Affinity Chromatography of Thymidine Kinase from a Rat Colon Adenocarcinoma

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Abstract
Thymidine kinase from a transplantable colon adenocarcinoma, induced by 1,2-dimethylhydrazine and maintained in CDF rats, was purified by affinity chromatography using thymidine-3''(4-aminophenylphosphate) coupled to carboxyhexyl-Sepharose. Most of the contaminating protein passed through the column; non-specifically adsorbed protein was washed from the column by 0.1 M KCl in 0.01 M Tris-HCl, pH 7.5. Thymidine kinase was eluted with 0.1 mM thymidine, 0.1 M KCl in 0.01 M Tris-HCl, pH 7.5. The purified enzyme accounted for .apprx. 26% of the applied activity. The specific activity of the purified material (peak fraction) was 3500 nmol TMP formed/mg protein per 10 min, a 1800-fold purification of the applied extract. The preparation is free of nucleoside phosphotransferase, but contains other protein impurities. Purification was completed in < 1 h, making this a useful procedure for isolation of this unstable enzyme.