Enzymatic Assay of Chymotrypsin (EC 126.96.36.199)
The scope of this procedure includes products that have a specification for Chymotrypsin activity. This assay procedure is not to be used to assay Chymotrypsin, Insoluble, Sigma-Aldrich Product Numbers C9134 and C7260, and Chymotrypsin, Acrylic Beads, Sigma-Aldrich Product Number C5407.
3.1. Purified Water - water from a deionizing system, resistivity > or = 18MΩ•cm @ 25ºC
3.2. Unit Definition – One unit will hydrolyze 1.0 µmole of BTEE per minute at pH 7.8 at 25ºC.
3.3. BTEE - N-Benzoyl-L-Tyrosine Ethyl Ester
T = 25ºC, pH = 7.8, A256nm, Light path = 1 cm
Continuous Spectrophotometric Rate Determination
7.3.1. 80 mM Tris HCl Buffer, pH 7.8 at 25ºC (Buffer)
Prepare a 9.69 mg/ml solution in purified water using Trizma® Base, Sigma-Aldrich Product Number T1503. Adjust the pH of this solution to 7.8 at 25°C.
7.3.2. 1.18 mM N-Benzoyl-L-Tyrosine Ethyl Ester Solution (BTEE)
188.8.131.52. Weigh 37 mg of N-Benzoyl-L-Tyrosine Ethyl Ester, Sigma-Aldrich Product Number B6125, into a 100 mL Class A volumetric flask.
184.108.40.206. Dilute the BTEE in 63.4 ml of Methanol, Sigma-Aldrich Product Number M1775, with swirling.
220.127.116.11. QS this solution to 100 ml using purified water. Invert the flask several times to ensure complete mixing.
7.3.3. 2 M Calcium Chloride Solution (CaCl2)
Prepare a 294 mg/ml solution in purified water using Calcium Chloride Dihydrate, Sigma-Aldrich Product Number C3881.
7.3.4. 1 mM Hydrochloric Acid Solution (HCl)
Prepare a solution in purified water by diluting 0.10 ml of Hydrochloric Acid Solution, Sigma-Aldrich Product Number H3162, to 100 ml in a 100 ml Class A volumetric flask. Mix by inversion and place on ice.
7.3.5. Chymotrypsin Enzyme Solution
18.104.22.168. Immediately before use prepare a solution containing 2-5 chymotrypsin units per milliliter in cold Reagent 7.3.4 (HCl).
22.214.171.124. For chymotrypsin impurity testing, prepare samples at 10 mg/ml in cold Reagent 7.3.4 (HCl).
7.4.1. Pipette (in milliliters) the following reagents into suitable quartz cuvettes:
|Reagent 7.3.1 (Buffer)||1.42||1.42|
|Reagent 7.3.2 (BTEE)||1.40||1.40|
|Reagent 7.3.3 (CaCl2)||0.08||0.08|
7.4.2. Mix by inversion and equilibrate to 25ºC using a suitably thermostatted spectrophotometer. Monitor the A256nm until constant.
7.4.3. Add the following (in milliliters) running each blank or test level one at a time:
|Reagent 7.3.4 (HCl)||0.10||-----|
|Reagent 7.3.5 (Test)||-----||0.10|
7.4.4. Immediately mix by inversion and record the increase in A256nm for approximately 3-5 minutes.
7.4.5. Repeat steps 7.4.3 and 7.4.4 until all blank and test reactions are complete.
7.4.6. Obtain the ΔA256nm/minute for both the blank and test reactions using the maximum linear rate over a one minute interval using at least 4 points.
|7.5.1.||Units/ml Enzyme =||(ΔA256 Test - ΔA256 Blank) X (3) X (df)|
|(0.964) X (0.10)|
3 = Volume (in milliliters) of reaction mix
df = Dilution factor
0.964 = Millimolar extinction coefficient of BTEE at 256nm
0.10 = Volume (in milliliters) of test used in assay
|7.5.2.||Units/mg Solid =||units/mL Enzyme|
|mg Solid/mL Enzyme|
|7.5.3.||Units/mg Proteinb=||units/mL Enzyme|
|mg Protein/mL Enzyme|
7.6. FINAL ASSAY CONCENTRATION
In a 3.00 mL reaction mix, the final concentrations are 38 mM Tris, 0.55 mM N-Benzoyl-L-Tyrosine Ethyl Ester, 14% (v/v) Methanol, 53mM Calcium Chloride, 0.03 mM Hydrochloric Acid, and 0.2 - 0.5 units of Chymotrypsin.