Fluorimetric assay for terminal deoxynucleotidyl transferase activity |
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| Authors: | M R Deibel C G Liu M D Barkley |
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| Institution: | 1. Morton Plant Hospital, BayCare Health System, Clearwater, FL, USA;2. University of Florida College of Pharmacy, Gainesville, FL, USA;3. Morton Plant Mease Health Care, Consultants in Infectious Diseases, Inc., Clearwater, FL, USA;1. Aalto University, Department of Chemistry, Laboratory of Analytical Chemistry, P.O. Box 16100, FI-00076 Aalto, Finland;2. University of Helsinki, Department of Physics, P.O. Box 64, FI-00014, Finland;1. Institute of Bioinformatics and Medical Engineering, School of Electrical and Information Engineering, Jiangsu University of Technology, Changzhou, Jiangsu 213001, China;2. Department of Physics, Department of Biochemistry, and Informatics Institute, University of Missouri, Columbia, MO 65211, USA |
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| Abstract: | A fluorimetric assay for measuring terminal deoxynucleotidyl transferase activity in purified and crude enzyme preparations has been developed. Etheno-substituted deoxynucleotides are shown to be substrates of the enzyme. The assay involves polymerization of the fluorescent nucleotide 1,N6-ethenodeoxyadenosine triphosphate (epsilon dATP) on an oligodeoxynucleotide initiator, poly(deoxyadenylic acid) with an average chain length of 50 residues] under the reaction conditions used in the standard radiometric assay. The incorporation of epsilon dATP into polymer is quantitated by fluorescence after isolation and nuclease digestion of the product. The enzymological properties of etheno substrates were also determined. Epsilon dATP binds about twofold tighter than dATP to terminal transferase, but has a twofold-lower catalytic rate. However etheno substitution does not affect initiator binding. The fluorimetric assay is suitable for clinical analysis of terminal transferase in human leukemias, and may be a useful adjunct to recently developed immunochemical methods which detect protein, not activity. |
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