55] dCTP Deaminase from Salmonella typhimurium

Abstract

Publisher Summary This chapter describes the purification of deoxycytidine triphosphate (dCTP) deaminase enzyme from Salmonella typhimurium organism. The assay employed for the purification of dCTP deaminase is based on the difference in molar extinction coefficients between dCTP and deoxyuridine tri phosphate (dUTP). In this assay, the dCTP deaminase activity is measured as [ 3 H]dUTP formed from [ 3 H]dCTP with time. Because the assay is based on a chromatographic separation of dCTP and dUTP, it is essential that the enzyme preparation be free of enzymes that degrade these compounds. The sensitivity of the assay is limited by the absorbance of the substrate dCTP and by the fact that the assay is based on the measurements of decreases in absorbancies. The advantage of this assay over the spectrophotometric assay is that it employs much less enzyme due to its larger sensitivity and the smaller assay volume. The molecular weight of dCTP deaminase as determined by gel filtration on Sephadex G-200 is about 82,000.