A rapid and sensitive assay for quantifying the activity of both aerobic and anaerobic ribonucleotide reductases acting upon any or all substrates
Fig 6
The use of internal standards eliminates the need for same-day standard curves.
(A) Standard curves of the same amount of dC (0–1 nmole) run on the mass spectrometer on two different days (day 1 and day 2). (B) Standard curves run on day 1 and day 2 after internal standard correction (the area under the MRM curve of the heavy [13C/15N] dC nucleoside). (C) Activity assay data for EcNrdA and EcNrdB with nmoles dC calculated after correction of both standard and sample dC integration using a heavy dC internal standard. Both assays were run on day 2 along with the day 2 standard curve used for day 2 activity calculation; day 1 standard curve used for day 1 activity calculation was run on a previous day. (D) Calculation of activity from curves in (C) with EcNrdA or EcNrdB as the limiting reagent. NrdA activity was determined to be 2000 ± 300 nmoles dC/mg-min using the day 2 standard curve to calculate activity and 2200 ± 400 nmoles dC/mg-min using the day 1 standard curve to calculate activity. NrdB activity was determined to be 8000 ± 600 nmoles dC/mg-min using the day 2 standard curve to calculate activity and 9000 ± 700 nmoles dC/mg-min using the day 1 standard curve to calculate activity. The above conditions contained NrdA (0.1 μM dimer for NrdA condition and 0.5 μM dimer for NrdB condition), NrdB (0.5 μM dimer for NrdA condition and 0.1 μM dimer for NrdB condition), ATP (3000 μM), CDP (1000 μM), Trx (30 μM), TrxR (0.5 μM), and NADPH (200 μM).