Dowex formate columns

The combined organic acid containing effluents may be passed through a Dowex formate column for separation (steps 4-1 through 4-57) without further treatment. 

Prepare Dowex formate columns according to steps 4-1 to 4-8,4-16, and 4-17 to 4-23. 

Ion-exchange chromatography is used most frequently, because of its simplicity and low cost. Extracts usually containing [ HJinositol-labeled inositol phosphates are placed on a Dowex formate column from which GPI, IP, IP2 (inositol bisphosphate), and IP3 are eluted sequentially with buffers of ammonium formate of increasing molarity. 

Figure 4>11. Data obtained through use of Dowex-1-formate columns for the separation of succinate, malate, and isocitrate. [From T. G. Cooper and H. Beevers, /. Biol. Chem., 244 3507-3513 (1969).]...

NH2), pK2 6.4 (P04 ). Purification can be carried out by passage through a DEAE-cellulose formate column, then through a Dowex 50 (H+) column to remove Na ions, concentrated by lyophilisation and redissolved in H2O. It is commercially available as a solution of 0.05g/mL of H2O. The concentration of acetoacetylcoenzyme A is determined by the method of Stem et al. [J Biol Chem 221 15 1956]. It is stable at pH 7-7.5 for several hours atO (half-life ca l-2hours). At room temperature it is hydrolysed in ca l-2hours at pH 7-7.5. At pH 1.0/20° it is more stable than at neutrality. A solution of the trisodium salt (0.05g/mL H2O) adjusted to pH 5 with 2N NaOH can be stored frozen for several weeks. It is stable at pH 2-3/-17° for at least 6 months. [Hersch Jencks J Biol Chem 242 3468 1967, Clikenbeard et al. J Biol Chem 250 3108 1975, Simon Shemin J Am Chem Soc 75 2520 1953, Moffatt KhoranaSl 1265 1959, Salem et al. Biochem J 258 563 1989, Beilstein 26 III/IV 3668.]... 

To prepare the [m-(acetylpyridinio)-n-alkyl]adenosine pyrophosphates, 10 mmoles of acetylpyridinio-n-alkylphosphoric acid and 10 mmoles of adenosine 5 -phosphoromorpholidate (as salt of 4-morpholine iV,iV -dicyclohexylcarboxamidine) are dissolved in 20 ml of freshly distilled o-chlorophenol. The mixture is stored at room temperature for 7 days. Progress of the condensation reaction is checked daily by paper electrophoresis (0.1 M Tris chloride at pH 8.1, 30 V/cm). After the reaction is completed, 60 ml of water are added to the mixture and the suspension is extracted 3 times, each with 200 ml of ethyl ether. The first ether extract obtained is reextracted with 30 ml of water. The combined aqueous phases are reduced to a volume of 5 ml under reduced pressure and are charged on to a Dowex 1X8 column (formate form, 2 X 50 cm, 100-200 mesh). The column is washed with 6.5 liters of water, and the coenzyme analogs are eluted from the resin with a convex gradient of formic acid ... 

One millimole of 9-/3-D-ribofuranosyl-6-methylthiopurine (298 mg) (methylthioinosine, MTI) is allowed to react with 1.1 mmoles of periodic acid (HjlOs, 251 mg) in 11 ml of distilled water in the dark for 30 min. The solution is rapidly pipetted onto a Dowex 1-formate column (10 X 30 mm), and the self-eluate and two water washes are collected in a 100-ml round-bottom flask. The solution of MMPR-OP, approximately 30 ml, is frozen in a thin film around the sides of the flask by rotation in a Dry Ice-acetone mixture, and the frozen solution is immediately lyophilized to give a white fluffy powder. A yield of 70-80% can be expected. The MMPR-OP should be stored dry at —20°. Solutions of... 

Periodate oxidation to give the final product, [ S]MMPR-0P, is carried out as follows. [ S]MTI, 0.475 mmole (142 mg), is allowed to react with 0.52 mmole (120 mg, 1.1 equivalents) of periodic acid in 5.20 ml of water in the dark for 30 min. The reaction mixture is pipetted onto a Dowex 1-formate column, 10 X 30 mm, and the eluate and two washes are collected in a 50-ml round-bottom flask and immediately lyophilized to yield about 100 mg of pure [ SjMMPR-OP. The initial specific activity is about 7000 cpm/nmole. All these reactions may be scaled up or doym, as long as the correct proportions described here are held constant. 

Dowex 1-formate columns should be washed with concentrated formic acid and then eluted with distilled water until neutral before use for purifying MMPR-OP. The formic acid wash produces a milky solution at the solvent front that would otherwise contaminate the MMPR-OP. 

Figure 3 shows the elution profile of NMN, nicotinic acid, NAD, NaMN, NaAD, quinolinic acid, NADP, and ADP-ribose on a Dowex 1-X2 formate column (29 cm X 0.8 cm i.d.) (22,23). The elution was carried out by application of a formic acid concave-concentration gradient 0.01 N -> 0.25 N -> 2 N -> 6 N. The mixing chamber initially contained 250 mL of water. The detection was at 260 nm. 

Separation of Niacin Metabolites on a Dowex 1-Formate Column... 

Figure 4 shows the elution profile of MNA, N -methylnicotinic acid, nicotinamide Al-oxide, 4-Py, 2-Py, nicotinamide, 6-hydroxynicotinamide, nicotinic acid, nicoti-nuric acid, and 6-hydroxynicotinic acid on a Dowex 1-X4 formate column (73.6 cm X 0.9 cm i.d.) (24). Gradient elution was done with ammonium formate-formic acid of various pHs and ionic strengths. Detection was performed by UV absorption at 254 nm. 

This technique has been widely used in early studies to isolate and purify biotin and biotin vitamers and their metabolites from the culture filtrate of many microorganisms. Ogata (6) used a Dowex 1X2 formate column. After washing with deionized water, the biotin vitamers (biotin, biotin sulfoxides, and dethiobiotin) were eluted with formic acid (0.012 M). The same Dowex 1X2 chromatography technique proved to be very good to monitor and to titrate biotin and vitamers in... 

Figure 14 Elution pattern from column chromatography of [ H]dethiohiotin (50 mg, SA = 7400 MBq/mmol) and [ C]hiotin (5 mg, SA =1110 MBq/mmol). The mixture was poured over a Dowex 1X2 (formate) column 1.5 X 16.5 cm, and eluted with a hnear gradient of formic acid (0 to 1 M, 600 mL). Fractions (12 mL) were collected. (From F. Frappier, personal communication, 1985.)...

Dowex (2.5 g per column) is prewashed in a sintered glass funnel with distilled water. Disposable columns (5 ml) are filled with 2 ml bed volume of prewashed Dowex. Columns are washed with 6 ml ice-cold distilled water. A 60-pl aliquot of each standard (NeuAc-2, NeuAc-5, and NeuAc-10) and a water blank are applied to the column. For diagnostic samples, patients and controls apply 200 pi to the column. Wash all columns three times with 2-ml ice-cold ammonium acetate buffer. Elute NeuAc by applying 3 x 2 ml and 1 x 1 ml ice-cold ammonium formate buffer. Close the collection tubes (50-ml volume) with a pierced cap and centrifuge fast to spin down droplets from the wall of the tube. Freeze the eluates in an upright position in liquid nitrogen. 

The most recent modification of the NBD-Cl method involves a further improvement in its qualitative support (616). It involves the infusion of the extract employed for thin-layer chromatography via an electrospray interface into a mass spectrometer operating in the multiple-stage mass spectrometry mode, thus allowing confirmation of suspect results. The cleanup of the thyroid gland samples was also performed with a selective extraction procedure, based on the specific complex formation of the thiouracil, methylthiouracil, propylthiouracil and phenylthiouracil, tapazole, and mercaptobenzimidazole residues with mercury ions bound in a Dowex 1-X2 affinity column. 

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