immobilized many classes of pyrimidine compounds on the carrier Sepharose^® 4B via alkyl spacers by constructing various spacers using cyanogen bromide activation of the carrier. A crude enzyme solution of Escherichia coli with cytosine- and 5-fluorocytosine-deaminating activity was studied by adsorption and desorption chromatography with columns of the sixty-eight kinds of gels we made. Gels made with the following five ligands were effective. 2-Mercaptopyrimidine or 2-thiobarbituric acid, when coupled with 1, 6-diaminohexane and then with the activated carrier, was suitable. So was 2-amino-4, 6-dihydroxypyrimidine or 5-aminouracil, when linked by carbodiimide coupling to a carrier coupled with 6-aminohexanoic acid. Orotic acid, when linked in the same way with a carrier coupled with 1, 4-diaminobutane, was also effective.