Neutral Protease (Dispase) - Assay

The nature of this enzyme requires the substrate to be prepared accurately. Take care to follow the instructions below.

1M Tris

Buffer-Substrate (2% casein in 0.05M Tris buffer): Dissolve 1.0gm casein (USB grade or equivalent) in 40.0ml reagent grade water by stirring for approximately twenty minutes. Add 1.0ml of 1N NaOH and 2.5ml of 1.0M Tris. Allow substrate to stir until casein has gone into solution. pH to 7.8 with dilute phosphoric acid and continue stirring for 5 minutes. Bring to a final volume of 50.0ml with reagent grade water. Prior to pipetting substrate make sure substrate is homogeneous throughout and casein has not fallen out of solution. If casein appears to have fallen out of solution, do not use substrate for assay.

Enzyme activator - 0.01 M CaCl₂: Dissolve 147 mg CaCl₂ in 100 ml reagent grade water. When not used keep in refrigerator.

5% TCA: Dissolve 5 gms TCA in 100 ml reagent grade water. Keep at room temperature.

Tyrosine standard: Prepare 10 ml at a concentration of 10 μ moles/ml in reagent grade water. (181μg tyrosine = 1μmole). Weigh 18.1 mg tyrosine and dissolve in 10ml reagent grade water by stirring to initial boil. Remove from heat and cool to approximately 40°C for assay.

0.5 M NaOH: Dissolve 1 gm. in 50 ml reagent grade water, or make a dilution from 1M or 10M NaOH.

Folin & Ciocalteus Phenol Reagent, use neat