Retreiver 500

Pyridine dinucleotides 25–35 were desalted and obtained in the ammonium form using a simple gradient in a volatile NH₄HCO₃ buffer followed by repeated lyophilization. A Bio-Rad Econo-Column (2.5 × 8 cm) was filled with a slurry of DE53 cellulose and allowed to form a packed column of absorbent 3 cm high. Water (25 mL) was passed through the column until the effluent pH was neutral. The sample of dinucleotide was applied to the column as a dilute solution at pH ≥ 7.5 and properly adsorbed. The separation was developed by applying a linear gradient formed between water (150 mL) and 0.5 M NH₄HCO₃ (150 mL). Application of a gradient (0–0.5 M NH₄HCO₃) allows proper binding of the dinucleotide to the column. A flow rate of about 1–2 mL/min is achieved using a peristaltic pump to produce a slight positive pressure. Contaminating salts such as NaCl, ammonium chloride, or sodium trifluoroacetate elute into the low ionic strength buffer ahead of the dinulcleotide. NAADP derivatives generally elute into the mobile phase at concentrations of NH₄HCO₃ > 200 mM. Fractions are collected automatically with the aid of a fraction collector (Teledyne Isco, Retreiver 500, 100 drops/tube) and the dinucleotide detected by its absorption at 254 nm. Repeated lyophilization of the sample from water ensures complete removal of volatile NH₄HCO₃, leaving pure, salt free dinucleotide.

NAADP derivatives can be separated from NADP, ADP-ribose phosphate, and other nucleotides by anion exchange chromatography on DEAE cellulose. DE-52 cellulose was slurry packed into glass Bio-Rad Econo-Columns (2.5 × 50 cm) to form a resin bed approximately 42 cm high. Water (100 mL) was passed through the column to ensure the pH of the effluent from the column was neutral. The dinucleotide mixture was applied to the column as a dilute solution at pH 7.5, and washed into the bed with 10–20 mL of distilled water. The separation was developed by the application of a linear gradient formed between water (350 mL) and 0.6 M NH₄HCO₃ (350 mL). A flow rate of about 1–2 mL/min is achieved using a peristaltic pump (Teledyne-Isco, TRIS) to produce a slight positive pressure. The separation allows purification of compounds based on charge and NAADP derivatives usually elute into the middle of the gradient, into approximately 320 mM NH₄HCO₃. Fractions were collected using an automatic fraction collector (Teledyne- Isco, Retreiver 500, 200 drops/tube), and the dinucleotides detected by their UV absorbance (ISCO UA-6 UV/Vis Detector). UV absorbing peaks were combined, frozen, and lyophilized. Repeated lyophilization of the sample from water ensures complete removal of volatile NH₄HCO₃, leaving pure, salt free dinucleotide.