A new method for quantitative determination of tritium-labeled nucleoside kinase products adsorbed on DEAE-cellulose.
The counts of tritiated compounds--adsorbed to paper disks, paper chromatograms, electrophoretograms or TLC plates--can be strongly affected by extended self-absorption of tritium beta-particles on the matrix, due to their low energy. Therefore fully quantitative results can be obtained only by elution of the substances or decomposition of the matrix and subsequent counting in homogeneous solution. In this study we describe a new method for fast and proper decomposition of cellulose matrices by cellulase digestion prior to scintillation counting. This new approach yields up to 98% recovery. For method validation recombinant herpes simplex type 1 thymidine kinase was characterised kinetically. The Km of 0.2 microM remained the same as expected but Vmax was considerably higher yielding 1050 pmol/microgram/min.
Gerber
S
S
Department of Pharmacy, Swiss Federal Institute of Technology, Zurich, Switzerland.
Folkers
G
G
D000327:Adsorption; D001610:Beta Particles; D002480:Cellulase; D002848:Chromatography, DEAE-Cellulose / Q000379:methods; D007700:Kinetics; D010770:Phosphotransferases / Q000378:metabolism*; D011865:Radioisotope Dilution Technique; D011994:Recombinant Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D018139:Simplexvirus / Q000201:enzymology; D013936:Thymidine / Q000032:analysis* / Q000378:metabolism*; D013937:Thymidine Kinase / Q000378:metabolism*; D013938:Thymidine Monophosphate / Q000032:analysis* / Q000378:metabolism; D014316:Tritium
D011994:Recombinant Proteins; D014316:Tritium; D013938:Thymidine Monophosphate; D010770:Phosphotransferases; D013937:Thymidine Kinase; C021892:nucleoside phosphotransferase; D002480:Cellulase; D013936:Thymidine