B12 METABOLISM IN HUMANS By NICOLE AURORA LEAL A ...

extracts of ATR 239K (160 mg protein) and 239M (180 mg protein) were diluted to 1
mg/ml in 50 mM sodium phosphate (pH 7.0), 300 mM NaCl. Then, ultra-pure
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ammonium sulfate was added as a fine powder with stirring. Precipitated proteins were
centrifuged for 15 min at 12,000 x gmax using a Beckman JLA-10.500 rotor. The protein
pellets were resuspended in 4 ml of 5 mM potassium phosphate (pH 6.8), and passed
through a 0.45 µm filter. ATR 239K (60 mg) and 239M (35 mg) were applied to a 60 ml
ceramic hydroxyapatite column (Bio-Rad) equilibrated with 5 mM potassium phosphate
(pH 6.8). The column was washed with 60 ml equilibration buffer and eluted with a 600
ml linear gradient of 5 mM to 200 mM potassium phosphate (pH 6.8) at a flow rate of 5
ml/min. Fractions containing ATR of the highest purity were pooled and exchanged into
10 mM potassium phosphate (pH 7.0), 50 mM KCl using a Vivaspin centrifugal
concentrator with a molecular weight cutoff of 10,000 Daltons (Viva Science, Binbrook,
UK). Samples were filtered through a 0.45 µm pore-size membrane and further purified
using a Mono Q HR 10/10 anion exchange column (Amersham Pharmacia Biotech,
Piscataway, NJ). ATR 239K (10 mg) and 239M (9 mg) were applied to the column
which had been equilibrated with 10 mM potassium phosphate (pH 7.0), 50 mM KCl.
Then, the column was washed with 8 ml equilibration buffer and eluted with a 160 ml
linear gradient from 50 to 1000 mM KCl in 10 mM potassium phosphate (pH 7.0) at a
flow rate of 4 ml/min. Protein elution was monitored by following the absorbance of the
column effluent at 280 nm and 8 ml fractions were collected. Purified ATR was
concentrated and stored in 10 mM phosphate buffer (pH 7.0), 130 mM KCl, 50%
glycerol at -20°C.