A rapid and sensitive assay for quantifying the activity of both aerobic and anaerobic ribonucleotide reductases acting upon any or all substrates
Fig 3
Measurement of RNR activity for a single nucleotide under a single condition.
(A) Generation of a dC standard curve. A multiple reaction monitoring (MRM) curve for dC (m/z 228.2 > 112.1) is extracted from the initial total ion chromatogram (TIC) and the peaks are integrated (all in triplicate; one set of standards is shown above for the TIC and MRM) to generate a standard curve that relates the area under the dC MRM peak to the nmoles dC injected. The peak in the TIC at approximately 1.4 mins corresponds to the desired dephosphorylated product (dC) whereas the peak at approximately 3.5 mins corresponds to the dephosphorylated effector (A) in this experiment; note the MRM peak for dC is at the same retention time as the dC peak in the TIC. (B) Calculation of sample activity. Another set of MRM curves are extracted and integrated from TICs for each of the time points of the assay. The integrated MRM values are then converted to nmol dC / mg NrdA by using the standard curve to calculate nmol dC injected and then dividing by the mg NrdA in each injection. The slope of the line (in nmol/mg-min; after the aforementioned has been completed in triplicate) is the activity of the protein. All reactions are done in triplicate and are shown as mean ± standard error of the mean. The above condition contained NrdA (0.1 μM dimer), NrdB (0.5 μM dimer), ATP (3000 μM), CDP (1000 μM), Trx (30 μM), TrxR (0.5 μM), and NADPH (200 μM).