as broadening of peaks, consider regenerating the column
(go tostep 9).- Column storage: Purge the system. Flush the column in 5%
(v/v) acetonitrile at 0.2 ml/min for 30 min. Purge the system.
Flush the column in 70% (v/v) acetonitrile at 0.2 ml/min for
30 min. Stop the system, disconnect the column, and close it
tight. Store at room temperature. - Column regeneration: The column can be regenerated if the
sharpness of the nucleotide separation is reduced. Fix the col-
umn to the system in the opposite direction of the usual flow
and flush with 30% (v/v) methanol at 0.2–0.4 ml/min for
~1 h. Next, wash the column with mQ water by gradually
increasing the flow rate until 0.8 ml/min for ~30 min, followed
with overnight equilibration with pyrophosphate buffer, as
described in Subheading3.3.
3.6 Calculations The nucleotides in sample extracts are identified by their retention
times in comparison with standards (these later eluted in sharp
peaks;seeFig. 1c).
- Calculate nucleotide concentrations (inμmol) using the fol-
lowing formula:
½nucleotideðÞ¼μmol
ðÞVol:SNþVol:KOMO
Vol:SN
ðÞVol:bufferþVol:sample
Vol:buffer
½Std AUCnucleotides
AUCStd:Vol. SN is the volume of extract supernatant inμl.
Vol. KOMO is the volume of KOH/MOPS used for neutralization
inμl.
Vol. buffer is the volume of pyrophosphate buffer inμl.
Vol. sample is the volume of neutralized sample inμl.
[Std] is the standard concentration inμM.
AUC nucleotide is the area under the curve in mV * min for the
specific nucleotide.
AUC Std is the area under the curve in mV * min for the
corresponding nucleotide standard.
The result can be expressed inμmol/million cells,μmol/mg tissue,
orμmol/mg protein (if cell count, tissue weight, or protein
amount, respectively, were measured in parallel).- Calculate the so-called adenylate energy charge, which mainly
reflects the energy supply-to-demand relationship, using the
following equation:
adenylate energy charge¼ðÞ½þATP 0 : 5 ½ADP
ðÞ½þATP ½þADP ½AMPDetermination of Adenine Nucleotides by HPLC 235