- KOH/MOPS solution: 2 N KOH/0.3 M 3-(N-morpholino)
propanesulfonic acid (MOPS). Add ~70 ml of mQ water to a
glass beaker containing a magnet. Weigh 11.22 g of KOH and
6.28 g of MOPS, and transfer to the glass beaker. Mix gently
until complete dissolution. Make up to 100 ml, transfer in a
glass bottle, and store at 4C.
2.3 HPLC Buffers 1. Pyrophosphoric acid solution: 1 M H 4 P 2 O 7. Pre-warm pyro-
phosphoric acid bottle at 37C in a water bath. Weigh 17.8 g
of pyrophosphoric acid in a glass container. Add ~90 ml of mQ
water to a glass beaker containing a magnet. Add pyropho-
sphoric acid step-by-step in water under moderate stirring in a
flow hood. Make up to 100 ml and transfer in a glass bottle.
- Pyrophosphate (saturated) solution. Add sodium pyrophos-
phate decahydrate in a glass container containing a magnet
and ~25 ml mQ water until dissolution is not possible
anymore. - Pyrophosphate buffer: 25 mM sodium pyrophosphate,
pH 5.75. Add ~1.8 L of mQ water to a glass beaker containing
a magnet. Weigh 25 g of sodium pyrophosphate decahydrate
and transfer to the glass beaker. Mix gently until complete
dissolution. Add 50 ml of pyrophosphoric acid solution. Adjust
pH to 5.75 with a solution of saturated sodium pyrophosphate
decahydrate. Make up to 2 L, filter (0.22μm;seeNote 1), and
transfer in a glass bottle. - Acetonitrile: 70% (v/v). Add 700 ml of acetonitrile in a glass
cylinder and complete to 1 L with mQ water. - Acetonitrile: 5% (v/v). Add 50 ml of acetonitrile in a glass
cylinder and complete to 1 L with mQ water. - Methanol: 30% (v/v). Add 300 ml of methanol absolute in a
glass cylinder and complete to 1 L with mQ water.
2.4 Adenine
Nucleotide Standards
- ATP: 1 mM adenosine 5^0 -triphosphate (ATP), pH 7.4. Add
~20 ml of mQ water to a glass beaker containing a magnet.
Weigh 15.1 mg of ATP disodium salt trihydrate and transfer to
the glass beaker container. Mix and adjust pH to 7.4 with
Tris–HCl powder. Make up to 25 ml with mQ water. Aliquot
(200μl) in 0.5 ml tubes and store at 80 C. - ADP: 1 mM adenosine 5^0 -diphosphate (ADP), pH 7.4. Add
~20 ml of mQ water to a glass beaker containing a magnet.
Weigh 10.7 mg of ADP sodium salt and transfer to the glass
beaker container. Mix and adjust pH to 7.4 with Tris–HCl
powder. Make up to 25 ml with mQ water. Aliquot (200μl)
in 0.5 ml tubes and store at 80 C. - AMP: 1 mM adenosine 5^0 -monophosphate (AMP), pH 7.4.
Add ~20 ml of mQ water to a glass beaker containing a magnet.
Determination of Adenine Nucleotides by HPLC 231