Table I.
Step | Protein | Total Activity | Specific Activity | Yield | Purification Factor |
---|---|---|---|---|---|
mg | nmol h−1 | nmol mg−1 h−1 | % | ||
Crude extract | 2,175 | 16,706 | 7.7 | 100 | 1 |
DEAE-Sepharose | 681 | 16,154 | 24 | 97 | 3 |
S-300 HR | 162 | 4,128 | 25 | 25 | 3.2 |
MONO-Q | 12.6 | 1,073 | 85 | 6.4 | 11 |
Phenyl-superose | 2.1 | 430 | 205 | 2.6 | 27 |
The activity was determined through the chymotrypsin-like activity of the 20S proteasome, with N-succinyl-L-L-V-Y-7-amido-4-methylcoumarin (Suc-L-L-V-Y-AMC) as substrate (final concentration 100 μm), as described in “Materials and Methods.”