An assay for ribonucleotide reductase based on ion-exchange chromatography of the reaction product.
A rapid and convenient assay for ribonucleotide reductase has been developed in which the reaction product, deoxycytidine diphosphate (dCDP), is isolated without further conversion. The enzymatic reaction is terminated by the addition of ethanol and the sample is chromatographed on a single, small, and disposable column of polyethylenimine cellulose. A two-step elution is conducted with buffers containing 25% ethanol. First, contaminants and byproducts such as cytidine and its monophosphate are removed at low ionic strength while the diphosphates are retained. Then dCDP is selectively eluted as a sharp peak with a strong borate buffer. Under these conditions, the excess substrate, cytidine diphosphate, remains on the column, presumably as the borate complex. The assay is linear with time for 15 min at 25 degrees C and linear with the amount of enzyme even at very low concentrations. With slight modifications, the assay seems applicable to the use of UDP or ADP as substrates. The method is not suitable for samples which contain nucleotide kinase or other interfering enzymes which convert a significant amount of dCDP into byproducts. However, another chromatographic system based on similar principles has been found which could be used to measure any dCTP produced in this way.
Narine
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Bacchetti
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Chan
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D000818:Animals; D002482:Cellulose / Q000031:analogs & derivatives; D055598:Chemical Phenomena; D002621:Chemistry; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D003565:Cytidine Diphosphate / Q000302:isolation & purification; D003570:Cytidine Triphosphate / Q000302:isolation & purification; D003845:Deoxycytosine Nucleotides / Q000302:isolation & purification; D004229:Dithiothreitol; D007668:Kidney; D011094:Polyethyleneimine / Q000031:analogs & derivatives; D012264:Ribonucleotide Reductases / Q000032:analysis*; D018139:Simplexvirus; D013379:Substrate Specificity; D014776:Virus Cultivation
D003845:Deoxycytosine Nucleotides; C018367:polyethyleneimine-cellulose; D003565:Cytidine Diphosphate; D003570:Cytidine Triphosphate; C045813:2'-deoxycytidine diphosphate; D011094:Polyethyleneimine; D002482:Cellulose; D012264:Ribonucleotide Reductases; D004229:Dithiothreitol