298 Sweeney and Walker
3.6.2. Clostripain
Note that the enzyme may need to be activated prior to use (see Section
2.2.7.). Dissolve the substrate protein at 1-10 mg/mL in 50 mM ammo-
nium bicarbonate, 0.2 mM calcium acetate, and 2.5 mM DTT. Add
clostripain at 1-2% (w/w), and incubate for 2-3 h at 37°C. More extensive
incubations can result in some cleavage at lysine residues (75).
3.6. 3. Elastase
Dissolve the substrate at 1-10 mg/mL in 100 mM ammonium bicar-
bonate at 37°C. Add elastase at 1-2% (w/w), and incubate for 4 h (76).
3.6.4. Endoproteinase Arg-C
Dissolve the substrate in 1% ammonium bicarbonate, pH 8.0. Add
enzyme at 2% (w/w), and incubate at 37°C for 8 h (77).
3.6.5. Endoproteinase Asp-N
Dissolve the substrate in 50 mM phosphate buffer, pH 7.0, at 37°C. Add
enzyme at 5% (w/w) in the same buffer, and incubate for 4-18 h (78).
3.6.6. Endoproteinase Glu-C
For cleavage at glutamate residues, dissolve the proteins substrate
in 0.1M ammonium bicarbonate, pH 8.0, at a concentration of about
10 mg/mL, and add enzyme at 3% (w/w). Incubate at 37°C for 2-18
h. To extend the cleavage to aspartate residues, perform the same
incubation, but using 0.1M sodium phosphate buffer, pH 7.8 (79).
3.6. Z Endoproteinase Lys-C
Dissolve the protein substrate in 0.1M ammonium bicarbonate, pH
8, at 10 mg/mL. Add enzyme at 2% (w/w), and incubate at 37°C for 2
h. The same amount of enzyme is again added, and after 22 h, the
digestion is stopped (80,81).
3.6.8. Pepsin
Dissolve the substrate in 0.01M HC1, and adjust the pH to 2.0. Add
pepsin to 1% (w/w), and incubate for 1 h at 25°C (82).
3.6.9. Thermolysin
Dissolve the substrate protein in 0.1M ammonium bicarbonate con-
taining 5 mM CaC12. Add enzyme to 2% (w/w), and incubate at 37°C
for 1 h (83).