To standardize a procedure for the enzymatic assay of Chymotrypsin.
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
BTEE + H2O chymotripsin > N - Benzoyl - L - Tyrosine + Ethanol
It is the responsibility of all trained Analytical Services laboratory personnel to follow this protocol as written.
Refer to the Material Safety Data Sheet (MSDS) for hazards and appropriate handling precautions.
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)
126.96.36.199. Weigh 37 mg of N-Benzoyl-L-Tyrosine Ethyl Ester, Sigma-Aldrich Product Number B6125, into a 100 mL Class A volumetric flask.
188.8.131.52. Dilute the BTEE in 63.4 ml of Methanol, Sigma-Aldrich Product Number M1775, with swirling.
184.108.40.206. 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
220.127.116.11. Immediately before use prepare a solution containing 2-5 chymotrypsin units per milliliter in cold Reagent 7.3.4 (HCl).
18.104.22.168. 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)
|Reagent 7.3.2 (BTEE)
|Reagent 7.3.3 (CaCl2)
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)
|Reagent 7.3.5 (Test)
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.
||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
||Units/mg Solid =
||mg Solid/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.
8. REFERENCES & ATTACHMENTS
Wirnt, R., Bergmeyer, H. U., ed., Chymotrypsin, Methods of Enzymatic Analysis, New York (1974), 1009-1012.
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