Purification of paraoxonase in rabbit serum
| Purification step | Fraction2-a | Protein | Activitycnmol p-NP/30 min/ml fraction | Yield (%) | Specific activity nmol p-NP/30 min/mg protein | Purification factor | ||||
|---|---|---|---|---|---|---|---|---|---|---|
| Ca++ | EDTA | Ca++ | EDTA | Ca++ | EDTA | Ca++ | EDTA | |||
| dep | res | dep | res | dep | res | dep | res | |||
| mg protein/ml fraction | ||||||||||
| Serum | – | 70.3 | 5,850 | 150 | 100 | 100 | 83.2 | 2.1 | 1.0 | 1.0 |
| First centrifugation | L | 0.9 | 3,640 | ND | 62 | – | 4,044 | ND | 49 | – |
| S | 41.9 | 910 | 120 | 16 | 80 | 21.7 | 2.9 | 0.3 | 1.3 | |
| Second centrifugation | 430 | 110 | 7 | 73 | 11 | 2.8 | 0.1 | 1.3 | ||
| SS | 39.0 | |||||||||
| Sephacryl-DEAE | SS2 | 16.0 | 15 | 90 | 0.3 | 60 | 0.9 | 5.6 | 0.0 | 2.6 |
| SS25 | 11.8 | 40 | 20 | 1.0 | 13 | 3.4 | 1.7 | 0.8 | 0.8 | |
| SS26 | 10.0 | ND2-b | 70 | – | 47 | ND | 7.0 | – | 3.3 | |
The purification process was independently performed in triplicate, after which the three groups of fractions were pooled and paraoxonase and amount of protein were assayed. The fractions were obtained as explained in Materials and Methods. The purification factor was calculated from the ratio between the specific activities of each fraction and the specific activity of serum. Calcium-dependent activity (Ca++ dep) was calculated as the difference between the activity exhibited by the samples in the presence of 2.5 mM Ca++ and that recorded in the presence of 5 mM EDTA. EDTA-resistant activity (EDTA res) was regarded as the activity exhibited by the samples in the presence of 5 mM EDTA.