Enzymatic Assay of Trypsin
1. OBJECTIVE
To standardize a procedure for the enzymatic assay of Trypsin at Sigma-Aldrich St. Louis.
2. SCOPE
The scope of this procedure is for all products that have a specification for Trypsin activity using Nα-Benzoyl-L-Arginine Ethyl Ester as a substrate.
3.1 Purified Water = water from a deionizing system, resistivity > or = 18MΩ•cm @ 25ºC
3.2 BAEE - Nα-Benzoyl-L-Arginine Ethyl Ester
3.3 Unit Definition - One BAEE unit will produce a ΔA253nm of 0.001 per minute with BAEE as substrate at pH 7.6 at 25ºC in a reaction volume of 3.20 mL.
4. DISCUSSION
BAEE+ H2O Trypsin > Nα-Benzoyl-L-Arginine + Ethanol
5. RESPONSIBILITIES
It is the responsibility of trained Analytical Services laboratory personnel to follow this procedure as written.
6. SAFETY
Refer to Material Safety Data Sheets (MSDS) for hazards and appropriate handling precautions.
7.1 CONDITIONS:
T = 25°C, pH = 7.6, A253nm, Light path = 1 cm
7.2 METHOD:
Continuous Spectrophotometric Rate Determination
7.3 REAGENTS:
7.3.1 67mM Sodium Phosphate Buffer, pH 7.6 at 25°C (Buffer)
Prepare a 8.04 mg/ml solution in purified water using Sodium Phosphate, such as Sigma-Aldrich Product Number , S0751. Adjust to pH 7.6 at 25°C with 1 N NaOH.
7.3.2 0.25mM Nα-Benzoyl-L-Arginine Ethyl Ester (BAEE)
Prepare a 0.086 mg/ml solution in Reagent 7.3.1 (Buffer) using Nα-Benzoyl-L-Arginine Ethyl Ester, such as Sigma-Aldrich Product Number , B4500.
7.3.3 1 mM Hydrochloric Acid Solution (HCl)
Prepare a 1:1000 dilution in purified water using a 1 N HCl solution such as Sigma-Aldrich Product Number , 318949.
7.3.4 Trypsin Enzyme Solution (Trypsin)
Immediately before use, prepare a solution containing 425-575 units/mL of Trypsin in cold Reagent 7.3.3 (HCl).
7.4 TEST METHOD
7.4.1 Pipette (in milliliters) the following reagents into suitable quartz cuvettes:
| Blank | Test-1 | Test-2 | Test-3 | |
| Reagent 7.3.2 (BAEE) | 3.00 | 3.00 | 3.00 | 3.00 |
| Reagent 7.3.3 (HCl) | 0.20 | 0.10 | 0.05 | ---- |
7.4.2 Mix by inversion and equilibrate to 25°C. Monitor the A253nm until constant, using a suitably thermostatted spectrophotometer. Then add:
| Reagent 7.3.4 (Trypsin) | ---- | 0.10 | 0.15 | 0.20 |
7.4.3 Immediately mix by inversion and record the increase in A253nm for 5 minutes. Using a 1 minute time period and a minimum of 4 data points, obtain the A253nm/min using the maximum linear rate for both the Test and Blank.
| 8.1 | BAEE units/ml = | (ΔA253nm/min Test - ΔA253nm/min Blank)(df) |
| (0.001)(VE)(1) |
| 8.2 | BAEE units/mg solid = | BAEE Units/mL enzyme)(df) |
| mg solid/mL enzyme |
where:
df = Dilution Factor
0.001= The change in A253nm/minute per unit of Trypsin at pH 7.6 at 25ºC in a 3.2 ml reaction mix
VE = Volume (in milliliters) enzyme used in step 7.4.2
1 = Volume of Reaction Mixture per unit definition
8.3 FINAL ASSAY CONCENTRATION :
In a 3.20 mL reaction mix, the final concentrations are 62.8 mM Sodium Phosphate, 0.23 mM Nα-Benzoyl-L-Arginine Ethyl Ether, 0.031 - 0.063 mM Hydrochloric Acid, 42.5 – 115.0 units of Trypsin.
9.1 Bergmeyer, H.U., Gawehn, K., and Grassi, M. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H.U., ed) Volume I, 2nd ed., 515-516, Academic Press, Inc., New York, NY.
9.2 Replaces PROC-CHR-OP-002136 (v1) for Analytical Services use.
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