TABLE 1.
Purification of AOH2 from HGsa
| Stepb | Vol (ml) | Protein amt (mg) | Activity (units) | Sp act (units/mg) | Fold purification | % Yield |
|---|---|---|---|---|---|---|
| Ultra | 22 | 409 | 123.9 | 0.3 | 1 | 100 |
| 55°C | 22 | 245 | 95.3 | 0.4 | 1.3 | 77 |
| PD10 | 28 | 112 | 83.1 | 0.7 | 2.3 | 67 |
| Benz. Seph. | 15 | 0.33 | NA | NA | NA | NA |
| MonoQ | 1.4 | 0.06 | 6.2 | 103.8 | 346 | 5 |
a
HGs (3.4 g) were isolated, homogenized in 27 ml of buffer, and ultracentrifuged to obtain a cytosolic extract, which was processed as indicated. Enzymatic activity was measured as the ability of the various purification fractions to oxidize RAL into ATRA. One unit of enzymatic activity is defined as 1 nmol of RAL oxidized/min. The results are representative of two separate AOH2 preparations. NA, not applicable.
b
Ultra, ultracentrifugation; 55°C, treatment at 55°C; PD10, size exclusion column; Benz. Seph., benzamidine-Sepharose affinity column chromatography; MonoQ, MonoQ anion-exchange column chromatography.