Journal
ANALYTICAL BIOCHEMISTRY
Volume 295, Issue 1, Pages 82-87Publisher
ACADEMIC PRESS INC
DOI: 10.1006/abio.2001.5191
Keywords
continuous spectrophotometric assay; thymidine kinase; herpes simplex virus type 1; catalytic turnover constant; gene therapy; antiviral
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A simple method to determine the in vitro catalytic turnover constant of several substrates of herpes simplex virus type I thymidine kinase is presented in this study. The method is based on a continuous spectroscopic enzyme-coupled assay and allows one to monitor the herpes simplex virus type 1 thymidine kinase activity in the presence of unlabeled substrates. A clear correlation between the catalytic turnover constant and the rate of decrease in absorbance over time during the assay has been demonstrated. Exploiting this correlation, this method has been used to determine rapidly and precisely the catalytic turnover constant of antiviral lead compounds not readily available in the radioactive labeled form. (C) 2001 Academic Press.
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