Dry bath incubator

1,N⁶-etheno-ATP (eATP), 1,N⁶-etheno-ADP (eADP), 1,N⁶-etheno-AMP (eAMP), and 1,N⁶-etheno-adenosine (eADO) were prepared according to a modified method of Levitt et al., 1984 (link). Briefly, 180 µl of a citrate phosphate buffer (pH 4.0) containing 62 parts 0.1 M citric acid and 38 parts 0.2 M Na₂HPO₄, was added to 150 µl of 200 µM authentic purine (i.e., ATP, ADP, or AMP) in an Eppendorf tube. 2-Chloroacetaldehyde was synthesized according to a modified method of Levitt et al., 1984 (link): equal amounts of 10-fold diluted H₂SO₄ in distilled H₂O and chloroacetaldehyde dimethyl acetal were added to a round bottom boiling flask. The mixture was distilled slowly under a fume hood and the distillate fraction containing approximately 1.0 M 2-chloroacetaldehyde was collected at 79–82°C. The reagent was stored at −20°C when not in use. Twenty µl 2-chloroacetaldehyde was added to the Eppendorf tube containing the mixture of authentic purine and citric buffer in a fume hood and then heated for 40 min at 80°C in a dry bath incubator (Fisher Scientific, United States) to produce 1,N⁶-etheno-purine substrates and 1,N⁶-etheno-purine standards (Bobalova et al., 2002 (link)).

Red blood cell ghosts were prepared with a modification of a previously described method [25 (link)]. Aliquots of BM and MVM were washed three times by centrifugation-resuspension at 47,500 ×g for 45 min. The membrane pellet was resuspended with a solution containing 250 mM sucrose and 10 mM Tris-Hepes (pH 7.2) and the protein concentration was adjusted to 1.5 mg/mL. A 200 μL aliquot of the membrane suspension was mixed with 500 μL of 70% nitric acid and placed in a heating block (Fisher Scientific Dry Bath incubator) at 80°C for 24 h. After 24 h digestion, 200 μL of 60% perchloric acid was added, and the digestion was continued for 24 h at 80°C. Finally, the whole mixture was suspended with 2 mL of MQ water. The Ca²⁺ and Mg²⁺ contents of the sample were measured by inductively coupled plasma spectroscopy (Perkin Elmer Optima 3000 DV ICP system).